Study of Z-Disc-Associated Signaling Networks in Skeletal Muscle Cells by Functional and Global Phosphoproteomics

PHDTHESIS Study of Z-disc-associated Signaling Networks in Skeletal Muscle Cells by Functional and Global Phosphoproteomics

Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau

vorgelegt von

Lena Reimann geboren in Bielefeld Freiburg im Breisgau 01.08.2016

Angefertigt am Institut für Biologie II AG Biochemie und Funktionelle Proteomforschung zellulärer Systeme unter der Leitung von Prof. Dr. Bettina Warscheid Dekan der Fakultät für Biologie: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Stefan Rotter

Betreuer der Arbeit: Prof. Dr. Bettina Warscheid

Referent: Prof. Dr. Bettina Warscheid Koreferent:Prof. Dr. Jörn Dengjel Drittprüfer: Prof. Dr. Gerald Radziwill

Datum der mündlichen Prüfung:21.10.2016 ART IS I, science is we. - Claude Bernard

Zusammenfassung

Als essentielle, strukturgebende Komponente des Sarkomers spielt die Z-Scheibe eine maßge- liche Rolle für die Funktionalität der quergestreiften Muskulatur. Die stetige Identifizierung von neuen Z-Scheiben-lokalisierten Proteinen, sowie deren Relevanz in muskulären Krankheits- bildern, hat die Z-Scheibe zunehmend in den Fokus der aktuellen Forschung gerückt. Neben ihrer strukturgebenden Funktion zeigen neuere Studien, dass die Z-Scheibe ein Hotspot für Signalprozesse in Muskelzellen ist. Bisher gibt es jedoch keine globalen Untersuchungen zur Aufklärung der komplexen Signalwege assoziiert mit dieser Struktur. Um Z-Scheiben-assoziierte Signalprozesse näher zu charakterisieren, wurde im ersten Teil dieser Arbeit eine großangelegte Phosphoproteomstudie mit ausdifferenzierten, kontrahierenden C2C12 Myotuben durchgeführt. Zu diesem Zweck wurden die tryptisch ver- dauten Proteine mittels SCX-Chromatographie fraktioniert. Die anschließende Phosphopep- tidanreicherung erfolgte mit Titandioxid, gefolgt von einer hochauflösenden massenspek- trometrischen Analyse. Insgesamt wurden 11.369 Phosphorylierungsstellen, darunter 586 in sarkomerischen Proteinen gefunden. Von diesen wurden 70% in Z-Scheiben-lokalisierten Proteinen identifiziert. Des Weiteren konnte Filamin c (FLNc) zusammen mit mehreren seiner bekannten Interaktionspartner als Phosphorylierungshotspot in der Z-Scheibe identifiziert werden. Die drei C-terminalen FLNc Phosphorylierungsstellen S2621, S2625 und S2633 in der Hinge 2-Region sowie in Domäne 24 wurden durch in silico Analysen als Substrate der Proteinkinase C (PKC) vorhergesagt. Radioaktive und MS-basierte in vitro Kinaseexperimente bestätigten S2625 als PKC-Substrat. Um die Prognose zu testen, dass FLNc im Bereich der Hinge 2-Region durch die Cysteinprotease Calpain 1 geschnitten wird, wurde die phospho- rylierungsabhängige Prozessierung dieser beiden Proteine mittels top down Massenspektrome- trie näher untersucht. Dabei wurde erstmalig gezeigt, dass sich der Calpain-induzierte Schnitt an FLNc Y2626 in direkter Nachbarschaft zur Phosphorylierungsstelle befindet. Des Weitern konnte durch quantitative Western Blot-Analysen und Co-Immunopräzipitationsexperimente (Co-IP) gezeigt werden, dass die Phosphorylierung das Protein vor der Calpainolyse schützt. Im zweiten Teil der Arbeit wurde der Pi3k/Akt Signalweg in kontrahierenden Myotuben durch Stimulation mit IGF-1 und Inhibierung mit LY294002 in einer globalen quantitativen Phosphoproteomstudie untersucht. Dazu wurde die Funktionalität des Ansatzes zunächst über quantitative Western Blot-Analysen mit phosphospezifischen Antikörpern, die gegen bekannte Substrate des Signalweges gerichtet sind, bestätigt. Für die anschließende Phospho- proteomanalyse wurden die Peptidproben zunächst mittels SCX-Chromatographie fraktioniert. Anschließend wurden die Fraktionen mittels Titandioxid für Phosphopeptide angereichert und massenspektrometrisch analysiert. Insgesamt wurden 16.633 Phosphorylierungsstellen identifiziert, von denen 13.225 in zwei von drei Replikaten quantifiziert wurden. Motif-X Analysen der 243 signifikant herunterregulierten Phosphopeptide nach LY294002 Behandlung zeigte eine 148-fache Anreicherung des verlängerten, bisher unbekannten basophilen Motives RxRxxpSxxS in 20 Peptiden. In 19 dieser Peptide wurde auch das zweite Serin innerhalb dieses Motives als phosphoryliert identifiziert, was die Frage nach den Kinasen, die für diese Phosphorylierungen verantwortlich sind, aufwarf. In silico Vorhersagen und in vitro Kinase- experimente zeigten, dass S2234 und S2237 in FLNc von den Kinasen AKT beziehungsweise PKC phosphoryliert werden. Darüber hinaus konnte mithilfe von pull-down- und Co-IP- Experimenten gezeigt werden, dass diese beiden Phosphorylierungen die Interaktion zu dem neu identifizierten FLNc-Interaktionspartner FILIP1 verringern. Im letzten Teil dieser Arbeit wurde gezeigt, dass für die exakte Lokalisation einer Phospho- rylierung zum Teil andere Proteasen als Trypsin eingesetzt werden müssen. Obwohl Trypsin die am Weitesten verbreitete Protease in der Proteomforschung ist, eignet sie sich nicht, wenn sich die potentielle Phosphorylierunsgsstelle direkt C-terminal zu einem Arginin oder Lysin befindet. Am Beispiel von Myl12b wurde gezeigt, dass Thermolysin geeignet ist, zwei benachbarte Phosphorylierungsstelle C-terminal zu Arginin eindeutig zu identifizieren. Summary

Recent studies have questioned the role of the sarcomeric Z-disc as a solely rigid and structure- bearing component of the sarcomere. An increasing number of identified Z-disc proteins and the involvement of numerous Z-disc proteins in diseases and signaling pathways have emphasized the role of the Z-disc as a signaling hot spot in cross-striated muscle cells. Even though there have been studies addressing the role of several proteins associated with the myofibrillar Z-disc, no study globally investigating signal transduction networks associated with this structure has been published so far. With this background, the first part of this thesis focuses on the analysis of the global phosphoproteome in contracting C2C12 myotubes by large-scale phosphoproteomics. To this end, myotubes were lysed and proteins were digested using trypsin. Following SCX chromatography, phosphopeptides were enriched by titanium dioxide and analyzed by high resolution LC-MS. In total, 11,369 phosphosites, including 586 sarcomeric phosphosites, could be identified. Out of the sarcomeric sites, 70% were located on Z-disc and Z-disc- associated proteins, highlighting the Z-disc as signaling hot spot. Furthermore, the Z-disc- associated protein filamin c (FLNc) and several of its known interaction partners were found to be highly phosphorylated. In FLNc, three phosphosites, namely S2621, S2625 and S2633, were identified in its hinge 2 region and the beginning of its Ig-like domain 24. In silico kinase prediction proposed these sites to be substrates of protein kinase C (PKC). To test this hypothesis, radioactive and MS-based in vitro kinase assays were combined to obtain site-resolved data. The assays revealed that PKCα phosphorylates FLNc S2623 in a concentration-dependent manner. As the hinge 2 region and the Ig-like domain 24 were proposed to interact with the cysteine protease calpain, the phosphorylation-dependent association of FLNc and calpain was further analyzed. An MS-based top down approach was developed, which unequivocally identified FLNc Y2626 as calpain cleavage site for the first time. Based on the close proximity of the PKC phosphosite and the calpain cleavage site, the role phosphorylation was further analyzed using phospho-specific site mutants for quantitative Western blotting and co-immunoprecipitation experiments. The results revealed that phosphorylation of S2625 by PKCα effectively protects FLNc from calpainolysis. In the second part of this work, the quantitative changes in the phosphoproteome of contracting C2 myotubes were measured by large-scale phosphoproteomics in response to activation and inhibition of the Pi3k/Akt pathway. For this purpose, C2 myotubes were incubated either with IGF-1 or with LY294002 to activate or block the Pi3k/Akt pathway. Subsequently, cells were lysed and protein digests were fractionated by SCX chromatography, followed by enrichment of phosphopeptides by titanium dioxide and LC-MS analysis. In total, 16,633 phosphosites were identified of which 13,225 phosphopeptides could be quantified in two of three biological replicates. Motif-X analysis of the 243 significantly down-regulated peptides following treatment with LY294002 revealed a 148-fold enrichment of an extended, so far unknown basophilic motif RxRxxpSxxS in 20 peptides. As 19 of these 20 peptides were also phosphorylated at the second serine, it raised the issue which two kinases might be involved in the phosphorylation in such close proximity. One of the proteins comprising this phosphorylation motif was FLNc phosphorylated at S2234 and S2237. In silico kinase prediction and in vitro kinase assays revealed that AKT is phosphorylating S2234, while PKCα was shown to mediate phosphorylation of S2237. Furthermore, pull-down and co- immunoprecipitation experiments demonstrated, that these phosphosites are mandatory to decrease FLNc binding to the newly identified interaction partner Filip1. In the last part of this work, different proteases were tested with the aim to accurately localize phosphosites. Even though trypsin is the most commonly used protease in bottom-up proteomics, it hampers the site-specific localization of phosphosites C-terminal to arginine or lysine. In this work, thermolysin was proven to be a suitable protease to unequivocally identify two neighboring phosphosites C-terminal to arginine in Myl12b. Declaration

1. Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

2. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

3. Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität Freiburg sind mir bekannt, insbesondere weiß ich, dass ich vor Vollzug der Promotion zur Führung des Doktortitels nicht berechtigt bin.

Ort, Datum Unterschrift

Contents

1. Introduction1 1.1. The Muscle ...... 1 1.1.1. The Z-disc ...... 4 1.1.2. Filamins ...... 7 1.2. Phosphoproteomics ...... 12 1.2.1. Protein phosphorylation ...... 12 1.2.2. Phosphoproteomics workﬂow ...... 15 1.2.3. Large-scale skeletal muscle phosphoproteomic studies ...... 20

2. Aims of this study 23

3. Results and Discussion 25 3.1. Identification of the myofibrillar Z-disc as hot spot of protein phosphorylation 25 3.1.1. Optimization of lysis conditions for muscular proteomics ...... 25 3.1.2. Formation of mature Z-discs in cultured myotubes ...... 28 3.1.3. Characterization of the basal phosphoproteome of fully differentiated, contracting C2C12 myotubes ...... 29 3.1.4. The Z-disc is a sarcomeric phosphorylation hot spot ...... 33 3.1.5. Identification of filamin C as target of PKCα ...... 38 3.1.6. Establishment of an on-bead calpain cleavage assay ...... 44 3.1.7. Tyrosine C-terminally to the PKCα substrate site is the main calpain 1 cleavage site in FLNc ...... 45 xii Contents

3.1.8. PKCα-mediated phosphorylation of S2623/S2624 protects human FLNc from limited proteolysis by calpain 1 in C2 cells ...... 48 3.1.9. PKC modulates the dynamic behavior of human FLNc through phosphorylation of S2623/S2624 in skeletal myotubes ...... 49 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes . . . . 53 3.2.1. Characterization of different Pi3k/Akt inhibitors in contracting C2 myotubes ...... 53 3.2.2. Study of the Pi3k/Akt signaling pathway by large-scale quantitative phosphoproteomics ...... 56 3.2.3. Characterization of the Pi3k-dependent phosphoproteome ...... 58 3.2.4. The unique insertion in Ig-like domain 20 of filamin C is multiply phosphorylated in myotubes ...... 62 3.2.5. Identification of Filip1 as a novel FLNc interaction partner ...... 65 3.2.6. Identification of R1751 as calpain cleavage site in the hinge 1 region of FLNc ...... 69 3.3. New insights into smooth muscle myosin phosphorylation ...... 73

4. Material and Methods 77 4.1. Cell lysis optimization for phosphoproteomic studies ...... 77 4.2. Establishment of different proteases for improved sequence coverage in LC- MS/MS analyses ...... 79

5. Final remarks and outlook 81

6. Publications and manuscripts 87 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot with PKCα modulating protein dynamics ...... 87 6.1.1. Abstract ...... 91 6.1.2. Introduction ...... 92 6.1.3. Experimental procedures ...... 95 Contents xiii

6.1.4. Results ...... 115 6.1.5. Discussion ...... 126 6.2. Quantitative Phosphoproteomics and Proximity Proteomics Reveals FILIP1 as a Phosphorylation-dependent Interactor of FLNc in Skeletal Muscle Cells 198 6.2.1. Abstract ...... 202 6.2.2. Introduction ...... 203 6.2.3. Experimental procedures ...... 206 6.2.4. Results ...... 219 6.2.5. Discussion ...... 228 6.3. New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation ...... 256 6.3.1. Abstract ...... 257 6.3.2. Introduction ...... 257 6.3.3. Material and methods ...... 259 6.3.4. Results ...... 261 6.3.5. Discussion ...... 265

Bibliography 271

Appendix 307

A. Figures 307 A.1. Supplemental figures for identification of Z-disc as myofibrillar phosphorylation hot spots ...... 307 A.2. Supplemental figures for quantitative analysis of the myofibrillar phosphoproteome ...... 312 xiv Contents

B. Tables 317 B.1. Supplemental tables for identiﬁcation of Z-disc as myoﬁbrillar phosphorylation hot spots ...... 317 B.2. Supplemental tables for characterization of Pi3k/Akt-mediated signaling events in myotubes ...... 319 B.3. Supplemental tables for new insights into smooth muscle myosin phosphorylation ...... 321

C. Vector Maps 323 C.1. Vector maps of vector backbones ...... 323

D. Alignments 329 D.1. Alignment of ﬁlamin isoforms ...... 329

E. Parameters 333 E.1. Mascot parameters and results for standard protein proteolysis ...... 333 E.2. MaxQuant parameters ...... 348

Publications and Poster 351 Nomenclature xv

Nomenclature amino acids

All amino acid names used in this work will be termed in the one letter code according to ’IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature and symbolism for amino acids and peptides. Recommendations 1983’. European Journal of Biochemistry, vol. 138(1): pp.9-37. gene names

All mouse genes names used in this work will be following the ’Rules for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and Rat’ (2005) by the International Committee on Standardized Genetic Nomenclature for Mice (http://www. informatics.jax.org/mgihome/nomen/gene.shtml). Thus, they are generally italicized with only the ﬁrst letter in uppercase and the remaining letters in lowercase. protein names

Unless otherwise stated all mouse protein names used in this work will be following the ’Rules for Nomenclature of Genes, Genetic Markers, Alleles, and Mutations in Mouse and Rat’ (2005) by the International Committee on Standardized Genetic Nomencla- ture for Mice. Thus, they are written with only the ﬁrst letter in uppercase and the remaining letters in lowercase. Nomenclature for human proteins and genes (e.g. for antibodies which are directed against a human epitope) are following the HUGO Gene Nomenclature Committe (HGNC) (http://www.genenames.org/). xvi Acronyms

Acronyms

ABC ammonium bicarbonate buffer ABD actin-binding domain ADP adenosine diphosphate Ampk 5’ adenosine monophosphate-activated protein kinase Ankrd2 ankyrin repeat domain-containing protein 2 ATP adenosine triphosphate Bag3 Bcl2-associated athanogene 3 BioID proximity-dependent biotin identification BP biological process c-Myc Myc proto-oncogene protein CamkII Ca2+/calmodulin-dependent protein kinase II CASA chaperone-assisted selected autophagy CC cellular component CID collision-induced dissociation CNBr cyanogen bromide co-IP co-immunoprecipitation DAPI 4’,6-diamidino-2-phenylindole DHB 2,5-dihydroxybenzoic acid DMSO dimethyl sulfoxide Dusps dual-specificity phosphatases Edc3 enhancer of mRNA-decapping protein 3 Eef2 eukaryotic elongation factor 2 Eef2k eukaryotic elongation factor 2 kinase EGFP enhanced green fluorescence protein Eif4b eukaryotic translation initiation factor 4B Acronyms xvii

Enah enabled homolog EPS electrical pulse stimulation ERLIC electrostatic repulsion-hydrophilic interaction chromatography ETD electron transfer dissociation FASP filter-aided sample preparation FILIP1 filamin a-interacting protein 1 FLNa filamin A FLNb filamin B FLNc filamin C FRAP fluorescence recovery after photobleaching Gapdh glyceraldehyde-3-phosphate dehydrogenase GO gene ontology

Gsk3β glycogen synthase kinase-3 β Gtf2f1 general transcription factor 2f HCD higher-energy collisional dissociation hFLNc human ﬁlamin c HILIC hydrophilic interaction chromatography hpH high pH hpH-RP high pH reversed phase chromatography Hspb1 heat shock protein beta-1 Ig-like immunoglobulin-like IGF-1 insulin-like growth factor 1 Ilk integrin-linked kinase IMAC ion metal afﬁnity chromatography IMMs immortalized mouse skeletal myoblasts iTRAQ isobaric tags for relative and absolute quantitation Larp1 La-related protein 1 xviii Acronyms

LC-MS liquid chromatography-mass spectrometry LC-MS/MS liquid chromatography tandem mass spectrometry Ldb3 LIM domain binding protein 3 LIT linear ion trap Map3k3 mitogen-activated protein kinase kinase kinase 3 Mapk mitogen-activated protein kinase MF molecular function mFLNc mouse filamin c MOAC metal oxide affinity chromatography MS mass spectrometry MSA multi-stage activation mTOR mammalian target of rapamycin mTORC1 mTOR complex 1 mTORC2 mTOR complex 2 Myl12b myosin regulatory light chain 12B m/z mass to charge N-terminal amino-terminal PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline Pfkfb2 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 Pi3k phosphatidylinositol-3 kinase PKA protein kinase A PKB protein kinase B PKC protein kinase C PLA proximity ligation assay PPI protein-protein interaction PTMs post-translational modifications Acronyms xix

RP reversed phase Rsk p90 ribosomal S6 kinase S6k p70 S6 kinase SAX strong anion exchange SCX strong cation exchange SDS sodium dodecyl sulfate SEM standard error of the mean Sgk1 serum- and glucocorticoid-induced kinase 1 SILAC stable isotope labeling by amino acids in cell culture SPECHT single-stage phosphopeptide enrichment and stable-isotope tagging Src proto-oncogene tyrosine-protein kinase Src SVM support vector machine Synpo synaptopodin Synpo2 myopodin Synpo2l tritopodin/synaptopodin 2-like Tbc1d4 TBC1 domain family member 4 TFA triﬂuoroacetic acid

TiO2 titanium dioxide TMT tandem mass tags WT wildtype Xirp1 Xin actin-binding repeat-containing protein 1 Xirp2 Xin actin-binding repeat-containing protein 2 Xirps Xin actin-binding repeat-containing proteins

CHAPTER 1

Introduction

1.1 The Muscle

There are over 600 muscles in the human body (Marieb and Hoehn, 2007) which account for up to 40-50% of the total human body weight (reviewed by Sandri, 2010) and are therefore one of the most abundant tissues in humans and animals. Muscles are not only responsible for body movement, but also play an important role in maintaining and stabilizing the posture, functionality of inner organs and generating the heart beat. This wide variety of functions is covered by three sub-types of muscles: smooth, cardiac and skeletal. Smooth and cardiac muscles are responsible for involuntary contractions of inner organs, such as the gastrointestinal tract and the heart, whereas skeletal muscles are controlled by the somatic nervous system. Thus they are responsive to reﬂexes and controlled, voluntary impulses which enable motion (Pollard et al., 2008).

Both cardiac and skeletal muscles are classiﬁed as striated muscles due to their X-ray diffraction pattern ﬁrst described by Bear (1944) even though they meet two different require- ments with regard to contraction intensity and repetitions. In electron microscopy, striated muscle appears as an alternating striated highly structured pattern of bright isotropic I-bands and dark, anisotropic A-bands with a middle line, called M-line (Gilëv, 1962) (Figure 1.1). The darker region within the I-band, the Z-disc (derived from the German word zwischen, 2 1.1. The Muscle

A Sarcomere I-band A-band I-band

Z-disc M-line Z-disc B

thick filament titin thin filament nebulin α-actinin

Figure 1.1.: Electron micrograph and schematic overview of the structural elements of one sarcomere. (A) Detailed view of a single sarcomere, the basic unit of contraction with the substructures Z-disc and I-band as well as M-line and A-band (modified from Bennett et al., 1986). The I-band comprises the thin filaments whereas the A-band reflects the overlap of thin and thick filaments. (B) The schematic overview reveals the main structural components of the Z-disc. While titin is spanning the whole sarcomere, α-actinin is forming the backbone of the Z-disc. The thick filaments consist of myosin and the thin filaments of actin and nebulin, respectively.

between) borders the ends of one sarcomere, which is the basic unit of contraction within the muscle. Sarcomeres are composed of the four filamentous systems actin, myosin, titin and nebulin (Gregorio et al., 1999). The latter two are responsible for the balance of elasticity and stiffness of the sarcomere and act as its organization template and length-determining factor (Fürst et al., 1988; Wang and Wright, 1988). In contrast, the actin and myosin-filaments are in charge of the sarcomeric contraction. As the muscle is stimulated via Ca2+ release from the sarcoplasmic reticulum in response to a neuronal stimulus, myosin heads can bind to their myosin-binding sites on actin due to the hydrolysis of adenosine triphosphate (ATP) to organic phosphate and adenosine diphosphate (ADP). Thereby, a conformational change in myosin triggers the sliding of thick and thin filament against each other, thus pulling the 1.1. The Muscle 3

Z-disc towards the center of the sarcomere (Ebashi and Endo, 1968; Huxley and Niedergerke, 1954; Huxley and Hanson, 1954; Spudich and Watt, 1971). While M-lines are stretched and ﬂexible during this process the stiffness of the Z-discs during contraction are at least two-fold increased in comparison to the M-lines (reviewed by Gautel, 2011).

sinew

tendon bone fascia skeletal muscle epimysium

perimysium nerve, artery, vein

fascicle

endomysium muscle ber I-band A-band

myo bril Figure 1.2.: Schematic overview of the composition of a skeletal muscle. The tendon connects the muscle to the bone. Skeletal muscle is surrounded by fascia tissue and consists of multiple muscle ﬁber bundles (fascicles), which are wrapped by the perimysium. Individual muscle ﬁbers are surrounded by the endomysium. The bundle of fascicles representing the individual muscle is enclosed by the epimysium. Adapted from McKinley and O’Loughlin (2009). 4 1.1. The Muscle

Sarcomeres are only the smallest subunits of a skeletal muscle. Hundreds of sarcomeres together form one myofibril, which is in turn organized together with other arranged myofibrils in parallel to muscle fibers. Each muscle fiber is wrapped by the endomysium and bundles of muscle fibers build one fascicle, which is in turn surrounded by the perimysium (Figure 1.2). Together with nerves, arteries and veins a cluster of fascicles, covered by the epimysium form a skeletal muscle (Mayne and Sanderson, 1985). When an electrical impulse is transferred from a neuron via the neuromuscular junction to the muscle fiber, the muscle is contracting via shortening of the sarcomeres and thus the produced force is transduced from the myotendinous junctions to tendons and thereby to bones (reviewed by Berthier and Blaineau, 1997).

In order to study muscular processes ex vivo, Yaffe and Saxel (1977) isolated C2 cells from mouse limb muscle and generated the C2 cell line and their sub-clone the C2C12 cell line. Both cell types are today the most widely accepted and most frequently used ex vivo models for skeletal muscle cells and their differentiation. For example, Nedachi et al. (2009) and Nedachi et al. (2008) characterized the ability of contracting C2C12 myotubes to secrete various chemokines/cytokines in response to electrical pulse stimulation (EPS), thus certifying them as adequate in vitro muscle model system. Moreover, Manabe and colleagues showed that EPS of C2C12 myotubes induces and activates the same signaling pathways as observed in in vivo studies (Manabe et al., 2012). Furthermore, C2C12 myotubes represent a well-established and widely used cell system to study a variety of different mechanisms such as mammalian target of rapamycin (mTOR) independent autophagy (Mordier et al., 2000), 5’ adenosine monophosphate-activated protein kinase (Ampk) dependent changes in signaling pathways and protein names synthesis (Williamson et al., 2006) and TNF-α-induced muscle atrophy via regulation of the Akt/mTOR/FoxO1 pathway (Wang et al., 2014).

1.1.1 The Z-disc

As borders of the sarcomere, Z-discs have come into focus of diverse studies, revealing not only their role in the transduction of the mechanical force during muscle contraction but also their increasing importance in sarcomeric signaling (reviewed by Frank et al., 2006). Originally identified as Krause’s membrane (Jordan, 1933; Schafer, 1890), the Z-disc was 1.1. The Muscle 5 long seen as a rather rigid assembly of structural proteins (Forbes and Sperelakis, 1980). α- actinin, the first protein which was found to be specifically localized at the Z-disc (Maruyama and Ebashi, 1965) is defining its width by the number of layers varying from ~50 nm in fast twitch fibers to 100 nm in slow and cardiac fibers (Rowe, 1973). Together with the important structural proteins nebulin, titin, CapZ and obscurin (reviewed by Luther, 2009; Pyle and Solaro, 2004) α-actinin forms the structural backbone of Z-discs. Moreover, the protein- protein interaction (PPI) between α-actinin and CapZ is creating an anchoring complex for actin filaments which is thus building the connection from the Z-disc to the thin filaments (Papa et al., 1999).

The importance of the Z-disc for sarcomeric function and integrity has become evident in the last two decades as mutations and dysfunction of several structural and further newly identiﬁed Z-disc proteins were found to play a major role in different kinds of muscular dystrophies (reviewed by Frank et al., 2006; Knöll et al., 2011). Moreover, Z-discs not only house and anchor structural proteins but also a still increasing number of proteins with diverse functions in mechanosensation and signaling (reviewed in Faulkner et al., 2001; Frank and Frey, 2011; Luther, 2009) (Figure 1.3). Currently, according to the NCBI gene database1 over 100 gene products are associated with the Z-disc in human (111), rat (113) and mouse (109). However, these annotations have to be treated with caution, since the correct allocation of proteins to the Z-disc structure is a challenging task for three reasons: giant proteins like titin (Labeit et al., 1992) and obscurin (Young et al., 2001) are (i) only partly located at the Z-disc as they are spanning the whole sarcomere. Additionally, several proteins are only located (ii) at different cellular states and physiological situations or (iii) temporarily at the Z-disc.

Examples of proteins which are predominantly expressed at the Z-disc but are able to shuttle to the nucleus upon different stimuli are synaptopodin-2 (myopodin) (Linnemann et al., 2010)) and ankyrin repeat domain-containing protein 2 (Ankrd2) (Kojic et al., 2004)). Faul et al. (2007) demonstrated that phosphorylation of myopodin by protein kinase A (PKA)

1 http://www.ncbi.nlm.nih.gov/gene/?term=z-disc 6 1.1. The Muscle

Figure 1.3.: Schematic overview of the Z-disc. The sarcomeric border structure not only comprises structural proteins, but also a number of shuttling, signaling proteins such as calcineurin and protein kinase C. Adapted from Pyle and Solaro (2004).

or Ca2+/calmodulin-dependent protein kinase II (CamkII) mediates binding of 14-3-3 proteins and subsequent nuclear import. Even though the exact mechanism is still unrevealed for Ankrd2, Tsukamoto et al. (2008) showed, that Ankrd2 is translocated to the nucleus in sarcomere-damaged myoﬁbrils and interacts there with the transcriptions factors YB-1, PML and p53. A family of proteins that is only located at the Z-disc under different damage conditions are the Xin actin-binding repeat-containing proteins (Xirps) Xin, XIRP1 and XIRP2 (van der Ven et al., 2006). Otten et al. (2012) and Eulitz et al. (2013) described that XIRPs translocate to the Z-disc in early developmental states, after exercise-induced myoﬁbril damage and during remodeling processes within the muscle.

Z-disc proteins were not only shown to fulﬁll an important role in mechano-sensing (Hoshijima, 2006), but are also involved in autophagy and degradation processes. Several proteins were found to be connected to the Z-disc under stress conditions, thus they are serving as inner cellular control for damaged proteins. Among these are proteins, such as the E3 ubiquitin ligases MuRF1 (Centner et al., 2001) and MuRF3 (Spencer et al., 2000) as well as 1.1. The Muscle 7

Calpain-1 (Raynaud et al., 2006)) and protein complexes like the components of the chaperone- assisted selected autophagy (CASA) machinery (Arndt et al., 2010; Ulbricht et al., 2013). Besides the degradation of damaged proteins these processes also affect different signaling pathways ultimately regulating proliferation, adaption to stress and apoptosis (reviewed by Willis et al., 2010).

Among the proteins that are only temporarily or partially located at or in the Z-disc are several proteins involved in developmental and cellular signaling processes. These are not only kinases, such as protein kinase C (PKC) (Gu and Bishop, 1994), PAK1 (Ke et al., 2004), integrin-linked kinase (Ilk) (Hannigan et al., 1996) and mitogen-activated protein kinase (Mapk) (Purcell et al., 2004; Sheikh et al., 2008; Vahebi et al., 2007) but also the phosphatase calcineurin (Heineke et al., 2005) indicating the involvement of the Z-disc in phosphorylation-mediated signaling pathways.

1.1.2 Filamins

Filamins are a family of actin-binding proteins consisting of the three isoforms filamin A (FLNa), filamin B (FLNb) and filamin C (FLNc), encoded by the three different gene names FLNA, FLNB and FLNC, respectively. All three isoforms share a sequence homology of 60-80% (reviewed by Stossel et al., 2001) and besides their function to bind actin, filamins interact with over 90 proteins, including intracellular signaling factors, membrane receptors, transcription factors and cytoskeletal components (reviewed by Nakamura et al., 2011; Popowicz et al., 2006).

All ﬁlamins consist of an amino-terminal (N-terminal) actin-binding domain (ABD), followed by 24 highly homologous immunoglobulin-like (Ig-like) domains of 93-103 amino acids length (Thompson et al., 2000) (Figure 1.4). For FLNa it was demonstrated that the Ig-like domains, 16-17, 18-19 and 20-21 are arranged in pairs (Lad et al., 2007). These Ig-like domain dimers are also suggested for FLNb and FLNc (Heikkinen et al., 2009) and function as an auto-inhibited force-activatable mechano-sensor thus regulating the binding of several interaction partners, such as β-integrin (Rognoni et al., 2012). 8 1.1. The Muscle

ABactin-binding dimerization domain domain

2 hinge 2

Ig-like in domain 1 od do ma r m o a d in 1 d o r rod Ig-like domain 15 domain 1 90° F-actin

hinge 1 hinge 1 calpain rod cleavage domain 2 sites hinge 2 actin-binding domain Ig-like domain 24 dimerization domain

Figure 1.4.: Schematic illustration of the arrangement of the FLNa domains and the suggested model for the interaction of FLNa with filamentous actin. (A) Like demonstrated exemplary for FLNa, all filamins contain an N-terminal ABD followed by 24 Ig-like domains of ∼96 amino acids each. The two domains rod 1 (Ig-like domain 1-15) and rod 2 (Ig-like domain 16-23) are interconnected by the ∼35 amino acid long hinge 1 region. A second hinge region is located between the rod 2 domain and Ig-like domain 24 which functions as dimerization domain. Both hinge regions have been suggested to be targets of calpain cleavage in FLNa and FLNc. Modified from Stossel et al. (2001). (B) The dimeric structure of filamin a is able to cross-link actin filaments mediated by the binding of the ABD to two orthogonally arranged strains of filamentous actin. The two hinge region facilitate binding due to their flexibility. Modified from Zhou et al. (2010).

The domain structure of all filamins is interconnected by two flexible hinge regions positioned between the Ig-like domains 15 and 16 (hinge 1) and d23 and d24 (hinge 2), respectively (reviewed by Stossel et al., 2001). These hinge regions are characterized by a high sequence divergence between the isoforms and a reduced sequence homology of 45 % (van der Flier and Sonnenberg, 2001). Even though the two hinge regions show minor sequence homology between the different filamins, they were suggested to be a target of calpain cleavage in all isoforms (Gorlin et al., 1990; Tigges et al., 2003). Moreover, the hinge 2 region was suggested to fulfill an important regulatory role together with domain 24 for the dimerization of all proteins within the filamin family (Himmel et al., 2003). Due to their Y-shaped form, the dimers are capable of cross-linking actin-filaments via their ABDs (Pudas et al., 2005; Sjekloca´ et al., 2007) (Figure 1.4 B). Additionally, hinge 1 was shown to be important for the flexibility of 1.1. The Muscle 9 the protein and the maintenance of the viscoelastic functionality of the cross-linked actin networks against stresses (Gardel et al., 2006). Depending on the differentiation state and the muscle cell type, the hinge 1 region may be removed by alternative splicing events in FLNc and FLNb, thus reducing the flexibility of the protein (van der Flier et al., 2002; Xie et al., 1998). However, the molecular mechanisms leading to this splicing are not revealed yet.

While FLNa and FLNb are ubiquitously expressed and localized to actin networks (Xie et al.,1998, Chiang and Greaser, 2000, Thompson et al., 2000), FLNc is predominantly expressed in skeletal and cardiac muscle cells (Maestrini et al., 1993). Its expression is increased early during myoﬁbrillar development (Goetsch et al., 2005) thus helping Z-disc assembly by acting as a molecular scaffold protein (van der Ven et al., 2000a). Moreover, even in in fully developed sarcomeres the major cellular pool of FLNc is located at the edges of the Z-disc with only a minor portion being at the sarcolemma in association with the cortical actin cytoskeleton, intercalated discs and myotendinous junctions, respectively (Thompson et al., 2000; van der Ven et al., 2000b).

Within the Z-disc, FLNc plays a central role in PPIs and signaling events (Fujita et al., 2012). First identified as chicken gizzard filamin by Wang et al. (1975) and Hartwig and Stossel (1975) the proteins name convention changed from actin-binding protein 280 like (ABP-L), γ-filamin and filamin-2 (FLN-2) to finally FLNc. FLNc features a unique insertion of 82 amino acid within domain 20 which is conserved up to almost 100% among mammals. This region is supposed to be responsible for the inner-sarcomeric targeting to the borders of the Z-discs (van der Ven et al., 2000a). Noteworthy, this domain was shown to be essential for the binding of FLNc to other Z-disc proteins such as myotilin (van der Ven et al., 2000a), aciculin (Molt et al., 2014), myopodin (Linnemann et al., 2010), KY protein (Beatham et al., 2004) and the two isoforms Xin and Xin actin-binding repeat-containing protein 2 (Xirp2) from the family of Xirps (van der Ven et al., 2006) (Figure 1.5). Additionally, FLNc is amongst others a target of the kinases protein kinase B (PKB) (Murray et al., 2004b), PKC (Kawamoto and Hidaka, 1984; Tigges et al., 2003), Ampk (Wallach et al., 1977; Wallach et al., 1978) and CamkII (Ohta and Hartwig, 1995). Just recently, it was shown that FLNc interacts 10 1.1. The Muscle with aciculin in regions of myofibrillar microdamage and is characterized by extremely fast fluorescence recovery after photobleaching (FRAP) dynamics, an indicator for its role as a fast accommodating signaling protein (Leber et al., 2016).

Aciculin F-actin Myopodin Myotilin Xin -integrin Ky-protein FATZ/Myozenins/Calsarcins Calpain -,-sarcoglycans, dimerization

Figure 1.5.: Schematic overview of FLNc’s domain structure and a selection of its domain- specific interaction partners. Like the other filamins, FLNc contains an ABD, which mediates binding to filamentous actin followed by 24 Ig-like domains. In contrast to the other filamins, FLNc features a unique 82 amino acid comprising insertion within Ig-like domain 20, depicted in green. Most of the FLNc specific binding partner interact with the protein by this specific region. Modified from van der Ven et al. (2006).

Consistent with its central function as signaling hub, mutations in the gene coding for FLNc were identiﬁed to cause severe muscle diseases, termed ﬁlaminopathies (reviewed by Fürst et al., 2013). The large variety of diseases are generally characterized by progressive muscle weakness and have been extensively studied in the last decade to reveal the mechanistic role of FLNc (Bönnemann et al., 2003; Duff et al., 2011; Guergueltcheva et al., 2011; Vorgerd et al., 2005). For Duchenne muscular dystrophy and limb-girdle muscular dystrophy patients it was shown that FLNc is no longer located at the Z-disc, but instead at the sarcolemma (Thompson et al., 2000; van der Ven et al., 2000b). In contrast, patients with mutations in the two rod domains or the dimerization domain 24 often show large protein aggregates in the cytoplasm (Vorgerd et al., 2005). Within these plaques, accumulations of FLNc and its interaction partners Xin and myotilin, as well as Z-disc-associated proteins, desmin and αB-crystallin have been found (Kley et al., 2007; Luan et al., 2010; Nilsson et al., 2013; 1.1. The Muscle 11

Shatunov et al., 2009).

Although FLNc is described to be the muscle-speciﬁc ﬁlamin isoform it is also expressed in other tissues, such as lung, liver and kidney2. Moreover, FLNc was just recently found to have a functional role as tumor suppressor in gastric cancer cells (Qiao et al., 2015). While gene silencing of endogenous FLNc in tumor tissue enhances cell migration and invasion of neighboring tissues, less invasive carcinomas are characterized by higher FLNc expression levels.

2 http://www.proteinatlas.org/ENSG00000128591-FLNC/tissue 12 1.2. Phosphoproteomics

1.2 Phosphoproteomics

1.2.1 Protein phosphorylation

Today, more than 400 post-translational modifications (PTMs) are known, which increase the complexity of the human proteome dramatically. This modifications allow the cells to adapt dynamically to changing environmental conditions (reviewed by Hart and Ball, 2013; Wilkins et al., 1996). Due to its importance in diseases and signaling, phosphorylation of amino acids is one of the best studied PTMs (Cohen, 2001). About 30% of the whole proteome is suggested to be modified by phosphorylation (reviewed by Cohen, 2002b). From 20 essential amino 3- acids, nine can be modified with a phosphate group (PO4 ) by four different mechanisms (Lottspeich and Engels, 2005). Namely, these are (i) N-phosphorylation of the amino group of the three positively charged amino acids lysine, arginine and histidine, (ii) acylphosphorylation of the two negatively charged amino acids aspartate and glutamate, (iii) S-phosphorylation of cysteine thiol groups as well as (iv) O-phosphorylation of the amino acids tyrosine, serine and threonine. Of these, the latter represents the most important phosphorylation mechanism in eukaryotes (Lottspeich and Engels, 2005).

In contrast to irreversible modiﬁcations such as oxidation of cysteine to sulphinic, sulphenic or sulphonic acid, protein phosphorylation is a reversible modiﬁcation (reviewed by Mann and Jensen, 2003). Addition of a single phosphate group from an ATP molecule to an amino acid residue is mediated by protein kinases, an enzymatic sub-class that is encoded by 518 different genes in the human genome (reviewed by Manning et al., 2002). As antagonists of kinases, protein phosphatases catalyze the removal of the phosphorylation in the presence of

H2O (Figure 1.6).

The knowledge about kinase-speciﬁc phosphorylation motifs has increased signiﬁcantly over the past decades (Hutti et al., 2004; Songyang et al., 1994) and has resulted in several 1.2. Phosphoproteomics 13

ATP ADP

protein PO 2- kinase 3 OH O

phosphoprotein protein

protein phosphatase

Pi H2O

Figure 1.6.: Illustration of kinase-mediated O-phosphorylation and phosphatase-dependent de-phosphorylation. Protein kinases can catalyze the transfer of a phosphate group from ATP to the hydroxy group of serine, threonine and tyrosine, respectively. The respective amino acid of a given protein is modified with a doubly negatively charged phosphorylation and ADP is released. In contrast, protein phosphatases mediate the removal of the phosphate group in the presence of H2O, thereby generating a de-phosphorylated protein and inorganic phosphate (Pi). kinase-substrate prediction tools such as ScanSite3, NetworKIN4 and NetPhorest5. Moreover, the relevance of substrate docking sites for the interaction of kinase and target protein has been discovered (reviewed by Ubersax and Ferrell Jr, 2007). Even though their importance has been known for decades (reviewed by Hunter, 1995), comparatively less is known about the specificity of the ∼150 protein phosphatases, of which only 40 were identified as specific serine and threonine phosphatases (Cohen, 2002a). However, in the last years especially the role of dual-specificity phosphatases (Dusps) as critical regulators of cell signaling and cancer signaling has moved into focus (Bermudez et al., 2010; Patterson et al., 2009; Wang et al., 2015), thus opening a new research field.

The addition of one or even more doubly negatively charged phosphate groups can have a signiﬁcant effect upon the modiﬁed protein and its biological function in signal transduction and has been discussed in numerous reviews such as Mann and Jensen (2003), Seet et al.

3 http://scansite3.mit.edu 4 http://networkin.info 5 http://netphorest.info 14 1.2. Phosphoproteomics

(2006), Thapar (2015) and Thorner et al. (2014). In summary, the variety of biological processes meditated through phosphorylation of the target protein can be reduced to ﬁve major alterations (Figure 1.7). These changes concern (i) the protein activity, (ii) the protein turnover and degradation, (iii) the binding capacity of the protein, (iv) the protein folding and conformation and (v) the localization of the protein.

P P 2 1

3 P P P 4

Figure 1.7.: Schematic illustration of phosphorylation-mediated signaling processes. Protein phosphorylation can mediate various signaling processes by the change of the protein activity (1), the protein turnover (2), the protein interaction (3), the protein conformation (4) and the protein localization (5).

Protein kinases are a well studied example for the change in the protein activity by phosphorylation. While several kinases such as Akt (Franke et al., 1995) and B-raf (Chong et al., 2001) can be activated by phosphorylation of speciﬁc residues, there are other examples for instance glycogen synthase kinase-3 β (Gsk3β) (Cross et al., 1995) and eukaryotic elongation factor 2 kinase (Eef2k) (Knebel et al., 2001) which are inactivated by the addition of a phosphate group. Moreover, phosphorylation can induce protein degradation and turnover, as it was shown for the degradation of Myc proto-oncogene protein (c-Myc) by an F-box protein (Yada et al., 2004). Additionally, the turnover of paxilin in focal contacts was shown to be dependent on the phosphorylation state of two tyrosine phosphorylation sites (Qin et al., 2015). An example for phosphorylation induced interaction is the group of 14-3-3 proteins. The different isoforms of this protein family were characterized to bind their targets, such as C-raf 1.2. Phosphoproteomics 15

(Raf-1), only in the phosphorylated isoform (Muslin et al., 1996). Phosphorylation-dependent intra-molecular interaction, for instance the binding of the SH2 domain to a phosphorylated tyrosine residue within the amino acid sequence of the proto-oncogene tyrosine-protein kinase Src (Src), serves as model for a phosphorylation-mediated conformational change (reviewed by Seet et al., 2006). Last, phosphorylation of the Z-disc protein myopodin (also called synaptopodin-2, SYNPO2) was illustrated to mediate its shuttling from the Z-disc to the nucleus (Faul et al., 2007).

However, phosphorylation of a substrate protein usually results in the combination of several of the described mechanisms. For instance the phosphorylation of myopodin by PKA/CamkII is initially leading to the binding of 14-3-3β and thus a released interaction to the Z-disc core component α-actinin. Subsequently, importin-α can bind the protein complex and induce the import into the nucleus (Faul et al., 2007). Moreover, phosphorylation of FLNa was shown to have a protective function, leading to a weaker binding of ﬁlamin a-interacting protein 1 (FILIP1) thus to less decreased FLNa cleavage by calpain 1 (Vadlamudi et al., 2002 and reviewed by Zhou et al., 2010).

1.2.2 Phosphoproteomics workﬂow

Even though protein phosphorylation is known for over 100 years (Levene and Alsberg, 1906), the lack of suitable large-scale approaches hampered in-depth analyses for a long time. This drawback has been overcome by the combined development of fast high-resolution mass spectrometers and latest computer improvements (reviewed by Olsen and Mann, 2013). With these improvements, the methods to study the phosphoproteome and its dynamic changes have been optimized substantial in the last years (Grimsrud et al., 2010). However, the reduction of sample complexity and efﬁcient enrichment of phosphopeptides is still mandatory for in-depth large-scale phosphoproteomics (reviewed by Dunn et al., 2010). For this reason, the most commonly used techniques for large-scale phosphoproteomic analyses will be described brieﬂy in the following. 16 1.2. Phosphoproteomics

1.2.2.1. Reduction of sample complexity

High sample complexity, several order of magnitude in dynamic range and low abundance of phosphoproteins represent a challenging task for phosphoproteomics (Song et al., 2009). To this end, separation techniques to reduce the sample complexity have been established. Originally, gel-based approaches, such as 1DE- and 2DE-polyacrylamide gel electrophoresis (PAGE) were the methods of choice for the reduction of sample complexity. However, the limit in resolution power to approximately 1,000 proteins in combination with the low abundance of phosphoproteins led to the development of gel-free techniques (reviewed by Rabilloud et al., 2010). Nowadays, off-line chromatography approaches of digested samples prior to low pH reversed phase (RP) liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis are the method of choice in the ﬁeld of phosphoproteomics. As these pre-fractionations aim to increase the number of identiﬁed (phospho)proteins, a maximal orthogonality to the following LC-MS method is used (Mohammed and Heck, 2011), which uses the chemical changes in the net charges of peptides under different pH conditions.

The most widely used technique is the separation by strong cation exchange (SCX) chromatography, which is based on the different net charge states of modified peptides. Most unmodified tryptic peptides are doubly positively charged at low pH conditions, due to a protonation at the N-terminal amino group and the C-terminal amino acids (lysine or arginine) (Mohammed and Heck, 2011). In contrast, a phosphorylation is introducing one negative - charge into the peptide by addition of a phosphate group (PO3H ). As separation by SCX chromatography is based on the total net charge of a peptide, phosphorylated peptides elute earlier than non-phosphorylated peptides in an increasing salt gradient and can thus be separated from their non-modified counterparts (Essader et al., 2005). However, the non-binding capacity of highly phosphorylated peptides to the SCX column and the co-elution of phosphorylation isoforms at the beginning of the gradient reduce the orthogonality of this method (Gilar et al., 2005).

Recently, Batth et al., 2014 introduced a novel chromatographic technique for the pre- fractionation of peptides which is based on RP-chromatography at high pH (hpH). In contrast 1.2. Phosphoproteomics 17 to SCX chromatography, hpH-RP chromatography is characterized by a higher resolution, due to a better resolving power of hydrophobic interactions. Moreover, RP fractionation at hpH is maximizing the orthogonality to low pH RP-LC-MS methods. By using this method, Batth et al. (2014) claim that they could increase the number of localized phosphopeptides with a MaxQuant localization probability ≥ 0.75 by a factor of four.

Additional methods for the pre-fractionation of phosphopeptides, which will not be further discussed are strong anion exchange (SAX) (Dai et al., 2007), hydrophilic interaction chromatography (HILIC) (Boersema et al., 2007; Gilar et al., 2005) and electrostatic repulsion- hydrophilic interaction chromatography (ERLIC) (Hao et al., 2010).

1.2.2.2. Phosphopeptide enrichment

Sub-stoichiometric occurrence and low abundance, raise a special need for phosphopeptide enrichment prior to LC-MS analysis (reviewed by Mann et al., 2002). One method for the enrichment are antibodies, which target a modiﬁed site, such as phospho tyrosine or a speciﬁc motif, such as the basophilic recognition motif RxRxxpS. However, these antibodies show often a limited sensitivity and demand high amounts of starting material for the analysis (reviewed by Zhao and Jensen, 2009).

A more wildly distributed method for large-scale phosphoproteomic analyses is the enrichment of peptides modified with a negatively charged phosphate group via their interaction to immobilized metal ions (reviewed by Zhao and Jensen, 2009). Currently, there are two major approaches, ion metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC).

IMAC was initially described by Porath et al. (1975) for the enrichment of histidine and cysteine containing proteins which can bind to Zn2+ and Cu2+ immobilized agarose. For the enrichment of phosphopeptides, trivalent cations such as Al3+, Fe3+, Ga3+ haven been shown to be more suitable (reviewed by Block et al., 2011). As also non-phosphorylated peptides contain multiple acidic amino acids residues, which can interact with the IMAC material, binding of peptides to the resin should be performed below pH 1.9 to reduce unspeciﬁc peptide 18 1.2. Phosphoproteomics

binding (reviewed by Thingholm and Jensen, 2009). Recently, tetravalent Ti4+-IMAC has been introduced as novel phosphopeptide enrichment resin which outperforms the previously known materials in terms of speciﬁcity for peptides containing multiple basic residues (de Graaf et al., 2014).

In 2004, Pinkse and co-workers introduced MOAC by titanium dioxide (TiO2) as an efficient method for the enrichment of synthetic phosphopeptides (Pinkse et al., 2004). Exchange of acetic acid to trifluoroacetic acid (TFA) and the addition of 2,5-dihydroxybenzoic acid (DHB) to the loading buffer improved the method as it decreased the binding affinity for non-phosphorylated peptides (Larsen et al., 2005). Additionally, a shift of the ammonia elution to pH > 11 instead of the previously used elution with ammonium bicarbonate at

pH 9 led to an improved phosphopeptide recovery (Larsen et al., 2005). As TiO2 enrichment is more robust to higher salt concentrations and thus needs no further desalting step it is more often used in combination with SCX fractionation than IMAC (reviewed by Thingholm and Larsen, 2009).

1.2.2.3. Mass spectrometry-based phosphopeptide identiﬁcation

A phosphate group adds an additional mass of 80 Da to the modified amino acid in comparison to the unphosphorylated peptide. This mass difference can be measured by the detection of mass to charge (m/z) ratios with high resolution mass spectrometry (MS). Since the ionization efficiency of phosphopeptides in a positive MS ion mode is lower compared to the non- phosphorylated counterpart peptide, enrichment prior to LC-MS/MS analysis is beneficial for the identification of phosphopeptides (Pan et al., 2013).

While collision-induced dissociation (CID) is the most commonly used fragmentation technique for shotgun proteomics, it is not feasible for efﬁcient phosphopeptide fragmentation. Addition of a phosphate group to serine or threonine results in a bond, that is more labile than the protonated peptide bond and is thus fragmenting more easily at low energy CID. This bond breakage is resulting in the loss of phosphoric acid and in MS/MS spectra with dominating neutral loss peaks. As a consequence, only little to no sequence and no unambiguous site 1.2. Phosphoproteomics 19 localization information is obtained from this type of spectra (reviewed by Boersema et al., 2009).

To overcome this drawback of limited sequence information in neutral loss spectra, multistage activation (MSA) also called pseudo-MS3 was invented (Olsen and Mann, 2004; Schroeder et al., 2004). During MSA, the neutral loss peaks (at -98 Da, -49Da and -32.7 Da) are triggered at the same time for fragmentation as the parent ion. Hence, product ions of the neutral loss fragments are detected along with the initial MS/MS product ions, resulting in one composite spectrum without the required additional time for a MS3 isolation cycle (Ulintz et al., 2008).

In contrast to CID fragmentation higher-energy collisional dissociation (HCD) preserves the labile phosphate groups on the peptide backbone during fragmentation. Thus HCD allows for a conﬁdent unambiguous phosphosite assignment (reviewed by Tsiatsiani and Heck, 2015). An additional beneﬁt in contrast to CID fragmentation is the detection of fragment ions in the orbitrap mass analyzer instead of the linear ion trap (LIT) analyzer, allowing for a "high-high" mass detection of both MS1 and MS/MS spectra (Nagaraj et al., 2010).

Another "gentle" fragmentation approach is electron transfer dissociation (ETD) (Syka et al., 2004). Contrary to the three previously mentioned fragmentation techniques, this method is based on the interaction of radical ﬂuoranthene anions with multiple protonated peptides

(Martens et al., 2016). Hence, the N-Cα bond is weakened resulting in the fragmentation of the peptide into c- and z-ions without the loss of the phosphate group (reviewed by Boersema et al., 2009). However, this method is limited to multiple charged peptides ≥ 3+, as electron transfer to lower charged peptides might occur without dissociation.

Comparing the fragmentation techniques of ion trap CID/neutral loss-triggered ETD and MSA, Wiese et al. (2014) could show that the different methods provided highly complementary phosphopeptide identifications with an overlap of only 12%. For this reason they argue, that additional efforts in terms of measurement and data analysis time might be worthwhile for an improvement in phosphosite localization. Taken together, all the advancements in pre-fractionation, phosphopeptide enrichment and MS-identification led to an significant 20 1.2. Phosphoproteomics

increase in phosphoproteomic data (reviewed by Lemeer and Heck, 2009).

1.2.3 Large-scale skeletal muscle phosphoproteomic studies

Improvements of mass spectrometers and phosphorylation enrichment techniques since the beginning of the second decade of the 21st century have raised the possibility to study phosphoproteomics at a large scale. Nonetheless, Geiger et al. (2013) pointed out, that muscle (phospho)proteomics still represents a challenging task, due to a small number of highly abundant ﬁbrillar structure proteins, which account for over 50% of the total tissue mass. As an example, tryptic digestion of the 3.6 MDa protein titin with one allowed misscleavage site results in the theoretical number of 34,350 peptides. Hence, these highly abundant high molecular mass proteins result in large numbers of highly intensive peptide peaks. Today, there are ﬁve large-scale phosphoproteomic studies which focus on the analysis of the phosphoproteome in skeletal muscle cells/tissue. Namely, these are the studies by Højlund et al. (2009), Hou et al. (2010), Drexler et al. (2012), Kettenbach et al. (2015) and Hoffman et al. (2015). To place the data from this thesis in the context of these studies, their main achievements will be shortly summarized in the following.

Højlund and co-workers were the ﬁrst to publish a large-scale in vivo phosphoproteomic study of human skeletal muscle. They lysed 3-5 mg human tissue sample in a HEPES buffer system, performed tryptic digestion followed by manual step-gradient SCX chromatography

resulting in 9 fractions. These were further enriched by TiO2 and eluates from the enrichment were analyzed by an LTQ-FT with a 85 min gradient and a TOP10 CID fragmentation method. Thus they were able to identify 306 phosphosites in 127 muscle proteins (Højlund et al., 2009).

Hou and colleagues could increase the number of identiﬁed phosphosites and proteins by the factor of almost 10 one year later. By using 4.5 mg protein lysate from L6 myotubes they were able to identify 2,230 phosphopeptides in 1,195 proteins. For this purpose, they

performed a nine step-gradient pre-fractionation with self-packed RP C18 columns, subjected

the fractions to TiO2 enrichment and analyzed it by a 5h 2D SCX-RP-nano LC gradient, 1.2. Phosphoproteomics 21 coupled online to an LTQ-FT. For peptide fragmentation a TOP5 CID method was applied (Hou et al., 2010).

Drexler et al. were the first who performed 2012 quantitative muscle analysis mice labeled by stable isotope labeling by amino acids in cell culture (SILAC) to reveal the differences between slow and fast muscle fibers. Therefore, they used between 8.5 and 13 mg of muscle wet weight processed it with a Lys-C filter-aided sample preparation (FASP) digestion protocol and performed SCX chromatography in combination with TiO2 enrichment of the peptides. Subsequently, they used for the first time a TOP5 HCD fragmentation for the identification of phosphosites in muscle peptides on an LTQ-Orbitrap Velos by a 150 min LC-gradient. Thus, they identified 973 proteins, comprising 2,573 phosphosites of which 1,040 were not reported before at that time-point.

In 2015, Kettenbach and co-workers introduced a new method called single-stage phosphopeptide enrichment and stable-isotope tagging (SPECHT) for the analysis of phosphopeptides from muscle tissue (Kettenbach et al., 2015). Thereby, they wanted to overcome the draw- backs of expensive SILAC labeling of mice. To this end, they performed the TiO2 enrichment of 2 mg tryptically digested proteins prior to dimethyl labeling of the samples and performed subsequent SCX chromatography with the mixed light and heavy samples. To verify their workflow, they threated either mice or SILAC labeled C2C12 myotubes with insulin and compared the results from three biological replicates each time with a 90 min LC-MS/MS run on a Q-Exactive Plus system with a TOP10 method. By their SILAC approach they were able to quantify 2,437, 2,937 and 2,470 phosphopeptides, respectively in three biological replicates. For their SPECHT experiment with dimethyl labeled mouse samples, they were able to increase this number to 3,015, 3,092 and 3,084 phosphopeptides, respectively. Among these, they identified various phosphopeptides from proteins of the contractile apparatus, such as actinin-3, obscurin, nebulin, myomesin 2 and myotilin to be significantly increased in response to insulin stimulation of mice.

The largest, muscle proteomics study so far published was performed by Hoffman et al. (2015). By studying the pathway downstream of exercise-activated AMPK signaling, they 22 1.2. Phosphoproteomics were able to identify 1,004 unique exercise-regulated phosphosites on 562 proteins from

human muscle biopsies following phosphopeptide enrichment with TiO2 and HILIC. In total, they quantiﬁed 8,511 phosphosites in two out of four biological replicates from the human tissue which was either labeled with isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT) in a 100 min gradient on a Q-Exactive system with a TOP20 method. Moreover, they screened for Ampk targets in SILAC labeled L6 myotubes, followed

by SCX fractionation and TiO2 enrichment and quantiﬁed thus 7,421 phosphorylation sites in two out of four biological replicates. CHAPTER 2

Aims of this study

In the last decade, the essential function of the sarcomeric Z-disc as anchor for intracellular signaling processes occurring in response to mechanical stress and physiological stimuli has increasingly been recognized (reviewed by Pyle and Solaro, 2004). Moreover, several Z-disc proteins have been shown to play a role in diseases such as myofibrillar myopathies and muscular dystrophies (reviewed by Fürst et al., 2013). These findings considerably changed the view of the Z-disc as rigid, structure-bearing component to a highly dynamic, large protein interaction (reviewed by Sanger and Sanger, 2008). Even though significant effort has been made to improve our knowledge about molecular processes involved in Z-disc assembly, function, and maintenance in-depth (phospho)proteomic analyses are deemed essential reveal the underlying regulatory mechanisms.

Therefore, the first aim of this thesis is to globally characterize the basal phosphoproteome of contracting C2C12 mouse myotubes. To this end, (i) a cell culture-based workflow to obtain myotubes with mature Z-disc as well as (ii) a large-scale phosphoproteomic workflow was established. In order to analyze myogenesis and Z-disc formation, cell fusion and sarcomeric assembly were monitored by light and fluorescence microscopy. Furthermore, an electrical pulse stimulation protocol was developed to enhance sarcomere formation through induction of muscle contraction. Subsequently, an experimental workflow ranging from cell lysis conditions, pre-fractionation techniques, and phosphopeptide enrichment to LC-MS and 24 Aims of this study

large-scale data analysis methods was established. Data evaluation was performed with a focus on phosphorylation events occurring in sarcomeric and Z-disc proteins in particular. Identiﬁed phosphorylation sites were further subjected to in silico kinase prediction analyses. Predicted kinase-substrate relationships were validated using radioactive and site-resolving MS-based in vitro kinase assays. The biological function of the selected phosphorylation sites was further investigated with appropriate biochemical and MS-based methods.

The second aim of this work is to characterize changes in the Pi3k/Akt pathway of contracting C2 myotubes. To this end, a SILAC-based quantitative approach was pursued to identify substrates of the Pi3k/Akt pathway following treatment of myotubes with different chemical components known to inhibit or stimulate the Pi3k/Akt pathway such as IGF-1, LY294002, wortmanin, Torin-1 and rapamycin. Their effects on known substrates of the Pi3k/Akt signaling cascade were monitored by Western blotting using phospho-specific antibodies. Once an appropriate treatment was found, the changes in phosphorylation level were mapped by (i) quantitative Western blot analysis and (ii) large-scale quantitative phosphoproteomic analysis. Subsequently, the phosphoproteomics data were analyzed with regard to (i) significantly regulated phosphopeptides, (ii) enriched phospho-motifs and (iii) kinase-substrate relationships. These relationships were characterized by in vitro kinases assay. Furthermore, potential phosphorylation-dependent protein-protein interactions were identified by MS-based methods and validated by biochemical and cell culture-based experiments, such as in vitro pull-down assays and cell culture-based co-immunoprecipitation experiments.

The last aim of this work is the characterization of different proteases for the site-speciﬁc determination of phosphorylated amino acid residues. To this end, the proteases chymotrypsin, trypsin in combination with cyanogen bromide, Lys-C, elastase, trypsin and thermolysin were characterized for their proteolytic efﬁciency. The established digestion protocol was used to study phosphorylation in Myl12b at two amino acid residues C-teminal to an arginine. CHAPTER 3

Results and Discussion

3.1 Identiﬁcation of the myoﬁbrillar Z-disc as hot spot of protein phosphorylation Original publication: Reimann et al., under revision (see chapter 6.1)

One decade ago Frank et al. (2006) and Sanger and Sanger (2008) highlighted the sarcomeric Z-disc as "nodal point in signaling and disease" and a highly dynamic region within the muscle that functions as scaffold for numerous proteins. Since that time, various publications have focused on the identiﬁcation of new Z-disc and Z-disc associated proteins (Bang et al., 2001; Gregorio et al., 1998; Zieseniss et al., 2008) or protein-protein interactions within this important structure (Linnemann et al., 2010; Molt et al., 2014; van der Ven et al., 2000a). However, there has been no study speciﬁcally addressing the complexity of global signaling events within this structure published so far.

3.1.1 Optimization of lysis conditions for muscular proteomics

In order to globally characterize the phosphorylation landscape of skeletal muscle myotubes, the first aim of this thesis was to maximize the amount of detectable muscle (phospho)proteins by MS-based proteomics methodology. The composition of the muscle with a small number 26 3.1. Identification of the myofibrillar Z-disc as hot spot of protein phosphorylation

of highly abundant contractile proteins, which account for over 50% of the total tissue mass, present a considerable challenge for MS-based methods applied to muscle tissue (Geiger et al., 2013). First studies focusing on the improvement of phosphoproteomic methods were reported by Højlund et al. (2009), who identiﬁed 127 phosphoproteins comprising 306 phosphorylation sites in two out of three human skeletal muscle biopsies and, Lundby et al. (2012), who identiﬁed 4,346 phosphosites in 1,108 phosphoproteins of mouse muscle tissue.

To further increase the coverage of (phospho)proteins in muscle myotubes, five different lysis buffers were tested for the capability to solubilize sarcomeric proteins in this work (see table 4.1 in chapter 4.1). To this end, differentiated, contracting C2C12 myotubes were lysed with (i) phosphate buffered saline (PBS) containing 1% Triton X-100, (ii) urea buffer, (iii) urea buffer containing 1% sodium dodecyl sulfate (SDS), (iv) modified RIPA buffer, and (v) sucrose-Tris buffer. Subsequently, proteins ere digested with trypsin and analyzed with a 3 h gradient on an LTQ Orbitrap XL system. Resulting raw data were searched with the MaxQuant embedded Andromeda algorithm using the parameters listed in table E.1. Subsequently, the MaxQuant output protein groups file was processed with Perseus and total intensities were

log10 transformed, z-scored and clustered by hierarchical cluster analysis (Figure 3.1).

In sum, 1,799 protein groups were identified, which were sub-divided into six distinct clusters. The highest signal intensities and the highest number of protein identifications were obtained with PBS containing 1% Triton X-100 (1,760 proteins) and the urea buffer (1,629 proteins). The lower number of protein identification along with lower signal obtained intensities with the other buffer systems was most likely caused by the application of an additional detergent removal step. Overall, 1,121 proteins were identified when using the urea buffer containing 1% SDS 1,491 protein groups with the modified RIPA buffer and 1,240 proteins with the sucrose-Tris buffer. gene ontology (GO) enrichment analysis with the Cytoscape plugin ClueGO revealed that Z-disc proteins were enriched the most in cluster 2 (p-value 1.2E- 4), cluster 3 (p-value 1.4E-7) and cluster 6 (p-value 5.1E-7). Within cluster 5 the GO-terms ’nucleosome’ (p-value 7.2E-39), ’chromosome’ (p-value 7.5E-21) and ’spliceosomal complex’ (p-value 9.9E-15) were the top enriched categories. In contrast, the GO-terms ’mitochondrial 3.1. Identification of the myofibrillar Z-disc as hot spot of protein phosphorylation 27

mod. RIPA Sucrose- Urea SDS Urea PBS Tween buffer Tris buffer buffer buffer buffer 1.5 1

2 1.0

0.5

0.0

4 -0.5 transformed z-scored intensities transformed z-scored 10 log 5 -1.0

-1.5

Figure 3.1.: Hierarchical cluster analysis for the normalized intensities of identified proteins from C2C12 myotubes obtained with different lysis buffers. Total intensities were log10 transformed, z-scored, filtered for at least one valid value and clustered by euclidean average k-means cluster analysis. The identified 1799 protein groups were divided into six clusters, containing 174 (cluster 1, gray), 334 (cluster 2, purple), 542 (cluster 3, red), 262 (cluster 4, blue), 151 (cluster 5, rose) and 339 (cluster 6, marine) proteins, respectively. 28 3.1. Identification of the myofibrillar Z-disc as hot spot of protein phosphorylation

envelope’ (p-value 4.7E-8) and ’focal adhesion’ (p-value 5.5E-5) were enriched in cluster 4.

As the acetone precipitation and resuspension of samples solubilized with PBS buffer containing 1% Triton X-100 was seen as critical step for reproducible analyses, the high salt urea buffer in combination with a desalting step after protein digestion was chosen as buffer system of choice. Thus, to globally map the phosphoproteome, myotubes were lysed on ice with the urea buffer in further work.

3.1.2 Formation of mature Z-discs in cultured myotubes

A further critical step for the analysis of the Z-disc associated phosphoproteome was the formation of fully differentiated, contracting myotubes, comprising mature, functional sarcomeric structures (including I-band, Z-disc, A-band and M-line). As these structures are not found in proliferating myoblasts and many proteins are only expressed after the initiation of myoblast differentiation, EPS of differentiated myotubes was established to enhance sarcomeric assembly (Fujita et al., 2007). While mild pacing conditions were shown to enhance sarcomeric formation, more intense stimuli were reported to cause sarcomeric lesions and damage (Orfanos et al., 2016).

In this work, a differentiation and EPS protocol was established for C2 and C2C12 cells in order to obtain a maximal number of functional sarcomeres. Cells were grown on collagen coated cover slips, stimulated with different EPS conditions, ﬁxed with ice-cold methanol acetone (50:50, v:v) and subjected to immuno-histochemical staining. To characterize the differentiation stage of the cells and evaluate the grade of sarcomere formation, antibodies directed against the Z-disc backbone protein α-actinin (a653) (van der Ven et al., 2000b) and the Z-disc-associated end of titin (T12) (Fürst et al., 1988) were used (Figure 3.2).

Without EPS the cells did not contract by themselves and, thus, no or only few Z-disc structures were observable in these myotubes (Figure 3.2 I). In contrast, if pacing conditions were too harsh myotubes were mechanically stressed as revealed by α-actinin-enriched stress ﬁbers (Figure 3.2 III). Best sarcomeric formations were observed with voltages between 10 and 12 V (see Figures 3.2 II and A.1), with a stimulation duration of at least 4 h for C2 3.1. The basal phosphoproteome of contracting C2C12 myotubes 29

-actinin -actinin -actinin titin T12 titin T12 Z-discs Z-disc

stress fibers

III2 m 2 m III 20 m

Figure 3.2.: Staining of C2C12 myotubes for α-actinin and the Z-disc associated end of titin (T12). While no mature Z-disc formation is observable without EPS (I), complete sarcomeric formation was detected for electrical pulse stimulation (EPS) at 10 V, 4 ms and 0.05-1 Hz (II). In contrast, voltage numbers of 15 V and above caused stress ﬁber formation in the myotubes (III). Scale bar: I-II, 2 µm; III, 20 µm myotubes and 16 h for C2C12 myotubes.

3.1.3 Characterization of the basal phosphoproteome of fully differentiated, contracting C2C12 myotubes

As most of the phosphorylation sites annotated in public databases like PhosphoSitePlus 6 derive from non-muscular cell culture or tissue experiments, the next aim of this thesis was to identify the basal phosphoproteome of fully differentiated, contraction myotubes. To this end, C2C12 mouse myoblasts were grown to a conﬂuence of 90%, differentiated by serum reduction to 2%, and stimulated for 16 h with the previously established EPS protocol (Figure 3.3A I-III). Cell fusion during differentiation and formation of sarcomeric structures with mature Z-discs was observed by ﬂuorescence microscopy with the T12 antibody directed against the Z-disc-associated end of titin and 4’,6-diamidino-2-phenylindole (DAPI) for chromatin staining within the nucleus (Figure 3.3 A IV-VI).

Cell lysis was performed on ice using high-urea buffer (table 4.1e) supplemented with phosphatase inhibitors. Subsequently, tryptic digests of proteins were desalted, separated by SCX chromatography and enriched for phosphorylated peptides with TiO2 (Figure 3.3 B). Eluates from TiO2 enrichment and a small, non-enriched aliquot of each SCX fraction to improve peptide and protein identiﬁcations were analyzed by high-resolution liquid chromatography-mass

6 http://www.phosphosite.org/ 30 3.1. The basal phosphoproteome of contracting C2C12 myotubes

A I II III

IV DAPI VVIDAPI DAPI T12 T12 T12

Thermo Q EXACTIVE O O S c i e n t i f i c O Ti Ti O

PO3

differentiated, cell lysis, strong cation enrichment of LC-MS/MS contracting tryptic digestion, exchange phosphorylated analysis and C2C12 cells and desalting chromatography peptides bioinformatics

Figure 3.3.: Cell culture and phosphoproteomic workflow for global analysis of the C2C12 myotube phosphoproteome. (A) Formation of contracting sarcomeres in C2C12 skeletal muscle cells studied by light (I-III) and fluorescence (IV-VI) microscopy. Cell fusion was demonstrated by DAPI staining of nuclei (blue) and sarcomere formation by using a T12 antibody (red) (IV-VI). Mono-nuclear myoblast at 50% confluence (I, IV) were grown to 90% confluence before cell fusion was induced by serum reduction. Following differentiation, cells were fused to poly-nuclear myotubes (II, V). To generate contracting myotubes, cells were electrically stimulated for 16-24 h. Sarcomere formation was confirmed by observing the typical striated pattern in fluorescence microscopy image (VI). Scale bar: I-III, 100 µm; IV-VI, 10 µm. (B) Phosphoproteomics of differentiated, contracting skeletal muscle cells. C2C12 myotubes were lysed using high-urea buffer in the presence of phosphatase inhibitors. Following digestion of proteins using trypsin and desalting, peptide mixtures were fractionated by SCX chromatography. A minor aliquot of each of the 55 SCX fractions was directly analyzed by LC-MS/MS, whereas the major part was enriched for phosphopeptides by TiO2. Data analysis was performed with the MaxQuant and Perseus software. 3.1. The basal phosphoproteome of contracting C2C12 myotubes 31 spectrometry (LC-MS) on an LTQ Orbitrap-XL system. While non-enriched samples were subjected to CID for MS/MS analysis, phosphopeptide fractions were fragmented by MSA for enhanced site-specific information about phosphorylated amino acid residues (Schroeder et al., 2004). With this workflow, a total of 6,808 proteins were identified in two independent biological replicates (Figure 3.4 A).

AB1000 1,385 proteins # Proteins 6,808 750 529 proteins # Phosphoproteins 2,941 500

# Phosphosites 11,369 250

# Localized p-sites # of phosphoproteins 8,175 0 1 2 3 4 5 6-10 11-15 16-20 >20 # of p-sites per protein CD

nucleus (2021) nucleus (931)

mitochondrion (916) CC Z-disc (23) CC gene Z-disc (33) expression (471) gene expression (1100) phosphorylation (246) BP

BP protein (477) cytoskeleton (98) transport organization nucleic acid (1019) kinase activity (235) binding cytoskeletal (267) cytoskeletal (163)

MF protein binding protein binding MF kinase activity (410) PKC binding (12)

0 50 >100 050>100 -log (corr. p-value) proteins 10 -log10 (corr. p-value) phosphoproteins

Figure 3.4.: Global overview of the contracting myotube (phospho)proteome. (A) Numbers of (phospho-)proteins and phosphosites (p-sites) identified in contracting C2C12 myotubes. Localized p-sites were identified with a MaxQuant localization probability score ≥ 0.75. (B) Distribution of p-sites according to the number of sites per protein. (C) (D) GO-term enrichment analysis of the C2C12 myotube proteome (C) and phosphoproteome (D) dataset established in this work. P-values after Benjamini-Hochberg false discovery rate (FDR < 0.05) correction were plotted against their corresponding GO-terms from the three main domains cellular component (CC), molecular function (MF) and biological process (BP). Numbers of identified proteins for each term are shown in brackets.

Out of these 6,808 proteins, 43% (2,941) were modiﬁed by phosphate groups including 529 proteins, each comprising more than 6 phosphosites per protein (Figure 3.4 B). In total, 32 3.1. The basal phosphoproteome of contracting C2C12 myotubes

11,369 identified phosphorylation sites. The overlap of identified proteins and phosphoproteins between the two replicates was 83% and 63%, respectively. As the aim of this study was to map myotube-specific phosphosites, the results were further filtered by applying a MaxQuant localization probability of ≥ 0.75. Overall, 8,175 sites (72%) fulfilled this criterion and were thus deemed as localized myotube phosphorylation sites.

To identify functional features of the myotube proteome in comparison to its phosphoproteome GO enrichment analysis was performed with the Cytoscape plug-in BiNGO (Figure 3.4 C-D). In both datasets, the terms ’Z-disc’, ’nucleus’, ’gene expression’, ’kinase activity’ and ’cytoskeletal protein binding’ were significantly enriched, whereas ’mitochondrion’ was only overrepresented in the proteome data. In total, 916 annotated mitochondrial proteins were identified. Even though, this amount is comparable to numbers found in purified mitochondria of 1,098 proteins out of 14 different mouse tissues (Pagliarini et al., 2008) and 654 proteins out of single muscle fibers (Murgia et al., 2015), none of the mitochondrial proteins were identified to be phosphorylated. Moreover, the percentage of annotated mitochondrial phosphoproteins in comparison to published proteins is rather small, suggesting a comparable low extent of kinase-dependent phosphorylation in the organelle. While Deng et al. (2011), Grimsrud et al. (2012), and Lee et al. (2007) identified 228, 102 and 62 mitochondrial phosphoproteins, respectively out of rat/mouse liver tissue, 77 phosphorylated proteins were published from purified skeletal muscle mitochondria (Zhao et al., 2011).

In contrast, biological processes related to phosphorylation and cytoskeletal organization and ’PKC binding’ as molecular function of were specifically enriched in the phosphoproteome (Figure 3.4 D). Moreover, 70% (23) of the annotated Z-disc proteins (33) identified here were phosphorylated supporting the notion that the Z-disc acts as an important signaling platform within the sarcomere (Frank and Frey, 2011; Frank et al., 2006; Pyle and Solaro, 2004). However, GO-based annotation of only 33 Z-disc and Z-disc associated proteins was considered to be insufficient for a detailed analysis. To this end, a list of 105 proteins that are part of or associated with the sarcomeric Z-disc was generated in the framework of this thesis based on literature work and with the help of expert knowledge from Dr. Peter MF van der 3.1. The basal phosphoproteome of contracting C2C12 myotubes 33

Ven and Dr. Dieter O. Fürst (Institute for Cell Biology, University of Bonn).

This list (see supplemental table S4 in section 6.1) contains 42 true Z-disc proteins as well as 21 proteins directly connected with the Z-disc (Z-disc-associated and peripheral) and further 42 proteins connected to the Z-disc (membrane-associated, intercalated disc, intermediate ﬁlament). The latter group comprises also proteins from the boundaries of the Z-disc, including those which are connecting myoﬁbrils with the sarcolemma, e.g. via costameric proteins like the dystrophin-associated protein complex. Thus, this list builds the basis for further analyses of the sarcomeric (phospho)proteome.

3.1.4 The Z-disc is a sarcomeric phosphorylation hot spot

Based on the defined Z-disc and Z-disc associated proteome, the (phospho)proteomic dataset was further evaluated. With the exception of a few proteins only expressed in cardiac muscle fibers such as nebulette (Moncman and Wang, 1995) and Fbxl22 (Spaich et al., 2012) or only located at the Z-disc under distinct conditions such as Xin (name derived from the Chinese word for heart, Hawke et al., 2007) during myofibrillar repair or remodeling processes, the dataset virtually covers the complete so far known Z-disc proteome (see supplemental table S4 in section 6.1).

Remarkably, 532 out of 589 phosphosites (91%) in sarcomeric proteins were identified in 66 proteins of the I-band, whereas only 9% of the identified sarcomeric phosphosites were located in A-band proteins (Figure 3.5 A). Moreover, the majority of 77% (412 phosphosites) was identified in 31 genuine Z-disc proteins (encoded by 29 genes) and 21 proteins (encoded by 15 genes) peripherally associated with this structure (Figure 3.5 A and D). Thus, 69% of Z-disc and 71% of Z-disc-associated proteins were found to be phosphorylated, underscoring the results of the GO enrichment analysis. Furthermore, the majority of localized phosphosites (264, 83%) in proteins associated with the myofibrillar Z-disc were assigned with high confidence (probability ≥ 0.9) and further 53 sites (17%) were scored with probabilities between 0.75 and 0.9 (Figure 3.5 C).

Interestingly, GO enrichment analysis also identiﬁed the molecular function of ’PKC 34 3.1. The basal phosphoproteome of contracting C2C12 myotubes

A BC 53 Z-disc Z-disc p-sites A-band associated 233 p-sites 54 p-sites 179 p-sites

I-band 264 p-sites 532p-sites non-Z-disc Σ 586 120 p-sites Σ 532 Σ 317 p-sites p-sites p-sites

D Itga Itgb Itga Itgb Ankrd2 Ankrd2

P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Dst P P P P Plec P P P Ctnna1 P Des P P P P P P P P P P P P P Des P P P P P P P P Flnc Ctnnb1 P P P P Pgm5 P P P Lasp1 P P P P P P P EnahP Tln2 P P P P Capzb P P P P P P P P P P P P P P P P P P P P P Tcap Sorbs2 P P Mypn P P P P P P P P P P Cryab P P P Neb P P P P P P P P Hspb1P P Sync P P P P P P P P P P P P P P P P P P P Pxn P P P P P P P P P P P P P Actn3 P P P P P P P Nexn P P Bag3 P P P P P P P P P P P P P P P P P P P P P P Capzb P Synpo2lP P P P P P P P P P P P P P Pdlim1 Evl P P P P P P P P P P Rock1 P P P P P P P P P PP P Xirp1 P P P P P P P P P Vcl P PP P P P P P P P P P PSynpo2 P Flnc P P P P Vasp P Pdlim3 P Ttn P P P P P P P Capn3 P P P PP P P P P PTcap P Obscn P P Synpo P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Ldb3 Capzb P P P 3425401B19Rik P P P P P P P P P P P P P P Pdlim5 P P P Hspb8 P Myot P P Epb41l1 P P Myoz2 Flnc P P P P P Obsl1 P P P P P P P Capn3 PTcap P P P P P P P P Zyx P P P P P P Capzb P

Figure 3.5.: The Z-disc is the main site of protein phosphorylation in sarcomeres. (A) Dis- tribution of phosphosites (p-sites) identified in sarcomeric proteins of the I or A-band (A). 532 p-sites (91%) were identified in proteins of the I-band. Of these, 412 p-sites (77%) were identified in true Z-disc and Z-disc-associated proteins, which is an essential structural part of the I-band (B). (C) Classification of p-sites localized in 44 Z-disc-associated proteins. 264 p-sites (83%) were identified with high confidence with a MaxQuant localization probability ≥ 0.90 (red). Further 53 p-sites were localized with a probability ≥ 0.75 and < 0.90 (gray). (D) Schematic overview of the Z-disc phosphoproteome. 44 genes (encoding for 51 proteins) were found to be modified by a single or multiple phosphate groups accounting for 70% of the known Z-disc proteome. Phosphosites localized in the Z-disc-associated regions of the two structural proteins titin (12 p-sites) and desmin (14 p-sites) are not depicted. 3.1. The basal phosphoproteome of contracting C2C12 myotubes 35 binding’ to be significant increased within the phosphoproteins. Among the phosphorylated Z-disc and Z-disc-associated proteins LIM domain binding protein 3 (Ldb3) also called cypher or ZASP (Arimura et al., 2004; Yamashita et al., 2014; Zhou et al., 1999) and enigma homologue protein ENH (PDLIM5) (Nakagawa et al., 2000) were found, which together with calsarcin-1 (also called moyzenin-2 or FATZ-2) (Faulkner et al., 2000) form a complex with an important role in linking the Z-disc to the extra-cellular matrix via the multi-adapter protein FLNc (Cheng et al., 2010).

Remarkably, numerous Z-disc and Z-disc-associated proteins such as FLNc, Bcl2-associated athanogene 3 (Bag3), Ldb3, enabled homolog (Enah), Xin actin-binding repeat-containing protein 1 (Xirp1), heat shock protein beta-1 (Hspb1), and the three members of the podin protein family synaptopodin (Synpo), myopodin (Synpo2) and tritopodin/synaptopodin 2-like (Synpo2l) were found to be phosphorylated at multiple amino acid residues with up to a total number of 25 distinct phosphosites in Synpo2l (Figure 3.6).

The identified phosphosites often occurred in clusters within one structural subunit or between two domains, as observed in the podin protein family, Bag3 and FLNc. This observation is in accordance with Iakoucheva et al. (2004) who identified intrinsic disorder to be an essential common feature of all eukaryotic serine, threonine and tyrosine phosphorylations. Moreover, Collins et al. (2008) predicted that 86% of all phosphorylation sites investigated in their study were located in regions of sequence disorder, thus linking phosphorylation to protein-protein interaction, which also occurs preferential in these regions (Sickmeier et al., 2007 and reviewed by Tompa and Fuxreiter, 2008). Additionally, Buljan et al. (2012) describe that tissue-specific alternative splicing events include unique disordered segments which result in binding regions for interaction partners that are distinct in these tissues.

Interestingly, two phosphorylation clusters with two and three phosphorylation sites, respectively, were identified within the uniquely expressed region of the muscle-specific filamin isoform FLNc. The first two sites, S2234 and S2237, of mouse FLNc were identified in a 82 amino acid-long isoform-specific insertion within Ig-like domain 20. Three further sites were localized in the hinge 2 region of the protein. The latter region of FLNc comprises the 36 3.1. The basal phosphoproteome of contracting C2C12 myotubes

Flnc Bag3 ABD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 WW PxxP BAG

M1 P2726 M1 P577 S138 S177 S179 S270 S274 S285 S382 S390 S404 S2633 S1528 S2621 S2625 S2234 S2237 Ldb3 Enah PDZ LIM LIM LIM WH1/EVH1 PxxP VASP M1 V723 M1 A802 T736 T732 T734 S98 T119 S121 S123 S177 S144 S255 S351 S354 S355 S362 S637 S740 Xirp1 Hspb1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 HSP20-like α-crystallin M1 L1129 M1 K209 S13 S15 S86 S102 S180 S203 S332 S139 S205 S208 S295 S533 Synpo isoform 2 PPxY PxxP M1 K901 S19 T583 S534 Y536 S240 S284 S296 S433 S505 S524 S585 S789 S803 S808 S824 S892 S249 S273 S450 S501 T530 S589 S594 S819 Synpo2 PDZ PxxP PxxP M1 E1087 T897 T715 S540 S543 S545 S546 S596 S895 S899 S618 S767 S771 S903 S719 S727 Synpo2l PDZ PxxP PxxP M1 Q975 S266 S368 T891 T912 T926 S371 T138 T559 T469 T702 T718 T791 S890 S435 S438 S163 S175 S140 S177 S667 S675 S699 S787 S793 S789

Figure 3.6.: Schematic illustration of highly phosphorylated Z-disc proteins. Localized phosphosites (mouse) are color-coded based on information provided by the PhosphoSitePlus database (version 07.01.2016) with black for known sites, blue for phosphosites known by similarity to orthologues sites in human proteins, and red for newly identiﬁed sites. Information about protein domains was retrieved from the UniProt and InterPro database. Immunoglobulin-like domains in FLNc and XIRP1 are numbered. phosphosites S2621, S2625 and S2633, and displays a reduced sequence homology of 45% in comparison to the other two ﬁlamin isoforms, FLNa and FLNb (reviewed by van der Flier and Sonnenberg, 2001). Of these sites, phosphorylation of S2621 has not been reported before in mouse cells.

Ig-like domain 20 of FLNc was proposed to be sufficient for the targeting of the protein to the Z-disc in neonatal rat cardiomyocytes and differentiated C2C12 cells (van der Ven et al., 2000b). Previous work of Murray et al. (2004b) identified the phosphorylation site corresponding to S2234 in mouse FLNc to be a target of AKT-mediated phosphorylation in skeletal rabbit muscle. This site was not only confirmed to be phosphorylated in contracting 3.1. The basal phosphoproteome of contracting C2C12 myotubes 37

C2C12 mouse myotubes, but was also identiﬁed to occur in a doubly phosphorylated peptide together with phosphorylation of mouse S2237.

Remarkably, binding of several FLNc interaction partners such as aciculin (also called phosphoglucomutase-like protein 5, PGM5), Xirp1 and XIRP2 to FLNc requires the insertion in Ig-like domain 20 (Molt et al., 2014; Kley et al., 2013). Moreover, also the Z-disc proteins myopodin (Linnemann et al., 2010) and myotilin (van der Ven et al., 2000b) were reported to speciﬁcally bind this FLNc region. With the exception of XIRP2, all these FLNc interactors were also identiﬁed to be highly phosphorylated proteins (Figure 3.6). In this context, it is interesting to note that phosphorylation of FLNc, aciculin, and Xin was shown to increase within a few minutes after inducing pressure overload in mouse hearts (Chang et al., 2013). Thus, all three proteins were proposed to interact closely in signaling pathways underlying the adaptation of striated muscle to mechanical stress. For this reason, phosphorylation of FLNc at S2234 and S2237 may provide a mechanism for controlling dynamic and competing protein interactions depending on the prevailing physiological condition.

The second phosphorylation cluster was identiﬁed in the hinge 2 region at the C-terminal end of FLNc between Ig-like domain 23 and 24. In previous work, it was suggested that the C-terminal part of FLNc is a target of PKCα phosphorylation (Tigges et al., 2003). However, the exact site remained elusive. In this work, S2621 and S2625, both located in the hinge 2, as well as S2633 in the N-terminus of Ig-like domain 24 were identiﬁed to be phosphorylated in FLNc (Figure 3.6).

As all three phosphorylation sites were located in the serine-rich amino acid stretch

2618SSSSRGASYSSIPKFSSDASK2638, manual spectra interpretation was used for unambiguous site-specific identification of the phosphate groups. All three phosphorylation sites were annotated in a singly phosphorylated peptide (Figure 3.7 A-C). Additionally, pS2621 and pS2625 were identified in a doubly phosphorylated peptide (Figure 3.7 D), indicating that FLNc can be concurrently phosphorylated at these serine residues in vivo.

Similar to Ig-like domain 20, the C-terminal region of FLNc plays an important role in several protein-protein interactions. Among others, the hinge 2 region together with domain 38 3.1. The basal phosphoproteome of contracting C2C12 myotubes

A B 2+ [M+3H] - H3PO4 100 689.26 100 382.43 pS2625 pS2621 S S S S R G A S Y S S I P K G A S Y S S I P K

y11-H2O 2+ [M+3H] - H2O 1214.62 729.43 y11 y11*-H2O b * 12 1232.57 1116.60 1152.57 y11* b12 1134.68 1250.57 b -H O 4 2 y2 b * 313.18 y10-H2O 7 244.24 648.32 y2 1047.61 b4* b132+ b9-H O 244.32 331.32 b 2 b 4 674.30 y 981.44 y72+ Relative Abundance 3 7 429.25 y10 Relative Abundance 262.15 y3 b5* 781.39 2+ b9* b9 1065.81 b7 y5 357.33 487.39 y6 y7 y 2+ 865.55 909.63 531.37 y * 10 477.24 694.34 7 861.51 b4* 630.54 763.47 533.48 b * 420.52 728.33 3 b3 361.83 494.76 198.19 573.86 296.19 684.28 779.07 0 0 500 1000 150 500 1000 C m/z D m/z y9* 100 948.49 100 680.41 pS2633 pS2621, pS2625 G A S Y S S I P K F S S D A S K S S S S R G A S Y S S I P K

729.48 y9 1046.40

y11-H2O y9-H2O b ** 1196.71 b7-H2O 12 y11* b12* 930.56 1134.58 648.23 y8 1214.63 1232.59 y * 2+ y12* y 2+ 6 y14 y * y14* 11 b11* y 798.55 11 1478.62 656.95 y7* 11 y6-NH b7 y2 1312.64 Relative Abundance 3 b * b14* Relative Abundance y10-H O 666.28 b11* 12 244.27 674.48 2 b 2+ 630.34 y 769.28 12 b9 10 b13* b15* y9 838.53 b4-H O y ** b4-H O y7* b10 1309.65 y 2 b5* b5 b * 11 2 y10* b 13 313.15 y y 9 1116.65 y y5 1061.66 y11 13 y 5 7 945.28 3 y4 1148.65 y121407.70 b b y3 4 531.37 861.55 1294.50 507.36 14 15 1030.65 305.14381.15 1333.56 1565.87 357.25444.35 0 0 500 1000 1700 500 1000 1500 m/z m/z

Figure 3.7.: FLNc is phosphorylated in its hinge 2 region at S2621, S2625, and S2633 in mouse C2C12 myotubes. (A)-(D), Annotated fragmentation spectra of phosphorylated peptides from mouse FLNc. In vivo phosphosites were assigned to S2621 (A), S2625 (B), S2633 (C) and in case of the doubly phosphorylated peptide 2618SSSSRGASYSSIPK2631 to both S2621 and pS2625 (D). Fragment ions exhibiting a neutral loss of phosphoric acid (H3PO4; 97.9768 u) are marked with an asterisk (*); loss of water (H2O) and ammonia (NH3) as indicated. Phosphorylated serines are depicted in red; b- and y-ion series in red and blue, respectively. m/z, mass-to-charge ratio

24 was shown to function as dimerization region leading to the cross-linking ability of the protein (Himmel et al., 2003; Pudas et al., 2005; Sjekloca´ et al., 2007). Moreover, domain 23 facilitates binding of calsarcin-2 (Takada et al., 2001) and domain 24 enables the interaction with β- and γ-sarcoglycan (Thompson et al., 2000). Cleavage of FLNc in the hinge 2 region by the muscle-speciﬁc cysteine protease calpain 3 abolishes the interaction to both sarcoglycans (Guyon et al., 2003).

3.1.5 Identiﬁcation of ﬁlamin C as target of PKCα In order to further investigate kinases that are mediating phosphorylation of FLNc at the amino acid residues S2621, S2626 and S2633, in silico kinase prediction analysis employing 3.1. The basal phosphoproteome of contracting C2C12 myotubes 39 the algorithms NetPhosK and NetworKIN was performed (Figure 3.8 A). The results indicate that all three phosphorylated residues are putative substrate sites of PKC/PKCα.

A M1 P2726 ABD 1 2 15 16 17 18 19 20 21 22 23 24

P P P S2621 S2625 S2633

VTKSSSS RGAS YSSIPKFS SDASKVV 2615 2640

CaMKIIα 1.90 - PKCα 3.57 NetworKIN 3.0 PAK1 1.41 - PKC 0.59 RSK 0.60 PKC 0.66 NetPhosK 1.0 Cdc2 0.50 PKC 0.52 B C mFLNc d23-24 hFLNc d23-24 WT AD WT AA DD PKC ++++++ +++ PKC ++ ++++++ +++ 80 kDa 80 kDa - PKC - -PKC 60 kDa - -  60 kDa- 29 kDa - 29 kDa- -FLNc -FLNc 24 kDa - 24 kDa- 29 kDa- 29 kDa- FLNc -FLNc 24 kDa - - 24 kDa-

Figure 3.8.: Mouse and human FLNc domain 23-24 are phosphorylated by PKCα in a concentration dependent manner. (A) Kinase prediction analysis employing the algorithms Net- worKIN 3.0 and NetPhosK 1.0. Scores are given for individual kinases predicted per site. -, no kinase predicted. A minimum score threshold was set to 1.4 for NetworKIN and to 0.5 for NetPhosK. (B) and (C) Radioactive in vitro kinase assays. Recombinant wildtype and phosphosite mutants of mouse (m; B) and human (h; C) FLNc Ig-like domains 23-24 (d23-24) were treated with PKCα in the presence of [γ-33P]ATP and analyzed by SDS-PAGE followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. As a control, PKCα was incubated in [γ-33P]ATP-containing kinase buffer without hFLNc d23-24 (B). WT, wildtype; A, alanine; D, aspartate; +, 10 ng PKCα; ++, 20 ng PKCα

To test this hypothesis, wildtype (WT) and phosphosite mutants of FLNc Ig-like domains 23-24 (Figure A.2) were recombinantly expressed in E. coli, puriﬁed by Ni2+-NTA-agarose and dialyzed for subsequent in vitro kinase assays. As the amino acid sequence in the region of interest differs between mouse FLNc 2618SSSSRGASYSSIPK2631 and human FLNc 40 3.1. The basal phosphoproteome of contracting C2C12 myotubes

2617SSSSRGSSYSSIPK2630 in a simple amino acid residue, both protein fragments were included in the analysis. Moreover, both serines at position S2623 and S2624 were mutated to A and D in human filamin c (hFLNc), whereas in mouse filamin c (mFLNc) only S2625 was exchanged. Subsequent to dialysis, mFLNc, hFLNc and the respective site mutants were incubated with 10 ng or 20 ng PKCα in the presence of [γ-33P]ATP and analyzed by SDS-PAGE followed by auto-radiography and Coomassie staining. Autoradiograms revealed that for all six constructs the intensity of the phosphorylation was dependent on the kinase concentration (Figure 3.8 B and C). Generally, signal intensities observed for the site mutants were lower but not abolished. This observation may suggest, that an additional site within the C-terminal end of both mFLNc and hFLNc becomes phosphorylated under in vitro conditions. Auto-phosphorylation of the FLNc constructs or co-purification of E. coli-specific kinases was excluded, as no signals were observable following their incubation with [γ-33P]ATP without PKCα (Figure A.3).

Even though it was shown that PKCα is targeting the C-terminal region of mFLNc and hFLNc, the exact PKC target site remained elusive in the radioactive in vitro kinase assay. To overcome this drawback, an MS-based assay was established. Following in vitro phosphorylation using non-radiolabeled ATP, FLNc constructs were digested in solution, followed by

phosphopeptide enrichment via TiO2 and subsequent analysis by LC-MS using complementary fragmentation techniques for MS/MS to obtain unambiguous phosphosite information. The resulting MS1 spectra of equal protein amounts (see Figure A.3) were quantiﬁed using Skyline (MacLean et al., 2010) and the mean intensities of biological triplicates plus the standard error of the mean (SEM) were calculated (Figure 3.9 A and B).

By this comprehensive analysis, phosphorylation of S2625 could be unequivocally confirmed as main PKCα substrate site in mFLNc, accounting for ∼ 46% of the total phosphopeptide intensity (Figure 3.9 A, left panel and 3.9 C). Additionally, phosphorylation of S2603 and S2633 was also identified in the in vitro analysis, albeit to a lesser extent. As these latter two sites were not found to be phosphorylated in vivo, they were classified as artificial in vitro sites. For the two mouse phosphosites mutants (Figure 3.9 A, middle and right panel), 3.1. The basal phosphoproteome of contracting C2C12 myotubes 41

A mFLNc d23-24

WT A100 D

60 80 40 1.5 E10 1.5 E10 60 1.5 E10

40 KVVTR KVVTR KVVTR S S S LH... 40 LH... LH... 20 S S S YSSIPK 20 S SRGADYSSIPK SRGAAYSSIPK FSSDA FSSDA S S 20 FSSDA GA LSGGH SS SS LSGGH normalized intensity [%] 0 0 0 LSGGH 20 S2603 S2625 S2637 S2603 S26 S2637 S2603 S2620 S2637

B hFLNc d23-24 WT AA DD 100 80 40 80

YSSIPK 60 1E10 1E10 1E10

SS 60 YSSIPK G | KVVTR KVVTR KVVTR S S S S

S 40

20 LH... G

| 40 LH... S LH... S S SRGAAYSSIPK SSSSR SRGDDYSSIPK 20 S FSSDA S FSSDA FSSDA 20 SS SSSSR SS LSGGH LSGGH normalized intensity [%] 0 LSGGH 0 0 S2602 S2623 S2623 S2636 S2602 S2619 S2636 S2602 S2619 S2636 S2624

C y2 D [M+3H]3+ 244.17 504.02 100 100

G A S Y S S I P K S S S S R G S S Y S S I P K

2+ [M+3H] - H3PO4 a1 z5 698.53 101.07 515.41

c5 522.52 z6 678.40 b2 z122+ 129.07 y 660.23 c7 y Relative Abundance 5 8 y -H O Abundance Relative c z+2 7 2 c 531.45 746.28 z 948.46 11 13 y y5-H O 745.39 c 4 8 c 1408.69 1 2 3 c7* 932.42 9 1170.52 147.08 513.30 279.17 366.15 648.37 z b4* y6-H O y * y z9 996.57 10 c y3 361.15 y 2 7 3 y c6 989.50 1145.62 13 4 y5 676.37 359.30 4 c8 b3* 357.25 b * 763.40 444.39 579.33 c10 1380.64 444.28 5 531.30b * z4 833.38 z11 198.09 b 448.18 6 y y 1083.53 z12 3 535.21 b7* 6 y7 2 z3 428.24 1232.69 296.06 648.30 694.38 861.38 244.34 341.29 1319.56 0 0 100 550 1000 100 500 1000 1500 m/z m/z

Figure 3.9.: S2625 and S2623/S2624 are the specific substrates sites of PKCα in the hinge 2 region of mFLNc and hFLNc, respectively. (A) and (B) MS-based in vitro kinase assays. MS data from three independent kinase experiments for mFLNc d23-24 WT, A and D sites mutants (D) and hFLNc WT, AA, and DD site mutants (E) were quantified. Intensities of phosphopeptides distinctive for a specific phosphorylation site (red) were added up per experiment and represented as normalized mean ± SEM. (C) and (D) Fragmentation spectra of mono-phosphorylated peptides of mouse (C) and human (D) FLNc d23-24 WT forms. PKCα-dependent phosphorylation of mFLNc-S2625 and hFLNc-S2623 was determined by higher-collisional dissociation and electron transfer dissociation, respectively. Fragment ions exhibiting a neutral loss of phosphoric acid (H3PO4; 97.9768 u) are marked with an asterisk (*); loss of water (H2O) as indicated. Phospho- rylated residues are depicted in red; b/c- and y/z-ion series in red and blue, respectively. WT, wildtype; A, alanine; D, aspartate 42 3.1. The basal phosphoproteome of contracting C2C12 myotubes

phosphorylation shifted to S2620, within a cluster of four serines in -6 to -3 position of the initial phosphorylation site. Virtually the same phosphorylation pattern was observed for the WT and the corresponding double site mutants forms of hFLNc(Figure 3.9 B). However, in contrast to mFLNc, the main target site of PKCα in hFLNc was S2623 (Figure 3.9 D). In addition, concurrent phosphorylation at S2623/S2624 was observed.

To conﬁrm the results, that mFLNc S2625 is an in vivo substrate of PKCα in skeletal muscle cells, Anna Lena Fricke (Department of Biochemistry and Functional Proteomics, University of Freiburg) performed a quantitative phosphoproteomic MS-analysis of SILAC labeled, contracting C2 myotubes (see Figure 4 G in section 6.1). To study the PKCα-dependent

phosphorylation of the FLNc phosphopeptide 2623GApSYSSIPK2631, she treated the myotubes with with PMA, an activator for the conventional PKC isoforms α, β, γ or Gö6976, an inhibitor for PKCα and PKCβ1 in comparison to DMSO-treated cells. Following cell lysis, tryptic protein digestion, high pH reversed phase chromatography (hpH-RP) chromatography, she

enriched the samples for phosphopeptides with TiO2 and analyzed them by quantitative LC-MS. As result, she could show, that phosphorylation of mFLNc S2625 to be signiﬁcantly decreased upon treatment of C2C12 myotubes with Gö6976 (see Figure 4 H in section 6.1).

In contrast to FLNc, the filamin family protein FLNa was found to be phosphorylated at four sites, positioned at S968, S1084, S1459 and S2327. All these were not located in the hinge 2 region of FLNa. However, an earlier large-scale phosphoproteomic analysis in non-muscle mouse tissue proposed that FLNa can be phosphorylated in its hinge 2 region and Ig-like domain 24 at the sites S2523, S2526 and T2599 (Huttlin et al., 2010). Sequence alignment of the filamin isoforms (see appendix section D.1) shows that FLNa S2523 has no conserved site in FLNc. Moreover, the homologous site to FLNa S2526 and T2599 (hFLNc 2602 and T2677) were also not identified in the in vivo study under the conditions tested. Thus it can be assumed, that on the one hand the phosphorylation-dependent mechanisms differ between the filamin family isoforms and on the other hand, that phosphorylation occurs tissue specific.

To sum up, the MS-based in vitro kinase assays revealed that S2625 and S2623/S2624 3.1. The basal phosphoproteome of contracting C2C12 myotubes 43 localized in the hinge 2 region of mFLNc and hFLNc, respectively are specific targets of PKCα in vitro. An involvement of PKCα in the phosphorylation of the other two sites that were found to be located at S2603 and S2633 within the C2C12 phosphoproteome study of contracting myotubes could not be verified. Furthermore, S2625 of mFLNc was identified to be significantly down-regulated upon Gö6976 treatment in contracting C2 myotubes. With this comprehensive analyses new information is gained about FLNc phosphorylation. The current knowledge about FLNc phosphorylation in PhosphoSitePlus is based on the publications of Klammer et al. (2012) and Zhou et al. (2012) and Rigbolt et al. (2011). Even though these studies have improved the understanding of the phosphoproteome substantially, none of them focused on phosphorylation events in muscle cells. While the first two analyzed the phosphoproteome of cancer cells, the latter studied changes in phosphorylation in human embryonic stem cell differentiation. Moreover, analysis of the supplemental data revealed that Klammer et al. (2012) and Rigbolt et al. (2011) reported hFLNc S2624 to be phosphorylated, in contrast to Zhou et al. (2012) who reported hFLNc S2623 to be the modified residue. Thus, this thesis successfully eliminated previous ambiguities about the localization of FLNc phosphorylation sites and added knowledge muscle-specific phosphorylations.

The sequence alignment also shows that the serine-rich amino acid stretch 2621SRGASYSSI

PKFSS2633 in the hinge 2 region is uniquely expressed in FLNc, allowing for the assumption that it may have an isoform-speciﬁc function. Previous studies suggested this region to be a substrate of calpain 1 proteolysis (Raynaud et al., 2006), a hypothesis that was strengthened by the observation that calpain 1 and FLNc co-localize at the edges of Z-discs (Raynaud et al., 2006; van der Ven et al., 2000a). For FLNa it was reported that its phosphorylation by PKA leads to an increased resistance against calpain 1 cleavage, thereby inhibiting cytoskeletal reorganization (Chen and Stracher, 1989). Even though Raynaud et al., 2006 proposed a similar PKC-dependent mechanism for the protection of FLNc from calpain 1 cleavage, the speciﬁc sites mediating this process for both, phosphorylation and calpain cleavage remained undiscovered. Interestingly, active PKC itself is further cleaved to a constitutively active form by calpain, resulting in a positive feedback loop which might increase FLNc phosphorylation 44 3.1. The basal phosphoproteome of contracting C2C12 myotubes

and, thus, its resistance to calpain-mediated proteolysis (Goll et al., 2003; Kishimoto et al., 1989).

3.1.6 Establishment of an on-bead calpain cleavage assay

Calpains are a family of Ca2+-activated cysteine proteases with a wide range of targets comprising cytoskeletal and membrane proteins (e.g., ankyrin, desmin, titin), kinases (e.g., PKC) and phosphatases (e.g., protein tyrosine phosphatase 1B) as well as proteins that regulate cell cycle, gene expression and apoptosis (reviewed by Goll et al., 2003). However, despite the fact that several substrates are known, the exact site(s) of calpain cleavage is difﬁcult to predict (Figure A.5 as highlighted in recent work (Shinkai-Ouchi et al., 2016).

Even though FLNc was predicted to be a target of calpain processing over a decade ago, the exact cleavage site remained elusive and its biological relevance undiscovered. While Guyon et al. (2003) suggested that the "10-kDa size difference indicates that C3 [calpain 3] cleaves FLNC near the hinge [2] region", Raynaud et al. (2006) proposed that "γ-ﬁlamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2" is the calpain cleavage site. To overcome these ambiguity, an on-bead in vitro calpainolysis assay which could be coupled to a subsequent MS-analysis was developed in this work.

These predictions indicate that the calpain cleavage site may be in the region of the identiﬁed PKCα-mediated phosphorylation at hFLNc S2623/2624. To identify (i) were the exact calpain cleavage site is located in hFLNc and (ii) whether the PKCα-mediated phosphorylation at hFLNc S2623/S2624 has an effect on the cleavage efﬁciency of calpain, an on-bead calpainolysis assay was established. To this end, FLNc d23-24 WT and its site mutants were recombinantly expressed in E. coli, immobilized on Ni2+-agarose beads and incubated with recombinant calpain 1. Supernatants after calpain incubation and remaining samples on the beads were subjected to SDS-PAGE followed by Western blot analysis using an antibody

directed against the C-terminal His6-tag of the hFLNc constructs (Figure 3.10 A and B).

The results indicate, that all ∼25 kDa hFLNc constructs are cleaved in their hinge 2 region

between the two Ig-like domains, resulting in two fragments. While the C-terminal His6- 3.1. The basal phosphoproteome of contracting C2C12 myotubes 45

ABFLNc 23-24 FLNc 23-24 WT AA DD WT AA DD SB SB SB SB SB SB Calpain + + + + + + Calpain + + + + + + 68 kDa 68 kDa 53 kDa 53 kDa 41 kDa 41 kDa 32 kDa 32 kDa 23 kDa 23 kDa

14 kDa # 14 kDa # ~ * # * * * * * 10 kDa 10 kDa

Figure 3.10.: Establishment of an on-bead calpainolysis assay for hFLNc d23-24. (A) Coomassie stain of FLNc d23-24 WT, AA and DD mutant with and without the incubation with recombinant calpain 1. Supernatants [S] were separated from the beads [B] by a centrifugation step. (B) Corresponding Western blot results for the analysis of calpain using an antibody directed against the C-terminal His6-tag. # N-terminus, * C-terminus, ∼ lysozyme contamination tagged fragments had a molecular mass of ∼12 kDa (Figure 3.10 B), the molecular mass of the N-terminal fragments in the supernatants were ∼13 kDa (Figure 3.10 A). This approach revealed, that the N-terminal calpain cleavage product of hFLNc d23-24 was cut from the bead-bound C-terminal end and could be separated from the beads by a centrifugation step in spin-down columns. In order to identify the exact calpain cleavage site in the hFLNc fragments, the supernatants were acidiﬁed with TFA and subjected to a top-down LC-MS/MS analysis with a combined CID/ETD fragmentation approach.

3.1.7 Tyrosine C-terminally to the PKCα substrate site is the main calpain 1 cleavage site in FLNc

As the Western blot analysis detected a calpain cleavage site in the hinge 2 region, hFLNc d23-24 (Figure A.2) was expressed and used for the top-down-based identification of the specific proteolysis site. For hFLNc d23-24, the cleavage site could be unequivocally mapped to Y2625, resulting in a 13,355.66 kDa fragment whose sequence was confirmed by CID fragmentation (Figure 3.11 A-C). Additional cleavage products (Figure A.6) indicated that also S2618 and S2626 are calpain target sites, however to a minor extent. The main cleavage site Y2625 is in direct neighborhood to the identified PKCα substrate site. For this reason, 46 3.1. The basal phosphoproteome of contracting C2C12 myotubes the effect of the phosphorylation sites on the calpain cleavage was investigated by using the phosphosite mutants of hFLNc d23-24, that were already used in the in vitro kinase assay (Figure 3.11 C-D).

C hFLNc d23-24 WT A 60 MTRGEQSQAG DPGLVSAYGP GLEGGTTGVS SEFIVNTLNA 50 GSGALSVTID GPSKVQLDCR ECPEGHVVTY TPMAPGNYLI 40 AIKYGGPQHI VGSPFKAKVT GPRLSGGHSL HETSTVLVET 30 VTKSSSSRGS SYSSIPKFSS DASKVVTRGP GLSQAFVGQK 20 NSFTVDCSKA GTNMMMVGVH GPKTPCEEVY VKHMGNRVYN 10 VTYTVKEKGD YILIVKWGDE SVPGSPFKVK VPVDHHHHHH normalized intensity [%] 0 EEF S2618 Y2625 S2626

D hFLNc d23-24 AA 60 y 5+ y 6+ B 61 y 4+ 87 1268.66 58 1526.44 50 100 b 1510.10 10 4+ 2+ 1030.45 y57 b 40 y 5+ 1486.54 32 62 1547.21 1288.86 y 4+ 30 y 7+ b15 b 61 82 16 1585.57 1234.91 1483.71 1554.75 8+ 20 y113 5+ y58 1448.73 10 4+ 1208.83 y62 y 4+ y 3+ 1610.85 47 42 normalized intensity [%] 0 1213.38 1440.08 b 2+ y 3+ b S2618 A2623 Y2625 S2626 b 2+ 33 47 17 24 y 3+ 1603.76 1690.87 1717.81 2+ 1143.05 41 b21 1407.07 E 1021.50 y10 b14 y 3+ hFLNc d 23-24 DD 1045.48 1396.68 50 60 6+ 1733.56 2+ y68 b18 1183.61 50 Relative Abundance 887.91 b13 b18 y8 y 1297.61 1774.83 830.36 9 40 917.40 2+ b 2+ y 3+ b22 y13 34 52 1086.52 1346.64 1653.29 1795.28 30 b7 y11 787.37 1146.54 20

0 10 1000 1500 2000 m/z normalized intensity [%] 0 S2618 Y2625 S2626

Figure 3.11.: Y2625 is the major calpain 1-dependent cleavage site in the hinge 2 region of hFLNc. Top-down MS analysis of recombinant hFLNc d23-24 fused C-terminally to a His6- and EEF-tag. Puriﬁed protein was treated with recombinant human calpain 1 and N-terminal cleavage products were subjected to LC-MS/MS using low-energy collision-induced dissociation (CID) as fragmentation method. (A) Amino acid sequence of hFLNc d23-24-His6-EEF. (B) Top down CID spectrum of the main cleavage product observed at m/z 13,355.66; sequence and fragmentation pattern are depicted in black in (A) with the calpain 1-dependent cleavage site in red. (C-E) Determination of the main calpain 1 cleavage sites. Data from three independent experiments for hFLNc d23-24 WT (C) and S2623/S2624 phosphosite mutants [AA mutant, (D); DD mutant, (E)] were quantiﬁed and represented as normalized mean ± SEM. WT, wildtype; A, alanine; D, aspartate

Interestingly, the AA and DD site mutants showed virtually identical cleavage patterns with 3.1. The basal phosphoproteome of contracting C2C12 myotubes 47 an additional cleavage site at S2623 only occurring in the non-phosphorylatable AA variant. For all mouse FLNc d23-24 constructs, the same tendency was detected (Figure A.7) with one exception. For the S2625D mutant, S2627 was detected as an additional cleavage site. However, this altered speciﬁcity is most likely the consequence of a considerably reduced efﬁciency of calpain 1 for cleaving this variant as observed by the reduced signal intensity in the MS-analysis.

To test the hypothesis that the amino acid sequence has an effect on the cleavage efficiency and may thus be an indicator of an effective site-specific protection against calpainolysis, the bead-bound samples after the cleavage assay were investigated by quantitative Western blot analysis (Figure 3.12). The calculated percentages of processed protein showed that ∼25% of hFLNc d23-24 WT were cleaved by calpain 1. In contrast, both mutants were characterized by a significantly decreased calpain cleavage efficiency. While the amount of cleaved hFLNc AA was reduced to 13%, only 3% of hFLNc DD were proteolyzed by calpain. This results are in accordance with the in silico prediction score for the hFLNc constructs (see table B.1 in sectionB).

AB hFLNc d23-24 40 WT AA DD *** ** Calpain 1 + + + *** 30 24 kDa - -FLNc 20

10 14 kDa - -FLNc* 0 αEEF-tag % cleaved hFLNc d23-24 WT AA DD Figure 3.12.: Calpain 1 cleavage efficiency at Y2625 in hFLNc is sequence-dependent. (A) Determination of cleavage efficiency following the calpainolysis assay with recombinantly expressed hFLNc d23-24 and the respective site mutants AA and DD analyzed by immunoblotting. # C-terminal cleavage product. (B) Quantification of data shown in (A) for n=3 experiments. Error bars represent the SD, a two-tailed Student’s t test was used. **p ≤ 0.0034, ***p ≤ 0.0003; WT, wildtype; A, alanine; D, aspartate

These results led to the assumption that the amino acid exchange of two hydrophilic 48 3.1. The basal phosphoproteome of contracting C2C12 myotubes

serine to two negatively charged aspartate might result in a changed conformation in the cleavage area thus reducing the calpain 1 binding and cleavage efﬁciency. This hypothesis is strengthened by the ﬁnding, that the polypeptide conformation has indeed more effect on the substrate proteolysis than the linear amino acid sequence surrounding the potential cleavage site (Goll et al., 2003).

3.1.8 PKCα-mediated phosphorylation of S2623/S2624 protects human FLNc from limited proteolysis by calpain 1 in C2 cells

To address the question whether PKC-mediated phosphorylation of hFLNc-S2623/S2624 is the determining factor for protection against calpain 1 cleavage under cellular conditions, we transiently expressed N-terminally tagged hFLNc d22-24 in C2 mouse skeletal muscle cells and treated the cells either with PMA to stimulate classical PKC isoforms, or with Gö6976 to inhibit PKC isoforms α and β1. 48 h after transfection cells were lysed with a calpain reaction buffer (Mandic et al., 2002) and subsequently equalized cell lysates were treated with recombinant calpain 1 in the absence or presence of calpain inhibitor IV. As control for unphosphorylated hFLNc d22-24, the according sample was incubated with λ-phosphatase prior to treatment with recombinant calpain 1. Protein levels of hFLNc d22-24 and cleavage products thereof were detected by Western blot analysis directed against the N-terminal Myc-tag of hFLNc d22-24.

As expected, efficient proteolysis of hFLNc d22-24 following treatment of C2 lysates with calpain 1 was observed and this effect was abolished with calpain inhibitor IV (Figure 3.13, lanes 1-3). Pre-treatment of lysates with the PKC inhibitor Gö6976 slightly increased proteolysis, whereas incubation with the PKC stimulant PMA reduced the levels of cleavage products in the analysis. Consistently, cleavage of hFLNc d22-24 by activated calpain 1 was more than two-fold reduced following activation of PKC by PMA. Inhibition of PKCα by Gö6976 resulted in a slight, although not significant increase in the amount of cleavage products. The latter finding can be explained by assuming that only a rather small fraction of over expressed hFLNc d22-24 is phosphorylated by endogenous PKC in C2 myoblast cells, underscoring the strong dependence of FLNc’s susceptibility to calpain 1 on its phosphorylation status. 3.1. The basal phosphoproteome of contracting C2C12 myotubes 49

+ + + Gö 6976 ++ ++PMA + + + + Calpain inhibitor IV + + + + ++ + Calpain 1 + -Phosphatase 68 kDa - -FLNc αMyc-tag FLNc* 53 kDa- - 93 kDa- -Calpain 68 kDa- 41 kDa- -GAPDH 32 kDa-

Figure 3.13.: PKC-mediated phosphorylation protects FLNc from calpain 1-dependent proteolysis in C2 cells. C2 cells expressing hFLNc d22-24 were treated with Gö6976 or PMA as indicated. Subsequently, human calpain 1 was added to cell lysates supplemented with 5 mM CaCl2 in the presence or absence of calpain inhibitor IV as indicated. Treatment with λ-phosphatase was used to mimic the non-phosphorylated state. Samples were analyzed by immunoblotting. FLNc*, N-terminal cleavage product.

In the framework of the publication Reimann et al. (2016, under revision), Anja Nicole Schwäble (Department of Biochemistry and Functional Proteomics, University of Freiburg) could conﬁrm the PKCα-mediated protection of FLNc from calpain 1 cleavage by quantitative Western blot analysis in HEK293 cells (see Figure 6 A-C in section 6.1). Thereby, she could show, that PMA stimulation of the cells is resulting in signiﬁcantly reduced cleavage of FLNc. Moreover, she performed a comparative quantitative analysis of calpain 1-dependent cleavage products of hFLNc d22-24 WT and S2623/S2624 site mutants in HEK293 cells (see Figure 6 D and E in section 6.1). By this analysis, she demonstrated that the phosphomimetic DD variant was almost completely protected from calpainolysis.

3.1.9 PKC modulates the dynamic behavior of human FLNc through phosphorylation of S2623/S2624 in skeletal myotubes

Based on the ﬁndings, that PKCα is phosphorylating hFLNc at S2623/S2624 and thus protecting the protein from calpainolysis, Yvonne Leber and Anne Rohland from the group of Dieter O. Fürst (Institute for Cell Biology, University of Bonn), performed FRAP analyses. 50 3.1. The basal phosphoproteome of contracting C2C12 myotubes

Thereby, they wanted to decipher whether PKC-mediated phosphorylation and calpain 1- dependent proteolysis of hFLNc have a physiological effect on its dynamic behavior. To this end, they used immortalized mouse skeletal myoblasts (IMMs) expressing full length hFLNc fused to enhanced green fluorescence protein (EGFP) and differentiated the cells into myotubes. To detect changes, that were induced by the phosphorylation sites or the calpain cleavage of the protein they used mutant forms in which S2623/S2624 were changed to alanine (AA) or aspartate (DD) or the Ig-like domain 24 (amino acids 2628-2725) was deleted. Additionally they treated the myotubes with the PKCα and β1 specific inhibitor Gö6976. The FRAP recovery profiles of all mutants, the WT and the Gö6976 treated WT were analyzed and the protein halftimes were calculated.

The results (see Figure 7 in section 6.1) revealed, that the mean halftimes were signiﬁcantly increased for the AA mutant. This effect was further increased following the inhibition of classical PKC activity in myotubes expressing hFLNc WT. In contrast, the DD mutant of hFLNc exhibited signiﬁcantly shorter mean halftimes compared to the WT form. The same

effect was observed for the MIg-like d24 mutant, mimicking the FLNc protein after cleavage of calpain. For the changes in the mobile fraction no signiﬁcant changes were detected with

the exception for the MIg-like d24 mutant, that showed an increase in the mobile fraction in comparison to FLNc WT. The considerably shorter halftime of the MIg-like d24 mutant along with an increase in mobility, may be explained by the inability of the truncated hFLNc form to dimerize.

In summary, combined analysis of kinase assays, calpainolysis assays and FRAP experiments suggests that PKCα precisely controls the dynamics of hFLNc in skeletal myotubes through phosphorylation of S2623/S2624 (Figure 3.14). Thus, this ﬁnding is a paradigmatic example of a phosphorylation-dependent regulation of two proteins identiﬁed by a follow-up study based on a qualitative large-scale phosphoproteomic analysis. 3.1. The basal phosphoproteome of contracting C2C12 myotubes 51

PKC PKC S2623

P Y2625 FLNc CAPN P CAPN 24

Figure 3.14.: Predicted mechanism for competing calpain 1 and PKCα interaction with FLNc. When PKC is phosphorylation the hinge 2 region of hFLNc at S2623, calpain binding and proteolysis is effectively blocked (left side). In contrast, blocking of PKC by e.g. Gö6976 results in non-phosphorylated hFLNc at S2623. Subsequently, calpain can bin to the protein an cleave it in its hinge 2 region at Y2625. Thus, the protein is losing its dimerization Ig-like domain 24 and is characterized by a higher mobility in FRAP analyses.

3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 53

3.2 Characterization of Pi3k/Akt-mediated signaling events in myotubes Original manuscript: Reimann et al., (see chapter 6.1.5)

The phosphatidylinositol-3 kinase (Pi3k)/Akt pathway is one of the key signaling pathways in cells which regulates, among others, cell growth and proliferation as well as cell survival (reviewed by Manning and Cantley, 2007). In skeletal muscle cells, the pathway was shown to induce myotube hypertrophy (Rommel et al., 2001), characterized by an elevated protein synthesis rate resulting in an higher number of sarcomeres of increased diameter and length (reviewed by Glass, 2003 and Schoenfeld, 2010). Even though Akt is known to phosphorylate its substrates at the basophilic motif RxRxxpS (Alessi et al., 1996), only little is known about its muscle-speciﬁc targets. One challenging aspect in the analysis of Akt substrates is that the basophilic recognition motif is not exclusive for the kinase. In fact, also mTOR/p70 S6 kinase (S6k), serum- and glucocorticoid-induced kinase 1 (Sgk1) and Mapk/p90 ribosomal S6 kinase (Rsk) phosphorylate their targets in this motif (Alessi et al., 1996; Moritz et al., 2010 and reviewed by Manning and Cantley, 2007).

3.2.1 Characterization of different Pi3k/Akt inhibitors in contracting C2 myotubes

To identify conditions under which substrates of the Pi3k/Akt pathway can be analyzed in contracting C2 myotubes, the cells were treated for different time periods with chemicals to stimulate or inhibit this signaling pathway at distinct points. To this end, cells were either treated with (i) insulin-like growth factor 1 (IGF-1) to induce Pi3k/Akt signaling, (ii) LY294002 to inhibit Pi3k, (ii) wortmanin to block Pi3k downstream targets, (iv) Torin-1 or (v) rapamycin to inhibit either both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) or only mTORC1. Subsequently, cells were lysed with modiﬁed RIPA buffer (table 4.1 c in chapter 4.1) in the presence of phosphatase inhibitors at different time points (e.g. 10 min, 30 min, 1 h, 2 h), lysates were equalized according to protein concentrations 54 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

determined by Bradford assay and subjected to PAGE followed by Western blot analysis directed against known substrates of the Pi3k/Akt pathway.

To characterize the effect of the different chemicals on the Pi3k/Akt pathway, the activation status of Akt and S6k was monitored using antibodies directed against AKT phosphorylated at T308 and S473 and S6K phosphorylated at S389, respectively (Figure 3.15). As control, antibodies detecting the total protein amount of the two kinases and the protein amount of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were used.

10 ng ml-1 10 µM 10 nM 10 nM 10 nM IGF-1 LY294002 Wortmanin Torin-1 Rapamycin

control10 min30 min1.0 h 2.0 h control10 min30 min1.0 h 2.0 h 10 min1.0 h 2.0 h control10 min30 min1.0 h2.0 h 10 min1.0 h 2.0 h - AKT - AKT (Thr308) - AKT (Ser473) - p70 S6K - p70 S6K (Thr389) - GAPDH

Figure 3.15.: Time-dependent inhibitor screen in differentiated, contracting C2 myotubes. Myotubes were starved over night, stimulated with EPS and incubated either with IGF-1 or the inhibitors LY294002 (Pi3k), wortmanin (Pi3k), Torin-1 (mTORC1 and mTORC2) and rapamycin (mTORC1) for the indicated time periods. Pathway activity was mapped by the phospho-speciﬁc antibodies of AKT and p70 S6K, respectively.

Under basal conditions (control) phosphorylation signal was detected in the controls for all phospho-speciﬁc antibodies. IGF-1 stimulation led to a considerable increase in phosphorylation of both Akt and S6k with a maximum after 1 h of treatment. In contrast, LY294002, wortmanin and Torin-1 effectively abolished phosphorylation of S6k and Akt phosphorylated at S473. Interestingly, rapamycin had no effect on the Akt phosphosites but reduced completely phosphorylation of S6K at T389 after 1 h. This observation can be explained by the fact that rapamycin inhibits only mTORC1, which phosphorylates its targets downstream from Akt. In contrast, inhibition of mTORC2 by Torin-1 blocks Sin1-mediated phosphorylation of AKT at S473 (Humphrey et al., 2013; Liu et al., 2013; Yang et al., 2006). As the phospho-speciﬁc signals showed highest intensity after 1 h of stimulation with IGF-1, this time point was used to quantitatively study Pi3k-mediated signaling with IGF-1 and 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 55

LY294002 used for activation or inactivation of the pathway.

To this end, C2 cells were differentiated for four days, serum-starved over night and stimulated by EPS for 4 h. Subsequently, the cells were treated for 1 h with 10 ng µl−1 IGF-1 as inducer of the Pi3k/Akt pathway and 10 µM LY294002 to block signaling downstream of Pi3k. As control, myotubes were mock-treated with dimethyl sulfoxide (DMSO). Equalized protein amounts were subjected to SDS-PAGE followed by quantitative Western blot analyses with antibodies directed against main targets of the Pi3k/Akt pathway (Figure 3.16 A). To detect changes upon treatment, ratios of phosphorylation-speciﬁc signals to total protein amounts were calculated for each condition and plotted as mean ratio ± SEM (Figure 3.16 B).

A B 5 4 3 * ** * IGF-1 – + – 4 3 LY294002 – – + 2 3 AKT - 2 2 1 AKT-pT308 - 1

1 AKT-p473/AKT

AKT-pS473 p70 S6K-pT389/S6K - 0 0 0 control IGF LY control IGF LY control IGF LY eEF2 - 4 ** * * *  5 eEF 2-pT56 - 1.5 * 3 4 GSK3 - 1.0 3 2 GSK3-pS9 -S9/GSK3 -  2 1 0.5 1

Rictor eEF2-pT56/eEF2 - GSK3 eEIF4B-S406/eEIF4B 0 0 0 Rictor-pT1135 - control IGF LY control IGF LY control IGF LY

p70 S6K - 4 ** 1.5 * p70 S6K-pT389 - 3 1.0 eEIF4B - 2

eEIF4B-pS406 - GAPDH 0.5 1 GAPDH - Rictor-pS1135/Rictor0 0.0 AKT-p308/AKT control IGF LY control IGF LY

Figure 3.16.: Quantitative substrate mapping of the Pi3k pathway in contracting C2 myotubes. (A) Western blot analysis of Pi3k pathway substrates. Total protein amounts were detected in comparison to phosphorylation-speciﬁc signals. (B) Quantiﬁcation of Western blot results from (b). Calculated intensities were normalized to the control and two-tailed Student’s t-test was performed (n=4, *p ≤ 0.05, **p ≤ 0.01)

Quantiﬁcation revealed that all tested kinases and substrates were signiﬁcantly regulated with regard to their phosphorylation status, either upon IGF-1stimulation, LY294002 treatment 56 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

or both. Interestingly, even though myotubes were starved for at least 16 h, AKT exhibited some activity, characterized by phosphorylation of T308 and S473 detected in control samples. Nevertheless, AKT phosphorylation was signiﬁcantly increased by IGF-1 stimulation and almost completely abolished upon inhibition of Pi3k with LY294002. Phosphorylation of a direct (GSK3β, S9) and indirect Akt substrates, (Rictor, T1135; p70 S6K, T389; EIF4B S406) was also observed under control conditions. Furthermore, these substrates exhibited the same response to IGF-1 and LY294002 treatment as phospho-AKT. As expected, inhibitory phosphorylation of eukaryotic elongation factor 2 (Eef2) at T56, which is mediated by Eef2k was found to be regulated the opposite way. This is due to the fact, that p70 S6K phosphorylates eEF2K at Ser366, thereby blocking its activity (Knebel et al., 2001).

In sum, quantitative Western blot analysis demonstrated that treatment of contracting myotubes with IGF-1 and LY294002 is a suitable setup to study Pi3k/Akt-mediated signaling events. By detecting changes in the phosphorylation pattern of the substrate pathway members Gsk3β (Cross et al., 1995), p70 S6K (Sancak et al., 2007) and the mTORC2 subunit Rictor (Julien et al., 2010) the functionality of this approach was shown. Interestingly, this analysis revealed basal phosphorylation of all investigated targets in fully differentiated and contracting C2 myotubes following 16 h of starvation (Figure 3.16 A, ﬁrst lane). This ﬁnding is in contrast to previously published studies using C2C12 myoblasts (Murray et al., 2004b; Wan et al., 2007) or differentiated and starved myotubes without EPS (Rommel et al., 1999; Rommel et al., 2001; Tong et al., 2009). However, this basal phosphorylation activity is comparable to the phosphorylation level from human muscle biopsies (Hoffman et al., 2015), underlining the importance to apply EPS to myotubes for the analysis of muscle signaling processes in vitro.

3.2.2 Study of the Pi3k/Akt signaling pathway by large-scale quantitative phosphoproteomics

To globally study the Pi3k/Akt signaling network in contracting C2 myotubes, a quantitative large-scale phosphoproteomics study was designed (Figure 3.17 A). C2 myoblasts were labeled for at least nine cell doublings with SILAC, differentiated into myotubes for 4 days, 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 57 serum-starved for 16 h, stimulated with EPS for 4 h and treated for 1 h either with IGF-1, LY294002 or DMSO (see Figure A.8). Subsequently, cells were lysed on ice using high urea buffer (table 4.1e) in the presence of phosphatase inhibitors, mixed in a 1:1:1 ratio according to protein concentration, digested with trypsin, desalted and dried by lyophilization over night. To reduce the complexity of peptide mixtures, samples were fractionated by SCX chromatography and enriched for phosphorylated peptides by TiO2 prior to LC-MS/MS using MSA and HCD as fragmentation methods. Additionally, a small aliquot of each fraction was directly analyzed by LC-MS/MS using CID fragmentation to improve protein identiﬁcation.

A B IGF-1 + + LY294002 # Proteins 7,202

# Phosphoproteins 3,490 Lys 0 Lys 4 Lys 8 Arg 0 Arg 6 Arg 10 # Phosphosites 16,633

PI3K # Quantiﬁed p-sites 13,225 LY294002 # Localized p-sites 11,493

Akt Pathway Inhibition C replicate 1 SCX/TiO2 (total 8,685) 1,969 680 p-sites p-sites

Enrichment 397 L RARLHpSDQGK p-sites H replicate 2 (total 10,323) 1,825 5,639 p-sites p-sites 1,523 M p-sites Intensity replicate 3 (total 9,053) 1,192 m/z p-sites

LC/MS & MaxQuantcontrol SILAC  13,225 quantiﬁed inhibiton stimulation phosphosites

Figure 3.17.: Quantitative phosphoproteomics analysis of Pi3k/Akt signaling in contracting C2 myotubes. (A) Experimental design. For large-scale quantitative phosphoproteomics, contracting C2 myotubes were labeled using SILAC followed by IGF-1 and LY294002 treatment. Three independent biological replicates were analyzed with label switch. (B) Overview of the number of identified proteins and phosphoproteins, including the number of counted, quantified and localized (MaxQuant localization probability score ≥ 0.75) phosphosites obtained by SILAC-MS analyses. (C) Overlay of quantified peptides according to the number of phosphorylation sites (p-sites) in three biological replicates. 69% of all phosphopeptides were quantified in two out of three experiments. 58 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

MS data processing of all acquired 270 raw files from three biological replicates was performed with MaxQuant and Perseus. Almost half (48%) of the 7,202 proteins identified in the dataset were found to be phosphorylated (Figure 3.17 B). These 3,490 phosphoproteins comprised a total number of 16,633 phosphosites of which 69% (11,493 sites) were assigned to a distinct amino acid with a MaxQuant localization probability ≥ 0.75. A total of 80% (13,255 sites) was quantified in at least one biological replicate and of these 70% (9,197 sites) were quantified in at least two out of three biological replicates (Figure 3.17 C). Pearson correlation analysis confirmed the reproducibility of each treatment with a coefficient of at least 0.66 (Figure A.9 A-C). The majority of identified phosphate groups were located at serine residues (80%). Moreover, half of the identified tryptic phosphopeptides were doubly phosphorylated and triply charged (Figure A.9 D-F).

With a number of 9,197 phosphorylation sites quantified, this this dataset provides the so far largest muscle-specific phosphoproteome dataset. While Kettenbach et al. (2015) quantified 2,530 phosphosites in two out of three biological replicates of SILAC-labeled C2C12 myotubes, Lundby et al. (2013) quantified 8,518 phosphosites in heart muscle tissue of mice . In a recent muscle-specific large-scale phosphoproteomic study focusing on Ampk- mediated signaling events 7,421 phosphorylation sites were quantified in two out of four replicates of SILAC-labeled L6 rat myotubes and 8,511 phosphosites in biopsies from human femoral muscle (Hoffman et al., 2015).

3.2.3 Characterization of the Pi3k-dependent phosphoproteome

The majority of the quantiﬁed phosphopeptides remained unaltered in response to IGF-1 (97.6%) or LY294002 (96.9%) treatment (Figure A.9 G). To identify signiﬁcantly regulated

phosphopeptides, the mean log2 ratio of the three biological replicates were calculated and

plotted against the -log10 transformed p-value of the student’s t-test for each treatment

(Figure 3.18 A and B). All peptides with a p-value ≤ 0.05 and a ≥ 1.5-fold mean log2 ratio change were considered to be significantly regulated. For the treatment with IGF-1, these criteria resulted in the identification of 149 and 97 phosphopeptides that were significantly up and down-regulated, respectively (Figure 3.18 A). Additionally, 243 phosphopeptides were 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 59 significantly down-regulated after LY294002 incubation, while 80 phosphopeptides showed a significant increase in their phosphorylation level (Figure 3.18 B).

AB4 6 pSP RxRxxpS RxRxxpSxxS pSxxxS

Junb

Gtf2f1 Rere 4 Pom121 Helb Srpk2 Papd5 Kif26b Phldb2 Larp1 Patl1 Larp1 Sdpr Irs1 Flnc Znf516 2 Nup153 Brd1 Pfkfb2 Foxk1 Macf1 Cep170 Flnc Map3k3Nup153 Mrgbp Lmod1 Hdgfrp2 Bcl6 (p-value) (p-value) Arfgef2 Zc3hav1 Cep170b Erbb3 Pphln1 Bclaf1 Jun Zc3h4 Bcl9l Rptor 10 10 Edc3 C78339 Mrgbp Cic Agfg1 Bad Gsk3b Foxk2 Synpo Tsc2 Prrc2c Mllt4 Prrc2c Gtf2f1 Srrm2 Abcf1 Zfp36l2 2 Ndrg2Zbtb21 Znf516 Helb Rbl2 Chd8 Ndrg1 Papd5 Gtf2f1 -log -log Bclaf1 Nup153 Ankrd17 Samd4aBad Mast2Ccdc6 Pds5b Smap2 Prrc2b Tom1 Pcif1 Cdc42bpa Itsn1 Brpf1 Pphln1 Eif4b Zzef1 Tsr3 Prrc2a Klc2 Chd4 Tbc1d4 Tp53bp1

0 0 -2 0 2 -4 -2 0 2

mean log2 ratio IGF-1 mean log2 ratio LY294002

RA C PF S V E SSS D PSS SDP SS VK K D Q GVDA E STPG P E F S Q V A G A V AP S S S CE P SQ L P T G N RLK P G EA K GA I PR SD KA N S E I E HPM S R I V up KE LR AK I APQH QE PGAP G LP ADGR S RL T I R G D SG RT D NKV G DDR R R F ET AE FN AVL V L G QQEEK E TR C T T ATGH R RLT FD D T G YDEKL HV EFFM GAA A LA NHGN HDH S D GN L CA HPDHGNM T V M I ER I I R I LM LGE Q HS N AE I AL I H P QQGTLLS SQR QHL

MT P HS LNPLKVT LY294002 TRVVYNT TVT YTN IGF-1 up QV Q MYSYYQPQWV -7 -5 -3 -1S 1 3S 5 7 -7 R-5 R-3 -1 1 3 5 7 ASP P S I PGGL R H SEST PKA K APP PP S L G ESEQ G PP SA KQ S D G R R L P EG VS I AA I K A RA E P T RPS N RD D I K LA F LAGL AQ SS R G TGS G E SK L R G Q VHPP S TGA S P G A I F VL I E R A T VA SLV A L TE S V AS W HKRG EDQ F I P N N F G CD A KMHH FEE HSQ T M GL K R KFH K A E M H H KN E MNKK R V R down NL I ALTL VFFKQN N L NT H NPLR Q Q S MKFNGM FH LAS D Q Q Q Q TNNLQNR GLRQDT F P V LM R S YQVQTVT QRTVEV VM Q RY PP RVTV LY294002 LY294002 IGF-1 down S -7 -5 -3 -1 1 3 5 7 -7 -5 -3 -1P 1 3 5 7 R R S S

Figure 3.18.: Determination of regulated phosphopeptides and enriched phospho-motifs in C2 myotubes following stimulation or inhibition of Pi3k/Akt signaling. (A-B) Volcano plots of log2 transformed mean ratios (control/treatment) of all quantified phosphopeptides with a MaxQuant localization probability ≥ 0.75 plotted against the -log10 p-value. Significantly regulated phosphopeptides (p-value ≤ 0.05, n = 3, two-tailed Student’s t-test; mean log2 ratio change ≥ 1.5-fold) are shown as open blue circles. Peptides of interest are labeled according to their phosphorylation motif, regions of interest highlighted in light orange (≥ 1.5 fold-change) and orange (≥ 3.0 fold-change). (C-D) Motif-X analysis of significantly regulated phosphopeptides against the whole dataset as background reveals an enrichment of the basophilic motif RxRxxpS (58-fold) and the proline-directed motif pSP (5-fold) for the IGF-1 treatment (C) and of the pSxxxS motif (4-fold) and the basophilic motif RxRxxpSxxS (148-fold) for LY294002 treatment (D). x, any amino acid

For further characterization of the involved signaling network, motif-X analysis was performed with a ± 15 amino acid sequence window for all signiﬁcantly regulated serine phosphosites searched against the complete mouse proteome as background (Schwartz and Gygi, 2005). With a 58-fold increase, the basophilic RxRxxpS-motif was the top up-regulated motif after treatment of C2 myotubes with IGF-1 (Figure 3.18 C, upper panel). In comparison, 60 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

the proline-directed motif pSP was the top identiﬁed motif (5-fold enrichment) among the 97 down-regulated phosphopeptides following IGF-1 stimulation (Figure 3.18 C, lower panel). In contrast, treatment of C2 myotubes with LY294002 resulted in a 4-fold increase of the pSxxxS motif (Figure 3.18 D, upper panel). Most interestingly, Pi3k inhibition by LY294002 treatment resulted in the identiﬁcation of 20 peptides comprising the extended basophilic motif RxRxxpSxxS accounting for an 148-fold increase of this motif compared to the background, while the classical RxRxxpS motif was only 40-fold enriched (Figure 3.18 D, lower panel). In order to decipher the role of these motifs, the results from the large-scale phosphoproteomic analysis were mapped to the canonical Pi3k/Akt pathway (Figure 3.19).

IGF-1

S25 S9 S474 Akt2 S478 Pdk1 S664 PIP3 S336 S325 S307 S302 Gys1 Gsk3β S136 Bad S112 T448 Irs1 S343 Shc-1 S318 Ppp1ca S439 S444 Glycogenesis T104 S88 Pi3K Phgk1 S469 S94 Grb2 Pfkfb2 Yap1 Irs2 S486 T1249 S573 S303 S590 S1122 S2234 T247 S2237 LY294002 Sos1 Pygm Glycolysis T2239 Flnc Akt1s1 S532 S538 T763 S450 S184 Actin S543 T635 Ulk1 Regulation organization S1469S1138 T1135 S1068 S721 S863 Nras Ulk2 of autophagy S1468 Rictor Mlst8 TSC1 Raptor Mlst8 S1032 S362 Prkca Rheb T503 S523 S366 mTOR Deptor TSC2 mTOR Deptor Braf S367 S333 S2481 T747 T762 S664S330 S981 S1443 T421 S429 S2481 Larp1 S357 S2478S2454 mTORC2 mTORC1 Ndrg1 S2478 S2454 S138 S751 S743 T765 Rps6kb1 Map2k1 S664S350 Map3k3 Ndrg2 Eef2k S418 Sgk1 S166 S424 T69 Y205 Map2k2 T461 T36 T57 T203 Mapk3 Eef2 Rps6 Eif4ebp1 T59 Eif4b S409 RxRxxSxxS S235 T241 Mapk1 motif p-site S406 T412 T422 T183 Y185 Rps6ka3 Mnk1 IGF-1 LY294002 S399 S406 S427 T408 Rps6ka4 Mknk2 > 3.0 S414 Foxk1 S431 > 1.5 S402 S243 S229S235 Eif4e S225 T437 S436 S433 not signiﬁcant < - 1.5 S431 Gtf2f1 T389 S377 T384 Protein synthesis < - 3.0

Figure 3.19.: Pathway analysis of proteins with regulated phosphosites. Excerpt from the canonical IGF-1-activated signaling pathway, comprising the downstream signaling branches of the Pi3k/Akt and Mapk cascade. Proteins are represented by their gene names according to Uniprot annotation. Interactions were curated from literature and the KEGG database. Signiﬁcantly regulated (p ≤ 0.05, two-tailed Student’s t-test; mean log2 ratio change ≥ 1.5-fold) phosphorylation sites are color coded according to their regulation factor shown in the legend. Proteins not present in the dataset are shown in light gray.

The analysis revealed the three proteins N-Myc downstream regulated gene 1 and 2 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 61

(Ndrg1, Ndrg2) and mitogen-activated protein kinase kinase kinase 3 (Map3k3), all targets of Sgk1 were identified to contain phosphopeptides comprising the motif RxRxxpSxxS. In addition, peptides of La-related protein 1 (Larp1), general transcription factor 2f (Gtf2f1), eukaryotic translation initiation factor 4B (Eif4b) and the muscle-specific scaffold protein FLNc comprised phosphopeptides with the enriched RxRxxpSxxS motif. Except for TBC1 domain family member 4 (Tbc1d4), all identified sequences mapping the motif were also found to be phosphorylated at the second serine. Moreover, the doubly phosphorylated peptides of Eif4b, NDRG1 and FLNc were also significantly regulated. Interestingly, in FLNc two out of three identified regulated phosphosites, namely S2234 and S2237, were found to be located within the RxRxxpSxxS motif.

The “classical” basophilic motif RxRxxpS has been reported to be strictly required for Akt-mediated phosphorylation (Alessi et al., 1996). Since its discovery it has been in the focus of various studies (Dobson et al., 2011; Moritz et al., 2010; Sakamaki et al., 2011). In contrast, the here identified extended motif RxRxxpSxxS has not been described before. Even though Humphrey et al. (2013) identified an higher preference of Akt for substrates with a serine/threonine in +1 and +2 positions in comparison to mTOR/Pi3k in adipocytes, the evidence for a serine in +3 position was not provided in their analysis. By mapping the appropriate motifs to the canonical Pi3k/Akt pathways no distinct kinase was identified to mediate this motif.

A closer look at the 20 peptides comprising the RxRxxpSxxS motif (see Table B.2) revealed that all were phosphorylated at the serine residue in the +3 position, except for Tbc1d4. This indicated a possible involvement of an additional kinase in the phosphorylation of peptides at the RxRxxpSxxS motif. In Eif4b, Ndrg1 and FLNc these doubly phosphorylated peptides were also significantly regulated. All other identified phosphopeptides were only regulated in their singly phosphorylated form. For Ndrg2, Gtf2f1, Map3k3, Larp1, Edc3 and Itsn1 the phosphorylation site in +3 position did not fulfill the filter criteria of a MaxQuant localization probability ≥ 0.75. Most probably this lower localization probability is caused by a cluster of serine residues in the +2 to +4 position of the respective peptides hampering the clear 62 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

assignment of the phosphate group to a distinct serine.

For seven proteins, the kinase mediating the phosphorylation of the ﬁrst serine residue in this motif is known, whereas no information is available for the other proteins. While hFLNc-pS2233, enhancer of mRNA-decapping protein 3 (Edc3)-pS161 and 6-phosphofructo- 2-kinase/fructose-2,6-biphosphatase 2 (Pfkfb2)-pS464 were shown to depend on Akt (Larance et al., 2010; Murray et al., 2004b; Novellasdemunt et al., 2013), Eif4b-pS406 was suggested to be mTORC-dependent (Gorp et al., 2009). Moreover, Gsk3 and Sgk1 were reported to phosphorylate S330 in Ndrg1 (Murray et al., 2004a), while phosphorylation of Ndrg2 at S350 were proposed to depend on Rsk1 (Murray et al., 2004a) and Map3k3 at S166 on Sgk1 (Chun et al., 2003), respectively.

In contrast to these ﬁndings, nothing is known about the kinase(s) mediating phosphorylation in the +3 position. Which kinases might be involved in the phosphorylation of the +3 position and what may be the role of two phosphorylation sites in such close proximity? Kinase prediction and the matching PKC motif with a preference for arginine residues in the -7 to -3 position and a glycine residue in the -1 position (Nishikawa et al., 1997) identiﬁed 6 proteins to be potential classical PKC substrates.

3.2.4 The unique insertion in Ig-like domain 20 of ﬁlamin C is multiply phosphorylated in myotubes

To decipher the kinases which phosphorylate the RxRxxpSxxpS motif in mFLNc, the two identified phosphorylation sites S2234 and S2237 were analyzed for their regulation upon IGF-1 or LY294002 treatment of C2 myotubes (Figure 3.20 A). Both the singly phosphorylated peptide at S2234 as well as the doubly phosphorylated peptides (S2234/S2237 and S2234/T2239) were significantly down-regulated upon LY294002 treatment. In contrast, the singly S2237 phosphorylated peptide was found to be significantly down-regulated following IGF-1 stimulation.

Even though mFLNc-S2234 has been described as Akt target over a decade ago (Murray et al., 2004b) and was identiﬁed in numerous studies since then (Hou et al., 2010; Huttlin et al., 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 63

AB IGF-1 LY294002 *** LGSFGpSITR * S2237 IGF-1 – + – 1.5 LY294002 – – + * LGpSFGSIpTR S2234 T2239 FLNc - 1.0 S2234 FLNc-pS2234 * LGpSFGpSITR - 0.5 S2237 GAPDH - FLNc-pS2234/FLNc * LGpSFGSITR S2234 0 control IGF LY

-2 -1 0 1

Mean log2 ratio

Figure 3.20.: The FLNc unique 82 aa insertion within Ig-like domain 20 is a phosphorylation hot spot. (A) The mouse peptide LGSFGSITR, singly and doubly phosphorylated at S2234 and S2237/T2239 is signiﬁcantly regulated upon IGF-1 and LY294002 treatment in C2 myotubes. (B) Western blot analysis and quantiﬁcation of mFLNc phosphorylated at S2234 in comparison to total FLNc level.

2010; Phanstiel et al., 2011; Rigbolt et al., 2011; Wu et al., 2012), its biological relevance remained elusive. A proximity ligation assay (PLA) of differentiated C2 myotubes with an antibody directed against phospho-FLNc S2234 and an antibody against AKT revealed, that the two proteins are co-localizing at the Z-disc after 1 h of IGF-1 stimulation (see Figure A.10 A and B). In contrast, PLA of cells treated with LY294002 for 1 h showed less interaction of phospho-FLNc S2234 with AKT at the Z-disc (see Figure A.10 C and D).

A comparison of the measured MS-intensities revealed that phosphorylation of mFLNc S2234 was two orders of magnitude more abundant than the other two sites (S2237 and T2239) (Figure A.11). To validate the large-scale phosphoproteomic results, samples from the MS-approach were quantified with a site-specific phospho-FLNc antibody, which uniquely identifies mFLNc-S2234 and hFLNc-S2233 phosphorylation, respectively (Figure 3.20 B). Quantitative Western blot analysis verified the tendency identified for mFLNc phosphorylation levels in the large-scale phosphoproteomics study and showed a significant down-regulation of FLNc S2234 phosphorylation upon LY294002 treatment of the myotubes.

All three sites are located in the isoform-speciﬁc 82 amino acid insertion within Ig-like domain 20 of FLNc. Blast analysis7 (data not shown) conﬁrmed this amino acid stretch to be

7 http://www.uniprot.org/blast/ 64 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes almost 100% conserved among mammals. In silico kinase prediction using the algorithms NetworKIN 3, NetPhorest and Scansite 3 consistently identiﬁed human FLNc S2233 as a substrate of PKB/Akt (Figure 3.21 A) and S2236 and T2238 as putative substrate sites of PKC/PKCα.

A B

mFLNc 2231RLGS FGS I T R 2240 hFLNc d18-21 WT hFLNc 2230RLGS FGS I T R 2239 ATP + +++ 82 aa domain 20 insert PKCα + + AKT1 + + 68 kDa - PKC/ T2238 S2233 S2236 53 kDa - AKT/PKB CaMKII NetworKIN 3.0 n. a. NetPhorest 41 kDa Scansite 3 - αEEF-tag

C 60 AKT1 PKC 40 20 PKC+ AKT1

10 normalized intensity [%] 0 T2179 S2233 S2236 S2233 S2182 S2236

Figure 3.21.: S2233 and S2236 of human FLNc are specific substrate sites of Akt and PKCα, respectively. (A) Kinase prediction with NetworKIN 3.0, NetPhorest and Scansite revealed the conserved domain 20 to be a target of the two kinases Akt/PKB and PKC/PKCα. A minimum score threshold was set to 0.2 for NetworKIN 3.0 and NetPhorest. (B) Phosphorylation-dependent mobility shift analysis by PhosTag-PAGE following in vitro phosphorylation. Reactions were performed using human FLNc d18-21 and unlabeled ATP in the presence of AKT1, PKCα or a combination of the two kinases at a concentration of 10 ng/100 µg protein. Western blot analysis of PhosTag gel with an antibody directed against the C-terminal EEF-tag reveals multiple shifted bands of human FLNc d18-21. (C) MS-based quantification of the in vitro kinase assays. Reactions were performed as described in (B). MS data from three independent kinase experiments with hFLNc d18-21 WT were quantified using Skyline. Intensities of phosphopeptides distinctive for a specific phosphorylation site were added up per experiment and represented as normalized mean ± SEM. No phosphorylation of hFLNc T2238 was detected. WT, wildtype.

The human sites S2233, S2236 and T2238 (corresponding to S2234, S2237 and T2239 in mFLNc) were consistently identiﬁed as in vivo phosphosites in HeLa and K562 cells (Zhou et al., 2012). Based on the kinase prediction and the presence of a PKCα consensus site motif 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 65

(Nishikawa et al., 1997), it was hypothesized that PKCα phosphorylates human FLNc at S2236. To conﬁrm this hypothesis, in vitro kinase assays were performed with recombinantly expressed FLNc domain 18-21 wildtype using the kinases AKT and/or PKCα in the presence of ATP. Samples were subjected to PhosTag-PAGE followed by Western blot analysis directed against the C-terminal EEF-tag (Figure 3.21 B). While a weak band shift was observed for hFLNc D18-21 upon incubation with PKCα, approximately 50% of the protein shifted following its reaction with AKT. Two shifted bands were observed after the incubation with both kinases (Figure 3.21 B lane 5). In contrast, no band shifts were detectable in control experiments without ATP or the kinases.

To obtain residue-resolved information about hFLNc d18-21, in vitro kinase assays were coupled to LC-MS/MS analysis. Experiments were performed in triplicates using equal protein amounts, followed by phosphopeptide analysis using complementary fragmentation methods and MS1-based quantification using Skyline. This comprehensive analysis confirmed S2233 in hFLNc as substrate site of AKT. As observed in vivo, in vitro phosphorylation of hFLNc d18-21 by Akt resulted in efficient phosphorylation of S2233 (Figure 3.21 C). In contrast, incubation with PKCα resulted in phosphorylation of T2179, S2182, S2233, and S2236, all four sites which are located in the unique insertion of hFLNc. As the first two phosphosites have not been identified in vivo, it was assumed that they occurred non-specifically under in vitro conditions. Thus, it was hypothesized that S2236 is the predominant substrate of PKCα. Accordingly, incubation with both kinases led to an increase of the doubly phosphorylated peptide modified at the residues S2233 and S2236.

3.2.5 Identiﬁcation of Filip1 as a novel FLNc interaction partner

The unique 82 amino acid insertion Ig-like domain 20 of FLNc is lacking in filamin A and B and has previously been shown to fulfill an important role in the interaction of FLNc with several other sarcomeric proteins including aciculin (Molt et al., 2014), Xin/Xirp1 (van der Ven et al., 2006), myopodin (Linnemann et al., 2010) and myotilin (van der Ven et al., 2000b). In order to identify new putative interaction partners of FLNc d18-21, Anna Lena Fricke (Department of Biochemistry and Functional Proteomics, University of Freiburg) established 66 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes proximity-dependent biotin identification (BioID) (Roux et al., 2012) using SILAC-labeled contracting C2 myotubes in her bachelor thesis (supervised by Lena Reimann) (Figure 3.22).

A B Mean log10 FLNc/BirA* Mean log10 FLNc/FLNc - 1 + BirA* + + BirA*FLNc + + Biotin 2 LMH SILAC

B B B Enrichment

RLGSFGSITR H L 3 M MaxQuant Intensity LC-MS/MS &

m/z

C 2 1: BirA* FLNc 2: Ldb3 1 D 3: Bag3 Z disc M band 4: Filip1 myoﬁbril 5: Xirp1 sarcomere 6: FLNc 2 A band 7: PGM5 3 1 4 muscle myosin myosin II 5 complex complex 6 I band contractile ﬁber part H zone 7

FLNc d18-21 + biotin cell-substrate /BirA* + biotin 10 adherens junction myosin complex 0 stress ﬁber actin ﬁlament

Mean log bundle actin myosin ﬁlament cytoskeleton focal adhesion 012 actomyosin

Mean log10 BirA* FLNc d18-21 +biotin/-biotin Figure 3.22.: Identiﬁcation of putative FLNc interaction partners by proximity-dependent biotinylation. (A) Experimental design of SILAC-based proximity-dependent biotinylation experiment. Differentiated and EPS-treated C2 cells transiently expressing the promiscuous biotin ligase BirA* and BirA*FLNc d18-21 were incubated with 50 mM biotin. Cell lysates were mixed in a 1:1:1 ratio, biotinylated proteins were enriched via streptavidin, tryptically digested and analyzed by LC-MS/MS. (B) Log10 transformed SILAC ratios of two biological and 2 technical replicates obtained from MaxQuant analysis were processed with Perseus by hierarchical cluster analysis. (C) Analysis of biotinylated proteins identiﬁed highly enriched (orange, cluster 1), moderately enriched (blue, cluster 2) and non-enriched (gray, cluster 3) proteins. Together with known FLNc binding partners, Filip1 is in the orange cluster and thus a potential novel FLNc interacting protein. MaxQuant ratios were log10 transformed and used for hierarchical cluster analysis with Perseus (n= 3 cluster). Proteins of interest are labeled, regions of interest highlighted in gray (>5 fold change). D: domain; BirA*: promiscuous biotin ligase. (D) Gene Ontology over-expression analysis for cellular components of the orange and the blue cluster from the hierarchical cluster analysis performed with the Cytoscape plugin ClueGO. 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 67

To this end, SILAC labeled C2 cells transiently expressing BirA*FLNc d18-21 or BirA* were differentiated into contracting myotubes and incubated with biotin for 24 h prior to cell lysis (Figure 3.22 A). Cells expressing BirA* following the addition of biotin and cells expressing BirA*FLNc d18-21 without the addition of biotin were used as negative controls. Following enrichment of biotinylated proteins by streptavidin beads, tryptic on bead digestion and quantitative LC-MS analysis, the SILAC ratios of all 463 identified proteins were log10 transformed. Subsequently, hierarchical cluster analysis was performed to identify significantly enriched proteins (Figure 3.22 B). As a result 63 highly enriched (cluster 1, orange), 95 moderately enriched (cluster 2, blue) and 305 non-enriched (cluster 3, gray) proteins (Figure 3.22 C). Heatmap cluster analysis in combination with scatter plot analysis revealed the enrichment of the known FLNc interaction partners Bag3, Xirp1 and aciculin (PGM5). Furthermore, GO enrichment analysis demonstrated a over-representation of the terms ’Z-disc’, II-band’, ’sarcomere’, ’myofibril’ and ’contractile fiber part’ for the highly enriched proteins of the orange cluster. Additionally, GO-terms associated with the ’A-band’, ’actin cytoskeleton’ and ’cell-substrate adherens junction were identified for the moderately enriched proteins, proving that this method is suitable to identify and enrich proteins from complex sub-structures within the myofibril.

Furthermore, FILIP1, a known interaction partner of FLNa (Nagano et al., 2002) was identiﬁed among the highly enriched proteins. The interaction between FLNc d18-21 and the C-terminus of FILIP1 could be conﬁrmed by our collaboration partners (Dieter O. Fürst, Institute for Cell Biology, University of Bonn). To this end, they used yeast two hybrid screens, dot-blot overlay assays and pull- down experiments with different domains of FLNc to verify the domain 20-21 as binding region with FILIP1 C-term (see Figure 5 in section 6.1.5).

As the regulated phosphorylation sites S2233 and S2236 are located in the unique 82 amino acid insert of domain 20 of hFLNc, pull-down experiments of human FLNc and its phosphosite mutants with FILIP1 CT were performed to reveal whether this interaction is phosphorylation-dependent. To this end, recombinantly expressed bead-bound GST-FILIP1

C-term was incubated with recombinantly expressed and puriﬁed His6-tagged FLNc d18-21 68 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

WT, its phosphosite mutants and FLNc d1-3 as control. In FLNc d18-21, either S2233 or S2233 and S2236 were mutated to aspartate (D) or alanine (A) to mimic its constitutively phosphorylated or non-phosphorylated state. The amount of FLNc isoforms bound to FILIP1

C-term was detected by Western blot analysis directed against the His6-tag (Figure 3.23 A). The results of four independent replicates were quantified and the amount of each bound FLNc isoform was normalized to FLNc d18-21 WT (Figure 3.23 B). This analysis revealed, that the FLNc d18-21 phosphomimetic DD mutant had a significant reduced binding affinity to FILIP1 C-term in comparison to the other FLNc d18-21 isoforms.

AB GST-FILIP 1.5 * FLNc d1-3 d18-21 WT WT D DD A AA 53 kDa 1.0

41 kDa anti-HIS Eluate

53 kDa 0.5 anti-HIS

41 kDa bound to GST-FILIP nom. amount of FLNc d18-21 Input 0.0 68 kDa anti-FILIP WT D DD A AA 53 kDa FLNc d18-21

Figure 3.23.: S2233 and S2236 have regulatory function in the interaction of FLNc with Filip1. (A) Western blot analysis of pull-down assay with GST-FILIP1 C-terminus (CT) as bait and FLNc d18-21, its phosphomutants and FLNc d1-3 as prey, respectively. (B) Quantiﬁcation of pull-down experiments normalized to the wildtype. Phosphosite mutants were generated as described in (A). Calculated intensities were normalized to the control, SEMs were calculated and two-tailed Student’s t-test was performed (n=4; *p-value ≤ 0.05). Phosphosite mutants were generated from the respective WT construct by exchange of one or two serine residues at the position 2233 and 2236 to alanine (A) or aspartate (D) as indicated. WT, wildtype; d, domain.

In order to conﬁrm the results from the pull-down experiments, Anja Nicole Schwäble (Department of Biochemistry and Functional Proteomics, University of Freiburg) performed co-immunoprecipitation (co-IP) experiments in HEK293 cells. To this end, she co-expressed Filip1 CT-GFP with either Myc-tagged FLNc d22-24, FLNc d18-21 or the respective phosphosite mutants and performed anti-Myc co-IPs. By this approach she could reveal, that the 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 69

FLNc d18-21 A and AA mutant had a signiﬁcant lower binding afﬁnity to Filip1 CT-GFP in comparison to the FLNc d18-21 WT as well as the D and DD mutant (see Figure 6 in section 6.1.5).

These results suggest that non-phosphorylated FLNc is interacting with FILIP1 C-term. Furthermore, phosphorylation of hFLNc at S2236 by PKCα appears to signiﬁcantly regulate this interaction. This is surprising given the fact that FLNc S2233 phosphorylation by AKT was found to be considerably more abundant that phosphorylation of S2236 by PKCα.

3.2.6 Identiﬁcation of R1751 as calpain cleavage site in the hinge 1 region of FLNc

For FLNa, the interaction with Filip1 is proposed to induce calpain binding leading to FLNa degradation in COS7 cells (Vadlamudi et al., 2002 and reviewed by Zhou et al., 2010). Upon binding of Filip to FLNa, calpain is interacting with the protein complex and is inducing cleavage in the hinge 1 region of FLNa, resulting in a C-terminal 110 and an N-terminal 170 kDa fragment. Subsequently, the C-terminal fragment can further be cut in the hinge 2 region and the resulting FLNa fragment d16-23 can translocate to the nucleus, where it was shown to interact with the androgen receptor. (Loy et al., 2003). Gorlin et al. (1990) identiﬁed the cleavage site in the hinge 1 region of FLNa to be located at Y1761.

The predominantly expressed FLNc isoform in skeletal muscle cells contains no hinge 1 region (Xie et al., 1998). However, (unpublished data from the group of Dieter O. Fürst, Institute for Cell Biology, University of Bonn) FLNc containing the hinge 1 region is suggested to be the predominantly expressed protein isoform in proliferating myoblasts. To validate, whether calpain is also cleaving FLNc in its hinge 1 region, the top-down MS-based calpainolysis assay was performed with a FLNc d15-21 fragment including the hinge 1 region. As control for the functionality of the assay a FLNc construct comprising d16-24 with the previously described cleavage site in the hinge 2 region was used (see section 3.1.7).

Both fragments were recombinantly expressed in E. coli, immobilized on Ni2+-NTA- agarose beads and incubated with recombinant calpain 1. Supernatants together with remaining sample on the beads were subjected to SDS-PAGE followed by Western blot analysis using 70 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes

an antibody directed against the C-terminal His6-tag (Figure 3.24 A and B). Resulting N- terminal cleavage products in the supernatant were analyzed as intact protein fragments by high resolution LC-MS following a top-down approach on a Velos Orbitrap Elite system (Figure 3.24 C).

A FLNc FLNc B FLNc FLNc d15-21 d16-24 d15-21 d16-24 ++Calpain 1 ++Calpain 1 170 kDa - 125 kDa - 104.08 kDa 125 kDa - 93 kDa * 85.21 kDa 93 kDa 104.08 kDa - - 85.21 kDa 68 kDa - # 68 kDa - # 53 kDa - 53 kDa - 41 kDa - 41 kDa - 32 kDa - 32 kDa - 23 kDa - 23 kDa - 14 kDa - # 14 kDa 10 kDa - # - * 10 kDa - C coomassie stain αHis-tag

hinge 1 1735CDPLPHEEEPSEVPQLR|QPYAPPR1758

His- EEF- hFLNc d15-21 1516 17 18 19 20 21 tag tag M1 F793

FLNA_HUMAN 1739 TALAGDQPSVQPPLRSQQLAPQYTYA--QGGQQTWAPERPLVGVNGLDVTSLRPFDLVIP 1796 FLNB_HUMAN 1703 MATDGEVTAVEEAPVN------ACPPGFRPWVTEEAYVPVSDMNGLGFKPFDLVIP 1752 FLNC_HUMAN 1733 LACDPLP-HEEEPSEVPQLRQPYAPPRPGARPTHWATEEPVVPVEPME-SMLRPFNLVIP 1790

D His- EEF- hFLNc d16-24 16 17 18 19 20 21 21 23 24 tag tag M1 F972 hinge 2 2593TGPRLSGGHSLHETSTVLVETVTKSSSSRGSSY|SSIP2629

FLNA_HUMAN 2450 YLISIKYGGPYHIGGSPFKAKVTGPRLVSNHSLHETSSVFVDSLTKATCAPQHG--APGP 2507 FLNB_HUMAN 2495 YLISVKYGGPNHIVGSPFKAKVTGQRLVSPGSANETSSILVESVTRSST--ETCYSAIPK 2552 FLNC_HUMAN 2571 YLIAIKYGGPQHIVGSPFKAKVTGPRLSGGHSLHETSTVLVETVTKSSSSRGSSYSSIPK 2630

Figure 3.24.: Analysis of calpain 1 cleavage sites in human FLNc domains. (A) Coomassie stain of FLNc domains 15-21 and 16-24 with and without the incubation with recombinant calpain 1. (B) Corresponding Western blot results for the analysis of calpain using an antibody directed against the C-terminal His6-tag. # C-terminus, * N-terminus. (C) and (D) Results from cleavage site identification in FLNc d15-21 and 16-24 by top-down mass spectrometry and sequence alignment of filamin family members. Identified calpain cleavage site in FLNc d15-21 between R1751 and Q1752 as well as literature-based cleavage site in hinge 1 region of FLNa (Gorlin et al., 1990) and the previously identified cleavage site in the hinge 2 region are marked in red.

Western blot-based analysis revealed one main C-terminal cleavage product for each construct (Figure 3.24 B). For FLNc d15-21, the cleavage product had a reduced molecular 3.2. Characterization of Pi3k/Akt-mediated signaling events in myotubes 71 mass of ∼ 10 kDa, indicating that the cleavage must have occurred in the hinge 1 region. In contrast, the observed resulting C-terminal protein fragment from calpain cleavage of FLNc d16-24 had a remaining size of ∼ 13 kDa, conﬁrming the results of the calpain cleavage experiments with FLNc d23-24.

The subsequent MS-based analysis of the resulting N-terminal fragments identiﬁed FLNc R1751 as calpain cleavage site in FLNc d15-21. In contrast, no N-terminal fragment could be detected by top-down MS for FLNc d16-24. In this way, no cleavage site could be identiﬁed even though Western blot analysis demonstrated, that FLNc d16-24 is cleaved by calpain. As an explanation, it was assume that the rather large molecular weight of ∼ 90 kDa from the fragment that was subjected to top-down analysis was not suitable for the LC-MS setup. Comparison of the FLNc hinge 1 cleavage site with the published calpainolysis site in FLNa revealed that both sites are located in the hinge 1 region even though this amino acid stretches share no sequence homology.

By this MS-coupled assay, the exact calpain cleavage site in FLNc hinge 1 could be determined for the ﬁrst time. Moreover, this method may serve as a valuable screening technique to systematically identify calpain cleavage sites in substrates and thereby would allow for the development of important prediction algorithms (Franco and Huttenlocher, 2005). However, the establishment revealed, that the currently used LC-MS setup is not suitable for the analysis of calpain cleavage fragments in the molecular mass range of 90 kDa. To this end, the method has to be optimized for larger molecular mass proteins. Thus, this technique could then be used to reﬁne prediction models based on experimental data.

3.3. New insights into smooth muscle myosin phosphorylation 73

3.3 New insights into smooth muscle myosin phosphorylation

Original publication: Puetz et al., (see chapter 6.3)

Classical bottom-up shotgun (phospho)proteomic approaches use trypsin as protease of choice as its cleavage results in an average peptide length of 14 amino acids with a standard deviation of 20 amino acids for one allowed miss cleavage (reviewed by Vandermarliere et al., 2013). Furthermore, metabolic labeling strategies such as SILAC are designed for the combination with tryptic digestion, as each resulting tryptic peptide contains one labeled arginine or lysine. However, 56% of the generated tryptic peptides are shorter than six amino acids (Swaney et al., 2010). Hence, they are too small to be detected by standard LC-MS methods and therefore limit the sequence coverage that can be obtained for a protein of interest.

Tryptic peptides produce y-ion rich spectra and high scoring identiﬁcations with CID/HCD fragmentation because the y-ions contain the basophilic C-terminal arginine or lysine (Tabb et al., 2004). This dominant y-ion series represents a major drawback when the phosphorylation site of interest is located at the position P1 or P2 of the peptide as almost no b-ions are detected.

Additionally, small ions such as b2-, y1- and y2-ions are not in the region of stable oscillation in a 3D ion trap (Mathieu equation, Douglas et al., 2005; "one-third-rule", Louris et al., 1987). Thus, no or only insufﬁcient site-speciﬁc information is available for phosphorylation at the N-terminal end of a peptide.

To overcome this drawback, alternative proteases can be used, which allow for the generation of in differently cleaved peptides. Ideally, the phosphorylation site is no longer located at the P1 or P2 position of the peptide. To test this approach, the proteases chymotrypsin, trypsin in combination with cyanogen bromide (CNBr), Lys-C, elastase, trypsin, and thermolysin were used to digest gel bands of five different proteins (see section 4.2 for method and table B.2 for digestion rules of different proteases). Peptides obtained from each digestion were analyzed in an one hour LC-gradient on an LTQ Orbitrap XL system with a TOP5 CID 74 3.3. New insights into smooth muscle myosin phosphorylation fragmentation method. Generated raw files were analyzed with Mascot Perkins et al., 1999 searched against the NCBI database without taxonomy filter. The resulting sequence coverage of each protein was mapped for each protease (Figure B.3). As result, trypsin digestion (mean 67.8%) and the incomplete thermolysin digestion (mean 90.8%) performed best in terms of sequence coverage.

100 -casein -casein myoglobin 80 -lactoglobolin ovalbumin mean coverage 60

40 sequence coverage [%] sequence coverage 20

0 Chymo- Trypsin trypsin C(Br)N Lys-CElastase Trypsin Thermolysin

Figure 3.25.: Sequence coverage of standard proteins digested with different proteases. The proteases chymotrypsin, trypsin in combination with CNBr, Lys-C, elastase, trypsin and thermolysin were used to digest destained gel band from the proteins α-casein, β-casein, myoglobin, β-lactoglobin and ovalbumin. Resulting raw ﬁles were analyzed with Mascot and the sequence coverage of each protein, as well as the mean sequence coverage for all protein digested with one protease were plotted.

To investigate whether thermolysin digestion is also suitable for the unambiguous identiﬁ- cation of phosphorylation sites, the digesting method was tested on a protein, in which tryptic digestion failed to unambiguously identify two potential neighboring phosphorylation sites. In mouse myosin regulatory light chain 12B (Myl12b), also called myosin light chain 20, the two neighboring amino acids at position T18 and S19 were both reported to be phosphorylated in smooth muscles. While none of these sites are phosphorylated under resting conditions both isometric contraction and electrical ﬁeld stimulation induced relaxation result in protein mono-phosphorylation. In order to identify which site is phosphorylated under which 3.3. New insights into smooth muscle myosin phosphorylation 75 condition, mono-phosphorylated spots of Myl12b were cut from 2-DE PAGE gels, digested with thermolysin and subjected to LC-MS/MS analysis. As the phosphosites of interest are located in a +2 and +3 position to an arginine in the sequence 10TKKRPQRATSNVFAM25, the use of trypsin was not considered to be expedient. Protein digestion with thermolysin and subsequent analysis of resulting peptides by MSA fragmentation on an LTQ Orbitrap XL system revealed that in fully isometric contracted smooth muscle cells S19 of Myl12b is unambiguously phosphorylated phosphorylated (Figure 3.26 A).

A 2+2+ 2+ b - H3POPO4 [M+2H] - H O - H PO b - NNHH3 7 443.79 2 3 4 7 100 361.30 820.4

HPO3 b 2+2+- NNHH 7 3 QRATS NVF

b 2+2+ 7 2+ b 419.20 [M+2H] - H2O 7 492.85 837.4 b - H POPO - NNHH 6 3 4 3 y 1 b - NHNH b - H POPO 3 3 6 3 4 b 166.08 6 347.46 b - NNHH3 b - H POPO 6 b 5 3 4 3 Relative Abundance Relative y 721.38 y 4 623.26 y 2 b 526.35 640.44 7 265.20 4 y 546.28y 6 874.2 0 5 500 m/z 1000

100 355.15 QRAT* S # NVFAMOx

2+ [M+2H] - 2x H2O - H3PO4 [M+2H]2+- H O - H PO b - H POPO 2 3 4 9 3 4 552.93 2+2+ b - H POPO *b*b *b*b - H POPO 8 3 4 - NNHH 4 4 3 4 544.10 [M+2H]2+- 2x H O b 3 b - NNHH 2 8 9 3 # 2+ y b [M+2H] - H2O b - NNHH 967.53 1038.49 3 4 7 3 384.32 601.84 820.32 b b y 8 9 Relative Abundance Relative 2 461.35 b 984.56 1055.52 b 7 237.14 2 b b y 837.56 285.07 3 6 6 404.23 534.60 886.67 0 500 m/z 1000

Figure 3.26.: Analysis of phosphorylation sites in myosin light chain 20 (Mlc20) by high resolution mass spectrometry. (A) Fragmentation spectrum of the mono-phosphorylated peptide QRATSNVFAMOx of MYL12B from fully isometric contracted smooth muscle cells. (B) Site- speciﬁc fragment ions of relaxed muscle by electrical ﬁeld stimulation point to phosphorylation events at T18 (*) or S19 (#).

In contrast, the analysis of the mono-phosphorylated 2-DE spot obtained from electrical field stimulation induced relaxation identified no distinct phosphorylation site. Ion series 76 3.3. New insights into smooth muscle myosin phosphorylation evidences for both isoforms phosphorylated at T18 or S19 were identified in the MSA spectrum. Thus, it was concluded that mono-phosphorylation of T18 rather than S19 is responsible for relaxation by electrical field stimulation. CHAPTER 4

Material and Methods

4.1 Cell lysis optimization for phosphoproteomic studies

C2C12 myoblasts were cultured in high glucose DMEM medium (Life Technologies, Darm- stadt, Germany) supplemented with 15% FCS (PAA, GE Healthcare Life Sciences, Freiburg, Germany), 1% non-essential amino acids, 1% penicillin/streptomycin and 1% sodium pyruvate (all Life Technologies) in six well plates (Techno Plastic Products AG, Trasadingen, Switzerland) to a conﬂuency of approximately 90%. Differentiation was induced by reduction of the FCS content to 2% (Ong et al., 2002) in the absence of sodium pyruvate. Differentia- tion medium was changed every 48 h until complete myotube formation was observed (day 5-6). After myotube development, sarcomere formation was improved by electrical pulse stimulation (0.5 Hz, 4 ms, 10-12 V) with a C-Pace EP Culture Pacer (IonOptix, Milton, USA) for 16-24 h.

Differentiated, contraction myotube were lysed on ice using the ﬁve different lysis buffer listed in table 4.1. Subsequent to cell lysis, cells were scraped from the dish and soniﬁed 2x for 10 s on ice for complete cell lysis. Insoluble material was removed by centrifugation for 20 min at 21,000 x g and 42 ◦C. Supernatants were equalized to a protein concentration of 100 µg by using the Bradford assay (BioRad, München, Germany). The resulting volume was diluted 1:4 with 50 mM ammonium bicarbonate buffer (ABC) solution and digested with 78 4.1. Cell lysis optimization for phosphoproteomic studies

sequencing grade trypsin (1:50) (Promega) for 3.5 h at 200 rpm and 42 ◦C.

Table 4.1. Summary of buffer compositions that were used for the establishment of myotube cell lysis.

(a) Phosphate buffered saline (PBS) buffer (b) Urea buffer with 1% sodium dodecyl sulfate. supplemented with Triton X-100. 1% SDS Urea buffer (pH 8.5) PBS-T buffer (pH 7.4) 30 mM Tris 27 mM KCl 7 M Urea 1.37 M NaCl 3 M Thiourea 100 mM Na2HPO4 1% SDS 18 mM KH2PO4 0.1 % Triton X-100

(c) Modiﬁed RIPA buffer. (d) Sucrose-Tris buffer. modiﬁed RIPA buffer (pH 7.6) Sucrose-Tris buffer (pH 8.0) 50 mM Tris 0.25 M Sucrose 150 mM NaCl 50 mM Tris 1% NP-40 1 mM EDTA 0.25% Na-Desoxycholat 1 mM β-mercaptoethanol

(e) High salt urea buffer.

Urea buffer (pH 8.5) 30 mM Tris 7 M Urea 3 M Thiourea

Triton X-100 was removed before protein digestion by a precipitation step in the six-fold volume of acetone over night. Subsequently, precipitated samples were washed twice and resuspended with 50 mM ABC and subjected to tryptic digestion. SDS was removed after the digestion by a three extractions steps against the equal volume of butnaol/n-heptane (1:4, v/v). Detergents of the sample lysed in modiﬁed RIPA buffer were removed with detergent 4.2. Establishment of different proteases for improved sequence coverage in LC-MS/MS analyses 79 removal columns (Pierce, Thermo Scientiﬁc) according to the manufacturer’s protocol.

Afterwards, all peptides were desalted using an Oasis HLB cartridge (Waters Corporation, Milford, USA) according to the manufacturer’s protocol. Eluates were aliquoted, lyophilized and stored at -80 ◦C. For LC-MS/MS analysis, samples were resolved in 150 µl 0.1% TFA and 1/10 of the sample was analyzed on the LTQ Oribtrap XL using a 3 h LC-gradient with a TOP5 CID fragmentation method. Generated Raw ﬁles were searched with MaxQuant 1.5.2.8 embedded Andromeda algorithm against the mouse Proteomeset isoform database (version 01.12.2015; 57,275 entries) with the parameters listed is table E.1 in section E.2.

4.2 Establishment of different proteases for improved sequence coverage in LC-MS/MS analyses

The proteases trypsin , chymotrypsin, elastase (all three Promega), LysC (Wako chemicals) and thermolysine (R&D diagnostics) were tested for their proteolytic digestion efficiency in combination with LC-MS/MS analysis in order to maximize the sequence coverage of proteins and samples of interest. To this end, stock solutions (2 µg µl−1) of the five commercially available proteins α-casein, β-casein, myoglobin, β-lactoglobulin and ovalalbumin (all Sigma Aldrich) were prepared. 2 µg per protein were loaded onto a 12% BisTris gel followed by PAGE, staining of proteins with colloidal Coomassie, cutting of protein bands from the gel and destaining according to standardized protocols. Afterwards, each protein was digested with trypsin, chymotrypsin, elastase, LysC and thermolysine, respectively. The digests were performed as described in the following table 4.2. For trypsin proteolysis in combination with subsequent chemical CNBr digest, gel pieces were further processed according to a protocol from Montfort et al. (2002) with slight modification. Briefly, tryptic peptides were extracted from the gel piece by using 100% acetonitrile, gel pieces were dried in vacuo, CNBr (one crystal) was resuspended in 70% TFA, added to the gel pieces and incubated for 14 h at room temperature in the dark. Peptide extraction digests was performed two times with 0.1% TFA, 95% acotonitrile in an ice-cooled ultrasonic bath for 15 min. Supernatants were collected, dried in vacuo and stored at -80 ◦C 80 4.2. Establishment of different proteases

Table 4.2. Summary of different digestion conditions.

Concentration Enzyme to Enzyme Digestion buffer Duration Temperature ng µl−1 protein ratio

Elastase 100 100 mM ABC 1:20 over night 37 °C Thermolysine 20 50 mM ABC 1:50 2 h 60 °C LysC 5 25 mM Tris, 1mM EDTA (pH 7.8) 1:100 over night 37 °C Trypsin 50 50 mM ABC 1:20 over night 37 °C Chymotrypsin 25 100 mM Tris, 10 mM CaCl (pH 7,8) 1:50 over night 37 °C

prior to LC-MS/MS analysis.

Samples were analyzed in an one hour gradient on an LTQ Orbitrap XL system, coupled online to an UltiMate 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher Scientific, Dreieich, Germany). The UHPLC system was equipped with two C18 µ-precolumns (ID: 0.3 mm x 5 mm; PepMapTM, Thermo Fisher Scientific) and an Acclaim® PepMapTM analytical column (ID: 75 µm x 250 mm, 2 µm, 100 Å, Dionex LC Packings/Thermo Fisher Scientific). The high-resolution MS instrument was externally calibrated using standard compounds and equipped with a nanoelectrospray ion source and distal coated SilicaTips (FS360-20-10-D, New Objective, Woburn, USA). MS/MS analyses were generally performed on multiply charged peptide ions applying a normalized collision energy of 35% with an activation q of 0.25 and an activation time of 30 ms and an exclusion time of 45 s for a TOP5 CID fragmentation method. Generated Raw files were searched with Mascot against the NCBI database with no taxonomy restriction using parameters listed in the appendix E.1. CHAPTER 5

Final remarks and outlook

The first part of this work aimed at the characterization of the basal phosphoproteome of contracting C2C12 myotubes with the focus on sarcomeric and Z-disc-specific phosphorylation events. This extensive analysis identified the Z-disc as phosphorylation hotspot within the sarcomere accounting for 70% of the total identified sarcomeric phosphosites. Interestingly, the identified phosphosites often occurred in clusters within structural subunits or between two domains. One example for this observation is the cluster of phosphorylation sites in the C-terminal region of the Z-disc-associated protein FLNc. In silico kinase prediction reveled that all three mFLNc phosphorylation sites at S2621, S2625 and S2633 are putative PKC/PKCα substrate sites. That mFLNc as well as hFLNc d23-24 are indeed substrates of PKCα in a concentration-dependent manner was confirmed by radioactive in vitro kinase assays. However, to identify individual phosphorylation sites, a non-radioactive in vitro kinase assay was coupled to in-solution tryptic digestion, followed by TiO2 phosphopeptide enrichment and LC-MS/MS analysis with varying fragmentation techniques. By this approach, S2625 of mFLNc and S2623/S2624 of hFLNc could unequivocally be identified as PKCα substrates. This example shows, that the combination of phosphosite identification by large- scale phosphoproteomics with subsequent in silico kinase prediction and MS-based in vitro kinase assays is an effective method to study kinase-substrate relationships. Furthermore, the use of specific site mutants was revealed to be beneficial for unambiguous annotation of the 82 4.2. Establishment of different proteases

phosphate group to a distinct amino acid.

Once the kinase mediating the phosphorylation of mFLNc S2625 and hFLNc S26623/S2624 was identiﬁed, it raised the issue about the biological function of this phosphorylation site in the hinge 2 region of FLNc. This hinge 2 region of FLNc is proposed to be a substrate of calpain cleavage in literature. As classical bottom-up shotgun proteomics failed to identify the exact calpain cleavage site in the FLNc hinge 2 region, a top-down MS-based approach 2+ was developed. By coupling FLNc d23-24 via a C-terminal His6-tag to Ni -NTA beads, the calpainolysis could be performed on the beads and the proteolysed N-terminal fragment could be separated from the beads. Subsequent top-down MS-analysis of the N-terminal fragment with an Orbitrap Elite instrument unambiguously identiﬁed Y2626 and Y2625 as main calpain cleavage sites in mFLNc and hFLNc, respectively.

The adjacent localization of the phosphorylation site and calpain cleavage site raised to the question, whether these sites affect each other. Quantitative Western blot analysis of in vitro and ex vivo calpainolysis assays in cell lysates from C2 and HEK293 cells treated with an activator (PMA) or inhibitor (Gö6796) of PKCα activity unequivocally showed that phosphorylation by PKCα protects FLNc from calpain cleavage. This targeted follow-up study is showing the potential of large-scale phosphoproteomic data. Furthermore, the result shows that the top-down MS-based approach for the identiﬁcation of calpain cleavage sites is as promising method which may serve as a valuable screening technique to systematically identify calpain cleavage sites in substrates. Thereby it would allow for the subsequent development of important prediction algorithms.

In the second part of this work, the quantitative changes in the phosphoproteome of contracting C2 myotubes were analyzed following activation and inhibition of the Pi3k/Akt pathway by IGF-1 and LY294002, respectively. Quantitative Western blot analysis with antibodies directed against phosphorylated key substrates of the Pi3k/Akt pathway in combination with a SILAC-based, large-scale phosphoproteomic analysis provided to be a suitable method to detect quantitative changes following the inhibition or activation of the pathway. Moreover, motif-X analysis of significantly regulated phosphopeptides was enabled to identify enriched 4.2. Establishment of different proteases 83 motifs and their corresponding kinases. Interesting, beside the known basophilic RxRxxpS motif, the previously unknown substrate motif RxRxxpSxxS, which was termed "extended basophilic motif" was enriched the most. The finding that 19 out of the 20 phosphopeptides comprising this motif were also identified to be phosphorylated at the +3 serine raised the issue which kinases might be involved in the phosphorylation of two serine residues in such close proximity. For seven proteins, kinases were described in the literature for the first serine, however, nothing was found to be known about kinases mediating the phosphorylation at the +3 serine residue.

Interestingly, an extended basophilic motif was identified in the unique insertion within Ig- like domain 20 of the Z-disc protein mFLNc at positions S2234 and S2237. Kinase prediction predicted the second serine residue within the motif to be a potential PKC substrate while the first site was predicted and previously proven to be an AKT substrate. To test the assumption that both kinases are mandatory for the generation of the doubly phosphorylated peptide of hFLNc at position S2233 and S2236, a combined in vitro kinase assay was performed. The MS-based analysis confirmed that AKT predominantly phosphorylates hFLNc at S2233, while PKCα mediated phosphorylation at S2236. Strikingly, the simultaneous incubation with both kinases resulted in the detection of the doubly phosphorylated peptide at S2233 and S2236.

To reveal the biological role of these two phosphorylation sites, new FLNc interaction partners were searched by a proximity-dependent proteomic BioID approach. The functionality of the method was shown by the identification of several known FLNc interaction partner such as Bag3, Xirp1 and aciculin. In addition, GO term enrichment analysis confirmed the specific enrichment of I-band and Z-disc components. Following analysis of the BioID dataset, FILIP1, a previously published FLNa interaction partner was identified to be also a putative FLNc interacting protein. The interaction of hFLNc d18-21 with the C-terminal end of FILIP1 was verified by classical biochemical interaction studies, such as yeast-two-hybrid analysis, overlay assays and pull-down assays. Moreover, the usage of hFLNc S2233 and S2236 phosphosite mutants in pull-down and co-IP experiments together with FILIP1 could 84 4.2. Establishment of different proteases prove a direct phosphorylation-dependent interaction of these two proteins.

The experiments with the phosphosite mutants clearly showed, that phosphorylation of both hFLNc sites S2233 and S2236 are necessary to prevent binding to FILIP1. This ﬁnding is another paradigmatic example of a phosphorylation-dependent protein-protein interaction. However, the biological relevance of this mechanism will have to be revealed in upcoming experiments. For FLNa it was demonstrated that the interaction with FILIP1 induces a calpain-mediated proteolysis in the hinge 1 region of the protein. Moreover, this mechanism is supposed to be regulated by phosphorylation of S2152 in FLNa. Whether the homologous site in FLNc, S2146 has also an effect will be examined in the future. Moreover, the mutual regulation of FLNc, FILIP1 and calpain will have to be analyzed in cell culture-based co- transfection experiments.

In the last past of this work, the use of different proteases than was shown to be beneﬁcial for the localization of phosphorylation sites C-terminal to arginine or lysine. From the three proteases tested besides trypsin, incomplete thermolysin digestion performed best, with a mean sequence coverage of 90.8%. The functionality of thermolysin for the identiﬁcation of phosphorylation sites was demonstrated for the two neighboring phosphosites T19 and S20 of Myl12b. However, the incomplete digestion was seen as major drawback for complex samples, as the computation time would increase immensely.

To sum up, this work showed phosphorylation dependent interaction studies based on large-scale qualitative as well as quantitative data. Moreover, in silico kinase prediction in combination with MS-based in vitro kinase assays were shown to be a suitable method to obtain site-resolved kinase substrate information. However, in future work, support vector machine (SVM) learning algorithms should be established to perform kinase-substrate identi- ﬁcation based on large-scale phosphoproteomic SILAC ratios. Moreover, a suitable method to match the detected phosphosites of this work to phosphosites of the human proteome should 4.2. Establishment of different proteases 85 be developed, as algorithms such as PhosphOrtholog8 and DAPPLE29 are not applicable for this propose. This matching would not only be beneﬁcial to compare human and mouse large-scale phosphoproteomic datasets, but would also allow for the application of useful follow-up bioinformatic platforms, such as PhosphoSiteAnalyzer.

Last, state-of-the-art MS-based follow-up studies, such as BioID analysis and top-down proteomics, in combination with classical biochemical methods, such as pull-down assay and co-IPs were proven as reliable techniques to uncover phosphorylation-dependent biological functions. Furthermore, the vast amount of identified and regulated phosphorylation sites uncovered in the two large-scale phosphoproteomic studies are an invaluable basis for identification of further muscle-specific phosphorylation-dependent mechanisms.

8 http://www.phosphortholog.com/ 9 http://saphire.usask.ca/saphire/dapple2/index.html

CHAPTER 6

Publications and manuscripts

6.1 Myoﬁbrillar Z-discs are a protein phosphorylation hot spot with PKCα modulating protein dynamics

Contributions:

• Establishment of C2/C2C12 cell culture growth and differentiation conditions, SILAC labeling and immunohistochemical staining of the cells.

• Adaption of phosphoproteomics workﬂow, subsequent phosphopeptide enrichment, MS-based analysis as well as data analysis.

• Literature recherches for deﬁnition of the Z-disc proteome.

• Adaption of radioactive in vitro kinase assay and development of MS-based in vitro kinase assay.

• Development of top down method for the identiﬁcation of calpain cleavage sites, establishment of in vitro calpain assay and contributory performance of cell culture based calpain assay.

• Participation in experimental design, preparation of ﬁgure 1-6 and contribution to the writing of the manuscript. 88 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Myofibrillar Z-discs are a protein phosphorylation hot spot with PKC

modulating protein dynamics

Lena Reimann1, Heike Wiese1,4, Yvonne Leber3, Anja N. Schwäble1, Anna L. Fricke1,

Anne Rohland3, Bettina Knapp1, Christian D. Peikert1, Friedel Drepper1, Peter F.M.

van der Ven3, Gerald Radziwill1,2, Dieter O. Fürst3, Bettina Warscheid1,2*

1Department of Biochemistry and Functional Proteomics, Institute of Biology II,

Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany

2BIOSS Centre for Biological Signalling Studies, University of Freiburg

3Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn,

53121 Bonn, Germany

4Present address: Institute of Pharmacology and Toxicology, University of Ulm,

89081 Ulm, Germany

*to whom correspondence should be addressed:

[email protected]

Key words: skeletal myotubes, sarcomeric Z-disc, phosphoproteomics, top-down

mass spectrometry, filamin C, protein kinase Cα, calpain 1

Running title (max. 60): The phosphoproteome of the myofibrillar Z-disc

1 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 89

Abbreviations page

ABD, actin-binding domain

AGC, automatic gain control

BAG, Bcl2-associated athanogene

BP, biological process

CC, cellular component

D, domain

ENAH, enabled homolog

ETD, electron transfer dissociation

FA, formic acid

FDR, false discovery rate

FLNc, filamin C

FRAP, fluorescence recovery after photobleaching

GO, gene ontology

HCD, higher-energy collisional dissociation

HEK293, human embryonic kidney 293

hpH-RP, high pH reversed phase chromatography

HRP, horseradish peroxidase HSPB1, heat shock protein beta-1 Ig, immunoglobulin IMMs, immortalized mouse skeletal myoblasts IPTG, isopropyl -D-1-thiogalactopyranoside LDB3, LIM domain binding 3 LIT, linear ion trap MF, molecular function MSA, multi-stage activation NL, neutral loss PDZ, post synaptic density protein, Drosophila disc large tumor suppressor, and

2 90 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

zonula occludens-1 protein PEI, polyethylenimine PKC, protein kinase C PMA, phorbol-12-myristat-13-acetat PPxY, proline-proline-x-tyrosine PxxP, proline‐rich ROI, regions of interest SCX, strong cation exchange SD, standard deviation SEM, standard error of the mean SYNPO, synaptopodin SYNPO2, myopodin SYNPO2L, tritopodin (CHAP; synaptopodin 2-like) TiO2, titanium dioxide VASP, vasodilator-stimulated phosphoprotein WH1, WASP-homology/EVH1, Ena/VASP homology 1 WT, wildtype XIRP1, Xin actin-binding repeat containing protein 1

3 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 91

Abstract

The Z-disc is a protein-rich structure critically important for myofibril development

and integrity. Since a role of the Z-disc for signal integration and transduction was

recently suggested, its precise phosphorylation landscape warranted in-depth

analysis. We therefore established a site-resolved protein phosphorylation map of

the Z-disc in skeletal myocytes and found that it is a phosphorylation hotspot in living

cells, underscoring its functions in signaling and disease-related processes. In an

exemplary fashion, we analyzed the actin-binding multi-adaptor protein filamin C

(FLNc), which is essential for Z-disc assembly and maintenance, and found that

PKC phosphorylation at distinct serine residues in its hinge 2 region prevents its

cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after

photobleaching experiments indicated that this phosphorylation modulates FLNc

dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited

remarkably fast kinetics and exceedingly high mobility. Our dataset provides an

invaluable resource for further identification of kinase-mediated changes in

myofibrillar protein interactions, kinetics and mobility that will greatly advance our

understanding of Z-disc dynamics and signaling.

4 92 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Introduction

The highly regular organization of myofibrils, the contractile organelles of cross-

striated muscle cells, gives rise to the typical banding pattern of skeletal and cardiac

muscle fibers. Myofibrils are mainly composed of an almost crystalline array of thin

and thick filaments based on actin and myosin, respectively. The repeating

contractile units of myofibrils are the sarcomeres, which are flanked by Z-discs. The

latter protein-rich structures provide an essential structural framework by tethering

actin filaments at their barbed ends, cross-linking them by antiparallel dimers of

α-actinin and linking them to the giant protein titin at its aminoterminus. Z-discs not

only define the lateral boundaries of adjacent sarcomeres, but also help to connect

myofibrils to each other, e.g. via intermediate filaments. In addition, they are involved

in linking the contractile apparatus to the sarcolemma and the extracellular matrix via

large, membrane-associated protein complexes, the costameres. The function of the

Z-disc is not only limited to force transmission, but it is also an important hub for

signal transduction events. To fulfil its dual role, Z-discs have to be dynamic and at

the same time have to encompass numerous structural proteins.

Over the last years, the number of proteins with functions in mechanosensing and

signal transduction identified to localize at least temporarily to the Z-disc has steadily

increased [reviewed in (1-3)]. To date, over 100 gene products are linked to the term

“Z-disc” in the human or mouse NCBI gene database

(http://www.ncbi.nlm.nih.gov/gene/). However, its precise protein inventory and

phosphorylation landscape have not been coherently analyzed. Numerous signaling

proteins such as protein kinase C (PKC) (4) and the protein phosphatase calcineurin

(5) were shown to dynamically localize to the Z-disc. Notably, kinase- and

phosphatase-mediated phosphorylation and dephosphorylation events may likely

5 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 93

control the dynamic shuttling of proteins in and out of the Z-disc as recently revealed

for myopodin (6), a protein interacting with F-actin, α-actinin, and filamin C (FLNc) (7,

8). The large cytoskeletal protein FLNc, in turn, constitutes an important hub in the Z-

disc interactome with manifold binding partners such as myotilin (9), nebulette (10),

the Xin actin-binding repeat containing proteins Xin (11) and XIRP2 (12), and the

calsarcins/myozenins/FATZ proteins (13-15).

Distinct from its two other ubiquitously expressed family members FLNa and FLNb,

FLNc is mainly expressed in cross-striated muscles (16). In healthy muscle, it

predominantly localizes at Z-discs, whereas a minor portion is found beneath the

sarcolemma in association with the dystrophin-associated glycoprotein complex (17).

During myofibril development, FLNc assists in Z-disc assembly by acting as a

molecular scaffold (18). Mutations in its gene cause severe myopathies and

cardiomyopathies [reviewed in (19)].

All filamin isoforms feature an aminoterminal actin-binding domain (ABD) and a rod

of 24 immunoglobulin-like (Ig-like) domains. Flexibility is mainly provided by hinge

regions between Ig-like domains 15 and 16 (hinge 1) and 23 and 24 (hinge 2).

Depending on cell type and differentiation stage, alternative splicing may remove

hinge 1 in FLNc and FLNb (20, 21). The carboxyterminal Ig-like domain 24 mediates

homodimerization, resulting in filamin dimers capable of cross-linking actin filaments

(22-24), whereas hinge 2 was suggested to fulfil a regulatory role in dimerization

(22). FLNc features a unique insertion of 82 amino acids in Ig-like domain 20, which

is sufficient for Z-disc targeting (18). This insert is also likely important for

establishing diverse protein interaction and scaffolding functions for cytoplasmic

signaling processes. Compatible with its role in intracellular signaling events, FLNc

was proposed to shuttle between the Z-disc and the sarcolemma or other

6 94 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

compartments, particularly in disease states (17, 18, 25, 26). Phosphorylation events

and calpain-dependent cleavage of FLNc may likely be involved in regulating

functions and localization of the protein (27-29), however precise information

concerning phosphorylation and cleavage sites and the physiological effects thereof

have remained obscure.

In this work, we identified the myofibrillar Z-disc as major site of protein

phosphorylation in contracting C2C12 myotubes by large-scale phosphoproteomics.

By establishing a site-specific phosphorylation map of the Z-disc proteome, we

identified a large number of highly phosphorylated proteins including FLNc. We

focused on a phosphosite cluster in the hinge 2 region of FLNc for a detailed

analysis. Through in vitro kinase assays coupled to high resolution MS we precisely

mapped serine residues serving as specific PKC substrate sites. These data were

further confirmed by an in vivo approach. In a newly established top-down MS-based

approach, we determined a distinct tyrosine residue positioned directly

carboxyterminal to these phosphorylation sites as the major calpain 1 cleavage site

in human and mouse FLNc. PKC-mediated phosphorylation not only controls

cleavage by calpain 1, but also modulates FLNc dynamics. Interestingly, an FLNc

variant lacking Ig-like domain 24, thus mimicking calpain 1 cleaved FLNc, exhibited

remarkably fast kinetics and exceedingly high mobility.

7 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 95

Experimental Procedures Cell Culture

C2C12 and C2 myoblasts were cultured in high glucose DMEM medium (Life

Technologies, Darmstadt, Germany) supplemented with 15% FCS (PAA, GE

Healthcare Life Sciences, Freiburg, Germany), 1% non-essential amino acids, 1%

penicillin/streptomycin and 1% sodium pyruvate (all Life Technologies) in six-well

plates (Techno Plastic Products AG, Trasadingen, Switzerland) to a confluency of

approximately 90%. Differentiation was induced by reduction of the FCS content to

2% (30) in the absence of sodium pyruvate. Differentiation medium was changed

every 48 h until complete myotube formation was observed (day 5-6). After myotube

development, sarcomere formation was improved by electrical pulse stimulation (0.5

Hz, 4 ms, 10 - 12 V) with a C-Pace EP Culture Pacer (IonOptix, Milton, USA) for

16-24 h. SILAC labeling of C2C12 myoblasts was performed with high glucose

SILAC-DMEM medium (GE Healthcare Life Sciences, Freiburg, Germany)

supplemented with dialyzed 15% FCS (PAA), 1% non-essential amino acids,1%

sodium pyruvate, 1% proline (all Life Technologies), 84 mg/l arginine and 146 mg/l

lysine (Cambridge Isotope Laboratories Inc., Tewksbury, USA) for at least nine cell

doublings. Light, medium, and heavy stable isotope labeling by amino acids in cell

13 12 12 culture (SILAC) was performed with C6 L-arginine and C6 L-lysine, C6 L-arginine

13 15 13 15 and D4 L-lysine, and C6 N4 L-arginine and C6 N2 L-lysine, respectively. Labeled

myoblasts were seeded into six-well plates and differentiation was induced by

reduction of the dialyzed FCS content to 2% in the absence of sodium pyruvate (30).

Immortalized mouse skeletal myoblasts (IMMs) were cultivated and transfected as

described before (31, 32). IMMs with passage numbers of up to 40 were used for

experiments. Human embryonic kidney cells (HEK293) were cultured in DMEM

8 96 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

supplemented with 10% FCS and 1% sodium pyruvate. Transient transfections of

HEK293 and C2 cells were performed using polyethylenimine (PEI, 1 g/l

Polysciences Europe GmbH) and Lipofectamine2000 (Invitrogen, Darmstadt,

Germany), respectively. To this end, cells were grown to a confluence of 70% in 6

cm dishes and for each transfection, a total of 4.5 g DNA was used. PEI was mixed

in a 3:1 ratio with DNA in a 6-fold volume of Opti-MEM (Life Technologies) and

incubated for 15 min. C2 cells were transfected in solution according to the

manufacture’s protocol. Transfected cells were grown for 24 h before cell lysis. All

cell lines were regularly tested to be mycoplasma-negative.

Cell Lysis and Sample Preparation

For lysis of C2C12 myotubes, plates were placed on ice and cells were washed

twice with ice-cold PBS (CM-PBS; 0.75 mM CaCl2, 0.75 mM MgCl2, 155 mM NaCl2,

2.7 mM KCl, 2 mM KH2PO4, 10 mM Na2HPO4, pH 7.4). 150 l precooled lysis buffer

(7 M urea, 2 M thiourea, 1 mM sodium orthovanadate, 10 mM -glycerophosphate,

9.5 mM sodium fluoride, 10 mM sodium pyrophosphate) were added per well, cells

were scraped from the dish and sonified 2x for 10 s on ice for complete cell lysis.

Insoluble material was removed by centrifugation for 20 min at 21,000 x g and 4°C

and the protein concentration was estimated using the Bradford assay (BioRad,

München, Germany). Reduction and alkylation of proteins from replicate 2 and

SILAC-labeled samples was performed as described (33) with slight modifications.

Each reaction was carried out for 30 min using a final concentration of 1 mM

dithiothreitol (DTT) and 55 mM 2-chloroacetamide before alkylation was quenched

with a final concentration of 5 mM DTT. A lysate volume equal to a total protein

amount of 7 mg (replicate 1), 8.1 mg (replicate 2), and 2 mg (for each SILAC

9 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 97

replicate) was diluted 1:4 with 50 mM ammonium bicarbonate solution and digested

with sequencing grade trypsin (1:50) (Promega) for 3.5 h at 200 rpm and 42°C.

Peptides were desalted using an Oasis HLB cartridge (Waters Corporation, Milford,

USA) according to the manufacturer’s protocol. Eluates were aliquoted, lyophilized

and stored at -80°C.

Strong Cation Exchange Chromatography

Tryptic digests were dissolved in 200 l SCX buffer A [5 mM potassium dihydrogen

phosphate, 20% acetonitrile (ACN, v/v), pH 2.8]. The supernatant was loaded onto a

Polysulfoethyl-A column (Ø 4.6 mm, 20 cm, 5 m, 200 Å, PolyLC, Columbia, USA)

equilibrated with SCX buffer A using a Dionex Ultimate 3000 UHPLC system.

Peptides were separated at a flow rate of 700 l/min applying a linear gradient of 0-

30% SCX buffer B [5 mM potassium dihydrogen phosphate, 20% ACN (v/v), 500 mM

KCl, pH 2.8] in 50 min starting 10 min after sample injection, followed by a gradient

of 30-50% and 50-100% of solvent B in 10 min each. The column was washed with

100% B for 5 min and re-equilibrated for 20 min with 100% buffer A. 3 min fractions

were collected during the gradient and 10 l of each SCX fraction were dried in

vacuo for LC/MS analysis. The remaining volume of each fraction was used for TiO2

enrichment. A total of 55 SCX fractions were collected for replicates 1 and 2 and

further processed for phosphoproteomics analysis in a randomized procedure. Non-

enriched and TiO2-enriched peptide samples were dried in vacuo and stored

at -80°C for further analysis.

High-pH Reversed-Phase Chromatography

10 98 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

For each SILAC replicate, 2 mg of total peptide were resolved in 200 l buffer A (10

mM ammonium hydroxide, pH 10.5), sonicated for 3 min, centrifuged at 20,000 x g

for 4 min and filtrated into a sample vial by using a 0.2 m syringe filter

(Phenomenex, Aschaffenburg, Germany). High-pH reversed phase (hpH-RP)

chromatography was performed using a Dionex Ultimate 3000 equipped with a RP

Gemini C18 column (∅ 4.6 mm × 25 mm, 3 , 110 Å, Phenomenex) and operated at

a flow rate of 750 l/min. Peptide separation was carried out using a binary solvent

system (A: 10 mM ammonium hydroxide, pH 10.5; B: 10 mM ammonium hydroxide,

pH 10.5, 90% acetonitrile) at a temperature of 40°C. Peptide loading and binding

was performed at 1% B for 5 min, followed by peptide elution by increasing buffer B

to 20% in 35 min and further to 45% in 20 min. Ninety fractions were collected in a

96 deep well plate at 50 s intervals from minute 2 to 77. Every 30th fraction was

pooled and acidified with TFA to a final concentration of 1%. Fractions 19-20 were

used for subsequent TiO2 enrichment.

Titanium Dioxide Enrichment

Phosphopeptides were enriched using titanium dioxide (TiO2) spherical beads as

described before (34) with slight modifications. Briefly, 30 l of TiO2 material (TiO2,

5m, GL Science Inc.) resuspended in ACN 1:1 (v/v) were used for SCX fractions

and hpH-RP fractions, while 15 l were used for single protein digests. The TiO2

material was washed 2x with 200 l washing buffer (80% ACN, 0.1% TFA) and 1x

with 50 l loading buffer [20% (v/v) acetic acid, 20 mg/ml 2,3-dihydroxybenzoic acid,

420 mM octasulfonic acid, 0.1% (v/v) heptafluorobutyric acid]. SCX fractions or

protein digests from in vitro kinase assays were mixed 1:1 (v/v) with 2x loading

buffer, added to the prepared TiO2 material, vortexed and incubated for 20 min at

11 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 99

4°C with slight agitation. Each sample was washed twice with 200 l washing buffer.

Subsequently, 50 l elution buffer (50 mM ammonium dihydrogen phosphate, pH

10.5) were added to each sample followed by incubation for 10 min. Subsequently,

50 l ACN were added, the suspension was loaded onto a gel loader tip (GE

Healthcare) supplied with a 1 mm Teflon capillary and peptides were eluted by

centrifugation at 5,000 rpm. Eluates were placed on ice, acidified with 8 l TFA and

dried in vacuo.

Liquid Chromatography and High Resolution Mass Spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed

using the UltiMateTM 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher

Scientific, Dreieich, Germany) coupled online to either a Velos Orbitrap Elite, Q

Exactive Plus, or LTQ-Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany)

instrument. UHPLC systems were equipped with two C18 -precolumns (Ø 0.3 mm x

5 mm; PepMapTM, Thermo Fisher Scientific) and an Acclaim® PepMapTM

analytical column (ID: 75 m x 250 mm, 2 m, 100 Å, Dionex LC Packings/Thermo

Fisher Scientific). High-resolution MS instruments were externally calibrated using

standard compounds and equipped with a nanoelectrospray ion source and distal

coated SilicaTips (FS360-20-10-D, New Objective, Woburn, USA). MS/MS analyses

were generally performed on multiply charged peptide ions applying a normalized

collision energy of 35% with an activation q of 0.25 and an activation time of 30 ms

unless otherwise stated.

For large-scale phosphoproteome analysis, peptide mixtures from SCX fractions

before and after TiO2 enrichment were separated using a binary solvent system

consisting of 0.1% formic acid (FA, solvent A, "A") and 0.1% FA/86% ACN (solvent

12 100 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

B, "B"). Samples were washed and pre-concentrated on a C18 -precolumn with

0.1% TFA for 30 min before switching the column in line with the analytical column.

Replicate 1 was analyzed applying a gradient of 78 min at a flow rate of 300 l/min,

replicate 2 with a 75-min gradient at 250 l/min. Peptide samples from replicate 1

were eluted with a gradient of 5-30% B in 60 min, 30-50% B in 20 min and 50-95% B

in 5 min. After each gradient, the analytical column was washed with 95% B for 3

min and re-equilibrated for 5 min with 5% B. For replicate 2, the gradient used was

as follows: 5-42% B in 65 min and 42-95% B in 5 min followed by 95% B for 5 min

and 5% B for 15 min for re-equilibration. Analyses were performed on an LTQ-

Orbitrap XL using CID for SCX fractions and multi-stage activation (MSA) with

neutral loss (NL) masses of 32.7, 49 and 98 Da for TiO2-enriched samples. The

instrument parameters were as follows: spray voltage 1.5 kV, capillary voltage 44,

capillary temperature 200°C, tube lens voltage 100 V. Data dependent acquisition

was performed using the software XCalibur 2.1.0 SP1.1160. Mass spectra were

acquired in a m/z range of 370-1,700 with a resolution of 60,000 at m/z 400.

Automatic gain control (AGC) was set to 2 x 105 (CID) and 5 x 105 ions (MSA) and a

maximum (max.) fill time of 500 ms. Multiply charged ions were selected for

fragmentation and detection in the linear ion trap (LIT) using a TOP5 method with the

following parameters: dynamic exclusion time: 45 s, AGC: 10,000 ions, max. fill time:

400 ms.

Samples from in vitro kinase assays were enriched for phosphopeptides by TiO2 and

analyzed by LC-MS. RPLC separations were performed as described above with

slight modifications. A binary solvent system consisting of 0.1% FA (solvent A) and

0.1% FA/86% ACN (solvent B) either with 4% DMSO or without was used in a 1 h

LC gradient. Eluting peptides were analyzed on a Velos Orbitrap Elite system

13 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 101

operated with the following parameter settings: spray voltage 1.8 kV, capillary

temperature 200°C. Mass spectra were acquired from m/z 370-1,700 with a

resolution of 60,000 and 120,000 (at m/z 400) for MSA and electron transfer

dissociation (ETD), respectively. AGC was set to 1 x 106 ions at a max. fill time of

200 ms. For MSA (NL of 32.7, 49 and 98 Da), a TOP10 method with a dynamic

exclusion time of 45 s and an AGC of 10,000 ions with a max. fill time of 200 ms was

used. For ETD, a TOP6 method with an exclusion time of 30 s and an AGC of 5,000

ions with a max. fill time of 100 ms and an activation time of 150 ms was employed.

MS/MS analyses employing higher-energy collisional dissociation (HCD) were

performed on a QExactive Plus instrument with the following parameters: spray

voltage 1.8 kV, capillary temperature 200°C. Mass spectra were acquired from m/z

375-1,700 with a resolution of 70,000 at m/z 200. The AGC was set to 3 x 106 ions

with a max. fill time of 60 ms. For HCD, a TOP10 method was used with an

exclusion time of 30 s and an AGC of 1 x 106 ions with a max. fill time of 120 ms and

a normalized collision energy of 30 eV.

Enriched phosphopeptide mixtures resulting from hpH-RP fractionation and

subsequent TiO2 enrichment were analyzed on a Velos Orbitrap Elite. Samples were

separated on the analytic column using a binary solvent system consisting of 0.1%

FA (solvent A#) and 0.1% FA/ 30%ACN/ 50% MeOH (solvent B#) supplemented with

4% DMSO in a 2 h gradient. Eluting peptides were directly transferred to the

electrospray source of a Velos Orbitrap Elite system operated with the following

parameter settings: spray voltage 1.8 kV, capillary temperature 200°C. Mass spectra

were acquired from m/z 370-1,700 with a resolution of 60,000 (at m/z 400) and an

AGC target of 1 x 106 ions with a max. fill time of 200 ms. A TOP15 MSA

14 102 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

fragmentation method (NL of 32.7, 49 and 98 Da) with a dynamic exclusion time of

45 s and an AGC of 1 x 105 ions with a max. fill time of 200 ms was used.

For top-down analyses, LC-MS measurements were performed on a Velos Orbitrap

Elite coupled online to an U3000 HPLC system (Thermo Fisher Scientific) equipped

with one C18 -precolumn (Ø 0.3 mm x 5 mm; PepMapTM, Thermo Fisher

Scientific) and an Acclaim PepMap300 analytical column (ID: 75 m x 150 mm, 5

m, 300 Å, Thermo Fisher Scientific). 5 l acidified sample were injected and

separated using a binary solvent system consisting of 0.1% FA (solvent A) and 0.1%

FA in 100% CAN (solvent B) with a gradient from 5% B to 90% B in 25 min at a flow

rate of 313 l/min. Subsequently, the column was washed for 2 min with 90% B and

re-equilibrated with 5% B for 13 min. The MS instrument was operated applying a

spray voltage of 1.8 kV and a capillary temperature of 200°C. Data-dependent

acquisition was performed using the software XCalibur 2.1.0 SP1.1160. Mass

spectra were acquired in an m/z range of 300-2,000 with a resolution of 60,000 at

m/z 400 and an AGC target value of 5 x 105 at a max. ion time of 200 ms. Multiply

charged ions ≥ 3+ were selected for fragmentation and detection in the orbitrap mass

analyzer. A TOP5 method was applied using CID and ETD fragmentation with the

following parameters: dynamic exclusion time: 5 s, AGC: 1 x 106 ions, max. fill time:

500 ms, isolation width: m/z 4, CID activation time: 10 ms, ETD activation time: 100

ms with a fixed first mass at m/z 100.

Bioinformatics

For global phosphoproteomics data, Andromeda integrated in MaxQuant 1.3.0.5 (35)

was used to search peak lists against the UniProt ProteomeSet mouse database

(release 01.12.2015, 57,276 protein entries). The precursor mass tolerance was set

15 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 103

to 20 ppm for the first search and to 4.5 ppm for the main search. For MSA/CID data,

the fragment mass tolerance was set to 0.5 Da. Trypsin was set as proteolytic

enzyme allowing up to two missed cleavages. Oxidation of methionine and

phosphorylation of serine, threonine and tyrosine were set as variable modifications

and cysteine carbamidomethylation as fixed modification in replicate 2. A false

discovery rate (FDR) of 1% was applied on both peptide (on modified peptides

separately) and protein lists. Numbers of unique phosphopeptides were counted

based on the MaxQuant peptide ID in the Phospho(STY) sites table. Phosphosites

scored with a MaxQuant localization probability of ≥ 0.75 were deemed “localized”,

while sites with a localization probability of < 0.75 were counted as putative sites

given that the aa sequence in combination with the number of phosphate groups was

not identified with localized sites elsewhere in the dataset.

For analysis of MS data from in vitro kinase assays, raw files were processed using

Andromeda embedded in MaxQuant 1.4.1.2 and searched against the sequences of

mouse and human FLNc d23-24 and their respective phosphosite mutants

generated in this work using the IPI human decoy database (version 3.76,178,826

entries) as background for correct FDR calculation. Precursor and fragment mass

tolerances were set to 10 ppm and 0.5 Da, respectively. Search parameters were as

follows: proteolytic enzyme: trypsin, max. number of missed cleavages: 2, and

variable modifications: methionine oxidation and phosphorylation of serine, threonine

and tyrosine. Result files from SILAC data were processed using MaxQuant 1.5.2.8

and searched against the UniProt ProteomeSet mouse database (release

02.01.2016, 58,238 protein entries). The parameters were set to the same value as

for the in vitro kinase assay with the addition of carbamidomethylation as fixed

modification and multiplicity was set to three with Arg0, Lys0, Arg6, Lys4, Arg10 and

16 104 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Lys8. MaxQuant msms.txt files, all raw files and FLNc isoform sequences were

imported into Skyline 2.6.0 (36). MS1 intensities were calculated as described by

(37) using the MS1 filtering tutorial provided by the software developers. Skyline

peptide settings were as follows: tryptic peptides with 1 missed cleavage, a time

window of 3 min, min. and max. peptide length 8 and 30 aa, respectively, exclusion

of cysteine-containing peptides, phosphorylation of serine, threonine and tyrosine

and oxidation of methionine as variable modifications, and max. number of variable

modifications and neutral losses 3 and 1, respectively. Orbitrap default parameters

were used for transition settings. Extracted ion chromatograms of the imported

peptides were manually inspected for correct peak picking and peak integration was

adjusted by hand, if necessary. Total MS1 areas for all peptides with an error less or

equal to 3 ppm were exported into a pivot table and processed using Excel2010 and

Origin 9.1. The mean and the standard error of the mean were calculated within the

three biological replicates first and afterwards within technical replicates. Intensities

of all phosphopeptides were summed separately for human and mouse FLNc

isoforms and phosphopeptides were normalized by the respective calculated

summed intensity.

For top-down experiments, raw data were converted into 32-bit mzXML files using

the MSConvert tool embedded in ProteoWizard 3.0.6965 (38) with default

parameters. mzXML files were further processed with MSDeconv (39) into an

msalign file with standard parameters and used for peptide search with TopPIC (40).

Search parameters were as follows: fragmentation mode read from the input file, no

cysteine protection group, an error tolerance of 10 ppm, an e-value of 0.01 as cut-off

and a maximal PTM mass of 1,000. Searches of the FLNc isoforms were performed

against FASTA files containing the sequences of the corresponding proteins. For

17 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 105

Gene Ontology (GO) enrichment analysis, the Cytoscape 3.2.1 plugin BiNGO (41)

was used. Enrichment was analyzed for the three main GO domains cellular

component (CC), molecular function (MF) and biological process (BP). Benjamini-

Hochberg FDR correction at a significance level of 0.05 was used for p-value

correction after the hypergeometric statistic test. Overrepresented categories after p-

value correction in comparison to the Mus musculus background dataset were

evaluated for major differences between the two protein groups. Calpain cleavage

sites were predicted using the program GPS-CCD with a cut-off score of 0.654 (42).

Mass Spectrometric Data Deposition

All raw data and original MaxQuant result files have been deposited to the

ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the

PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/login) (43) with the

dataset identifiers PXD003211 (large-scale phosphoproteomics analysis),

PXD003185 (in vitro kinase assay) PXD003216 (top-down experiments) and

PXD004151 (SILAC-mFLNc S625). Processed data of kinase assays and SILAC

data analyzed with Skyline as well as their results are available on PanoramaWeb

interface (44) https://panoramaweb.org/labkey/invitro_FLNc.url and

https://panoramaweb.org/labkey/invivo_mFLNc.url.

Design of cDNA Constructs and Site-Directed Mutagenesis

Site-directed mutagenesis was performed using the QuikChange® Lightning Site-

Directed Mutagenesis Kit (Stratagene/Agilent Technologies, Waldbronn, Germany)

as described in the manual. The AA (S2623/S2624 to alanine) and DD

(S2623/S2624 to aspartate) mutants of hFLNC d23-24 in pET23a/EEF were

18 106 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

obtained by PCR using the primer pairs

CCTCAAGCCGGGGCGCCGCCTACAGCTCCATCC with

GGATGGAGCTGTAGGCGGCGCCCCGGCTTGAGG, and

CCTCCTCAAGCCGGGGCGACGACTACAGCTCCATCCCCA with

TGGGGATGGAGCTGTAGTCGTCGCCCCGGCTTGAGGAGG, respectively, using

the non-mutant variant as template.

Site-directed mutagenesis of mFLNC d23-24 and hFLNc d22-24 was performed by

PCR in a two-step procedure. First, the primers GCGGTGCCGGCTACAGTTCC and

GGAACTGTAGCCGGCACCGC were used for mFLNc23-24. Second, the primer

pair GCGGTGCCGCCTACAGTTCC and GGAACTGTAGGCGGCACCGC or

GCGGTGCCGACTACAGTTCC and GGAACTGTAGTCGGCACCGC was used to

obtain the S2625A or S2625D point mutant of mFLNc d23-24. To generate the

hFLNc d22-24 S2623/2624D mutant, the primers CGGGGCGACAGCTACAGCTC

and GAGCTGTAGCTGTCGCCCCG were used and then the primer pair

CGGGGCGACGACTACAGCTC and GAGCTGTAGTCGTCGCCCCG. For the

hFLNc d22-24 S2623/2624A point mutant, GGGGCGCCAGCTACAGCTC and

GAGCTGTAGCTGGCGCCCC were used followed by GGGCGCCGCCTACAGCTC

and GAGCTGTAGGCGGCGCCC. To obtain full-length mutant FLNc variants in

pEGFP vectors for FRAP, the wildtype (WT) Ig-like d23-24 were replaced by the

respective mutant variant taking advantage of a unique AgeI restriction site in the

cDNA encoding d23 and the SalI site in the multiple cloning site of the vector.

Similarly, the cDNA encoding d23 and d24 was replaced by cDNA lacking the

sequences encoding aa 2628-2725 (i.e. part of hinge 2 and complete Ig-like d24) to

obtain an FLNc variant (∆Ig-like d24) similar to the calpain 1-digested full-length

FLNc protein, but still retaining the calpain 1 cleavage site.

19 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 107

Recombinant Protein Expression and Purification

Expression of His6-tagged mouse and human FLNc fragments in E. coli BL21 (DE3)

CodonPlus cells (Stratagene, Santa Clara, CA, USA) and their purification using

Ni2+- NTA agarose (Qiagen, Venlo, the Netherlands) was performed essentially as

described (7). Briefly, 50-100 ml of E. coli culture was allowed to express the

recombinant proteins by addition of isopropyl -D-1-thiogalactopyranoside (IPTG).

Cells were centrifuged and the resulting pellet was resuspended in 4 ml lysis buffer

(50 mM NaHPO4, 300 mM NaCl, 10 mM imidazole, 1 mg/ml lysozyme, pH 8.0) for 30

min on ice followed by ultrasonication for complete cell lysis. Cell debris was

removed by centrifugation for 30 min at 4,500 rpm and 4°C and the supernatant was

added to 500 l Ni2+-NTA agarose prepared according to the manufacturer’s

protocol. The slurry was incubated for 2 h at 4°C before the supernatant was

removed following a centrifugation step. Subsequently, Ni2+-NTA agarose-coupled

His6-tagged proteins were washed twice with precooled washing buffer (50 mM

NaHPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) on a pre-equilibrated gravity-flow

column (Mobicol, Boca Scientific, Boca Raton, FL, USA). Proteins were eluted by

adding elution buffer (50 mM NaHPO4, 300 mM NaCl, 250 mM imidazole, pH 8.0)

and stored at 4°C.

In Vitro Kinase Assays

Recombinantly expressed and purified mouse and human FLNc d23-24 WT and

phosphosite mutants were dialyzed overnight at 4°C in dialysis buffer (1 mM DTT,

100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2). For MS-coupled in vitro kinase

assays, a sample volume corresponding to 100 g protein was added to a volume of

20 108 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

200 l H2O and mixed with 5x PKC buffer [25 mM DTT, 100 mM KCl, 20 mM HEPES

pH 7.4, 10 mM MgCl2, 0.5 mM CaCl2, 2.5 mM ATP, sodium fluoride, sodium

pyrophosphate, sodium orthovanadate, -glycerophosphate, 0.15% CHAPS, 1x PKC

lipid activator (Merck Millipore, Darmstadt, Germany)]. The assay was started by

adding 200 ng PKC (Sigma-Aldrich) and the reaction was performed under shaking

at 200 rpm for 20 min at 30°C for each FLNc construct. For further analysis, samples

of three independent replicates were diluted 1:4 (v/v) with 50 mM ammonium

bicarbonate and subjected to in-solution digestion using sequencing grade trypsin

(1:50) (Promega) for 3.5 h at 200 rpm and 42°C. Single protein digests were acidified

with TFA [final concentration 1% (v/v)], subjected to TiO2 enrichment, and the

resulting phosphopeptide-enriched fractions analyzed by LC-MS/MS using

alternative fragmentation methods.

Radioactive in vitro kinase assays were performed with 1-2 g protein in 1x PKC

buffer. Samples were incubated with 5-10 Ci [-33P] ATP and 1 mM ATP for 20 min

at 30°C. In vitro phosphorylation was started by adding 10 ng or 20 ng PKC.

Subsequently, 4x Laemmli buffer was added to the samples followed by SDS-PAGE

and autoradiography.

SILAC Analysis

SILAC-labeled C2C12 myotubes were starved overnight and subjected to electrical

pulse stimulation for 4 h. Subsequently, myotubes were subjected to treatment with

phorbol-12-myristat-13-acetat (PMA, 200 M, Sigma Aldrich) for 15 min, Gö6976 (10

M, Merck) for 1 h, or DMSO for 1 h (control). A label switch was performed for each

of the three biological replicates analyzed in this work. Protein concentrations were

measured using the Bradford assay (BioRad, München, Germany) and control, PMA

21 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 109

and Gö6976 treated samples were mixed in a 1:1:1 ratio. Protein digestion was

performed as described above, peptides were separated using hpH-RP

chromatography, and fractions 19-20 were subjected to TiO2-based phosphopeptide

enrichment followed by LC-MS/MS analysis.

Calpainolysis Assays

In vitro calpainolysis assays were performed essentially as described (45). In brief,

recombinantly expressed mouse and human FLNc d23-24 WT and phosphosite

2+ mutants with a carboxyterminal EEF- and His6-tag were coupled to Ni -NTA

agarose beads and washed 2x with 1 ml calpain reaction buffer (20 mM HEPES, pH

7.4, 50 mM KCl, 2 mM MgCl2, 5 mM CaCl2, 1 mM DTT). Subsequently, the bound

protein fragments were incubated for 30 min with 5 g calpain 1 (Merck) in 200 l

reaction buffer at 37°C and 200 rpm on a Thriller Thermoshaker (Peqlab, Erlangen,

Germany). Pre-equilibrated gravity-flow columns were used to separate the beads

from the supernatant. For top down analysis of FLNc proteolysis products,

supernatants were acidified to a final concentration of 0.1% TFA. The remaining

agarose beads were incubated with 150 l Laemmli sample buffer for 5 min at 95°C

and samples were stored for Western blot analysis.

For further calpainolysis assays, HEK293 cells transiently expressing hFLNc d22-24

WT and the AA and DD phosphorylation site mutants were used or C2 cells

expressing hFLNc d22-24 WT. Prior to lysis, cells were treated with PMA (200 nM for

15 min) or Gö6976 (10 M for 1 h) as indicated. Cell lysis was performed 24 h after

transfection on ice with pre-cooled lysis buffer (10 mM Tris HCl, pH 7.4, 100 mM

NaCl, 0.1% Triton X-100, 2 mM EGTA, sodium fluoride, sodium pyrophosphate,

sodium orthovanadate, -glycerophosphate). Calpain inhibitor IV (Merck) was

22 110 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

directly added to the lysis buffer at a final concentration of 50 M. Subsequent to

lysis, cells were incubated in an ultrasonic bath for 20 min at 8°C and centrifuged for

5 min at 1,500 x g. Supernatants were adjusted to the same protein concentration

and calpainolysis was started by addition of 5 mM CaCl2 and 0.85 g calpain 1

(Merck) per 100 l reaction volume. Samples were incubated for 20 min at 37°C and

800 rpm and the reaction was stopped with the according volume of 5x Laemmli

buffer. Cleavage products from three independent assays were analyzed by SDS-

PAGE and immunoblotting. The percentage of cleaved hFLNc d23-24 WT and

hFLNc d23-24 mutants was calculated based on quantified immunoblot signals of

uncleaved hFLNc and the respective ~13 kDa cleavage products.

Fluorescence Recovery After Photobleaching and Data Analysis

Fluorescence recovery after photobleaching (FRAP) experiments were performed

with a Cell Observer Spinning Disk microscope (Carl Zeiss, Jena) equipped with an

external 473 nm diode laser (Rapp OptoElectronic, Hamburg) allowing bleaching of

precisely defined regions of interest (ROI) using a Plan-Apochromat 63x/1.4 oil

objective. Cells were kept at 37°C under 5% CO2. Image processing was carried out

using Zen 2012 software (Carl Zeiss, Jena, Germany). For FRAP analyses, 15-30

experiments were performed. For each cell, 1-3 ROIs were chosen for bleaching with

each region limited to a single Z-disc of neighboring myofibrils. Photobleaching was

performed with 100% intensity of the 473-nm laser with a pulse time of 1 ms with 8

iterations. Images were taken before and immediately after bleaching. The

fluorescence recovery was monitored for 400 s with an interval time of 0.1- 5 s.

FRAP experiments were performed with a Cell Observer SD (Carl Zeiss, Jena)

equipped with an external 473 nm laser coupled via a scanner (UGA-40, Rapp

23 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 111

OptoElectronic, Hamburg) allowing bleaching of precisely defined regions of interest

(ROI), using a Plan-Apochromat 63x/1.4 oil objective. Cells were permanently kept at

37°C and 5% CO2. Image processing was carried out using Zen2012 software (Carl

Zeiss, Jena). For FRAP analyses 15-30 experiments were performed. For each cell,

1-3 ROIs were chosen for bleaching with areas of bleaching limited to one

sarcomere. Photobleaching was done with 100% intensity of a 473-nm laser for a

pulse time of 1 ms with 8 iterations. Images were taken before and immediately after

bleaching. The fluorescence recovery was monitored for 400 s interval time of 0.1 to

2 s. The ImageJ package Fiji (46) was used to determine fluorescence intensity of

bleached and unbleached areas at each time point. Normalized FRAP curves were

generated from raw data as previously described (47). FRAP data are displayed as

mean of 10-15 individual experiments. To generate corrected FRAP curves, the

intensity in the bleached ROI () and in the whole cell excluding the bleached

area () at each time point were initially subtracted by the corresponding

background intensity () and the fraction formed.

Subsequently, normalization to the prebleach intensity was performed according to

the following equitation:

where is the normalized and corrected intensity, the corrected

intensity before photobleaching and the corrected first data point after

photobleaching. Normalized fluorescence intensity versus time was plotted using

Prism 4.0 (GraphPad Software). All results are expressed as mean ± SD. Before

24 112 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

treatment cells were starved for 2 h in serum-free medium. For photobleaching upon

inhibition of PKC activity, 10 M Gö6976 inhibitor was added to the cell culture

medium 2 h before starting the FRAP experiments and cells were kept in the

medium throughout the analysis.

Antibodies

Mouse monoclonal antibody T12 labels a titin epitope close to the Z-disc (48). Rabbit

polyclonal antisera recognizing the carboxyterminal 16 amino acids of FLNc (49) and

FLNc d22-24 were custom-made by BioGenes, Berlin, Germany. The latter

antiserum was extensively cross-absorbed against recombinantly expressed FLNa

and FLNb d22-24 to ascertain specificity for FLNc. Anti-GAPDH (#2118) and

anti-calpain 1 (#2556) were purchased from Cell Signaling Technology (Leiden, the

Netherlands), anti-Myc-tag (66004-1-Ig) from Proteintech (Manchester, UK).

Horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulins and

AlexaFluor596-conjugated rabbit anti-mouse immunoglobulins were purchased from

Sigma Aldrich and Life Technologies (Darmstadt, Germany), respectively.

Immunofluorescence Staining

For characterization of different cell stages, C2C12 myoblasts were seeded on

collagen-coated cover slips (Thermo Fischer Scientific) and cultivated for different

time periods. Immunolocalization studies were performed 2 d after seeding of cells,

5-6 d of myocyte differentiation and additional 16-24 h of electrical stimulation of

myotubes. Cell fixation was carried out for 10 min in methanol/acetone (1:1, v/v)

at -20°C. Cover slips were washed with CM-PBS, blocked with 10% normal goat

serum and 1% bovine serum albumin for 20 min and stained with T12 antibody

25 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 113

(1:20) for 45 min. After a washing step, samples were incubated with AlexaFluor596-

conjugated anti-mouse immunoglobulins (1:250) for 45 min. DAPI staining (1:1,000)

was performed for 2 min before cells were mounted with ProLongGold (Life

Technologies). Optical analysis of cell differentiation was performed using a Nikon

Eclipse TS 100 fluorescence microscope equipped with NIS Elements Basic

Research 4.00.03 software.

Miscellaneous

Information about the experimental design and statistical rationale for different

analyses performed in this work are provided within the respective subsections in

Material and Methods. Number of sample size, replicates, controls, and statistical

tests were chosen according to published data with comparable methodology and

generally accepted standards in the field. SDS-PAGE and Western blotting were

performed by standard protocols; signals were detected using HRP-coupled

secondary antibodies and an enhanced chemiluminescence system (Thermo Fisher

Scientific) with a ChemoCam (Intas, Göttingen, Germany) equipped with a full-frame

3.2 megapixel Kodak KAF-3200ME camera. No image processing, other than

cropping, scaling and contrast adjustment was applied. Quantification of Western

blot signals was performed with Quantity One 4.6.9 (Bio-Rad, Hercules, CA, USA).

For statistical analysis, two sample t-tests were performed using OriginPro 9.1

(OriginLab, Northampton, MA, USA). All quantitative Western blot data are

presented as mean ± standard error of the mean (SEM) or standard deviation (SD).

To minimize the effects of subjective bias, Western blot data were generated and

analyzed by two different experimentators. The sample size was estimated on

26 114 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

previous experiments and statistical tests were chosen based on published data with

comparable methodology.

27 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 115

Results

Analysis of the phosphoproteome of contracting myotubes.

With the aim to obtain further insight into signaling processes in sarcomeres, we

studied the phosphoproteome of differentiated, contracting skeletal muscle cells. To

this end, we used mouse myoblasts C2C12 cells that were grown to 90% confluence

and differentiated for 5-6 days by serum reduction (Figure 1A, I, II, IV and V). To

increase formation of contractile myofibrils with mature Z-discs, differentiated

polynuclear C2C12 cells were electrically stimulated (Figure 1A, III and VI,

MovieEV1). Subsequent to cell lysis using high-urea buffer, SCX-based separation

of tryptic digests and phosphospecific enrichment by titanium dioxide (TiO2),

phosphopeptides were analyzed by high resolution liquid chromatography-mass

spectrometry (LC-MS) (Figure 1B). In addition, a small aliquot of each SCX fraction

was directly analyzed to support peptide and protein identifications. From the

analysis of two biological replicates, we identified a total of 6,808 proteins comprising

2,941 (43%) phosphoproteins (Figure 1C, Table S1). The overlap of identified

proteins and phosphoproteins between the two replicates was 83% and 63%,

respectively. The total number of phosphosites was 11,369 with 8,175 (72%)

allocated to a distinct amino acid with a probability ≥ 0.75 (Table S2). The large

majority (1,914, 65%) of phosphoproteins comprised more than a single phosphosite

with 529 proteins modified by more than five distinct phosphate groups (Figure 1D).

The Z-disc is a hotspot of protein phosphorylation in sarcomeres

We assessed which and to what extent sarcomeric proteins contribute to the

phosphoprylation landscape of contracting skeletal myotubes. Remarkably, 532

(91%) of all the 586 phosphosites in sarcomeric proteins were identified in 66

28 116 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

proteins of the I-band (Figure 2A, Table S2). Within the I-band, 412 (77%) of all

phosphosites were found in 31 genuine Z-disc proteins and 21 proteins peripherally

associated with this structure (Figure 2B), underscoring the suggested outstanding

signaling capacity of Z-disc proteins in striated muscle cells (50, 2). The majority of

localized phosphosites (264, 83%) in proteins associated with the myofibrillar Z-disc

were assigned with high confidence (probability ≥ 0.9) and further 53 sites (17%)

were scored with probabilities between 0.75 and 0.9 (Figure 2C). Of these, 89 sites

were not reported in the PhosphositePlus database (version 07.01.2016, Table S2).

We employed gene ontology (GO) enrichment analysis to reveal functional features

overrepresented in the myofibrillar proteome (Figure 2D, Table S3) and

phosphoproteome (Figure 2E, Table S3). In both datasets, the terms “Z-disc”,

“nucleus”, “gene expression”, “kinase activity” and “cytoskeletal protein binding” were

significantly enriched, whereas “mitochondrion” was only overrepresented in the

proteome data. Interestingly, PKC-binding and processes related to phosphorylation

and cytoskeletal organization were specifically enriched in the phosphoproteome

(Figure 2E). In summary, our data revealed the Z-disc as the major myofibrillar site

of protein phosphorylation, supporting the notion that it acts as an important

signaling platform within the sarcomere (2, 50).

A detailed view on the Z-disc phosphorylation landscape

In the framework of this study, we defined a list of 42 true Z-disc proteins as well as

21 proteins directly associated with the Z-disc (associated and peripheral) and

further 42 proteins connected to the Z-disc (membrane-associated, intercalated disc,

intermediate filament) based on literature and expert knowledge (Table S4). The

latter group comprises peripheral Z-disc proteins, including those connecting

29 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 117

myofibrils with the sarcolemma, e.g. via costameric proteins like the dystrophin-

associated protein complex. With the exception of a few Z-disc proteins only

expressed in distinct muscle fibers such as nebulette and Fbxl22 in cardiac muscle

(51, 52) or only located at the Z-disc under distinct conditions such as during

embryonic development or during myofibrillar repair or remodeling processes (53),

our data cover the complete Z-disc proteome known to date. This protein

compendium includes a total of 31 specific (29 genes, 69%) and 21 associated (15

genes, 71%) Z-disc components with confident phosphosite localizations (Figure

3A, Table S2). Remarkably, numerous Z-disc and -associated proteins such as

FLNc, BCL2-associated athanogene 3 (BAG3), LIM domain binding 3 (LDB3),

enabled homolog (ENAH, also referred to as MENA or ENA), Xin actin-binding

repeat containing 1 (XIRP1), heat shock protein beta-1 (HSPB1), and the three

members of the podin protein family synaptopodin (SYNPO), myopodin (SYNPO2)

and tritopodin (CHAP; synaptopodin 2-like, SYNPO2L) were phosphorylated at

multiple amino acid residues with up to a total number of 25 distinct phosphosites in

SYNPO2L (Figure 3B, Table S2). Interestingly, phosphosites often occurred in

clusters, as observed in the podin protein family and FLNc.

The carboxyterminal part of filamin C is multiply phosphorylated in myotubes

In this study, we identified six distinct phosphosites in mouse FLNc, two of which

(pS2234, pS2237) were located in the isoform-specific insertion within the FLNc

Ig-like domain 20 and three (pS2621, pS2625, pS2633) were clustered in its

carboxyterminus (Figure 3B, Table S2). Phosphorylation of FLNc at S2621 is

reported for the first time in this work. Previous work suggested FLNc to be a

potential substrate of PKC in its carboxyterminal region (28). This prompted us to

30 118 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

further analyze phosphorylation of FLNc at S2621 and S2625, both located in the

hinge 2, as well as S2633 in the aminoterminus of Ig-like domain 24 (Figure 3B). We

confirmed the correct assignment of these phosphosites located in a highly serine-

rich stretch of amino acids (2618SSSSRGASYSSIPKFSSDASK2638) by manual

interpretation of fragmentation spectra (Figure S1A-C). For pS2621 and pS2625, we

also identified the doubly phosphorylated peptide (Figure S1D), indicating that FLNc

can be concurrently phosphorylated at these serine residues in vivo. Kinase

prediction analysis employing the algorithms NetPhosK and NetworKIN indicated

that all three phosphorylated residues are putative substrate sites of PKC/PKC

(Figure S1E). However, the exact PKC target site in the carboxyterminal part of

FLNc remained ambiguous.

Mouse FLNc S2625 and human FLNc S2623/S2624 are specific substrate sites

of PKC in vitro

Our combined MS-based phosphoproteomics and kinase prediction data revealed

for mouse FLNc (mFLNc) three in vivo sites potentially phosphorylated by

PKC/PKC (pS2621, pS2625 and pS2633). In human FLNc (hFLNc), the

orthologous residues were consistently identified as in vivo phosphosites as well

(54). Based on the presence of a PKC consensus site motif (55), we hypothesized

that PKC likely phosphorylates mFLNC at S2625 and hFLNc at S2623 and/or

S2624 (Figure S2A). To test our hypothesis, we performed in vitro kinase assays

using recombinantly expressed wildtype (WT) and phosphosite mutants of Ig-like

domains 23-24 (d23-24) of mouse and human FLNc, each with a His6 and EEF tag

at their carboxyterminal end (Figure S2B). In mFLNc, we mutated S2625 to

aspartate (D) or alanine (A) to mimic its constitutively phosphorylated or non-

31 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 119

phosphorylated state, whereas in hFLNc the orthologous residues S2623 and S2624

were mutated. Incubation with PKC in the presence of [-33P]-ATP showed that

both WT constructs were phosphorylated by PKC in a concentration-dependent

manner, whereas signal intensities for the respective site mutants were considerably

decreased, but not abolished (Figure 4A and 4B). This indicated that PKC is

capable of unspecifically phosphorylating additional sites in this in vitro assay.

Phosphorylation of mFLNc d23-24 and hFLNc d23-24 WT and their respective

phosphosite mutant forms was not observed following their incubation with [-33P]-

ATP in the absence of PKC (Figure S3A).

To obtain residue-resolved information about PKC-mediated phosphorylation of

FLNc, we performed in vitro kinase assays in conjunction with quantitative MS.

Experiments were performed in triplicates using equal protein amounts (Figure S3B)

followed by phosphopeptide analysis using complementary fragmentation methods

and MS1-based quantification using Skyline (36) (Table S5). This comprehensive

analysis unequivocally established PKC-mediated phosphorylation of mFLNc at

S2625 (Figure 4C, left panel and Figure 4E). In addition, we found that PKC also

phosphorylated S2603 and S2637 in vitro, although to a lesser extent (Figure 4C,

left panel). The latter two phosphosites have not been identified in vivo, suggesting

that they occurred non-specifically under in vitro conditions. Interestingly,

phosphorylation was partially “swapped” to S2620 when S2625 was mutated to

alanine or aspartate in mFLNc (Figure 4C, middle and right panel). For hFLNc

d23-24 WT and the respective AA and DD mutants, we observed largely identical

PKC-dependent phosphorylation patterns (compare Figure 4D and 4C). Here,

S2623 served as main substrate site of PKC, but phosphorylation of both S2623

and S2624 was observed to a minor extent as well (Figure 4D, left panel and

32 120 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Figure 4F). In summary, we revealed S2625 and S2623/S2624 localized in the

hinge 2 region of mouse and human FLNc, respectively, as specific substrate sites of

PKC in vitro.

FLNc S2625 is an in vivo substrate of PKC in mouse skeletal muscle cells

To demonstrate that mFLNc S2625 is a substrate of PKC in vivo, we determined the

relative abundance of the FLNc phosphopeptide 2623GApSYSSIPK2631 in C2C12

myotubes treated with PMA, an activator for the conventional PKC isoforms  or

Gö6976, an inhibitor for PKC and PKC1 (56) in comparison to DMSO-treated cells

(control). To this end, we performed SILAC labelling of C2C12 cells and specifically

analyzed the mFLNc phosphopeptide 2623GApSYSSIPK2631 by quantitative MS

following TiO2 enrichment (Figure 4G). The experiment was performed in three

biological replicates including a label swap to determine significant changes in

S2625 phosphorylation. As a result, we found phosphorylation of mFLNc S2625 to

be significantly decreased upon treatment of C2C12 myotubes with Gö6976 (Figure

4H, Figure S3C). Since PKC is the conventional PKC isoform predominantly

expressed (97%) in mouse skeletal muscle cells (57), these data strongly indicate

that FLNc S2625 is phosphorylated by PKC in vivo. However, we cannot exclude

that PKC1 may further contribute to mFLNc S2625 phosphorylation in vivo. Our

data also indicate that PKC is active in contracting myotubes as we only observed

a slight increase in FLNc S2625 phosphorylation in PMA-treated cells (Figure 4H,

Figure S3C). In summary, SILAC-MS analysis shows that FLNc is a target of PKC

at S2625 in mouse skeletal muscle cells.

33 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 121

Tyrosine carboxyterminally to the PKC substrate site is the main calpain 1

cleavage site

The carboxyterminal part of FLNc was reported to be a substrate of the proteases

calpain 3 and calpain 1 (also referred to as -calpain) active in skeletal muscle fibers

(29, 58, 59). In silico data indicated that calpain 1 preferentially cleaves hFLNc

carboxyterminally to Y2625 in addition to S2626 as suggested previously (29) and,

correspondingly, mFLNc at Y2626 and S2627 (Table S6). Interestingly, mutation of

the neighboring PKC substrate sites S2623/S2624 (hFLNc) and S2625 (mFLNc) to

alanine or aspartate decreased the cleavage probability at several sites. For the

phosphomimetic mutants, this effect was even more pronounced and also led to a

clear decrease in the number of predicted calpain 1 cleavage sites.

To unequivocally identify which site is the main cleavage site in FLNc, we

established a novel site-resolving MS-based in vitro calpainolysis assay. To this end,

carboxyterminally His6- and EEF-tagged d23-24 constructs of human and mouse

FLNc WT and the respective phosphosite mutants (Figure S2B) were recombinantly

expressed, immobilized on Ni2+-NTA beads and incubated with recombinant

calpain 1. Resulting aminoterminal cleavage products were characterized as intact

protein fragments by high resolution LC-MS following a top-down approach. For

hFLNc d23-24 WT, we determined Y2625 as the main calpain 1 cleavage site,

whereas cleavage at S2618 and S2626 was only minor (Figure 5A-C, Figure S4A,

Table S7). The AA and DD site mutants showed virtually identical cleavage patterns

with an additional cleavage site only occurring in the non-phosphorylatable AA

variant (Figure 5D and 5E, Figure S4B and S4C, Table S7). Consistent with these

findings, top-down MS analysis of calpain 1-dependent aminoterminal cleavage

products of mFLNc revealed Y2626 as main cleavage site (Figure S5A-G, Table

34 122 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

S7). For the S2625D mutant, we observed S2627 as an additional prominent

cleavage site. However, this altered specificity is most likely the consequence of a

considerably reduced efficiency of calpain 1 for cleaving this variant as observed by

immunoblot analysis using an antibody against the His6-tag to detect both intact (~24

kDa) and cleaved forms (~13 kDa) (Figure S5H). Thus, a lower cleavage specificity

may be indicative of an effective site-specific protection against proteolysis.

In both human and mouse FLNc, the main calpain 1 cleavage site (hFLNc-Y2625

and mFLNc-Y2626) was found in direct vicinity of the PKC substrate site(s)

identified in this work. Since the introduction of negative charges at these sites might

affect the susceptibility of the respective substrate to calpain 1 cleavage, we studied

the efficiency of calpain 1-dependent cleavage of all hFLNC variants by SDS-PAGE

and immunoblotting (Figure 5F, Figure S2B). We quantified immunoblot signals

from three independent in vitro calpainolysis assays to calculate the percentage of

cleaved hFLNc (Figure 5G). Approximately 25% of hFLNc d23-24 WT was cleaved

by calpain 1, whereas the phosphomimetic DD mutant was almost completely

protected from proteolysis, and the AA mutant appeared only slightly less

susceptible to degradation. These data suggest that exchange of two hydrophilic by

two negatively charged/phosphorylated residues causes a conformational change

around the cleavage site, rendering the substrate less prone to calpain 1 binding.

Indeed, the rate of calpain cleavage directly depends on polypeptide conformation

rather than the linear sequence of amino acids surrounding a cleavage site (60).

Taken together, we showed that Y2625 and Y2626 are the major calpain 1 cleavage

sites in human and mouse FLNc d23-24, respectively, whereas the introduction of

negative charges at the aminoterminal serine residues (i.e. S2623/S2624 or S2625)

significantly reduces its susceptibility to cleavage.

35 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 123

PKC-mediated phosphorylation of S2623/S2624 protects human FLNc from

limited proteolysis by calpain 1

To address the question whether PKC-mediated phosphorylation of hFLNc-

S2623/S2624 is the determining factor for protection against calpain 1 cleavage

under cellular conditions, we transiently expressed aminoterminally tagged hFLNc

d22-24 in HEK293 or C2 mouse skeletal muscle cells (Figure S2C) and treated the

cells either with PMA to stimulate classical PKC activity, or with Gö6976 to inhibit

PKC. Subsequently, cell lysates were treated with recombinant calpain 1 in the

absence or presence of calpain inhibitor IV and intact hFLNc d22-24 and cleavage

products thereof were detected by immunoblotting using antibodies directed against

either d22-24 or the last carboxyterminal 16 amino acids of hFLNC. As expected, we

observed efficient proteolysis of hFLNc d22-24 in both HEK293 and C2 cells

following treatment of lysates with calpain 1 and this effect was abolished with

calpain inhibitor IV (Figure 6A, lanes 1-3; Figure S6, lanes 1-3). Pretreatment of

lysates with the PKC inhibitor Gö6976 slightly increased proteolysis, whereas

incubation with the PKC stimulant PMA reduced the levels of cleavage products

(Figure 6A, lane 5 and 8¸ Figure S6, lane 5 and 8). Consistently, cleavage of

hFLNc d22-24 by activated calpain 1 was more than two-fold reduced following

activation of PKC by PMA (Figure 6B and 6C). Inhibition of PKC by Gö6976

resulted in a slight, although not significant increase in the amount of cleavage

products. The latter finding can be explained by assuming that only a rather small

fraction of overexpressed hFLNc d22-24 is phosphorylated by endogenous PKC in

HEK293 cells, underscoring the strong dependence of FLNc’s susceptibility to

calpain 1 on its phosphorylation status. Moreover, comparative quantitative analysis

36 124 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

of calpain 1-dependent cleavage products of hFLNc d22-24 WT and S2623/S2624

site mutants (DD, AA; Figure S2C) expressed in HEK293 cells demonstrated that

the phosphomimetic DD variant was almost completely protected from proteolysis

(Figure 6D and 6E). In line with the effect observed for the recombinant hFLNc d23-

24 AA variant (Figure 5F and 5G), mutation of S2623/S2624 to alanine resulted in a

lower percentage of cleaved hFLNc d22-24 in HEK293 cells (Figure 6D and 6E).

PKC modulates the dynamic behavior of human FLNc through

phosphorylation of S2623/S2624 in skeletal myotubes.

FLNc fulfils important roles in the assembly, repair and maintenance of myofibrils

(17, 32, 61), functions that suggest that FLNc must be highly dynamic and mobile

under distinct physiological conditions. We therefore continued to investigate

whether PKC-mediated phosphorylation and calpain 1-dependent proteolysis of

hFLNc have a physiological effect on its dynamic behavior. To study the mobility and

dynamics of hFLNc in living cells, we employed fluorescence recovery after

photobleaching (FRAP) using immortalized mouse myoblasts expressing hFLNc WT

fused to EGFP or mutant forms in which S2623/S2624 were changed to alanine (AA)

or aspartate (DD) or the Ig-like domain 24 (amino acids 2628-2725) was deleted

(Figure S2D). Following photobleaching of defined regions of myotubes expressing

EGFP-fusion proteins in a cross-striated pattern (Figure 7A), the recovery profiles of

hFLNC WT (in cells treated with or without the PKC inhibitor Gö6976) and mutant

forms were recorded. hFLNc-EGFP recovery profiles were biphasic with a lateral

diffusion-dependent initial, fast recovery as determined by diffusion testing (Figure

S7). The subsequent slower phase of the recovery profile depends on the exchange

process of bound protein with the soluble fraction and, thus, allows for the calculation

37 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 125

of protein halftimes as shown in Figure 7B. Mean halftimes were significantly

increased for the AA mutant (54 s) and, to an even higher extent, following inhibition

of PKC activity (93 s) in myocytes expressing hFLNc WT. In stark contrast, the DD

and Ig-like d24 mutants of hFLNc exhibited significantly shorter mean halftimes (11

and 10 s) compared to the WT form (25 s). Remarkably, we did not observe changes

in the mobile fraction of hFLNc when cells were treated with Gö6976 to inhibit PKC

or double site mutants were expressed (Figure 7C). The only exception was the Ig-

like d24 mutant of hFLNc showing an increase in the mobile fraction (92%)

compared to FLNc WT (78%), which points to a lower affinity of this truncated FLNc

form to Z-disc structures. In summary, our data suggest that PKC precisely controls

the dynamics of hFLNc in skeletal myotubes through phosphorylation of

S2623/S2624. Removal of Ig-like domain 24 mimicking cleavage by calpain 1 led to

a considerably shorter halftime along with an increase in mobility, possibly due to the

inability of the truncated hFLNc form to dimerize.

38 126 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Discussion

In this work, we present a comprehensive site-resolved protein phosphorylation map

of contracting cultured skeletal myotubes with 8,175 localized sites in 2,941 proteins

(Figure 1C, Table S1 and S2). Evaluation of our global phosphoproteome dataset

revealed the Z-disc as major protein phosphorylation hot spot in myofibrils, whereas

only 9% of the identified sarcomeric phosphosites were located in A-band proteins

(Figure 2A, 2B, and 2E). The vast majority of genuine Z-disc and Z-disc-associated

proteins (44, 70%) were modified by a single or even multiple phosphate groups,

establishing phosphorylation of Z-disc proteins as a central theme for intracellular

signal transduction processes in myofibrils (Figure 3A). Remarkably, we found

clustered multisite phosphorylations in numerous Z-disc-associated proteins,

including FLNc, BAG3, ENAH, LDB3, and the three podin protein family members

SYNPO, SYNPO2, and SYNPO2L (Figure 3B). These findings hint at distinct

regulatory regions that may control their protein interactions, dynamics, localization

and/or other functions. Our data strengthen the emerging view of the Z-disc as a way

station and molecular scaffold for protein kinases and phosphatases (2, 50, 62).

GO enrichment analysis revealed PKC signaling as a significant feature in the

phosphoproteome of skeletal myotubes (Figure 2E, Table S3). Several proteins

were reported to anchor PKC at the Z-disc following activation, highlighting the

importance of PKC signaling in the regulation of highly dynamic Z-disc protein

assemblies essential for skeletal and cardiac muscle function and growth (50, 63,

64). For example, the actin-capping protein CapZ anchors PKC to myofilaments in

cardiomyocytes (65). Under hypertrophic conditions, PKC signaling decreases the

binding affinity of CapZ to actin filaments, thereby destabilizing the actin filaments

and allowing sarcomere remodeling (66). Further PKC anchoring proteins at the

39 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 127

Z-disc are LDB3, also called cypher or ZASP (67-69), and enigma homologue

protein ENH (PDLIM5) (70), which together with calsarcin-1 (MYOZ2) form a

complex with an important role in linking Z-discs to the extracellular matrix via the

multi-adaptor protein FLNc (71). All these proteins were characterized as

phosphoproteins in striated muscle cells in this work (Figure 3A, Table S1). A

detailed analysis of FLNc revealed phosphorylation at six distinct serine residues

(Figure 3B, Table S2). Two of these phosphosites are located in the unique 82

amino acid insert within Ig-like domain 20, which facilitates targeting of FLNc to the

Z-disc (9). Earlier work identified FLNc as a bona fide substrate of PKB/Akt at S2233

in rabbit skeletal muscle (72). Here we consistently identified phosphorylation of

murine FLNc at S2234 (corresponding to S2233 in human FLNc) (72) as well as

concurrent phosphorylation of S2234 and S2237 in mouse myotubes. Interestingly,

binding of aciculin (also called PGM5; a dystrophin-binding protein involved in

myofibril development, maintenance and repair) to FLNc requires the insertion in Ig-

like domain 20 (32). Likewise, only FLNc containing this insertion interacts with Xin

(XIRP1) and XIRP2 (12). Other proteins reported to specifically bind this FLNc region

include myopodin (7) and myotilin (9). With the exception of XIRP2, we identified all

these FLNc interactors also as strongly phosphorylated proteins (Figure 3A and

3B). In this context, it is interesting to note that FLNc, aciculin, and Xin are proteins

whose phosphorylation increased within a few minutes after inducing pressure

overload in mouse hearts (73), suggesting their close interplay in signaling pathways

underlying the adaptation of striated muscle to mechanically induced stress. Thus,

phosphorylation of FLNc at S2234/S2237 may provide a mechanism for controlling

such dynamic and competing protein interactions depending on the physiological

condition.

40 128 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

In addition, we identified a cluster of three phosphosites in the carboxyterminus of

FLNc in hinge 2 and at the beginning of repeat 24 (Figure 3B and S1). Similar to Ig-

like domain 20, this region of FLNc is involved in multiple protein interactions,

including dimerization [domain 24; (22, 23)], binding to calsarcin-2 [domain 23; (15)]

and - and -sarcoglycans [domain 24; (17)]. The interaction with both sarcoglycans

is abolished by calpain 3-dependent cleavage of FLNc at its hinge 2 region (58).

Moreover, phosphorylated FLNc was found in consequence of a phospholipid-

dependent interaction of PKC via its carboxyterminal end (28). However, PKC-

dependent phosphorylation sites in FLNc and their physiological relevance have

remained uncharacterized so far.

Our in vitro kinase assays revealed S2625 and S2623/S2624, located in the

carboxyterminus of mouse and human FLNc, respectively, as specific PKC sites

(Figure 4, Table S5). In contrast, phosphorylation events of mFLNC at S2621 and

S2633 were not mediated by PKC, implying involvement of additional kinases.

Selective PKC signaling to the carboxyterminal Ig-like domain 24 of FLNa and

FLNc, but not FLNb was proposed previously (28). Here we unequivocally show that

PKC targets the hinge 2 region of FLNc in vitro. This serine-rich sequence stretch

(2621SRGASYSSIPKFSS2633) is uniquely present in FLNc, implying that the PKC site

in FLNa differs. Previous work reported three phosphosites (p2523, pS2526, and

pS2599) in hinge 2 and Ig-like domain 24 of FLNa in non-muscle mouse tissues (74).

We here identified four FLNa phosphosites (pS968, pS1084, pS1459, pS2327), all

not residing within Ig-like domains 23 to 24 (Table S2), indicating that only the

carboxyterminus of FLNc is specifically targeted by PKC in skeletal muscle cells

under the conditions tested.

41 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 129

What may the functional impact of PKC phosphorylation of FLNc’s hinge 2 region

be? The carboxyterminus of FLNc was identified as substrate of calpain 1 (29), and

FLNc and calpain 1 colocalize at the edges of the Z-disc (18, 29). FLNa

phosphorylated by PKA was more resistant to calpain 1 cleavage, thereby inhibiting

cytoskeletal reorganization (75). A similar PKC-dependent mechanism was

proposed for protecting FLNc from calpain 1 cleavage (29). Interestingly, active

PKC itself is further cleaved to a constitutively active form by calpain, which might

increase FLNc phosphorylation and its resistance to calpain cleavage (60, 76).

Chicken gizzard filamin is rapidly degraded to 240- and 10-kDa fragments by

calpain, thereby severing its cross-linking ability, however the exact cleavage site

remained unidentified (77). The detection of calpain cleavage sites is generally

hampered by the lack of known consensus sequences, rendering predictions

unreliable (78). To overcome this issue, we combined here for the first time in vitro

proteolysis assays with top-down MS to accurately determine calpain 1 cleavage

sites in FLNc. Our in vitro data show that the main cleavage site is a tyrosine residue

located directly carboxyterminally to the PKC substrate site(s) in the hinge 2 region

of FLNc (Figure 5A-C, Table S7). This cleavage specificity is retained for the

respective phosphosite mutants (Figure 5D and 5E, Table S7), but its efficiency was

significantly decreased in the phosphomimetic form (Figure 5F and 5G, Table S7).

Our experimental data generally support earlier findings suggesting that calpain

preferentially cleaves in disordered segments between structured domains between

a tyrosine (P1) and a carboxyterminal serine (P1') with proline in position P2'- P4' or P3

(79).

Calpains were suggested to play a role in integrin signaling (80) by cleaving

specifically -integrin family members at a conserved NPXY/NXXY motif in the

42 130 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

carboxyterminal half of the cytoplasmic domains involved in talin and filamin binding

(81). Involvement of calpains in signaling has been emphasized by in vitro studies

revealing kinases (e.g. focal adhesion kinase, FAK), phosphatases (e.g. protein

tyrosine phosphatase 1B, PTP1B) and cytoskeletal proteins (e.g. paxillin, vinculin) as

substrates (60). Nonetheless, virtually no information on how calpain cleavage

modulates distinct signaling events in vivo is available to date. In this work, we

showed that stimulation of PKC activity by PMA significantly attenuates the

susceptibility of FLNc to cleavage by calpain 1 (Figure 6A-C, Figure S6). The effect

was even more pronounced for a FLNc variant in which the serine residues identified

as substrate sites of PKC (S2623/2624) were mutated to aspartic acid, thus

mimicking human FLNc in its constitutively phosphorylated form (Figure 6D and 6E).

We conclude that phosphorylation of these serines is the mechanism by which FLNc

is protected from limited proteolysis by calpain 1 in its hinge 2 region.

Finally, we used FRAP experiments to study the effect of phosphorylation and

calpain 1 cleavage on FLNc dynamics in living skeletal muscle cells. Most

importantly, an FLNc variant mimicking calpain 1-cleaved FLNc showed a highly

significant reduction of the mean halftime and an increase of the mobile fraction

(Figure 7B and 7C). This indicates that FLNc without its dimerization domain, but

including all its known Z-disc targeting domains, is still targeted to Z-discs. The lack

of Ig-like domain 24 apparently reduces the stability of the interactions with its Z-disc

ligands, rendering the protein less stable (82).

Our FRAP data also revealed a highly significant decrease in the mean halftime for

the phosphomimetic (S2623D/S2624D) variant of FLNc. Consistently, the effect was

reciprocal for the double site mutant mimicking FLNc in its dephosphorylated form

(S2623A/S2624A), whereas inhibition of PKC even further increased the mean

43 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 131

halftime (Figure 7B). The latter observation may indicate that α phosphorylates

FLNc at an additional site, potentially beyond Ig-like domains 23-24, which further

affects its dynamics. A strong candidate is S2237, located in the insertion of Ig-like

domain 20, which is involved in targeting FLNc to Z-discs (9). These data, in

accordance with the observation that binding of phosphorylated filamin to isolated

myofibrils is significantly lower (83), convincingly show that phosphorylation of FLNc

is important for increasing its mobility, a prerequisite for initiating signaling

processes.

In summary, our extensive analysis of the phosphoproteome of the sarcomeric

Z-disc identified this structure as a myofibrillar phosphorylation hotspot. Our data

confirm recent suggestions that the Z-disc is highly involved in signaling processes.

Our finding that calpain 1 cleavage of FLNc is regulated by phosphorylation of one

(mouse) or two (human) serine residues directly flanking the cleavage site is a

paradigmatic example of a phosphorylation-dependent mechanism identified by a

follow-up study based on these data. We expect that the vast amount of

phosphorylation sites uncovered in this work will lead to identification of further

events regulated by protein phosphorylation.

44 132 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Acknowledgements

We thank Andreas Schummer, Caroline Gerbeth and Kurt Lobenwein for technical

assistance and Zacharias Orfanos and Chris Meisinger for discussions. Deposition

of the data to the ProteomeXchange Consortium was supported by the PRIDE

Team, EBI, and publication to the Panorama public server was supported by the

Skyline/Panorama Team. This research was supported by the Deutsche

Forschungsgemeinschaft (FOR 1352 to B.W. and D.O.F., and FOR 1228 to D.O.F.)

and the Excellence Initiative of the German Federal & State Governments (EXC 294

BIOSS to BW).

Author Contributions

L.R. performed phosphoproteomics, mass spectrometric and biochemical analyses

with the support of H.W., B.K., A.F. and A.S. FRAP measurements were performed

by Y.L. and A.R. All authors analyzed data, designed and interpreted experiments.

B.W. and D.F. supervised the study. B.W. conceived the project and wrote the

manuscript with the input of other authors.

Conflict of Interest

The authors declare that they have no conflict of interest.

45 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 133

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Figure Legends

Figure 1. Global phosphoproteomic analysis of C2C12 myotubes.

A, Formation of contracting sarcomeres in C2C12 skeletal muscle cells studied by

light (I-III) and fluorescence (IV-VI) microscopy. Cell fusion was demonstrated by

DAPI staining of nuclei (blue) and sarcomere formation by staining using a T12

antibody (red) directed against the Z-disc-associated end of titin (IV-VI).

Mononuclear myoblast at 50% confluence (I, IV) were grown to 90% confluence

before cell fusion was induced by serum reduction. Following five to six days of

differentiation, cells were fused to polynuclear myotubes with a diameter of 20-50 m

and a length of up to 1-2 mm (II, V). To generate contracting myotubes, cells were

electrically stimulated for 16-24 h. Sarcomere formation was confirmed by observing

the typical striated pattern in fluorescence microscopy (VI); no morphological

changes were observed in light microscopy (III). Scale bar: I-III, 100 m; IV-VI, 10

m.

B, Phosphoproteomics of differentiated, contracting skeletal muscle cells. C2C12

myotubes were lysed using high-urea buffer in the presence of phosphatase

inhibitors. Following digestion of proteins using trypsin and desalting, peptide

mixtures were fractionated by strong-cation exchange (SCX) chromatography. In

total, 55 SCX fractions were collected and a small aliquot of each fraction was

directly analyzed by LC-MS/MS. From each sample, phosphopeptides were enriched

by titanium dioxide (TiO2) followed by LC-MS/MS analysis. Datasets of two biological

replicates were jointly analyzed using MaxQuant and Perseus software.

55 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 143

C, Numbers of (phospho-)proteins and phosphosites (p-sites) identified in

contracting C2C12 myotubes. Localized p-sites were identified with a MaxQuant

localization probability score ≥ 0.75.

D, Distribution of phosphosites (p-sites) according to the number of sites per protein.

Figure 2. The Z-disc is the main site of protein phosphorylation in sarcomeres.

A, B, Distribution of phosphosites (p-sites) identified in sarcomeric proteins of the

I- or A-band (A). 532 p-sites (91%) were identified in proteins of the I-band. Of these,

412 p-sites (77%) were identified in true Z-disc and Z-disc-associated proteins,

which is an essential structural part of the I-band (B).

C, Classification of p-sites localized in 44 Z-disc-associated proteins. 264 p-sites

(83%) were identified with high confidence with a MaxQuant localization probability ≥

0.90 (red). Further 53 p-sites were localized with a probability ≥ 0.75 and < 0.90

(grey).

D, E, GO-term enrichment analysis of the C2C12 myotube proteome (D) and

phosphoproteome (E) dataset established in this work. P-values after Benjamini-

Hochberg false discovery rate (FDR < 0.05) correction were plotted against their

corresponding GO-terms from the three main domains cellular component (CC),

molecular function (MF) and biological process (GO BP). Numbers of identified

proteins for each term are shown in brackets.

Figure 3. A site-specific protein phosphorylation map of the myofibrillar Z-disc

proteome from mouse skeletal muscle cells.

A, Schematic overview of the Z-disc phosphoproteome. 44 genes (encoding for 51

proteins) were found to be modified by a single or multiple phosphate groups

56 144 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

accounting for 70% of the known Z-disc proteome. Phosphosites localized in the

Z-disc-associated regions of the two structural proteins titin (12 p-sites) and desmin

(14 p-sites) are not depicted.

B, Schematic illustration of highly phosphorylated Z-disc proteins. Localized

phosphosites (mouse) are color-coded based on information provided by the

PhosphoSitePlus database (version 07.01.2016) with black for known sites, blue for

phosphosites known by similarity to orthologues sites in human proteins, and red for

newly identified sites. Information about protein domains was retrieved from the

UniProt and InterPro database. Immunoglobulin-like domains in FLNc and XIRP1 are

numbered. ABD, actin-binding domain; BAG, Bcl2-associated athanogene; PDZ,

post synaptic density protein, Drosophila disc large tumor suppressor, and zonula

occludens-1 protein; PxxP, proline‐rich; PPxY, proline-proline-x-tyrosine; VASP,

vasodilator-stimulated phosphoprotein; WH1, WASP-homology/EVH1, Ena/VASP

homology 1.

Figure 4. S2625 and S2623/S2624 are the specific substrates sites of PKC in

the hinge 2 region of mFLNC and hFLNc, respectively.

A, B, Radioactive in vitro kinase assays. Recombinant wildtype and phosphosite

mutants of mouse (A) and human (B) FLNc Ig-like domains 23-24 (d23-24) were

treated with PKC in the presence of [-33P]ATP and analyzed by SDS-PAGE

followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was

replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. As a control,

PKC was incubated in [-33P]ATP-containing kinase buffer without hFLNc d23-24

(B). WT, wildtype; A, alanine; D, aspartate; +, 10 ng PKC; ++, 20 ng PKC

57 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 145

C, D, MS-based in vitro kinase assays. Reactions were performed as described in

(A, B) using unlabeled ATP and PKC. MS data from three independent kinase

experiments for mFLNc d23-24 WT, A and D sites mutants (C) and hFLNc WT, AA,

and DD site mutants (D) were quantified. Intensities of phosphopeptides distinctive

for a specific phosphorylation site (red) were added up per experiment and

represented as normalized mean ± SEM.

E, F, Fragmentation spectra of mono-phosphorylated peptides of mouse (E) and

human (F) FLNc d23-24 WT forms. PKC-dependent phosphorylation of mFLNc-

S2625 and hFLNc-S2623 was determined by higher-collisional dissociation and

electron transfer dissociation, respectively. Fragment ions exhibiting a neutral loss of

phosphoric acid (H3PO4; 97.9768 u) are marked with an asterisk (*); loss of water

(H2O) as indicated. Phosphorylated residues are depicted in red; b/c- and y/z-ion

series in red and blue, respectively.

G, Experimental workflow to study mouse FLNc S2625 phosphorylation in C2C12

myotubes by SILAC and quantitative MS. Three biological replicates were performed

including label-swaps.

H, Changes in the phosphorylation of mouse FLNc S2625 in C2C12 myotubes

treated with PMA or Gö6976 in comparison to control. Top: SILAC triplet of the

mouse FLNc phosphopeptide 2623GApSYSSIPK2631. Bottom: Relative quantification

of the mouse FLNc phosphopeptide 2623GApSYSSIPK2631 using Skyline. Shown is

the mean log2 ratio of the area under the curve ± SEM. **p ≤ 0.006, n=3

Figure 5. Y2625 is the major calpain 1-dependent cleavage site in the hinge 2

region of hFLNc.

58 146 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

A, B, Top-down MS analysis. Recombinant hFLNc d23-24 fused carboxyterminally

to a His6- and EEF-tag was treated with recombinant human calpain 1 and

aminoterminal cleavage products were subjected to LC-MS/MS using low-energy

collision-induced dissociation (CID) as fragmentation method. (A) Amino acid

sequence of hFLNc d23-24-His6-EEF. (B) Top down CID spectrum of the main

cleavage product observed at m/z 13,355.66; sequence and fragmentation pattern

are depicted in black in (A) with the calpain 1-dependent cleavage site in red.

C-E, Determination of the main calpain 1 cleavage site. In vitro calpain 1 assays and

top-down analyses were performed as in (A, B). Data from three independent

experiments for hFLNc d23-24 WT (C) and S2623/S2624 phosphosite mutants [AA

mutant, (D); DD mutant, (E)] were quantified and represented as normalized mean ±

SEM. Calpain 1-dependent cleavage sites are denoted.

F, Samples were analyzed by immunoblotting. FLNc*, carboxyterminal cleavage

product

G, Quantification of data shown in (F) for n=3 experiments. Error bars represent the

SD, a two-tailed Student’s t test was used. **p ≤ 0.0034, ***p ≤ 0.0003

Figure 6. PKC protects hFLNc from calpain 1-dependent proteolysis through

phosphorylation of S2623/S2624

A, HEK293 cells transiently expressing hFLNc d22-24 were treated with Gö6976 or

PMA as indicated. Subsequently, cell lysates supplemented with 5 mM CaCl2 were

incubated with human calpain 1 and the calpain inhibitor IV as indicated. Samples

were analyzed by immunoblotting. FLNc*, aminoterminal cleavage product; FLNc#,

carboxyterminal cleavage product

59 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 147

B, HEK293 cells expressing hFLNc d22-24 were treated with Gö6976 or PMA as

indicated. Calpain 1 was added to cell lysates and activated with CaCl2 or not as

indicated. Samples were analyzed by immunoblotting. FLNc*, aminoterminal

cleavage product; FLNc#, carboxyterminal cleavage product

C, Quantification of data shown in (B). Percentage of cleaved hFLNc d22-24 was

calculated for n=3 independent experiments. Shown is the mean ± SEM. *p = 0.0442

D, HEK293 cells were transfected with constructs for hFLNc d22-24 WT and

respective S2623/S2624 phosphosite mutants (AA, DD mutant). Calpain 1 was

added to cell lysates (containing 5 mM CaCl2) as indicated and samples were

analyzed by immunoblotting. FLNc*, aminoterminal cleavage product; FLNc#,

carboxyterminal cleavage product

E, Quantification of data shown in (D). Percentage of cleaved hFLNc d22-24 was

calculated for n=3 independent experiments. Shown is the mean ± SEM. *p =

0.0245, **p ≤ 0.0078

Figure 7. Dynamics and mobility of hFLNc is controlled by phosphorylation

and cleavage of its Ig-like domain 24.

A, Immortalized mouse myoblasts (IMMs) were transfected with constructs for

EGFP-tagged full-length hFLNc WT, S2623/2624 phosphosite mutants (AA, DD), or

hFLNc lacking Ig-like domain 24. IMMs expressing full-length hFLNc WT were

treated with PKC inhibitor Gö6976 for 1 h or not before fluorescence recovery after

photobleaching (FRAP) analysis. Shown is a representative FRAP experiment with

IMMs after 5 days of differentiation before bleaching (prebleach), immediately

(postbleach), 5 and 20 s after bleaching, and after full recovery (recovery). Boxes

indicate the region bleached. Scale bar: 5 m

60 148 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

B, Quantification of data from FRAP studies described in (A). Statistical data are

depicted in box and whisker plots. Each calculated median halftime is shown as a

line surrounded by a box, which represents the interquartile range comprising the

median ± 25% of the data. Whiskers extend at most two standard deviations from

the median. Data from n ≥ 10 independent experiments are shown. **p ≤ 0.0011,

***p ≤ 0.0003

C, Percentage of mobile fractions calculated from FRAP studies described in (A).

Shown is the mean ± SD. n ≥ 10, *p = 0.0233

61 CD B A # Phosphoproteins # Localizedp-sites ..MobilrZdssaeapoenpopoyainhtso 149 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. IV I differentiated, #Phosphosites C2C12 cells contracting # Proteins tryptic digestion, and desalting cell lysis, 2,941 DAPI T12 6,808 8,175 VV II 11,369 chromatography strong cation Figure 1 exchange 62

# of phosphoproteins 1000 500 250 750 0

-01-51-0>20 16-20 11-15 6-10 5 4 3 2 1 DAPI T12 phosphorylated enrichment of O O 1,385 proteins peptides Ti PO # ofp-sitesperprotein III Ti 3 I O O bioinformatics 529 proteins analysis and LC-MS/MS Thermo S cientf Q

E XACTIVE DAPI T12 150 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

A BC 53 Z-disc Z-disc p-sites A-band associated 233 p-sites 54 p-sites 179 p-sites

I-band 264 p-sites 532p-sites non-Z-disc Σ 586 120 p-sites Σ 532 Σ 317 p-sites p-sites p-sites

nucleus (2021) nucleus (931)

mitochondrion (916) CC Z-disc (23) CC gene Z-disc (33) expression (471) gene expression (1100) phosphorylation (246) BP

BP protein (477) cytoskeleton (98) transport organization nucleic acid (1019) kinase activity (235) binding cytoskeletal (267) cytoskeletal (163)

MF protein binding protein binding MF kinase activity (410) PKC binding (12)

0 50 >100 050>100 -log (corr. p-value) proteins 10 -log10 (corr. p-value) phosphoproteins

Figure 2

63 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 151

A Itga Itgb Itga Itgb Ankrd2 Ankrd2

B FLNc BAG3 ABD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 WW PxxP BAG

M1 P2726 M1 P577 S138 S177 S179 S270 S274 S285 S382 S390 S404 S1528 S2234 S2237 S2621 S2625 S2633 LDB3 ENAH PDZ LIM LIM LIM WH1/EVH1 PxxP VASP M1 V723 M1 A802 T736 T732 T734 S98 T119 S121 S123 S177 S144 S255 S351 S354 S355 S362 S637 S740 XIRP1 HSPB1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 HSP20-like α-crystallin M1 L1129 M1 K209 S13 S15 S86 S102 S180 S203 S332 S139 S205 S208 S295 S533 SYNPO isoform 2 PPxY PxxP M1 K901 S19 T583 S534 Y536

S240 S249 S273 S284 S296 S433 S450 S501 S505 S524 T530 S585 S589 S594 S789 S803 S808 S819 S824 S892 SYNPO2 PDZ PxxP PxxP M1 E1087 T897 T715 S540 S543 S545 S546 S596 S618 S767 S771 S895 S899 S903 S719 S727 SYNPO2L PDZ PxxP PxxP M1 Q975 S266 S368 S371 T891 T912 T926 T138 T469 T559 T702 T718 T791 S435 S438 S890 S140 S163 S175 S177 S667 S675 S699 S787 S789 S793

Figure 3 64 152 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

mFLNc d23-24

A mFLNc d23-24 C WT A 100 D

WT AD 60 80 PKCα +++++++++ 40 80 kDa - 1.5 E10 1.5 E10 1.5 E10 -PKCα 60 60 kDa - 40 29 kDa S KVVTR S KVVTR - S KVVTR FLNc 40

- S LH...

24 kDa 20 S LH... S LH...

- YSSIPK

S 20 SRGADYSSIPK SRGAAYSSIPK FSSDA FSSDA S S 29 kDa - FSSDA 20 GA LSGGH SS -FLNc SS LSGGH 24 kDa normalized intensity [%] - LSGGH 0 0 0 S2603 S2625 S2637 S2603 S2620 S2637 S2603 S2620 S2637

hFLNc d23-24 hFLNc d23-24 D WT AA DD B 100 80 WT AA DD 40 PKCα ++ ++++++ +++ 80 YSSIPK

1E10 60 1E10 1E10 80 kDa - -PKCα 60 kDa - SS 60 YSSIPK G 29 kDa | - S KVVTR S KVVTR S KVVTR -FLNc S 40 20 G 24 kDa - | 40 S LH... S LH... S LH... SRGAAYSSIPK SSSSR SRGDDYSSIPK S FSSDA S FSSDA 29 kDa - FSSDA 20 20 SS SSSSR 24 kDa -FLNc SS - LSGGH LSGGH normalized intensity [%] 0 LSGGH 0 0 S2602 S2623 S2623 S2636 S2602 S2619 S2636 S2602 S2619 S2636 S2624 E y2 F [M+3H]3+ 244.17 504.02 100 100

G A S Y S S I P K S S S S R G S S Y S S I P K

2+ [M+3H] - H3PO4 a1 z5 698.53 101.07 515.41

c5 522.52 z6 678.40 b2 z122+ 129.07 660.23 c Relative Abundance y 7 y

Relative Abundance 8 y -H O 5 746.28 c z+2 7 2 c 531.45 z8 948.46 11 13 y1 y5-H2O 745.39 c3 4 c * c9 1170.52 1408.69 513.30 279.17 366.15 7 932.42 147.08 b4* y6-H O y * y 648.37 z9 996.57z10 c y3 361.15 2 7 3 y c6 989.50 1145.62 13 y4 y 676.37 359.30 4 c8 b * 357.25 5 763.40 444.39 579.33 c10 1380.64 3 444.28 b5* 531.30b * z 833.38 z11 198.09 b 448.18 6 y y 4 1083.53 z12 3 535.21 b7* 6 y7 2 z3 428.24 1232.69 296.06 648.30 694.38 861.38 244.34 341.29 1319.56 0 0 100 550 1000 100 500 1000 1500 m/z m/z

1.2E5 495.220 G + PMA H 499.227 GApSYSSIPK + Gö6976

Lys 0 Lys 4 Lys 8 6.0E4 495.722 497.233 499.729 Arg 0 Arg 6 Arg 10

Intensity 497.736 500.231 496.264 498.235 496.728 498.726 O O O Ti Ti O 0.0 496 498 500 PO3 m/z Enrichment

Gö6976 ** L GApSYSSIPK H PMA M Intensity -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2

m/z LC/MS & MaxQuant SILAC Mean log2 ratio Figure 4 65 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 153

A C hFLNc d23-24 WT MTRGEQSQAG DPGLVSAYGP GLEGGTTGVS SEFIVNTLNA 60 GSGALSVTID GPSKVQLDCR ECPEGHVVTY TPMAPGNYLI 50

AIKYGGPQHI VGSPFKAKVT GPRLSGGHSL HETSTVLVET 40 VTKSSSSRGS SYSSIPKFSS DASKVVTRGP GLSQAFVGQK 30 NSFTVDCSKA GTNMMMVGVH GPKTPCEEVY VKHMGNRVYN VTYTVKEKGD YILIVKWGDE SVPGSPFKVK VPVDHHHHHH 20

EEF 10 normalized intensity [%] y 5+ y 6+ B 61 4+ 87 1268.66 y58 1526.44 0 100 b 1510.10 S2618 Y2625 S2626 10 4+ 2+ 1030.45 y57 b y 5+ 1486.54 32 62 1547.21 1288.86 y 4+ y 7+ b15 b 61 82 16 1585.57 1234.91 1483.71 1554.75 hFLNc d23-24 AA 8+ D y113 60 5+ 1448.73 y58 1208.83 y 4+ 4+ 3+ 62 50 y47 y42 1610.85 1213.38 1440.08 2+ y 3+ b b 2+ b33 47 17 40 24 y 3+ 1603.76 1690.87 1717.81 2+ 1143.05 41 b21 1407.07 1021.50 y10 b14 y 3+ 30 1045.48 1396.68 50 6+ 1733.56 2+ y68 b18 20

Relative Abundance 1183.61 887.91 b13 b18 y8 y 1297.61 1774.83 830.36 9 917.40 2+ 2+ 3+ 10 b y normalized intensity [%] b22 y13 34 52 b 1086.52y 1346.64 1653.29 1795.28 7 11 0 787.37 1146.54 S2618 A2623 Y2625 S2626

0 1000 1500 2000 m/z hFLNc d 23-24 DD E 60 G F hFLNc d23-24 50 40 WT AA DD *** ** 40 Calpain 1 + + + *** 30 30 24 kDa - -FLNc 20 20

10 10

14 kDa - normalized intensity [%] -FLNc* 0 0 αEEF-tag % cleaved hFLNc d23-24 WT AA DD S2618 Y2625 S2626

Figure 5

66 154 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

+ + + Gö 6976 AB+ Gö 6976 ++ PMA + PMA + + + + Calpain inhibitor IV + +++ Calpain 1 + + + + ++ Calpain 1 + ++ CaCl2 68 kDa - -FLNc αFLNc 68 kDa - - FLNc 53 kDa -FLNc* d22-24 - 53 kDa - FLNc* 68 kDa FLNc - αFLNc - - d22-24 53 kDa- αFLNc 14 kDa - # - FLNc 14 kDa- C-term16aa 10 kDa - 10 kDa- -FLNc# - Calpain 68 kDa Calpain* 41 kDa - - - -GAPDH 41 kDa - - GAPDH CD E * WT AA DD ** 40 + + + Calpain 1 * 68 kDa- 30 30 - FLNc αFLNc 53 kDa FLNc* - - d22-24 20 20 14 kDa - - FLNc# 10 kDa- 10 10 68 kDa- -Calpain % cleaved hFLNc d22-24 % cleaved hFLNc d22-24 41 kDa- GAPDH 0 - 0 control Gö PMA WT AA DD

Figure 6

67 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 155

A pre-bleaching post-bleaching 5 s 20 s recovery

BC* *** 100 100 *** 80 80 *** 60 60 ** 40 40 halftime [s] 20 20 mobile fraction [%]

0 0

hFLNc+ Gö6976 wt hFLNc AA hFLNc wt hFLNc DD domain 24 hFLNc+ Gö6976 wt hFLNc AA hFLNc wt hFLNc DD domain 24 hFLNc ΔIg-like hFLNc ΔIg-like

Figure 7

68 156 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Supplementary Figure Legends

Supplemental Figure 1. FLNc is phosphorylated in its hinge 2 region at S2621,

S2625, and S2633 in mouse C2C12 myotubes.

A-D, Annotated fragmentation spectra of phosphorylated peptides from mouse

FLNc. In vivo phosphosites were assigned to S2621 (A), S2625 (B), S2633 (C) and

in case of the doubly phosphorylated peptide 2618SSSSRGASYSSIPK2631 to both

S2621 and pS2625 (D). Fragment ions exhibiting a neutral loss of phosphoric acid

(H3PO4; 97.9768 u) are marked with an asterisk (*); loss of water (H2O) and

ammonia (NH3) as indicated. Phosphorylated serines are depicted in red; b- and y-

ion series in red and blue, respectively. m/z, mass-to-charge ratio

E, Kinase prediction analysis employing the algorithms NetworKIN 3.0 and

NetPhosK 1.0. Scores are given for individual kinases predicted per site. -, no

kinase predicted. A minimum score threshold was set to 1.4 for NetworKIN and to

0.5 for NetPhosK.

Supplemental Figure 2. Overview of mouse and human FLNc constructs.

A, Schematic illustration of full-length mFLNc and hFLNc. The expanded views show

the serine-rich stretches of amino acids located in the hinge 2 region. FLNc

comprises 24 immunoglobulin (Ig)-like domains (grey). ABD represents the

aminoterminal actin-binding domain, Ig-like domain 20 is isoform-specific, and the

carboxyterminal Ig-like domain 24 is the dimerization domain of FLNc.

B, Schematic illustration of mFLNc and hFLNc d23-24 constructs comprising a His6

and EEF tag at the carboxyterminal end. Phosphosite mutants were generated from

1 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 157

the respective WT construct by exchange of one or two serine residues (S) to

alanine (A) or aspartate (D) as indicated. WT, wildtype; d, domain

C, Schematic illustration of mFLNc and hFLNc d22-24 constructs comprising a BirA*

sequence and 6xHis tag at the N-terminus. Phosphosite mutants were generated

from the respective WT construct as described in (B). BirA*, promiscuous biotin

ligase; WT, wildtype; d, domain

D, Schematic illustration of full-length hFLNc construct comprising an EGFP tag at

the carboxyterminal end. Phosphosite mutants were generated from the WT

construct as described in (B). WT, wildtype

A-D, The underlined amino acids represent the PKC phosphorylation site motif in

FLNc and site mutant variants thereof mimicking the non-phosphorylated (A, AA) or

constitutively phosphorylated (D, DD) form. A, alanine; D, aspartate

Supplemental Figure 3. Controls for the radioactive in vitro kinase assays, MS-

based in vitro kinase assay and extracted ion chromatogram of the mouse

FLNc phosphopeptide GApSYSSIPK from SILAC analysis.

A, Recombinant wildtype and phosphosite mutants of mouse and human FLNc Ig-

like domains 23-24 (d23-24) were treated with [-33P]ATP in the absence of PKC

and analyzed by SDS-PAGE followed by autoradiography or Coomassie staining.

S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24

by AA or DD. (B). WT, wildtype; A, alanine; D, aspartate

B, Abundance of recombinant mouse and human FLNc d23-24 wildtype and

phosphosite mutants in mass spectrometry-based in vitro kinase assays. Shown are

the mean values of the total peptide intensity measured for each of the FLNc d23-24

constructs (see Figure S2B). Data derived from three independent experiments per

2 158 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

construct. Error bars represent the SEM. WT, wildtype; A, alanine, D, aspartate; n.s.,

not significant

C, Extracted ion chromatogram of the endogenous mFLNc phosphopeptide

GApSYSSIPK from triple SILAC-labeled C2C12 myotubes following treatment with

PMA (light), Gö6976 (medium) and DMSO (heavy, control).

Supplemental Figure 4. Intact mass measurements of calpain 1-dependent

aminoterminal cleavage products of human FLNc d22-23 WT and phosphosite

mutants.

A-C, Shown are sections of representative deconvoluted full MS spectra from intact

mass measurements of aminoterminal cleavage products of hFLNc d23-24 WT (A),

AA (B) and DD (C) mutants (see Figure S2B). Sequence range and molecular

masses of cleavage products are depicted. WT, wildtype; A, alanine, D, aspartate;

m/z, mass-to-charge ratio

Supplemental Figure 5. Y2626 is the main calpain 1-dependent cleavage site in

the hinge 2 region of mouse FLNc.

A, Amino acid sequence of mFLNc d23-24 wildtype (WT) form fused to a His6 and

EEF tag at the carboxyterminal end used as recombinant protein in in vitro

proteolysis assays with recombinant human calpain 1. The sequence of the major

aminoterminal cleavage product and the corresponding main calpain 1 cleavage site

are depicted in black and red, respectively.

B-D, Sections of representative deconvoluted full MS spectra from intact mass

measurements of calpain 1-dependent aminoterminal cleavage products of mFLNc

d23-24-His6-EEF WT (B), S2625A (C) and S2625D (D) phosphosite mutants (see

3 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 159

Figure S2B). Sequences of cleavage products identified by top-down MS analyses

are indicated. WT, wildtype; A, alanine, D, aspartate; S, serine; m/z, mass-to-charge

ratio; (ox.), oxidized methionine was identified in the sequence

E-G, Quantification of data shown in (B-D). MS signals from three independent

experiments were quantified and represented as normalized mean ± SEM. Calpain

1-dependent cleavage sites are denoted.

H, Immunoblot showing the efficiency of cleavage of mFLNc d23-24 WT, S2625A

and S2625D site mutants by calpain 1. FLNc*, carboxyterminal cleavage product

Supplemental Figure 6. PKC-mediated phosphorylation protects FLNc from

calpain 1-dependent proteolysis in C2 cells

A, C2 cells expressing hFLNc d22-24 were treated with Gö6976 or PMA as

indicated. Subsequently, human calpain 1 was added to cell lysates supplemented

with 5 mM CaCl2 in the presence or absence of calpain inhibitor IV as indicated.

Treatment with phosphatase was used to mimic non-phosphorylated state.

Samples were analyzed by immunoblotting. FLNc*, aminoterminal cleavage product.

Supplemental Figure 7. Diffusion test revealed that the initial fast recovery of

FLNc-EGFP depends on lateral diffusion.

FRAP experiments were carried out on full-length hFLNc-EGFP (see Figure S2D)

expressing immortalized mouse myotubes (day 5 of differentiation). Bleach field

length was kept constant comprising the whole cell, whereas the bleach field width

was varied between 0.3 and 5.1 m.

A, Recovery curves were biphasic. Increasing the width of the bleached area

resulted in slower increase during the initial recovery but little variation in the final

4 160 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

phase. From this, we conclude that the initial, fast recovery is at least partially due to

lateral diffusion of unbound labeled proteins whereas the later, slower phase

indicates the exchange process of bound proteins with the soluble fraction.

B, Scatter plot confirming the correlation between initial fast recovery halftime and

bleach field width (n=34).

5 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 161

A B 2+ [M+3H] - H3PO4 100 689.26 100 382.43 pS2625 pS2621 S S S S R G A S Y S S I P K G A S Y S S I P K

y11-H2O 2+ 1214.62 [M+3H] - H2O 729.43 y11 y11*-H2O b * 12 1232.57 1116.60 1152.57 y11* b12 1134.68 1250.57 b -H O 4 2 y2 b * 313.18 y10-H2O 7 244.24 648.32 y2 1047.61 b4* b132+ b9-H O 244.32 331.32 b 2 b 4 674.30 y 981.44 y72+ Relative Abundance 3 7 429.25 y10 Relative Abundance 262.15 y3 b5* 781.39 2+ b9* b9 1065.81 b7 y5 357.33 487.39 y6 y7 y 2+ 865.55 909.63 531.37 y * 10 477.24 694.34 7 861.51 b4* 630.54 763.47 533.48 b * 420.52 728.33 3 b3 361.83 494.76 198.19 573.86 296.19 684.28 779.07 0 0 500 1000 150 500 1000 m/z m/z C D y9* 100 948.49 100 680.41 pS2633 pS2621, G A S Y S S I P K F S S D A S K pS2625 S S S S R G A S Y S S I P K

729.48 y9 1046.40

y11-H2O y9-H2O b ** 1196.71 b7-H2O 12 y11* b12* 930.56 1134.58 648.23 y8 1214.63 1232.59 y * 2+ y12* y 2+ 6 y14 y * y14* 11 b11* y 798.55 11 1478.62 656.95 y7* 11 y6-NH b7 y2 1312.64 Relative Abundance Relative 3 b * b14* Relative Abundance y10-H O 666.28 b11* 12 244.27 674.48 2 b 2+ 630.34 y 769.28 12 b9 10 b13* b15* y9 838.53 b4-H O y ** b4-H O y7* b10 1309.65 y 2 b5* b5 b * 11 2 y10* b 13 313.15 y y 9 1116.65 y y5 1061.66 y11 13 y 5 7 945.28 3 y4 1148.65 y121407.70 b b y3 4 531.37 861.55 1294.50 507.36 14 15 1030.65 305.14381.15 1333.56 1565.87 357.25444.35 0 0 500 1000 1700 500 1000 1500 m/z m/z

E M1 P2726 ABD 1 211121314 15 16 17 18 19 20 21 22 23 24

P P P S2621 S2625 S2633

VTKSSSS RGAS YSSIPKFS SDASKVV 2615 2640

CaMKIIα 1.90 - NetworKIN 3.0 PKCα 3.57 PAK1 1.41 - PKC 0.59 RSK 0.60 NetPhosK 1.0 PKC 0.66 Cdc2 0.50 PKC 0.52

Figure S1

6 162 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

A mFLNc SSSSRGASYSSIPKFSSDASK 2618 2638

M1 P2726 ABD 1 211121314 15 16 17 18 19 20 21 22 23 24

M1 P2725 hFLNc SSSSRGSSYSSIPKFSSDASK 2617 2637

B mFLNc d23-24 WT SSSSRGASYSSIPKFSSDASK 124 144 A SSSSRGAAYSSIPKFSSDASK 124 144 D SSSSRGADYSSIPKFSSDASK 124 144

M1 F243 His- EEF- 23 24 tag tag M1 F243 hFLNc d23-24 WT SSSSRGSSYSSIPKFSSDASK 124 144 AA SSSSRGAAYSSIPKFSSDASK 124 144 DD SSSSRGDDYSSIPKFSSDASK 124 144

Myc- C BirA* hFLNc d22-24 tag 22 23 24 M1 E664 WT SSSSRGSSYSSIPKFSSDASK 554 574 AA SSSSRGAAYSSIPKFSSDASK 554 574 DD SSSSRGDDYSSIPKFSSDASK 554 574

D hFLNc EGFP ABD 1 211121314 15 16 17 18 19 20 21 22 23 24 EGFP- tag

M1 P2725 WT SSSSRGSSYSSIPKFSSDASK 2617 2637 AA SSSSRGAAYSSIPKFSSDASK 2617 2637 DD SSSSRGDDYSSIPKFSSDASK 2617 2737 ∆ Ig 24 SSSSRGSSYSS 2617 2627

Figure S2

7 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 163

A B mFLNc d23-24 hFLNc d23-24 n.s. 6.0E10 WT ADWT AA DD PKCα ------ATP +++ +++ 4.0E10 n.s. 29 kDa - - FLNc 24 kDa - Intensity 2.0E10 29 kDa - - FLNc 24 kDa -

0.0

Nc AA LNc A hFLNc WThFL hFLNc DDmFLNc WTmF mFLNc D

C 8x105 PMA - GAS*YSSIPK - 495.22++ Gö6976 - GAS*YSSIPK - 497.23++ control - GAS*YSSIPK - 499.22++ 6x105

4x105 Intensity

2x105

0 55.5 56.0 56.5 57.0 57.5 Retention Time [min]

Figure S3

8 164 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

AB hFLNc d23-24 AA 2TRG...AAY132 hFLNc d23-24 WT 2TRG...SSY132 100 13323.72 100 13355.66 2TRG...AAY(ox.)132 2TRG...SSY(ox.)132 13338.69 13370.68 2TRG...KSS125 50 12631.37 2TRG...AYS133 50 2TRG...SYS133 13410.74 2TRG...KSS125 13442.68 12631.37 1MTR...SSY132 1MTR...AAY132 13485.72 13454.70 Relative Abundance Relative Abundance Relative 13574.71 0 0 12500 13000 13500 12500 13000 13400 m/z m/z

C hFLNc d23-24 DD 2TRG...DDY132 13411.64 100

2TRG...DDY(ox.)132 13427.66

2TRG...DYS133 2TRG...KSS125 13498.64 50 12631.15 1MTR...DDY132 13667.65 Relative Abundance 0 12500 13000 13500 m/z

Figure S4

9 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 165

A 1 MTRGEQSQAG DPGLVSAYGP GLEGGTTGVS SEFIVNTQNA GSGALSVTID 50 51 GPSKVQLDCR ECPEGHVVTY TPMAPGNYLI AIKYGGPQHI VGSPFKAKVT 100 101 GPRLSGGHSL HETSTVLVET VTKSSSSRGA SYSSIPKFSS DASKVVTRGP 150 151 GLSQAFVGQK NSFTVDCSKA GTNMMMVGVH GPKTPCEEVY VKHMGNRVYN 200 201 VTYTVKEKGD YILIVKWGDE SVPGSPFKVN VPVDHHHHHH EEF 243 BE mFLNc WT 2TRG...ASY132 13354.66 100 40 2TRG...SYS133 13441.69 1MTR...ASY132 13485.68

50 20

13572.73

Relative Abundance 2TRG...KSS(ox.)125 TRG...SSS 2 127 normalized intensity [%] 12646.33 13104.54 0 0 12500 13000 13500 m/z S2619 Y2626 S2627 CF60 mFLNc A 2TRG...AAY132 100 13338.66 2TRG...AYS133 13425.68 40 1MTR...AAY132 13469.68

50 20

Relative Abundance TRG...SSS 2TRG...KSS(ox.)125 2 127 13556.71 12646.33 13104.55 normalized intensity [%] 0 0 12500 13000 13500 m/z S2619 Y2626 S2627

DG40

mFLNc D 2TRG...DYS133 13469.67 100 2TRG...ADY132 1MTR...ADY132 13382.64 13513.67 30 1MTR...DYS133 13600.68 20 50

Relative Abundance 2TRG...KSS(ox.)125 12646.38 normalized intensity [%] 0 0 12500 13000 13500 S2619 Y2626 S2627 m/z

H mFLNc d23-24 WT A D Calpain 1 + + + 41 kDa - 32 kDa - 23 kDa - -FLNc

14 kDa - FLNc 10 kDa - - * αHis6-tag

Figure10 S5 166 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

+ + + Gö 6976 ++ ++PMA + + + + Calpain inhibitor IV + + + + ++ + Calpain 1 + λ-Phosphatase 68 kDa - FLNc - αMyc-tag -FLNc* 53 kDa- 93 kDa- -Calpain 68 kDa- 41 kDa- -GAPDH 32 kDa-

Figure S6

11 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 167

A B 1.00 25

20 0.75 15 0.50 0.8 µm 10

0.25 2.2 µm fast halftime [s] halftime fast 5 4.2 µm normalized intensity normalized

0.00 0 0 25 50 75 100 125 150 0 5 10 15 20 25 30 time [s] squared bleach ﬁeld width [µm2]

Figure S7

12 168 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

Supplementary Table Legends

Supplemental Table 1. Proteome dataset. Excerpts from the MaxQuant output files

"proteinGroups" (spreadsheet proteins) and "peptides.txt" (spreadsheet peptides)

derived from the global proteome analysis of C2C12 myotubes; contains information

about the identification of proteins/protein groups and peptides. Contaminants and

reversed entries were removed from the lists.

Supplemental Table 2. Global phosphoproteome dataset. Excerpts from the

MaxQuant output file "PhosphoSTY" derived from the global phosphoproteome

analysis of C2C12 myotubes; contains information about phosphopeptide

identification and phosphosite localization in the spread sheets “phospho sites >0.75”

and “phospho sites <0.75”. Contaminants and reversed entries were removed from

the lists.

Supplemental Table 3. Result files from GO enrichment analysis of the C2C12

proteome and phosphoproteome dataset. Enrichment analysis was performed

with the Cytoscape 3.0.2 plugin BiNGO. Enrichment was analyzed for the three main

GO domains cellular component (CC), molecular function (MF) and biological

process (BP). Benjamini-Hochberg false discovery rate correction at a significance

level of 0.05 was used for p-value correction after the hypergeometric statistic test.

Supplemental Table 4. The Z-disc protein compendium.

Supplemental Table 5. Quantitative MS data from in vitro kinase assays using

protein kinase C and mouse/human filamin C d23-24 wildtype and phosphosite

1 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 169

mutants. Label-free quantification was performed using the Skyline software.

Phosphopeptides from each of the three independent experiments were analyzed by

LC-MS using multi-stage activation (MSA), electron transer dissociation (ETD) and

higher-energy collisional dissociation (HCD).

Supplemental Table 6. In silico calpainolysis data. Predicted calpain 1-dependent

cleavage sites in human and mouse FLNc d23-24 wildtype and respective

phosphosite mutants and full-length FLNc. Serine residues which were mutated are

depicted in blue and the corresponding point mutations in red. Predictions were

performed using the algorithm GPS-CCD with a cut-off score of 0.654. Cleavage

site,; n.p., not predicted; n.a., not applicable.

Supplemental Table 7. Top-down MS data from in vitro calpainolysis assays.

Excerpts from the TopPIC output result files derived from top-down MS analysis of

calpain 1-dependent amino-terminal cleavage products of human and mouse FLNc

d23-24 wildtype and respective phosphosite mutants.

2 7 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 170

Supplemental Table S4: The Z-disc protein compendium.

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein Protein 3425401 x x 154.48 Z-disc CEFIP Dierck et al, 2013 3425401B19Rik B19Rik Alpha-actinin skeletal muscle isoform 2, F- Alpha-actinin-2 Actn2 x 103.83 Z-disc Ribeiro et al, 2014 actin cross-linking protein Alpha-actinin skeletal Alpha-actinin-3 Actn3 x x 103.04 Z-disc Beggs et al, 1992 muscle isoform 3 membrane- Ankyrin-B, Brain Ankyrin-2 Ank2 x 432.86 Mohler et al, 2005 associated ankyrin Ankyrin repeat ardiac ankyrin repeat domain-containing Ankrd1 x 36.00 Z-disc protein, CARP, Bang et al, 2001 protein 1 MCARP Ankyrin repeat ARPP, Skeletal Kojic et al, 2004; Z-disc also shuttling domain-containing Ankrd2 x x 39.86 muscle ankyrin Jasnic-Savovic et al, 2015; associated to nucleus protein 2 repeat protein Tsukamoto et al, 2002 BAG family molecular Bcl-2-associated

Bag3 x x 61.86 Z-disc Homma et al, 2006 chaperone athanogene 3, BIS regulator 3 Voltage-dependent L-type calcium membrane- Cacna1c x 240.14 CACNL1A1, Cav1.2, Christel et al, 2012 channel subunit associated alpha-1C Voltage-gated Voltage-dependent calcium channel L-type calcium membrane- subunit alpha Cav1.3, Cacna1d 247.06 Lu et al, 2007 channel subunit associated Calcium channel, L alpha-1D type, alpha-1 polypeptide isoform 2 ..MobilrZdssaeapoenpopoyainhtso 171 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein Calcium-activated Z-disc neutral proteinase 3, Calpain-3 Capn3 x x 94.24 Goll et al, 1991 associated LGMD2A, Calpain p94 F-actin-capping protein subunit Capza1 x 32.94 Z-disc CapZ alpha-1 Casella et al, 1987 alpha-1 F-actin-capping protein subunit Capza2 x 32.97 Z-disc CapZ alpha-2 Casella et al, 1987 alpha-2 F-actin-capping protein subunit Capzb x x 31.35 Z-disc CapZ beta Caldwell et al, 1989 beta membrane- Calmitin, calcium- sarcoplasmic MacLennan & Wong, 1971; Calsequestrin-1 Casq1 x 46.38 associated sequestering protein reticulum Scott et al, 1988 membrane-

Caveolin-1 Cav1 x 20.54 BSCL3 caveolae Williams & Lisanti, 2004 associated Alpha(B)-crystallin, Alpha-crystallin B Z-disc also shuttling Cryab x x 20.07 Heat shock protein Koh & Escobedo, 2004 chain associated to nucleus beta-5, HspB5 Alpha E-catenin, Catenin alpha-1 Ctnna1 x x 100.11 peripheral Cadherin-associated Costamere Chopra et al, 2012 protein

Catenin beta-1 Ctnnb1 x x 85.47 peripheral beta-catenin, CTNNB Costamere Kurth et al, 1996 Dystrophin- membrane- Dystroglycan Dag1 x 96.90 associated Durbeej et al, 1998 associated glycoprotein 1 intermediate CMD1I, CSM1, Richardson et al, 1981; Desmin Des x 53.50 filament CSM2, Desmin Clemen et al, 2013 7 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 172

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein membrane- Duchenne muscular

Dystrophin Dmd 425.83 Belkin & Burridge, 1994 associated dystrophy DJ4, DnaJ homolog DnaJ homolog Z-disc subfamily B member subfamily B Dnajb6 x 39.81 Sarparanta et al, 2012 associated 6, Heat shock protein member 6 J2, MSJ1 BPAG1, Bullous pemphigoid antigen 1, Dystonia Steiner-Champliaud et al, Dystonin Dst x x 870.52 peripheral musculorum protein, 2010, Boyer et al, 2010 Hemidesmosomal plaque protein membrane- Dystrophin-related Dystrobrevin alpha Dtna x x 84.07 Peters et al, 1998 associated protein 3 membrane- Dystrobrevin beta Dtnb x x 74.40 - Peters et al, 1998 associated membrane- Fer-1-like protein 1, Matsuda et al, 2001; Dysferlin Dysf x 237.91 associated LGMD2B Minetti et al, 1992 Protein enabled ENA, MENA, Protein Enah x x 83.98 Z-disc Benz et al, 2013 homolog enabled homolog Protein 4.1 Epb41 95.91 peripheral 4.1R, Band 4.1 Pinder et al, 2012

Band 4.1-like 4.1N, Band 4.1-like Epb41l1 x x 98.31 peripheral Pinder et al, 2012 protein 1 protein 1 Ena/vasodilator- Ena/VASP-like

Evl x x 44.34 Z-disc stimulated van der Ven et al, 2006 protein phosphoprotein-like F-box and leucine- Z-disc F-box/LRR-repeat Fbxl22 26.32 Spaich et al, 2012 rich protein 22 associated protein 22 ABP-280-like protein,

Filamin-C Flnc x x 291.12Z-disc Thompson et al, 2000 FLN2, gamma filamin ..MobilrZdssaeapoenpopoyainhtso 173 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein Glutamate receptor N-methyl D-aspartate membrane- ionotropic, NMDA Grin2b 165.96 receptor subtype 2B, Seeber et al, 2000 associated 2B NR3, NR2B Homer protein membrane-

Homer1 41.41 Homer-1 Stiber et al, 2008 homolog 1 associated Stress-responsive Heat shock protein Z-disc Hspb1 x x 23.014 protein 27, Hsp25, Koh & Escobedo, 2004 beta-1 associated HSP27 Alpha-crystallin C chain, Protein kinase Heat shock protein Z-disc Hspb8 x x 21.53 H11, Small stress Baker et al, 2010 beta-8 associated protein-like protein HSP22 Immunoglobulin- like and fibronectin membrane- KY-interacting type III domain- Igfn1 303.65 Baker et al, 2010 associated protein 1 containing protein 1 Integrin beta-1- membrane- Itgb1bp2 x 38.77 Melusin Brancaccio et al, 1999 binding protein 2 associated Integrin beta-1- membrane- CHORDC3, costamere Itgb1bp2 x 38.77 Brancaccio et al, 1999 binding protein 2 associated ITGB1BP, Melusin region membrane- JP1, Junctophilin Ito et al, 2001; Junctophilin-1 Jph1 x x 71.90 associated type 1 Takeshima et al, 2015 Takeshima et al, 2000; membrane- JP2, Junctophilin Junctophilin-2 Jph2 x x 74.69 Minamisawa et al, 2004; associated type 2 Takeshima et al, 2015 Catenin gamma, Butz & Larue, 1995; Junction membrane- CTNNG, can shuttle to Cifuentes-Diaz et al, 1998; Jup x x 81.8 plakoglobin associated Desmoplakin-3, the nucleus Zhou et al, 2007; Desmoplakin III, DP3 Cohen et al, 2014 7 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 174

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein Potassium voltage- MinK, Delayed gated channel membrane-

Kcne1 14.58 rectifier potassium Furukawa et al, 2001 subfamily E associated channel subunit IsK member 1 Keratin, type I intermediate Krt19 44.54 cytokeratin19 Ursitti et al, 2004 cytoskeletal 19 filament

Keratin, type II intermediate Krt8 54.56 cytokeratin8 Ursitti et al, 2004 cytoskeletal 8 filament Kyphoscoliosis Z-disc Ky 75.09 - Baker et al, 2010 peptidase associated Metastatic lymph LIM and SH3

Lasp1 x x 29.99 peripheral node gene 50 Li et al, 2004 domain protein 1 protein, MLN50 ORACLE, PDLIM6, Protein cypher, LIM domain- Ldb3 x x 76.43 Z-disc ZASP, Z-band Faulkner et al. 1999 binding protein 3 alternatively spliced PDZ-motif protein LIM and senescent Particularly cell antigen-like- interesting new Cys-

Lims1 x 44.34 peripheral Meder et al, 2011 containing domain His protein 1, protein 1 PINCH1 57 kDa cytoskeletal protein, LGMD1A,

Myotilin Myot x x 55.32 Z-disc Salmikangas et al, 1999 Myofibrillar titin-like Ig domains protein Calsarcin-2, FATZ, Filamin-, actinin-and Faulkner et al, 2000; Myozenin1 Myoz1 31.46 Z-disc telethonin-binding Frey et al, 2000 protein ..MobilrZdssaeapoenpopoyainhtso 175 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein

Calsarcin-1, FATZ-

Myozenin-2 Myoz2 x x 29.76 Z-disc Frey et al, 2000 related protein 2,

Calsarcin-3, FATZ-

Myozenin-3 Myoz3 26.98 Z-disc Frey & Olson, 2002 related protein 3

145 kDa sarcomeric

Myopalladin Mypn x x 144.11Z-disc Bang et al, 2001 protein

Protein Neb Neb x x 828.65 Z-disc NEM2 Wang & Wright, 1988

LIM-nebulette, LASP2, LIM and SH3 Nebulette Nebl 52.20 Z-disc domain protein 2, Zieseniss et al, 2008)

Actin-binding Z-disk protein F-actin-binding

Nexilin Nexn x x 72.11 Z-disc Hassel et al, 2009 protein, Nelin

Nebulin-related- intercalated

Nrap x 195.75 Zhang et al, 2001 anchoring protein disc

Obscurin-myosin

Obscurin Obscn x x 815.63 Z-disc light chain kinase, Young et al, 2001

Obscurin-RhoGEF

Protein Obsl1 Obsl1 x x 197.93Z-disc Geisler et al, 2007

Alpha-PAK, p21- p21 protein activated kinase 1, (Cdc42/Rac)- membrane- Pak1 x x 60.607 p65-PAK, PAKalpha, also Z-disc Ke et al, 2004 activated kinase associated Serine/threonine- 1 protein kinase PAK 1 7 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 176

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein PNCA1, Sarcoma embryonic Z-

Palladin Palld x x 152.13Z-disc Parast & Otey, 2000 antigen NY-SAR-77 disc Actopaxin, Calponin- like integrin-linked kinase-binding Olski et al, 2001; membrane- Alpha-parvin Parva x x 42.33 protein, Matrix- Chen et al, 2005; associated remodeling- Sepulveda & Wu, 2006 associated protein 2, MXRA2

membrane- also Yamaji et al, 2001; Beta-parvin Parvb x 41.67 Affixin associated costamere Sepulveda & Wu, 2006

CLIM1, C-terminal LIM domain protein PDZ and LIM Pdlim1 x x 35.77 Z-disc 1, Elfin, hCLIM1, LIM Kotaka et al. 2000 domain protein 1 domain protein CLP- 36 Actinin-associated LIM protein, ALP, PDZ and LIM

Pdlim3 x x 34.30 Z-disc Alpha-actinin-2- Xia et al, 1997 domain protein 3 associated LIM protein ENH1, Enigma PDZ and LIM homolog, Enigma-like

Pdlim5 x x 63.30 Z-disc Nakagawa et al, 2000 domain protein 5 PDZ and LIM domains protein Aciculin, Phosphoglucomuta Molt et al, 2014; Pgm5 x x 62.22 Z-disc Phosphoglucomutase se-like protein 5 Belkin & Burridge, 1995 -related protein Phosphatidylinosit

ol 4,5- Pik3ca x 124.41 Z-disc - Waardenberg et al, 2011 bisphosphate 3- ..MobilrZdssaeapoenpopoyainhtso 177 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein kinase catalytic subunit alpha isoform intercalated

Plakophilin 2 Pkp2 x 88.10 - also periperhy Goossens et al, 2007 disc Hemidesmosomal

Plectin Plec x x 517.28 peripheral Hijikata et al, 1999 protein 1 Serine/threonine- Calmodulin- protein dependent phosphatase 2B Ppp3ca x 57.61 Z-disc calcineurin A subunit Frey et al. 2000 catalytic subunit alpha isoform, CAM- alpha isoform PRP catalytic subunit membrane-

Presenilin-1 Psen1 x x 52.64 AD3, FAD, PSNL1 Sakuma et al, 2006 associated membrane-

Presenilin-2 Psen2 49.98 Ad4h, Ps-2, Psnl2 Takeda et al, 2005 associated

Paxillin Pxn x x 64.51 peripheral - Gehmlich et al, 2007 Ryanodine membrane-

Ryr2 564.82 - Lanner et al, 2010 receptor 2 associated also Sodium channel intercalated

Scn1a 19.37 Nav1.1 membrane Haufe et al, 2005 protein disc associated 50 kDa dystrophin- membrane-

Alpha-sarcoglycan Sgca x 43.29 associated Wakayama et al, 1996 associated glycoprotein, Adhalin 43 kDa dystrophin- membrane- associated

Beta-sarcoglycan Sgcb x 34.87 Durbeej et al, 2000 associated glycoprotein, LGMD2E membrane- 35 kDa dystrophin-

Delta-sarcoglycan Sgcd x 32.13 Nigro et al, 1996 associated associated 7 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 178

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein glycoprotein, LGMD2F Epsilon- membrane-

Sgce x 53.53 - Ettinger et al, 1997 sarcoglycan associated 35 kDa dystrophin- Gamma- membrane- associated

Sgcg x 32.08 Hack et al, 2000 sarcoglycan associated glycoprotein, LGMD2C SH3 domain-

binding glutamic Sh3bgr 23.10 Z-disc SH3BGR protein Jang et al, 2015 acid-rich protein Band anion intercalated Solute carrier family 4

Slc4a1 103.14 Moura Lima et al, 2003 transport protein disc member 1 Sodium/calcium membrane-

Slc8a1 108.04 Ncx1 Mohler et al, 2005 exchanger 1 associated 59 kDa dystrophin- membrane-

Alpha-1-syntrophin Snta1 x x 53.66 associated protein A1 Peters et al, 1998 associated acidic component 1 also Isoform 2 of Sorbin Arg/Abl-interacting costamere Rönty et al, 2005; and SH3 domain- Z-disc protein 2, Sorbin and Sorbs2 x x 145.01 region and Wang et al, 1997; containing protein associated SH3 domain- membrane- Sanger et al, 2010 2 containing protein 2 associated membrane- Sarcoplasmic

Sorcin Sri x 21.63 22 kDa protein, CP22 Meyers et al, 1995 associated reticulum SYNC1, Syncoilin also Kemp et al, 2009; Syncoilin Sync x x 53.63 Z-disc intermediate filament sarcolemma Moorwood, 2008 1 intermediate

Synemin Synm x 173.21 Carlsson et al, 2000 filament ..MobilrZdssaeapoenpopoyainhtso 179 spot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein

Synaptopodin Synpo x x 96.25 Z-disc Linnemann et al, 2010

Myopodin,

Synaptopodin-2 Synpo2 x x 116.53Z-disc genethonin-2, Linnemann et al, 2010

fesselin Synaptopodin 2-

Synpo2l x x 103.27 Z-disc Tritopodin, CHAP Beqqali et al, 2010 like protein LGMD2G, T-cap,

Telethonin Tcap x x 19.07 Z-disc Gregorio et al, 1998 Titin cap protein

Talin-2 Tln2 x x 271.66 peripheral - Senetar et al, 2007 Tripartite motif- MURF-3, Muscle- containing protein Trim54 x 41.13 Z-disc specific RING finger Gregorio et al, 2005

54 protein, aa 1-700 in Z-

Titin Ttn x x 3716 Z-disc Connectin, LGMD2J Labeit et al, 1992 disc Dmdl protein, membrane- Utrophin Utrn x x 392.70 dystrophin-related sarcolemma Helliwell et al, 1992 associated protein Vasodilator-

stimulated Vasp x x 39.67 Z-disc - van der Ven et al, 2006 phosphoprotein Shear & Bloch, 1985; Minetti Vinculin Vcl x x 116.72peripheral Metavinculin et al, 1992 Vacuolar protein

sorting-associated Vps18 x 110.22 Z-disc hVPS18 van der Ven et al, 2006 protein 18 homolog Wiskott-Aldrich

syndrome protein Was 54.19 Z-disc Wasp Takano et al, 2010 homolog 8 ..MobilrZdssaeapoenpopoyainhtspot hot phosphorylation protein a are Z-discs Myoﬁbrillar 6.1. 180

identified murine identified as MW sarcomeric protein names gene in this additional name note references phospho [kDa] subunit names study protein

Xin actin-binding Cardiomyopathy-

repeat-containing Xirp1 x x 123.43 Z-disc associated protein 1, van der Ven et al, 2006 protein 1 CMYA1, XIN Cardiomyopathy- Xin actin-binding associated protein 3, repeat-containing Xirp2 428.26 Z-disc Huang et al, 2006 CMYA2, Beta-xin, protein 2 Xeplin, Myomaxin

Zyxin Zyx x x 60.55 peripheral - Crawford et al, 1992

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6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 195

WT AA DD WT AA DD + ++Calpain 1 + ++Calpain 1

WT AA DD + ++Calpain 1

Original data 5 G 196 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot

αFLNc d22-24 GAPDH

+ Gö 6976 + Gö 6976 + PMA + PMA + +++ Calpain 1 + +++ Calpain 1 + + + CaCl2 + + + CaCl2

93 kDa 41 kDa 68 kDa 32 kDa 53 kDa

+ Gö 6976 + Gö 6976 + PMA + PMA + +++ Calpain 1 + +++ Calpain 1 + + + CaCl2 + + + CaCl2 93 kDa 41 kDa 68 kDa 32 kDa

53 kDa

+ Gö 6976 + Gö 6976 + PMA + PMA + +++ Calpain 1 + +++ Calpain 1 + + + CaCl2 + + + CaCl2

93 kDa 41 kDa 68 kDa 32 kDa 53 kDa

+ Gö 6976 + Gö 6976 + PMA + PMA + +++ Calpain 1 + +++ Calpain 1 + + + CaCl2 + + + CaCl2 93 kDa 41 kDa 68 kDa

53 kDa 32 kDa

Original data 6 C 6.1. Myoﬁbrillar Z-discs are a protein phosphorylation hot spot 197

αFLNc d22-24 GAPDH

WT AA DD WT AA DD + ++Calpain 1 + ++Calpain 1 93 kDa 41 kDa 68 kDa

32 kDa 53 kDa

23 kDa

WT AA DD WT AA DD + ++Calpain 1 + ++Calpain 1 93 kDa 41 kDa

68 kDa 32 kDa 53 kDa

23 kDa

WT AA DD WT AA DD + ++Calpain 1 + ++Calpain 1 93 kDa 41 kDa

68 kDa

32 kDa 53 kDa

WT AA DD WT AA DD + ++Calpain 1 + ++Calpain 1 93 kDa 41 kDa

68 kDa

32 kDa 53 kDa

Original data 6 E 198 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

6.2 Quantitative Phosphoproteomics and Proximity Proteomics Reveals FILIP1 as a Phosphorylation-dependent Interactor of FLNc in Skeletal Muscle Cells

Contributions: • Establishment of quantitative signaling studies of SILAC C2 myotubes with subsequent Western Blot analysis.

• Optimization of quantitative phosphoproteomics workﬂow, subsequent phosphopeptide enrichment, MS-based analysis as well as data analysis.

• Supervision of quantitative BioID experiments and data analysis.

• Implementation of pull down assay with phosphosite mutants.

• Participation in experimental design, preparation of ﬁgure 1-4 and 6 and writing of the manuscript with input from co-authors. 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 199

Quantitative Phosphoproteomics and Proximity Proteomics Reveals Filamin A-

Interacting Protein 1 as a Phosphorylation-dependent Interactor of Filamin C in

Skeletal Muscle Cells

Lena Reimann1, Anja N. Schwäble1, Anna L. Fricke1, Heike Wiese1,4, Marlene

Elsässer1,5, Bettina Knapp1, Christa Reichenbach1, Christian D. Peikert1, Friedel

Drepper1, Peter F.M. van der Ven3, Gerald Radziwill1,2, Dieter O. Fürst3, Bettina

Warscheid1,2*

1Department of Biochemistry and Functional Proteomics, Institute of Biology II,

Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany

2BIOSS Centre for Biological Signalling Studies, University of Freiburg

3Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn,

53121 Bonn, Germany

4Present address: Institute of Pharmacology and Toxicology, University of Ulm,

89081 Ulm, Germany

5Present address: Institute of Crop Science and Resource Conservation (INRES)-

Chemical Signalling, University of Bonn, 53113 Bonn, Germany

*to whom correspondence should be addressed:

[email protected]

Key words: skeletal myotubes, phosphoproteomics, PI3K/Akt pathway, filamin C,

filamin A-interacting protein 1, protein kinase Cα

Running title (max. 60): Phosphorylation-dependent interaction of FILIP1 with FLNc

1 200 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Abbreviations page

ABD, actin-binding domain

AGC, automatic gain control

Akt1s1, proline-rich Akt1 substrate 1

BAD, Bcl2-associated agonist of cell death

BAG, Bcl2-associated athanogene

CC, cellular component

Co-IP, Co-immunoprecipitation

CT, carboxy-terminus

d, domain

DMSO, dimethyl sulfoxide

eIF4B, eukaryotic translation initiation factor 4B

ETD, electron transfer dissociation

EPS, electrical pulse stimulation

FA, formic acid

FDR, false discovery rate

FILIP1, filamin A-interacting protein 1

FLNc, filamin C

GO, gene ontology

GTF2F1, general transcription factor IIF

HCD, higher-energy collisional dissociation

HEK293, human embryonic kidney 293

HRP, horseradish peroxidase Ig, immunoglobulin IGF-1, insulin growth factor 1 IPTG, isopropyl -D-1-thiogalactopyranoside LARP1, La-related protein 1

2 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 201

LDB3, LIM domain binding 3 LIT, linear ion trap MAP3K3, mitogen-activated protein kinase kinase kinase 3 MSA, multistage activation mTORC1/2, mammalian target of rapamycin complex 1/2 NL, neutral loss NDRG1/2, N-myc downstream regulated gene 1/2 PEI, polyethylenimine PGM5, phosphoglucomutase-like protein 5 (aciculin) PI3K, phosphatidylinositide 3-kinase PKC, protein kinase C PMA, phorbol-12-myristate-13-acetate SCX, strong cation exchange SD, standard deviation SEM, standard error of the mean Tbc1d4, TBC1 domain family member 4

TiO2, titanium dioxide TSC2, tuberous sclerosis complex 2 WT, wildtype

3 202 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Summary

Skeletal muscle is known to adapt dynamically to changes in workload by regulatory

processes under the control of the PI3K/Akt pathway. To globally study the PI3K/Akt

signaling network in contracting skeletal myotubes, we followed a quantitative

phosphoproteomics approach using IGF-1 for activation and LY294002 for inhibition

of PI3K signaling. The here established substrate network comprises 261 proteins

with regulated phosphosites. For a subset of 20 proteins, we identified the novel

extended basophilic motif RxRxxpSxxS with a decrease in phosphorylation following

inhibition of PI3K by LY294002. Several substrates were found to be additionally

phosphorylated at the serine residue in the +3 position, but exact information about

the involved kinases is missing. Among these proteins, the phosphorylation level of

the multi-adaptor protein filamin C (FLNc) was regulated at both serine residues in

the RxRxxSxxS motif located in the unique 82 amino acid insert of its

immunoglobulin (Ig)-like domain 20. While Akt phosphorylates FLNc at the first

serine, we identified PKC as the kinase phosphorylating the second serine residue.

Both sites were found to be required for the regulation of FLNc binding to filamin A-

interacting protein 1 (FILIP1) identified in this work as new interaction partner by

quantitative proximity proteomics and biochemical assays. Here, the C-terminal

region of FILIP1 interacts with FLNc Ig-like domains 20-21 in a phosphorylation-

dependent manner. Our data provide a valuable basis for a better understanding of

how FLNc interactions with multiple proteins via its Ig-like domain 20 are controlled

by its integration into kinase signaling networks in skeletal muscle cells.

4 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 203

Introduction

The PI3K/Akt signaling pathway plays an important role in cell growth and

proliferation as well as cell survival (reviewed by Manning & Cantley, 2007). Skeletal

muscle fibers are known to adapt dynamically to changes in workload by the

PI3K/Akt pathway (Bodine et al, 2001), resulting in an increase in fiber size (Rommel

et al, 2001). However, the exact molecular mechanisms are poorly understood and

are controversially discussed. Following long time resistance training, which can lead

to muscle hypertrophy, Akt phosphorylation as well as phosphorylation levels of its

substrates GSK3b and mTOR are reported to be significantly increased in human

muscle biopsies (Léger et al, 2006). Furthermore, short time resistance training was

shown to have the same effect under high-carbohydrate dietary in human muscles

(Creer et al, 2005). In contrast, acute exercise training resulted in a decrease in

phosphorylation levels of Akt, Akt1s1 and TCB1D4 in muscle biopsies from untrained

humans (Hoffman et al, 2015). Even though these studies report phosphorylation of

several downstream targets of the PI3K/Akt pathway, there is a need to expand

current knowledge about the associated substrate network in skeletal muscle cells.

Within the PI3K/Akt pathway, multiply kinases, namely Akt, SGK1 and S6K share the

basophilic recognition motif RxRxxpS/pT, making the assignment of a substrate to a

specific kinase challenging (reviewed by Manning & Cantley, 2007).

Recently, we highlighted role of the sarcomeric Z-disc as signaling hot spot within

myofibrils (Reimann et al, 2016, in revision). The muscle-specific scaffold protein

filamin c (FLNc) and several of its interaction partners, such as Bag3, XIRP1 and the

three isoforms of the podin family were identified to be highly phosphorylated. The

unique 82 amino acid comprising insert in Ig-like domain 20 of FLNc was shown to

contain an Akt-mediated phosphorylation site (Murray et al, 2004b).

5 204 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Stimulation of the PI3K/Akt pathway to study muscle-specific substrates is achieved

by stimulation with insulin-like growth factor (IGF-1), whereas LY294002 is blocking

the activity of PI3K (Vlahos et al, 1994). Upon stimulation with IGF-1, PDK1

downstream of PI3K phosphorylates Akt-pT308 (Alessi et al, 1996a). Subsequently,

Akt is phosphorylating, amongst others, mammalian target of rapamycin complex 2

(mTORC2), which can in turn phosphorylate Akt-pS473, resulting in fully activated

Akt (Alessi et al, 1996a). Subsequently, Akt phosphorylates, for example, Bcl2-

associated agonist of cell death (BAD)-pS136 (del Peso et al, 1997), glycogen

synthase kinase 3 (GSK3)-pS21/-pS9 (Cross et al, 1995) and proline-rich Akt1

substrate 1 (Akt1s1)-pT246 (Kovacina et al, 2003). Akt further promotes cell growth

by activation of the mammalian target of rapamycin complex 1 (mTORC1) via an

inhibitory phosphorylation of its negative regulator tuberous sclerosis complex 2

(TSC2)-pT1462 (Manning et al, 2002). Following TSC2 inhibition, mTORC1

phosphorylates p70 S6K-pT389 (Dufner & Thomas, 1999), resulting in eukaryotic

translation initiation factor 4B (eIF4B)-pS422 phosphorylation and initiation of

translation (Gingras et al, 2001; van Gorp et al, 2009).

In this work, we followed a large-scale quantitative phosphoproteomics approach to

study the PI3K/Akt substrate network in contracting C2 myotubes. By focusing on

regulated phosphopeptides in response to IGF-1 or LY294002 treatment, we

identified a novel extended basophilic motif RxRxxpSxxS enriched among the down-

regulated phosphopeptides following PI3K inhibition by LY294002. In our further

analysis, we concentrated on the two FLNc phosphosites in the unique 82 amino

acid insert in Ig-like domain 20 comprising this motif. Through in vitro kinase assays

coupled to high resolution MS, we identified Akt and PKC to phosphorylate the first

and the second serine in this motif. By quantitative proximity proteomics, we

6 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 205

identified filamin A-interacting protein 1 (FILIP1) as novel interaction partner of FLNc

d18-21. Biochemical experiments with phospho-mutants of FLNc demonstrated that

both phosphorylation sites in the RxRxxpSxxS motif are required for the regulation of

FILIP1 binding.

7 206 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Experimental Procedures

Cell Culture

For metabolic labeling using the SILAC technology (Ong et al, 2002), C2 myoblasts

(Nudel et al, 1988) were cultured in high glucose SILAC-DMEM medium (PAA, GE

Healthcare Life Sciences, Freiburg, Germany) supplemented with dialyzed 15% FCS

(PAA), 1% non-essential amino acids, 1% penicillin/streptomycin,1% sodium

pyruvate, 1% proline (all Life Technologies), 84 mg/l arginine and 146 mg/l lysine

(Cambridge Isotope Laboratories Inc., Tewksbury, USA) for at least 9 cell doublings.

13 12 Light, medium and heavy labeling of cells was performed with C6 L-arginine/ C6 L-

12 13 15 13 15 lysine, C6 L-arginine/D4 L-lysine and C6 N4 L-arginine/ C6 N2 L-lysine,

respectively. Stable isotope-labeled myoblasts were seeded into six-well plates

(Corning Incorporated, New York, USA) and grown to a confluency of approximately

90%. Differentiation was induced by reduction of the dialyzed FCS content to 2%

(Ong et al, 2002) in the absence of sodium pyruvate. Differentiation medium was

changed every 48 h until day 4. Subsequently, cells were serum-starved for 16 h

overnight and sarcomere formation was improved by electrical pulse stimulation

(EPS, 0.05 Hz, 4 ms, 10 V) for 4 h using a C-Pace EP Culture Pacer (IonOptix,

Milton, USA). One hour prior to cell lysis, cells were either treated with 10 ng/ml

insulin growth factor-1, 10 µM LY294002 or dimethyl sulfoxide (DMSO). SILAC

experiments were generally performed in three independent biological replicates with

a label switch.

Human embryonic kidney 293 (HEK293) cells were cultured in DMEM supplemented

with 10% FCS and 1% sodium pyruvate. Transient transfections of HEK293 cells

and C2 cells were performed as described before (Reimann et al, 2016, in revision).

8 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 207

All cell lines were regularly tested for mycoplasma contamination and found to be

mycoplasma-negative.

Cell Lysis and Sample Preparation

Cell lysis of C2 myotubes was performed as described before (Reimann et al, 2016,

in revision) in the presence of 1 mM sodium orthovanadate, 10 mM

β-glycerophosphate, 9.5 mM sodium fluoride, 10 mM sodium pyrophosphate. Protein

concentrations were equalized using the Bradford assay (BioRad, Munich, Germany)

and control, IGF-1- and LY294002-treated samples were mixed in a 1:1:1 ratio equal

to a total protein amount of 9 mg. Reduction and alkylation of proteins was

performed as described (Reimann et al, 2016, in revision) with slight modifications.

Each reaction was carried out for 20 min using a final concentration of 5 mM tris(2-

carboxyethyl)phosphine (TCEP) and 50 mM 2-chloroacetamide before alkylation was

quenched with dithiothreitol (DTT) used at a final concentration of 5 mM. For tryptic

digestion, samples were diluted 1:4 (v/v) with 50 mM ammonium bicarbonate

solution and incubated with sequencing grade trypsin (Promega) in a 1:50 (w/w) ratio

for 3.5 h at 42°C and 200 rpm. Peptides were desalted using an Oasis HLB cartridge

(Waters Corporation, Milford, USA) according to the manufacturer’s protocol. Eluates

were aliquoted, lyophilized and stored at -80°C. In addition, cells in one well of a 6-

well plate were lysed with 150 µl modified RIPA buffer (50 mM Tris, 150 mM NaCl,

1% NP-40, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 10 mM

β-glycerophosphate, 9.5 mM sodium fluoride, 10 mM sodium pyrophosphate, pH 7.6)

for subsequent Western blot analysis of control, IGF-1- and LY294002-treated

samples.

9 208 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Strong Cation Exchange Chromatography

Tryptic digests were dissolved in 400 µl SCX buffer A [5 mM potassium dihydrogen

phosphate, 20% acetonitrile (ACN, v/v), pH 2.8]. The supernatant was loaded onto a

Polysulfoethyl-A column (Ø 4.6 mm, 20 cm, 5 µm, 200 Å, PolyLC, Columbia, USA)

equilibrated with SCX buffer A using a Dionex Ultimate 3000 UHPLC system.

Peptides were separated at a flow rate of 700 µl/min applying a linear gradient with

SCX buffer B [5 mM potassium dihydrogen phosphate, 20% ACN (v/v), 500 mM KCl,

pH 2.8] as introduced before (Reimann et al, 2016, in revision). Fractions were

collected every three minutes during the gradient and 10 µl of each SCX fraction

were dried in vacuo for LC-MS analysis. The remaining volume of each fraction was

used for TiO2-based enrichment of phosphopetides. A total of 90 SCX fractions were

collected (30 fractions per biological replicate) and processed for phosphoproteomics

analysis in a randomized procedure. Non-enriched and TiO2-enriched peptide

samples were dried in vacuo and stored at -80°C for LC-MS analysis.

Titanium Dioxide Enrichment

Phosphopeptides were enriched using TiO2 spherical beads as described before

(Reimann et al, 2016, in revision; Wiese et al, 2014) with slight modifications. Briefly,

30 µl of TiO2 material (TiO2, 5 µm, GL Science Inc.) resuspended in ACN 1:1 (v/v)

were used for SCX fractions. The TiO2 material was washed twice (80% ACN, 0.1%

TFA) and pre-equilibrated with 50 µl loading buffer [20% (v/v) acetic acid, 20 mg/ml

2,3-dihydroxybenzoic acid, 420 mM octasulfonic acid, 0.1% (v/v) heptafluorobutyric

acid]. SCX fractions were mixed 1:1 (v/v) with 2x loading buffer, added to the

prepared TiO2 material, vortexed and incubated for 20 min at 4°C with slight

agitation. After incubation, the material was washed twice as before and again

10 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 209

incubated with 50 µl elution buffer (50 mM ammonium dihydrogen phosphate, pH

10.5) for 15 min. Subsequently, 50 µl ACN were added and supernatants were

acidified with 8 µl TFA on ice, split in two equal parts, and dried in vacuo.

Liquid Chromatography and High Resolution Mass Spectrometry

Reversed-phase liquid chromatography-mass spectrometry was performed using the

UltiMateTM 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher Scientific,

Dreieich, Germany) coupled online to a Velos Orbitrap Elite (Thermo Fisher

Scientific, Bremen, Germany) instrument. The UHPLC system was equipped with

two C18 µ-precolumns (Ø 0.3 mm x 5 mm; PepMapTM, Thermo Fisher Scientific)

and an Acclaim® PepMapTM analytical column (ID: 75 µm x 500 mm, 2 µm, 100 Å,

Dionex LC Packings/Thermo Fisher Scientific). The MS instrument was externally

calibrated using standard compounds and equipped with a nanoelectrospray ion

source and a stainless steel emitter (Thermo Fischer Scientific). MS/MS analyses

were generally performed on multiply charged peptide ions applying a normalized

collision energy (NCE) of 35% with an activation q of 0.25 and an activation time of

30 ms unless otherwise stated. For global quantitative phosphoproteomics, peptide

mixtures from SCX fractions of three independent biological replicates were

analyzed by LC-MS/MS applying a 2 h LC gradient and collision-induced dissociation

(CID) for peptide fragmentation. SCX fractions enriched for phosphopeptides by TiO2

were analyzed in two technical replicates applying the same 2 h LC gradient and

multistage activation (MSA) with neutral loss (NL) masses of 32.7, 49 and 98 Da for

technical replicate #1 and higher-energy collisional dissociation (HCD) with a NCE of

28% for technical replicate #2.

11 210 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Samples from in vitro kinase assays enriched for phosphopeptides by TiO2 were

analyzed by LC-MS applying a 1 h LC gradient with MSA, HCD or electron transfer

dissociation (ETD) for peptide fragmentation. The activation time for ETD

fragmentation was set to 100 ms.

Samples from BioID SILAC approaches were analyzed by LC-MS/MS in a 3 h

gradient on a Velos Orbitrap Elite instrument for two independent biological

replicates. For each biological replicate two technical replicates were analyzed

utilizing CID and the third one using MSA with NL masses of 32.7, 49.0 and 98.0 Da.

Bioinformatics

For quantitative phosphoproteomics data, Andromeda integrated in MaxQuant

1.5.1.0 (Cox et al, 2011) was used to search peak lists against the UniProt

ProteomeSet mouse database (release 01.11.2014, 52,489 protein entries). The

precursor mass tolerance was set to 20 ppm for the first search and to 4.5 ppm for

the main search. For MSA/CID data, the fragment mass tolerance was set to 0.5 Da.

Trypsin was set as proteolytic enzyme allowing up to two missed cleavages.

Oxidation of methionine and phosphorylation of serine, threonine and tyrosine were

set as variable modifications and cysteine carbamidomethylation as fixed

modification. A false discovery rate (FDR) of 1% was applied on both peptide (on

modified peptides separately) and protein lists. Numbers of unique phosphopeptides

were counted based on the MaxQuant peptide ID in the Phospho(STY) sites table.

Phosphosites scored with a MaxQuant localization probability of ≥ 0.75 were

deemed “localized”, while sites with a localization probability of < 0.75 were counted

as putative sites given that the amino acid (aa) sequence in combination with the

12 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 211

number of phosphate groups was not identified with localized sites elsewhere in the

dataset.

For analysis of MS data from in vitro kinase assays, raw files were processed using

Andromeda embedded in MaxQuant 1.4.1.2 and searched against the sequences of

human FLNc d18-21 and their respective phosphosite mutants generated in this

work using the UniProt ProteomeSet mouse database (release 01.08.2014, 51,550

protein entries) as background for FDR calculation. Precursor and fragment mass

tolerances were set to 10 ppm and 0.5 Da, respectively. Search parameters were as

follows: proteolytic enzyme: trypsin, max. number of missed cleavages: 2, and

variable modifications: methionine oxidation and phosphorylation of serine, threonine

and tyrosine. MaxQuant msms.txt files, all raw files and FLNc isoform sequences

were imported into Skyline 2.6.0 (MacLean et al, 2010). MS1 intensities were

calculated as described by (Schilling et al, 2012) using the MS1 filtering tutorial

provided by the software developers. Skyline peptide settings were as follows: tryptic

peptides with 1 missed cleavage, a time window of 3 min, min. and max. peptide

length 8 and 30 aa, respectively, exclusion of cysteine-containing peptides,

phosphorylation of serine, threonine and tyrosine and oxidation of methionine as

variable modifications, and max. number of variable modifications and neutral losses

3 and 1, respectively. Orbitrap default parameters were used for transition settings.

Extracted ion chromatograms of the imported peptides were manually inspected for

correct peak picking and peak integration was manually adjusted, if necessary. Total

MS1 areas for all peptides with a mass error of ≤ 3 ppm were exported into a pivot

table and processed using Excel2010 and Origin 9.1. The mean and the standard

error of the mean (SEM) were first calculated for the three biological replicates and

afterwards for the technical replicates. Intensities of all phosphopeptides were

13 212 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

summed separately for human and mouse FLNc isoforms and phosphopeptides

were normalized by the respective calculated summed intensity.

Raw files from LC-MS/MS analyses of BioID assays were searched against the

Uniprot Proteome Set mouse database (release 01.03.2016, 58,790 protein entries)

supplemented with the fusion protein BirA*FlnC d18-21. Trypsin was set as

proteolytic enzyme allowing up to two missed cleavages. Carbamidomethylation of

cysteine residues was set as fixed modification. N-terminal acetylation, methionine

oxidation and phosphorylation at serine, threonine and tyrosine residues were

allowed as variable modifications. The precursor mass tolerance for first search was

set to 20 ppm and to 4.5 ppm for main search. The mass tolerance of fragment ions

was set to 0.5 Da. A FDR of 1% was applied on both peptide (on modified peptides

separately) and protein lists and a minimum of one unique peptide was enabled for

protein identification. The protein groups file was processed using Perseus 1.5.2.6

and OriginPro 9.0. Reverse and potential contaminant hits as well as proteins

positive for “only identified by site” were removed. Data were filtered for at least three

SILAC ratios in each biological replicate and means were calculated for log10-

transformed SILAC ratios over biological and technical replicates. The mean log10

ratios were subjected to hierarchical clustering by euclidean average k-means

cluster analysis. Gene Ontology (GO) analysis for cellular component (release

30.4.2015) was performed using the Cytoscape 3.3.0 plugin ClueGO 2.1.7 (Bindea

et al, 2009).

Mass Spectrometric Data Deposition

All raw data and original MaxQuant result files have been deposited to the

ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the

14 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 213

PRIDE partner repository (Vizcaíno et al, 2013) with the dataset identifiers PXDxxxx

(large-scale quantitative analysis), PXDxxxx (in-vitro kinase assay), PXDxxxx (BioID

experiments) and PXDxxxx (SILAC phosphopeptide analysis).

Proximity-dependent Biotin Identification

SILAC-encoded C2 cells transiently expressing BirA* or BirA* FLNc d18-21 were

differentiated for 4 days and subjected to EPS (4 ms, 10 V, 0.05 Hz) for 4 h prior to

cell lysis. To enhance biotinylation, 50 M biotin (Amresco, Solon, USA) were added

to the culture medium for 24 h prior to cell lysis. One set of cells expressing BirA*

FLNc d18-21 were cultured without adding biotin and served as an additional control.

Cell lysis was performed as described (Roux et al, 2012), protein concentrations

were equalized using the Bradford assay followed by mixing of samples in a 1:1:1

ratio to a final protein amount of 900 g per replicate. Coupling of biotinylated

proteins to streptavidin Dynabeads (Invitrogen, Karlsruhe, Germany) and

subsequent washing steps were performed as described (Roux et al, 2012). For

tryptic on bead digestion, samples were washed twice with 50 mM ammonium

bicarbonate solution and incubated with sequencing grade trypsin (Promega) in a

1:50 (w/w) ratio for 3.5 h at 42°C and 200 rpm. Supernatants were acidified to a final

concentration of 1% TFA on ice, split in four equal parts and dried in vacuo.

In Vitro Kinase Assay

Recombinantly expressed and purified mouse and human FLNc d18-21 WT and

phosphosite mutants were dialyzed overnight at 4°C in dialysis buffer (1 mM DTT,

100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2). For MS-coupled kinase assays,

H2O was added to 100 µg protein to a total volume of 200 µl and mixed with 10 x

15 214 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

kinase buffer (NEB, Frankfurt, Germany). The assay was started by adding 200 ng

Akt (Proteinkinase, Kassel, Germany) and/or 200 ng PKC (Sigma-Aldrich) in the

presence of 1x PKC lipid activator (Merck Millipore, Darmstadt, Germany). The

reaction was carried out for 20 min at 30°C and 200 rpm. One tenth of each of the

three independent replicates was used for gel-based analyses. The remaining

sample was diluted 1:4 (v/v) with 50 mM ammonium bicarbonate and subjected to in-

solution digestion using sequencing grade trypsin (1:50) (Promega) for 3.5 h at 42°C

and 200 rpm. Single protein digests were acidified with TFA [final concentration 1%

(v/v)], subjected to TiO2-based phosphopeptide enrichment, and then analyzed by

LC-MS/MS using MSA, HCD and ETD fragmentation.

Yeast two-hybrid assays

A human FLNc cDNA fragment encoding Ig-like domains 17-19 was cloned into a

modified pLex vector and a human heart muscle cDNA library (BD Biosciences

Clontech) was screened for interaction partners. Direct yeast two-hybrid experiments

were performed by co-transforming yeast cells with different cDNA fragments of

hFLNc cloned into pLex and a FILIP1 prey construct found in the screen.

Transformation into L40 cells, culturing on selective plates and testing for -

galactosidase activity was performed as described previously (van der Ven et al.,

2000b).

Pull-down Assays

Expression in E. coli and purification of recombinant polyhistidine- or GST-fusion

proteins were carried out according to standard protocols described before

(Linnemann et al, 2010). For pulldown experiments, GST-FILIP1 CT fusion protein

16 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 215

immobilized on glutathione-agarose beads was incubated with purified EEF-tagged

FLNc fragments (FLNc d1-3, FLNc d18-21 WT, A, AA, D, DD) under constant

agitation at 4°C for 1 h. Beads were washed with GST washing buffer (50 mM Tris-

HCl, pH 8.0, 150 mM NaCl) and samples were eluted by addition of Laemmli sample

buffer at 95°C. Subsequently, samples were analyzed by SDS-PAGE followed by

quantitative Western blot analysis using antibodies directed against the respective

tag of the proteins.

Alternatively, His6-tagged FILIP1-2 CT was used to pull-down putative binding

partners. Mouse skeletal muscle extracts were prepared by grinding the tissue in a

mortar under liquid nitrogen. Muscle powder was dissolved in lysis buffer (50 mM

Na2HPO4, 300 mM NaCl, 20 mM Imidazol, 0.5 % (w/v) Tween-20, pH 8.0). Similarly,

extracts from differentiated human skeletal muscle cells were prepared by directly

lysing a cell pellet from 10 culture dishes (10 cm) with differentiated human skeletal

muscle cells in lysis buffer. Samples were extensively sonified (UP50H, Hielscher,

Teltow, Germany), and insoluble material was removed by centrifugation. Cleared

lysates were incubated while rotating for 1 h at 4°C with empty Ni2+-NTA-agarose

beads or beads with bound FILIP1-2 CT. As a control, beads with bound FILIP1-2

CT were incubated with lysis buffer only. After extensive washing (50 mM Na2HPO4,

300 mM NaCl, 40 mM imidazole, pH 8.0), FILIP1-2 CT and associated proteins were

eluted from the beads by addition of elution buffer (50 mM Na2HPO4, 300 mM NaCl,

200 mM imidazole, pH 8.0). Eluted proteins were analyzed by SDS-PAGE. Bands

observed only in the samples obtained from FILIP1-2 CT-coated beads incubated

with cell or tissue extracts were cut from the gel and analyzed by mass spectrometry.

Co-Immunoprecipitation and In Vitro Binding Assays

17 216 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

HEK293 cells were co-transfected with FILIP1-GFP CT and Myc-hFLNc d1-4, Myc-

hFLNc d18-21 WT or the corresponding phosphorylation site mutants. Cells were

lysed on ice 48 h after transfection using co-immunoprecipitation (co-IP) lysis buffer

[100 mM NaCl, 20mM Tris, 1 % Triton X-100, 1 mM sodium orthovanadate, 10 mM

β-glycerophosphate, 9.5 mM sodium fluoride, 10 mM sodium pyrophosphate,

protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany), pH 7.6].

Lysates were centrifuged for 5 min at 10,000 x g. 800 µl of each supernatant were

incubated with 10 µl anti-c-Myc Magnetic Beads (Thermo Scientific) for 3 h at 10°C

on a rotating wheel. Samples were washed three times with 300 µl co-IP lysis buffer

using a magnetic rack, and bead-bound proteins were resuspended in 50 µl Laemmli

buffer for Western blot analyses.

Co-immunoprecipitation and dot-blot overlay experiments using bacterially

expressed proteins were performed as described (Linnemann et al, 2010; Obermann

et al, 1997).

Antibodies

Anti-Akt pan (#4691), anti-Akt T308 (#2965), anti-Akt S473 (#4060), anti-eEF2 pan

(#2332), anti-eEF T56 (#2331), anti-eIF4B pan (#3592), anti-eIF4B S422 (#3591)

(anti-GAPDH (#2118), anti-GSK-3β pan (#12456), anti-GSK-3α/β S21/9 (#8566),

anti-p70S6K pan (#2708), anti-p70S6K T289 (#9206), anti-Rictor pan (#9476) and

anti-Rictor T1135 (#3806) were purchased from Cell Signalling Technology (Leiden,

Netherlands). FLNc antibody RR90 was used as described before (van der Ven et al,

2000a), phospho-specific FLNc S2233 antibody was purchased from Kinasource

Limited (Dundee, UK). A novel rabbit antiserum against FILIP1 was raised against

the recombinantly expressed carboxy-terminus of FILIP1 (BioGenes, Berlin,

18 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 217

Germany). The serum was affinity purified and preabsorbed against the highly

homologous FILIP1L to avoid cross-reactivity. Horseradish peroxidase (HRP)-

conjugated anti-rabbit immunoglobulins and AlexaFluor596-conjugated rabbit anti-

mouse immunoglobulins were purchased from Sigma Aldrich and Life Technologies

(Darmstadt, Germany), respectively.

Design of cDNA Constructs and Site-Directed Mutagenesis

All constructs used in this work were obtained by cloning PCR products amplified

with primers including restriction sites. Amplicons were cloned in the appropriate

vectors and used for expression in HEK293 or L40 yeast cells, or in E.coli. Site-

directed mutagenesis was performed using the QuikChange® Lightning Site-

Directed Mutagenesis Kit (Stratagene/Agilent Technologies, Waldbronn, Germany)

as described in the manual. The AA (S2233/S2236 to alanine) and DD

(S2233/S2236 to aspartate) mutants of hFLNC d18-21 in pET23a/EEF were

obtained by PCR using the primer pairs

GGGATCCTTCGGCGCCATCACCCGGCAG with

CTGCCGGGTCATGGCGCCGAAGGATCCC, and

TGGGATCCTTCGGCGACATCACCCGGCAGC with

GCTGCCGGGTGATGTCGCCGAAGGATCCCA, respectively, using the non-mutant

variant as template.

The A (S2233 to alanine) and D (S2233 to aspartate) mutants of hFLNC d18-21 in

pET23a/EEF and pcDNA3.1/Myc-His were obtained by single strand site-directed

mutagenesis. Single strand PCR of the non-mutant plasmid was performed using

primer pairs CTGGGAGCCTTCGGCAG, GATGCTGCCGAAGGCTCCCAG and

CCTGGGAGACTTCGGCAG, CTGCCGAAGTCTCCCAGGCG, respectively.

19 218 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Subsequently, complementary PCR products were mixed and incubated for 5 min at

98°C. The PCR mixtures were purified using the mi-PCR Purification Kit (Metabion

International AG, Planegg/Steinkirchen, Germany). To remove template DNA,

samples were digested using DpnI and transformed into E. coli (DH5 alpha strain).

Miscellaneous

Information about the experimental design and statistical rationale for different

analyses performed in this work are provided within the respective subsections in the

results. Number of sample size, replicates, controls, and statistical tests were chosen

according to published data with comparable methodology and generally accepted

standards in the field. SDS-PAGE and Western blotting were performed using

standard protocols; signals were detected using HRP-coupled secondary antibodies

and an enhanced chemiluminescence system (Thermo Fisher Scientific) with a

ChemoCam (Intas, Göttingen, Germany) equipped with a full-frame 3.2 megapixel

Kodak KAF-3200ME camera. No image processing, other than cropping, scaling and

contrast adjustment, was applied. Quantification of Western blot signals was

performed with Quantity One 4.6.9 (Bio-Rad, Hercules, CA, USA). For statistical

analysis, two sample t-tests were performed using OriginPro 9.1 (OriginLab,

Northampton, MA, USA). All quantitative Western blot data are presented as mean ±

SEM or standard deviation (SD). To minimize the effects of subjective bias, Western

blot data were generated and analyzed by two different experimenters.

20 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 219

Results

Quantitative analysis of PI3K/Akt signaling in contracting C2 myotubes.

To study the PI3K/Akt-associated signaling network in contracting skeletal myotubes,

we designed a triple SILAC approach allowing for the quantification of changes in the

phosphoproteome following treatment with IGF-1 to stimulate the PI3K/Akt pathway

or the inhibitor LY294002 (LY) to block PI3K and downstream signaling (Figure 1A).

The experiment was performed in three independent biological replicates, including a

SILAC label switch. To verify the treatment design, samples were analyzed by

quantitative immunoblotting using pan- and phospho-specific antibodies directed

against well-established substrates of the PI3K/Akt pathway (Figure 1B and 1C). As

shown by the levels of pS308 and pS473, IGF-1-mediated stimulation strongly

induced Akt activity, which was effectively blocked by LY. Interestingly, we observed

some Akt activity already under basal condition, i.e. in serum-starved C2 myotubes.

This finding is consistent with data obtained from human muscle biopsies showing

active Akt in the non-exercise state (Hoffman et al, 2015). The activity profile of Akt

was confirmed by its direct target GSK3-pS9 used as readout. Consistently, p70

S6K-pT389 and eIF4B-pS406 as readouts for TORC1 and Rictor-pT1135 and Akt-

pS473 for mTORC2 were induced by IGF-1, while LY abolished their activity. As

expected, reversed levels were determined for the inhibitory phosphorylation of eEF2

at T56 mediated by eukaryotic elongation factor 2 kinase (eEF2k).

SILAC-based, global phosphoproteome analysis of control, IGF-1- and LY-treated

C2 myotubes was performed with SCX chromatography (30 fractions per replicate,

n=3) and phospopeptide enrichment using TiO2 followed by high resolution LC-MS

(Figure 1A). To improve phosphosite localization, all fractions were measured in

duplicates with MSA and HCD fragmentation, respectively. Furthermore, a small

21 220 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

aliquot of each SCX fraction was analyzed by CID to determine changes in protein

abundance (n=3). Data acquired in 270 LC-MS runs were jointly processed with

MaxQuant resulting in 7,202 protein identifications including 3,490 (48%)

phosphoproteins (Figure S1A, Table S1). The total number of phosphosites was

16,633, of which 11,493 (69%) were localized (probability ≥ 0.75) and 13,255 (80%)

quantified (Figure S1A, Table S1 and S2). The distribution of pS:pT:pY was

79.9:18.3:1.8 with approximately 40% singly, 50% doubly, and 10% triply

phosphorylated peptides predominantly present at charge state +2 (36.16%) and +3

(53.46%) (Figure S1B-D). Data reproducibility was verified by the quantification of

70% (9,197) of all phosphosites in ≥ 2 biological replicates (Figure S1E) and

Pearson correlation coefficients between replicates of ≥ 0.66 (Figure S1F-H). As

expected, the majority of phosphopeptides did not change in response to IGF-1 or

LY treatment (Figure S1I).

Filamin C is part of the PI3K/Akt-dependent signaling network in myotubes.

To identify significantly regulated phosphopeptides, Volcano plots were generated by

plotting the mean log10 SILAC ratios of phosphopeptides of control versus IGF-1 or

LY-treated C2 myotubes against the respective negative log10 p-values (Figure 2A-

B, Table S2). In response to IGF-1, 149 and 97 phosphopeptides were significantly

up- or down-regulated (minimum fold-change of  1.5, p-value < 0.05, n=3), while

inhibiting PI3K resulted in 243 down- and 80 up-regulated phosphopeptides. To

assess kinase-substrate relationships, regulated phosphopeptides were analyzed for

phosphorylation-specific sequence motifs using motif-X (Schwartz & Gygi, 2005). In

the IGF-1 dataset, the basophilic motif RxRxxpS (58-fold) and the proline-directed

motif pSP (5-fold) were found to be overrepresented (Figure 2C), while the motifs

22 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 221

pSxxxS (4-fold) and RxRxxpSxxS (148-fold) were enriched following inactivation of

PI3K by LY (Figure 2D). Interestingly, the extended basophilic motif RxRxxpSxxS

was identified in 20 unique, LY-downregulated phosphopeptides and in fact more

prominent than the classical RxRxxpS motif with an enrichment of 40-fold in the

same dataset. Mapping these results to the canonical PI3K/Akt pathway (Figure 2E)

revealed the RxRxxpSxxS phospho-motif in the N-myc downstream regulated gene

1 and 2 (NDRG1, NRDG2) and in the mitogen-activated protein kinase kinase kinase

3 (MAP3K3), which are known targets of the serine/threonine-protein kinase SGK1.

In addition, phosphopeptides with the enriched RxRxxpSxxS motif were found for La-

related protein 1 (LARP1), the general transcription factor IIF (GTF2F1), eIF4B, the

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKGB2), and the muscle-

specific scaffold protein FLNc. Except for TBC1 domain family member 4 (Tbc1d4),

we also identified the serine residue at position +3 to be phosphorylated in these

proteins. For EIF4B, NDRG1, and FLNc, peptides phosphorylated at one or both

serine residues in the RxRxxpSxxS motif were found to be regulated. Interestingly,

FLNc comprised not only these two strongly regulated phosphosites (pS2234,

pS2237) but also another regulated phosposite at T2239 which are all located in the

unique 82 amino acid insert of its Ig-like domain (d) 20, which is sufficient for its

localization at the Z-disc in sarcomeres.

Filamin C is a substrate of Akt and PKC in its unique insert in Ig-like domain

20.

Based on our quantitative phosphoproteomics data, we examined the regulation of

the FLNc in vivo phosphosites S2234, S2237, T2239 located in the isoform-specific

insert of its Ig-like domain 20 in more detail (Figure 3A). While the level of FLNc-

23 222 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

pS2237 was reduced upon stimulation with IGF-1, levels of FLNc-pS2234 as well as

FLNc-pS2234/pS2237 and -pS2234/pT2239 were decreased upon inhibition of PI3K

by LY. Of these sites, FLNc-S2234 was approximately two orders of magnitude more

abundant (Figure S2A). Furthermore, it was already reported as Akt substrate site in

vivo (Murray et al, 2004b), but its biological relevance remained unknown. Our

finding that this site was regulated in a peptide with two other phosphosites in vivo

prompted us to further analyze kinase-mediated phosphorylation of FLNc at S2234,

S2237 and T2239. To this end, we used a phospho-specific antibody recognizing

mouse (m) FLNc-pS2234 and human (h) FLNc-pS2233 (Figure 3B and S2B).

Quantitative Western blot analysis confirmed our MS data and showed a significant

down-regulation of mFLNc-S2234 phosphorylation upon LY treatment (Figure 3B).

Blast analysis of the unique 82 amino acid insert showed an almost 100%

conservation of this region among mammalians. In silico kinase prediction further

revealed hFLNc-S2236/T2238 (mFLNc-S2237/T2239) as potential substrate sites of

PKC/PKC (Figure 3C). In addition, hFLNc was shown to be phosphorylated at

these sites in vivo based on phosphoproteomics studies in HeLa and K562 cells

(Zhou et al, 2013). Based on our prediction data and the presence of a PKC

consensus site motif (Nishikawa et al, 1997), we performed in vitro kinase assays

using recombinantly expressed hFLNc d18-21 in its wildtype form (Figure S3A)

incubated with purified PKCand/orAkt in the presence of ATP. PhosTag PAGE

analysis revealed distinct phosphorylation-dependent electrophoretic mobility shifts

of hFLNc d18-21 (Figure 3D). While hFLNc d18-21 exhibited a moderate band shift

upon incubation with PKC, the reaction with Akt led to a shift of approximately 50%

of the protein. Consistently, two shifted bands were observed following incubation of

hFLNc d18-21 with both kinases. To obtain residue-resolved information, we

24 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 223

additionally performed in vitro kinase assays coupled to LC-MS (n=3) with

complementary fragmentation methods and MS1-based quantification using Skyline.

This analysis confirmed hFLNC-S2233 as prominent substrate site of Akt, while

PKC preferentially phosphorylated S2236 (Figure 3E, Table S3). Accordingly,

incubation with both kinases led to an increase of doubly phosphorylated (pS2233

and pS2236) of hFLNc d18-21 (Figure 3E).

Characterization of the in vivo protein nano-environment of FLNc

Since FLNc is known to interact with a number of proteins via its C-terminal region

comprising Ig-like domain 20 (van der Ven et al, 2006), we reasoned that dynamic

FLNc interactions may dependent on its phosphorylation status controlled by Akt

and/or PKC. To delineate the in vivo protein nano-environment of FLNc around Ig-

like domain 20, we performed quantitative proximity proteomics using SILAC

following the basic concept of proximity-dependent biotin identification (BioID, Roux

et al, 2012). To this end, we performed triple SILAC experiments (n=3) using

contracting C2 myotubes transiently expressing BirA*FLNc d18-21 or BirA* in the

presence or absence of biotin as indicated (Figure 4A). In vivo biotinylated proteins

were enriched, digested with trypsin, and peptides analyzed by LC-MS. The mean

log10 transformed SILAC ratios of BirA*FLNc d18-21 + biotin versus the two controls

(BirA* + biotin and BirA*FLNc d18-21 - biotin) of 463 quantified proteins were

calculated (Table S4) and subjected to hierarchical cluster analysis to determine

groups of strongly enriched (cluster 1, orange, 63 members), moderately enriched

(cluster 2, blue, 95 members) and non-enriched (cluster 3, grey, 305 members)

proteins (Figure 4B). In addition to endogenous mFLNc, several Z-disc and Z-disc-

associated proteins such as LIM domain binding 3 (LDB3), dystonin, titin and the

25 224 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

known FLNc binding partners BCL2-associated athanogene 3 (BAG3) and Xin actin-

binding repeat containing protein 1 (XIRP1) were identified among the highly

enriched proteins (Figure 4C, Table S4). Further myofibrillar or myofibril-associated

proteins in the group of enriched proteins were enabled homolog (ENAH, also

referred to as MENA or ENA), nebulin, obscurin, obscurin 1-like protein, desmin,

plectin and the FLNc interaction partner aciculin (Phosphoglucomutase-like protein

5, Pgm5, Molt et al, 2014). Gene Ontology (GO) enrichment analysis of cluster 1 and

2 using the Cytoscape plugin ClueGo identified an over-representation of sarcomeric

I-band and Z-disc ontologies in cluster 1 and numerous proteins associated with the

sarcomeric A-band and M-band in cluster 2 (Figure 4D). Among the highly enriched

proteins also FILIP1, a known interaction partner of filamin A (FLNa) (Nagano et al,

2002), was identified.

FILIP1 is a novel FLNc interaction partner

Together with a previous finding that a C-terminal fragment (CT) of a novel isoform

of FILIP1 interacts with FLNc d17-19 in a yeast two hybrid screen using a human

heart cDNA library, this prompted us to further investigate the interaction between

FILIP1 and FLNc. The newly identified FILIP1 isoform ("isoform 3", referred to as

FILIP1-3 in this work) differs from FILIP1 isoform 2 listed in Uniprot (Q7Z7B0-2,

FILIP1-2) in its C-terminal region, i.e. the C-terminal amino acid sequence stretch

SGQDGSSQRPTPTRIPMSKESIIIHQLRMNSR is replaced by KASSFTSYE. Due to

alternative splicing, exon 6 is completely missing from this novel isoform (Figure

5A). Further direct yeast two hybrid analyses not only confirmed binding of FLNc

d17-19 to FILIP1-3, but also showed a strong interaction with FLNc d18-21 and

FLNc d20-21. By contrast, only a weak interaction was observed with FLNc d16-19,

26 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 225

while FLNc d18-19 showed no interaction (Figure 5B). This prompted us to

biochemically analyze the interaction of FILIP1-3 CT with FLNc and FLNa. Dot-blot

assays confirmed strong binding of FILIP1-3 CT to FLNc d20-21 and d18-21. Other

constructs did not bind in this assay (Figure 5C, left panel). Next, we tested the

homologous regions of FLNa for binding to FILIP1-3 CT, and we found interaction

with FLNa d18-21 and d20-21, but not with d18-19 (Figure 5C, right panel). These

data were confirmed by Western blot overlay and co-immunoprecipitation

experiments (not shown). Furthermore, identical results were obtained with FILIP1-2

CT (not shown), indicating that the interaction is not isoform-specific.

To analyze expression of FILIP1 in differentiating cultured myotubes and to compare

its expression with that of FLNa and FLNc, we used a novel affinity-purified

antiserum raised against the carboxy-terminus of FILIP1-3 CT. When compared to

FLNc, FILIP1 expression was induced somewhat later and started two days after

induction of differentiation. By contrast, FLNa expression was strongly reduced with

increasing differentiation (Figure 5D).

The specificity of the antiserum was tested by blocking its reactivity by addition of the

T7-tagged antigen to the incubation mixture (Figure 5E). Whereas without antigen,

reactivity was observed in mouse skeletal muscle and cultured human skeletal

muscle cell extracts, addition of antigen completely blocked the signal. Instead a

signal appeared in the high molecular mass region above 250 kDa. Incubation with

anti-T7-tag antibody confirmed the presence of the recombinant antigen at this

position (Figure 5E). We interpreted these data as binding of the antigen/antibody

complex to a ligand of FILIP1. Incubation with anti-FLNc antiserum confirmed the

presence of FLNc at this position (Figure 5E).

27 226 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

To further confirm binding of FILIP1 CT to FLNc biochemically, we applied a pull-

down assay. His6-tagged FILIP1-2 CT was bound to a column, extract from mouse

skeletal muscle or cultured human myotubes was added and bound proteins were

analyzed by PAGE followed by mass spectrometry (Figure 5F). In both cases, FLNc

was identified as specifically bound protein with up to 70 unique peptides and 34%

sequence coverage.

FLNc S2233 and S2236 phosphorylation reduces FILIP1 binding

To test whether the regulated phosphorylation sites S2233 and S2236 of hFLNc

have an impact on its interaction with FILIP1, pull down experiments were performed

with recombinant His6- and EEF-tagged hFLNc d18-21 WT and phosphosite mutants

and FLNc d1-3 as control (Figure S3B). In hFLNc d18-21, we mutated either S2233

or S2233 and S2236 to aspartate (D) or alanine (A) to mimic its constitutively

phosphorylated or non-phosphorylated form. For pull-down assays, we incubated

bead-bound GST-FILIP1 CT (Figure S3C) with the hFLNc d18-21 variants and

analyzed bound proteins by Western blotting (Figure 6A). Quantitative analysis

(n=4) revealed a significantly lower affinity of the double phosphomimetic mutant

(DD) in comparison to the WT and the other phosphosite mutant forms of hFLNc

d18-21 (Figure 6B). To verify these results in human cells, we co-expressed FILIP1

CT-GFP (Figure S3C) with either Myc-tagged hFLNc d22-24, hFLNc d18-21 or the

respective phosphosite mutants in HEK293 cells and performed an anti-Myc co-

immunoprecipitation (Figure 6C). Quantitative Western blot analysis revealed an

increase in the binding of FILIP1 CT-GFP to the S2233A mutant and a significant

increase to the double S2233/2236A mutant in comparison to the WT FLNc d18-21.

In contrast, no difference was detectable with the S2233D and S2233/2236D mutant.

28 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 227

These findings suggest the presence of in vivo phosphorylated hFLNc d18-21 WT in

HEK293 cells (not shown).

29 228 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Discussion

In this work, we characterized the phosphoproteome of contracting C2 myotubes in

response to IGF-1 stimulation or treatment with LY by a global quantitative

phosphoproteomics supported by quantitative immunoblotting in order to identify

novel muscle specific PI3K/Akt substrates. Our quantitative Western blot data

demonstrated the effective activation or inhibition of Akt by IGF-1 or LY294002

treatment, respectively. Accordingly, a number of well-known substrates within the

PI3K/Akt pathway were found to be significantly regulated, e.g. GSK3β at S9 (Cross

et al, 1995), p70 S6K at T389 (Sancak et al, 2007) and mammalian target of

rapamycin (mTOR) subunit Rictor at S1135 (Julien et al, 2010). Moreover, we

showed that EPS-treated and starved contracting C2 myotubes exhibit basal Akt

activity (Figure 1B). Since Akt was found to be inactive in undifferentiated C2C12

myoblasts (Murray et al, 2004b; Wan et al, 2007) as well as differentiated and

starved myotubes without EPS (Rommel et al, 2001; Rommel et al, 1999; Tong et al,

2009), mild EPS as performed in this study may likely lead to slight activation of Akt.

In contrast, our data on basal Akt phosphorylation levels are highly similar to the

basic phosphorylation activity in a human in vivo study (Hoffman et al, 2015),

highlighting the importance of contractility for the analysis of muscle signaling

processes in vitro.

Our global phosphoproteomics approach enabled the identification of 16,633 unique

phosphosites of which 9,179 were quantified in at least two out of three biological

replicates (Figure S1B). In comparison, in two other studies, 2,530 phosphosites

were quantified in C2C12 myotubes (Kettenbach et al, 2015) and 8,518

phosphosites were identified in mouse heart muscle tissue (Lundby et al, 2013). In

the largest muscle-specific phosphoproteome study reported so far, 8,511

30 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 229

phosphosites in human muscle biopsies and 7,421 phosphosites in two out of four

replicates of L6 rat myotubes were quantified (Hoffman et al, 2015).

Most importantly, our study identified significant phosphorylation-dependent changes

in 149 and 97 peptides upon inhibition (LY) and activation (IGF-1) of PI3K/Akt

signaling, respectively (Figure 2A-B). While well-known substrates of the canonical

PI3K/Akt pathway were found to be significantly regulated in our phosphoproteomics

approach, no effect on substrates of the MAPK/ERK pathway was observed (Figure

2E). Among the down-regulated sites following LY treatment of C2 myotubes, we

identified the phosphorylation motif RxRxxpSxxS to be 148-fold enriched in

comparison to the background (Figure 2D, lower panel). While the “classical”

basophilic motif RxRxxpS strictly required for Akt-mediated phosphorylation (Alessi

et al, 1996b) has been extensively studied (Dobson et al, 2011; Moritz et al, 2010;

Sakamaki et al, 2011), the RxRxxpSxxS motif is reported here for the first time. Even

though an increased preference of Akt for targets with a serine/threonine in +1 and

+2 positions in comparison to mTOR/PI3K substrates was identified in adipocytes

(Humphrey et al, 2013), the evidence for a serine in +3 position did not show up in

their analysis. By mapping the appropriate motifs to the canonical PI3K/Akt signaling

network, we were not able to map one distinct kinase to this motif. However, a closer

look at the 20 motifs revealed that all except the one in TBC1 domain family member

4 (Tbc1d4) were phosphorylated at the +3 serine. In EIF4B, NDRG1 and FLNc the

doubly phosphorylated peptides were also significantly regulated, whereas all other

identified phosphopeptides were only regulated in the singly phosphorylated form.

For the sites in the proteins NDRG2, GTF2F1, MAP3K3, LARP1, EDC3 and ITSN1,

the +3 phosphosite did not fulfill our localization criteria of ≥ 0.75. Most probably this

low localization probability is caused by the presence of additional serine residues in

31 230 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

the +2 to +4 position in the respective phosphopeptides, hampering unambiguous

phosphosite localization. For seven out of the 20 motifs, the kinases mediating the

first phosphosite are known, whereas no information is available for the others 13

serines. While FLNc-pS2233, EDC3-pS161 and PFKFB2 were shown to be

phosphorylated by Akt (Larance et al, 2010; Murray et al, 2004b; Novellasdemunt et

al, 2013), EIF4B-pS406 phosphorylation was suggested to be mTORC-dependent

(van Gorp et al, 2009). Moreover, GSK3 and SGK1 phosphorylate S330 in NDGR1,

while NDGR2 phosphorylation at S350 was proposed to be RSK1-dependent

(Murray et al, 2004a) and SGK1 phosphorylates MAP3K3 at S166 (Chun et al,

2003).

Which kinases might be involved in the phosphorylation of the serine residues in the

+3 position and what may be the role of two phosphorylation sites in close proximity?

Kinase prediction and the matching PKC motif with a preference for arginine

residues in the -7 to -3 position and a glycine residue in the -1 position (Nishikawa et

al, 1997) identified 6 out of the 20 sites to be potential classical PKC substrates.

Mouse FLNc S2234 (hFLNc-S2233) is known to be phosphorylated by Akt (Murray

et al, 2004b), the physiological relevance, however, remained uncharacterized so

far.

To identify the kinases involved in the phosphorylation of hFLNC at S2233 and

S2236, we performed in vitro kinase assays using recombinantly expressed hFLNc

d18-21 with Akt and/or PKC. Mobility shift analysis and quantitative MS data

confirmed hFLNc S2233 and S2236, located in the unique 82 amino acid insert in Ig-

like domain 20 of FLNc, as specific Akt and PKC sites, respectively (Figure 3C-E).

This region of FLNc, which is lacking in FLNA and FLNB, was previously shown to

fulfill an important role in the interaction of FLNc with several other sarcomeric

32 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 231

proteins such as aciculin (Molt et al, 2014), Xin/XIRP1 (van der Ven et al, 2006),

myopodin/SYNPO2, (Linnemann et al, 2010) and myotilin (van der Ven et al, 2000b).

To identify novel putative FLNc interaction partners of this region, we used a SILAC-

based quantitative proximity proteomics approach (Figure 4A). By this approach, we

identified 463 proteins, of which 63 were highly enriched (Figure 4B-D, orange

cluster). In this protein cluster, we found FLNc itself and the known FLNc interaction

partners BAG3 (Ulbricht et al, 2015), LDB3/ZASP (Faulkner et al, 1999), Xin/XIRP1

(van der Ven et al, 2006), XIRP2 (Kley et al, 2013), and titin (Labeit et al, 2006)

which confirms the effectiveness of our approach. GO enrichment analysis revealed

the terms “Z-disc”, “sarcomere”, I-band”, “contractile fiber part” and “myofibril” to be

over-represented in the here established protein nano-environment of FLNc. This

finding is in accordance with the observation that ~ 97% of the FLNc pool in skeletal

muscle is located at the sarcomeric Z-disc (Thompson et al, 2000). Moreover, we

identified a second network including the terms “stress fiber”, “focal adhesion”,

”actomyosin”, “actin filament bundle” and “cell-substrate adherens junction” in the 95

proteins showing moderate enrichment in this proximity-based analysis (Figure 4 B-

D, blue cluster). One of the proteins found in this cluster is aciculin (also called

PGM5), which was shown to directly interact with FLNc in muscle repair processes

(Leber et al, 2016; Molt et al, 2014).

Surprisingly we also identified in the cluster of highly enriched proteins FILIP1, a

known interaction partner of FLNa that was predicted to interact also with FLNc but

was never examined (Nagano et al, 2002). FILIP1 was originally identified as a

protein with increased expression in postmitotic neurons in the ventricular zone that

start to migrate and was proposed to regulate the function of FLNa via calpain-

mediated degradation, thus resulting in reduced cell migration (Nagano et al, 2004;

33 232 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Nagano et al, 2002). Phosphorylation of FLNa pS2152 was demonstrated to have a

protective function, leading to weaker FILIP1 binding and hence to decreased FLNa

cleavage by calpain (Vadlamudi et al, 2002, reviewed by Zhou et al, 2010). The

kinases PKA (Chen & Stracher, 1989), RSK (Ohta & Hartwig, 1996; Woo et al,

2004), PAK1 (Vadlamudi et al, 2002) and Akt (Li et al, 2015) have been proposed to

phosphorylate FLNa pS2152, but the precise kinase regulating this mechanism has

remained elusive. It is tempting to speculate that the upregulation of FILIP1 in

differentiating myotubes, coinciding with the downregulation of FLNa, relies on a

similar mechanism.

In the original publication, the FILIP1-binding site of FLNa was localized to a region

comprising Ig-like domains 16 and 17 plus an N-terminally truncated Ig-like domain

15 and an N-terminal fragment of domain 18, including the calpain cleavage site in

the hinge1 region. Accordingly, we initially found FILIP1 in a yeast two-hybrid screen

as binding partner of FLNc d17-19. We could, however, not confirm this interaction in

vitro. Instead, strong interaction with FLNc d20-21 and also FLNa d20-21 was

observed, indicating that similar to migfilin and -integrins (Ithychanda & Qin, 2011;

Lad et al, 2008), FILIP1 can bind filamins at multiple sites. Migfilin, e.g., binds FLNa

at at least four positions with d21 as the most strongly interacting domain (Lad et al,

2008). A similar mechanism might explain the differences in our FILIP1/filamin

interaction assays in yeast cells and in vitro experiments. Most interestingly in the

context of this study, our quantitative pull down and co-IP analyses demonstrated the

interaction of FILIP1 and FLNc to be regulated by phosphorylation of pS2233 and

pS2236 (Figure 6 A-D), both localized in the unique insertion in FLNc d20. It should

be noted that bacterially expressed FLNc d18-21 WT is unphosphorylated, whereas

it is phosphorylated (and thus similar to the S2233D/S2236D variant) in HEK293

34 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 233

cells, explaining the variability of these proteins in binding capacity as shown in Fig.

6. These findings on a phosphorylation-regulated interaction of FLNc with FILIP1

therefore demonstrate in an exemplary fashion the enormous potential to exploit our

phosphoproteome data set for future mechanistic studies.

In summary, our quantitative analysis of the phosphoproteome of differentiated,

contracting myotubes identified a total of 246 peptides with regulated

phosphorylation upon IGF-1 stimulation and LY294002 treatment. Our data confirm

hFLNc phosphorylation at S2233 to be Akt-dependent in vitro and in vivo. Moreover,

we identified the phosphorylation motif RxRxxpSxxpS and showed that in FLNc the

phosphorylation at the second site is mediated by PKC. Our finding that these two

sites have an effect on the binding to the newly identified FLNc interaction partner

FILIP1 is a paradigmatic example of a phosphorylation-dependent protein-protein

interaction. The vast amount of regulated phosphorylation sites uncovered in this

work are therefore an invaluable basis for identification of further events regulated by

protein phosphorylation particularly in contracting striated muscle cells.

35 234 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

Acknowledgements

We thank A. Schuster, M. Saynisch, D. Serwas, T. Leuning and P. Ruttig for help

with biochemical experiments on FILIP1 and the University of Cologne for help with

MS analyses from FILIP1 CT pull-downs. This research was supported by the

Deutsche Forschungsgemeinschaft (FOR 1352 to B.W. and D.O.F., and FOR 1228

to D.O.F.) and the Excellence Initiative of the German Federal & State Governments

(EXC 294 BIOSS to BW).

Author Contributions

L.R. performed phosphoproteomics, mass spectrometric and biochemical analyses

with the support of A.S., A.F. and B.K. All authors analyzed data, designed and

interpreted experiments. B.W. and D.F. supervised the study. B.W. conceived the

project and B.W. and L.R. wrote the manuscript with the input of other authors.

Conflict of Interest

The authors declare that they have no conflict of interest.

36 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 235

Figure Legends

Figure 1. Experimental setup to study PI3K/Akt signaling in contracting C2

myotubes.

A, Following triple SILAC labeling, contracting C2 myotubes were treated for 1 h with

IGF-1 or LY294002 to stimulate or inhibit PI3K/Akt signaling. SILAC experiments

were performed in three biological replicates including a label switch. Lysates were

equally mixed, proteins digested with trypsin, and peptides fractionated by SCX

chromatography. For each biological replicate, 30 SCX fractions were collected with

the main part of each fraction being enriched for phosphopeptides using TiO2 beads.

Non-enriched SCX fractions were analyzed by LC-MS using CID and

phosphopeptide-enriched SCX fractions by MSA and HCD in two technical

replicates.

B, Western blot analysis of PI3K/Akt pathway substrates. Total protein amounts

were detected in comparison to phosphorylation-specific signals.

C, Quantification of Western blot data from (B). Calculated intensities were

normalized to the control and two-tailed Student’s t-test was performed (*p ≤ 0.05,

**p ≤ 0.01; n=3)

Figure 2. The PI3K/Akt-dependent signaling network in skeletal myotubes.

A, B, Volcano plots of log2 transformed mean SILAC ratios (control/treatment) of all

localized phosphopeptides plotted against the -log10 p-value. Significantly regulated

phosphopeptides (p < 0.05; n=3; two-tailed Student’s t-test; min 1.5-fold ratio

change) are shown as open blue circles. Peptides of interest are labeled according

37 236 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

to their phosphorylation sequence motif, regions of interest highlighted in light

orange (≥ 1.5-fold) and orange (> 3.0-fold), respectively.

C, D, Motif-X analysis of significantly regulated phosphopeptides reveals an

enrichment of the basophilic motif RxRxxpS (58-fold) and the proline-directed motif

pSP (5-fold) for the IGF-1 treatment (C) and of the pSxxxS motif (4-fold) and the

basophilic motif RxRxxpSxxS (148-fold) for LY294002 treatment. x, any amino acid.

E, Excerpt from the canonical IGF-1 activated signaling pathway, comprising the

downstream signaling branches of the PI3K/Akt and MAPK signaling cascade.

Proteins are represented by their Uniprot mouse gene names. Interactions were

curated from the literature and the KEGG database. Significantly regulated

phosphorylation sites (p < 0.05; n=3; two-tailed Student’s t-test; min. 1.5-fold ratio

change) identified in our quantitative phosphoproteomics study are color-coded

according to their regulation factor as shown in the legend. Proteins not identified

within the dataset are shown in light gray.

Figure 3. FLNc is a substrate of Akt and PKC in its unique 82 amino acid

insert located in Ig-like domain 20.

A, The FLNc phosphopeptides with the sequence LGSFGSITR, either singly and

doubly phosphorylated at S2234 and S2237 or doubly phosphorylated at S2234 and

T2239, were found to be significantly regulated (*p ≤ 0.05; n=3) in vivo upon

treatment of C2 myotubes with LY294002 or IGF-1 by global phosphoproteomics.

B, Western blot analysis and quantification of immunoblot signals (***p ≤ 0.001; n=3)

detected for FLNc-pS2234 in comparison to total FLNc following treatment of

skeletal myotubes with IGF-1 or LY294002.

38 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 237

C, Kinase prediction data obtained with NetworKIN 3.0, NetPhorest and Scansite.

The conserved 82 amino acid insert of mouse (m) and human (h) FLNc Ig-like

domain 20 is predicted to be a target of Akt/PKB and PKC/PKC distinct sites. The

minimum score threshold was set to 0.2 for NetworKIN and NetPhorest.

D, In vitro kinase assay coupled to phosphorylation-dependent mobility shift analysis.

Reactions were performed using recombinant hFLNc d18-21 in the presence of ATP,

Akt (10 ng) and/or PKCα (10 ng) as indicated. Western blot analysis performed with

an antibody directed against the EEF-tag that was fused to the carboxy-terminus of

hFLNc d18-21 revealed multiple shifted bands, indicating kinase-dependent

phosphorylation events. h, human; wt, wild-type

E, In vitro kinase assays coupled to quantitative MS analysis for site determination.

Reactions were performed as described in (D). MS data from three independent

experiments for each kinase were quantified using Skyline. Intensities of

phosphopeptides distinctive for a specific phosphorylation site in hFLNc d18-21 were

added up per experiment and represented as normalized mean ± SEM.

Figure 4. Characterization of the in vivo protein nano-environment of FLNc

d18-21 in C2 myotubes.

A, Experimental design of the triple SILAC-based quantitative proximity proteomics

approach. Differentiated and EPS-treated C2 myotubes transiently expressing the

promiscuous biotin ligase BirA* or BirA*FLNc d18-21 were incubated with 50 mM

biotin as indicated. Cell lysates were mixed in a 1:1:1 ratio, biotinylated proteins were

enriched via streptavidin, digested using trypsin and analyzed by LC-MS.

Experiments were performed in two biological and two technical replicates.

39 238 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

B, Hierarchical cluster analysis of mean log10 transformed SILAC ratios of proteins

quantified in the proximity proteomics approach. Cluster 1, orange; cluster 2, blue;

cluster 3, gray

C, Classification of quantified proteins according to cluster analysis. Gray, non-

enriched proteins; blue, moderately enriched, and orange, highly enriched proteins.

Annotated proteins include the bait, endogenous FLNc, known binding partner and

the new putative binding partner FILIP1.

D, Gene Ontology enrichment analysis. Shown are the terms for cellular components

that were found to be enriched among the proteins in cluster 1 (yellow) and cluster 2

(blue).

Figure 5. FLNc interacts with the carboxy-terminus of FILIP1.

A, A yeast two hybrid screen identified a novel isoform (FILIP1-3) as binding partner

of FLNc d17-19. Shown are exons and slicing events. Alternative splicing produces

this isoform lacking exon 6. FILIP1-1 and FILIP1-2 represent the Uniprot isoforms

Q7Z7B0-1 and Q7Z7B0-2, respectively.

B, Direct yeast two hybrid assays indicate the presence of at least one additional

binding site. -: no binding; +: weak binding; ++: moderate binding; +++: strong

binding.

C, Dot-blot overlays showing binding of different fragments of FLNc and FLNa as

indicated on top to the carboxy-terminus of FILIP1-3 and FILIP1-2. Binding of d20-21

and d18-21 confirms yeast two hybrid results. Domains 16-19 do, however, not bind

in this assay. FLNa d20-21 and d18-21 also interact with both FILIP1 isoforms.

Staining with T7 antibody confirms spotting of protein. Staining with anti-EEF

40 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 239

antibody shows binding of EEF-tagged proteins. -, no overlaid protein. n.d., not

determined.

D, Analysis of the expression of FLNa, FLNc and FILIP1 in differentiating cultured

human myotubes confirms reduced expression of FLNa and increased expression of

FLNc during differentiation. FILIP1 expression is only detected in cells differentiated

for at least two days. The numbers on top indicate the differentiation in days. -

Tubulin (-TUB) was used as loading control.

E, Western blots show expression of FILIP1 in mouse skeletal muscle and cultured

human myotubes. Addition of T7-tagged FILIP1 CT to the antibody solution ("block")

abolishes binding (lanes 2 and 4), showing specificity of the antiserum. The

supplementary bands on top of these lanes indicate binding of the antibody/antigen

complex to a specific protein. Staining with T7-antibody confirms presence of FILIP1

CT. Staining with anti-FLNc indicates that the antibody/antigen complex probably

bound FLNc.

F, Pull-down experiments using Ni2+-NTA bound FILIP1 CT. Left lane: no cell extract

added; middle lane: pull down from cultured human myotubes; right lane: pull down

using empty Ni2+-NTA agarose beads. The arrow indicates the excised band that

was identified as FLNc by MS.

Figure 6. S2233 and S2236 have a regulatory function in the interaction of

FLNc with FILIP1.

A, Western blot analysis of pull-down assay with GST-FILIP1-CT as bait and hFLNc

d18-21, its phosphomutants and hFLNc d1-3 as prey, respectively. WT, wildtype.

Phosphosite mutants in A-D: D, S2233D; DD, S2233D/S2236D; A, S2233A; AA,

S2233A/S2236A. WT, wildtype; d, domain.

41 240 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

B, Quantification of pull-down experiments normalized to the wildtype. Phosphosite

mutants were as in (A). Calculated intensities were normalized to the control, SEMs

were calculated and two-tailed Student’s t-test was performed (n=4; *p ≤ 0.05).

C, Co-immunopurification experiments in HEK293 cells transiently expressing FILIP-

GFP, hFLNc d22-24, hFLNc d18-21 wildtype (WT) and the corresponding

phosphosite mutants, respectively.

D, Quantification of co-immunopurification experiments. Calculated intensities were

normalized to the control, SEMs were calculated and two-tailed Student’s t-test was

performed (n=4; *p ≤ 0.05).

42 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 241

Supplementary Figure Legends

Supplemental Figure 1. Overlap, reproducibility and characterization of the

quantitative phosphoproteome data.

A, Overview of the number of identified proteins and phosphoproteins, including the

number of counted, quantified and localized (MaxQuant localization probability score

≥ 0.75) phosphosites obtained by SILAC-MS analyses.

B, Modified residue distribution among phosphopeptides identified by SCX-

fractionation, subsequent TiO2-based phosphopeptide enrichment and LC-MS/MS

fragmentation with MSA and HCD.

C, Distribution of singly, doubly and triply phosphorylated peptides enriched in our

large-scale SILAC dataset.

D, Distribution of the charge states of identified phosphopeptides.

E, Reproducibility of quantified peptides over the three biological replicates. 69% of

all peptides were quantified in two out of three experiments.

F-H, Multi-scatter plots and Pearson correlation analysis of the three different

treatments within the tree biological replicates of the large-scale SILAC experiment.

I, Box-plot analysis of phosphopeptide ratios reveals that only a minority of peptides

is significantly regulated (log2 ratio ≤0.584 or ≥-0.584).

Supplemental Figure 2. Abundance of endogenous FLNc phosphopeptides

determined by global quantitative phosphoproteomics and specificity of the

antibody directed against human FLNc phosphorylated at S2233.

43 242 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

A, Shown are the mean intensities of the phosphopeptides modified at the given

residue for each treatment in the large-scale SILAC experiment. Data derived from

three independent experiments. Error bars represent the SEM.

B, Western blot analysis of C2 cells transiently expressing human FLNc d-18-21 and

the respective site mutants. IGF-1 stimulation and LY294002 treatment was used to

stimulate and inhibit phosphorylation at S2233, respectively. Amount of

phosphorylation was mapped with the phospho-specific FLNc S2233 antibody from

Kinasource Limited. Phosphosite mutants were generated from the respective WT

construct by exchange of one or two serine residues at the position 2233 and 2236

to alanine (A) or aspartate (D) as indicated. WT, wildtype; d, domain.

Supplemental Figure 3. FLNc phosphosite mutants generated in this work.

A, Schematic illustration of hFLNc d18-21 constructs used for bacterial

transformations comprising a 6xHis tag and an EEF tag at the N-terminus.

Phosphosite mutants were generated from the respective WT construct by exchange

of one or two serine residues (S) to alanine (A) or aspartate (D) as indicated. WT,

wildtype; d, domain.

B, Schematic illustration of hFLNc d18-21 constructs used for mammalian cell

transfection comprising a BirA* sequence a 6xHis tag and an EEF tag at the N-

terminus. Phosphosite mutants were as described in (B). BirA*, promiscuous biotin

ligase; WT, wildtype; d, domain,

C, Schematic illustration of GST-FILIP CT and FILIP CT-GFP used for bacterial

transformation and mammalian cell transfection, respectively.

44 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 243

A B IGF-1 + LY294002 + IGF-1 – + – LY294002 – – + AKT -- Lys 0 Lys 4 Lys 8 Arg 0 Arg 6 Arg 10 AKT-pT308 - AKT-pS473 - PI3K eEF2 - LY294002 eEF 2-pT56 -

Akt GSK3 - Pathway Inhibition GSK3-pS9 -

SCX/TiO2 Rictor - Rictor-pT1135 - Enrichment L RARLHpSDQGK p70 S6K - H p70 S6K-pT389 -

M eEIF4B - Intensity eEIF4B-pS406 - m/z GAPDH -

LC/MS & MaxQuantcontrol SILAC inhibiton stimulation

C 5 4 3 * 4 ** ** * * 4 3 3 2 3 2 2 2 1 1 1 1 AKT-p308/AKT AKT-p473/AKT p70 S6K-pT389/S6K 0 0 0 eEIF4B-S406/eEIF4B 0 control IGF LY control IGF LY control IGF LY control IGF LY

* * 4 ** 1.5 5 * 1.5 * 4 3 1.0 1.0 3 2 -S9/GSK3 

2 GAPDH 0.5 0.5 1 1 eEF2-pT56/eEF2 GSK3 

0 0 Rictor-pS1135/Rictor 0 0.0 control IGF LY control IGF LY control IGF LY control IGF LY

Figure 1

45 244 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

AB4 6 pSP RxRxxpS RxRxxpSxxS pSxxxS

Junb

0 0 -2 0 2 -4 -2 0 2

mean log2 ratio IGF-1 mean log2 ratio LY294002

E IGF-1

Figure 2

46 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 245

IGF-1 LY294002 AB*** IGF-1 – + – LGSFGpSITR * S2237 1.5 LY294002 – – + * LGpSFGSIpTR S2234 FLNc - 1.0 T2239 FLNc (pS2234) - 0.5 * LGpSFGpSITR S2234 S2237 GAPDH - pFLNc S2234/FLNc 0 * LGpSFGSITR S2234 control IGF LY

-2 -1 0 1

Mean log2 ratio

CD mFLNc 2231RLGS FGS I T R 2240 hFLNc d18-21WT hFLNc 2230RLGS FGS I T R 2239

ATP + +++ domain 20 82 aa insert PKCα + + AKT1 + + 68 kDa - PKC/ T2238 AKT/PKB S2233 S2236 53 kDa - CaMKII NetworKIN 3.0 n. a. NetPhorest Scansite 3 41 kDa - αEEF-tag

E 60 AKT1 PKC 40 20 PKC+ AKT1

10 normalized intensity [%] 0 T2179 S2233 S2236 S2233 S2182 S2236

Figure 3

47 246 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

A B Mean log10 FLNc/BirA* Mean log10 FLNc/FLNc - 1 + BirA* + + BirA*FLNc + + Biotin 2 LMH SILAC

B B B Enrichment

RLGSFGSITR H L 3 M MaxQuant Intensity LC-MS/MS &

m/z

FLNc d18-21 + biotin cell-substrate /BirA* + biotin 10 adherens junction myosin complex 0 stress ﬁber actin ﬁlament

Mean log bundle actin myosin ﬁlament cytoskeleton focal adhesion 012 actomyosin

Mean log10 BirA* FLNc d18-21 +biotin/-biotin

Figure 4

48 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 247

AB FLNc FLNc FLNc FLNc FLNc FLNc FILIP1-1 d16-19 d16-17 d17-19 d18-21 d18-19 d20-21 FILIP1-2 lacZ + -+++ +++ - ++

FILIP1-3 His3 +++ + +++ +++ - ++

C FLNa D FLNc domains domains 0186421 0

--250 kDa FLNa 16-19 16-17 17-19 18-21 18-19 20-21 18-21 18-19 20-21 FLNc FILIP1-3 250 kDa

FILIP1-2 130 kDa FILIP1 55 kDa -TUB T7-tag EEF-tag

FILIP1 block FILIP1 block T7-tag FLNc no extract pull down empty beads

250 kDa 180 kDa 130 kDa 100 kDa 130 kDa 70 kDa 55 kDa 100 kDa

70 kDa 35 kDa

55 kDa 25 kDa

muscle myotubes

Figure 5

49 248 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

AB GST-FILIP CT 1.5 * hFLNc d1-3 d18-21 WT WT D DD A AA 53 kDa 1.0

41 kDa anti-HIS Eluate

53 kDa 0.5 anti-HIS 41 kDa bound to GST-FILIP CT Input nom. amount of hFLNc d18-21 0.0 68 kDa anti-FILIP WT D DD A AA 53 kDa hFLNc d18-21

C FILIP-GFP CT D hFLNc 22-24 18-21 3.0 * WT WT D DD A AA 68 kDa 2.5 anti-FILIP 53 kDa 2.0 93 kDa 1.5 68 kDa anti-myc IP:anti-Myc 53 kDa 1.0

0.5 68 kDa anti-FILIP bound to myc-hFLNc d18-21 nom. amount of FILIP-GFP CT 0.0 WT D DD A AA Input 32 kDa anti-GAPDH hFLNc d18-21

Figure 6

50 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 249

Supplementary Table Legends

Supplemental Table 1. Proteome dataset. Excerpts from the MaxQuant output

files "proteinGroups" (spreadsheet proteins) and "peptides.txt" (spreadsheet

peptides) derived from the quantitative proteome analysis of C2 myotubes; contains

information about the identification of proteins/protein groups and peptides.

Contaminants and reversed entries were removed from the lists.

Supplemental Table 2. Global phosphoproteome dataset. Excerpts from the

MaxQuant output file "PhosphoSTY" derived from the quantitative phosphoproteome

analysis of C2 myotubes; contains information about phosphopeptide identification

and phosphosite localization in the spread sheets “phospho sites >0.75” and

“phospho sites <0.75”. Contaminants and reversed entries were removed from the

lists.

Supplemental Table 3. Quantitative MS data from in vitro kinase assays using

Akt, PKC and human FLNc 18-21.

Label-free quantification was performed using the Skyline software.

Phosphopeptides from each of the three independent experiments were analysed by

LC-MS using multi-stage activation (MSA), electron transfer dissociation (ETD) and

higher-energy collisional dissociation (HCD).

Supplemental Table 4. Quantitative MS data from BioID experiments using

BirA*FLNc d18-21. Excerpts from the MaxQuant output files "proteinGroups"

(spreadsheet proteins) and "peptides.txt" (spreadsheet peptides) derived from the

51 250 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc

quantitative proximity-dependent biotinylation-enriched proteome analysis of C2

myotubes; contains information about the identification of proteins/protein groups

and peptides. Contaminants and reversed entries were removed from the lists.

52 6.2. Phosphorylation-dependant interaction of FILIP1 with FLNc 251

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57 256 6.3. New insights into myosin phosphorylation

6.3 New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation

Contributions:

• Establishment of thermolysin digestion for phosphopeptides from 2DE spots.

• Contribution to design of ﬁgure 2. 6.3. New insights into myosin phosphorylation 257

J Muscle Res Cell Motil (2012) 33:471–483 DOI 10.1007/s10974-012-9306-9

ORIGINAL PAPER

New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation

Sandra Puetz • Mechthild M. Schroeter • Heike Piechura • Lena Reimann • Mona S. Hunger • Lubomir T. Lubomirov • Doris Metzler • Bettina Warscheid • Gabriele Pﬁtzer

Received: 14 February 2012 / Accepted: 25 May 2012 / Published online: 19 June 2012 Ó The Author(s) 2012. This article is published with open access at Springerlink.com

Abstract Nitrovasodilators and agonists, via an increase in Thr18 is significantly monophosphorylated during sustained intracellular cyclic nucleotide levels, can induce smooth relaxation. We therefore suggest that (i) monophosphoryla- muscle relaxation without a concomitant decrease in phos- tion of Thr18 rather than Ser19 is responsible for the phosphorylation of the regulatory light chains (RLC) of myosin. phorylation rebound during sustained EFS-induced relaxation However, since cyclic nucleotide-induced relaxation is asso- of mouse gastric fundus, and (ii) that relaxation can be ascri- ciated with a decrease in intracellular [Ca2?], and hence, a bed to dephosphorylation of Ser19, the site considered to be decreased activity of MLCK, we tested the hypothesis that the responsible for regulation of smooth muscle tone. site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were iso- Keywords Myosin light chain phosphorylation Smooth metrically contracted with ET-1 which induced an increase in muscle relaxation cAMP cGMP Murine gastric fundus monophosphorylation from 9 ± 1 % under resting conditions NANC neurons NO (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation Abbreviations with a half time of 8 s, which was associated with an initial Tris 2-Amino-2-(hydroxymethyl)-propan- decline in RLC phosphorylation to 18 % within 2 s and a 1,3-diol rebound to 34 % after 30 s whereas relaxation was sustained. HEPES N-(2-hydroxyethyl)piperazine-N0- In contrast, phosphorylation of RLC at Ser19 probed with (2-ethanesulfonic acid) phosphospecific antibodies declined in parallel with force. DTT Dithiothreitol LC/MS and western blot analysis with phosphospecific anti- RLC 20 kDa regulatory myosin light chain bodies against monophosphorylated Thr18 indicate that m-phosphorylation Monophosphorylation ET-1 Endothelin 1 EDTA Ethylenediaminetetraacetic acid Sandra Puetz and Mechthild M. Schroeter contributed equally EFS Electrical field stimulation to this study. L-NAME Nx-nitro-L-arginine methylester hydrochloride S. Puetz M. M. Schroeter M. S. Hunger L. T. Lubomirov D. Metzler G. Pfitzer (&) MLCK Myosin light chain kinase Institute of Vegetative Physiology, University of Cologne, MLCP Myosin light chain phosphatase Robert-Koch-Str. 39, 50931 Cologne, Germany e-mail: gabriele.pfi[email protected]

H. Piechura L. Reimann B. Warscheid Faculty of Biology and BIOSS Centre for Biological Signalling Introduction Studies, University of Freiburg, 79104 Freiburg, Germany There is general consent that phosphorylation of the M. S. Hunger 20 kDa regulatory light chain (RLC) of myosin at Ser19 is Clinics for Anesthesiology and Surgical Intensive Care, University of Cologne, Cologne, Germany a prerequisite for the actin activation of MgATPase activity 123 258 6.3. New insights into myosin phosphorylation

472 J Muscle Res Cell Motil (2012) 33:471–483

of smooth muscle myosin (reviewed in Kamm and Stull et al. 1989; reviewed in Ba´rańy and Ba´rańy 1996b). Sim- 1985; Pfitzer 2001; Somlyo and Somlyo 2003). RLC ilarly, dephosphorylation of RLC paralleled forskolin- phosphorylation is regulated by the balance of two induced relaxation of precontracted tracheal smooth mus- opposing enzymes, the Ca2?–calmodulin-activated MLCK cle (de Lanerolle 1988). Direct evidence that dephospho- and MLCP, a type 1 phosphatase. Structural studies suggest rylation of RLC initiates relaxation was obtained in that the actin-activated MgATPase activity of unphos- skinned fibres incubated with a myosin phosphatase (e.g. phorylated myosin is low because of an asymmetric Bialojan et al. 1987; Shirazi et al. 1994; reviewed in interaction between the two myosin heads (Sellers and Hartshorne et al. 1998). Knight 2007). This inhibitory interaction is relieved by However, matters again are more complex for two rea- phosphorylation of RLC at Ser19. sons: (i) the rate of relaxation can be accelerated even at Kate and Michael Ba´rańy were among the first who basal or near basal levels of RLC phosphorylation (Fischer carried the idea that activation of smooth muscle myosin and Pfitzer 1989; Gerthoffer et al. 1984), and (ii) in dif- requires phosphorylation of RLC to smooth muscle tissue. ferent types of tonic smooth muscles, relaxation could In two seminal reports, they showed that contractions occur without dephosphorylation of RLC (Ba´rańy and elicited by norepinephrine and K? in 32P labelled intact Ba´rańy 1993; Gerthoffer 1986; Ishibashi et al. 1995; Kat- carotid arteries were associated with a concomitant och 1992; Katoch et al. 1997; McDaniel et al. 1992; increase in RLC phosphorylation (Barron et al. 1979) Rembold et al. 2001; Steusloff et al. 1995; Tansey et al. which is reversed upon relaxation (Barron et al. 1980). 1990). Time course measurement in NO-relaxed arteries These proof-of-concept yet correlative studies were com- indicated that RLC are dephosphorylated during the initial plemented by skinned fibre experiments in which a con- phase of the relaxation but rebound to the level before traction could be elicited in the absence of Ca2? with a addition of NO during maintained relaxation (Rembold constitutive active fragment of MLCK showing that RLC et al. 2001; Kitazawa et al. 2009). These findings gave rise phosphorylation is sufficient to induce a contraction to the hypothesis that contraction may be turned off inde- (Walsh et al. 1982). Subsequently, many laboratories pendent of RLC dephosphorylation by additional regula- showed that contractions elicited by a variety of agonists in tory mechanisms (Brophy et al. 1999; Rembold et al. 2000; different types of smooth muscle from different species are Pfitzer et al. 1993). associated with an increase in RLC phosphorylation The uncoupling between relaxation and RLC dephos- (reviewed in Arner and Pfitzer 1999; Kamm and Stull phorylation was particularly evident when relaxation was 1985; Kim et al. 2008; Somlyo and Somlyo 2003). actively induced by agents that increase intracellular cGMP However, this simple concept was soon challenged by (McDaniel et al. 1992; Rembold et al. 2001; Kitazawa et al. the observations of the laboratory of Murphy (Dillon et al. 2009) or cAMP levels (Ba´rańy and Ba´rańy 1993). 1981). They found in tonic vascular smooth muscle that Although this uncoupling is the apparently most striking RLC phosphorylation was transient, i.e. it rose initially feature, we consider the observation that RLC phosphory- during the rising phase of the contraction but declined to lation remains elevated, the even more puzzling phenom- lower yet suprabasal values during the sustained phase. enon. This is because two processes should synergistically This finding was confirmed by many other laboratories in a favour dephosphorylation of RLC: (i) MLCK should be wide range of smooth muscle tissues and contractile stimuli inactive due to a decrease in cytosolic [Ca2?] (DeFeo and (summarized in Ba´rańy and Ba´rańy 1996b; Kamm and Morgan 1989; McDaniel et al. 1992), and (ii) cGMP and Stull 1985). Interestingly, shortening velocity declined cAMP enhance the activity of MLCP (Etter et al. 2001; concomitant with the decline in phosphorylation suggest- Kitazawa et al. 2009; Lubomirov et al. 2006). Therefore, ing that the cross-bridge cycling rate declines during a we hypothesized that Ser19, which is predominantly sustained contraction, i.e. the so called latch state (Dillon phosphorylated by MLCK and is associated with activation et al. 1981). Although several models have been put for- of smooth muscle contraction, is in fact dephosphorylated ward to account for the latch state it is still a matter of during relaxation and that the phosphorylation rebound is debate how it is regulated (Hai and Murphy 1989; Somlyo due to phosphorylation of one of the other sites. The other et al. 1988; Butler and Siegman 1998; Vyas et al. 1994; sites include Ser1, Ser2, Thr9 and Thr18. Thr18 is phos- Pfitzer et al. 2005). phorylated by MLCK albeit at a much lower rate than In keeping with the phosphorylation theory of smooth Ser19 whereby phosphorylation occurs at random, i.e. muscle contraction, relaxation of different types of smooth phosphorylation of Ser19 is not a prerequisite for Thr18 muscle induced by washout of the contractile agent or phosphorylation (Bresnick et al. 1995). Thr18 and Ser19 spontaneous relaxation of rhythmically active smooth are phosphorylated with equal efficiency by integrin-linked muscle was associated with concomitant dephosphoryla- kinase, ILK (Wilson et al. 2005), and ZIP kinase (Niiro and tion of RLC (e.g. Gerthoffer and Murphy 1983; Driska Ikebe 2001; Walsh 2011 for review). Ser1 and 2, Thr9 are 123 6.3. New insights into myosin phosphorylation 259

J Muscle Res Cell Motil (2012) 33:471–483 473 phosphorylated by PKC (Bengur et al. 1987). Phosphory- Animal Care and Use Committee of the University of lation of Thr 9 has been suggested to be involved in the Cologne. The stomach was quickly removed and trans- relaxant effect of okadaic acid in basilar arteries (Obara ferred to PSS with low calcium (in mM): 118 NaCl, 5 KCl, et al. 2008). 1.2 Na2HPO4, 1.2 MgCl2, 0.16 CaCl2, 24 Hepes, 10 glu- To test the hypothesis, that phosphorylation of Ser19 cose, pH 7.4 at 37 °C and bubbled with 100 % O2. Murine does not account for the uncoupling of stress from RLC gastric fundi were freed of mucosa, cut in the direction of dephosphorylation, we chose smooth muscle from gastric the circular muscle layer into strips (2 mm wide) and fundus as a model. This preparation can be relaxed by mounted vertically in Ca2?-free PSS in an organ bath electric field stimulation (EFS) within seconds. It is well between two platinum electrodes. Slack length was deter- established that EFS elicits relaxation by activating non- mined in Ca2?–free PSS, the fundus strips were then adrenergic non-cholinergic (NANC) neurons of the plexus stretched to 167 % of slack length in low calcium PSS. myentericus which in turn release NO and vasointestinal Calcium was slowly increased up to 1.6 mM and a control peptide (VIP) leading to an increase in cGMP and cAMP contraction was induced by depolarization with levels in the fundus smooth muscle cells (reviewed in 45 mM K?, replacing Na? in the PSS solution. Force was Lefebvre et al. 1995). An advantage of this preparation is recorded with a type Q11 transducer (Hottinger Baldwin that relaxation is not diffusion limited which is different Messtechnik, Germany). Adrenergic/cholinergic transmis- from vascular smooth muscle, in which relaxation is sion as well as prostaglandins were inhibited by addition of induced by NO-donors or agonists. Furthermore, relaxation atropine (10 lM), propranolol (1 lM), phentolamine occurs within seconds compared to minutes in the vascular (1 lM) and indomethacin (1 lM) to PSS for 20 min. This tissue allowing us to determine whether uncoupling of inhibitor cocktail was present throughout the experiment. relaxation from dephosphorylation is independent of the Contractions were elicited with 5 nM endothelin-1 (ET-1). time scale at which relaxation occurs. Electrical field stimulation (EFS) was performed with custom-made stimulators with trains of pulses at 10 Hz [30 V, pulse width 0.5 ms] for 30 s. The strips were snap frozen within 100 ms with liquid nitrogen precooled tongs Materials and methods at distinct time points of EFS (0, 2, 5 and 30 s) and fixed in dry-ice precooled 15 %TCA/acetone. Materials Determination of the intracellular Ca2?-transient The used primary antibodies were rabbit polyclonal anti- phospho-myosin light chain Ser-19 (pSer19), Rockland, For the simultaneous determination of force and intracellular PA, USA; rabbit polyclonal anti-phospho-MLC20 Thr-18 [Ca2?], the muscle strips were mounted horizontally in a (pThr18), Santa Cruz, CA, USA; anti-phospho-myosin myograph on the stage of an inverted Zeiss microscope light chain 2 pThr18/pSer19, Pierce Biotechnology, (Axiovert 35, Zeiss, Jena) using the cuvette of the confocal Rockford, IL, USA; and mouse monoclonal anti-MLC20 wire myograph system 120CW (DMT, Aarhus Denmark). antibody for total MLC20 (RLC), Sigma, Germany. The Thestripswereequilibratedasabove.AftertheK?-induced secondary antibodies were IRDye conjugated goat anti- test contraction, the strips were loaded with fura-2 for 4 h with mouse (IRDye680) and goat anti-rabbit (IRDye800), LI- 1 lM acetoxy methylester fura-2 (fura-2 AM) dissolved in COR, NE, USA and horse radish peroxidase-conjugated DMSO premixed with pluronic FF127 (1 lM final; Lucius donkey anti-mouse and donkey anti-rabbit both from Dia- et al. 1998). After loading, the strips were washed 2 9 15 min nova, Germany. Thermolysin came from R&D Systems, with PSS to remove extracellular fura-2 AM. The fluorescence Minneapolis, USA. The SyproÒ Ruby, Silver SnapÒ stain signal was recorded during the endothelin-induced contrac- and Bio-Rad Protein AssayÒ, were from Bio-Rad Labora- tion and EFS-induced relaxation with an imaging system from tories GmbH, Germany; the enhanced chemiluminescence TILL Photonics (Planegg, Germany). In brief, UV-light of Kit (Pierce ECL Western Blotting Substrate) from Perbio alternating wave lengths (340 and 380 nm) was obtained by a Science/Thermo Fisher Scientific, Germany; and the Phos- fast monochromator positioned in front of a high pressure tag kit from Wako Chemicals GmbH, Germany. All other xenon lamp passed with light guides and focused onto the chemicals were from Sigma or AppliChem, Germany. muscle strip through the quartz window on the bottom of the cuvette. The fura-2 emission (510 nm) was passed through the Muscle strip preparation objective lens (fluor 910, Zeiss, Jena), collected with a CCD camera (TILL Photonics Imago PCO Imaging, SensiCam) Male mice, 12 weeks old, were killed by cervical dislo- and analysed using TILL vision image 3.0 software. Force cation following procedures approved by the Institutional was recorded with MyoDaq (DMT). Synchronization of the 123 260 6.3. New insights into myosin phosphorylation

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force and the ﬂuorescence signal was performed with home- Nitrocellulose membranes were ﬁrst probed with anti- made software. In some experiments the fura-2 signal was bodies against pSer19, pThr18, or RLC total followed by calibrated according to the protocol of Himpens and Somlyo incubation with the appropriate secondary antibody and

(1988). In brief, the minimal ratio, Rmin was determined after visualization of the immunoreactive signal with enhanced incubating the strips with 10 lM ionomycin for 10 min in chemiluminescence ECL. Blocking of non-speciﬁc binding Ca2?-free calibration buffer containing ((in mM) 140 KCl, 2 sites and densitometric evaluation of immunoreactive

EGTA, 24 HEPES, 1.2 MgCl2,1.2.Na2HPO4, pH 6.8). Then bands were performed as in Lubomirov et al. (2006). When 2? 10 mM Ca was added to obtain the maximal ratio, Rmax, using the Odyssey system (LI-COR) for visualization of the followed by addition of 20 mM MnCl2 to record background signal, the blots were first probed with the phosphospecific values. From these values, [Ca2?] was calculated using the antibodies (pSer19, pThr18, pTrh18/pSer19) and the formula and an apparent dissociation constant of the Ca2?– appropriate IRDye conjugated secondary antibodies. fura 2 complex of 224 nM given by Grynkiewicz et al. (1985). Thereafter, the same blot was reprobed with a monoclonal mouse antibody against total RLC. For Odyssey imaging, Two dimensional gel electrophoresis membranes were blocked in 2 % milk/TBS (10 mM Tris pH 8.0, 150 mM NaCl) and probed with primary antibody TCA was removed from the strips by several acetone in 2 % milk/TBST (TBS with 0.1 % Tween 20 (v/v)), in washes, afterwards the strips were air-dried. The strips case of pThr18 in 1 % BSA/TBST. Detection and quanti- were homogenized in 50-ll sample buffer (10 mM Tris– fication of the infrared signals were performed using the HCl, pH 7.5, 9.2 M urea, 3 % ampholines pH 4.5–5.4, Odyssey system software. 10 mM DTT and 0.0001 % bromphenol blue). The lysates were subjected to 2D-PAGE and the silver or Sypro RubyÒ Protein digestion and mass spectrometry stained gels were analysed as described using phoretix software (Biostep, Germany) (Lucius et al. 1998). Regu- Following staining with colloidal Coomassie Brilliant Blue larly 3 major spots can be resolved. It is generally accepted G-250, protein spots of interest were cut from the gel, that the most basic spot (spot 1 in Fig. 1c) and the adjacent, destained by alternated incubation with 20 llof10mM

more acidic spot (spot 2 in Fig. 1c) represent unphos- NH4HCO3 and 5 mM NH4HCO3/50 % acetonitrile (ACN) phorylated and monophosphorylated RLC, respectively. (v/v) for 10 min and dried in vacuo. In-gel digestion of Spot 3 is considered to represent diphosphorylated RLC proteins was performed for 2 h at 60 °C using 2 ng

and unphosphorylated non-muscle RLC (Gagelmann et al. thermolysin dissolved in 5 llof50mMNH4HCO3. Pro- 1984; Gaylinn et al. 1989). teolytic peptides were extracted by incubating the gel spots twice with 10 ll of 95 % ACN (v/v)/0.1 % trifluoroacetic SDS-PAGE/western blotting acid (TFA) (v/v) for 15 min. Extracts were combined and dried in vacuo. For mass spectrometric analysis, peptides TCA was removed as above. For separation by SDS- were re-dissolved in 15 ll 0.1 % TFA (v/v). PAGE, the strips were prepared and subjected to SDS- Online reversed-phase nano-HPLC separations were per- PAGE as described by Lubomirov et al. (2006). The sep- formed using the UltiMate 3000 RSLC System (Dionex/Thermo arated proteins were transferred to nitrocellulose according Fisher, Idstein, Germany) equipped with two precolumns to Towbin et al. (1979). To use the intensity of desmin as (AcclaimÒ PepMap l-Precolumn Cartridge; 0.3 mm 9 5mm, an additional internal loading control, the gel area with particle size 5 lm)andanAcclaimÒ PepMap RSLC analytical proteins of molecular masses between 40 and 60 kDa was column (75 lm 9 25cm,C18,2lm, 100 A˚ ). Peptides were not transferred but stained with Coomassie R-250Ò, and the preconcentrated on the precolumn and washed for 5 min using desmin band was evaluated densitometrically using the 0.1 % (v/v) TFA at a flow rate of 30 ll/min. Subsequently, Odyssey Infrared Imaging System (LI-COR). peptides were separated at a flow rate of 300 nl/min using a For separation by Phos-tag gel electrophoresis, the binary solvent system consisting of solvent A [0.1 % formic acid fundus strips were agitated for 2 h in 120 ll sample buffer (v/v)] and solvent B [0.1 % FA (v/v), 84 % ACN (v/v)]. The B (65 mM Tris–HCl pH 6.8, 4 % SDS, 100 mM DTT, 5 % following gradient was used: 5–40 % solvent B in 30 min and glycerol, 0.04 % bromphenol blue), boiled and centrifuged 40–95 % solvent B in 5 min. The column was then washed for as described above. The protein concentration was deter- 5 min with 95 % solvent B and equilibrated with 5 % solvent B mined in the supernatant according to Bradford (1976) for 15 min. using BSA as standard. Equal amounts of protein were The LTQ Orbitrap XL instrument was equipped with a subjected to Phos-tag SDS-acrylamide electrophoresis and nanoelectrospray ion source (Thermo Fisher Scientific) and transferred to nitrocellulose as described by Takeya et al. distal coated SilicaTips (FS360-20-10-D, New Objective, (2008). Woburn, USA). The instrument was externally calibrated 123 6.3. New insights into myosin phosphorylation 261

J Muscle Res Cell Motil (2012) 33:471–483 475 using standard components. The general mass spectro- (n = 16). This frequency was used throughout. The time metric parameters were as follows: spray voltage, 1.5 kV; course of relaxation was biphasic starting with a lag period capillary voltage, 45 V; capillary temperature, 200 °C; and of *1.2 s followed by an exponential decay (Fig. 1e, upper tube lens voltage, 120 V. For data-dependent MS/MS panel). Half time of relaxation (T1/2) was 8.0 ± 0.13 s, and analyses, the software XCalibur 2.0.7 (Thermo Fisher relaxation was stably maintained for at least 30 s. ET-1- Scientific) was used. Full scan MS spectra were recorded induced contraction was associated with an increase in from m/z 370 to 1700 and acquired in the Orbitrap with the intracellular [Ca2?] from *142 nM under basal conditions Automatic Gain Control (AGC) set to 5 9 105 ions and a (PSS) to *310 nM. Upon EFS, intracellular [Ca2?] maximum fill time of 500 ms. The five most intense mul- decreased in parallel with relaxation and remained low tiply charged ions were selected for fragmentation by during maintained relaxation (Fig. 1b). multistage activation (MSA). MSA scans were performed In the next series of experiments, we investigated in the linear ion trap with an AGC set to 10,000 ions and a whether EFS-induced relaxation was associated with maximum fill time of 400 ms. Fragmentation was carried respective changes in phosphorylation of RLC using 2D- out using the following parameters: normalized collision PAGE. As predicted by the increase in intracellular [Ca2?], energy, 35 %; activation q, 0.25; activation time, 30 ms; RLC mono- (m-) phosphorylation (spot 2 in Fig. 1c) ion selection threshold, 2500; dynamic exclusion, 45s; increased from 9 ± 1%(n = 3) under resting conditions multistage activation enabled; and listed neutral losses (PSS) to 36 ± 1 % of RLCtotal (n = 4, p = 0.003). The m/z 98, 49, 32.6. time point chosen was such that it represented the phos- Mass spectrometric data were processed using Mascot phorylation status just preceding EFS-induced relaxation, Deamon v. 2.3.0 and searched against the NCBI database i.e. during the tonic phase of the contraction. EFS-induced (taxonomy filter Mus musculus, 143,284 sequences) using relaxation was preceded by a decline in m-phosphorylation the MASCOT algorithm v. 2.3.02 (Perkins et al. 1999). For to 18 ± 1 % of RLCtotal (p = 0.004) within 2 s after database searches, no enzyme was specified and mass tol- starting EFS. Thereafter, RLC were rephosphorylated erance was set to 5 ppm and 0.4 Da for peptide and frag- (Fig. 1c, e, middle panel); 30 s after starting EFS RLC ment ion masses, respectively. Oxidation of methionine phosphorylation was similar to the value of the ET-1 and phosphorylation of serine, threonine and tyrosine were contracted tissue. Thus, despite the fact that relaxation as considered as variable modifications. Proteins above a well as the decay in intracellular [Ca2?] was sustained, MASCOT significance threshold of 0.05 were considered dephosphorylation of rRLC was only transient suggesting unambiguously identified. Common contaminants (i.e. that force is uncoupled from m-phosphorylation during keratin) were excluded. Fragmentation spectra of phos- cyclic nucleotide-mediated relaxation. A similar observa- phopeptides were manually annotated and validated. tion has been made before in uterine smooth muscle relaxed by isoprenaline (Ba´rańy and Ba´rańy 1993) and Statistical analysis arterial smooth muscle relaxed by NO-donors (e.g. McDaniel et al. 1992; Rembold et al. 2000). All data are mean ± SEM; n is the number of strips. Sta- Under resting conditions, as well as in ET-1 contracted tistical significance was determined using unpaired t test and EFS-relaxed preparations, a third spot could be with Welch’s correction for unequal variances or ANOVA resolved in the 2D-PAGE (c.f. Fig. 1c). This spot was followed by Bonferroni post test for multiple comparisons extensively characterized by Ba´rańy and Ba´rańy (1996a) when applicable (Graph Pad software). The level of sig- and corresponds to spot 2 in their nomenclature. They nificance was set at p \ 0.05. proposed that it contains a mixture of diphosphorylated and non-phosphorylated, non-muscle RLC and estimated that the contribution of non-muscle RLC was between 8 and Results 16 % (Mougios and Ba´rańy 1986). In our experiments, the intensity of this spot (14 % in PSS, 13.8 ± 0.4 % in ET-1 Uncoupling of cyclic nucleotide-mediated relaxation (n = 3) and 12 ± 1 in 30 s EFS-treated tissues (n = 4) of from RLC dephosphorylation RLCtotal) did not differ significantly between the different treatments suggesting that only a small amount of RLC was Isolated smooth muscle strips from gastric fundus responded di-phosphorylated. This conclusion was confirmed by to ET-1 with a stable tonic contraction amounting to western blots probed with phosphospecific antibodies 7.45 ± 0.44 mN (n = 41), and to EFS with a TTX sensitive directed against di-phosphorylated RLC (Thr18/Ser19), in relaxation. The relaxation amplitude increased with which no immunoreactive signal was detected (c.f. increasing frequencies and was maximal at 10 Hz, which Fig. 3c). We therefore focused on the m-phosphorylation in relaxed the ET-1 precontracted strips by 80 ± 2% the further analysis. 123 262 6.3. 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Fig. 1 Phosphorylation transients during electrical-field-induced stimulation, ET-1 plateau of contraction just prior to EFS and 2, 5 and relaxation of endothelin-1 (ET-1) precontracted smooth muscle strips 30 s after starting EFS. d Western blots with pSer19 and RLC total from mouse gastric fundus. a Representative force tracing of ET-1- antibody, left panel separation of proteins with PhosTag gels and induced contraction and EFS-induced relaxation (10 Hz, 0.5 ms visualization of immunoreactivity with the odyssey system; right pulses, duration 30 s) and force recovery. b Simultaneous determi- panel separation by 15 % SDS-PAGE and visualization of the nation of intracellular Ca2?-transients and force in ET-contracted (left immunoreactivity with ECL. e Summary of time courses of EFS- panels) and EFS-relaxed fundus tissues. The ratios of the fluorescence induced relaxation (upper panel), m-phosphorylation of RLC deter- signals excited by 340 nm, and 380 nm (F340/F380) were used as mined by 2D-PAGE (middle panel) and m-phosphorylation of Ser19 indicator of intracellular [Ca2?]; representative tracings from 6 normalized to immunoreactive signal obtained with the total RLC independent experiments. c Determination of RLC phosphorylation antibody and expressed in % of the ET-1 value (lower panel). A by 2D-PAGE; spot 1 and 2 refer to, respectively, unphosphorylated similar result was obtained when pSer19 was expressed relative to the and monophosphorylated RLC. The intensity of spot 3 amounts to Coomassie stained desmin band. Symbols represent mean ± SEM of *14 % of total RLC and does not change significantly in contracted 4–9 determinations and relaxed preparations. PSS resting conditions prior to ET-1

It was proposed that the neurotransmitters responsible as DEA–NO (300 lM) and VIP (1 lM) relaxed the prep- for EFS-induced relaxation involve NO and VIP and their arations by 90 ± 3 and 80 ± 5%(n = 6) with a half time respective downstream signals cGMP–PKG and cAMP– of *30 s. RLC m-phosphorylation during sustained PKA (Lefebvre et al. 1995). Exogenous application of NO relaxation induced by DEA–NO and VIP was not

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J Muscle Res Cell Motil (2012) 33:471–483 477 significantly different from the value of ET-1 contracted multistage activation as an effective method for fragmen- fundus [DEA–NO: 28 ± 4%,n = 5, p = 0.14 vs. ET-1 tation of phosphopeptides, no phosphorylation was detec- and VIP: 33 ± 4%, n = 8, p = 0.75 vs. ET-1]. Thus, ted in the protein spot assigned non-phosphorylated, although relaxation induced by exogenously added neuro- whereas phosphorylated peptide species were reliably transmitters was much slower, it was also uncoupled from detected in the spots of phosphorylated RLC from both ET- RLC m-phosphorylation in a similar manner as with EFS- 1 and 30 s EFS-treated samples. In the ET-1 treated sam- induced relaxation irrespective of whether the neurotrans- ple, Ser19 was determined to be the major phosphorylated mitters acted through cGMP or cAMP. NO–cGMP sig- residue in RLC by MS/MS (Fig. 2b) confirming our results nalling may predominate under our conditions since L- with the pSer19 antibody. NAME inhibited relaxation by *75 % (data not shown), We then hypothesized that the N-terminal PKC site, which is in line with an earlier report showing that EFS- Thr9, might be m-phosphorylated in EFS-treated samples induced relaxation in PKG knock-out mice is significantly because this site was found to be phosphorylated during blunted (Pfeifer et al. 1998). okadaic acid-induced relaxation of smooth muscle (Obara et al. 2008) reported to occur without dephosphorylation of Uncoupling of force from RLC m-phosphorylation is RLC (Tansey et al. 1990). However, no peptides were not due to phosphorylation of Ser19 detected, which were phosphorylated at this site. Rather, m-phosphorylated peptide species of RLC were identified It has been implicitly assumed that m-phosphorylation is with evidence for Thr18 and Ser19 as the specific site of due to Ser19 phosphorylation. However, since intracellular phosphorylation (Fig. 2c). These data indicate that the [Ca2?] was low during maintained relaxation suggesting level of Thr18 phosphorylation was higher in EFS than low MLCK activity, we gave consideration to the possi- ET-1 treated samples. Since these reversible phosphoryla- bility that m-phosphorylation at 30 s was not caused by tion events take place at neighbouring amino acid resi- Ser19 phosphorylation, the major MLCK site. Indeed, dues (Ser19 and Thr18), it was not possible to separate western blot analysis with pSer19 phosphospecific anti- the corresponding phosphopeptide isoforms of RLC pres- bodies revealed that pSer19 immunoreactivity was high in ent in the m-phosphorylated 2-D gel spot. For the very lysates from ET-1 contracted fundus strips and rapidly N-terminus of the protein containing Ser1 and Ser2, which declined in EFS-treated preparations in parallel with are phosphorylated by PKC (Bengur et al. 1987), no pep- relaxation (Fig. 1d, e, lower panel). During the sustained tide could be detected, and hence, no evidence for further phase of relaxation pSer19 immunoreactivity was reversible phosphorylation events at these specific sites was 18 ± 7%(p \ 0.01, n = 9) of the value before starting retrieved in this work. EFS in ET-1 contracted strips which was taken as 100 %. Based on these results, we reasoned that m-phosphory- For comparison, under resting condition (PSS before lation of Thr18 significantly contributes to the phosphor- addition of ET-1) it was 11 ± 4 % of ET-1 (n = 3). Using ylation rebound in the EFS-relaxed tissue. This hypothesis Phos-tag gels which allow to separate m- from di-phos- was tested with western blot analysis using a commercially phorylated phospho-species of RLC (Takeya et al. 2008), available antibody against m-phosphorylated Thr18. While we confirmed that the antibody only detected monopho- the commercially available antibody against pSer19 has sphorylated RLC (c.f. Fig. 3). These results gave rise to the been widely used, we are aware of only few studies which interesting possibility that m-phosphorylation during sus- used this pThr18 antibody (e.g. Getz et al. 2010). There- tained relaxation involves a site different from Ser19. fore, we first assessed its specificity in an ELISA assay using differently phosphorylated peptides derived from Determination of the m-phosphorylated site RLC (aa 11–26). Figure 3a shows that the antibody reacted during relaxation neither with the non-phosphorylated nor with the Ser19 m- phosphorylated peptide whereas it recognized the Thr18 To determine which site of RLC is m-phosphorylated in m-phosphorylated and with a much lower affinity the 30 s EFS-relaxed strips, the unphosphorylated (spot 1 in diphosphorylated peptide. As shown in Fig. 3b and to our Fig. 1c) and m-phosphorylated spot (spot 2 in Fig. 1c) surprise, immunoreactivity with this antibody in lysates were cut out from 2D-PAGE from ET-1 and 30 s EFS- from ET-1 treated samples was frequently higher than treated samples and subjected to high resolution liquid expected from our MS/MS data and from the literature chromatography tandem mass spectrometry (LC/MS/MS). (Ba´rańy and Ba´rańy 1996b for review). The reason for this Following digestion of protein spots with thermolysin, discrepancy is not clear at present. We cannot exclude the myosin regulatory light chain polypeptide 9 (Mus muscu- possibility that there is some crossreactivity with pSer19 lus), i.e. RLC, was unambiguously identified in all spots which was not detected with the short RLC peptides used with a sequence coverage of 89–97 %. Furthermore, using in the ELISA assay. Compared to ET-1, pThr18 123 264 6.3. New insights into myosin phosphorylation

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Fig. 2 Identification of RLC and fragment spectra of phosphopep- phosphorylation site is localized to S19. c MS/MS spectrum of the tides. a Sequence of RLC. Amino acid sequences identified by MS are monophosphorylated peptide QRATSNVFAMOx (m/z 610.7601) of marked in black. In total, a sequence coverage of 97 % was achieved. myosin regulatory light chain from muscle treated with EFS for 30 s. Phosphorylation of RLC at Thr18/Ser19 (boxed gray letters) was The fragment ions observed in the spectrum support monophosph- confirmed by multiple peptides (inset). b MS/MS spectrum of the orylation events at T18 (*) as well as S19 (#). The corresponding monophosphorylated peptide QRATS*NVF (m/z 501.7239) of myo- survey scans for each peptide are displayed as zoom-in views sin regulatory light chain from fully contracted muscle. The

immunoreactivity during the initial phase of relaxation (2 with Phos-tag gels and the pThr18/pSer19 dual phosphor- and 5 s) was lower (Fig. 3b, d). The signal intensity ylation antibody (Fig. 3c). Taking together the results with increased again and was signiﬁcantly higher in prepara- the pSer19 and the pThr18 antibodies, we propose that tions relaxed for 30 s compared ET-contracted preparations rephosphorylation of RLC during sustained relaxation can (Fig. 3d). We conﬁrmed that the rise in pThr18 immuno- be ascribed to m-phosphorylation of pThr18 whereas reactivity was not due to an increase in diphosphorylation m-phosphorylation of pSer19 remains low.

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Fig. 3 Determination of m-phosphorylation of Thr18 during EFS- b Representative western blots with phosphospecific antibodies induced relaxation using phosphospecific antibodies. a Assessing against m-phosphorylated Thr18, and c pTrh18/pSer19 diphosphory- specificity of anti-phospho-antibody p-MLC (Thr18)-R (Santa Cruz lated RLC (left panel) and m-phosphorylated Ser19 (RLC) and total Biotechnology #sc-19848R) by ELISA. As substrate a differently RLC. The different phosphospecies of RLC were separated by Phos- phosphorylated peptide identical to amino acid residues 11-26 of tag gels, lane 1 mouse tail artery arteries incubated at pCa 6.8 and in myosin light chain polypeptide 9 Mus musculus (NCBI Protein Data the presence of 10 lM microcystin, lane 2–4 gastric fundus Bank NP_742116) was used. The antibody exhibited a higher affinity contracted with ET-1 (lane 2), relaxed with EFS for 5 s (lane 3) against monophosphorylated Thr18 (closed circles) than against and 30 s (lane 4). d Summary of changes of m-pThr18 during EFS- diphosphorylation at Ser19 and Thr18 (closed squares). The immu- induced relaxation, pSer19 is replotted from Fig. 1. Bars represent noreactivity against m-phosphorylation at Ser19 (closed diamonds) box plots of n = 4 pThr18 and n = 10 pSer19 determinations, and the nonphosphorylated peptide (open circles) was very poor and ** p \ 0.01 came close to the one measured against ovalbumin (open squares).

Discussion blot analysis, we suggest that m-phosphorylation of Thr18 increases during sustained relaxation. Hence, we propose Although not unequivocally found several authors reported that the rebound in RLC phosphorylation is due to a that relaxation mediated by NO-donors and isoprenaline of redistribution of phosphorylated residues in favour of different types of smooth muscle tissues from different m-phosphorylation of Thr18. species occurs without dephosphorylation of RLC or that Only a limited number of investigations determined the dephosphorylation was only transient (reviewed in Pfitzer phosphorylated residues in intact tissue stimulated with 2001) leading to the statement of Kate and Michael different agonists using phosphopeptide mapping in com- Ba´rańy: 0it can be concluded that RLC dephosphorylation bination with phosphoamino acid analysis with 32P. To the is not a prerequisite of smooth muscle relaxation0 (Ba´rańy best of our knowledge, our study is the first to apply MS/ and Ba´rańy 1993). Determination of RLC phosphorylation MS analysis. According to Ba´rańy and Ba´rańy (1993), the with 2D-PAGE during relaxation of gastric smooth muscle m-phosphorylated spot contained pSer and pThr and sug- induced by activation of NANC neurons corroborates these gested a pSer to pThr ratio of 6:1 (Csabina et al. 1986). conclusions. However, by analysing which amino acids are Others confirmed that the major 32P-labelled amino acid phosphorylated, we present evidence that this conclusion residue was Ser (e.g. McDaniel et al. 1992; D’Angelo et al. has to be modified. The novel finding of our study is that 1992). The findings are in accordance with the biochemical dephosphorylation of pSer19 does correlate with relaxa- experiments which showed that physiological activities of tion, and hence, this site cannot account for the rebound in MLCK predominantly phosphorylate Ser19 and also with RLC m-phosphorylation. Based on LC/MS and western experiments in Ca2?-activated skinned fibres (Haeberle

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et al. 1988). Our MS/MS data and western blot analysis western blots in particular with the pThr18 phosphospecific showing that pSer19 is the predominant m-phosphospecies antibody, and (iii) that the phosphospecific antibodies only of RLC in ET-1 contracted strips and that it is rapidly allow to determine the relative intensity changes between dephosphorylated during relaxation when intracellular different treatments for a given antibody but not to reliably [Ca2?], and hence, MLCK activity is low are consistent calculate the ratio between m-pThr18 and m-pSer19 during with these earlier reports. In addition, they are in keeping ET-1 and EFS. For instance, if one takes into account the with reports that Ser19 phosphorylation is low in swine relative changes of the immunoreactive signals during EFS, carotid arteries relaxed with forskolin (Meeks et al. 2008) then the maximally *1.7 fold increase in Thr18 would call as well as in a-toxin permeabilized mouse tail arteries for a ratio of pSer to pThr *1:1 in ET-1 treated prepara- relaxed with urocortin and cAMP (Lubomirov et al. 2006). tions which is clearly at variance with our MS/MS data and As we have no indication for pThr9 phosphorylation shown with previous investigations which showed that Thr18 was to be involved in relaxation of vascular smooth muscle the minor phosphorylated site and reported a pSer19/ (Obara et al. 2008), our results suggest that relaxation is pThr18 ratio of 6:1 in 32P labelled smooth muscle tissue due to dephosphorylation of Ser19. (Csabina et al. 1986). We note, however, that m-pThr18 The surprising finding was, that our MS/MS analyses was observed in phosphopeptide maps and amino acid indicated that the m-phosphorylated RLC from 30 s relaxed analysis of gizzard myofibrils phosphorylated in the preparations contained pThr18 (c.f. Fig. 2) and that western absence of Ca2? (Weber et al. 1999). As mentioned, blots with phosphospecific antibodies suggest that m-phos- quantitative information regarding the different RLC phorylation of Thr18 increased during sustained relaxation phosphospecies is very limited in intact smooth muscle and (c.f. Fig. 3). We comprehensively identified RLC with a it might be argued that the diverging results are due to the sequence coverage of up to 97 % and determined phosphor- fact that investigations with 32P labelled tissue can detect ylation sites at Thr18 and Ser19 via LC/MS. Phosphate only phosphorylation turnover, whereas non-radioactive incorporation into Thr18 has been described before but typi- methods like LC/MS/MS and western blots with phos- cally as pThr18/pSer19 diphosphorylation generated by the phospecific antibodies detect relative changes induced by action of Ca2?-independent RLC kinases such as integrin- different agonists and also permanent phosphorylations but linked kinase, ILK (Wilson et al. 2005), and ZIP kinase (Niiro not phosphorylation turnover. Thus, phosphoamino acid and Ikebe 2001;reviewedinWalsh2011). Compared to analysis with 32P may not detect pThr18 if it is not or only vascular smooth muscle (Weber et al. 1999, c.f. Fig. 3c, lane very slowly turning over compared with pSer19. Never- 1), diphosphorylation of intestinal smooth muscle by Ca2?- theless our MS/MS data also do not support such a high independent RLC kinase was much lower (Ihara et al. 2009; level of Thr18 phosphorylation in ET-1 treated prepara- Shcherbakova et al. 2010). In the EFS-relaxed fundus smooth tions, whereas they support the increase in pThr18 in EFS- muscle diphosphorylation was below the detection limit of the treated preparations. Thus, while we cannot give at present pThr18/pSer19 dual phosphorylation antibodies (c.f. Fig. 3c, the stoichiometries for the different phosphospecies in lanes 2–4). Notably, we also did not detect the corresponding m-phosphorylated RLC, our results support the idea that diphosphorylated peptide species in the m-phosphorylated there is a redistribution during maintained relaxation in spot by LC/MS providing a high mass accuracy of 1–3 ppm in favour of pThr18. In this context, it is of interest that MS survey scans. Hence, we argue that pThr18 phosphory- phosphorylation of Ser19 is not a prerequisite for Thr18 lation is not due to a contamination with diphosphorylated phosphorylation (Bresnick et al. 1995). RLC. Taken together our MS/MS and western blot data Our findings raise the question as to which kinase(s) or indicate that the phosphorylation rebound seen in 2D-PAGE is phosphatase(s) are responsible for m-phosphorylation of a consequence of intricate phosphorylation events taking Thr18 at low intracellular [Ca2?] and whether EFS acti- place at these two amino acid residues. Since western blot vates such a kinase. Of the Ca2?-independent RLC kinases analyses suggest that phosphorylation of Ser19 declines to currently in question, ILK rather than ZIP kinase appears to about 20 % of the ET-1 value, we propose that Thr18 be an attractive candidate. This is because in Ca2?-sensi- m-phosphorylation largely accounts for the m-phosphoryla- tized ileal smooth muscle ILK was proposed to be down- tion rebound. It is not clear at present whether the residual stream of PKC (Ihara et al. 2009), and PKC may still be Ser19 phosphorylation is due to residual activation of MLCK active during EFS-induced relaxation because of the con- or Rho kinase which is known to phosphorylate Ser19 Ca2?- tinued presence of ET-1. In contrast, Ca2?-sensitization independently and with higher efficacy than Thr18 (Kureishi attributed to ZIP kinase was not blocked by inhibition of et al. 1995). PKC in vascular smooth muscle (Choi et al. 2011). How- There are several limitations of our study: (i) we do not ever, these Ca2?-independent RLC kinases phosphorylate know whether phosphorylation of Ser1 and 2 contribute to Thr18 and Ser19 with equal efficiencies whereas our results m-phosphorylation, (ii) the semiquantitative nature of ask for a kinase with a preference for Thr18 during cyclic 123 6.3. New insights into myosin phosphorylation 267

J Muscle Res Cell Motil (2012) 33:471–483 481 nucleotide-induced relaxation. Another possibility would the release of inhibitory neurotransmitters from intrinsic be that Ser19 is preferentially dephosphorylated in the neurons was associated with a decline in m-phosphoryla- relaxed preparations. However, although several phospha- tion of RLC. However, during the sustained phase of tases have been isolated from smooth muscle tissues, there relaxation phosphorylation rebound to the values before is at present no evidence for a differential dephosphoryla- starting relaxation. This suggested that stress is uncoupled tion of Ser19 over Thr18 (Ikebe et al. 1986; Feng et al. from RLC dephosphorylation as has been observed in 1999). Thus, the mechanism that accounts for the shift in vascular smooth muscle relaxed by NO (Rembold et al. the phosphorylated residues is currently unclear. 2001). Determining the sites phosphorylated during the In recent years, pThr18 phosphorylation has gained sustained phase of relaxation revealed that the phosphor- attention as an index of the action of Ca2?-independent ylation rebound is mainly due to m-phosphorylation of kinases and was mainly considered to be present in diph- Thr18 whereas Ser19 is dephosphorylated consistent with osphorylated RLC. Whereas diphosphorylated myosin current concepts of the regulation of smooth muscle con- enhanced actin-activated MgATPase activity in solution traction. Finally, the combination of Phos-tag gels with (Ikebe and Hartshorne, 1985), it had no additional effect on western blotting should rapidly advance our understanding force in skinned fibres (Haeberle et al. 1988). In contrast, of the contribution of different phosphospecies of RLC to the actin-activated myosin ATPase activity of myosin the regulation of smooth muscle function. m-phosphorylated on Thr18 is *15-fold lower than that phosphorylated on Ser19 (Bresnick et al. 1995). Surpris- Acknowledgments This paper is dedicated to the memory of ingly, Thr18 m-phosphorylated myosin was able to move Michael and Kate Ba´rańy and in addition to Ma Jun. Ma Jun was a very gifted PhD student and she generated some of the shown 2D- actin filaments in the in vitro motility assay similar to that PAGE data and pharmacological characterizations of EFS-induced m-phosphorylated on Ser19 or both Thr18/Ser19. We are relaxation. Sadly, Ma Jun succumbed to a serious disease before not aware of an investigation of the effect of Thr18 finishing her PhD thesis. Specificity of the anti-pThr antibody was m-phosphorylation on force. However, our results suggest tested by Peptide Specialty Laboratories, Heidelberg, Germany. This work was supported by the Medical Faculty of the University of that pThr18 m-phosphorylated myosin does not support Cologne (Ko¨ln Fortune to GP), grants from the Deutsche Fors- force. Exchanging endogenous RLC with m-phosphory- chungsgemeinschaft (SFB 612 to GP and FOR1352 to BW), and lated at Thr18 in skinned fibres should help to resolve this Excellence Initiative of the German Federal & State Governments question. Thus, it is also not known whether m-phosphor- (EXC 294 BIOSS) to BW. ylation of this site influences relaxation or whether the Open Access This article is distributed under the terms of the phosphorylation rebound is a paraphenomenon of high NO Creative Commons Attribution License which permits any use, dis- activation. As pThr18 m-phosphorylated myosin is capable tribution, and reproduction in any medium, provided the original for dimerization and filament formation, it is tempting to author(s) and the source are credited. speculate that it may help to stabilize myosin filaments at low levels of RLC phosphorylation at Ser 19. Further studies are required to assess the functional relevance of References pThr18 m-phosphorylation during relaxation. Assuming that pSer19 regulates attachment of cross- Albrecht K, Schneider A, Liebetrau C, Ruëgg JC, Pfitzer G (1997) bridges and that the remaining phosphorylation of Ser19 is Exogenous caldesmon promotes relaxation of guinea-pig below the threshold for activation of contraction, dephos- skinned taenia coli smooth muscles: inhibition of cooperative reattachment of latch bridges? 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In: Ba´rańyM(ed)Biochemistryofsmoothmuscle situation, additional mechanisms may still be necessary contraction. Academic Press, San Diego, pp 321–339 which either increase the net detachment rate of dephos- Barron JT, Ba´rańy M, Ba´rańy K (1979) Phosphorylation of the phorylated crossbridges or inhibit the cooperative reat- 20,000-dalton light chain of myosin of intact arterial smooth tachment of dephosphorylated crossbridges (Albrecht et al. muscle in rest and in contraction. J Biol Chem 254:4954–4956 Barron JT, Ba´rańy M, Ba´rańy K, Storti RV (1980) Reversible 1997, Malmqvist et al. 1997; reviewed in Kim et al. 2008). phosphorylation of the 20,000-dalton light chain of myosin In conclusion, the initial phase of cGMP/cAMP-medi- during the contraction-relaxation-contraction cycle of arterial ated relaxation of gastric fundus smooth muscle induced by smooth muscle. J Biol Chem 255:6238–6344 123 268 6.3. New insights into myosin phosphorylation

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1.1. Electron micrograph and schematic overview of the structural elements of one sarcomere...... 2 1.2. Schematic overview of the composition of a skeletal muscle...... 3 1.3. Schematic overview of the Z-disc...... 6 1.4. Schematic illustration of the arragement of the FLNa domains and the suggested model for the interaction of FLNa with ﬁlamentous actin. . . . .8 1.5. Schematic overview of FLNc’s domain structure and a selection of its domain-speciﬁc interaction partners...... 10 1.6. Illustration of kinase-mediated O-phosphorylation and phosphatase-dependent dephosphorylation...... 13 1.7. Schematic illustration of phosphorylation-mediated signaling processes. . 14

3.1. Hierarchical cluster analysis for the normalized intensities of identiﬁed proteins from C2C12 myotubes obtained with different lysis buffers. . . . 27 3.2. Staining of C2C12 myotubes for α-actinin and the Z-disc-associated end of titin (T12)...... 29 3.3. Cell culture and phosphoproteomic workﬂow for global analysis of the C2C12 myotube phosphoproteome...... 30 3.4. Overview of the contracting myotube (phospho)proteome...... 31 3.5. The Z-disc is the main site of protein phosphorylation in sarcomeres. . . . 34 3.6. Schematic illustration of highly phosphorylated Z-disc proteins...... 36 302 List of Figures

3.7. FLNc is phosphorylated in its hinge 2 region at S2621, S2625, and S2633 in mouse C2C12 myotubes...... 38 3.8. Mouse and human FLNc domain 23-24 are phosphorylated by PKCα in a concentration dependent manner ...... 39 3.9. S2625 and S2623/S2624 are the specific substrates sites of PKCα in the hinge 2 region of mFLNC and hFLNc, respectively...... 41 3.10. Establishment of an on-bead calpainolysis assay for hFLNc d23-24. . . . . 45 3.11. Y2625 is the major calpain 1-dependent cleavage site in the hinge 2 region of hFLNc...... 46 3.12. Calpain 1 cleavage efficiency at Y2625 in hFLNc is sequence-dependent. . 47 3.13. PKC-mediated phosphorylation protects FLNc from calpain 1-dependent proteolysis in C2 cells...... 49 3.14. Predicted mechanism for competing calpain 1 and PKCα interaction with FLNc...... 51 3.15. Time-dependent inhibitor screen in differentiated, contracting C2 myotubes. 54 3.16. Quantitative substrate mapping of the Pi3k pathway in contracting C2 myotubes...... 55 3.17. Quantitative phosphoproteomics analysis of Pi3k/Akt signaling in contracting C2 myotubes...... 57 3.18. Determination of regulated phosphopeptides and enriched phospho-motifs in C2 myotubes following stimulation or inhibition of Pi3k/Akt signaling. 59 3.19. Pathway analysis of proteins with regulated phosphosites...... 60 3.20. The FLNc unique 82 amino acid insertion within Ig-like domain 20 is a phosphorylation hot spot...... 63 3.21. S2233 and S2236 of human FLNc are specific substrate sites of Akt and PKCα, respectively...... 64 3.22. Identification of putative FLNc interaction partners by proximity-dependent biotinylation...... 66 List of Figures 303

3.23. S2233 and S2236 have regulatory function in the interaction of FLNc with Filip1...... 68 3.24. Analysis of calpain 1 cleavage sites in human FLNc domains...... 70 3.25. Sequence coverage of standard proteins digested with different proteases. . 74 3.26. Analysis of phosphorylation sites in myosin light chain 20 (Mlc20) by high resolution mass spectrometry...... 75

6.1. Phosphorylation transients during electrical-field-induced relaxation of endothelin- 1 (ET-1) precontracted smooth muscle strips from mouse gastric fundus. . 262 6.2. Identification of RLC and fragment spectra of phosphopeptides...... 264 6.3. Determination of m-phosphorylation of Thr18 during EPS induced relaxation using phospho-specific antibodies...... 265

A.1. Staining of C2C12 myotubes for ﬁlamin C and the Z-disc associated end of titin (T12)...... 307 A.2. Overview of mouse and human FLNc constructs...... 308 A.3. Controls for the radioactive in vitro kinase assays...... 309 A.4. Controls for the MS-based in vitro kinase assays...... 309 A.5. Prediction motifs and position speciﬁc scoring matrix for in silico prediction of calpain cleavage sites...... 310 A.6. Intact mass measurements of calpain 1-dependent aminoterminal cleavage products of human FLNc d22-23 WT and phosphosite mutants...... 310 A.7. Y2626 is the main calpain 1-dependent cleavage site in the hinge 2 region of mFLNc ...... 311 A.8. Time line for differentiation of C2 myoblats into contracting myotubes for signaling studies...... 312 A.9. Reproducibility and characterization of the quantitative phosphoproteome in contracting C2 myotubes...... 313 A.10. Proximity ligation assay results for the interaction of phospho-FLNc S2234 with AKT...... 314 304 List of Figures

A.11. Abundance of endogenous FLNc phosphopeptides within the large-scale SILAC experiment ...... 315

C.1. Vector backbone of pET-23a(+) vector from Novagen...... 324 C.2. Vector backbone of pGEX vector from Amersham...... 325 C.3. Vector backbone of pcDNA3.1. (-) vector from Addgene...... 326 C.4. Vector backbone of pLenti vector from Origene...... 327 List of Tables

4.1. Summary of buffer compositions that were used for the establishment of myotube cell lysis...... 78 4.2. Summary of different digestion conditions...... 80

B.1. In silico calpainolysis prediction of human FLNc...... 318 B.2. List of the 20 phosphopeptides comprising the extendent basophilic motif RxRxxpSxxS...... 320 B.3. Speciﬁc protease cleavage rules according to expasy.org ...... 322

E.1. MaxQuant parameters for the analysis of lysis buffer optimization...... 349

APPENDIX A

Figures

A.1 Supplemental figures for identification of Z-disc as myofibrillar phosphorylation hot spots

I II III

FLNc titin T12 merge IV V VI

FLNc titin T12 merge

Figure A.1.: Staining of C2C12 myotubes for filamin C and the Z-disc associated end of titin (T12). Cells were either subjected to EPS protocol with 0.5 Hz, 4 ms, 10 V (I-III) or 0.5 Hz, 4 ms, 12 V (IV-VI). Co-localization of filamin c and titin (III and VI) was observable in Z-disc regions within the myotube. 308 A.1. Supplemental figures for Z-discs as myofibrillar phosphorylation hot spots

A mFLNc d23-24 WT SSSSRGASYSSIPKFSSDASK 124 144 A SSSSRGAAYSSIPKFSSDASK 124 144 D SSSSRGADYSSIPKFSSDASK 124 144

M1 F243 His- EEF- 23 24 tag tag M1 F243 hFLNc d23-24 WT SSSSRGSSYSSIPKFSSDASK 124 144 AA SSSSRGAAYSSIPKFSSDASK 124 144 DD SSSSRGDDYSSIPKFSSDASK 124 144

B Myc- BirA* hFLNc d22-24 tag 22 23 24 M1 E664 WT SSSSRGSSYSSIPKFSSDASK 554 574 AA SSSSRGAAYSSIPKFSSDASK 554 574 DD SSSSRGDDYSSIPKFSSDASK 554 574

Figure A.2.: Overview of mouse and human FLNc constructs. (A) Schematic illustration of mFLNc and hFLNc d23-24 constructs comprising a His6- and EEF-tag at the carboxyterminal end. Phosphosite mutants were generated from the respective WT construct by exchange of one or two serine residues (S) to alanine (A) or aspartate (D) as indicated. WT, wildtype; d, domain (B) Schematic illustration of mFLNc and hFLNc d22-24 constructs comprising a BirA* sequence and 6xHis tag at the N-terminus. Phosphosite mutants were generated from the respective WT construct as described in (B). BirA*, promiscuous biotin ligase; WT, wildtype; d, domain A.1. Supplemental ﬁgures for Z-discs as myoﬁbrillar phosphorylation hot spots 309

hFLNc d23-24 mFLNc d23-24 WT WT AA DD WT AD PKCα + ------ATP + + + + +++ 80 kDa- - PKC 60 kDa- 29 kDa-

24 kDa- - FLNc 29 kDa- - FLNc 24 kDa-

Figure A.3.: Controls for the radioactive in vitro kinase assays. Recombinant wildtype and phosphosite mutants of mouse and human FLNc Ig-like domains 23-24 (d23-24) were treated with [γ-33P]ATP in the absence of PKCα and analysed by SDS-PAGE followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. WT, wildtype; A, alanine; D, aspartate

n.s. 6.0E10

4.0E10 n.s.

Intensity 2.0E10

0.0

hFLNc WThFLNc AAhFLNc DDmFLNc WTmFLNc AmFLNc D

Figure A.4.: Controls for the MS-based in vitro kinase assays. Abundance of recombinant mouse and human FLNc d23-24 wildtype and phosphosite mutants in mass spectrometry-based in vitro kinase assays. Shown are the mean values of the total peptide intensity measured for each of the FLNc d23-24 constructs (see Figure S2B). Data derived from three independent experiments per construct. Error bars represent the SEM. WT, wildtype; A, alanine, D, aspartate; n.s., not significant 310 A.1. Supplemental figures for Z-discs as myofibrillar phosphorylation hot spots

Figure A.5.: Prediction motifs and position specific scoring matrix for in silico prediction of calpain cleavage sites according to www.calpain.org. (A) Sequence logo for the favored (upper half) and disfavored (lower half) amino acid in position -10 to +10 flanking the predicted calpain cleavage site. (B) Position specific scoring matrix for the amino acids favored and disfavored in position -10 to +10 flanking the predicted calpain cleavage site. Modified from www.calpain.org on 04.07.2016.

AB hFLNc d23-24 WT hFLNc d23-24 AA 2TRG...AAY132 2TRG...SSY132 100 13323.72 100 13355.66 2TRG...AAY(ox.)132 2TRG...SSY(ox.)132 13338.69 13370.68 2TRG...KSS125 50 12631.37 2TRG...AYS133 50 2TRG...SYS133 13410.74 2TRG...KSS125 13442.68 12631.37 1MTR...SSY132 1MTR...AAY132 13485.72 13454.70 Relative Abundance Relative Abundance 13574.71 0 0 12500 13000 13500 12500 13000 13400 m/z m/z

C hFLNc d23-24 DD 2TRG...DDY132 13411.64 100

2TRG...DDY(ox.)132 13427.66

2TRG...DYS133 2TRG...KSS125 13498.64 50 12631.15 1MTR...DDY132 13667.65 Relative Abundance 0 12500 13000 13500 m/z

Figure A.6.: Intact mass measurements of calpain 1-dependent aminoterminal cleavage products of human FLNc d22-23 WT and phosphosite mutants. (A)-(C) Shown are sections of representative deconvoluted full MS spectra from intact mass measurements of aminoterminal cleavage products of hFLNc d23-24 WT (A), AA (B) and DD (C) mutants (see figure A.2). Se- quence range and molecular masses of cleavage products are depicted. WT, wildtype; A, alanine, D, aspartate; m/z, mass-to-charge ratio A.1. Supplemental figures for Z-discs as myofibrillar phosphorylation hot spots 311

50 20

13572.73

Relative Abundance 2TRG...KSS(ox.)125 TRG...SSS 12646.33 2 127 normalized intensity [%] 13104.54 0 0 12500 13000 13500 m/z S2619 Y2626 S2627 CF60 mFLNc A 2TRG...AAY132 100 13338.66 2TRG...AYS133 13425.68 40 1MTR...AAY132 13469.68

50 20

Relative Abundance TRG...SSS 2TRG...KSS(ox.)125 2 127 13556.71 12646.33 13104.55 normalized intensity [%] 0 0 12500 13000 13500 m/z S2619 Y2626 S2627

DG40

mFLNc D 2TRG...DYS133 13469.67 100 2TRG...ADY132 1MTR...ADY132 13382.64 13513.67 30 1MTR...DYS133 13600.68 20 50

Relative Abundance 2TRG...KSS(ox.)125 12646.38 normalized intensity [%] 0 0 12500 13000 13500 S2619 Y2626 S2627 m/z Figure A.7.: Y2626 is the main calpain 1-dependent cleavage site in the hinge 2 region of mFLNc (A) Amino acid sequence of mFLNc d23-24 wildtype (WT) form fused to a His6 and EEF tag at the carboxyterminal end used as recombinant protein in in vitro proteolysis assays with recombinant human calpain 1. The sequence of the major aminoterminal cleavage product and the corresponding main calpain 1 cleavage site are depicted in black and red, respectively. (B-D) Sections of representative deconvoluted full MS spectra from intact mass measurements of calpain 1-dependent aminoterminal cleavage products of mFLNc d23-24-His6-EEF WT (B), S2625A (C) and S2625D (D) phosphosite mutants (see Fig. S2B). Sequences of cleavage products identified by top-down MS analyses are indicated. WT, wildtype; A, alanine, D, aspartate; S, serine; m/z, mass-to-charge ratio; (ox.), oxidized methionine was identified in the sequence. (G-H) Quantification of data shown in (B-D). MS signals from three independent experiments were quantified and represented as normalized mean ± SEM. Calpain 1-dependent cleavage sites are denoted. 312 A.2. Supplemental figures for quantitative analysis of the myofibrillar phosphoproteome

A.2 Supplemental ﬁgures for quantitative analysis of the myoﬁb- rillar phosphoproteome

d0 d1 d2 d3 d4

PM DMDM SM EPS contracting myoblasts cell fusion myotubes

Figure A.8.: Time line for differentiation of C2 myoblats into contracting myotubes for signaling studies. Myoblasts are grown to a confluence of ∼90% in proliferation medium (PM) containing 15% fetal calf serum (FCS). Cell fusion is induced by a change to differentiation medium (DM) containing 2% FCS or 2% horse serum (HS). DM is changed every 48 h. At the end of the third day (d3) myotubes are starved overnight in starvation medium (SM) without serum. 4 h prior to cell culture experiments myotubes are stimulated with EPS. Chemical components for signaling studies are provided in fresh SM and myotubes are continued to be stimulated with EPS during cell treatment. A.2. Supplemental figures for quantitative analysis of the myofibrillar phosphoproteome 313

A B C 10 10 10 0.785 0.668 0.767 0.664 0.775 0.666 10e 10e 10e 10 5e 10 10 5e 5e 10 10 10 0.785 0.669 0.767 0.665 0.775 0.66 10e 10e 10e 10 5e 10 10 5e 5e 10 10 10 0.668 0.669 0.664 0.665 0.666 0.66 10e 10e 10e 10 IGF-1 3 IGF-1 2 IGF-1 1 5e control 3 control 2 control 1 10 10 LY294002 3 LY294002 2 LY294002 1 5e 5e

5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 5e10 10e10 control 1 control 2 control 3 IGF-1 1 IGF-1 2 IGF-1 3 LY294002 1 LY294002 2 LY294002 3 D G 6 pT pS 18.28% 79.91% 4

2 pY 95% 95% 95% 95% 95% 95%

1.81% ratio 0 2 5% 5% E F 5% 5% 5% 5%

-log -2 3 pSTY 3+ 10.26%2 pSTY 53.46% -4

2 pSTY -6 48.57% 1 pSTY 2+ 41.57% 36.16% 5+ IGF-1 1 IGF-1 2 IGF-1 3 0.48% LY294002 1 LY294002 2 LY294002 3 4+ 9.90%

Figure A.9.: Reproducibility and characterization of the quantitative phosphoproteome in contracting C2 myotubes. (A-C) Multi-scatter plots and Pearson correlation analysis of the three different treatments, control DMSO, IGF-1 and LY294002 within the tree biological replicates of the large-scale SILAC experiment. (D) Distribution of modified amino acid residues among phosphopeptides identified by SCX-fractionation, subsequent TiO2 enrichment and LC-MS/MS fragmentation with MSA and HCD. (E) Distribution of singly, doubly and triply phosphorylated peptides enriched in our large-scale SILAC experiment. (F) Distribution of charge states of the analyzed phosphopeptides ionized via electro spray. (G) Box-plot analysis of phosphopeptide ratios reveals that only a minority of peptides is significantly regulated (1.5-fold SILAC ratio change) 314 A.2. Supplemental figures for quantitative analysis of the myofibrillar phosphoproteome

AB 1h IGF-1 actinin 1h IGF-1 5E4 PLA signal

2.5E4 intensity [~]

0 0 10203040 distance [µm] CD -actinin 1h LY294002 5E4 1h LY294002 PLA signal

2.5E4 intensity [~]

0 0 10203040 distance [µm]

Figure A.10.: Proximity ligation assay results for the interaction of phospho-FLNc S2234 with AKT. C2C12 myotubes were permeabilized, blocked with normal goat serum and incubated with antibodies directed against phospho-FLNc S2234 and pan AKT. Z-disc staining was performed with an antibody directed against α-actinin. (A) and (B) Fluorescence microscopy image and intensity distance measurement of a myotube after 1 h of IGF-1 stimulation. (C) and (D) Fluorescence microscopy image and intensity distance measurement of a myotube after 1 h of LY294002 treatment. Red dots indicate co-localization of phospho-FLNc S2234 and AKT antibodies, white bar: 40 µm. A.2. Supplemental ﬁgures for quantitative analysis of the myoﬁbrillar phosphoproteome 315

3x109

2x109

1x109

4x107 mean intensity 2x107

0 S2234 S2237 T2239 phosphorylation site

Figure A.11.: Abundance of endogenous FLNc phosphopeptides within the large-scale SILAC experiment. Shown are the mean intensities of the phosphopeptides modiﬁed at the given residue for each treatment in the large-scale SILAC experiment. Data derived from three independent experiments. Error bars represent the SEM.

APPENDIX B

Tables

B.2 Supplemental tables for characterization of Pi3k/Akt-mediated signaling events in myotubes 320 B.2. Supplemental tables for Pi3k/Akt-mediated signaling events S(0.189)GS(0.762)GMS(0.048)VIS(0.002)S(0.002)S(0.006)S(0.002)VDQRLPEE PS(0.338)S(0.651)EDEQQPEK Phospho (STY) Probabilities 2 IGF-1 Mean log 2 LY294002 Mean log within proteins Positions List of the 20 phosphopeptides comprising the extendent basophilic motif RxRxxpSxxS. Edc3Eif4b Q8K2D3Erbb3 161 Q8BGD9 -0.94075 406 Q61526Gtf2f1 0.19627 -0.98967 1091 HNS(1)WSSSSRHPNQATPK Q3THK3 0.30048 -1.10913 ERHPS(1)WRS(1)EETQERER 377 0.30743Map3k3 QT(0.101)S(0.876)ES(0.235)S(0.787)EGHVTGSEAELQER -1.20507Mast2 Q61084 0.66301 KPS(0.989)GGS(0.004)S(0.004)KGT(0.001)SRPGTPSAEAASTSSTLR 166 B1AST8Pfkfb2 -0.88913 969 0.46936Pom121 -0.90639 HLS(1)VS(0.78)S(0.119)QNPGRS(0.027)S(0.075)PPPGYVPER B2Z892 0.41392 H7BX32 464 LLS(1)GDS(1)IEKR 367 -1.55382Srrm2 -1.02777 0.57811 NYS(1)VGSRPLKPLSPLR 0.29783 Q8BTI8Tsr3 S(0.003)RT(0.005)S(0.022)S(0.97)VS(0.008)S(0.992)LASACTGGIPSSSR 946Zzef1 Q5HZH2 -0.67475 Q5SSH7-2 0.37939 21 S(0.048)RS(0.431)S(0.55)S(0.968)PDS(0.003)KMELGTPLR 1535 -1.16053 -1.32191 0.28998 0.47627 VRRPS(1)GRS(1)LDAFAEEVGAALR LLS(1)FRS(0.976)MEET(0.024)RPVPTVK Table B.2. Protein namesEnhancer of mRNA-decapping protein 3 Eukaryotic translation initiation factor 4B Receptor Gene tyrosine-protein names kinase erbB-3 Proteins Filamin-CGeneral transcription factor IIF subunit 1 Intersectin-1La-related protein 1Mitogen-activated protein kinase kinase kinase 3 FlncMicrotubule-associated serine/threonine-protein kinase 2 Protein NDRG1 Larp1 Itsn1 Q8VHX6Protein NDRG26-phosphofructo-2-kinase/ 2234fructose-2,6-bisphosphatase 2 Z4YJT3 E9Q3I8 -1.63064Nuclear envelope pore membrane protein 1032 POM 0.43807 121 315Protein PRRC2A Ndrg1 LGS(1)FGS(0.994)IT(0.006)R -1.27715Protein PRRC2B Ndrg2 -0.62421 0.64768Protein PRRC2C 0.53678 Serine/arginine CPS(0.994)QS(0.004)S(0.001)S(0.001)RPAT(0.001)GISQPPTTPTGQATREDAK Q62433 repetitive matrix Q9QYG0protein 2 330TBC1 domain family member 350 4 Prrc2aRibosome biogenesis protein TSR3 -0.85634 Prrc2bhomolog -0.75629 Tbc1d4 Prrc2cZinc -0.34036 ﬁnger ZZ-type and Q7TSC1 0.42221 EF-hand S(0.659)RT(0.341)AS(1)GSSVTSLEGTR domain-containing protein 1 F8WHT3 T(0.142)LS(0.825)QS(0.34)S(0.34)ES(0.34)GT(0.012)LPSGPPGHT(0.002)MEVSC H7BX82 761 Q3TLH4 415 348 -0.7223 901 -0.93159 -1.0768 0.45192 -2.32426 0.28744 ERSDS(1)GGS(0.058)S(0.902)S(0.039)EPFER 0.45509 ALS(1)LSSADSTDAKR 0.88925 HAS(1)APSHVQPSDSEKNR S(0.012)VS(0.988)HGS(0.999)NHAQNAEEQRNEPSVSIPK B.3. Supplemental tables for new insights into smooth muscle myosin phosphorylation 321

B.3 Supplemental tables for new insights into smooth muscle myosin phosphorylation 322 B.3. Supplemental tables for new insights into myosin phosphorylation if P is C-term to F, Y, W, ifN-term P to is Y Speciﬁc protease cleavage rules according to expasy.org Table B.3. Enzyme or ReagentTrypsin Cleavage sideElastaseLys CLys NCNBr C-terminal side of K or RArg C C-terminal side of R, V, I, L,Asp G N/ Lys C C-terminal sideAsp of N K Exceptions + N-terminal Glu N-terminal sideAsp of N-terminal N/ K side Glu of C D C-terminal or side E of M if N-terminal PChymotrypsin side is of C-term C-terminal D, to side C-terminal K of side or R of R K Glu C N-terminal side of D, C-terminalPepsin side (pH of C-terminal >2) E side of F, Y, W Proteinase KThermolysin C-terminal side of F, L, W, if Y, A, P C-terminal E, is side Q C-term of to E C-terminal R side Asp of N A, N-terminal F, side Y, of W, L, D N-terminal I, side V of A, F, I, L, M, V if D or E is N-term to A, F, if I, P L, is M, C-term V to E, or if E is C-term to E APPENDIX C

Vector Maps

C.1 Vector maps of vector backbones 324 C.1. Vector maps of vector backbones

Figure C.1.: Vector backbone of pET-23a(+) vector from Novagen. The backbone was used for cloning of all FLNc d1-3, d15-21, d16-24, d18-21 and d23-24 constructs used for recombinant expression in E. coli. C.1. Vector maps of vector backbones 325

(4848) BtgI Bf uAI - BspMI (62)

(4740) Bsu36I EcoNI (268)

lac operator MscI (465)

(4290) PluTI (4288) Sf oI BstBI (655) (4287) NarI* SwaI (685) (4286) KasI

(4153) HpaI

(4097) EcoRV

(4058) BssHII BamHI (930) AvaI - BsoBI - TspMI - XmaI (935) BmeT110I (936) SmaI (937) EcoRI (940) Pf oI (1015)

(3858) ApaI - BanII (3854) PspOMI Pf lFI - Tth111I (1118) (3828) BstEII BsaAI (1125)

ZraI (1222) pGEX-2T AatII (1224) 4 9 4 8 bp

(3647) MluI

(3330) BstAPI

(3229) Pf lMI

PstI (1901)

BsaI (2077) AhdI (2143)

(2622) AlwNI

Figure C.2.: Vector backbone of pGEX vector from Amersham. The backbone was used for cloning of FILIP1 C-terminal constructs used for recombinant expression in E. coli. 326 C.1. Vector maps of vector backbones

Figure C.3.: Vector backbone of pcDNA3.1. (-) vector from Addgene. The backbone was used for cloning of BirA*, BirA*FLNc d18-21 and d22-24 constructs used for transfection of C2 and HEK293 cells. C.1. Vector maps of vector backbones 327

Figure C.4.: Vector backbone of pLenti vector from Origene. The backbone was used for cloning of FILIP1-GFP C-term constructs used for transfection of C2 and HEK293 cells.

APPENDIX D

Alignments

D.1 Alignment of ﬁlamin isoforms 330 D.1. Alignment of ﬁlamin isoforms

P21333 FLNA_HUMAN 1 MSSSHSRAGQSAAGAAPGGGVDTRDAEMPATEKDLAEDAPWKKIQQNTFTRWCNEHLKCV 60 O75369 FLNB_HUMAN 1 ------MPVTEKDLAEDAPWKKIQQNTFTRWCNEHLKCV 33 Q14315 FLNC_HUMAN 1 ---MMNNSGY----SDAGLGLGDETDEMPSTEKDLAEDAPWKKIQQNTFTRWCNEHLKCV 53 ** ******************************

P21333 FLNA_HUMAN 61 SKRIANLQTDLSDGLRLIALLEVLSQKKMHRKHNQRPTFRQMQLENVSVALEFLDRESIK 120 O75369 FLNB_HUMAN 34 NKRIGNLQTDLSDGLRLIALLEVLSQKRMYRKYHQRPTFRQMQLENVSVALEFLDRESIK 93 Q14315 FLNC_HUMAN 54 GKRLTDLQRDLSDGLRLIALLEVLSQKRMYRKFHPRPNFRQMKLENVSVALEFLEREHIK 113 **: :** ******************:*:**.. **.****:***********:** **

P21333 FLNA_HUMAN 121 LVSIDSKAIVDGNLKLILGLIWTLILHYSISMPMWDEEEDEEAKKQTPKQRLLGWIQNKL 180 O75369 FLNB_HUMAN 94 LVSIDSKAIVDGNLKLILGLVWTLILHYSISMPVWEDEGDDDAKKQTPKQRLLGWIQNKI 153 Q14315 FLNC_HUMAN 114 LVSIDSKAIVDGNLKLILGLIWTLILHYSISMPMWEDEDDEDARKQTPKQRLLGWIQNKV 173 ********************:************:*::* *::*:***************:

P21333 FLNA_HUMAN 181 PQLPITNFSRDWQSGRALGALVDSCAPGLCPDWDSWDASKPVTNAREAMQQADDWLGIPQ 240 O75369 FLNB_HUMAN 154 PYLPITNFNQNWQDGKALGALVDSCAPGLCPDWESWDPQKPVDNAREAMQQADDWLGVPQ 213 Q14315 FLNC_HUMAN 174 PQLPITNFNRDWQDGKALGALVDNCAPGLCPDWEAWDPNQPVENAREAMQQADDWLGVPQ 233 * ******.::**.*:*******.*********::** .:** **************:**

P21333 FLNA_HUMAN 241 VITPEEIVDPNVDEHSVMTYLSQFPKAKLKPGAPLRP-KLNPKKARAYGPGIEPTGNMVK 299 O75369 FLNB_HUMAN 214 VITPEEIIHPDVDEHSVMTYLSQFPKAKLKPGAPLKP-KLNPKKARAYGRGIEPTGNMVK 272 Q14315 FLNC_HUMAN 234 VIAPEEIVDPNVDEHSVMTYLSQFPKAKLKPGAPVRSKQLNPKKAIAYGPGIEPQGNTVL 293 **:****:.*:***********************:: :****** *** **** ** *

P21333 FLNA_HUMAN 300 KRAEFTVETRSAGQGEVLVYVEDPAGHQEEAKVTANNDKNRTFSVWYVPEVTGTHKVTVL 359 O75369 FLNB_HUMAN 273 QPAKFTVDTISAGQGDVMVFVEDPEGNKEEAQVTPDSDKNKTYSVEYLPKVTGLHKVTVL 332 Q14315 FLNC_HUMAN 294 QPAHFTVQTVDAGVGEVLVYIEDPEGHTEEAKVVPNNDKDRTYAVSYVPKVAGLHKVTVL 353 : *.***:* .** *:*:*::*** *. ***:*. :.**::*::* *:*:*:* ******

P21333 FLNA_HUMAN 360 FAGQHIAKSPFEVYVDKSQGDASKVTAQGPGLEPSGNIANKTTYFEIFTAGAGTGEVEVV 419 O75369 FLNB_HUMAN 333 FAGQHISKSPFEVSVDKAQGDASKVTAKGPGLEAVGNIANKPTYFDIYTAGAGVGDIGVE 392 Q14315 FLNC_HUMAN 354 FAGQNIERSPFEVNVGMALGDANKVSARGPGLEPVGNVANKPTYFDIYTAGAGTGDVAVV 413 ****.* :***** * : ***.**:*:***** **:*** ***:*:*****.*:: *

P21333 FLNA_HUMAN 420 IQDPMGQKGTVEPQLEARGDSTYRCSYQPTMEGVHTVHVTFAGVPIPRSPYTVTVGQACN 479 O75369 FLNB_HUMAN 393 VEDPQGK-NTVELLVEDKGNQVYRCVYKPMQPGPHVVKIFFAGDTIPKSPFVVQVGEACN 451 Q14315 FLNC_HUMAN 414 IVDPQGRRDTVEVALEDKGDSTFRCTYRPAMEGPHTVHVAFAGAPITRSPFPVHVSEACN 473 : ** *: *** :* :*:..:** *:* * *.*:: *** * :**: * *.:***

P21333 FLNA_HUMAN 480 PSACRAVGRGLQPKGVRVKETADFKVYTKGAGSGELKVTVKGPKGEE-RVKQKDLGDGVY 538 O75369 FLNB_HUMAN 452 PNACRASGRGLQPKGVRIRETTDFKVDTKAAGSGELGVTMKGPKGLEELVKQKDFLDGVY 511 Q14315 FLNC_HUMAN 474 PNACRASGRGLQPKGVRVKEVADFKVFTKGAGSGELKVTVKGPKGTEEPVKVREAGDGVF 533 *.**** **********::*.:**** **.****** **:***** * ** :: ***:

P21333 FLNA_HUMAN 539 GFEYYPMVPGTYIVTITWGGQNIGRSPFEVKVGTECGNQKVRAWGPGLEGGVVGKSADFV 598 O75369 FLNB_HUMAN 512 AFEYYPSTPGRYSIAITWGGHHIPKSPFEVQVGPEAGMQKVRAWGPGLHGGIVGRSADFV 571 Q14315 FLNC_HUMAN 534 ECEYYPVVPGKYVVTITWGGYAIPRSPFEVQVSPEAGVQKVRAWGPGLETGQVGKSADFV 593 **** .** * ::***** * :*****:*. *.* **********. * **:*****

P21333 FLNA_HUMAN 599 VEAIGDDVGTLGFSVEGPSQAKIECDDKGDGSCDVRYWPQEAGEYAVHVLCNSEDIRLSP 658 O75369 FLNB_HUMAN 572 VESIGSEVGSLGFAIEGPSQAKIEYNDQNDGSCDVKYWPKEPGEYAVHIMCDDEDIKDSP 631 Q14315 FLNC_HUMAN 594 VEAIGTEVGTLGFSIEGPSQAKIECDDKGDGSCDVRYWPTEPGEYAVHVICDDEDIRDSP 653 **:** :**:***::********* :*: ******:*** * ******::*:.***: **

P21333 FLNA_HUMAN 659 FMADIRDAPQDFHPDRVKARGPGLEKTGVAVNKPAEFTVDAKHGGKAPLRVQVQDNEGCP 718 O75369 FLNB_HUMAN 632 YMAFIHPATGGYNPDLVRAYGPGLEKSGCIVNNLAEFTVDPKDAGKAPLKIFAQDGEGQR 691 Q14315 FLNC_HUMAN 654 FIAHILPAPPDCFPDKVKAFGPGLEPTGCIVDKPAEFTIDARAAGKGDLKLYAQDADGCP 713 ::* * * ** *:* ***** :* *:: ****:* : .**. *:: .** :*

P21333 FLNA_HUMAN 719 VEALVKDNGNGTYSCSYVPRKPVKHTAMVSWGGVSIPNSPFRVNVGAGSHPNKVKVYGPG 778 O75369 FLNB_HUMAN 692 IDIQMKNRMDGTYACSYTPVKAIKHTIAVVWGGVNIPHSPYRVNIGQGSHPQKVKVFGPG 751 Q14315 FLNC_HUMAN 714 IDIKVIPNGDGTFRCSYVPTKPIKHTIIISWGGVNVPKSPFRVNVGEGSHPERVKVYGPG 773 :: : . :**: ***.* * :*** : ****.:*.**:***:* ****::***:*** P21333 FLNA_HUMAN 779 VAKTGLKAHEPTYFTVDCAEAGQGDVSIGIKCAPGVVGPAEADIDFDIIRNDNDTFTVKY 838 O75369 FLNB_HUMAN 752 VERSGLKANEPTHFTVDCTEAGEGDVSVGIKCDARVLSEDEEDVDFDIIHNANDTFTVKY 811 Q14315 FLNC_HUMAN 774 VEKTGLKANEPTYFTVDCSEAGQGDVSIGIKCAPGVVGPAEADIDFDIIKNDNDTFTVKY 833 * ::****.***:*****:***:****:**** *:. * *:*****:* ********

P21333 FLNA_HUMAN 839 TPRGAGSYTIMVLFADQATPTSPIRVKVEPSHDASKVKAEGPGLSRTGVELGKPTHFTVN 898 O75369 FLNB_HUMAN 812 VPPAAGRYTIKVLFASQEIPASPFRVKVDPSHDASKVKAEGPGLSKAGVENGKPTHFTVY 871 Q14315 FLNC_HUMAN 834 TPPGAGRYTIMVLFANQEIPASPFHIKVDPSHDASKVKAEGPGLNRTGVEVGKPTHFTVL 893 .* .** *** ****.* *:**:::**:***************.::*** ********

P21333 FLNA_HUMAN 899 AKAAGKGKLDVQFSGLTKGDAVRDVDIIDHHDNTYTVKYTPVQQGPVGVNVTYGGDPIPK 958 O75369 FLNB_HUMAN 872 TKGAGKAPLNVQFNSPLPGDAVKDLDIIDNYDYSHTVKYTPTQQGNMQVLVTYGGDPIPK 931 Q14315 FLNC_HUMAN 894 TKGAGKAKLDVQFAGTAKGEVVRDFEIIDNHDYSYTVKYTAVQQGNMAVTVTYGGDPVPK 953 :*.***. *:*** . *:.*:*.:***.:* ::***** .*** : * *******:**

P21333 FLNA_HUMAN 959 SPFSVAVSPSLDLSKIKVSGLGEKVDVGKDQEFTVKSKGAGGQGKVASKIVGPSGAAVPC 1018 O75369 FLNB_HUMAN 932 SPFTVGVAAPLDLSKIKLNGLENRVEVGKDQEFTVDTRGAGGQGKLDVTILSPSRKVVPC 991 Q14315 FLNC_HUMAN 954 SPFVVNVAPPLDLSKIKVQGLNSKVAVGQEQAFSVNTRGAGGQGQLDVRMTSPSRRPIPC 1013 *** * *: *******:.** .:* **::* *:*.::******:: : .** :** D.1. Alignment of ﬁlamin isoforms 331

P21333 FLNA_HUMAN 1019 KVEPGLGADNSVVRFLPREEGPYEVEVTYDGVPVPGSPFPLEAVAPTKPSKVKAFGPGLQ 1078 O75369 FLNB_HUMAN 992 LVTPVTGRENSTAKFIPREEGLYAVDVTYDGHPVPGSPYTVEASLPPDPSKVKAHGPGLE 1051 Q14315 FLNC_HUMAN 1014 KLEPGGGAEAQAVRYMPPEEGPYKVDITYDGHPVPGSPFAVEGVLPPDPSKVCAYGPGLK 1073 : * * : ...:::* *** * *::**** ******: :*. * .**** *.****:

P21333 FLNA_HUMAN 1079 GGSAGSPARFTIDTKGAGTGGLGLTVEGPCEAQLECLDNGDGTCSVSYVPTEPGDYNINI 1138 O75369 FLNB_HUMAN 1052 GGLVGKPAEFTIDTKGAGTGGLGLTVEGPCEAKIECSDNGDGTCSVSYLPTKPGEYFVNI 1111 Q14315 FLNC_HUMAN 1074 GGLVGTPAPFSIDTKGAGTGGLGLTVEGPCEAKIECQDNGDGSCAVSYLPTEPGEYTINI 1133 ** .*.** *:*********************::** *****:*:***:**:**:* :**

P21333 FLNA_HUMAN 1139 LFADTHIPGSPFKAHVVPCFDASKVKCSGPGLERATAGEVGQFQVDCSSAGSAELTIEIC 1198 O75369 FLNB_HUMAN 1112 LFEEVHIPGSPFKADIEMPFDPSKVVASGPGLEHGKVGEAGLLSVDCSEAGPGALGLEAV 1171 Q14315 FLNC_HUMAN 1134 LFAEAHIPGSPFKATIRPVFDPSKVRASGPGLERGKVGEAATFTVDCSEAGEAELTIEIL 1193 ** :.********* : ** *** .******:...**.. : ****.** . * :*

P21333 FLNA_HUMAN 1199 SEAGLPAEVYIQDHGDGTHTITYIPLCPGAYTVTIKYGGQPVPNFPSKLQVEPAVDTSGV 1258 O75369 FLNB_HUMAN 1172 SDSGTKAEVSIQNNKDGTYAVTYVPLTAGMYTLTMKYGGELVPHFPARVKVEPAVDTSRI 1231 Q14315 FLNC_HUMAN 1194 SDAGVKAEVLIHNNADGTYHITYSPAFPGTYTITIKYGGHPVPKFPTRVHVQPAVDTSGV 1253 *::* *** *::. ***: :** * * **:*:****. **.**::::*:****** : P21333 FLNA_HUMAN 1259 QCYGPGIEGQGVFREATTEFSVDARALTQTGGPHVKARVANPSGNLTETYVQDRGDGMYK 1318 O75369 FLNB_HUMAN 1232 KVFGPGIEGKDVFREATTDFTVDSRPLTQVGGDHIKAHIANPSGASTECFVTDNADGTYQ 1291 Q14315 FLNC_HUMAN 1254 KVSGPGVEPHGVLREVTTEFTVDARSLTATGGNHVTARVLNPSGAKTDTYVTDNGDGTYR 1313 : ***:* : *:**.**:*:**:* ** .** *:.*:: **** *: :* *..** *:

P21333 FLNA_HUMAN 1319 VEYTPYEEGLHSVDVTYDGSPVPSSPFQVPVTEGCDPSRVRVHGPGIQSGTTNKPNKFTV 1378 O75369 FLNB_HUMAN 1292 VEYTPFEKGLHVVEVTYDDVPIPNSPFKVAVTEGCQPSRVQAQGPGLKEAFTNKPNVFTV 1351 Q14315 FLNC_HUMAN 1314 VQYTAYEEGVHLVEVLYDEVAVPKSPFRVGVTEGCDPTRVRAFGPGLEGGLVNKANRFTV 1373 *:** :*:*:* *:* ** :*.***:* *****:*:**:. ***:: . .** * ***

P21333 FLNA_HUMAN 1379 ETRGAGTGGLGLAVEGPSEAKMSCMDNKDGSCSVEYIPYEAGTYSLNVTYGGHQVPGSPF 1438 O75369 FLNB_HUMAN 1352 VTRGAGIGGLGITVEGPSESKINCRDNKDGSCSAEYIPFAPGDYDVNITYGGAHIPGSPF 1411 Q14315 FLNC_HUMAN 1374 ETRGAGTGGLGLAIEGPSEAKMSCKDNKDGSCTVEYIPFTPGDYDVNITFGGRPIPGSPF 1433 ***** ****:::*****:*:.* *******:.****: * *.:*:*:** :*****

P21333 FLNA_HUMAN 1439 KVPVHDVTDASKVKCSGPGLSPGMVRANLPQSFQVDTSKAGVAPLQVKVQGPKGLVEPVD 1498 O75369 FLNB_HUMAN 1412 RVPVKDVVDPSKVKIAGPGLGSG-VRARVLQSFTVDSSKAGLAPLEVRVLGPRGLVEPVN 1470 Q14315 FLNC_HUMAN 1434 RVPVKDVVDPGKVKCSGPGLGAG-VRARVPQTFTVDCSQAGRAPLQVAVLGPTGVAEPVE 1492 :***:**.* .*** :****. * ***.: *:* ** *:** ***:* * ** *:.***:

P21333 FLNA_HUMAN 1499 VVDNADGTQTVNYVPSREGPYSISVLYGDEEVPRSPFKVKVLPTHDASKVKASGPGLNTT 1558 O75369 FLNB_HUMAN 1471 VVDNGDGTHTVTYTPSQEGPYMVSVKYADEEIPRSPFKVKVLPTYDASKVTASGPGLSSY 1530 Q14315 FLNC_HUMAN 1493 VRDNGDGTHTVHYTPATDGPYTVAVKYADQEVPRSPFKIKVLPAHDASKVRASGPGLNAS 1552 * **.***:** *.*: :*** ::* *.*:*:******:****::***** ******.: P21333 FLNA_HUMAN 1559 GVPASLPVEFTIDAKDAGEGLLAVQITDPEGKPKKTHIQDNHDGTYTVAYVPDVTGRYTI 1618 O75369 FLNB_HUMAN 1531 GVPASLPVDFAIDARDAGEGLLAVQITDQEGKPKRAIVHDNKDGTYAVTYIPDKTGRYMI 1590 Q14315 FLNC_HUMAN 1553 GIPASLPVEFTIDARDAGEGLLTVQILDPEGKPKKANIRDNGDGTYTVSYLPDMSGRYTI 1612 *:******:*:***:*******:*** * *****:: ::** ****:*:*:** :*** *

P21333 FLNA_HUMAN 1619 LIKYGGDEIPFSPYRVRAVPTGDASKCTVTVSIGGHGLGAGIGPTIQIGEETVITVDTKA 1678 O75369 FLNB_HUMAN 1591 GVTYGGDDIPLSPYRIRATQTGDASKCLAT------GPGIASTVKTGEEVGFVVDAKT 1642 Q14315 FLNC_HUMAN 1613 TIKYGGDEIPYSPFRIHALPTGDASKCLVTVSIGGHGLGACLGPRIQIGQETVITVDAKA 1672 :.****:** **:*::* ******* .* * :. :: *:*. :.**:*:

P21333 FLNA_HUMAN 1679 AGKGKVTCTVCTPDGSEVDVDVVENEDGTFDIFYTAPQPGKYVICVRFGGEHVPNSPFQV 1738 O75369 FLNB_HUMAN 1643 AGKGKVTCTVLTPDGTEAEADVIENEDGTYDIFYTAAKPGTYVIYVRFGGVDIPNSPFTV 1702 Q14315 FLNC_HUMAN 1673 AGEGKVTCTVSTPDGAELDVDVVENHDGTFDIYYTAPEPGKYVITIRFGGEHIPNSPFHV 1732 **:******* ****:* :.**:**.***:**:*** :**.*** :**** .:***** * P21333 FLNA_HUMAN 1739 TALAGDQPSVQPPLRSQQLAPQYTYA--QGGQQTWAPERPLVGVNGLDVTSLRPFDLVIP 1796 O75369 FLNB_HUMAN 1703 MATDGEVTAVEEAPVN------ACPPGFRPWVTEEAYVPVSDMNGLGFKPFDLVIP 1752 Q14315 FLNC_HUMAN 1733 LACDPLP-HEEEPSEVPQLRQPYAPPRPGARPTHWATEEPVVPVEPME-SMLRPFNLVIP 1790 * : *. *. * *. :: ::**:****

P21333 FLNA_HUMAN 1797 FTIKKGEITGEVRMPSGKVAQPTITDNKDGTVTVRYAPSEAGLHEMDIRYDNMHIPGSPL 1856 O75369 FLNB_HUMAN 1753 FAVRKGEITGEVHMPSGKTATPEIVDNKDGTVTVRYAPTEVGLHEMHIKYMGSHIPESPL 1812 Q14315 FLNC_HUMAN 1791 FAVQKGELTGEVRMPSGKTARPNITDNKDGTITVRYAPTEKGLHQMGIKYDGNHIPGSPL 1850 *:::***:****:*****.* * *.******:******:* ***:* *:* *** ***

P21333 FLNA_HUMAN 1857 QFYVDYVNCGHVTAYGPGLTHGVVNKPATFTVNTKDAGEGGLSLAIEGPSKAEISCTDNQ 1916 O75369 FLNB_HUMAN 1813 QFYVNYPNSGSVSAYGPGLVYGVANKTATFTIVTEDAGEGGLDLAIEGPSKAEISCIDNK 1872 Q14315 FLNC_HUMAN 1851 QFYVDAINSRHVSAYGPGLSHGMVNKPATFTIVTKDAGEGGLSLAVEGPSKAEITCKDNK 1910 ****: *. *:****** :*:.** ****: *:*******.**:********:* **:

P21333 FLNA_HUMAN 1917 DGTCSVSYLPVLPGDYSILVKYNEQHVPGSPFTARVTGDDSMRMSHLKVGSAADIPINIS 1976 O75369 FLNB_HUMAN 1873 DGTCTVTYLPTLPGDYSILVKYNDKHIPGSPFTAKIT-DDSRRCSQVKLGSAADFLLDIS 1931 Q14315 FLNC_HUMAN 1911 DGTCTVSYLPTAPGDYSIIVRFDDKHIPGSPFTAKITGDDSMRTSQLNVGTSTDVSLKIT 1970 ****:*:***. ******:*:::::*:*******::* *** * *::::*:::*. :.*:

P21333 FLNA_HUMAN 1977 ETDLSLLTATVVPPSGREEPCLLKRLRNGHVGISFVPKETGEHLVHVKKNGQHVASSPIP 2036 O75369 FLNB_HUMAN 1932 ETDLSSLTASIKAPSGRDEPCLLKRLPNNHIGISFIPREVGEHLVSIKKNGNHVANSPVS 1991 Q14315 FLNC_HUMAN 1971 ESDLSQLTASIRAPSGNEEPCLLKRLPNRHIGISFTPKEVGEHVVSVRKSGKHVTNSPFK 2030 *:*** ***:: ***.:******** * *:**** *:*.***:* ::*.*:**:.**.

332 D.1. Alignment of ﬁlamin isoforms

P21333 FLNA_HUMAN 2037 VVISQSEIGDASRVRVSGQGLHEGHTFEPAEFIIDTRDAGYGGLSLSIEGPSKVDINTED 2096 O75369 FLNB_HUMAN 1992 IMVVQSEIGDARRAKVYGRGLSEGRTFEMSDFIVDTRDAGYGGISLAVEGPSKVDIQTED 2051 Q14315 FLNC_HUMAN 2031 ILVGPSEIGDASKVRVWGKGLSEGHTFQVAEFIVDTRNAGYGGLGLSIEGPSKVDINCED 2090 ::: ****** :.:* *:** **:**: ::**:***:*****:.*::********: **

P21333 FLNA_HUMAN 2097 LEDGTCRVTYCPTEPGNYIINIKFADQHVPGSPFSVKVTGEGRVKESITRRRRAPSVANV 2156 O75369 FLNB_HUMAN 2052 LEDGTCKVSYFPTVPGVYIVSTKFADEHVPGSPFTVKISGEGRVKESITRTSRAPSVATV 2111 Q14315 FLNC_HUMAN 2091 MEDGTCKVTYCPTEPGTYIINIKFADKHVPGSPFTVKVTGEGRMKESITRRRQAPSIATI 2150 :*****:*:* ** ** **:. ****:*******:**::****:****** :***:*.:

P21333 FLNA_HUMAN 2157 GSHCDLSLKIPEI------2169 O75369 FLNB_HUMAN 2112 GSICDLNLKIPEI------2124 Q14315 FLNC_HUMAN 2151 GSTCDLNLKIPGNWFQMVSAQERLTRTFTRSSHTYTRTERTEISKTRGGETKREVRVEES 2210 ** ***.****

P21333 FLNA_HUMAN 2170 ------SIQDMTAQVTSPSGKTHEAEIVEGE 2194 O75369 FLNB_HUMAN 2125 ------NSSDMSAHVTSPSGRVTEAEIVPMG 2149 Q14315 FLNC_HUMAN 2211 TQVGGDPFPAVFGDFLGRERLGSFGSITRQQEGEASSQDMTAQVTSPSGKVEAAEIVEGE 2270 . .**:*:******:. ****

P21333 FLNA_HUMAN 2195 NHTYCIRFVPAEMGTHTVSVKYKGQHVPGSPFQFTVGPLGEGGAHKVRAGGPGLERAEAG 2254 O75369 FLNB_HUMAN 2150 KNSHCVRFVPQEMGVHTVSVKYRGQHVTGSPFQFTVGPLGEGGAHKVRAGGPGLERGEAG 2209 Q14315 FLNC_HUMAN 2271 DSAYSVRFVPQEMGPHTVAVKYRGQHVPGSPFQFTVGPLGEGGAHKVRAGGTGLERGVAG 2330 . ::.:**** *** ***:***:**** *********************** ****. **

P21333 FLNA_HUMAN 2255 VPAEFSIWTREAGAGGLAIAVEGPSKAEISFEDRKDGSCGVAYVVQEPGDYEVSVKFNEE 2314 O75369 FLNB_HUMAN 2210 VPAEFSIWTREAGAGGLSIAVEGPSKAEITFDDHKNGSCGVSYIAQEPGNYEVSIKFNDE 2269 Q14315 FLNC_HUMAN 2331 VPAEFSIWTREAGAGGLSIAVEGPSKAEIAFEDRKDGSCGVSYVVQEPGDYEVSIKFNDE 2390 *****************:***********:*:*:*:*****:*:.****:****:***:*

P21333 FLNA_HUMAN 2315 HIPDSPFVVPVASPSGDARRLTVSSLQESGLKVNQPASFAVSLNGAKGAIDAKVHSPSGA 2374 O75369 FLNB_HUMAN 2270 HIPESPYLVPVIAPSDDARRLTVMSLQESGLKVNQPASFAIRLNGAKGKIDAKVHSPSGA 2329 Q14315 FLNC_HUMAN 2391 HIPDSPFVVPVASLSDDARRLTVTSLQETGLKVNQPASFAVQLNGARGVIDARVHTPSGA 2450 ***:**::*** : * ******* ****:***********: ****:* ***:**:****

P21333 FLNA_HUMAN 2375 LEECYVTEIDQDKYAVRFIPRENGVYLIDVKFNGTHIPGSPFKIRVGEPGHGGDPGLVSA 2434 O75369 FLNB_HUMAN 2330 VEECHVSELEPDKYAVRFIPHENGVHTIDVKFNGSHVVGSPFKVRVGEPGQAGNPALVSA 2389 Q14315 FLNC_HUMAN 2451 VEECYVSELDSDKHTIRFIPHENGVHSIDVKFNGAHIPGSPFKIRVGEQSQAGDPGLVSA 2510 :***:*:*:: **:::****:****: *******:*: *****:**** .:.*:*.****

P21333 FLNA_HUMAN 2435 YGAGLEGGVTGNPAEFVVNTSNAGAGALSVTIDGPSKVKMDCQECPEGYRVTYTPMAPGS 2494 O75369 FLNB_HUMAN 2390 YGTGLEGGTTGIQSEFFINTTRAGPGTLSVTIEGPSKVKMDCQETPEGYKVMYTPMAPGN 2449 Q14315 FLNC_HUMAN 2511 YGPGLEGGTTGVSSEFIVNTLNAGSGALSVTIDGPSKVQLDCRECPEGHVVTYTPMAPGN 2570 ** *****.** :**.:** .** *:*****:*****::**:* ***: * *******.

P21333 FLNA_HUMAN 2495 YLISIKYGGPYHIGGSPFKAKVTGPRLVSNHSLHETSSVFVDSLTKATCAPQHG--APGP 2552 O75369 FLNB_HUMAN 2450 YLISVKYGGPNHIVGSPFKAKVTGQRLVSPGSANETSSILVESVTRSST--ETCYSAIPK 2507 Q14315 FLNC_HUMAN 2571 YLIAIKYGGPQHIVGSPFKAKVTGPRLSGGHSLHETSTVLVETVTKSSSSRGSSYSSIPK 2630 ***::***** ** ********** ** . * .***:::*:::*::: :

P21333 FLNA_HUMAN 2553 GPADASKVVAKGLGLSKAYVGQKSSFTVDCSKAGNNMLLVGVHGPRTPCEEILVKHVGSR 2612 O75369 FLNB_HUMAN 2508 ASSDASKVTSKGAGLSKAFVGQKSSFLVDCSKAGSNMLLIGVHGPTTPCEEVSMKHVGNQ 2567 Q14315 FLNC_HUMAN 2631 FSSDASKVVTRGPGLSQAFVGQKNSFTVDCSKAGTNMMMVGVHGPKTPCEEVYVKHMGNR 2690 :*****.::* ***:*:****.** *******.**:::***** *****: :**:*.:

P21333 FLNA_HUMAN 2613 LYSVSYLLKDKGEYTLVVKWGDEHIPGSPYRVVVP 2647 O75369 FLNB_HUMAN 2568 QYNVTYVVKERGDYVLAVKWGEEHIPGSPFHVTVP 2602 Q14315 FLNC_HUMAN 2691 VYNVTYTVKEKGDYILIVKWGDESVPGSPFKVKVP 2725 *.*:* :*::*:* * ****:* :****::* ** APPENDIX E

Parameters

E.1 Mascot parameters and results for standard protein proteolysis 334 E.1. Mascot parameters and results for standard protein proteolysis

Chymotrypsin

CASA1_BOVIN Mass: 24513 Score: 143 Matches: 10(2) Sequences: 3(1) emPAI: 0.19 Alpha-S1-casein OS=Bos taurus GN=CSN1S1 PE=1 SV=2

Nominal mass (Mr): 24513; Calculated pI value: 4.98 NCBI BLAST search of CASA1_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Chymotrypsin: cuts C-term side of FLWY unless next residue is P Sequence Coverage: 15%

Matched peptides shown in Bold Red

1 MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE 51 KVNELSKDIG SESTEDQAME DIKQMEAESI SSSEEIVPNS VEQKHIQKED 101 VPSERYLGYL EQLLRLKKYK VPQLEIVPNS AEERLHSMKE GIHAQQKEPM 151 IGVNQELAYF YPELFRQFYQ LDAYPSGAWY YVPLGTQYTD APSFSDIPNP 201 IGSENSEKTT MPLW

CASB_BOVIN Mass: 25091 Score: 300 Matches: 35(8) Sequences: 7(4) emPAI: 0.99 Beta-casein OS=Bos taurus GN=CSN2 PE=1 SV=2

Nominal mass (Mr): 25091; Calculated pI value: 5.26 NCBI BLAST search of CASB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Chymotrypsin: cuts C-term side of FLWY unless next residue is P Sequence Coverage: 33%

Matched peptides shown in Bold Red

1 MKVLILACLV ALALARELEE LNVPGEIVES LSSSEESITR INKKIEKFQS 51 EEQQQTEDEL QDKIHPFAQT QSLVYPFPGP IPNSLPQNIP PLTQTPVVVP 101 PFLQPEVMGV SKVKEAMAPK HKEMPFPKYP VEPFTESQSL TLTDVENLHL 151 PLPLLQSWMH QPHQPLPPTV MFPPQSVLSL SQSKVLPVPQ KAVPYPQRDM 201 PIQAFLLYQE PVLGPVRGPF PIIV

MYG_HORSE Mass: 17072 Score: 271 Matches: 33(7) Sequences: 8(3) Myoglobin OS=Equus caballus GN=MB PE=1 SV=2

Nominal mass (Mr): 17072; Calculated pI value: 7.21 NCBI BLAST search of MYG_HORSE against nr Unformatted sequence string for pasting into other applications E.1. Mascot parameters and results for standard protein proteolysis 335

Taxonomy: Equus caballus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Chymotrypsin: cuts C-term side of FLWY unless next residue is P Sequence Coverage: 46%

Matched peptides shown in Bold Red

1 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL 51 KTEAEMKASE DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI 101 PIKYLEFISD AIIHVLHSKH PGDFGADAQG AMTKALELFR NDIAAKYKEL 151 GFQG

LACB_BOVIN Mass: 19870 Score: 236 Matches: 16(12) Sequences: 6(4) emPAI: 0.54 Beta- lactoglobulin OS=Bos taurus GN=LGB PE=1 SV=3

Nominal mass (Mr): 19870; Calculated pI value: 4.93 NCBI BLAST search of LACB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Chymotrypsin: cuts C-term side of FLWY unless next residue is P Sequence Coverage: 33%

Matched peptides shown in Bold Red

1 MKCLLLALAL TCGAQALIVT QTMKGLDIQK VAGTWYSLAM AASDISLLDA 51 QSAPLRVYVE ELKPTPEGDL EILLQKWENG ECAQKKIIAE KTKIPAVFKI 101 DALNENKVLV LDTDYKKYLL FCMENSAEPE QSLACQCLVR TPEVDDEALE 151 KFDKALKALP MHIRLSFNPT QLEEQCHI

OVAL_CHICK Mass: 42854 Score: 140 Matches: 39(5) Sequences: 12(4) emPAI: 0.43 Ovalbumin OS=Gallus gallus GN=SERPINB14 PE=1 SV=2

Nominal mass (Mr): 42854; Calculated pI value: 5.19 NCBI BLAST search of OVAL_CHICK against nr Unformatted sequence string for pasting into other applications

Taxonomy: Gallus gallus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Chymotrypsin: cuts C-term side of FLWY unless next residue is P Sequence Coverage: 30%

Matched peptides shown in Bold Red

1 MGSIGAASME FCFDVFKELK VHHANENIFY CPIAIMSALA MVYLGAKDST 336 E.1. Mascot parameters and results for standard protein proteolysis

51 RTQINKVVRF DKLPGFGDSI EAQCGTSVNV HSSLRDILNQ ITKPNDVYSF 101 SLASRLYAEE RYPILPEYLQ CVKELYRGGL EPINFQTAAD QARELINSWV 151 ESQTNGIIRN VLQPSSVDSQ TAMVLVNAIV FKGLWEKAFK DEDTQAMPFR 201 VTEQESKPVQ MMYQIGLFRV ASMASEKMKI LELPFASGTM SMLVLLPDEV 251 SGLEQLESII NFEKLTEWTS SNVMEERKIK VYLPRMKMEE KYNLTSVLMA 301 MGITDVFSSS ANLSGISSAE SLKISQAVHA AHAEINEAGR EVVGSAEAGV 351 DAASVSEEFR ADHPFLFCIK HIATNAVLFF GRCVSP

Elastase

CASA1_BOVIN Mass: 24513 Score: 312 Matches: 129(1) Sequences: 65(1) emPAI: 26.30 Alpha-S1- casein OS=Bos taurus GN=CSN1S1 PE=1 SV=2

Nominal mass (Mr): 24513; Calculated pI value: 4.98 NCBI BLAST search of CASA1_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 62%

Matched peptides shown in Bold Red

CASB_BOVIN Mass: 25091 Score: 731 Matches: 170(14) Sequences: 66(9) emPAI: 127.32 Beta-casein OS=Bos taurus GN=CSN2 PE=1 SV=2

Nominal mass (Mr): 25091; Calculated pI value: 5.26 NCBI BLAST search of CASB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 53%

Matched peptides shown in Bold Red

1 MKVLILACLV ALALARELEE LNVPGEIVES LSSSEESITR INKKIEKFQS 51 EEQQQTEDEL QDKIHPFAQT QSLVYPFPGP IPNSLPQNIP PLTQTPVVVP E.1. Mascot parameters and results for standard protein proteolysis 337

101 PFLQPEVMGV SKVKEAMAPK HKEMPFPKYP VEPFTESQSL TLTDVENLHL 151 PLPLLQSWMH QPHQPLPPTV MFPPQSVLSL SQSKVLPVPQ KAVPYPQRDM 201 PIQAFLLYQE PVLGPVRGPF PIIV

LACB_BOVIN Mass: 19870 Score: 651 Matches: 112(16) Sequences: 54(9) emPAI: 90.49 Beta- lactoglobulin OS=Bos taurus GN=LGB PE=1 SV=3

Nominal mass (Mr): 19870; Calculated pI value: 4.93 NCBI BLAST search of LACB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 70%

Matched peptides shown in Bold Red

MYG_HORSE Mass: 17072 Score: 95 Matches: 56(3) Sequences: 35(3) Myoglobin OS=Equus caballus GN=MB PE=1 SV=2

Nominal mass (Mr): 17072; Calculated pI value: 7.21 NCBI BLAST search of MYG_HORSE against nr Unformatted sequence string for pasting into other applications

Taxonomy: Equus caballus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 60%

Matched peptides shown in Bold Red

1 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL 51 KTEAEMKASE DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI 101 PIKYLEFISD AIIHVLHSKH PGDFGADAQG AMTKALELFR NDIAAKYKEL 151 GFQG

OVAL_CHICK Mass: 42854 Score: 49 Matches: 12(0) Sequences: 11(0) emPAI: 0.16 Ovalbumin OS=Gallus gallus GN=SERPINB14 PE=1 SV=2

Nominal mass (Mr): 42854; Calculated pI value: 5.19 338 E.1. Mascot parameters and results for standard protein proteolysis

NCBI BLAST search of OVAL_CHICK against nr Unformatted sequence string for pasting into other applications

Taxonomy: Gallus gallus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 16%

Matched peptides shown in Bold Red

1 MGSIGAASME FCFDVFKELK VHHANENIFY CPIAIMSALA MVYLGAKDST 51 RTQINKVVRF DKLPGFGDSI EAQCGTSVNV HSSLRDILNQ ITKPNDVYSF 101 SLASRLYAEE RYPILPEYLQ CVKELYRGGL EPINFQTAAD QARELINSWV 151 ESQTNGIIRN VLQPSSVDSQ TAMVLVNAIV FKGLWEKAFK DEDTQAMPFR 201 VTEQESKPVQ MMYQIGLFRV ASMASEKMKI LELPFASGTM SMLVLLPDEV 251 SGLEQLESII NFEKLTEWTS SNVMEERKIK VYLPRMKMEE KYNLTSVLMA 301 MGITDVFSSS ANLSGISSAE SLKISQAVHA AHAEINEAGR EVVGSAEAGV 351 DAASVSEEFR ADHPFLFCIK HIATNAVLFF GRCVSP

Lys-C

CASA2_BOVIN Mass: 26002 Score: 96 Matches: 14(3) Sequences: 6(2) emPAI: 1.06 Alpha-S2-casein OS=Bos taurus GN=CSN1S2 PE=1 SV=2

Nominal mass (Mr): 26002; Calculated pI value: 8.54 NCBI BLAST search of CASA2_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Lys-C: cuts C-term side of K unless next residue is P Sequence Coverage: 29%

Matched peptides shown in Bold Red

1 MKFFIFTCLL AVALAKNTME HVSSSEESII SQETYKQEKN MAINPSKENL 51 CSTFCKEVVR NANEEEYSIG SSSEESAEVA TEEVKITVDD KHYQKALNEI 101 NQFYQKFPQY LQYLYQGPIV LNPWDQVKRN AVPITPTLNR EQLSTSEENS 151 KKTVDMESTE VFTKKTKLTE EEKNRLNFLK KISQRYQKFA LPQYLKTVYQ 201 HQKAMKPWIQ PKTKVIPYVR YL

CASB_BOVIN Mass: 25091 Score: 202 Matches: 12(4) Sequences: 6(2) emPAI: 1.10 Beta-casein OS=Bos taurus GN=CSN2 PE=1 SV=2

Nominal mass (Mr): 25091; Calculated pI value: 5.26 NCBI BLAST search of CASB_BOVIN against nr E.1. Mascot parameters and results for standard protein proteolysis 339

Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Lys-C: cuts C-term side of K unless next residue is P Sequence Coverage: 32%

Matched peptides shown in Bold Red

MYG_HORSE Mass: 17072 Score: 1230 Matches: 108(47) Sequences: 14(9) Myoglobin OS=Equus caballus GN=MB PE=1 SV=2

Nominal mass (Mr): 17072; Calculated pI value: 7.21 NCBI BLAST search of MYG_HORSE against nr Unformatted sequence string for pasting into other applications

Taxonomy: Equus caballus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Lys-C: cuts C-term side of K unless next residue is P Sequence Coverage: 90%

Matched peptides shown in Bold Red

1 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL 51 KTEAEMKASE DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI 101 PIKYLEFISD AIIHVLHSKH PGDFGADAQG AMTKALELFR NDIAAKYKEL 151 GFQG

LACB_OVIMU Mass: 18139 Score: 288 Matches: 64(19) Sequences: 8(6) emPAI: 26.65 Beta- lactoglobulin OS=Ovis orientalis musimon GN=LGB PE=1 SV=1

Nominal mass (Mr): 18139; Calculated pI value: 5.26 NCBI BLAST search of LACB_OVIMU against nr Unformatted sequence string for pasting into other applications

Taxonomy: Ovis aries musimon

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Lys-C: cuts C-term side of K unless next residue is P Sequence Coverage: 28%

Matched peptides shown in Bold Red 340 E.1. Mascot parameters and results for standard protein proteolysis

1 IIVTQTMKGL DIQKVAGTWH SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGNLEILLQK WENGECAQKK IIAEKTKIPA VFKIDALNEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLACQ CLVRTPEVDN EALEKFDKAL KALPMHIRLA 151 FNPTQLEGQC HV

Trypsin

CASA1_BOVIN Mass: 24513 Score: 5635 Matches: 391(214) Sequences: 25(20) emPAI: 242.13 Alpha- S1-casein OS=Bos taurus GN=CSN1S1 PE=1 SV=2

Nominal mass (Mr): 24513; Calculated pI value: 4.98 NCBI BLAST search of CASA1_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 62%

Matched peptides shown in Bold Red

CASB_BOVIN Mass: 25091 Score: 2910 Matches: 121(79) Sequences: 14(7) emPAI: 7.38 Beta-casein OS=Bos taurus GN=CSN2 PE=1 SV=2

Nominal mass (Mr): 25091; Calculated pI value: 5.26 NCBI BLAST search of CASB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 33%

Matched peptides shown in Bold Red

201 PIQAFLLYQE PVLGPVRGPF PIIV

MYG_HORSE Mass: 17072 Score: 6496 Matches: 477(247) Sequences: 32(28) Myoglobin OS=Equus caballus GN=MB PE=1 SV=2

Nominal mass (Mr): 17072; Calculated pI value: 7.21 NCBI BLAST search of MYG_HORSE against nr Unformatted sequence string for pasting into other applications

Taxonomy: Equus caballus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 99%

Matched peptides shown in Bold Red

1 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL 51 KTEAEMKASE DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI 101 PIKYLEFISD AIIHVLHSKH PGDFGADAQG AMTKALELFR NDIAAKYKEL 151 GFQG

LACB_BOVIN Mass: 19870 Score: 3867 Matches: 254(134) Sequences: 20(15) emPAI: 108.13 Beta- lactoglobulin OS=Bos taurus GN=LGB PE=1 SV=3

Nominal mass (Mr): 19870; Calculated pI value: 4.93 NCBI BLAST search of LACB_BOVIN against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 67%

Matched peptides shown in Bold Red

OVAL_CHICK Mass: 42854 Score: 8137 Matches: 343(212) Sequences: 35(24) emPAI: 58.49 Ovalbumi n OS=Gallus gallus GN=SERPINB14 PE=1 SV=2

Nominal mass (Mr): 42854; Calculated pI value: 5.19 NCBI BLAST search of OVAL_CHICK against nr Unformatted sequence string for pasting into other applications 342 E.1. Mascot parameters and results for standard protein proteolysis

Taxonomy: Gallus gallus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 83%

Matched peptides shown in Bold Red

Trypsin-C(Br)N

gi|225632 Mass: 24420 Score: 302 Matches: 28(5) Sequences: 5(3) emPAI: 0.92 casein alphaS1

Nominal mass (Mr): 24420; Calculated pI value: 4.85 NCBI BLAST search of gi|225632 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Fixed modifications: Met->Hsl (C-term M) Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) Cleavage by CNBr+Trypsin, a mixture of enzymes: cuts C-term side of M cuts C-term side of KR unless next residue is P Sequence Coverage: 27%

Matched peptides shown in Bold Red

1 MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE 51 KVNELSKDIG SESTEDQAME DIKEMEAESI SSSGEIVPNS VEQKHIQKED 101 VPSERYLGYL EQLLRLKKYK VPQLEIVPNS AEERLHSMKE GIDAQQKEPM 151 IGVNQELAYF YPELFRQFYQ LDAYPSGAWY YVPLGTQYTD APSFSDIPNP 201 IGSENSEKTT MPLW

gi|223906 Mass: 23559 Score: 261 Matches: 29(15) Sequences: 4(2) emPAI: 0.40 casein beta

Nominal mass (Mr): 23559; Calculated pI value: 5.13 NCBI BLAST search of gi|223906 against nr Unformatted sequence string for pasting into other applications

E.1. Mascot parameters and results for standard protein proteolysis 343

Taxonomy: Bubalus bubalis

Matched peptides shown in Bold Red

1 RELEELNVPG EIVESLSSSE ESITHINKKI EKFQSEEQQQ TEDELQDKIH 51 PFAQTQSLVY PFPGPIPKSL PQNIPPLTQT PVVVPPFLQP EIMGVSKVKE 101 AMAPKHKEMP FPKYPVQPET ESQSLTLTDV ENLHLPPLLL QSWMHQPHQP 151 LPPTVMFPPQ SVLSLSQSKV LPVPEKAVPY PQRDMPIQAF LLYQEPVLGP 201 VRGPFPIIV gi|229460 Mass: 18355 Score: 581 Matches: 64(17) Sequences: 11(5) emPAI: 1.73 lactoglobulin beta

Nominal mass (Mr): 18355; Calculated pI value: 4.76 NCBI BLAST search of gi|229460 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Matched peptides shown in Bold Red

1 LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLQK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLVCQ CLVRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI gi|7546624 Mass: 16941 Score: 158 Matches: 9(0) Sequences: 4(0) emPAI: 0.17 Chain A, Myoglobin (Horse Heart) Wild-Type Complexed With Co

Nominal mass (Mr): 16941; Calculated pI value: 7.36 NCBI BLAST search of gi|7546624 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Equus caballus

Sequence Coverage: 28%

Matched peptides shown in Bold Red

1 GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK 51 TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP 101 IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG 151 FQG

gi|28566340 Mass: 42877 Score: 1425 Matches: 90(35) Sequences: 19(10) emPAI: 4.07 ovalbumin [Gallus gallus]

Nominal mass (Mr): 42877; Calculated pI value: 5.19 NCBI BLAST search of gi|28566340 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Gallus gallus

Matched peptides shown in Bold Red

1 MGSIGAASME FCFDVFKELK VHHANENIFY CPIAIMSALA MVYLGAKDST 51 RTQINKVVRF DKLPGFGDSI EAQCGTSVNV HSSLRDILNQ ITKPNDVYSF 101 SLASRLYAEE RYPILPEYLQ CVKELYRGGL EPINFQTAAD QARELINSWV 151 ESQXNGIIRN VLQPSSVDSQ TAXVLVNAIV FKGLWEKAFK DEDTQAMPFR 201 VTEQESKPVQ MMYQIGLFRV ASMASEKMKI LELPFASGTM SMLVLLPDEV 251 SGLEQLESII NFEKLTEWTS SNVMEERKIK VYFPRMKMEE KYNLTSVLMA 301 MGITDVFSSS ANLSGISSAE SLKISQAVHA AHAEINEAGR EVVGSAEAGV 351 DAASVSEEFR ADHPFLFCIK HIATNAVLFF GRCVSP

Thermolysin

gi|159793191 Mass: 23598 Score: 5446 Matches: 347(16) Sequences: 113(11) emPAI: 2086.84 alpha S1 casein [Bos taurus]

Match to: gi|159793191 Score: 5446 alpha S1 casein [Bos taurus] Found in search of C:\Program Files (x86)\Matrix Science\Mascot Daemon\mgf\20 110916-Thermolysin- NCBInr\mascot_daemon_merge.mgf

Nominal mass (Mr): 23598; Calculated pI value: 4.90 E.1. Mascot parameters and results for standard protein proteolysis 345

NCBI BLAST search of gi|159793191 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 88%

Matched peptides shown in Bold Red

1 LVAVALARPK HPIKHQGLPQ EVLNENLLRF FVAPFPEVFG KEKVNELSKD 51 IGSESTEDQA MEDIKQMEAE SISSSEEIVP NSVEQKHIQK EDVPSERYLG 101 YLEQLLRLKK YKVPQLEIVP NSAEERLHSM KEGIHAQQKE PMIGVNQELA 151 YFYPELFRQF YQLDAYPSGA WYYVPLGTQY TDAPSFSDIP NPIGSENSEK 201 TTMPLW

gi|225825 Mass: 23608 Score: 6506 Matches: 524(32) Sequences: 140(15) emPAI: 17196.96 beta casein

Nominal mass (Mr): 23608; Calculated pI value: 5.24 NCBI BLAST search of gi|225825 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Bos taurus Links to retrieve other entries containing this sequence from NCBI Entrez: gi|226030 from Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 91%

Matched peptides shown in Bold Red

1 RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH 51 PFAQTQSLVY PFPGPIHNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE 101 AMAPKHKEMP FPKYPVEPFT ESQSLTLTDV ENLHLPLPLL QSWMHQPHQP 151 LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGP 201 VRGPFPIIV gi|229460 Mass: 18355 Score: 4964 Matches: 308(15) Sequences: 99(11) emPAI: 2726.67 lactoglobulin beta

Match to: gi|229460 Score: 4964 lactoglobulin beta Found in search of C:\Program Files (x86)\Matrix Science\Mascot Daemon\mgf\20 110916-Thermolysin- NCBInr\mascot_daemon_merge.mgf

Nominal mass (Mr): 18355; Calculated pI value: 4.76 NCBI BLAST search of gi|229460 against nr Unformatted sequence string for pasting into other applications 346 E.1. Mascot parameters and results for standard protein proteolysis

Taxonomy: Bos taurus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 87%

Matched peptides shown in Bold Red

1 LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP 51 EGDLEILLQK WENDECAQKK IIAEKTKIPA VFKLDAINEN KVLVLDTDYK 101 KYLLFCMENS AEPEQSLVCQ CLVRTPEVDD EALEKFDKAL KALPMHIRLS 151 FNPTLQEEQC HI

gi|7546624 Mass: 16941 Score: 5997 Matches: 406(32) Sequences: 122(23) emPAI: 106385.26 Chain A, Myoglobin (Horse Heart) Wild-Type Complexed With Co Match to: gi|7546624 Score: 5997 Chain A, Myoglobin (Horse Heart) Wild-Type Complexed With Co Found in search of C:\Program Files (x86)\Matrix Science\Mascot Daemon\mgf\20 110916-Thermolysin- NCBInr\mascot_daemon_merge.mgf

Nominal mass (Mr): 16941; Calculated pI value: 7.36 NCBI BLAST search of gi|7546624 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Equus caballus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity Sequence Coverage: 100%

Matched peptides shown in Bold Red

1 GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK 51 TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP 101 IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG 151 FQG

gi|129293 Mass: 42854 Score: 10238 Matches: 477(66) Sequences: 186(46) emPAI: 21651.23 RecName: Full=Ovalbumin; AltName: Full=Allergen Gal d II; AltName: Full=Egg albumin; AltName: Full=Plakalbumin; AltName: Allergen=Gal

Nominal mass (Mr): 42854; Calculated pI value: 5.19 NCBI BLAST search of gi|129293 against nr Unformatted sequence string for pasting into other applications

Taxonomy: Gallus gallus

Variable modifications: Oxidation (M),Phospho (ST),Phospho (Y) No enzyme cleavage specificity E.1. Mascot parameters and results for standard protein proteolysis 347

Sequence Coverage: 88%

Matched peptides shown in Bold Red

348 E.2. MaxQuant parameters

E.2 MaxQuant parameters E.2. MaxQuant parameters 349

Table E.1. MaxQuant parameters for the analysis of lysis buffer optimization. Parameter Value Version 1.5.2.8 User name lreimann Machine name FPROT-BEAST Fixed modifications none Decoy mode revert Special AAs KR Include contaminants True MS/MS tol. (FTMS) 20 ppm Top MS/MS peaks per 100 Da. (FTMS) 12 MS/MS deisotoping (FTMS) True MS/MS tol. (ITMS) 0.5 Da Top MS/MS peaks per 100 Da. (ITMS) 8 MS/MS deisotoping (ITMS) False MS/MS tol. (TOF) 40 ppm Top MS/MS peaks per 100 Da. (TOF) 10 MS/MS deisotoping (TOF) True MS/MS tol. (Unknown) 0.5 Da Top MS/MS peaks per 100 Da. (Unknown) 8 MS/MS deisotoping (Unknown) False PSM FDR 0.01 Protein FDR 0.01 Site FDR 0.01 Use Normalized Ratios For Occupancy True Min. peptide Length 7 Min. score for unmodified peptides 0 Min. score for modified peptides 40 Min. delta score for modified peptides 6 Min. unique peptides 1 Min. razor peptides 1 Min. peptides 1 Use only unmodified peptides and True Modifications included in protein quantification Acetyl (Protein N-term);Oxidation (M) Peptides used for protein quantification Razor Discard unmodified counterpart peptides True Min. ratio count 2 Re-quantify False Use delta score False Match between runs True Matching time window [min] 0.7 Alignment time window [min] 20 Find dependent peptides False Fasta file UniProt ProteomeSet Mouse Isoform.fasta Labeled amino acid filtering True Site tables Oxidation (M)Sites.txt RT shift False Advanced ratios True

Publications and Poster

Parts of this work have previously been published/submitted to the following journals and conferences:

Scientiﬁc publications

Reimann, L., Wiese, H., Leber, Y., Schwäble, A. N., Fricke, A. L., Rohland, A., Knapp, B., Peickert, C. D., Drepper, F., van der Ven, P. M., Radziwill, G., Fürst, D. O., and Warscheid, B. (2016) Myoﬁbrillar Z-discs are a protein phosphorylation hot spot with PKCα modulating protein dynamics. (in revision).

Puetz, S., Schroeter, M. M., Piechura, H., Reimann, L., Hunger, M. S., Lubomirov, L. T., Metzler, D., Warscheid, B., and Pﬁtzer, G. (2012) New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation. Journal of Muscle Research & Cell Motility 33, 471-483.

Submissions to conferences

Reimann, L., Wiese, H., Fricke, A. L., Leber, Y., Radziwill, G., Van der Ven, P. F., Fürst, D. O., and Warscheid, B. (2014) Dissecting Signaling Processes In and Out of the Z-disc by Functional Proteomics. Special Interest Meeting - Molecular Insight into Muscle Function and Protein Aggregate Myopathies, Potsdam, Germany.

Reimann, L., Leber, Y., Wiese, H., Fricke, A. L., Orfanos, Z., Radziwill, G., van der Ven, P. F., Fürst, D. O., and Warscheid, B. (2014) Identiﬁcation of the Myoﬁbrillar Z-Disc as a Nodal Point in Skeletal Myocyte Signaling by Large-Scale Phosphoproteomics. 13th Human 352 Publications and Poster

Proteome Organization World Congress, Madrid, Spain.

Reimann, L., Wiese, H., Fürst, D. O., and Warscheid, B. (2013) Study of Z-Disc Proteins Involved in Hypertrophic Signaling Events. 7th Proteomics Advanced Summer School, Brixen, Italy.

Reimann, L., Wiese, H., Fürst, D. O., and Warscheid, B. (2013) Phosphoproteome Analysis of Z-Disc Proteins for Signalling Network Studies. International Symposium "Signaling and Sorting", Freiburg, Germany.

Reimann, L., Wiese, H., Fricke, A. L., Leber, Y., Orfanos, Z., Radziwill, G., van der Ven, P. F., Fürst, D. O., and Warscheid, B. (2013) Study of Signaling Processes In and Out of the Z-Disc. Myoﬁbrillar Z-disk Structure and Dynamics, EMBL Hamburg, Germany.

Reimann, L., Wiese, H., Wiese, S., Drepper, F., Fürst, D. O., and Warscheid, B. (2012) Phosphoproteome Analysis of Z-Disc Proteins in C2C12 Cells. MaxQuant Summer School 2012, Munich, Germany

Reimann, L., Piechura, H., Wiese, S., Cramer, S., Fürst, D. O., and Warscheid, B. (2011) Quantitative Proteomics of Z-disc Proteins for Protein Signaling Network Studies in C2 and C2C12 Cells. 5th Proteomics Basic Summer School, Brixen, Italy. Acknowledgments

Mein erster Dank gilt Prof. Dr. Bettina Warscheid, die mir vor fünf Jahren, als das Z- disc Projekt und das Labor noch in ihren Kinderschuhen steckten, ihr Vertrauen für dieses interessante und spannende Projekt geschenkt hat. Danke für die vielen konstruktiv, kritischen Anmerkungen, die mich stets dazu gebracht haben noch mehr aus den Daten herauszuholen und danke für die vielen Freiheiten, die du mir gelassen hast. Besonders möchte ich mich auch für die vielen Stunden an sorgfältigen Korrekturen dieser Arbeit sowie diverser Vorträge, Abstracts, Poster und Paper bedanken.

Ein ganz herzlicher Dank gilt Dr. Heike Wiese, die mir am Anfang meiner Doktorarbeit alles beigebracht hat, was man für den Einstieg in die large-scale phosphoproteomics Welt so wissen muss. Ich weiß, dass ich stets auf dich zählen konnte (auch nachdem ihr nach Ulm weiter gezogen wart), sei es bei den Arbeiten an den Manuskripten oder bei den Korrekturen dieser Arbeit.

Mein weiterer Dank gilt Prof. Dr. Gerald Radziwill für die vielen (montags morgens) Besprechungen, die meinen biologischen Horizont um so vieles haben wachsen lassen und meine Arbeiten stets auf ihren biologischen Hintergrund hinterfragt haben. Danke auch für die kritischen Korrekturen an den beiden Manuskripten und dieser Arbeit.

Ein großes Dankeschön geht auch an meine Kooperationspartner Prof. Dr. Dieter O. Fürst und Dr. Peter MF van der Ven. Ihr habt mich immer mit eurem Muskelfachwissen unter- stützt, uns und mir mit Rat und Tat beiseite gestanden und dazu beigetragen, dass diese Arbeit in dieser Form entstehen konnte. 354 Acknowledgments

Bei meinen Z-disc Sisters Anja Schwäble und Anna Lena Fricke möchte ich mich bedanken, dass sie in ihren jeweiligen Abschlussarbeiten so viel für mich und für das Z-disc Projekt getan haben, dass diese Arbeit ohne sie so nicht zustande gekommen wäre. Danke, dass ihr meine Begeisterung für das Projekt geteilt habt und euch vor Rückschlägen nicht habt abschrecken lassen. Ohne euch wären ich und das Projekt nie so weit gekommen, ihr seid der größte Teil des we von Claude Bernards Zitat - art is I, science is we - und ich weiß, dass das Muskelprojekt bei euch in guten Händen ist.

Bei Dr. Ida Suppanz und Christian Peikert möchte ich mich für die vielen gemeinsamen Arbeitsstunden vor unseren jeweiligen Rechnern im Büro bedanken. Schön, dass wir unser "Leid" von stundenlangen Skyline Auswertungen, unsere "Verzweiﬂung" über MaxQuant Ergebnistabellen und unsere "Kämpfe" mit dem Adobe Illustrator stets zusammen "durchlit- ten" haben. Und Danke, dass ihr mich in der Endphase so tatkräftig durch Korrekturarbeiten am Text und Quelltext dieser Arbeit unterstützt habt!

Insgesamt möchte ich dem Team der AG Warscheid danken, das die letzten Jahren so schnell hat verﬂiegen lassen und stets für eine angenehme Arbeitsatmosphäre gesorgt haben und immer für einen Grillabend oder ein Feierabendbier zu haben waren. Ohne Christa Reichenbach und ihre Unterstützung beim Blotten und so vielen anderen Kleinigkeiten und ohne Bettina Knapp samt ihres MS-Wissens und ihrer Hilfsbereitschaft bei allen massenspek- trometrischen Fragen wäre diese Arbeit in ihrer Form nicht möglich geworden. Besonderer Dank gilt auch Dr. Silke Oeljeklaus und Dr. Friedel Drepper, die immer ein offenes Ohr und einen guten Ratschlag für meine Fragen parat hatten; sei es hinsichtlich von Datenbanken, Auswertungen oder den Tücken der englischen Sprache gewesen.

Bei meinen BnB Mädels, Jana und Kirsten sowie Andi und Stefan möchte ich mich für die vielen kraftspendenden Wort und Taten bedanken. Ohne euch wäre mein Leben weniger bunt, laut und fröhlich und ich schätze mich glücklich mir eurer Unterstützung gewiss zu sein.

Zuletzt gilt mein größter Dank meiner Familie und Sebastian, die mir stets den Rückhalt gegeben haben, den ich gebraucht habe und mich wieder aufgebaut haben, wenn ich an einem Tiefpunkt angelangt war.