Medizinische Hochschule Hannover

Institut für Physiologische Chemie

Regulation and Function of ERK3/MK5-mediated Signaling

INAUGURAL – DISSERTATION Zur Erlangung des Grades eines Doktors der Naturwissenschaften - Doctor rerum naturalium – (Dr. rer. nat.)

vorgelegt von

Frank Brand

geboren am 19.11.1982 in Hoyerswerda

Hannover 2012

Angenommen vom Senat der Medizinischen Hochschule Hannover am 12.04.2012

Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover

Präsident: Prof. Dr. med Dieter Bitter-Suermann

Betreuer: Prof. Dr. rer. nat. Matthias Gaestel

Kobetreuer: Prof. Dr. rer. nat. Ernst Ungewickell

1. Gutachter: Prof. Dr. rer. nat. Matthias Gaestel

2. Gutachter: Prof. Dr. rer. nat. Ernst Ungewickell

3. Gutachter: Prof. Dr. rer. nat. Andreas Kispert

Tag der mündlichen Prüfung vor der Prüfungskommission: 12.04.2012

Prof. Dr. rer. nat. Matthias Gaestel

Prof. Dr. rer. nat. Matthias Gaestel

Prof. Dr. rer. nat. Ernst Ungewickell

Prof. Dr. rer. nat. Andreas Kispert

Abstract

Frank Brand

Title of dissertation: ‘Regulation and Function of ERK3/MK5-mediated Signaling’

The family of mitogen-activated protein kinase (MAPK)-activated kinases (MKs, or MAPKAPKs), including the three distinct kinases MK2, MK3, and MK5, are downstream targets of the cytokine- and stress-induced p38 MAP kinases. Interaction and activation of MKs by p38 MAP kinases have been demonstrated in vitro and in vivo. The physiological relevance of the MK5/p38-interaction is doubtful, since its activity could not be triggered by any of the known MAP kinase stimuli. An interaction screen using MK5 revealed a strong binding to the atypical member of MAPKs ERK3. From previous studies, it has been concluded that MK5 is the first ‘bona fide’ substrate of ERK3, thus forming a stable complex promoting their protein stability and kinase activation. The physiological function of the ERK3/MK5-module has not been established so far, but related knockout models suggest an important role of both proteins, since the knockout phenotypes are characterized by incomplete penetrance and neonatal lethality, respectively. Because of the absence of any known stimulus or downstream target of the ERK3/MK5-signaling complex, further investigation were required.

The aim of this study was the identification of novel interacting partners of ERK3 and MK5 by yeast-based screening methods to complete the atypical MAP kinase signaling pathway. The characterization of ERK3- and MK5-deficiency, by analysis of potential morphological and physiological differences of primary isolated cells, should contribute to the establishment of the molecular function of this complex. The investigation of the related knockout animals should verify a shared phenotype of the kinases. Since ERK3 protein is characterized by a high turnover, a possible regulation of the ERK3/MK5-complex by targeting ERK3 protein stability was also analyzed.

It is shown that the absence of ERK3 and MK5 affects cell growth, motility and organization of the actin filament in mouse embryonic fibroblasts. Analysis of ERK3 and MK5 tissue- specific protein expressions suggest an important function in the brain. Based on screening of brain-specific cDNA expression libraries, an interaction of ERK3 and MK5 with the septin isoform Sept7 or Sept8 could be elucidated. Septins are structural proteins, which can form heteropolymeric filaments that are involved in many biological processes, such as dendritic branching and spine formation in neurons. Interestingly, it has been shown that hippocampal neurons of adult MK5-deficient mice display less complexity due to a decreased number of dendritic spines. Overexpression studies in ex vivo cultures of primary hippocampal neurons demonstrated that the presence of ectopically expressed ERK3 and MK5 significantly increase the Sept7-induced higher complexity of transfected neurons, which has been reported before. Another MK5-specific interacting protein was identified, the Rho guanine nucleotide exchange factor Kalirin-7, which has been brought into connection with the regulation of spine dynamics and might have additional functional relevance in this process. This neuronal function of the ERK3/MK5-signaling module could explain the pleiotropic phenotypes of the ERK3 and MK5 knockout mice causing the increased mortality.

Keywords: atypical MAPKs, MAPKAP kinase 5, septins, hippocampal neuron

Deutsche Zusammenfassung

Frank Brand

Thema der Dissertationsarbeit: „Regulation and Function of ERK3/MK5-mediated Signaling”

Die Familie der Mitogen-aktivierten Protein Kinasen (MAPK)-aktivierten Kinasen (MK, auch MAPKAPK) wird durch p38 MAP-Kinasen-Kaskaden unter verschiedensten zellulären Einflüssen wie Stress oder Wachstumsfaktoren aktiviert. Die Aktivierung von MK2 und MK3 durch die p38 MAP-Kinasen wurde in Verbindung gebracht mit einer veränderten Zytokinproduktion, Zellmigration, Zytoskelett, Genexpression und einen veränderten Zellzyklus. Eine mögliche Aktivierung von der evolutionär verwandten MAPKAP-Kinase 5 (MK5) durch p38 MAP-Kinasen ist hingegen fragwürdig, da diese Kinase durch keinerlei bekannte MAP-Kinase-Stimulation aktiviert werden kann. Vielmehr zeigte es sich, dass MK5 ein Substrate der atypischen MAP-Kinase ERK3 ist. Dabei konnte sowohl in vitro als auch in vivo gezeigt werden, dass MK5 und ERK3 einen stabilen zytosolischen Komplex bilden, indem beide Kinasen in ihrer aktiven Form vorliegen. Untersuchungen an den jeweiligen knockout-Mausmodellen zeigten jedoch keinen auffälligen Phänotyp. ERK3-defiziente Tiere sterben in den ersten Minuten nach ihrer Geburt und MK5 knockout-Mäuse weisen eine erhöhte Sterblichkeit in der Embryonalentwicklung auf. Aufgrund der fehlenden Kenntnisse über mögliche Stimulationen oder Substrate dieses Signalkomplexes, sind weitere Experimente von Nöten.

Das primäre Ziel dieser Arbeit war die Entdeckung neuer Interaktionspartner von ERK3 und MK5 mittels Hefe-basierter Screening-Methoden, um die Funktion des Komplexes in Verbindung mit bekannten Signalwegen zu bringen und die atypische MAP Kinase-Kaskade zu vervollständigen. Eine genauere Untersuchung der MK5- und ERK3-Defizienz sollte einerseits auf der Ebene von isolierten Primärzellen als auch in den jeweiligen knockout- Modellen durchgeführt werden. Da ERK3 in Zellen kontinuierlich abgebaut wird, wurde ebenfalls eine mögliche Regulation durch Proteinstabilisierung bzw. Destabilisierung in Betracht gezogen.

Dabei konnte gezeigt werden, dass die Abwesenheit von ERK3 und MK5 zu einem veränderten Zellwachstum, Zellwanderung und Zytoskelett in embryonalen Fibroblasten der Maus führte. Bestimmungen der gewebespezifischen Proteinexpression beider Kinasen deuteten, aufgrund ihrer Anreicherung im Gehirn, auf eine mögliche Funktion in neuronalen Zellen hin. Interaktionsstudien mit MK5 und ERK3 in Hefe-basierten Screening-Verfahren, bei dem Gehirn-spezifische cDNA Bibliotheken analysiert wurden, ergaben für beide Kinasen eine mögliche Bindung an Septinen, wobei ERK3 speziell mit Sept7 und MK5 mit Sept8 interagierte. Septine sind Strukturproteine, die in der Lage sind filamentöse Einheiten zu bilden, welche vielerlei Funktionen in Säugerzellen übernehmen. Bereits beschrieben ist ihre Funktion als Stützskelett bei der Ausbildung von Dendriten und Spines in Nervenzellen. Weiterhin konnte gezeigt werden, dass erwachsene MK5-defiziente Tiere eine deutlich verringerte Anzahl an Spines in Neuronen des Hippocampus aufweisen. In ex vivo Kulturen primärer Neuronen konnte durch zusätzliche Überexpression von ERK3 und MK5 eine Sept7-bedingte Steigerung an neuronaler Komplexität bezüglich der Morphologie von Dendritenauswüchsen und Anzahl von Spines deutlich verstärkt werden. Eine mögliche

Beteiligung eines weiteren MK5-spezifischen Interaktionspartners sollte ebenfalls in Betracht gezogen werden. Der Rho guanine nucleotide exchange factor Kalirin-7 spielt eine wichtige Rolle bei der Ausbildung von Spines durch Umstrukturiering des Aktinfilaments in Nervenzellen. Die aufgezeigte neuronale Funktion des ERK3/MK5-Signalmoduls könnte durchaus eine Erklärung für die pleiotrophen Phänotypen der jeweiligen knockout-Modelle geben.

Abbreviations

Abbreviations Description aa Amino acid AP-1 Activating protein 1 ASK Apoptosis signal-regulated kinase ATF Activating transcriptional factor ATM Ataxia-telangiectasia mutated kinase ATR Ataxia-telangiectasia and Rad3-related BMK1 Big MAP kinase 1 CBP CREB-binding protein CDC Cell division control CDK Cyclin-dependent kinase CRE Cyclic AMP response element CREB Cyclic AMP response element binding protein DLK Dual leucine zipper-bearing kinase DUSP Dual specific protein phosphatase EGF Epidermal growth factor ELK1 E twenty-six-like transcription factor 1 ERK Extracellular signal-regulated kinase GEF Guanine nucleotide exchange factor GPCR G protein-coupled receptor Hspb1 Heat shock protein beta-1 JNK c-Jun N-terminal kinase MAPK Mitogen-activated protein kinase MBP Myelin basic protein MDM2 Murine double minute 2 MEF Mouse embryonic fibroblast MEK Mitogen activated protein kinase kinase MEKK Mitogen activated protein kinase kinase kinase MLK Mixed-lineage kinase MK Mitogen-activated protein kinase-activated kinase MKK MAPK kinase MKKK MKK kinase MNK MAPK-interacting protein kinase MPK MAP kinase phosphatase MSK Mitogen- and stress-activated kinase mTORC1 Mammalian target of rapamycin complex 1 NFκB Nuclear factor kappa-light-chain enhancer of activated B cells NES Nuclear export signal NGF Nerve growth factor NLS Nuclear localization signal

NLK Nemo-like kinase PRAK p38-regulated / activated kinase PDGF Platelet-derived growth factor PFA Paraformaldehyde PKA/B/C/D Protein kinase A/B/C/D PMA Phorbol myristate acetate PTB Phosphotyrosine-binding Raf kinase MKKK (rapidly accelerated fibrosarcoma) RSK p90 ribosomal S6 kinase SD Minimal medium SH2/3 Src homology 2/3 SOS Son of sevenless STAT Signal transducer and activators of transcription TAK Transforming growth factor-beta activated kinase TAO Thousand and one amino acid kinase Tpl-2 Tumor progression locus-2 kinase TSC2 Tuberous sclerosis complex 2 WT Wild type

Index Index

1 Introduction 1 1.1 Conventional MAP kinase signaling 1 1.1.1 ERK1/2-mediated cellular signaling 3 1.1.2 p38 MAP kinase-mediated cellular signaling 4 1.1.3 JNK and ERK5-mediated cellular signaling 6 1.2 Signaling pathways by the atypical MAP kinases 8 1.3 The atypical MAP kinases ERK3 and ERK4 9 1.3.1 Identification of ERK3 /4 9 1.3.2 Structural properties and activation of ERK3/4 10 1.3.3 The interaction between ERK3/4 and MK5 12 1.3.4 ERK3-interacting proteins and the biological relevance of the interactions 13 1.4 The MAPKAP kinases 14 1.4.1 Members of the MAPKAP kinase family 14 1.4.2 General functions of the MAPKAPKs 16 1.4.3 The subfamily of MKs 17 1.4.4 MK5-interacting proteins and the biological relevance of the interactions 19 1.5 The ERK3/MK5-signaling module 20 1.5.1 Potential function of the ERK3/MK5-signaling module 20 1.5.2 The ERK3/MK5-module as therapeutic target 21

2 Objectives 22

3 Materials and methods 23 3.1 Materials 23 3.1.1 Mouse strains and cell lines 23 3.1.2 Plasmids 24 3.1.3 Oligonucleotides 26 3.1.4 Antibodies 26 3.1.5 Further chemicals 28 3.1.6 Equipment 30 3.1.7 Kits and disposal materials 31 3.1.8 Frequently used buffers 32

3.2 Methods 33 3.2.1 Cloning and site-directed mutagenesis 33 3.2.2 Cell culture and transfection 34

Index 3.2.3 Tissue homogenization 35 3.2.4 Western blotting (conventional and multiplex) 36 3.2.5 Pull-down experiments 36 3.2.6 Immunofluorescence and confocal microscopy 37 3.2.7 MK5 substrate ELISA 38 3.2.8 Recombinant protein expression in E. coli 39 3.2.9 In vitro kinase assay 39 3.2.10 Cell counting and WST-1 proliferation assay 41 3.2.11 Propidium iodide staining for analysis of cell viability or cell cycle 42 3.2.12 Scratch wound healing assay 43 3.2.13 FACS analysis of GFP-ERK3 stably transfected PC-12 cells 43 3.2.14 In vitro screen of ERK3-activating kinase 44 3.2.15 Phosphoproteomics in tissues or immortalized MEF cells 45 3.2.16 Yeast two-hybrid screen 45 3.2.17 The ras recruitment system 46 3.2.18 Endogenous Sept7 solubility assay 47 3.2.19 Isolation of primary hippocampal neurons 47 3.2.20 Sholl analysis of transfected primary neurons 48 3.2.21 In vivo spine morphology 49

4 Results 50 4.1 Characterization of the ERK3/MK5-signaling module 50 4.1.1 Protein expression of ERK3 and MK5 in selected cell lines and murine tissues 50 4.1.2 Localization, interaction and activation of the ERK3/MK5-module 52 4.1.3 Growth analysis of ERK3/MK5-deficient MEFs 54 4.1.4 The impact of ERK3/MK5-deficiency on MEF migration 55 4.1.5 The actin filament in ERK3/MK5-deficient MEFs 56 4.2 Regulation of ERK3 protein stability by extracellular factors 56 4.2.1 NGF-stimulation of PC-12 cells 56 4.2.2 Rapid degradation of ERK3 occurs via the 26S proteasome 58 4.2.3 The extracellular pH regulates ERK3 protein levels 59 4.2.4 FACS-based method for real-time monitoring of ERK3 protein 59 4.2.5 Screen for the underlying mechanisms of ERK3 degradation 61

4.3 Interaction partners of the ERK3/MK5-signaling module 63

Index

4.3.1 In vitro screening for an ERK3/MK5-activating kinase 63 4.3.2 Influence of PAK activity on ERK3 phosphorylationand stability 66 4.3.3 Analysis of the phosphoproteome in MK5-deficient tissues 67 4.3.4 Analysis of the phosphoproteome in ERK3/MK5- deficient MEFs 68 4.3.5 Yeast two-hybrid screens for MK5-interacting partners 70 4.3.6 Screen for ERK3-interacting proteins using the ras recruitment system 80 4.3.7 The ERK3-interacting protein Septin7 81 4.4 The Neuronal function of the ERK3/MK5-signaling module 86 4.4.1 Regulation of septin organization via Borg proteins 86 4.4.2 Overexpression-dependent phenotype in ex vivo primary neurons 87 4.4.3 In vivo spinal phenotype in hippocampal neurons of MK5-deficient mice 90

5 Discussion 91 5.1 Functional aspects of ERK3/MK5-signaling module in MEFs 91 5.1.1 The implication in cell cycle progression 91 5.1.2 The impact on cell migration by alteration of actin filament 92 5.2 ERK3 acting as sensor for environmental changes 94 5.2.1 ERK3 protein levels are sensitive to extracellular pH changes 94 5.2.2 Possible mechanisms of the pH-dependent ERK3 degradation 95 5.3 Interacting partners of the ERK3/MK5-signaling module 97 5.3.1 In vitro activation of the ERK3/MK5-signaling module 97 5.3.2 The absence of ERK3 and MK5 lead to changes in the phosphoproteome 98 5.3.3 Evaluation of the biological relevance of the novel MK5- interacting proteins 99 5.3.4 Evaluation of the biological relevance of the novel ERK3- interacting proteins 104 5.4 The ERK3/MK5-signaling module affects neuronal morphology by reorganizing septin filaments 105

6 Conclusions and Perspective 109 7 References 111 8 Appendix 126 8.1 List of tables and figures 8.2 List of recombinant kinases (Proqinase GmbH) 8.3 Declaration (Eigenständigkeitserklärung) 8.4 Acknowledgements (Danksagung) 8.5 Curriculum vitae (Lebenlauf)

Introduction

1 Introduction

1.1 Conventional MAP kinase signaling

The discovery of a family of serine/threonine protein kinases, which are activated in response to a variety of extracellular stimuli, goes back to the early 90s. At that time, a group of scientists around Melanie H. Cobb and George D. Yancopoulos reported about three distinct kinases, which are activated and tyrosine phosphorylated in response to cellular stimulation with insulin and nerve growth factor (Boulton et al., 1991). They purified and cloned the first three mammalian mitogen-activated protein kinases (MAPKs), which they referred to as extracellular signal-regulated kinases (ERKs) 1, 2 and 3 (Boulton et al., 1990a, Boulton et al., 1990b). Besides, ERK activation by tyrosine receptor phosphorylation they could demonstrate the downstream activation of ribosomal protein S6 kinase and the subsequent phosphorylation of S6 protein. They created the first scheme of cellular signaling by activation of kinase cascades, including MAPKs (Cobb et al., 1991). Conventional MAPKs are activated by dual threonine/tyrosine phosphorylation of a conserved TXY-motif, which is phosphorylated by a MAPK kinase (MAPKK, MKK, MEK). MEK1 was the first isolated MAPK kinase, which phosphorylates both threonine and tyrosine residues of the TXY-motif (Nakielny et al., 1992a, Nakielny et al., 1992b). MEK1 is phosphorylated by an upstream kinase referred to as Raf1 (Dent et al., 1992), which is activated by the small GTPase Ras (Robbins et al., 1992). The Raf1-MEK1/2-ERK1/2 signaling cascade was the first MAP kinase-pathway, which was completely understood, and is one of the most extensively studied cellular pathways. The inactivation of MAPK occurs due to dephosphorylation of both phospho-sites by a single dual specific protein phosphatase (DUSP). The first identified mammalian MAPK phosphatase was designated as human VH1 phosphatase homolog (HVN1, DUSP1) (Zheng and Guan, 1993).

Currently, there are 14 distinct mammalian MAPKs identified, whereas ten of them showed the typical MAPKs activation via dual phosphorylation, and thus designated as conventional MAPKs. Besides the ERK1/2 kinase, these conventional MAP kinases are classified based on their homology into the subfamily of the p38 MAP kinases (α, β, γ, and δ), the c-Jun N- terminal kinases (1, 2, and 3) and ERK5 (also know as big MAPKs 1, BMK1). The distinct functions of the subfamilies of MAPKs will be discussed in the following chapters. The four other known MAPKs including ERK3/4, ERK7 and the Nemo-like kinase (NLK) do not share the conventional activation, because they do not exhibit a TXY-motif within their activation loop; hence they are referred to as atypical MAP kinases. The activation of the atypical MAP kinase ERK3 occurs upon single-site phosphorylation of serine at position 189 (Cheng et al.,

1 Introduction 1996b). The detected single phosphorylation of its SEG-motif, within the activation loop, is also conserved in ERK4 (S186) (Aberg et al., 2006, Kant et al., 2006). Initially, ERK4 has been demonstrated to be phosphorylated at unknown tyrosine residue(s) in response to growth factor treatment (Peng et al., 1996). ERK7 and its human homolog ERK8, as well as NLK, also contain a single phosphor-acceptor site TEY and TQE, respectively. The recent knowledge about atypical MAPKs signaling will be addressed in later parts of this introduction. So far, many different stimuli are known activators of conventional MAPKs cascades, which promote the regulation of various downstream targets. The following Fig. 1 summarizes conventional MAP kinases cascades and indicates several downstream targets regulating, among others, apoptosis.

Fig. 1. Cascades of conventional MAP kinase activation. MAPK signaling occurs via a conserved cascade of three distinct kinases, which are stepwise activated. Conventional MAPKs are the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1, 2, and 3 (JNK1/2/3), p38 MAP kinase α, β, γ, and δ (p38α/β/γ/δ), and ERK5. MAPK signaling is involved in lots of cellular mechanism, for example in apoptosis and stress-mediated signaling. Figure adapted from (Wada and Penninger, 2004). MAPK signaling is involved in many eukaryotic cell processes, including differentiation and proliferation, cell cycle progression and mitosis, apoptosis and cell survival, metabolism, as well as migration. The spectrum of extracellular signals regulating MAPK pathways ranges from growth factors and inflammatory cytokines, to UV irradiation, osmotic pressure, and mechanical stress. Activation of various MAPK pathways can occur autonomously or even

2 Introduction simultaneously by co-activation of the single kinase cascades. Two distinct MAPKs pathways could be stimulated at the same time, but can lead to opposite cellular behaviors. Since, several MAPK pathways are active at the same time, cellular behavior results from total signal integration. Among others, downstream substrates of MAP kinases are the family of MAPK-activated kinases (MAPKAPKs). This family is composed of the MAPK-activated protein kinases (MKs) (Gaestel, 2006), p90 ribosomal S6 kinases (RSKs) (Carriere et al., 2008b), mitogen- and stress-activated kinases (MSKs) (Arthur, 2008), and MAPK-interacting kinases (MNKs) (Buxade et al., 2008). The MAPKAPKs represent a further downstream step in MAPK signaling that mediates cellular response by affecting, among others, a broad spectrum of transcription factors.

1.1.1 ERK1/2-mediated cellular signaling

As described above, ERK1 and ERK2 kinase were the first discovered mammalian MAPKs. They display 83% sequence homology and both are ubiquitous expressed in tissues with highest abundance in the brain, skeletal muscle, thymus and the heart. In quiescent cells, ERK1/2 are negligibly phosphorylated, whereas the kinases can be rapidly activated in response to a large number of extracellular signals, such as growth factors, including the epidermal growth factor (EGF), the nerve growth factor (NGF), the platelet-derived growth factor (PDGF), and in response to insulin (Boulton et al., 1990b). The binding of growth factor dimers to their receptor leads to receptor dimerization and activation via tyrosine phosphorylation. These phospho-sites serve as binding motifs for proteins that contain a Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domain, including the growth factor receptor-bound protein 2 (Grb2) (Lowenstein et al., 1992). Binding of Grb2 induces the recruitment of another cytosolic protein referred to as the son of sevenless (SOS). The guanine nucleotide exchange factor (GEF) SOS is thereby activated and promotes GDP-to- GTP exchange within the membranous small GTPases of the Ras family (Bonfini et al., 1992). The activated Ras GTPases mediate, among others, activation of Raf kinases, which represents the MAPKKK in the ERK1/2 signaling cascade (Dent et al., 1992).

Moreover, activation of ERK1/2 kinase has also been shown to be downstream of active heterotrimeric G protein-coupled receptors, cytokines, osmotic stress and microtubule reorganization (Raman et al., 2007). In non-stimulated cells, all components of the ERK1/2 signaling cascade are cytosolic localized. ERK1/2 relocalize and accumulate in the nucleus in response to cascade activation. The function of this nuclear enrichment is still unknown (Chen et al., 1992, Lenormand et al., 1993). Downstream kinase targets of ERK1/2 are the

3 Introduction MNK1/2, MSK1/2, and RSK1-4, which phosphorylate various substrates. ERK1/2 can directly phosphorylate a high number of transcription factors like the CREB-binding protein (CBP), Elk1, c-Fos, c-Myc, nuclear factor of activated T-cells c4 (NFATc4), p53, Smad1-4, specificity protein 1 (SP1), and the signal transducer and activators of transcription (STATs), also several cytoskeletal proteins, such as calnexin, microtubule-associated proteins (MAP), paxillin, and tau. Even other signaling molecules, including epithelial growth factor receptor (EGFR), Grb2-associated binder 2 (Gab2), phospholipase C (PLC), small GTPase Rab4, and Tuberous sclerosis protein 2 (TSC2) are known targets [substrates reviewed by (Yoon and Seger, 2006)].

The diversity of the phosphorylated proteins indicates the complexity of ERK1/2 mediated signaling. Many biological processes are regulated by ERK1/2 kinase activity, including cell growth, mobility, proliferation and differentiation. In Fig. 2, selected biological functions are summarized.

Fig. 2. Overview of biological processes involving ERK1/2-mediated substrate phosphorylation. The figure describes selected ERK1/2 targets mediating cellular behavior, such as altered gene transcription, cytoskeletal assembly, apoptosis, proliferation, differentiation, oncogenic transformation, growth and metabolism in response to extracellular stimuli [substrates reviewed by (Yoon and Seger, 2006)]. Abbreviations: AML1, acute myeloid leukemia 1 protein; ATF, activating transcription factor; BCL6, B-cell lymphoma 6 protein; ER, estrogen receptor; HIF1α, hypoxia-inducible factor 1α; MCL1, induced myeloid leukemia cell differentiation protein; PLA2, phospholipase A2.

1.1.2 p38 MAP kinase-mediated cellular signaling

Extracellular signal-regulated pathways are indispensable for a living organism to react on environmental changes. At the cellular level, changes of the environment are usually

4 Introduction connected to stress, which can have multiple reasons, such as oxidative and mechanical stress, UV irradiation, changes in temperature, and osmotic pressure. One key molecule in stress-mediated signaling is the archetypal member of a further MAP kinase signaling cascade, which is designated, related to its molecular mass, as p38 MAP kinase. The family of p38 MAPKs share high homology with the product of the yeast hog1 gene, which is activated in response to hyperosmolarity (Han et al., 1994, Lee et al., 1994, Rouse et al., 1994). There are four different known isoforms referred to as p38α, β, γ, and δ. The p38α and p38β isoforms are found ubiquitous in nearly all tissues, whereas the p38γ and p38δ have more restricted expression patterns, which suggest a more specialized function. The p38α isoforms showed the highest expression levels and most of p38-mediated cellular processes are due to activation of the alpha isoform. In Fig. 3, selected isoform-specific targets of p38 are summarized.

Activation of p38 kinases occurs upon dual phosphorylation of the TGY-motif by the MAPKKs MKK3, MKK6 (Derijard et al., 1995) and occasionally by MKK4 (Meier et al., 1996). The p38 signaling cascades are activated by, among others, the Rho family GTPases Rac and Cdc42

Fig. 3. The p38 MAP kinases are activated in response to inflammatory cytokines, growth factors and environmental stress. The scheme shows representative targets of distinct p38 isoforms. Activation of p38 triggers altered transcription and translation, which regulates stress-mediated cellular behavior. Abbreviations: CHOP, C/EBP-homologous protein; DLK1, dual-leucine-zipper-bearing kinase 1; EEA1, early-endosome antigen 1; eEF2K, eukaryotic elongation factor 2 kinase; eIF4E, eukaryotic initiation factor 4E; HMG-14, high-mobility group 14; NHE-1, Na+/H+ exchanger 1; PSD95, postsynaptic density 95; Sap1, SRF accessory protein 1; TAO, thousand-and-one amino acid; TPL2, tumor progression loci 2; TTP, tristetraproline; ZAK1, leucine zipper and sterile-α motif kinase 1; ZNHIT1, zinc finger HIT-type 1. (Cuadrado and Nebreda, 2010)

5 Introduction (Bagrodia et al., 1995), GPCRs (Goldsmith and Dhanasekaran, 2007) and TRAF proteins bound to activated tumor necrosis factor receptor (TNFR) (Bradley and Pober, 2001). These stimuli lead to activation of MAPKKKs, such as MEKK1/2/3, MLK2/3, ASK1, Tpl2, TAK1 and TAO1/2 (Cuadrado and Nebreda, 2010), which promote MKK3 and MKK6 activity. Downstream of p38 MAP kinases various targets have been identified including kinases MNK1/2, MSK1/2, and MK2/3/(5), transcription factors ATF1, CREB, NFκB, p53, and STAT1/3, as well as the eukaryotic elongation factor 2K (Cuadrado and Nebreda, 2010). Beside the canonical activation, p38 can be activated by autophosphorylation partially promoted by Tyr323-phosphorylation due to ζ -chain-associated protein kinase of 70 kDa (ZAP70) and p56lck. This activation is independent to MKK3/6 activity and was found to play a functional role in T-lymphocytes (Salvador et al., 2005).

Like for ERK1/2, it has also been reported that due to activation of p38 it relocalizes to the nucleus (Raingeaud et al., 1995), whereas interaction of p38 with MK2/3 (Gaestel, 2006) and MK5 (Li et al., 2008) leads to nuclear export of stable complexes. A major function of p38 pathway is the regulation of the proinflammatory cytokine production (Karin, 2006). Moreover, p38 negatively regulates cell cycle progression by downregulation of cyclin expression and upregulation of cyclin-dependent kinases (CDKs)-inhibitors (Thornton and Rincon, 2009).

1.1.3 JNK- and ERK5-mediated cellular signaling

Another MAPK subfamily, which has been shown to be activated in response to stress, is referred to as JNK [also known as stress-activated protein kinases (SAPKs)]. The JNK1 has been co-purified with the c-Jun protein (Hibi et al., 1993), which explains the designation as c-Jun N-terminal kinase. Three distinct isoforms were purified, which share high homology (>85%) (Kyriakis et al., 1994). JNK1 and JNK2 are ubiquitous expressed, whereas JNK3 expression seems to be restricted to parts of the brain, testis and the heart (Bode and Dong, 2007). Similar to p38 MAP kinase, JNKs are activated in response to cellular stress, like heat shock, mechanical and oxidative stress, growth factors, cytokines, and partially by certain GPCR ligands (Bogoyevitch et al., 2010). Activation of JNK is triggered by dual phosphorylation of the TPY-motif by MKK4 and MKK7 (Lawler et al., 1998). The upstream MAPKKKs that phosphorylate MKK4/7 are MEK1-4, MLK1-3, Tpl-2, DLK, TAO1/2, TAK1, and ASK1/2 (Wagner and Nebreda, 2009). In general, activation of JNK1 and JNK2 promotes c-Jun expression and cell proliferation (Jaeschke et al., 2006). Several other

6 Introduction transcription factors are phosphorylated by JNKs, including ATF-2, Elk1, heat shock factor 1 (HSF-1), c-Myc, NFATc1, and STAT3 (Bogoyevitch et al., 2010). JNK can modulate the activator protein 1 (AP-1) complex formation, which has a strong impact on cell cycle progression (Sabapathy et al., 2004). The JNKs are involved in signaling pathways promoting to apoptotic response upon cellular stress stimuli (Dhanasekaran and Reddy, 2008). The inactivation of JNKs has been reported to inhibit cytochrome c release. Interestingly, no phosphorylation of MAPKAP kinases by JNK isoforms is known. JNK isoform-specific signaling pathways remain to be elusive.

The MAPKs ERK5 is characterized by its molecular mass, which is two times higher than that of other conventional MAPKs, thus the kinase is referred to as big MAP kinase 1 (BMK1). The kinase was discovered simultaneously by three independent groups (English et al., 1995, Lee et al., 1995, Zhou et al., 1995). ERK5 has a unique C-terminal part, containing a nuclear localization signal (NLS) and a proline-rich region. The kinase is ubiquitously expressed with highest abundance in the brain, thymus, and spleen (Yan et al., 2003). It is activated by dual phosphorylation of its TEY-motif by MEK5 (English et al., 1995), which is activated by MEKK2/3 (Wang et al., 2006). ERK5-activation has been shown to induce nuclear accumulation of the kinase (Wang et al., 2006). An ERK5-specific extracellular stimulus could not yet been shown, since MEKK2/3 also activates p38 and JNK MAPKs. Nevertheless, ERK5 has been demonstrated to be necessary during embryonic development (Yan et al., 2003). For instance, it is required for normal development of the vascular system (Regan et al., 2002) and indispensable for cell survival. Several substrates of ERK5 have been identified, including myocyte enhancer factor 2 (MEF2), Sap1a, c-Myc, serum- and glucocorticoid-induced protein kinase (SGK), connexion 43, and Bad (Hayashi and Lee, 2004, Wang et al., 2006). ERK5 activity is necessary for cell cycle progression, promoting G1/S phase transition through increased expression of cyclin D1 (Mulloy et al., 2003). It has also been reported that ERK5 can phosphorylate RSKs (Ranganathan et al., 2006), as it was shown for ERK1/2.

In contrast to ERK1/2, p38 MAPK, JNK and ERK5, four other MAP kinase family members are not activated by the ‘conventional way’. They are designated as atypical MAPKs representing three mostly unexplored MAPKs pathways, which will be discussed in the following chapter. Atypical MAPKs, except for ERK7, are characterized by a single phospho- acceptor site, suggesting that their activation is triggered by protein kinases different to common dual-specific MAPKK.

7 Introduction 1.2 Signaling pathways by the atypical MAP kinase

The atypical type of MAPKs will play an important role in this thesis. Four known atypical MAP kinases are designated as ERK3 and its nearest homolog ERK4, furthermore ERK7 (human homolog ERK8), as well as the Nemo-like kinase (NLK). The phylogenetic tree of the human kinome demonstrates that especially ERK7 (ERK8) and ERK3/4 are evolutionary separated from the other conventional MAPKs (Fig 4). While ERK3/4 will be in focus of the following sections, the other two atypical MAP kinase members will be introduced in this section.

ERK7 was cloned from rat cDNA, using degenerate primers from the ERK1 kinase domain (Abe et al., 1999). Later the human homolog ERK8 was purified by the same group (Abe et al., 2002). Especially the C-terminus differs from conventional MAP kinase and is responsible for subcellular localization and kinase autophosphorylation (Abe et al., 1999, Abe et al., 2001). ERK7 protein level is strongly regulated by N-terminal ubiquitination and subsequent degradation by the ubiquitin-proteasome pathway (Kuo et al., 2004). Since the dual-phosphoacceptor site is conserved in ERK7 kinase, a conventional activation pathway may exist, whereas no known MAPKK could phosphorylate Fig. 4. Phylogenetic tree of the human kinome. MAPKs belong to the group of CDK, MAPK, GSK and CLK kinases and activate this kinase. Interestingly, in (CMGC). Figure modified from (Manning et al., 2002). quiescent cells ERK7 seems to be constitutively active, which might indicate that its activity is primarily regulated by autophosphorylation (Abe et al., 1999, Abe et al., 2001). For the human homolog ERK8, it has been shown that stimulation with serum and

H2O2 leads to increased phosphorylation (Klevernic et al., 2006). So far, no in vivo substrate of ERK7/8 has been identified, whereas classical MAPK substrates like myelin basic protein (MBP), c-Myc, and c-Fos are in vitro phosphorylated by ERK7/8 (Abe et al., 1999). There is no evidence for MAPKAP kinase activation by ERK7.

8 Introduction Regarding to its biological function, it has been reported that ERK7 might be involved in estrogen-(Henrich et al., 2003) and glucocorticoid-mediated signaling (Saelzler et al., 2006) as well as cell proliferation (Abe et al., 1999).

The Nemo-like kinase (NLK) was identified using degenerate primers from conventional MAPK sequences (Brott et al., 1998). The NLK displays 45% homology with ERK2 and is characterized by N- and C-terminal extensions, which are not present in conventional MAPKs. The C-terminal extension is conserved from human to worm and might regulate NLK-interaction with various substrates (Ishitani et al., 1999). It has been demonstrated that NLK is downstream activated by Wnt-1 and Wnt-5a signaling (Kanei-Ishii et al., 2004), several cytokines, such as IL-6, granulocyte colony-stimulating factor (G-CSF), and transforming growth factor β (TGFβ) (Ohkawara et al., 2004, Kojima et al., 2005). It has been reported that the MAPKKK TAK1 promotes NLK activation. Unfortunately, no related MAPKK could be identified, which phosphorylate the TGE-motif in the activation loop of NLK. Evidence exist for the involvement of homeodomain-interacting kinase 2 (HIPK2) in binding and phosphorylation of NLK in vitro and in vivo, although it is not clear whether HIPK2 directly phosphorylates the activation-site of NLK, or it triggers NLK autophosphorylation by phosphorylation of an unknown site located within the C-terminus (Kanei-Ishii et al., 2004). Several NLK substrates were identified, including T-cell factor/lymphoid enhancer factor (TCF/LEF) family (Ishitani et al., 1999) and STAT3 (Kojima et al., 2005), regulating β-catenin pathways in both positive and negative directions (Ishitani et al., 1999, Smit et al., 2004). Currently, there is no evidence for MAPKAP kinase activation by NLK.

1.3 The atypical MAP kinases ERK3 and ERK4

1.3.1 Identification of ERK3/4

ERK3 or MAPK6 was identified together with ERK2 in a homology screen of a rat brain cDNA library using ERK1 degenerate primer (Boulton et al., 1991), identifying a 543 aa long protein. Later, also human (Zhu et al., 1994) and mouse (Turgeon et al., 2000) homologs have been described, which share about 90% sequence similarity with the rat protein, but additionally share a 178 aa long C-terminal extension, resulting in a molecular mass of about 100 kDa. The missing rat C-terminal extension could be explained by a missing nucleotide between codon 502 and 503 in the initial rat sequence. This leads to a frame shift resulting in a 720 aa protein, which shares 94% homology to human ERK3 (Turgeon et al., 2002).

9 Introduction Genomic analysis revealed six distinct human MAPK6 pseudogenes present on four different gene loci (Turgeon et al., 2002).

ERK4 was discovered in 1992 by using the same procedure as for ERK3. The kinase was initially designated as p63mapk, and the rat protein consists of 557 aa (Gonzalez et al., 1992). Later, the name has been changed to MAPK4/ERK4, because of sequence inconsistency showing a corrected molecular mass of about 70 kDa (587 aa). ERK4 shares 73% homology to ERK3. The high protein sequence similarity and the genomic organization of ERK3 and ERK4 might suggest that their related genes arose by duplication of a common ancestor. Interestingly, ERK3/4 genes are not found in plants or invertebrates, which suggests a vertebrate-specific function (Krens et al., 2006).

1.3.2 Structural properties and activation of ERK3/4

ERK3 and ERK4 kinases are characterized by C-terminal extensions, which contain a conserved region within the first part, whereas other regions of their C-terminus are more diverse. In contrast to conventional MAPKs, ERK3/4 contain a single phospho-acceptor site in their activation loop. The conventional MAPK conserved APE-site of the kinase subdomain VIII is replaced by an SPR-site (Kostich et al., 2002). The effect of this variation remains elusive, but computational modeling with the Swiss-Model sever, using ERK2 as template, revealed that the Ser199 of the ERK3 SPR-motif interacts with the Arg304 of the kinase subdomain XI (Fig. 5A). This interaction leads to stabilization of the C-terminal loop of ERK3. In conventional MAPKs, the glutamate residue of the APE-motif is responsible for subdomain XI interaction. It seems that the function of the SPR-motif is similar to the common APE- motif, whereas differences in affinity of hydrogen bonding cannot be excluded (Coulombe and Meloche, 2007).

Besides the ERK2 homology modeling, the crystal structure of a human ERK3 fragment (9- 327 aa), including the whole kinase domain (20-316 aa), was published in the RCSB protein data bank (DOI:10.2210/pdb2i6l/pdb; Filippakopoulos et al.; Fig. 5B). Currently, a crystal structure of the full-length protein is not available. It is known that full-length ERK3 protein is difficult to express in E. coli. Other systems, such as protein expression in fungal or insect cells, principally work, but enrichment of the protein to the required concentration for effective crystal formation was not reached, because of the low protein solubility (personal communication with A. Hennig, Institute of Biophysical Chemistry, Hannover Medical

10 Introduction School). Attempts for co-crystallization of the ERK3(4)/MK5-complex in collaboration with the MHH Biophysical chemistry department were unsuccessful.

A B SPR-motif Kinase domain ERK3 (9-327aa)

Fig. 5. Structural analysis of ERK3-related fragments. A) First structural investigation revealed that the S199 of ERK3 unique SPR-motif of kinase subdomain VIII could interact with the Arg304 of subdomain XI. This was shown by computational modeling using the Swiss-Model server and ERK2- data as template. Figure modified from (Coulombe and Meloche, 2007). B) Crystal structure of human ERK3 fragment (9-327aa) including the whole kinase domain (20-316aa) published in the RCSB protein data bank (DOI:10.2210/pdb2i6l/pdb; data by Filippakopoulos & colleagues).

ERK3 phosphorylation at Ser189 has been detected in quiescent cells (Boulton et al., 1991, Coulombe et al., 2003, Coulombe et al., 2004), whereas the mode of ERK3/4-activation remains unknown. Catalytically dead ERK3 kinase is also phosphorylated at S189 residue, which indicates that activation of ERK3 occurs in trans by a distinct kinase (Coulombe et al., 2004). A potential ERK3 activating kinase has been purified from crude rabbit muscle lysates (Cheng et al., 1996b). This kinase could not activate ERK1/2 that might confirm its specificity. No further efforts have been made to characterize the isolated kinase. Another kinase family, which might activate ERK3, are type I p21-activated kinases 1-3 (PAK1-3) (De la Mota- Peynado et al., 2011, Deleris et al., 2011). The interaction with PAKs was identified in both directions using a yeast two-hybrid screen and a peptide microarray approach, respectively. Both studies did not show activation and interaction on endogenous protein level. Increase in kinase phosphorylation of ERK3 at Ser189 has been observed in serum-starved cells, which could not be influenced by growth factors treatment or application to chemical stress (Deleris et al., 2008). Additional phosphorylation at four distinct C-terminal phospho-sites was detected after stimulation with anti-neoplastic agents, such as nocodazole, that trap cells in mitosis (Tanguay et al., 2010). It is an interesting question how activation loop phosphorylation of ERK3 is correlated with its enzymatic activity. It has also been published that ERK3 like ERK7 autophosphorylates in vitro (Cheng et al., 1996a).

11 Introduction Regarding to ERK4-activation, even less is known. Activation site phosphorylation at Ser186 is induced by interaction with the known ERK4 (also ERK3) substrate MK5 (MAPKAPK5, PRAK), whereas MK5 is not thought to be responsible for Ser186 phosphorylation, rather than triggering multiple phosphorylation at other unknown site(s) (Aberg et al., 2006, Kant et al., 2006). Conventional MAPK stimulations, such as calf serum, PMA, PDGF, H2O2, or UV-c, have no influence on Ser186 phosphorylation. (Perander et al., 2008a) In vitro, ERK4 can also be activated by PAKs. The in vivo relevance of ERK4 is questionable due to the absence of any functional or visible phenotype, except for an impaired depression-related behavior, in the ERK4 knockout mouse model (Rousseau et al., 2010). Thus, the research on ERK4-interaction with MK5 becomes less important for this study.

1.3.3 The interaction between ERK3/4 and MK5

In 2004, ERK3 was identified as MK5-interacting protein in a yeast two-hybrid screen. MK5 was used as bait, screening a day 11 mouse embryo cDNA library (Schumacher et al., 2004). The specificity of ERK3/MK5-interaction has been confirmed by another group, which analyzed the growth of yeast transformants with MK5 bait and several distinct MAPKs prey constructs (Seternes et al., 2004). First interaction studies indicated that both kinases are phosphorylated in response to binding each other. The ERK3 protein levels are stabilized by this interaction. The mode of activation was under controversial discussion. The group around Ole-Morten Seternes concluded that ERK3 directly phosphorylates the Thr182 in the activation loop of MK5, whereas our group could demonstrate that ERK3-binding mediates autophosphorylation of MK5 that induced further phosphorylation of MK5 by ERK3. ERK3 binding to MK5 is promoted by its activation site phosphorylation (Deleris et al., 2008), which might indicate the presence of a further ERK3-activating kinase. To target the interacting sites within ERK3 and MK5 both groups performed pull-down studies with deletion mutants, concluding that the protein binding occurs via their C-terminus. From ERK3 mutational studies, the region of MK5-binding to ERK3 could be localized to amino acid 301-357 (Schumacher et al., 2004) and 330-340 (Seternes et al., 2004), respectively. The ERK3- binding motif within MK5 seems to be located within amino acid 368-472 (Schumacher et al., 2004) and 423-472 (Seternes et al., 2004). Our group could also show that the catalytic activity of ERK3 and common MAPK-binding motif designated as D domain of MK5 (MK5: 361-364 aa) is not required for their interaction (Schumacher et al., 2004). ERK3 activity is necessary for significant MK5 activation (Seternes et al., 2004).

12 Introduction Later, Aberg and colleagues could define the MK5-binding domain in ERK4 by peptide array to FRIEDEIDDIV sequence (328-338 aa) within the L16 loop of ERK4. The corresponding motif in ERK3 is FHIEDEVDDIL (332-342 aa). The identified binding-motif has been designated as FRIEDE-domain, which represents a new mode of MAPK-MAPKAPK interaction. This motif is not conserved in other conventional MAPKs. (Aberg et al., 2009)

Intracellular ERK3 is evenly distributed in cytosol and nucleus, whereas MK5 is predominantly localized in the nucleus. Upon overexpression of ERK3 and MK5, both proteins are relocalized to the cytoplasm, whereas nuclear localization is completely abolished. Nuclear export of MK5 does not require activation loop phosphorylation at Thr182 and/or activity of ERK3, whereas the C-terminus of both proteins is indispensable (Schumacher et al., 2004).

MK5 was the first described bona fide substrate of ERK3/4. The interaction of ERK4 and MK5 could be shown on endogenous level (Kant et al., 2006, Perander et al., 2008a). The biological relevance of ERK3/MK5-interaction has not been established, but since that time further discoveries related to MK5-specific and ERK3-specific cellular function have been reported, which will be addressed in the next section.

1.3.4 ERK3-interacting proteins and the biological relevance of the interactions

ERK3 mRNA is ubiquitously expressed in all tissues with the highest abundance in the brain, skeletal muscle, and in the gastrointestinal tract. In mouse embryos, ERK3 expression is strongly increased between day 9 and day 11 (Turgeon et al., 2000). The first described potential biological function of ERK3 was as a regulatory protein in cell cycle progression. It has been reported that the kinase negatively regulates cell proliferation (Coulombe et al., 2003, Julien et al., 2003), and ERK3 has been shown to be necessary for G1/S phase transition by interacting with cyclin D3 and the phosphatase Cdc14 (Hansen et al., 2008). The regulation of ERK3 activity during mitosis might be regulated by cyclin-dependent kinase 1 (Cdk1) and Cdc14 phosphatase (Tanguay et al., 2010). In mouse oocytes, ERK3 is required for meiotic metaphase-anaphase transition (Li et al., 2010). During mitosis, ERK3 protein levels are stabilized, and multiple phosphorylations at the C-terminus could be observed (Tanguay et al., 2010). Increased protein expression of ERK3 has also been detected during differentiation in neuronal cells and myoblasts (Boulton et al., 1991, Coulombe et al., 2003).

13 Introduction ERK3 protein levels are strongly regulated by proteasomal degradation (Coulombe et al., 2003, Coulombe et al., 2004). In contrast, except for ERK7, MAPKs are stabile proteins, which are mainly regulated by activation-site phosphorylation and subcellular relocalization. ERK3 degradation upon N-terminal ubiquitination is independent of the activation loop phosphorylation, presence of the C-terminus and enzymatic activity (Coulombe et al., 2004).

The ERK3-deficient mouse model indicates an important role during embryogenesis. Knockout mice showed intrauterine growth restrictions and early neonatal death due to reasons, which are not fully understood (Klinger et al., 2009).

Recent findings suggest the interaction of ERK3 with type I p21-activated kinases 1-3 (PAK1- 3), indicating another interesting functional link between these two pathways. The PAKs are involved in the regulation of the cytoskeleton (Bokoch, 2003, Molli et al., 2009). For MK5, an implication in processes, leading to actin remodeling upon PKA-activation, has already been demonstrated (Gerits et al., 2007a, Kostenko et al., 2011b).

1.4 The MAPKAP kinases

1.4.1 Members of the MAPKAP kinase family

The family of the MAPK-activated kinases (MAPKAPKs) consists of 11 distinct kinases, which belong to the superfamily of calcium/calmodulin-dependent kinases (CAMK). MAPKAPKs are further divided into the ribosomal-S6-kinase subfamily (RSK1–4), the mitogen- and stress-activated-kinase subfamily (MSK1,2), MAPK-interacting kinases -1 and - 2 (MNK1,2), and the real MAPKAPKs or MKs (Roux et al., 2004, Gaestel, 2006) (Fig. 6). RSK and MSK families are characterized by the presence of an additional kinase domain, which is homologous to those of the superfamily of AGC kinases (main subfamilies PKA, PKG, and PKC).

The family of RSKs were the first described MAPKAPKs (Erikson and Maller, 1985), which are important downstream targets in ERK1/2-mediated signaling (Smith et al., 1999). As already mentioned, RSKs have two functionally distinct kinase domains, the N-terminal kinase domain (NTKD), which belongs to AGC family, and the C-terminal kinase domain (CTKD), which is homologous to CAMK-family (Jones et al., 1988). Multiple studies analyzing the protein expression profiles of RSK isoforms revealed that RSK1-3 is found ubiquitously, with spatiotemporal changes and mutually exclusive expressions during embryogenesis (Kohn et al., 2003). Even though RSK4 is also ubiquitous expressed, but the

14 Introduction observed protein amounts are almost close to the detection limit (Dummler et al., 2005). Activation of RSKs by ERK1/2 and probably also ERK5 is promoted by Thr573 phosphorylation within CTKD (Smith et al., 1999, Ranganathan et al., 2006), which leads to autophosphorylation of Ser380 (Vik and Ryder, 1997). By phosphorylation, a docking site for phosphoinositide-dependent protein kinase 1 (PDK1) is formed, which phosphorylates the Ser221 activation loop site of the NTDK (Richards et al., 1999).

Fig. 6. Phylogenetic tree of MAPK-activated protein kinases (MAPKAPKs). MAPKAPKs belong to the superfamily of calcium/calmodulin-dependent kinases (CAMKs) and consist of MAPK-activated kinases MK2, MK3 and MK5, ribosomal-S6-kinases 1-4 (RSK1–4), the mitogen- and stress-activated- kinases 1 and 2 (MSK1, 2), and MAPK-interacting kinases 1 and 2 (MNK1, 2). Figure from (Gaestel, 2006).

The MSK family of MAPKAPKs was identified by genome-wide homology analysis (Deak et al., 1998) as well as by yeast two-hybrid screens using p38α as bait (Pierrat et al., 1998). Both MSK isoforms (MSK1 and MSK2) are downstream targets of ERK1/2 and p38 MAPKs, which bind to the MAPK binding motif LAKRRK (Tomas-Zuber et al., 2001). MSKs are ubiquitously expressed with highest abundance in brain, heart, placenta, and skeletal muscle (Deak et al., 1998). Upstream MAPKs have been shown to phosphorylate MSKs at Ser360, Thr581 and Thr700 (McCoy et al., 2007). Unlike the activation of RSK, single CTKD phosphorylation at Thr581 seems to be sufficient for proper MSK activation (Chrestensen and Sturgill, 2002). Phosphorylation of Thr700 masks the autoinhibitory C-terminal sequence and leads to phosphorylation of Ser376, Ser381, and the NTKD activation site Ser212 (Vermeulen et al., 2009).

The MNK family of MAPKAPKs could be identified in two distinct ERK1/2-substrate screens (Fukunaga and Hunter, 1997, Waskiewicz et al., 1997). Besides ERK1/2, MNKs are also downstream targets of p38 isoforms. Each of the two isoforms has a long (MNK1/2A) and a short (MNK1/2B) splice variant (Slentz-Kesler et al., 2000, O'Loghlen et al., 2004). MNK1B and MNK2A are characterized by a high basal activity (Scheper et al., 2001, O'Loghlen et al., 2007), whereas MNK1A and MNK2B show lower basal activity. In case of MNK1A, kinase activity significantly increases after MAPK-dependent phosphorylation (Scheper et al., 2003).

15 Introduction 1.4.2 General functions of the MAPKAP kinase subfamily

It has been reported that mutations of the Rps6ka3 gene (RSK2) can cause Coffin-Lowry syndrome (CLS), which is an X-linked form of mental retardation characterized with facial, hand, and skeletal malformations (Trivier et al., 1996). RSK2 knockout mice display impaired cognitive functions, including poor coordination (Dufresne et al., 2001), as well as defects in osteoblasts activity known as osteopenia depending on altered regulation of c-Fos and ATF4 (David et al., 2005). Additionally, 15% reduction of body weight has been observed for RSK2 knockout animals, which is connected to the loss of white adipose tissue (El-Haschimi et al., 2003). Interestingly, RSK1/2/3 triple knockout mice are viable and without any further phenotype in comparison to single RSK2 KO (Dumont et al., 2005). Studies on the cellular level indicate an important role for RSKs in nuclear signaling, cell cycle progression and cell proliferation, cell growth and protein synthesis, and cell survival (Fig. 7). Nuclear signaling including regulation of immediately early (IE) genes, like c-Fos and serum- response factor (SRF), has already been Fig. 7. Substrate targets of ribosomal-S6-kinases 1-4 described (Ginty et al., 1994, Bruning et (RSK1–4), the mitogen- and stress-activated-kinases 1 and 2 (MSK1, 2), and MAPK-interacting kinases 1 al., 2000). The regulation of c-Fos and 2 (MNK1, 2). Activity of MAPKs downstream kinases effect gene transcription, cell growth, survival, and promotes cyclin D1 expression, which proliferation as well as mRNA translation. Figure modified from (Cargnello and Roux, 2011). Abbreviations: C/EBPβ, induces G1-phase progression (Chen et Ccaat-enhancer-binding protein β; DAPK, death- associated protein kinase; GSK3, glycogen synthase al., 1996). RSKs are also involved in cell kinase 3; LKB1, liver kinase B1; MAD1, mitotic spindle assembly checkpoint protein; Myt1, myelin transcription growth regulation by interfering with factor 1; nNOS, nitric oxide synthase; YB-1, Y box binding mammalian target of rapamycin (mTOR) protein. signaling pathway by phosphorylating TSC2 (Roux et al., 2004), Raptor (Carriere et al., 2008a), eukaryotic elongation factor 2 (eEF2) kinase (Wang et al., 2001), and eukaryotic translation initiation factor 4B (eIF4B) (Shahbazian et al., 2006). Other identified substrates are NHE-1 (Takahashi et al., 1999), cell adhesion molecule L1 (CHL1) (Wong et al., 1996), and Ran-binding protein 3 (RanBP3) (Yoon et al., 2008).

MSK-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced endotoxic shock (Ananieva et al., 2008). In general, MSKs predominantly phosphorylate nuclear substrates, which regulate gene expression at multiple levels, by directly interfering with transcriptional

16 Introduction regulation and chromatin remodeling (Vermeulen et al., 2009). MSKs have been shown to phosphorylate CREB with higher affinity than PKA, RSK2, and MK2 (Deak et al., 1998, Pierrat et al., 1998). Knockout of MSK1/2 results in 50% reduction in c-Fos and JunB mRNA under stress stimulation (Wiggin et al., 2002). MSKs are involved in NFκB signaling by phosphorylation of p65 (Vermeulen et al., 2003). It has been reported that under certain conditions MSK2 can suppress p53 transcriptional activity by a non-canonical pathway (Llanos et al., 2009). Primary roles of MSKs in chromatin remodeling are promotion of gene relaxation and activation, for example, by phosphorylation of high-mobility group 14 (HMG14) (Thomson et al., 1999).

MNKs are involved in regulation of protein synthesis via recruitment to eIF4F complex, phosphorylating eIF4E (Quan et al., 1995) and eIF4G (Pyronnet et al., 1999). Generally, there is evidence that MNKs negatively regulate protein synthesis under certain conditions, but can, on the other hand, positively affect mRNA translation and polysome assembly (Bianchini et al., 2008, Zhang et al., 2008). MNK1 and MNK2-deficient mice develop normally, and isolated cells have no defects in global protein synthesis (Ueda et al., 2004). Further described MNK substrates are (PTB)-associated splicing factor hnRNP A1 (Buxade et al., 2005), cytoplasmic phospholipase A2 (Hefner et al., 2000), and RTKs-regulating protein Sprouty 2 (Bundschu et al., 2006).

1.4.3 The subfamily of MKs

The first identified MK was designated as MK2, since MAPKAPK1 (a, b, c, d) has been used as the initial term for RSKs. It was discovered in 1992 as an ERK1/2-activatable protein kinase, which phosphorylates the heat shock protein beta-1 (Hspb1, also known as Hsp25/27) in response to stress (Stokoe et al., 1992). Two years later, it was shown by two distinct groups that MK2 is phosphorylated by p38 in response to stress stimuli (Freshney et al., 1994, Rouse et al., 1994). Another two years later, MK3 has been discovered by a p38 yeast two-hybrid screen (McLaughlin et al., 1996) and analysis of protein depletion in small- cell lung cancer (Sithanandam et al., 1996). MAPKAPK5 (MK5) originally designated as p38- regulated/activated kinase (PRAK, term commonly used for human homolog) was discovered in 1998 within two independent studies by Expressed Sequence Tags (EST) database analysis identifying homologous sequence to MK2. It has been shown that MK5 is in vitro activated by p38 and ERK1/2, whereas JNKs do not activate the kinase (New et al., 1998, Ni et al., 1998). A MK isoform designated as MK4 exists, but the protein is uniquely found in the sea urchin (Komatsu et al., 1997).

17 Introduction In contrast to the high homology of MK2 and MK3 (73%), MK5 is considerably unrelated sharing only 38% sequence homology to MK2. Homologous sequences of mammalian MKs are conserved in MK2 analogs of C. elegans and Drosophila. All MKs possess a kinase domain, which is closely related to those of the CAMKs family members. They contain a MAPK binding motif, a functional nuclear localization signal (NLS) and a nuclear export sequence (NES), which are overlapping in MK5. The NLS of MK5 also overlaps with the MAPK-binding domain, which might cause a weaker interaction to p38 MAP kinases (Seternes et al., 2002). It has already been published by our group that MK5 does not stabilize p38 protein levels in vivo (Shi et al., 2003), as it has been shown for MK2 and MK3 (Ronkina et al., 2007). MK5 lacks a SH3-binding motif, which is present in N-terminus of both MK2 and MK3. The proline-rich SH3-domain of MK2 has been demonstrated to interact with the SH3 of c-Abl in vitro (Plath et al., 1994). In contrast, MK5 carries a unique C-terminal extension of about 100 aa, which includes the binding motif of ERK3 and ERK4. The NES of MK5 has been shown to be functional and nuclear export is promoted by the chromosomal region maintenance 1 (CRM1), which can be blocked by treatment with leptomycin B (Seternes et al., 2002). Comparison of tissue-related mRNA levels of MK isoforms revealed that all mRNAs are found in most of the tissues, whereas MK2 and MK3 are highly abundant in heart, kidney and skeletal muscle (Stokoe et al., 1992, Engel et al., 1993, Sithanandam et al., 1996). MK5 is predominantly expressed in heart, skeletal muscle, lung, and pancreas (New et al., 1998, Ni et al., 1998, Perander et al., 2008a, Gerits et al., 2009). Both MK2 and MK3 genes give rise to at least two alternative splice variants (Stokoe et al., 1992, Chevalier and Allen, 2000, Moise et al., 2010). In case of MK5, two splice variants of 471 and 473 aa have been isolated from human, whereas in mice four distinct isoforms have been described (Dingar et al., 2010). The biological relevance of the two human isoforms remains dubiously. The murine isoforms are characterized by distinct subcellular localization, tissue expression, and catalytic activities.

Structural differences between MK2/3 and MK5 suggest an independent mode of activation. MK2 and MK3 are activated in response to various stress stimuli (Freshney et al., 1994, Rouse et al., 1994, McLaughlin et al., 1996, Guay et al., 1997), whereas MK2-activity is completely dependent on p38α (Clifton et al., 1996, Adams et al., 2000). The binding of p38α to MK2 promotes phosphorylation at Thr222 of the activation loop, at Ser272 located within kinase subdomain X, and within the autoinhibitory region of MK2 at Thr334 (Ben-Levy et al., 1995). Under certain conditions, p38 can also phosphorylate MK5 at the activation loop site Thr182 (Heimrich and Frotscher, 1991, New et al., 1998, Ni et al., 1998, Seternes et al., 2002, New et al., 2003). In vivo activation of MK5 by p38 is arguable, because it is not activated by strong p38 activating stimuli sorbitol and arsenite, and the interaction of endogenous p38 with MK5 has never been reported. Furthermore, MK5-deficient mice are

18 Introduction not resistant to LPS-induced endotoxic shock, and no altered cytokine production could be observed in the knockout mouse (Shi et al., 2003). Interestingly, none of the found properties of the MK2-deficient mouse model could be observed in the MK5 knockout mouse. In fact, no clear phenotype of MK5-deficient animals has been reported, so far. Our data revealed that targeted deletion of MK5 gene in C57BL/6 background leads to increased embryonic lethality at day 11.5 due to unknown reasons (Schumacher et al., 2004).

1.4.4 MK5-interacting proteins and the biological relevance of the interactions

The biological relevance of MK5 is unknown, but several studies indicating putative functions of MK5. This section will focus on published data, which showed MK2/3-related, but also unique functions for MK5.

In contrast to MK5, MK2 has been found to be involved in p38-mediated cytokine production (Han et al., 1994, Lee et al., 1994), cell migration (Piotrowicz et al., 1998, Hedges et al., 1999), actin remodeling (Rousseau et al., 1997), cell cycle control (Ambrosino and Nebreda, 2001), and gene expression (de Nadal and Posas, 2010). Control of cytokine production like TNFα and IL-6 occurs in a p38α-dependent manner by MK2 substrate phosphorylation of, among others, tristetraproline (TTP) and butyrate response factor 1 (BRF1) (Mahtani et al., 2001). MK2 has impact on the actin filament organization and has influence on cell mobility, mainly due to phosphorylation of the heat shock protein beta-1 (Hspb1; also known as Hsp25/27) (Lambert et al., 1999). MK2 can regulate the cell cycle via phosphorylation of CDC25B/C promoting G2/M phase arrest (Manke et al., 2005).

MK5 has also been demonstrated to be involved in actin remodeling, but downstream to a p38-independent pathway. Overexpression of MK5 in HeLa cells resulted in increased cellular F-actin amounts and higher cell mobility (Tak et al., 2007). Interestingly, interaction with 14-3-3ε protein can inhibit this effect by decreasing MK5-mediated phosphorylation of Hsp27. In PC-12 cells, forskolin-induced F-actin expression levels are reduced upon knockdown of MK5 (Gerits et al., 2007a). Later studies gave evidence for direct phosphorylation of MK5 by protein kinase A (PKA), which induce the rearrangement of actin filament. Phosphorylation at Ser115 by PKA promotes the subsequent nuclear export of MK5, which is independent of Thr182 phosphorylation (Kostenko et al., 2011b).

Another interesting function was described by Sun and colleagues. They reported that MK5 plays a role in oncogene-induced senescence. MK5-deficient mice are more susceptible to dimethylbenzanthracene (DMBA) - induced skin carcinogenesis as wild type animals. In

19 Introduction summary, they described a tumor suppressive function of MK5, which is involved in Ras- induced cellular senescence. They proposed a pathway of MK5-induced growth arrest, by direct phosphorylation of p53 at Ser73 inducing its transcriptional activity (Sun et al., 2007). The described phosphorylation site is not a typical MK5 motif. It is a proline-directed phosphorylation site, which is more likely a substrate-motif for MAPK- or CDK-mediated phosphorylation. The effect of p38 on p53 has not been examined in this study. It is known that p53 is an in vivo substrate of p38 and ERK1/2 (Milne et al., 1994, Yeh et al., 2004, Lindqvist et al., 2009). The impact of ERK3/4 was also not analyzed.

Experiments performed in the laboratory of Martin Eilers indicate an MK5-specific function in tumorigenesis. They could show a negative regulation of c-Myc protein expression by MK5. In colorectal tumorigenesis, MK5 expression is downregulated and the negative feedback loop on c-Myc expression via FoxO3a-miR-34b/c is disrupted (Kress et al., 2011). Again, this would underline possible function as a tumor suppressor.

Most recently it has been described that MK5 also functions in energy-depletion-induced suppression of the mTORC1 pathway, which is involved in regulation of cellular growth. In this regard, MK5, specifically activated by p38β, can phosphorylate the Ras homolog enriched in brain (Rheb) at Ser130. This represents a third independent pathway of mTORC1 inhibition, besides the AMP kinase-activated TSC2- and raptor-mediated pathways (Zheng et al., 2011).

1.5 The ERK3/MK5-signaling module

1.5.1 Potential function of the ERK3/MK5-signaling complex

In previous sections, the individual functional relevance of MK5 (section 1.4.4) and ERK3 kinases (section 1.3.4) was separately described. The published data does not clarify a possible ERK3/MK5-signaling pathway. The knockout models of both kinases manifest important functions, which cannot be compensated by alternative signaling pathways. Main discrepancy, which should be eliminated, is the influence of p38 on MK5 signaling to show a clear ERK3/MK5-dependent pathway, which is not interfering with p38-activation and the activation of MK2/3. This could be achieved by generating monospecific MK5- and ERK3- inhibitors or the use of existing inhibitors, such as the p38α/β inhibitor SB202190. The tissue- specific p38-MK5 pathways are not sufficiently investigated, so far.

20 Introduction 1.5.2 The ERK3/MK5-module as therapeutic target

At this point, clear statements about the use of inhibitors for MK5 or ERK3 as therapeutic targets are hard to define. The discovery of a clear phenotype forms the basis for further decisions. In case of ERK3, no inhibitor has been created until now, whereas in regarding to MK5 several agents are already successfully tested.

For MK5, small molecule inhibitors could be used, which have been synthesized by the pharmaceutical company- Pfizer as MK2 inhibitors (for example PF-3644022) (Gaestel et al., 2007). This group of inhibitors can block kinase activity of all MKs with different potency. The use of these inhibitors in wild type cells would not be meaningful, but they can be an adjuvant tool in MK2/3 knockout cells for the analysis of cellular functions requiring MK5-activity.

Another possible inhibitor, noroxoaconitine, has been shown to inhibit MK5 (IC50 81 nM) and

MK3 (IC50 210 nM), whereas not MK2 (Kostenko et al., 2011a). All these described inhibitors are competitors of ATP and interfere with the ATP-binding pocket in MK’s. The use of these inhibitors in animal models is not likely because of missing knowledge about off-target effects and especially for use of diterpenoid alkaloid noroxoaconitine the high toxicity.

Interestingly, a so called mode of action inhibitor of MK5 designated as GLPG0259 has already passed the second phase of a clinical trial done by the Galapagos NV company (Mechelen, Belgium). The GLPG0259 compound also acts as ATP-competitor with high potency to MK5 (IC50 24 nM). Inhibition of MK2 and MK3 has not been published by the company. Nevertheless, the inhibitor has been tested for the treatment of rheumatoid arthritis, a chronic inflammatory arthropathy, which is associated with cartilage erosion and bone destruction. Unfortunately, in human trials, where the GLPG0259 was exhibited to a small number of rheumatoid arthritis patients, no sufficient efficacy could be determined, whereas in vitro and ex vivo models revealed a reduction of inflammatory cytokines (IL-6), and altered NFκB signaling has been detected (European League Against Rheumatism Report 2011). At this point, it is not clear, whether MK2 and MK3 inhibition contributes to the therapeutic outcome of GLPG0259.

21 Objectives 2 Objectives

Since the interaction between the atypical MAP kinase ERK3 and the MAPKAP kinase MK5 has been confirmed in previous studies (Schumacher et al., 2004, Seternes et al., 2004), an in vivo function of this complex is considered. The published data strongly suggest MK5 as ‘bona fide’ substrate of ERK3, whereas no shared function has been established, so far. The two kinases form a stable complex that promotes their protein stability and kinase activity, respectively. Analysis of the related knockout models clearly showed a reduction of MK5 activity in ERK3-deficient cells (Aberg et al., 2006) and a decrease in ERK3 protein in cells lacking MK5 (Schumacher et al., 2004). The following experiments were performed to address the physiological relevance of the ERK3/MK5-signaling module.

1) This work primarily focused on the identification of novel interaction partners of ERK3 and MK5 to complete the atypical MAP kinase pathway. By the use of yeast-based screening approaches, several cDNA expression libraries should be screened. The identified positive clones should be confirmed by co-localization studies and pull- down assays in mammalian cells. The biological relevance of the confirmed interactions should be considered and approved by established methods.

2) Investigation of the phenotype of ERK3- and MK5-deficiency should be done on the level of primary isolated MEF cells and by investigation of the corresponding knockout mice. MEF cells were analyzed regarding to their growth and proliferation, motility and structure of their actin filament.

3) Regulation of cellular ERK3 protein stability should be analyzed on endogenous levels. A GFP-ERK3 stably transfected PC-12 cell line should also be used to investigate possible manipulation of ERK3 protein stability in living cells.

4) To identify the potential ERK3-activating kinase a panel of activated recombinant serine/threonine protein kinases should be utilized in combination with ERK3 and MK5 using a high-throughput format in vitro kinase assay approach.

5) Analysis of the phosphoprotein pattern in tissues and stimulated MEF cells with phospho-serine/threonine kinase substrate antibodies should contribute to the targeting of new substrates of ERK3 and MK5.

22 Materials

3 Materials and methods

3.1 Materials.

3.1.1 Mouse strains and cell lines

Tab. 1. Mouse strains

Mouse strain Description Reference / Source

B6.129-ERK3tm1MaI ERK3 knockout model (Klinger et al., 2009)

B6-MAPKAPK5tm1Mg1 MK5 knockout model (Shi et al., 2003)

B6.Cg-Tg(Thy1-YFPH)2Jrs/J Neuron-specific YFP expression Jackson Laboratory

BALB/cJ Inbred strain Jackson Laboratory

C57BL/6J Inbred strain Jackson Laboratory

Tab. 2. Cell lines

Cell line Description Reference source Human gastric adenocarcinoma AGS Ralf Gerald, MHH cells HEK293T Human embryonic kidney cells our group

HeLa Human epitheloid cervix carcinoma our group

HT29 Human colon adenocarcinoma Ursula Seidler, MHH

IMR-32 Human neuroblastoma (abdominal) Roland Seifert, MHH Mouse embryonic fibroblast; MEF immortalized with SV40 large T antigen our group (MK5 KO, ERK3 KO, ERK3/MK5 dKO) STA-NB11 Human neuroblastoma (adrenal) Roland Seifert, MHH

PC-12 Rat phaeochromocytoma cell Renate Scheibe, MHH Herbert Hildebrandt, SH-SY-5Y Human neuroblastoma (thorax) MHH SK-N-AS Human neuroblastoma (adrenal) Roland Seifert, MHH

SK-N-BE(2) Human neuroblastoma Roland Seifert, MHH

23 Materials

3.1.2 Plasmids

Tab. 3. Plasmids

Plasmid name Description Reference / Source pACT2 N-term myc-tag, yeast expr. Clontech pcDNA3-HA-p38a N-term HA-tag, mammalian our group pcDNA3-GFP-ERK3 C-term dsRed-tag, mammalian Sylvain Meloche pcDNA3-GFP-Sept7 N-term GFP-tag, mammalian Koh-ichi Nagata pcDNA3-RFP-ERK3 C-term dsRed-tag, mammalian Sylvain Meloche pcDNA3-RFP-ERK3 N-term dsRed-tag, mammalian our group pcDNA6/BioEase-ERK3 N-term. BioEase-tag, mammalian Schumacher et al., 2004 pcDNA6/BioEase-MK5 N-term. BioEase-tag, mammalian Schumacher et al., 2004 pCEP4-HA-Mtbp N-term HA-tag, mammalian Mark T. Boyd pd2EGFP Instable GFP, mammalian expr. Clontech pDEST17 N-term His-tag, E. Coli expr, Invitrogen pDEST27 N-term GST-tag, mammalian expr. Invitrogen pDEST27-MK5 Mouse MK5 full-length Schumacher et al., 2004 pDEST27-ERK3 Mouse ERK3 full-length Schumacher et al., 2004 pDEST27-ERK3ΔC1 1-471 aa deletion mutant Schumacher et al., 2004 pDEST27-ERK3ΔC2 1-357 aa deletion mutant Schumacher et al., 2004 pDEST27-ERK3ΔC3 1-301 aa deletion mutant Schumacher et al., 2004 pDEST27-Sept7 human Invitrogen pDEST27-Sept7ΔN 162-436 aa fragment mutant our group pDEST27-Sept7ΔC 1-325 aa fragment mutant our group pDEST27-Sept7ΔNΔC 162-325 aa fragment mutant our group pEAK10-6His-myc-Kal7 N-term 6His-myc-tag, mammalian Betty Eipper pEGFP(C1,C2,C3) N-term GFP-tag, mammalian expr. Clontech

24 Materials pEGFP-C1-MK2 Mouse MK2 full-length our group pEGFP-C1-MK5 Mouse MK5 full-length Schumacher et al., 2004 pEGFP-MK5L337A Mutation lysine 337 to alanine Ole-Morten Seternes pEGFP-MK5T182A Mutation threonine 182 to alanine Ole-Morten Seternes pEGFP-C1-ERK3 Mouse ERK3 full-length Schumacher et al., 2004 pENTR/D-TOPO-Sept7ΔN 162-436 aa fragment mutant our group pENTR/D-TOPO-Sept7ΔC 1-325 aa fragment mutant our group pGADT7 N-term myc-tag, yeast expr. Clontech pGBKT7-MK2 N-term HA-tag, yeast expr. our group pGBKT7-MK5 N-term HA-tag, yeast expr. Schumacher et al., 2004 pGBT9 Gateway homolog to pGBKT7 Manfred Koegl pGEX-2T-Borg1 N-term GST-tag, E.coli expr. Ian Macara pGEX-2T-Borg2 N-term GST-tag, E.coli expr. Ian Macara pGEX-2T-Borg3 N-term GST-tag, E.coli expr. Ian Macara pGEX-4T3-Kal7/SR3-6 N-term GST-tag, E. coli expr. Betty Eipper pGEX-4T3-Kal7/SR4-7 N-term GST-tag, E. coli expr. Betty Eipper p425-Met25-Ras(61)ΔF Bait Vector (RRS) Ami Aronheim pRK5-HA-Sept7 N-term HA-tag, mammalian expr. Koh-ichi Nagata pRK5-HA-Sept8 N-term HA-tag, mammalian expr. Koh-ichi Nagata pRK5-myc-Sept7 N-term myc-tag, mammalian expr. Koh-ichi Nagata pRK5-myc-Sept8 N-term myc-tag, mammalian expr. Koh-ichi Nagata pCMV-SPORT6-Sept7 IRAVp968C04100D6 RZPD pYes-M cDNA prey vector (RRS) Ami Aronheim

25 Materials 3.1.3 Oligonucleotides

Tab. 4. Oligonucleotides

Oligo name Sequence (5'-----3') Description (1) pACT2 FP CAC CAT GGC TTA CCC ATA CGA Y2H Prey cDNA subcloning TGT T (2) pACT2 RP TCA GTA TCT CTA CGA TTC ATA Y2H Prey cDNA subcloning

(3) 5’ Kalirin Mut GTC CTG CAG CGT CCC CTG GCC phosphorylated, S487A CCT GGG AAC mutation in Kal7/SR3-6 (4) 3’ Kalirin Mut ATC CAG CAG TGC TTT CCC ATC S487A mutation in CTG GCT GAC Kal7/SR3-6 (5) Kal7 Sequencing TAT GAG CAG GTG ACC CAG GCC Sequencing of S487A mutation in Kal7/SR3-6 (6) Sept7 FW CAC CAT GTC GGT CAG TGC GAG Full-length Sept7 ATC amplification from pCMV- SPORT6-Sept7 (7) Sept7 RW TTA AAAGAT CTT GCC TTT CT Full-length Sept7 amplification from pCMV- SPORT6-Sept7 (8) Sept7ΔC CTT GTT GTT ATC CAC TCC AT Sept7 deletion mutant

(9) Sept7ΔN CAC CTT ATA CTT CAT TGC TCC Sept7 deletion mutant TTC

3.1.4 Antibodies

Tab. 5. Antibodies

Antibody Description Reference / Source anti-ß-actin mouse, polyclonal, WB 1:1000 SCBT (sc-47778), C4 anti-ERK3 rabbit, polyclonal, WB 1:1000 CST (#4067) anti-HA rabbit, polyclonal, WB 1:1000 SCBT (sc-805), Y-11 anti-pHsp25 [S86] rabbit, polyclonal, ELISA 1:500, Biosource / Invitrogen WB 1:1000 (#44536) anti-GAPDH mouse, monoclonal, WB 1:5000 Millipore (MAB374), clone 6C5 anti-GFP mouse, monoclonal, WB 1:1000, IP SCBT (sc-9996), B2 1:500 anti-MAPKAPK5 rabbit, polyclonal, WB 1:1000, IP Sigma (HPA015515) 1:500 anti-hMTBP rabbit, polyclonal, WB 1:1000 Mark T. Boyd (non- commercial)

26 Materials anti-myc mouse, monoclonal, WB 1:1000, IF SCBT (sc-40), 9E10 1:200 anti-p38a rabbit, polyclonal, WB 1:1000 CST (#9228), L53F8 anti-PRAK sheep, polyclonal, WB 1:1000 P. Cohen (non- commercial) anti-pPRAK [T182] sheep, polyclonal, WB 1:500 P. Cohen (non- commercial) anti-P ATM/ATR substrate rabbit, polyclonal, WB 1:1000, CST (#2261) motif (s/t)Q anti-P CDK substrate motif rabbit, polyclonal, WB 1:1000, CST (#2324) (K/R)sPX(K/R) anti-P PKA substrate motif rabbit, monoclonal, WB 1:1000, CST (#9624), 100G7E (RRXS/T) anti-P PKB (Akt) substrate rabbit, monoclonal, WB 1:1000, CST (#9614), 110B7E motif (RXRXXS/T) anti-P PKC substrate motif rabbit, polyclonal, WB 1:1000, CST (#2261) (K/R)XsX(K/R) anti-P PKD substrate motif rabbit, polyclonal, WB 1:1000, CST (#4381) (LXRXX(s/t) anti-Sept7 rabbit, polyclonal, WB 1:1000, IF IBL Japan (#18991) 1:200 anti-α-Tubulin mouse, monoclonal, WB 1:1000 SCBT (sc-8035), TU- 02 anti-rabbit normal IgG (IP 1:500, ctrl Ab) SCBT (sc-2027) anti-goat HRP-conjugated, donkey Ab SCBT (sc-2033) anti-mouse HRP-conjugated, goat Ab SCBT (sc-2005) anti-mouse Alexa488-conjugatesd, goat Ab Invitrogen anti-mouse Alexa547-conjugated, goat Ab Invitrogen anti-rabbit HRP-conjugated, goat Ab SCBT (sc-2004) anti-rabbit Alexa488-conjugated, goat Ab Invitrogen anti-rabbit Alexa547-conjugated, goat Ab Invitrogen anti-sheep HRP-conjugated, rabbit Ab SCBT (sc-2924)

27 Materials 3.1.5 Further chemicals

Tab. 6. Further chemicals

Chemical compound Description Reference / Source

Antimycin A Cytochrome C reductase inhibitor Sigma-Aldrich

Anisomycin Inhibitor of protein synthesis Merck

[γ-32P]-ATP radiolabeled ATP, 800Ci/mmol Hartmann

[γ-33P]-ATP radiolabeled ATP, 3000Ci/mmol Hartmann

B-27 Supplement Primary neuronal culture Invitrogen

Protein Assay Bradford solution Bio-Rad

Bortezomib Proteasome inhibitor Merck

BSA Bovine serum albumin (fraction V) Carl Roth

Caliculin A Phosphatase inhibitor Sigma-Aldrich

Clean IP HRP-conjugated Antibody Thermo Scientific

CO2-independent medium pH stabile medium Invitrogen

Cobalt chloride inorganic compound Sigma-Aldrich

Collagen A 0.1% solution Biochrom AG

Complete Protease inhibitor cocktail Roche

Cyclosporine A (CsA), PP2B inhibitor Sigma-Aldrich

CsA peptide Autoinhibitory peptide Merck

DiR Lipophilic tracer (infrared) Invitrogen

DMEM Mammalian cell cultures Invitrogen

EcoRI R0101 NEB

Effectene Transfection agent Qiagen

Glutathione Sepharose 4B GST pull-down GE Healthcare

HBSS Hank’s balanced salt solution Invitrogen

HindIII R0104 NEB

28 Materials

HyperFect Transfection agent Qiagen

L-[35S]Methionine Radiolabeled methionine Hartmann

Microcystine-LR Phosphatase Inhibitor Sigma-Aldrich

MG132 Proteasome inhibitor Merck

N-2 Supplement Primary neuronal culture Invitrogen

Neurobasal medium Primary neuronal culture Invitrogen

NGF 7S nerve growth factor Merck

Nocodazole Inhibition of spindle formation Sigma-Aldrich

Nonfat dry milk powder Blocking agent Sucofin

Okadaic Acid phosphatase inhibitor Sigma Aldrich

Ouabain Sodium pump inhibitor SCBT o-vanadate Tyrosin phosphatase inhibitor Sigma-Aldrich

PAK18 p21-activated kinase inhibitor Merck

Paraformaldehyde Cell fixation Merck

Phalloidin Alexa647-conjugated Invitrogen o-Phenylenediamine HRP-substrate for ELISA R&D Systems

Phosphatase inhibitor Cocktail 1 + 2 Sigma-Aldrich

Phusion polymerase DNA polymerase NEB

PKI Protein kinase A inhibitory peptide SCBT

PMA Phorbol 12-myristate 13-acetate Merck

Polyethylenimine Macromolecular cation Sigma-Aldrich

Propidium iodide DNA intercalating compound Sigma-Aldrich

Protein G Sepharose Immunoprecipitation GE Healthcare

Radicicol HSP90 inhibitor Sigma-Aldrich

Rapamycin mTORC1 inhibitor Merck

SalI R0138 NEB siRNA scrambled AllStars Neg. Control (1027281) Qiagen

29 Materials siRNA Sept7 Hs_SEPT7_1_HP (1616498) Qiagen

Staurosporine Unspecific kinase inhibitor Merck West Pico Chemiluminescent SuperSignal Thermo Scientific Substrate T4 Ligase DNA ligase NEB

Wortmannin PI3K inhibitor Sigma-Aldrich

WST-1 vital cell staining for viability Roche

XhoI R0146 NEB

YNB medium powder Yeast Nitrogen Base BD Difco zVAD-Fmk Caspase inhibitor Merck

3.1.6 Equipment

Tab. 7. Laboratory Equipment

Equipment Description Reference Source

543 Slab-Gel Dryer Gel-dryer Bio-Rad

Biofuge Fresco Microcentrifuge Heraeus

C6 Flow Cytometer Flow cytometer BD Accuri

Countress Cell counter Invitrogen

FLA9000 Phospho-imager Fujifilm

Gene Pulser II Electroporator Bio-Rad

LAS3000 Intelligent dark box Fujifilm

Mini-Protean II Electrophoresis cell Bio-Rad

Neon Transfection System Mammalian cell electroporator Invitrogen

Odyssey Infrared scanner Li-cor Biosciences

PerfectBlue ExW S Electrophoresis cell Peqlab

PerfectBlue SEDEC M Semi-dry electroblotter Peqlab

30 Materials

PerfectBlue Web S/M Wet tank-electroblotter Peqlab

Stereomicroscope Leica

Storage Phosphor Screen 10 x 20 cm, Cyclone Packard

TCS SP2 Confocal microscope Leica

Victor2 Multilabel plate reader PerkinElmer

Vibratome Tissue-sections MICROM

3.1.7 Kits and disposal materials

Tab. 8. Kits

Kits Reference Source

FD Rapid GolgiStain Kit FD Neurotechnologies

Gateway pENTR/d-TOPO Cloning Kit Invitrogen

Gateway LR Clonase II Kit Invitrogen

Neon Transfection System 10 µl / 100 µl Kit Invitrogen

Nucleobond Xtra Mini Midi Maxi Macherey-Nagel

QIAquick PCR Purification Kit Qiagen

TNT Quick Coupled Transcription/Translation System Promega

Z’-Lyte Kinase Assay Kit (Peptide 4) Invitrogen

Plasticware for cell culture was purchase from TPP (Switzerland), common plasticware, like Petri dishes, reaction tubes, serological pipettes, and cell scrapers, was purchase from Sarstedt or Greiner Bio-One.

31 Materials 3.1.8 Frequently used buffers

Kinase Assay Lysis Buffer PLC Lysis Buffer

20 mM Tris-acetate pH 7.0 50 mM HEPES pH 7.5

0.1 mM EDTA 1 mM EGTA

1 mM EGTA 150 mM NaCl

1 mM Na3VO4 15 mM MgCl2

10 mM β-glycerophosphate 10% Glycerol

50 mM NaF 1% Triton X-100

5 mM Sodium pyrophosphate

1% Triton X-100

IP Buffer Laemmli Buffer (4x)

50 mM Tris/HCl pH 8.0 0.25 M Tris/HCl pH 6.8

50 mM NaF 4% (m/v) SDS

1 mM Na3VO4 4 mM EDTA

1% Triton X-100 0.25% (m/v) Bromophenol Blue

20% β-Mercaptoethanol

40% Glycerol

TAE buffer Towbin Buffer

40 mM Tris base 25 mM Tris base

1 mM EDTA 192 mM Glycine

0.114% (v/v) acetic acid (glacial)

32 Methods 3.2 Methods

3.2.1 Cloning and site-directed mutagenesis

To clone full-length Sept7 cDNA and its different deletion mutants into pENTR/d-TOPO vector (Invitrogen), the open reading frame of mouse septin7 cDNA (1-436 aa) was amplified from the pCMV-SPORT6-SEPT7 (IRAVp968C04100D6-RZPD) plasmid by PCR. For full- length amplification, the primer pair (6) and (7) was used. To target the ERK3-interacting site of Sept7, the following deletion mutants were generated. A Sept7 C mutant (1-325 aa) was amplified by using the primers (6) and (8), a Sept7ΔN mutant (162-436 aa) with the primer pair (7) and (9), and a Sept7ΔNΔC mutant (162-325 aa) using primer (8) and (9) (oligo’s described in section 3.1.3.). Cloning into pENTR vector was performed following the standard protocol of the Gateway pENTR/d-TOPO cloning kit. The insertion of the PCR amplified blunt-end cDNA fragment into the pENTR vector was achieved by a topoisomerase I- directional cloning, which detects the 5’ CACC sequence of the cDNA insert and ligates the insert with the complementary GTGG 3’ overhang of the pENTR vector. In a second step, ENTR clones of Sept7 were subcloned into mammalian expression vectors (destination vectors). In this regard, cloning was performed after the standard procedure of the Gateway LR Clonase II kit (Invitrogen). The cDNA of the pENTR clone is inserted into a destination vector via recombination of the att-sites of pENTR cDNA insert and a pDEST vector gateway cassette. The principle is based on natural recombination events of the chromosomes in the bacteriophage lambda and E. coli. Two specific destination vectors were used; the pcDNA6/BioEase and the pDEST27 leading to an N-terminal BioEase(BE)-tag and GST-tag (both purchased from Invitrogen). The BE-tag is a 72 aa long sequence, which is in vivo biotinylated, when expressed in mammalian cells. The generated tagged Sept7 and its deletion mutants were used for pull-down studies with ERK3. Both, deletion mutant clones of Sept7 and ERK3 were generated by Dr. Shashi Kant.

The cDNA fragments of ERK3-interacting prey clones received from a yeast-based cDNA screen following the ras recruitment system (RRS, section in chapter 3.2.17), were subcloned into the GFP-tag containing mammalian expression vector pEGFP-C1 (performed by Stefanie Schumacher). The cDNA fragments from the pYes-M prey vector were digested using EcoRI and XhoI restriction enzymes. The pEGFP-C1 vector was digested with EcoRI and the SalI (compatible with XhoI site). After in gel purification of the cDNA inserts, each of them was ligated with the linearized pEGFP-C1 backbone using the T4 Ligase for 2 h at 16°C. The resulting plasmids express an N-terminal GFP-tagged fusion protein. The following ERK3-interacting prey clones were identified by sequencing and subcloned into

33 Methods pEGFP: Cdc10 (Sept7), CDC25, Cop9 protein, MAP kinase phosphatase 6 (MKP6), and Ubiquitin-like protein 3 (Ubl3). The generated plasmids were used in a GST-ERK3 pull-down assay to approve interaction of ectopically expressed proteins in mammalian cell lysates (HEK293).

The same procedure was used for subcloning of the positive cDNA prey clones received from MK5 yeast two-hybrid screens of a pGADT7 normalized universal mouse cDNA library and a pACT2 brain-specific cDNA library (described in section 3.2.16). The following cDNA fragments were cloned into pEGFP-C1: sodium/potassium exchanging ATPase regulatory subunit beta 1 (VI/1), Rho GEF Kalirin7 (VI/84), Mdm2-binding protein (IV/20), and Septin8 (VI/13 and VI/67). The generated plasmids were also used for GST-MK5 pull-down experiments. The cDNAs were also cloned into pENTR vector as described above using the primer (1) and (2) (section 3.1.3).

For in vitro kinase assay (described in section 3.2.8) plasmids for expression of Kalirin7 (Kal7) fragments were provided by Dr. Betty Eipper (University of Connecticut, USA). The Kal7 fragment designated as KalSpec3-6 (416-870 aa) carries a putative MK5 phosphorylation site LQRPLS* (482-487aa), which was mutated to prove site-specific phosphorylation by MK5. Site-directed mutagenesis was performed using the following primer set (3) and (4) (section 3.1.3). Mutagenesis was done after the Phusion™ standard procedure. As template pGEX4T3-Kal7/Spec3-6 was used in PCR. One of the adjacent primers was phosphorylated in 5’ resulting in a linearized point-mutated PCR-product, which was purified from a gel and ligated using T4 ligase as described above. The presence of S487A mutation was approved by sequencing performed by GATC Biotech GmbH (Konstanz, Germany) using primer (5).

3.2.2 Cell culture and transfection.

Most of the cell lines (described in chapter 3.1.1) were cultured in DMEM supplemented with 10% fetal calf serum (PAA), 2 mM L-glutamine (PAA), 100 U of penicillin G/mL (PAA) and 100 µg/mL streptomycin (PAA). Neuroblastoma cell lines were cultured in DMEM/F-12 medium with 20% FCS. For PC-12 cells, addition of 10% horse serum (PAA) and 5% FCS was required for maintenance of these cells. To achieve adherence of PC-12 cells, culture dishes were coated with 10 µg/mL collagen A in PBS for 30 min at 37°C. Cell lines were commonly cultured in 10 cm cell culture Petri dishes using 10 mL medium. Cells were splitted by trypsinization using a 0.05% trypsin/EDTA solution. Passaging was performed

34 Methods once till three times a week related to the defined growth rates, which were frequently observed by an inverted microscope.

For transfection, cells were seeded 24 h before, to reach approximately 90% confluency. Generally, transfection was performed using polyethylenimine (PEI) at a ratio of 2:1 to the used plasmid DNA and the DNA-PEI-complex formation was done in OptiMEM for 15 min at RT. Further analysis of transfected cells was performed 24 h till 48 h post transfection. The procedure was optimized for transient overexpression in HeLa and HEK293 cells (plasmid transfection), required for pull-down experiments and immunofluorescence. To reach higher transfection efficiencies, transfection mediated by Lipofectamine (2000) was preferred. The Lipofectamine transfection was performed following the standard protocol. For PC-12 cell transfection, electroporation was most efficient, which was performed with cell suspension using the Neon Transfection System with the following parameters: 3 impulses with 1400 V for 10 ms using 100 µl gold-tips. The cell density was set to 5.0 x 106 vc/mL and a total plasmid DNA concentration of 40 µg/mL was used. For HeLa cell transfection with siRNA, the HyperFect transfection agent was used following the standard protocol. Transfection of primary hippocampal neurons was performed after the standard procedure of the Effectene Reagent.

3.2.3 Tissue homogenization

For generation of tissue lysates, mice were dissected after death through cervical dislocation or CO2 inhalation following the rules of the German animal welfare law. All animal experiments were registered by the appropriate authorities. Dissected organs were washed with cold PBS to remove adhering blood cells and stored on ice until lysis. The organ disruption was performed with the help of liquid nitrogen and a mortar. A fine tissue powder was generated, which was dissolved in kinase assay lysis buffer (recipe in section 3.1.5) containing Complete™ protease inhibitor and phosphatase inhibitor cocktail 1 & 2. The lysates were centrifuged at 16,000 x g for 1 h at 4°C. The supernatant was carefully removed using a microliter syringe and centrifuged, again. The clear supernatant was transferred into a new reaction tube and protein concentration was determined using the Bio-Rad protein assay. Protein concentrations were adjusted with 4x Laemmli buffer (recipe in section 3.1.5) and H2O. MK5 and ERK3 protein expression were analyzed by western blot.

35 Methods 3.2.4 Western blotting (conventional & multiplex)

The protein concentrations were determined using the Bradford assay to equalize protein amounts, before applying protein samples to the polyacrylamide-SDS gradient gels. Lysates were mixed with 4x Laemmli’s SDS sample buffer (recipe in section 3.1.8) and heated for 5 min at 96°C to denature the proteins. The samples were applied to 7.5-16% polyacrylamide-SDS gradient gels covered with a 3% polyacrylamide-SDS stacking gel and proteins were separated using a Protean II electrophoresis cell and Towbin buffer. Afterwards, separated proteins were blotted on a Hybond ECL nitrocellulose membrane using semi-dry electroblotter or wet blotter tank. The membranes were blocked in PBS with 0.1% Tween 20 containing 5% powdered skim milk or 5% bovine serum albumin for 1 h at RT to avoid non-specific antibody-binding. After three washes with PBS containing 0.1% Tween 20, the membranes were incubated with the primary antibody dissolved in blocking buffer for 16 h at 4°C or for 1 h at RT, followed by incubation with the horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Antigen-antibody complexes were detected with home-made enhanced chemiluminescence (ECL) detection solution (solution A: 1.2 mM luminol in 0.1 M Tris-HCl (pH 8.6); solution B: 6.7 mM p-coumaric acid in

DMSO; 35% H2O2 solution; ratio 3333 : 333 : 1) using the Luminescent Image Analyzer LAS- 3000. For multiplex western blotting, protein separation and membrane-blotting were done as described above, whereas the membranes were not stained with Ponceau and blocking as well as primary antibody incubation was performed with 1% BSA/PBS. Thereby, two primary antibodies (of different host species) were applied at the same time. As secondary antibodies Alexa647- and DiR-conjugated antibodies were applied. Incubation was done in the dark for 2 h at RT. Detection of the two distinct fluorophores with emission peaks at 700 nm and 800 nm was performed with the Odyssey Infra-red scanner. Infrared fluorescent detection is a direct, nonenzymatic approach that improves quantitative accuracy and reproducibility, and facilitates multiplexing. The fluorescent signal is stable and allows immediate detection and after extended storage.

3.2.5 Pull-down experiments

The following three types of pull-down experiments were performed during this study: an immunoprecipitation (IP) using protein G sepharose beads (GE healthcare) and functional antibodies, a GST pull-down of GST-tagged proteins, and pull-down of in vivo biotinylated BioEase-tagged proteins using streptavidine agarose beads. For immunoprecipitation cells

36 Methods were lysed in IP buffer (recipe in section 3.1.5) containing Complete protease inhibitor. Cells of one 10 cm cell culture plate (~107) were lysed in 500 µl buffer, and insoluble parts were removed by centrifugation at 16,000 x g for 20 min at 4°C. Clear supernatants were mixed with 1 µg of the appropriate antibody and incubated on a rotary shaker over night at 4°C. After antibody pre-incubation, 30 µl protein G sepharose beads (50% slurry) were equilibrated in 1 mL IP buffer and added to the lysate. The IPs were further incubated on a rotary shaker for 4 h at 4°C. Subsequently, beads were washed five times with IP buffer and finally mixed with Laemmli buffer (recipe in section 3.1.5) to denature the proteins. Samples were separated using SDS-PAGE and analyzed after western blot. To avoid background signal caused by the heavy and light chain of the antibody used for IP, another secondary antibody was applied, which only detects the native primary antibodies (Clean IP, Thermo Scientific).

For GST- and BE-pull-down, cells were lysed in PLC lysis buffer (recipe in section 3.1.5) and beads were added immediately after lysis and incubated on a rotary shaker over night (<16 h) at 4°C. After incubation, the beads were also washed five times with PLC buffer, the bound proteins were denatured in Laemmli buffer, and analyzed by western blotting. In general, BE- and GST-pull-down are more efficient than IP, which is limited by the applied antibody.

3.2.6 Immunofluorescence and confocal microscopy

For immunofluorescence cell were seeded on poly-L-lysine-coated glass coverslips and transfected following the standard protocol described before (section 3.2.2). Cells were washed three times with pre-warmed PBS (37°C) and fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at 4°C. Permeabilization was performed by incubation with 0.25% Triton X- 100 dissolved in PBS for 20 minutes at RT. Fixed cells were blocked with 10% serum or 6% BSA in PBS for 1 h at RT. Primary antibodies were applied at a dilution of 1:200 in 1% BSA/PBS for 1 h at RT. After three washing steps with PBS, fluorophore-conjugated secondary antibodies, Alexa488-conjugated streptavidine (Invitrogen), or Alexa647- conjugated phalloidin was added at a dilution of 1:500 in 1% BSA/PBS. Afterwards, antibody solution was removed and cells were treated with 4’,6-diamino-2-phenylindole (DAPI; 1:25000 in PBS) as counter staining for 5 min at RT. After three washing steps with PBS, coverslips were washed with H2O and the remaining water was removed using a tissue. Coverslips were placed upside down on a drop of mounting medium (Carl Roth or self-made

37 Methods

Mowiol/DABCO) on a glass slide. The mounting medium was dried over night at 4°C in the dark. Imaging was performed at the Leica TCS SP2 confocal microscope with comparable settings. For the observation of cytoskeleton or neuronal morphology, a multi-layered picture was taken using constant Z-width and number of optical slices. Multi-image stack was modulated into a 3D projection using the Leica LCS lyte software.

3.2.7 MK5 substrate ELISA

A 96-well ELISA plate was coated with 200 µl Hsp25 recombinant protein dissolved in a carbonate buffer (35 mM NaHCO3, 15 mM Na2CO3, 0.02%NaN3; adjusted pH 9.6 with HCl) over night at 4°C. As a control, 1% BSA was used for coating. After coating, each well was washed three times with PBS / 0.05% Tween 20 and incubated with 1% BSA/PBS solution for 30 min at RT, followed by three washing steps with PBS/Tween. Afterwards, 0.1 µg of the recombinant kinases MK5, ERK3, or both together were dissolved in 80 µl kinase assay buffer (75 mM HEPES, 3.75 µM ortho-vanadate, 1.5 mM DTT, 0.01% PEG40,000, 1% BSA; adjusted to pH 7.5 with NaOH) and added to Hsp25- and BSA-coated wells. Reactions were started by adding 20 µl of ATP solution (15 mM MnCl2, 15 mM MgCl2, 0.1 mM ATP). Kinase reaction was incubated for 2 h at RT, followed by three washing steps with PBS/Tween. After aspirating the kinase reaction solutions, blocking procedure with 1% BSA was repeated for 30 min at RT, followed by incubation with anti-pHsp25 [S86] in 100 µl of 1% BSA/PBS over night at 4°C. After incubation with primary antibody, wells were washed three times and the corresponding HRP-conjugated secondary antibody was applied in 1% PBSA (1:500) for 2 h at RT. The antibody solution was aspirated and wells were washed three times. Primary antibody-binding was indirectly analyzed by using colorimetric HRP-substrate from R&D Systems based on oxidation of o-phenylenediamine. In detail, 100 µl of Color Reagent A and B (1:1) were added to each well and the reaction were stopped after 20 min at RT by adding

20 µl of 2 N H2SO4 to each well. An intensively upcoming yellowish till orange color was photometrically observed by measuring absorbance at 450 nm. The signal was normalized to background-absorbance at 540 nm. The measurement was performed at the Victor2 multilabel plate reader (PerkinElmer). The total HRP signal depending on Hsp25 phosphorylation by MK5 was compared with similar treated BSA-coated wells, to discriminate non-specific antibody adsorption or non-specific antibody-binding to surface- bound proteins from recombinant kinase preparation.

38 Methods 3.2.8 Recombinant protein expression in E.coli

The Escherichia coli BL21 Rosetta (DE3) pLysS strain (Merck) was transformed with expression plasmids for GST-tagged kalirin7 fragments (SR4-7, SR3-6, and SR3-6 S487A), GST-tagged Borg1-3, and His-tagged Sept7 (ΔN or ΔC mutant). The Rosetta strain is characterized by expression of mammalian tRNAs, which are rarely found in E. coli (for codons: AGG, AGA, AUA, CUA, CCC, and GGA). These tRNAs are expressed by an additional pretransformed plasmid, which is selected by a chloramphenicol-resistance. A single colony of transformants was used for inoculation of a 25 mL LB medium over night culture, which was used for inoculation of the main culture (0.5-1 L). E. coli was incubated on a rotary shaker at 37°C until an optical density of 0.6 at 600 nm was reached. Protein expression was induced by the addition of 0.5 mM IPTG and culture broth was further cultivated for 3 till 4 h at 37°C. Afterwards, cells were pelleted by centrifugation at 4,000 x g for 30 min at 4°C. Pellet was washed once with H2O and stored at -20°C till lysis. The cell disruption was performed by sonification in PBS containing protease inhibitor. The IPTG- induced protein expression and the solubility of the protein were checked by SDS-PAGE. Depending on the used tag, the recombinant proteins were purified using glutathione- sepharose or Ni2+-NTA-superflow beads. An advantage of the His-tag purification is to enrich protein under denatured condition, which is required for proteins that are not soluble under native condition. The recombinant protein was restored on the beads in PBS / 0.04% NaN3 at 4°C. For extended storage, the protein was eluted from the beads, dialyzed to the storage buffer, and stored at -80°C.

3.2.9 In vitro kinase assays

In vitro kinase assays are important tools to analyze kinase kinetics in regarding to its activation, inhibition, and substrate targeting. The in vivo situation can not be mimicked, but many of in vitro shown kinase properties can be reflected on the endogenous kinases. The in vitro experiments are a useful tool for screening of kinase substrates, inhibitors or competing kinases. Several methods were established to measure kinase activity. Within this study, the following three methods have been utilized: using radiolabeled ATP (hot kinase assay), phospho-specific substrate antibodies (cold kinase assay), or a FRET-based artificial substrate peptide, which degradation is prevented by site-specific phosphorylation (Z’-Lyte kinase assay).

39 Methods Hot kinase assay

The hot kinase assay was performed using recombinant MK5 together with GST-tagged Borg protein, His-tagged Sept7ΔC or Sept7ΔN, and GST-tagged Kal7Spec3-6 or Kal7Spec4- 7, which were expressed in E. coli. For kinase assays, 15 µl of recombinant protein bond to glutathione-sepharose or Ni-NTA-superflow (dry bead volume) and 2.5 µl of buffer (500 mM sodium β-glycerophosphate, 1 mM EDTA; adjusted to pH 7.4 with HCl) and 2 µg recombinant untagged MK5, GST-tagged p38 or His-tagged ERK3 (Sf9 preparation from Dr Ole-Morten Seternes) were added in desired combinations. Reaction was filled up to a final volume of 20 µl. As positive controls, 10 µg of the known MK5-substrate Hsp25 or ERK3- substrate MBP was used in each experiment. Afterwards, 5 µl of hot ATP mixture (20 mM 33 32 MgCl2, 0.5 mM ATP, 0.1 µl of [γ- P]-ATP or [γ- P]-ATP) were added, and the reaction mix was incubated for 1 h at 30°C. Reaction was stopped by adding 8 µl 4x Laemmli buffer (recipe in section 3.1.5) and samples were heated to 96°C for 5 min. Protein samples were separated on a 7.5-22.5% polyacrylamide gradient gel. After separation, protein were fixed in the gel using a coomassie staining-solution (250 mg/L Coomassie G, 40% methanol, 10% glacial acetic acid). After destaining the appearing proteins bands were analyzed using the Odyssey infrared scanner. The gel was placed on a filter paper and dried for 2 h at 70°C using a gel dryer. The dried gel was covered in foil and place upside down on a Cyclone storage phosphor screen. Radioactivity incorporated into substrates was quantified by phosphoimaging using a FLA9000. The detection of substrate phosphorylation is based on changes in light emission from the screen, excited by a laser (633 nm), which is caused by ionizing radiation from samples with incorporated 32P or 33P.

Cold kinase assay

The procedure of cold kinase assay was preferred for measuring recombinant Hsp25 phosphorylation by ectopically expressed MK2 or MK5 purified from HEK293 lysates. The used anti-pHsp25 antibody (Biosource / Invitrogen) was raised against the phosphorylated MK5-like phosphorylation-motif of Hsp25 at Ser86. HEK293 cells were transfected with different combinations of kinases and their mutants. Afterwards, MK5 was purified by GST pull-down or GFP immunoprecipitation in the presence of appropriate inhibitors against proteases and phosphatases. Cold kinase assay was performed as described for hot kinase assay, whereas no additional radiolabeled ATP was added. Kinase reaction was done in the presence of kinase inhibitors against protein kinase A and C and Rho-dependent kinase (2.5 µM PKI, 400 nM H7, 400 nM HA1077). Kinase reaction was stopped by the addition of

40 Methods 4x Laemmli buffer. And samples were applied to a SDS-PAGE and transferred onto nitrocellulose membrane.

Z’-Lyte kinase assay

The Z’-Lyte kinase assay was developed by Invitrogen. The assay based on an artificial substrate peptide, which contains coumarin at N-terminus and fluorescein at the C-terminus. Both fluorescent dyes are kept in close proximity and are able to generate a FRET- (fluorescence resonance energy transfer) signal, which can be measured. The fluorophore coumarin can be excited with monochromatic light at 405 nm and emitted light at 445 nm, which can excite the fluorescein molecule, which emitted light at 520 nm (FRET signal). The Ser/Thr kinase substrate peptide 4 contains a MK-specific phosphorylation-motif (F/L/V/A-X- R-X-X-S/T-X-X). In the first step of the assay, recombinant kinase is incubated with the peptide and phosphorylation events take place. In a second step, the peptide is incubated with a protease, which specifically cleaves the unphosphorylated peptide. If the peptide is phosphorylated, it will not be degraded and the FRET signal will retain. The reaction volume was minimized to 20 µl and reaction were performed in 384-well black plates with low protein binding affinity, following the Z’-Lyte standard protocol. The FRET signal was determined with the Victor2 multilabel plate reader using sample excitation at 350 nm and measuring emission at 450 nm (coumarin) and 535 nm (FRET, fluorescein). As control, a completely phosphorylated peptide sample was measured to detect the maximum of FRET-signal.

3.2.10 Cell counting and WST-1 proliferation assay

Cell counting was performed using the Neubauer chamber or the Countress cell counter. In this regard, cells were also stained with trypan blue solution by the ratio of 1:1 with the cell suspension to discriminate dead and alive cells. The proliferation was determined using the vital staining WST-1 dye. This assay is based on the generation of colored formazan salts by reduction of WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzol- disulfonate). The reduction is caused by enzymes located outside of the plasma membrane of living cells. The more cells are viable the higher the creation of formazan. The product can be photometrically detected by measuring absorbance at 450 nm. The optimal number of

41 Methods cells, seeded in a 96-well plate, was previously determined. In general, it ranged between 102 and 103 cells per well. Each tested cell line was seeded in triplicates for each measured time point on a separate plate. To minimize evaporation of culture medium, the surrounding empty wells were filled with PBS. At the day of measurement, 10 µl WST-1 dye was added to the culture medium and absorbance at 450 nm was measured after 1, 2 and 4 h of ongoing cultivation within the incubator. Measurement was done using the Victor2 multilabel plate reader and the signal was normalized to background-absorbance at 630 nm. For reliable evaluation of the cell viability, it was important to keep the formazan signal in the linear range of absorbance for all samples. Whenever required, the culture medium was changed before adding WST-1 solution, which resulted in a decrease in formazan production (reduced signal intensity and absorbance at 450 nm), often necessary at later time points measuring high cell densities.

3.2.11 Propidium iodide staining for analysis of cell viability or cell cycle

Propidium iodide (PI) is a DNA-intercalating dye, which is used for fast detection of dead cells and for analysis of general DNA content of fixed cells. Staining of dead cells was achieved by the addition of propidium iodide to the culture-medium (final concentration 10- 40 µg/mL). The DNA binding dye can only pass the membrane of apoptotic or dead cells. Measurements can follow immediately after staining (5 min) by detection of emission at around 620 nm after excitation with 488 nm using the accuri C6 flow cytometer (FL-2 channel 585/40 nm). A positive PI signal can only be observed in case the dye is bound to DNA. For PI-staining of cellular DNA content, the cells were fixed with ice cold 70% ethanol. About 1- 2 x 106 cells were dissolved in 0.5 mL PBS and 4.5 mL 70% ethanol were added dropwise under continuous shaking to avoid cell aggregates. Cells were restored in ethanol at least 2 h at 4°C or for longer times at -20°C. For PI staining of fixed cells, the ethanol was removed by three washing steps with PBS, cells were pelleted by centrifugation at 300 x g for 5 min at 4°C, and resuspended in 100 µl propidium iodide staining solution (10 µg/mL propidium iodide, 4 mM sodium citrate, 0.01% Triton X-100, 100 µg/mL RNase A; adjust to pH 7.4). Staining procedure was performed for 30 min at 30°C. RNase activity was stopped by the addition of 100 µl salt solution (10 µg/mL propidium iodide, 0.01% Triton X-100, 400 mM NaCl).

42 Methods 3.2.12 Scratch Wound Healing Assay

A simple method for determination of cellular motility is the scratch wound healing assay. In our group, a modified protocol was established by Dr. Manoj B. Menon (Menon et al., 2009). In principle, the closure of an artificial ‘wound’ is analyzed, which is applied by scratching the cell monolayer of an adherent cell culture with a plastic tip. SV40 Large T antigen immortalized mouse embryonic fibroblasts (MEF) cells with following genotypes were investigated: wild type (WT), MK5-deficient (MK5 KO), ERK3-deficient (ERK3 KO), and ERK3/MK5 co-deficient (ERK3/MK5 dKO) MEFs (each at passage 12). Before seeding, cells were stained with the lipophilic tracer DiR dye (0.03% in growth medium) for 20 min at 37°C. The cells were seeded in quadruples in a 96-well format with 3.0 x 104 cells per well, leading to a confluent cell monolayer over night. The cells were incubated with 30 µg/mL mitomycin C for 1 h at 37°C to block proliferation, before the scratches were applied with a 10 µl tip (Sarstedt) to each well with high accuracy to restore reproducibility. The distribution of cells at time point zero were determined by scanning the emission at 800 nm of DiR, bound to the cell membranes, using the Odyssey scanner (Li-Cor). At this time, no cell should remain within the scratch area, which defines the area of possible infiltration. After 24 h of ongoing cultivation, the stained cell layer was scanned again, showing partial movement of cells into the region. Using the ImageJ software (NIH, Bethesda, USA), the relative closure of non-fluorescent ‘wound’ area was graphically determined.

3.2.13 FACS analysis of GFP-ERK3 stably transfected PC-12 cells

For stable plasmid DNA integration, PC-12 cells were transfected either with pEGFP-ERK3, pEGFP, or pd2EGFP by electroporation using the Neon transfection system. At least 1- 2 x 107 cells were required for each transfection to efficiently select enough stably transfected cells within two weeks by the addition of 600 µg/mL geneticin (G-418) in 10 mL growth medium. Afterwards, the geneticin concentration was reduced to 100 µg/mL and continuously supplied to avoid contamination with non-transfected PC-12 cells. To separate the GFP-positive subpopulation with comparable GFP-expression levels, the cells were sorted at the MHH cell sorter facility (Dr. Matthias Ballmaier). The resulting stably transfected cell lines were used for further experiment in regard to ERK3 stability upon extracellular pH changes. For each stimulation 2.0 x 105 PC-12 cells, stably expressing GFP-ERK3, GFP or an instable form of GFP with a half-life of 2 h (d2EGFP), were seeded in 12-well plates using

1 mL/well CO2-independent medium (Invitrogen) with 10% FCS over night at 37°C under

43 Methods atmospheric CO2. Next day, pH was changed by the addition of 6 µL 1 M HCl (pH 6.4), 12 µl

0.5 M NaOH (pH 8.4) or 12 µl H2O (pH 7.4). GFP-positives cells were determined immediately after changing the pH and after 30 min, 1 h, 2 h, 4 h, and 18 h. Percentage of GFP-positive subpopulation compared to non-fluorescent cells was determined in a FL1-FL2 plot to eliminate pH-dependent changes in autofluorescence. The influence of GFP-ERK3 stability by stimulation of PC-12 cells with a panel of inhibitors was performed in suspension using CO2-independent medium in 16 mL reaction tubes and a 37°C water bath. Experiments, done in the water bath, allow multiple stimulations of single samples without interferences through temperature changes. In the following experiment, several agents were screened to identify stimuli, which can prevent alkaline-induced degradation of GFP-ERK3. Among others, broad protein phosphatase and kinase inhibitors and selective stimulation and inhibitors of cellular MAP kinase signaling were tested. Another experiment focused on comparison of the stability of GFP-ERK3 and endogenous ERK3, which was analyzed by western blot and FACS analysis. The GFP-ERK3 stably transfected PC-12 cells were investigated either by western blotting with anti-GFP and anti-ERK3 or by determination of GFP-positive cells using FACS.

3.2.14 In vitro screen of ERK3-activating kinase

A screen to identify a potential ERK3-activating Ser/Thr protein kinase was performed following the Z’-Lyte kinase assay (described in chapter 3.2.9). The activation of ERK3 was indirectly measured by analyzing the resulting Z’-Lyte substrate peptide 4-phosphorylation by MK5. A set of 100 distinct recombinant active Ser/Thr protein kinases was generously provided by the Proqinase GmbH (Freiburg, Germany). A complete list of received kinases and related data is found in the appendix (section 8.2). In the assay, 0.3 µg of each supplied kinase were tested together with 0.5 µg recombinant ERK3 and MK5 (20 µl Z’-Lyte assay format). The substrate peptide 4-phosphorylation should be increased, if the kinase X can activate the ERK3/MK5-module. The threshold for a significant increase of MK5 activity was set to 40% of relative substrate phosphorylation. The MK5/ERK3 co-incubation in the Z’-Lyte assay results in about 20% of phosphorylated substrate, which means a significant increase should be around 200% of the basal level. As internal controls in vitro activated MK2, MK3 and MK5 was tested, which caused a strong phosphorylation of the substrate peptide 4 (nearly 100%). For all Ser/Thr protein kinases, which reach the threshold, further control experiments were performed. Control reactions were performed with single MK5, ERK3 or kinase X, in pairs of kinase X and ERK3 or MK5, and in combination of all three kinases. Each reaction was done in triplicates to increase the statistical relevance.

44 Methods 3.2.15 Phosphoproteomics in tissues or immortalized MEF cells

Phosphoproteomic analysis was performed using phospho-Ser/Thr kinase substrate antibodies against well-characterized kinases-specific phosphorylated substrate-motifs. The phosphorylation pattern were detected in lysates of specific tissues of wild type and MK5- deficient mice. The generation of tissue lysates is described in section 3.2.3. The antibodies, which were used in this study, are commercially available from Cell Signaling Technology. In detail, the following phospho-motifs were analyze in the tissue samples: RRXS/T (PKA motif), (K/R)XsX(K/R) (PKC motif), (K/R)sPX(K/R) (CDK motif), and (s/t)QG (ATM/ATR motif). Each tissue-specific lysate of three distinct wild-type and MK5-deficient animals was separated in the same gradient gel and blotted on nitrocellulose using the more efficient wet transfer. Uniform protein transfer was confirmed by Ponceau S staining (0.2% in H2O containing 5% acetic acid). Phospho-motif antibodies were applied in 5% BSA solution. Immortalized MEF cells were also analyzed depending on their ERK3/MK5 genotype-related phosphoproteome. In this regard, the deletion of ERK3 could also be investigated. Immortalized MEF cells deficient in ERK3 and MK5 are compared with retroviral ERK3/MK5- rescued MEFs. The MEF cells were stimulated with 650 nm phorbol 12-myristate 13-acetate (PMA) for 30 min at 37°C to activate PKC-dependent signaling, moreover stimulation with 50 µM pervanadate for 30 min at 37°C, which is a strong tyrosine protein phosphatase inhibitor, was use to increase the amount of phosphoproteins. Possible differences in phospho-pattern of ERK3/MK5 double knockout (dKO) and rescued (dRes) cells could arise, which cannot be observed in unstimulated cells. Beside the antibodies used for the tissue analysis also RXRXX(s/t) (PKB/Akt motif), LXRXX(s/t) (PKD motif) and PX(t)P (MAPKs/CDK motif) phospho-motif antibodies were used. Both MEF cell lines were stimulated as described before and both stimulation were analyzed on the same western blot.

3.2.16 Yeast two-hybrid screen

A pretransformed mouse brain MATCHMAKER pACT2-cDNA library (Clontech; MY4008AH) and a pretransformed normalized universal mouse MATCHMAKER pGADT7-cDNA library (Clontech), each in S. cerevisiae strain Y187 (MATα), were mated with pGBKT7-MK5- pretransformed yeast strain AH109 (MATa) and plated on selection plates. Transformation of bait vector was performed by the lithium acetate method described in the yeast protocols handbook (Clontech). Mating procedure was performed following the Clontech standard procedure for 24 h at 30°C. Diploid zygotes containing one copy of each bait and prey

45 Methods plasmid were harvested and washed properly with H2O to remove essential amino acids enriched in YPDA full-medium. The yeast culture broth was plated on minimal medium (SDΔAHLT) lacking tryptophan for bait plasmid selection, leucine for prey plasmid selection, adenine and histidine that are both selections for the transactivated Gal4-reporter ade1 and his3 genes induced by bait and prey interaction. The plates were incubated for 3–21 days at 30°C. Clones were selected and regrown on new selection plates. From the remaining yeast clones, plasmid DNA was isolated by alkaline lysis and DNA was retransformed into E. coli using electrocompetent cells and a Gene Pulser II. The prey plasmid was selected by ampicillin resistance and plasmids were analyzed after digestion with HindIII endonuclease. The cDNA inserts were compared in size and fragment pattern. Different prey clones were identified by sequencing using the pACT2-FW/RW standard primer (GATC Biotech GmbH; GATGATGAAGATACCCCAC; CAGTTGAAGTG-AACTTGC). The cDNA insert, which contains coding regions in frame were used for retransformation experiments to confirm bait- prey-interaction in yeast. Each prey plasmid was co-transformed either with empty bait vector, MK2-bait, or MK5-bait in S. cerevisiae AH109. For each prey clone, all combination were spread on SDΔLT plate and SDΔAHLT plate to confirm the specificity of MK5- interaction. Empty bait containing yeast clones, which grew on SDΔAHLT plates together with the specific prey clone, were classified as false positive. Found positive interacting prey fragments were subclone into pEGFP as wells as pENTR for further shuttling following the Gateway standard cloning procedure (described in section 3.2.1).

3.2.17 The ras recruitment system

This yeast screening system for analysis of protein-protein interaction was developed and provided by Dr Ami Aronheim (The Rappaport Institute, Israel). The method is based on an inactive cytosolic Ras mutant (lacking carboxy-terminal CAAX box), which is fused to the bait protein and unable to bind the plasma membrane (Broder et al., 1998). The Ras mutant can only be activated by interaction with a membrane-bound prey protein, which is permanently localized to the plasma membrane upon myristylation. Stringency for survival of yeast cells is achieved by using a yeast strain referred to as CDC25-2, which express a temperature- dependent active Ras mutant. ERK3 was cloned into p425-Met25-Ras(61)ΔF bait vector and used for stepwise transformation of CDC25-2 yeast cells together with a mouse brain cDNA library, which has been subcloned into pYes-M yeast expression vector. Yeast transformation was performed following the lithium acetate standard protocol as described in the yeast protocols handbook (Clontech). The screen using the ras recruitment system was performed by Stefanie Schumacher. Yeast clones were amplified in SDΔLU medium

46 Methods (minimal medium lacking leucine and uracil) containing glucose for 5 days at 24 °C and selected on SDΔLUM medium plates (lacking leucine, uracil, and methionine) with galactose for further 4 days at 36 °C. Selected yeast clones only survive at 36°C on SDΔLUM medium with galactose and not with glucose or additional methionine, respectively. From positive clones, the prey pYes-M plasmid was isolated and sequenced. For further analysis resulting cDNA fragments were cloned into the pEGFP mammalian expression vector (described in section 3.2.1) and used for pull-down interaction studies (described in section 3.2.5).

3.2.18 Endogenous Sept7 solubility assay

Solubility of filamentous and globular Sept7 was investigated in MEF cells deficient in ERK3/MK5 (dKO) and compared with ERK3/MK5-rescued MEFs (dRes). For analysis, two strategies were utilized. On the one hand, cells were treated with a permeabilization buffer

(10 mM PIPES, 0.015% digitonin, 10 mM NaCl, 10 mM PMSF, 5 mM EDTA, 1 mM MgCl2; adjusted to pH 6.8 with HCl) and centrifuged at 480 x g for 10 min at 4°C. The supernatant was mixed with 4x Laemmli buffer, whereas the pellet was dissolved in 1x Laemmli buffer (recipe in section 3.1.8). The soluble cytosolic fraction should pass the perforated plasma membrane and the higher molecular filamentous structures should retain within the cell. On the other hand, MEF cells were lysed by Triton X-100 using normal PLC lysis buffer (recipe in chapter 3.1.8). Higher molecular filamentous structures were separated by ultra- centrifugation at 50,000 x g for 30 min at 4°C. The supernatant was also mixed with 4x Laemmli buffer, whereas the pellet was dissolved in 1x Laemmli buffer. For quantitative analysis, the same cell number of each MEF cell line was seeded and the lysate volume was kept constant. For determination of protein concentration, quantitative western blot analysis was performed using fluorophore-conjugated secondary antibodies and the Odyssey infrared scanner (described in chapter 3.2.4). Septin7 and β-actin concentrations were analyzed in dKO and dRes lysates. As control α-tubulin was analyzed in all samples.

3.2.19 Isolation of primary hippocampal neurons.

For isolation of primary hippocampal neurons, mice were dissected after death through CO2 inhalation following the rules of the German animal welfare law. All animal experiments were registered by the appropriate authorities. Isolation was done using BALB/cJ mice at embryonic day 18 (E18.5). In detail, the head was cut of using scissors and the cranium was

47 Methods opened with a pair of fine forceps. The brain was gently removed and placed on 3% agarose covered with Hank’s balanced salt solution (HBSS). The cerebrum was separated into the two hemispheres and hippocampal formation was dissected from each using two fine forceps. To avoid contamination with neurofibroblasts, the surrounding meninges of the hippocampus were completely removed. Hippocampal parts of each mouse were collected in 2.5 mL HBSS solution. Dissected hippocampi were washed three times with HBSS before adding 100 µl of 2.5% trypsin solution. Trypsin digest was performed for 20 min at 37°C. After gently aspirating the trypsin/HBSS solution, 1 mL DMEM/FCS containing 10 µg/mL DNase I was added. The tissue-pieces were triturated using a modified glass Pasteur pipette with a constricted notch, prepared with a Bunsen burner. The tissue was continuously pipetted until a single-cell suspension was reached. Cell number was determined using the Countess and cells were seeded onto poly-L-lysine-coated lipid-free glass coverslips. The coating was done using 200 µg/mL poly-L-lysine dissolved in 100 mM borate buffer (pH 8.5), which was applied to the coverslips for 4 h at 37°C. Cells were seeded at different densities (1.0 x and 2.0 x105 cells per well in 12-well plate). After neuronal cells have been attached (~3 h), the medium was changed to serum-free neuronal differentiation medium (Neurobasal medium, B27 supplement, N2 supplement, 2 mM glutamine, and Pen/Strep, 100 ng/mL 7S NGF). Cells were grown for 5 or 10 days before transfection was performed (described in chapter 3.2.2). For maintenance culture medium was changed after 7 days and prior transfection.

3.2.20 Sholl Analysis of transfected primary neurons

Transfection of primary neurons was performed at DIV5 (‘days in vitro’) with 50 ng of pcDNA6/BioEase-ERK3, pcDNA3.1-HA-MK5, or pRSK5-myc-Sept7 following the standard protocol of the Effectene Reagent (Qiagen). For visualization of transfected neurons additionally 500 ng pEGFP (always 0.5 of 1 µg total DNA used in transfection) was co- transfected and each type of transfection was performed in triplicates. 72h post transfection neuronal cultures were fixed with 4% PFA for 15 min at 4°C. Imaging of GFP-positive neurons was done using the Leica TCS SP2 confocal microscope with constant settings. The GFP-positive neurons were scanned in several optical slices (constant parameters, 400x magnification). From the resulting stack series, 3D-projections were generated and analyzed using the ImageJ software with the Sholl analysis plugin tool (v1.0; University of California, San Diego). The received pictures were converted into 8-bit b/w data files. The Sholl analysis is performed by counting of intersection of neuritis on concentric cycles around the soma (Sholl, 1953). By counting intersections at several distances to the soma a neuron-specific

48 Methods graph is generated, which describes the dendritic complexity. For each transfection all coverslips were completely scanned for GFP-positive neurons. Numbers of intersections to the Sholl radius were statistically evaluated for each transfection. Another experiment focused on the morphology of dendritic spines due to overexpression with the same proteins. The primary neurons were grown ten days until transfection and 5 day after (DIV10+5). Analysis of spine morphology was done with the confocal microscope. A quantitative analysis was not possible, because of an insufficient number of transfected neurons.

3.2.21 Analysis of in vivo spines morphology

Experiments for study of in vivo spine morphology were performed in the lab of Prof. Stefan Britsch (University of Ulm, Germany). Overall, two strategies for visualization of in vivo morphology of hippocampal spine architecture were utilized: using a transgene YFP mouse strain or a vital neuronal staining referred to as Golgi-staining.

Brains of 2 month old MK5 WT and knockout animals carrying the Thy1-YFP transgene, obtained from breeding of C57BL/6J or B6-MAPKAPK5tm1Mg1 mouse with B6.Cg-Tg(Thy1- YFPH)2Jrs/J mouse (Jackson Laboratories) were isolated and fixed in 4% PFA overnight. Afterwards, the isolated brains were embedded in 4% low-melting agarose in PBS. For analysis, 100-150 µm Vibratom sections were prepared and placed on coverslips. Neuronal structures were determined by confocal imaging (Leica Sp5II) and spine number was determined using ImageJ software (NIH; Bethesda; USA). Received values were statistical evaluated.

As an alternative method, a Golgi staining of MK5 control and knockout animals were performed according to the manufacture instruction of the FD Rapid GolgiStain Kit (FD Neurotechnologies). The isolated brains were fixed for two weeks at 4°C in the dark using several fixing solutions, which were supplied by the company. The brains were also embedded in 4% low-melting agarose and 150 µm sections were made. Golgi staining was performed following the standard protocol of the used kit. After staining brain slices were dried and embedded with Entallan (Merck). Neuronal structures were analyzed by confocal microscopy and spine number were counted using ImageJ software.

49 Results 4 Results

4.1 Characterization of the ERK3/MK5-signaling module

4.1.1 Protein expression of ERK3 and MK5 in selected cell lines and murine tissues

Previous analysis of MK5 (New et al., 1998, Ni et al., 1998) and ERK3 mRNA levels (Turgeon et al., 2000) has demonstrated a ubiquitous distribution of both transcripts in all mouse tissues, whereas ERK3 mRNA could not be detected in liver, heart and spleen. Database searches revealed significant high mRNA levels in some neuroblastoma cell lines (Khan et al., 2001). To verify expression pattern on protein level both kinases have been analyzed by western blotting of adult murine tissue lysates (Fig. 8A/B) and lysates of well- established cell lines, including selected neuroblastoma lines (Fig. 8A). ERK3 is characterized by a high protein turnover and is known to be degraded upon N-terminal ubiquitination (Coulombe et al., 2003, Coulombe et al., 2004). The resulting protein levels in cell lines and tissues could strongly differ from previously measured mRNA amounts due to this posttranslational regulation by proteasomal activity. The protein expression of MK5 or ERK3 appeared stronger in highly proliferating cell lines compared to fully differentiated tissues. ERK3 and MK5 protein levels in tissues were close to the detection limit, hence a more sensitive HRP substrate (SuperSignal West Pico Chemiluminescent Substrate) had to be utilized (Fig. 8B). The ERK3 protein migrates with a molecular mass of 104 kDa, whereas also additional bands appeared, which might be non- specific (~80, 65, 60 kDa; * in Fig. 8A). Some of the additional bands correlated with ERK3- specific degradation products, which have been described earlier (Bind et al., 2004). The highest ERK3 protein level could be observed in the neuroblastoma cell line SH-SY5Y (Fig. 8A). In tissues, detectable amounts of ERK3 were found in the thymus and heart (Fig. 8B). MK5 protein, migrated with a molecular mass of 54 kDa, was detected in all tested tissues with highest abundance in thymus, spleen, lung, brain, and kidney (Fig. 8B). The detected protein amounts of MK5 were comparable in all analyzed cell lines. Interestingly, a second protein band with a molecular mass of around 70 kDa appeared that is present in all cell lines and most of the tissues. This protein band is completely lost in MK5-deficient cells/tissues and might signify a further MK5 isoform, which arose from unknown RNA processing. The MK5 antibody also revealed a non-specific staining pattern, especially in tissues. Some of the seemingly non-specific bands, which could only be found in MK5-deficient tissues, could also be truncated non-functional MK5 gene products, obtained as a result of the targeted deletion of exon 6, coding for a part of kinase subdomains VIa and VIb (Shi et al., 2003).

50 Results

A

B

Fig. 8. ERK3 and MK5 protein expression in selected cell lines (A) and adult mouse tissues (A/B). A) Western blot analysis was performed with well-established cell lines and mouse tissues using antibodies for ERK3 and MK5. ERK3 protein migrated with a molecular mass of about 104 kDa. Additional bands appearing at 60, 65, and 80 kDa were non-specific (*). MK5 protein migrated with a molecular mass of 54 kDa and a second 70 kDa band (‡) was also detected, which cannot be found in MK5-deficient cells (MEF dKO) and tissue (MK5-/- brain). B) A more sensitive ECL substrate (SuperSignal West Pico) revealed detectable amount of ERK3 protein in thymus and heart. MK5 protein was detected in all tested tissues with highest abundance in thymus, kidney, brain, lung, and spleen. Tissue-related protein expression was analyzed for ERK3 in three independent wild type mice (1-3) or in case of MK5 in two wild type (1-2) and one MK5-deficient animal (3). Some of the assumed non-specific bands, which were only found in MK5-deficient tissues, could also be truncated non- functional MK5 gene products, obtained by targeted deletion of exon 6 (†).

51 Results 4.1.2 Localization, interaction and activation of the ERK3/MK5-module

Interaction of MK5 and ERK3 has been identified in two independent yeast two-hybrid screens (Schumacher et al., 2004, Seternes et al., 2004). Co-localization studies in HEK293 cells demonstrated that GFP-MK5 is located in the nucleus and dsRed-tagged ERK3 is found ubiquitously in the cell. Simultaneous overexpression caused the nuclear export of MK5 and a predominant cytosolic localization of both kinases (Fig. 9A). The ERK3/MK5-interaction can be shown by co-immunoprecipitation (IP) using a MK5-specific antibody. Thus, ERK3 could be efficiently co-purified, whereas p38α could not be detected in the MK5-IP pellet. Non- specific binding of ERK3 could be excluded using unrelated anti-rabbit IgG (Fig. 9B). It has previously been reported that ERK3 and MK5 are activated by mutual interaction (Schumacher et al., 2004, Seternes et al., 2004). To analyze ERK3-dependent activation of MK5, a ‘substrate-ELISA’ assay has been established. Phosphorylation of the known in vitro MK5 substrate Hsp25 (Hspb1) was used as read-out of MK5 activity. Immobilized recombinant Hsp25 was incubated with recombinant ERK3, MK5 or in combination using kinase assay buffer, including ATP. The phosphorylation of Hsp25 was detected using the p[S86]-Hsp25-specific antibody and the corresponding secondary horseradish peroxidase (HRP)-conjugated antibody. Chromogenic detection of bound HRP showed the highest signal upon incubation of Hsp25 with both kinases, which clearly verified MK5-acitivation in the presence of ERK3 (Fig. 9C). The observed background signal was caused by antibody-binding to unphosphorylated Hsp25, since unrelated binding of the primary antibody could be excluded by investigation of BSA-immobilized control wells (Fig. 9C). ERK3- and p38α-mediated in vitro activation of MK2 and MK5 was analyzed in the following experimental setting. Combinations of kinases were transiently expressed in HEK293 cells, and MK2 or MK5 was purified from lysates by IP of the fused GFP-tag. The isolated MK2 or MK5 protein was subsequently used in a cold kinase assay together with the substrate Hsp25. The phosphorylation was observed by western blotting of the kinase assay samples using an anti-pHsp25 [S86] antibody. The constitutive active mutant MK5 L337A and the kinase dead mutant MK5 T182A (Seternes et al., 2002) were analyzed as controls. Single expressed MK2 or MK5 showed no detectable phosphorylation of Hsp25 (Fig. 9D). Upon co- expression of p38α and not by co-expression of ERK3, Hsp25-phosphorylation by GFP-MK2 could be detected, whereas MK5 has been activated by both kinases. The amounts of phosphorylated Hsp25 in the presence of MK5 were significantly higher due to co-expression of ERK3, while p38 induced the phosphorylation to a minor extent. The p38/ERK3-dependent changes of Hsp25 phosphorylation by constitutive active MK5 mutant (L337A) were similar to that of WT MK5, whereas the kinase dead mutant showed no detectable Hsp25 phosphorylation. These data suggest that the major in vitro activator of MK5 is ERK3.

52 Results

A

B C MK5-substrate ELISA

D

Hsp25

Fig. 9. Interaction of ERK3 and MK5 promotes activation of MK5 and cytosolic relocalization of both kinases. A) GFP-tagged MK5 and dsRed-ERK3 was transiently transfected alone or in combination in HEK293 cells and cellular distribution was observed B) Immunoprecipitation (IP) of endogenous MK5 in lysates of HEK293 cells revealed co-purification of endogenous ERK3 and not p38α. As negative control unrelated anti-rabbit IgG was used. C) MK5-substrate ELISA was performed to measure MK5-dependent phosphorylation of Hsp25 at Ser86 regarding to the presence of ERK3 D) GFP-tagged MK2, wild type MK5, constitutive active MK5 (L337A), and kinase dead MK5 (T182A) were overexpressed in HEK293 cells alone and in combination with p38α or ERK3. After IP using anti-GFP antibody, non-radioactive kinase assay was performed using recombinant Hsp25 as substrate. Hsp25 phosphorylation by MK2/MK5 at Ser86 was analyzed using phospho-specific antibody.

53 Results

4.1.3 Growth analysis of ERK3/MK5-deficient MEFs

A search for a phenotype of MK5- and ERK3-deficiency (dKO) has been performed on the level of isolated primary mouse embryonic fibroblasts (MEFs). The analysis of cell growth revealed a decrease in cell A proliferation within the first four days of MEF cells with a deletion of MK5 and/or ERK3 (Fig. 10A). The co- deletion of both ERK3 and MK5 (dKO) has an additive effect, resulting in a decreased proliferation rate even in later phases of the experiment. The B highest cell viability was measured for all genotypes after 6 days. MEFs with single ERK3- or MK5-deletion reached the similar viability after 6 days of cultivation like the wild type MEFs, whereas the viability of dKO cells was significantly lower at that time (Fig. 10A). The ERK3/MK5- dependent limitation in growth could

Fig. 10. Mouse embryonic fibroblasts (MEF) deficient in also be observed in SV40 large T MK5 and ERK3 (dKO) showed impaired proliferation and decreased sensitivity to glucose starvation. A) Primary antigen immortalized cells (Fig. 10B, isolated MEF cells of wild type, MK5 KO, ERK3 KO, and ERK3/MK5 dKO mice were cultivated for 11 days. WST-1 line plot). Double rescue of MK5 and assay was performed to measure cell viability at several time points of cultivation. B) SV40 large T antigen-immortalized ERK3 (dRes) increased the number of MEF cells of wild type, MK5 KO, MK5 KO with retrovirally rescued MK5 (MK5 Res), ERK3/MK5 dKO, and dKO with MEF cells detected after 120 h of retrovirally rescued ERK3 and MK5 (dRes) were cultivated for 120 h under glucose-free conditions. Afterwards, total cell glucose-free cultivation. It has been number (line plot) and percentage of dead cells (bar plot) were determined by propidium iodide (PI) staining. reported that MK5 is involved in energy-depletion-induced suppression of mTORC1 (Zheng et al., 2011). Analysis of sensitivity to glucose-starvation in immortalized MEF cell lines revealed less dead cells after 120 h of glucose-free cultivation in knockout MEFs (Fig. 10B¸ bar plot). The effect is diminished by rescue of the kinases.

54 Results

4.1.4 The impact of ERK3/MK5-deficiency on MEF cell migration

Closer microscopic observation of primary and immortalized ERK3/MK5-deficient (dKO) MEFs gave evidence of morphological differences, showing impaired cell shape and/or cell- cell contact. Deficient cells appeared more bulky, and the number of cell-to-cell contacts was decreased. These morphological differences could affect cell motility and might result from changes of the cytoskeleton. Analysis of cell migration of immortalized MEFs was performed by the scratch wound healing assay using a modified protocol (Menon et al., 2009). To avoid variations caused by distinct growth rate, the cell proliferation was inhibited with mitomycin C pretreatment (Fig. 11).

Fig. 11. ERK3/MK5 targeted deletion affects cell migration of immortalized MEFs. Analysis of cell migration with MEFs cells was performed using scratch wound healing assay. Cells were seeded in 96-well format visualized by the lipophilic tracer DiR dye. Protein biosynthesis was inhibited by pretreatment with mitomycin C. Cell monolayer was documented using Odyssey infrared scanner and wound closure of a scratch applied with a plastic pipette tip was measures graphically using ImageJ software.

Graphical data analysis demonstrated that lack of ERK3 and/or MK5 lead to increased migration of immortalized MEFs. Co-deletion of the ERK3 and MK5 gene showed an additive effect.

55 Results 4.1.5 The actin filament of ERK3/MK5-deficient MEFs

Investigations performed by the group of Ugo Moens suggested a direct role of MK5 activity in actin filament rearrangement, which is regulated by PKA-dependent activation of MK5 through phosphorylation at Ser115 (Gerits et al., 2007a, Kostenko et al., 2011b). Hence, the effect of MK5 deficiency on actin filament organization has been analyzed by phalloidin staining. In 4% PFA fixed cells, the actin filament was stained with Alexa647-conjugated phalloidin. Comparison of dKO MEFs with Fig. 12. The actin filament is less organized in immortalized ERK3/MK5-deficient MEFs. Cells were seeded on glass dRes cells revealed a reduced coverslips, fixed with 4% PFA, and stained with Alexa647- conjugated phalloidin. ERK3/MK5 dKO and dRes cells were formation of actin filaments in dKO analyzed regarding to their organization of globular actin (*), cortical actin (†), and microfilaments (‡) (scale bar 10 µm). cells (Fig. 12). Overall, the observed cell-to-cell variation of actin- organization was high. To get a more unbiased view of actin filament structures, their solubility in cell lysis buffer has been analyzed by quantitative western blotting. No significant differences could be observed by using a 0.015% digitonin/PIPES permeabilization buffer (section 4.3.6). This method has successfully been applied to analyze septin filament organization in K562 cells (Sellin et al., 2011).

4.2 Regulation of ERK3 protein stability by extracellular factors

4.2.1 NGF-stimulation of PC-12 cells

ERK3 has been identified together with ERK1/2 as NGF- and insulin-stimulated kinase in PC-12 cells (Boulton et al., 1991). This cell line is known to differentiate in response to nerve growth factor (NGF) stimulation. The kinetics of ERK3 activation-site phosphorylation upon NGF-stimulation of PC-12 cells were investigated. Interestingly, the amount of ERK3 protein was significantly increased after 3 h of NGF-treatment (Fig. 13A). Phosphorylation signal of the molecular mass-related protein band was already increased after 30 min. Another experiment demonstrated that the changes of ERK3 protein levels were independent of NGF-treatment (Fig. 13B). The disruption of continuous cultivation condition resulted in complete degradation of ERK3 protein within 30 min. The progress of degradation and stabilization of ERK3 protein is

56 Results illustrated in Fig. 13C. The protein seems to be restored after further 120 min under continuous cultivation within the incubator. At this point, it was not clear, which parameter induced the observed ERK3 degradation.

A

B

C

Fig. 13. ERK3 protein level is highly regulated in PC-12 cells. A) NGF-dependent phosphorylation and protein expression of ERK3 was analyzed in PC-12 cells by western blotting. B) Changes in ERK3 protein levels are not related to stimulation with NGF. C) For analysis of ERK3 degradation kinetics, PC-12 cells were either cultivated in single 3,5 cm Petri dishes (continuous cultivation) or in 2 wells of a 6-well test plates (discontinuous cultivation).

57 Results 4.2.2 Rapid degradation of ERK3 occurs via the 26S proteasome

ERK3 protein has been shown to be degraded by the proteasome upon N-terminal ubiquitination (Coulombe et al., 2003, Coulombe et al., 2004). Consistently, ERK3 degradation related to environmental changes can be clearly blocked by treatment with common proteasome inhibitors, which could be demonstrated by (pre-)incubation with bortezomib and MG132 (Fig. 14A). ERK3 degradation was completely blocked, and basal

A

B

Fig. 14. ERK3 degradation in response to environmental changes can be blocked by proteasomal inhibitors. A) PC-12 cells were seeded in 6-well test plates and cultivated for 24 h under normal conditions in DMEM. Afterwards, cells were pre-treated with 50 nM bortezomib, 25 µM MG132, or DMSO (CTRL) for 4 h. After pre-incubation the first sample was lysed and thereby the short time conditional change was applied to the other wells. Further samples were taken after 30 and 120 min. The influence of mechanical stress on ERK3 protein stability was analyzed by shaking of cell culture dishes within the incubator on a rotary shaker with 300 rpm for 5 min (done in duplicates). PC-12 were lysed after 30 min and 120 min. B) The cleavage of ERK3 protein is induced by pH changes of the DMEM caused by absence of 5% CO2. PC-12 cells were cultivated in 6-well plates with DMEM or CO2-independent medium (pH stable medium). Effects of short-time conditional changes and long-time environmental changes (light at 37°C water bath or incubation at RT in dark) were observed. levels were increased. The application of mechanical stress within the incubator resulted in only minor changes of ERK3 protein levels (Fig. 14A). Analysis of daylight, temperature, and pH as responsible factors clearly demonstrated that the changes in ERK3 protein were influenced by the extracellular pH of the culture medium. CO2-independent medium, which in contrast to DMEM does not require CO2 for efficient pH-buffering, stabilized the ERK3 protein

(Fig. 14B). Continuous alkaline pH of DMEM (~8.5) due to cultivation without 5% CO2 resulted in permanent loss of ERK3 protein in PC-12 cells (Fig. 14B).

58 Results 4.2.3 The extracellular pH regulates ERK3 protein levels

For analysis of ERK3 degradation upon alkalinization of the culture medium, experiments were performed using CO2-independent medium cultivating cells under atmospheric CO2. The applied pH changes were initially performed by complete exchange with medium adjusted to respective pH. The results of these experiments showed that medium exchange itself causes differences in the protein amounts of ERK3. In further experiments, the pH was directly adjusted in the culture medium by titration with HCl or NaOH. Analysis of MEF cells demonstrated that ERK3 protein levels are similarly regulated as in PC-12 cells (Fig. 15). It could also be demonstrated that ERK3 degradation was independent to MK5 protein expression. Additional MK5-rescue in dKO MEFs increased the basal protein levels of ERK3, but did not prevent complete cleavage of ERK3 after incubation of MEF cells with alkaline medium. The MK5 protein level is not influenced by pH changes.

Fig. 15. ERK3 protein levels are positively and negatively regulated through acidification and alkalinization of the culture medium. PC-12 and MEF cells with distinct genotype regarding to ERK3 and MK5 were seeded in 6-well cell culture dishes and cultivated in CO2-independent medium. After 24 h of cultivation the pH was changed by titration with HCl and NaOH and cells were lysed after 30 and 120 min. ERK3 and MK5 protein levels were analyzed by western blotting.

4.2.4 A FACS-based method for real-time monitoring of ERK3 protein levels

For analysis of ERK3 protein stability, a more flexible method was required, which allowed ‘real-time’ measurement of ERK3 protein levels in living cells. This method should be useful for the investigation of regulatory pathways, which mediate degradation of ERK3.

59 Results

A FACS-based approach for the determination of GFP-tagged protein has been established. PC-12 cell lines stably transfected with GFP-ERK3, GFP, and instable GFP (d2GFP) were generated. The N-terminal GFP-tagged ERK3 was preferably used for further experiments. An unstable form of GFP carrying the PEST domain of murine ornithine decarboxylase (d2GFP) was used as control, since wild type GFP was highly stable. Comparison of the detected protein degradation of GFP-ERK3 revealed similar protein cleavage upon alkalinization of the culture medium (Fig. 16A). In contrast to endogenous ERK3, the fluorescence of exogenous GFP-ERK3 did not completely diminished after alkalinization of the medium, indicating that protein is still present in these cells. It has been already demonstrated that specific GFP-ERK3-fragments can be found in the cell, which are not A B

C

Fig. 16. In stably transfected PC-12 cells GFP-ERK3 is degraded in response to alkaline extracellular pH. A) Gating of GFP-ERK3-positive cells (P7) in FL2-H/FL1-H plot. B) Analysis of GFP- ERK3 protein levels by western-blot and flow cytometry (FACS) showed similar results. C) PC-12 cells stably transfected with GFP-ERK3, GFP or an instable GFP were analyzed upon alkalinization and acidification of the culture medium. The percentage of GFP-positive cells was determined by FACS after 0.5, 1, 2, and 4 h.

60 Results products of proteasomal degradation (Bind et al., 2004). Cleavage of GFP-ERK3 is induced by alkaline pH, whereas GFP and instable GFP showed no significant response (Fig. 16B). Expression of d2GFP was increased under neutral and alkaline pH. Overall, degradation events of GFP-ERK3 were significantly slower than that detected with endogenous ERK3 (Fig. 16A). To get a more precise view the analysis was prolonged to a final measurement after 4 h.

4.2.5 Screen for the underlying mechanisms of ERK3 degradation

In the following experiments, different agents affecting activity of protein kinases and protein phosphatases, protein synthesis, proteasome, or cell cycle progression were applied to PC- 12 cells in combination with pH-changes to identify possible factors of ERK3 protein regulation. Alkaline pH-induced degradation is significantly inhibited by bortezomib, whereas increased GFP-signal upon acidic conditions might result from accumulation of newly synthesized GFP-ERK3 protein (Fig. 17).

Fig. 17. GFP-ERK3 protein cleavage can be influenced by bortezomib, nocodazole, PMA, and cobalt chloride. PC-12 cells stably transfected with GFP-ERK3 were treated with anisomycin (stimulation started 1 h before changing pH), bortezomib (1 h pretreatment), nocodazole (16 h pretreatment), phorbol-12-myristate-13-acetate (PMA), and cobalt chloride (1 h pretreatment) under acidic (6.4) and alkaline pH (8.4). GFP-signal of GFP-ERK3 stably transfected cells were detected by FACS after 30 and 240 min.

The highest GFP-ERK3 levels could be detected after stimulation with nocodazole and phorbol-12-myristate-13-acetate (PMA). The PMA-effect might be artificial by its influence on CMV-promoter activity (Loser et al., 1998). Nocodazole-dependent cellular arrest in mitosis increased the basal GFP-ERK3 protein levels, but did not prevent the alkaline-induced

61 Results degradation. Interestingly, cobalt chloride could also prevent the degradation of GFP-ERK3. This compound is frequently used for mimicking hypoxia in cells. In the next experiment, the effect of cobalt chloride inhibiting alkaline pH-dependent degradation of GFP-ERK3 was compared to other agents for targeted inhibition of distinct central signaling molecules (Fig. 18). Treatment with antimycin A, which blocks ATP synthesis, and staurosporine, which inhibits the activity of numerous protein kinases, completely prevent GFP-ERK3 degradation under alkaline conditions. No effects have been observed upon treatment with sodium pump inhibitor ouabain, the calcium- and magnesium-chelating compound EGTA, the pan caspase inhibitor zVAD-fmk, the mTORC1 inhibitor rapamycin, the HSP90 inhibitor radicicol, the PI3K inhibitor wortmannin or the protein kinase A inhibitor PKI.

Fig. 18. The pH-dependent cleavage of GFP-ERK3 can be blocked by cobalt chloride, antimycin A, and staurosporine. PC-12 cell stably transfected with GFP-ERK3 were treated with sodium pump inhibitor ouabain, cytochrome C reductase inhibitor antimycin A, cation chelator EGTA, pan capase inhibitor zVAD-fmk, inhibitor of mTORC1 rapamycin, unspecific protein kinase C inhibitor staurosporine, HSP90 inhibitor radicicol, PI3K inhibitor wortmannin, and proteinkinase A inhibitor PKI. Number of GFP-positive cells was described in relation to untreated cells cultivated four hours at pH 8.4.

Since broad inhibition of protein kinases by staurosporine seems to affect ERK3 degradation, several protein phosphatase inhibitors were also analyzed in this experimental setting. The cleavage of GFP-ERK3 is inhibited by high dosage of okadaic acid (1 µM) and after treatment with protein tyrosine phosphatase inhibitor ortho-vanadate (Fig. 19). Lower concentration of okadaic acid and other serine/threonine phosphatase inhibitors, cyclosporine A, microcystine-LR, calyculin A, and sodium fluoride, had no significant effect. The signaling cascades regulating ERK3 protein stability, which are induced by alkaline extracellular pH, remained elusive. Obviously, phosphorylation-dephosphorylation events of signaling molecules are required for the regulation of GFP-ERK3 degradation. The phosphorylation of ERK3 could also have functional relevance and should be further analyzed.

62 Results

Fig. 19. High dosage of okadaic acid and broad inhibition of protein tyrosine phosphatases can block degradation of GFP-ERK3. PC-12 cells stably transfected with GFP-ERK3 were stimulated under alkaline pH with PP2B inhibitor cyclosporine A (CsA), a related autoinhibitory CsA peptide, PP1/PP2A inhibitors okadaic acid, microcystine-LR, calyculin A, sodium fluoride or PTP inhibitor ortho- vanadate and a broad phosphatase inhibitory cocktail from Sigma-Aldrich. GFP-positive cells were analyzed by FACS after 4 h of stimulation.

4.3 Interaction partners of the ERK3/MK5-signaling module

There are two main questions, which are central for the investigation of the cellular function of the ERK3/MK5-signaling module: First, identification of extracellular stimuli, which activate the signaling complex and, second, the identification of cellular targets for the two kinases. Various efforts have been made to identify ERK3/MK5-interaction partners, which will be described here.

4.3.1 In vitro screening for an ERK3/MK5-activating kinase

The idea of the screen was the identification of a Ser/Thr protein kinase, which can activate the ERK3/MK5-signaling module in an in vitro kinase assay. As read-out of activation, MK5 kinase activity was measured using the Z’-Lyte kinase assay, including the verified MK- substrate peptide 4 (containing the phospho-site F/L/V/A-X-R-X-X-S/T-X-X). Kinase assay was performed with an E. coli-expressed GST-tagged MK5 and a Sf9-expressed His-tagged ERK3. Phosphorylation of the MK5 substrate peptide 4 upon single incubation with MK5 or

63 Results ERK3 was negligible (lanes 101 and 103 in Fig. 20). As possible candidates a panel of 100 recombinant Ser/Thr protein kinases (mostly Sf9-derived recombinant proteins), generously provided by the ProQinase GmbH (Freiburg, Germany), has been screened in combination with MK5 and ERK3 (Fig. 20). Relative substrate phosphorylation was normalized to unphosphorylated peptide (0%) and a completely phosphorylated peptide sample (100%). Substrate incubation with ERK3 and MK5 resulted in a final peptide phosphorylation of about 20% (basal level). The threshold for a significant increase in MK5-mediated phosphorylation was set to two times of basal level (40%). A reaction with MK5 and GST-p38α, which is a strong in vitro activator of MK5 (lane104) was included in the screen as well as in vitro activated MKs (lanes 63-65), which almost completely phosphorylated the substrate peptide. The additional incubations with the following kinases resulted in significant higher substrate phosphorylation: microtubule affinity-regulated kinase 1/3 (MARK1/3), p21-activated kinase 1/3 (PAK1/3), PDZ-binding kinase (PBK), and testis-specific kinase 2 (TSK2).

Fig. 20. In presence of ERK3, MK5-mediated substrate phosphorylation is significantly increased upon additional incubation with MARK1/3, PAK1/3, PBK, or TSK2. MK5 kinase activity was measured using the Z’-Lyte kinase assay with the corresponding substrate peptide 4. A panel of recombinant serine/threonine protein kinases was used together with recombinant inactive MK5 and ERK3. As internal control in vitro activated MK2, MK3, and MK5 was used. Abbreviations: MARK1/3, microtubule affinity-regulated kinase 1/3; PAK1/3, p21-activated kinase 1/3; PBK, PDZ-binding kinase, and TSK2, testis-specific kinase 2.

64 Results To ascertain reasons for increased peptide phosphorylations, further kinase assays have been performed (Fig. 21). The control reactions should clarify, whether increased substrate phosphorylation is specifically induced by kinase X-mediated ERK3-activation, which promotes MK5-activity and the subsequent phosphorylation of the substrate. The MARK proteins increased peptide phosphorylation together with MK5 alone or both ERK3 and MK5. Probably MARK1/3 and PBK might influence MK5-activity via direct interaction with MK5. In contrast, both p21-activated kinases, PAK1 and PAK3, directly phosphorylated peptide 4. Similar results could be obtained for TSK2.

Fig. 21. Verification of potential ERK3/MK5-complex activating kinases. Control reactions were performed to distinguish certain reasons of increased substrate peptide 4 phosphorylation.

65 Results

4.3.2 The influence of PAK activity on ERK3 phosphorylation and stability

To confirm phosphorylation of ERK3 by PAKs radioactive in vitro kinase assay was performed using recombinant GST-tagged PAKs and His-tagged ERK3 as previous in the Z’- Lyte kinase assay (Fig. 22A). Increased phosphorylation of ERK3 has been detected in the presence of group I PAK1-3, whereas PAK4 and 7 (also known as PAK5) did not affect ERK3 phosphorylation. Because of same molecular mass of His-ERK3 and GST-PAK6 the phosphorylation of ERK3 could not be determined, since all PAKs were also phosphorylated in the presence of ERK3. It is not clear whether the phosphorylation of the distinct PAK isoforms is directed by ERK3 or the interaction of ERK3 and PAKs promotes autophosphorylation. Since the data suggest functional relevance of PAKs in ERK3 phosphorylation, it was interesting to analyze whether PAK activity can regulate ERK3 stability. A PAK-specific inhibitory peptide referred to as PAK18 was used to stimulate PC-12 cells stably transfected with GFP-ERK3. The GFP-signal was determined by FACS upon alkaline-induced degradation of ERK3 (Fig. 22B). As a positive control staurosporine was used, since it has been shown to inhibit degradation of ERK3 at alkaline pH (Fig. 18). After 4 h at pH 8.4, PC-12 cells treated with PAK18 did not show higher GFP-signal compared to DMSO treated cells (CTRL).

A B

Fig. 22. Phosphorylation of ERK3 is increased in presence of PAK1, 2, and 3, whereas inhibition of PAKs does not influence ERK3 stability. A) In vitro kinase assay was performed using recombinant GST-tagged PAKs (Proqinase) together with His-tagged ERK3. Incorporated phosphate from γ-33P-ATP was detected by phospho-imaging. B) PC-12 cells stably transfected with GFP-ERK3 were treated either with DMSO (CTRL), staurosporine, or PAK18 and pH was changed to 8.4. GFP- positive cells were analyzed after 4 h of stimulation and values were normalized to control cells incubated at pH 7.4.

66 Results

4.3.3 Analysis of the phosphoproteome of MK5-deficient tissues

Phospho-motif antibodies, which detect phosphorylated kinase-specific substrates, are a useful tool to study the mammalian phosphoproteome, allowing broad analysis and quantification of kinase-specific phosphorylation pattern. They could be helpful for identification of novel kinase substrates. So far, there is no MK substrate motif antibody available, but the putative MK phosphorylation site [FVIL]-x-R-x-x-S is similar to the basophilic motifs of the AGC kinase family members PKA, PKB, PKC, and PKD. Here, an antibody sampler kit containing antibodies raised against the PKA, PKC, CDK and ATM/ATR substrate phospho-motif (Cell Signal Technologies) was used in western blot analysis. In Tab. 9, the results of the distinct antibody staining pattern after western blotting of several tissues obtained from wild type and MK5-deficient mice are summarized. The tissues of each genotype were analyzed in triplicates, and the received staining patterns were compared.

Tab. 9. Substrate phospho-motif antibody staining pattern of PKA, PKC, CDK, and ATM/ATR kinase of MK5-deficient compared to wild type tissues showed additional (A) or missing (B) phosphoprotein-bands. Tissue lysates were generated from each three MK5-deficient and wild type mice. Additional or lost bands were only considered if all samples of specific genotype showed the same pattern.

Sk. Phosphoband [#/(kDa)] Brain Heart Kidney Liver Lung Muscle Spleen Thymus A B A B A B A B A B A B A B A B 1 1 P- (Ser/Thr) PKA Substrate Ab (RRXS/T) X X X X X X X X X X X X X X (15) (110) 1 P-(Ser) PKC Substrate Ab (K/R)XsX(K/R) X X X X X X X X X X X X X X X (39) 1 1 P-(Ser) CDKs Substrate Ab (K/R)sPX(K/R) X X X X X X X X X X X X X X (25) (25) P-(Ser/Thr) ATM/ATR Substrate Ab (s/t)QG X X X X X X X X X X X X X X X X

Analysis of phospho-motif antibodies staining partially revealed differences within the same genotype. Only phospho-bands that appeared or disappeared in all three distinct lysates of the MK5-deficient tissue were taken into account (listed in the Tab.1). The resolution of the SDS PAGE was moderate, so that the number of detected phosphoproteins mostly remained below 30. Low expressed substrates or minor changes in phosphorylation could be overlooked. The strongest difference between wild type and MK5-knockout tissues has been detected in lysates of the heart using PKA/PKC phospho-motif antibodies. Additional substrates probably phosphorylated by PKA or PKC could be observed in heart tissue lacking MK5 (Fig. 23). This might indicate a negative regulation of MK5 in PKA/PKC- dependent signaling of cardiac cells. Moreover, one additional PKA phospho-substrate band has also been detected in spleen of MK5-deficient mice. The phosphoprotein migrates with a molecular mass of 110 kDa. The most convincing difference has been found in lysates of brain after staining with the CDK phospho-motif antibody (Fig. 23). A phospho-band

67 Results disappeared in MK5 knockout tissues, which migrated with a molecular mass of 25 kDa. This effect on SP-motif phosphorylation of a 25 kDa protein could also be indirectly affected by ERK3, which is reduced in MK5-deficient cells/tissues (Schumacher et al., 2004). CDKs and MAPKs are known to phosphorylate their substrates at proline-directed sites, indicating potential phosphorylation of a CDK-motif by ERK3 itself.

Fig. 23. Analysis of substrate phospho-motif pattern of PKA, PKC, CDK, and ATM/ATR in MK5- deficient mouse adult tissues. Lysates of brain, heart, kidney, liver, lung, skeletal muscle, spleen, and thymus were generated of each three wild type and MK5-deficient mice. The figure represents the western blots of lysates that showed a different pattern due to genotype. Changed phosphorylations are indicated with arrows, whereas asterisks mark the phospho-bands that are missed in the MK5- knockout. The phospho-motif pattern of ATM/ATR kinase was always equal in both genotypes.

4.3.4 Analysis of the phosphoproteome of ERK3/MK5-deficient MEFs

A disadvantage of tissue-related phosphoproteome analysis is the high variability in ‘basal’ phosphorylation between distinct animals sharing the same genotype. Phosphoproteomic analysis on the cellular level is more promising, since external influences can be minimized. Ex vivo cell cultures or established cell lines are homogenous cell populations, which can be

68 Results easily manipulated with high reproducibility by various stimuli under defined conditions. Phosphoproteome has been investigated in SV40 large T antigen immortalized ERK3/MK5 dKO MEFs compared to dRes MEFs. Cells were stimulated with phorbol 12-myristate 13- acetate (PMA, also known as TPA) or pervanadate to increase phosphoprotein levels due to PKC activation and inhibition of protein tyrosine phosphatases, respectively. To reach higher resolutions in protein separation precast SDS gradient gels were used. However, these gels were not suitable for separation of the required high protein amounts, hence running artifacts appeared (Fig. 24). Staining with PKA substrate phospho-motif antibody leads to an additional band with a molecular mass of 75 kDa in ERK3/MK5 rescued MEFs upon stimulation with PMA. The PKB (Akt) phospho-motif staining revealed an additional

Fig. 24. PKA, PKB (Akt), PKC, and PKD substrate phospho- phosphoprotein migrating with a motif antibody detection in immortalized dKO MEFs (1) compared to dRes MEFs (2). MEF cells were either stimulated molecular mass of 150-170 kDa in with 650 nM phorbol 12-myristate 13-acetate (PMA) or 50 µM pervanadate for 30 min at 37°C to increase general kinase dRes cells after both PMA and activity. For proper phosphoprotein signals high amounts of proteins were loaded, which caused partially artefacts. pervanadate treatment. The PKC Stimulated substrate-phosphorylations are indicated by an arrow. and the PKD phospho-motif staining showed an increased phosphorylation signal of a protein, which migrated with 35 and 50 kDa, respectively. These two phospho-bands are detected in knockout and rescued cells, but they appeared much stronger in rescued cells. Phospho- mapping with MAPK and CDK substrate phospho-motif antibodies showed no differences. Further analysis of the identified phosphoproteins should be performed, for instance, by 2D gel electrophoresis followed by mass spectrometry analysis of isolated gel spots.

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4.3.5 Yeast two-hybrid screens for MK5 interaction partners

The initial yeast two-hybrid (Y2H) screen, identifying ERK3 as MK5 interacting kinase, was performed in our lab using a mouse 11-day embryo MATCHMAKER cDNA library (Schumacher et al., 2004). Since that time, further screens have been performed to identify additional MK5 interaction partners. Two cDNA libraries were chosen for these screens: a mouse brain cDNA library, since MK5 and ERK3 are both expressed in the brain, and a normalized universal mouse cDNA library for identification of rarely expressed interacting partners, which are artificially increased in this library. In three independent screens, 3.2 x 106 clones of the brain library (two experiments) and 2.1 x 106 clones of the normalized universal library (one experiment) were analyzed. In Tab. 10 all identified positive interacting prey clones, which were confirmed by retransformation experiments in AH109 yeast strain (Fig. 25), are summarized. Interestingly, cDNA fragments of two distinct proteins were

Fig. 25. Reconstitution of MK5-bait interacting prey-vectors together with the empty, MK2-, or MK5- bait vector in yeast. Growth of co-transformants were analyzed either on minimal medium lacking leucine and tryptophan (SDΔLT; bait-prey selection) or on medium additionally lacking adenosine and histidine (SDΔAHLT; selection of Gal4 transactivation). frequently found in the brain-specific library: the regulatory subunit beta 1 of the sodium/potassium transporting ATPase (ATP1b1) and the structural protein Septin8 (Sept8). Since the interaction with septin isoforms could be found for both MK5 and ERK3 (section 4.3.6), a functional role of the ERK3/MK5-signaling module in regulating septin structures became highly probable. Further experiments have been performed to confirm MK5-specific interaction and to describe the biological relevance of these interactions by searching for shared functions of MK5 and the identified prey proteins.

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Tab. 10. Positive cDNA prey clones identified in three distinct yeast two-hybrid screens using MK5 as bait. Overall, 3.2 x 106 clones of a murine brain cDNA library and 2.1 x 106 clones of a mouse normalized universal cDNA library were screened and 188 clones were selected and further analyzed. Interactions with Septin8 (Sept8) and sodium pump regulatory subunit beta-1 (ATP1b1) were already known from previous screens (*), whereas interactions with the Mdm2-binding protein and the Rho nucleotide exchange factor kalirin7 (Kal7), the small nuclear ribonucleoprotein 48, the doublecortin-like kinase 1, the cell division cycle and apoptosis regulator protein 1, the keratin-like protein 222, the carboxypeptidase E, and the microtubule-associated protein 1a were unknown, so far. A gray background highlights the prey proteins, which were further analyzed in this study. cDNA Reconstitution of interaction with Protein Full name Clone number(s) Growth Expressed fragment library baits screening / empty MK2 MK5 [aa] clone# several ~67-484 (C- 1 Sept8 Septin 8 brain VI/ 4, 13, 31, 67 * fast x term.)

2 ATP1b1 Sodium/ potassium ATPase subunit beta 1 brain VI/1 * slow x x 204-304 (C-terminus)

3 Kal7 Rho nucleotide exchange factor kalirin 7 brain VI/ 84 slow x 588-941 (1663) norm. 4 Mtbp Mdm2-binding protein IV/20 fast x 431-894 (C-term) universal

5 snRNP48 Small nuclear ribonucleoprotein 48 brain VI/ 25, 51, 100 slow x several ~21-271 (337)

6 Dclk1 Doublecortin-like kinase 1 brain V/53 fast x 488-756 (C-terminus) Cell division cycle and apoptosis regulator 7 Ccar brain VI/41 fast x x 617-839 (1146) protein 1 8 Krt222 Keratin-like protein 222 brain VI/97 slow (x) x x full-length (294)

9 CPE Carboxypeptidase E brain VI/56 slow (x) x x 176-446 (476)

10 Mtap1a Microtubule-associated protein 1a brain V/40 fast x x x 2561-2677 (C-term)

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4.3.5.1 Septin8

The most frequently found prey protein using MK5 as bait by screening a mouse brain cDNA library was Septin8 (Sept8). The high number of different interacting Sept8 fragments allowed a first more detailed characterization of the MK5 binding site within Sept8 (Fig. 26). The interacting region is localized at the C-terminal part starting from amino acid 319.

*

*

Fig. 26. MK5 interacts with C-terminal part of Septin8 (Sept8). Two prey clones of Sept8 (VI/13 and VI/67, depicted in blue) were sequenced and compared with cDNA fragments received from a former screen (depicted in green). The MK5-binding region of Sept8 is localized at the C-terminal part starting from amino acid 319. MK5 was binding to a specific Sept8 splice variant, which is characterized by additional N- and C-terminal extensions (*). The tissue-specific splice variants were detected on protein level (AAH49819) or predicted by mouse chromosomal analysis (RP23-188H3.2-004).

Septins are known to have multiple splice variants, which show distinct tissue-specific expression patterns. MK5 interacted with a splice variant, which has not yet been purified at the protein level [RP23-188H3.2-004; (Osoegawa et al., 2000)]. This isoform has an additional C-terminal extension of 55 aa, whereas other parts are similar to the reviewed protein consisting of 429 aa (Blaser et al., 2003). Interestingly, the full-length clone (VI/67) was homologous to the 484 aa long isoform, but additionally contained a 29 aa long N- terminal extension, which is found within another 458 aa long splice variant [AAH49819; (Strausberg et al., 2002)]. Reconstitution of bait- and prey-plasmid, using the clones VI/13 and VI/67 for co-transformation of AH109 strain, was performed. Dilution series were spotted on minimal medium SDΔLT and SDΔAHLT agar plates for quantification of yeast growth. Under stringent conditions (SDΔAHLT) both Sept8 cDNA fragments showed comparable growth, which indicates that the C-terminal part of Sept8 is sufficient for MK5-interaction

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(Fig. 27A). The Sept8 coding fragments of the preys VI/13 and VI/67 were subcloned into pEGFP vector for further analysis. In GST-MK5 pull-down, both GFP-tagged Sept8 fragments could be co-purified, whereas no binding to GST has been observed (Fig. 27B). A B

Fig. 27. MK5 interacts with Sept8 fragments in yeast and HEK293 cell lysates. A) Reconstitution of bait and prey interaction was performed by co-transformation of S. cerevisiae AH109 with Sept8 prey cDNA clones VI/13 or VI/67 together with the empty, MK2-, or MK5-bait vector. B) GST-MK5 specifically interacts with GFP-tagged Sept8 fragments subcloned from the pACT2 prey vector into pEGFP vector.

The Sept8 localization studies using the GFP-tagged Y2H clones to transfect HEK293 cells showed that the protein fragments are exclusively cytosolic localized. By transfection of a myc-tagged full-length Sept8, provided by Dr. Koh-ichi Nagata, the localization could be confirmed (Aichi Cancer Center, Japan). The intracellular distribution of Sept8 was characterized by vesicle-like structures, which has been reported by the group of Nagata (Ito et al., 2009). The localization of Sept8 clearly differed from ectopically expressed GFP- or myc-tagged Sept7, identified as ERK3 interaction protein (section 4.3.7), that forms filamentous structures within the cytosol. The nuclear import of myc-Sept8 is partially induced by MK5 co-expression, whereas the nuclear export of MK5 has also been observed (Fig. 28). Interaction of MK5 and Sept8 has been verified by additional BioEase(BE)-tagged ERK3 co-expression. In the presence of ERK3, MK5 is known to be activated and subsequently exported from the nucleus (Schumacher et al., 2004, Seternes et al., 2004). In case of specific Sept8-MK5 interaction, Sept8 should be found exclusively in the cytosolic compartment upon co-expression of ERK3. Analysis of immunofluorescence images could confirm this idea, whereas a small part of myc-Sept8 is retained in the nucleus (Fig. 28). Another interesting question was, whether Sept8 also interacts with Sept7, showing the functional relevance in septin filament formation.

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Fig. 28. Sept8 is partially co-localized with MK5. HEK293 cell were transfected either with myc- tagged Sept8 or HA-tagged MK5 or both. As control additional expression of BE-ERK3, inducing nuclear export of HA-MK5 was also analyzed (5 µm scale bar).

The cells were transfected with HA-Sept7 and myc-Sept8 and immunostaining was performed. The Fig. 29 demonstrated that Sept8 is co-localized with Sept7 and both formed strong filamentous structures within the cytosol, which are similar to Sept7 homomeric filaments.

Fig. 29. Sept7 and Sept8 generated strong cytosolic filamentous structures. HA-tagged Sept7 and myc-tagged Sept8 were co-expressed in HEK293 cells and immunostained with the corresponding tag- specific antibodies (5 µm scale bar).

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4.3.5.2 Rho-GEF Kalirin7

Kalirin7 (Kal7) is a multidomain protein of 1663 amino acids. The MK5-interacting fragment identified in Y2H screen (amino acids 588-946) belongs to a part of nine distinct spectrin-like repeats (SR) (Fig. 30A). Interaction between MK5 and Kal7 was verified by co-purification of endogenous MK5 in a GST-pull-down from lysates of HEK293 cells with recombinant GST- fusion protein of Kal7 spectrin-like repeat SR4-7 (amino acids 517-976) and SR3-6 (amino acids 416–870), respectively. Both Kal7 fragments were able to bind endogenous MK5 in this pull-down (Fig. 30B) indicating that MK5 binds to the overlap region containing SR4-6.

A

B C

Fig. 30. Kal7 as in vitro substrate of MK5. A) Schematic representation of the mouse Kalirin7 (Kal7) domain structure and the MK5 interacting region. B) Pull-down of MK5 with GST-tagged fragments of Kal7 spanning spectrin-like repeats 3-6 or 4-7. C) Identification of the MK5 phospho-site within Kal7. The fragment SR3-6, but not the mutated fragment SR3-6-S487A is phosphorylated by MK5. Phosphorylation was detected by 32P incorporation and phospho-imaging. Serine 487 is located in an ideal MK5 consensus motif LXRXXS* and a putative SH3-binding PXS*P motif (cf. part C). Abbreviation: DH, Dbl homology; GEF, guanosine nucleotide exchance factor; PH, pleckstrin homology; PDZ, PDZ binding motif (Ser-Thr-Tyr-Val); sec14p, domain binds PIP2(3,5) and PIP3; and SR, spectrin-like repeat.

In vitro phosphorylation assays revealed that a Kal7 fragment containing SR3-6, but not the fragment with SR4-7, is strongly phosphorylated in the presence of MK5 and its in vitro activator p38α MAP kinase (Fig. 30C), indicating that the phosphorylation site for MK5 is located in SR3. The SR3 contains an ideal consensus phosphorylation motif for MK5 (LQRPLS*P, Fig. 30A) between amino acids 482-487. Mutation of S487 to alanine completely abolished in vitro phosphorylation of SR3-6 by MK5 demonstrating that S487 is the only phosphorylation site for MK5 in spectrin-like repeat 3-7. Interestingly, Kal7 S487 phosphorylation was already detected by mass spectrometry in mouse brain extracts (Gnad

75 Results et al., 2011). This phosphorylation site is located within an intramolecular PXXP SH3 domain of Kal7 and is proposed to stimulate GEF activity by preventing intramolecular SH3-PXXP interaction (Schiller et al., 2006). This indicates a possible role for the ERK3/MK5-complex in Kal7 regulation. To further demonstrate the in vivo relevance of the interaction between MK5 and Kal7, HA- tagged MK5 and myc-tagged Kal7 were co-expressed in HEK293 cells and their cellular distributions were analyzed (Fig. 31). MK5 and Kal7 showed partial co-localization in cytoplasmic compartments. Interestingly, localization of both proteins was completely changed to an exclusive cytosolic distribution, when ERK3 was co-expressed together with the two proteins.

Fig. 31. Co-localization studies revealed that Kal7 interacts with MK5. Myc-tagged Kal7, HA- tagged MK5 or in combination were overexpressed in HEK293 cells. Additional overexpression of BE- tagged ERK3 was performed to analyze Kal7-MK5-interaction after nuclear export of MK5 (5 µm scale bar).

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4.3.5.3 Mdm2-binding protein

Mdm2-binding protein (Mtbp) is known to inhibit autoubiquitination of murine double minute (MDM2), which enhances MDM2 stability. This promotes MDM2-mediated ubiquitination of p53 and its subsequent degradation (Boyd et al., 2000a, Brady et al., 2005). Interestingly, MK5 (PRAK) has previously been shown to have an effect on p53 stability via direct phosphorylation at Ser37 (Sun et al., 2007). Perhaps the interaction of MK5 and Mtbp alters their regulatory functions on p53. The Mtbp cDNA fragment was subcloned from pACT2 prey vector into a myc-tag containing mammalian expression vector and used for pull-down studies in HEK293 cell lysates. Interaction of MK5 with Mtbp could be shown by co-purification of a myc-Mtbp fragment in a GST-MK5 pull-down (Fig. 32A). Another experiment revealed that recombinant GST-MK5 can bind to in vitro expressed Mtbp, whereas also GST-MK2 and GST interacted with the Mtbp fragment (Fig. 32B), which indicated partial non-specific binding. By co-fractionation performed in the laboratory of Mark T. Boyd (Liverpool, United Kingdom), the interaction between MK5 and Mtbp could be confirmed. They could detect binding of MK5 from HEK293 lysates to a human hMTBP-immobilized matrix, by showing co-retention of hMTBP and MK5 from the column (Fig. 32C).

A B

C

Fig. 32. The Mdm2-binding protein (Mtbp) interacts with MK5 in vitro. A) In GST pull-down experiment using HEK293 lysates, myc-tagged Mtbp (Y2H fragment) and GST-tagged MK5 was co- expressed. As negative control myc-Mtbp was co-expressed with GST. B) The pGADT7-Mtbp prey vector was used for cell-free in vitro expression following the standard procedure of the TNT Quick Coupled Transcription/Translation System. GST pull-down was performed with Mtbp and either with recombinant GST, GST-MK2 or GST-MK5. C) HEK293 cells were transfected with MK5 and the lysates were applied to a sepharose column immobilized with recombinant hMTBP. Afterwards, hMTBP was eluted from the column and elution-fractions were analyzed regarding to hMTBP and MK5 by western blotting.

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A HA-tagged full-length Mtbp (plasmid provided by Mark T. Boyd) was used in co-localization studies. MK5 and Mtbp were localized in the nucleus and co-expression of both proteins did not alter the subcellular distribution (Fig. 33). Interestingly, Mtbp seems to inhibit the ERK3- induced nuclear export of MK5 (Fig.33, lower panel).

Fig. 33. Mtbp and MK5 are exclusively nuclear localized. HeLa cells were transiently transfected with plasmids containing HA-tagged Mtbp, GFP-tagged MK5, and dsRed-tagged ERK3, respectively. Immunostaining of Mtbp and MK5 with the corresponding tag-specific antibody was performed. For nuclear export of MK5 additional dsRed-tagged ERK3 was co-expressed to analyze changes in distribution of MK5 and Mtbp.

The reduced nuclear export of MK5 might result from competitive binding of ERK3 and Mtbp to MK5, even though MK5-Mtbp interaction is not clearly approved by this experiment.

4.3.5.4 Sodium/potassium transporting ATPase regulatory subunit beta-1

Interaction studies with the regulatory subunit of the sodium pump (Atp1b1) have recently been started, and binding of the Y2H fragment of Atp1b1 (204-304 aa) could be confirmed in HEK293 cell lysates. It is possible to enrich GFP-tagged Atp1b1 in pull-down using biotinylated MK5 (BE-MK5) and streptavidine-conjugated agarose beads (Fig. 34). Unfortunately, also GFP was partially co-purified in this experiment, whereas with lower extent. Additional expression of His-ERK3, which induces activation of BE-MK5, increased the binding of Atp1b1 to MK5. The identification of multiple Atp1b1 prey clones of distinct protein regions within two independent Y2H screens clearly supports the idea of a relevant interaction between MK5 and Atp1b1.

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Fig. 34. The sodium/potassium-transporting ATPase subunit beta-1 (Atp1b1) can be precipitated by MK5 in pull-down assay. BE-tagged MK5 and GFP-tagged Atp1b1 (Y2H fragment) was overexpressed in HEK293 cells. Biotinylated MK5 was purified using streptavidine-agarose beads. As negative control GFP was co-expressed with BE-MK5 and different plasmid concentrations of the MK5 plasmid were used in co-transfection together with GFP-Atp1b1. His-ERK3 was additionally co- transfected to investigate the role of MK5-activation in GFP-Atp1b1-binding.

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4.3.6 Screening for ERK3-interating proteins using the ras recruitment system

Since classical Gal4 promoter-based yeast two-hybrid screening methods with ERK3 failed, due to autoactivation of the Gal4 promoter, another screening approach was performed to indentify novel ERK3-interacting proteins. The ras recruitment system (RRS) developed by Ami Aronheim (Broder et al., 1998) was utilized. Because of high abundance of ERK3 in the brain, a brain-specific cDNA library, which was also provided by A. Aronheim, was preferred. In Tab. 3, the identified prey clones are reviewed. Tab. 11. Overview of positive clones obtained from yeast-based screen following the ras recruitment system (RRS). Thereby, 2.0 x 105 clones of a murine brain cDNA library were screened.

No Name Abbreviations Classification Reference

1 Ubiquitin-like protein 3 Ubl3, MUB membrane-anchored ubiquitin-fold protein Downes et al. 2006

2 Septin7 Sept7, Cdc10 filament-forming cytoskeletal GTPase Soulier and Vilotte, 1998

3 MAP kinase phosphatase 6 MKP6, Dusp14 dual-specific protein phosphatase Marti et al. 2001

4 Cop9 signalosome Cop9 multi-subunit protease Wei et al. 1998

5 Cdc25Mn Cdc25, Ras-GRF1 Ras-specific nucleotide exchange factor Martegani et al. 1992

ERK3 fusion protein with cytoplasmic mutant of Ras was generated as bait construct. In total, 2 x 105 prey clones, containing a membrane-localization sequence, were screened. Eight clones showed growth at 36 °C under galactose-inducing conditions and were selected as potential ERK3-interacting proteins (Tab. 11). The membrane bound nucleotide exchange factor Cdc25 could activate the yeast Ras independent of the bait protein and was regarded as possible false positive candidate. The cDNA inserts of Cdc25, Ubiquitin-like protein3, septin7, MAP kinase phosphatase 6, and Cop9 were subcloned into GFP- tag containing mammalian expression vectors, co-expressed with GST-ERK3 in HEK293 cells and Fig. 35. GST-ERK3 pull-down with GFP-tagged fragments of RRS-derived positive clones. The cDNA fragments of analyzed by GST pull-down isolated prey clones were subcloned into pEGFP vector and (performed by Stefanie Schumacher). overexpressed together with GST-ERK3 in HEK293 cells. After lysis GST pull-down was performed. GFP-tagged MK5 Only Cdc25, Ubl3, and Sept7 were was used as positive control and GFP as negative control in this assay. co-purified. As positive a control GFP-MK5 and as a negative control GFP were added. Compared to the non-specific background of GFP-binding to GST-ERK3, only Sept7 was sufficiently co-purified.

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4.3.7 The ERK3-interacting protein Septin7

To characterize the regions in septin7 (Sept7) and ERK3, which are responsible for their interaction, several deletion mutants of both proteins were generated (Fig. 36A and B). In case of Sept7, the C-terminal part carrying the variable coiled coil (CC) domain is necessary for binding of ERK3, since its deletion (SeptΔC) completely abolished interaction with BioEase(BE)-tagged ERK3 (Fig. 36C). The C-terminal extensions of ERK3 reduced (ERK3ΔC1 mutant) or almost completely abolished (ΔC2, ΔC3 mutants) interaction with BE-

Fig. 36. Pull-down studies with deletion mutants of Sept7 and ERK3, schematically represented in A and B, respectively, were performed to further characterize the interaction between both proteins. C) GST-tagged deletion mutants of Sept7, which lack either or both C- and N-terminus, were used in a GST pull-down of BE-tagged ERK3. Only Sept7 with the complete C-terminus can bind to ERK3. D) Pull-down of BE-tagged Sept7ΔN with GST-tagged C-terminal deletion mutants of ERK3. The region of ERK3 between amino acids 358-720 (ΔC2) is essential for effective interaction with Sept7ΔN. E) Pull-down of endogenous ERK3 by overexpression of GST-Sept7, but not by GST-Sept7ΔC, from lysates of HEK293 cells. F) Pull-down of endogenous Sept7 by GST-ERK3 from lysates of HEK293 cells.

81 Results tagged Sept7ΔN (full-length BE-tagged Sept7 is poorly expressed in HEK293 cells) (Fig. 36D). The binding region for MK5 in ERK3 has been localized to the FRIEDE-motif between amino acid 332-342 (Aberg et al., 2009). The ΔC2 mutant of ERK3 is still able to bind to MK5 (Seternes et al., 2004). Hence, Sept7 obviously binds to a more C-terminal part, of ERK3 (ΔC2: amino acids 358-720) and interaction of MK5 and Sept7 to neighboring but distinct regions in the C-terminus of ERK3 might occur. Depending on the level of expression and protein tags used, co-expression of interacting proteins may lead to false positive results. Hence, the interaction of the endogenous proteins with its tagged partner protein was analyzed by pull-down analysis. GST-Sept7, but not GST-Sept7ΔC, was able to pull-down endogenous ERK3 from lysates of HEK293 cells (Fig. 36E). Similarly, GST-ERK3 was also able to bind endogenous Sept7 from lysates of HEK293 cells (Fig. 36F). These results support the notion of an interaction of both proteins in cell lysates.

To prove the relevance of the Sept7- ERK3-interaction, the subcellular Fig. 37. ERK3 acts as a docking platform for MK5 and localization of both proteins were Sept7. A) HEK293 cells were transfected with myc-Sept7, BE-ERK3, or in combination to analyze their subcellular further analyzed. In HEK293 cells, myc- distribution. B) MK5 can bind to Sept7-bound ERK3 and re- localize to Sept7 filamentous structures (5 μm scale bar). tagged Sept7 was cytosolic localized in heterogeneous stick-like structures (Fig. 37A). BE-tagged ERK3 alone was diffusely distributed in the cytoplasm and nucleus, whereas together with myc-Sept7 nearly complete co-localization to the septin filament was observed (Fig. 37A). Furthermore, localization of HA-MK5 and myc-Sept7 in the absence and presence of ERK3 revealed that ERK3 acts as a docking platform for both MK5 and Sept7 in parallel (Fig. 37B). When co- expressed with myc-Sept7 alone, HA-MK5 displayed exclusive nuclear localization, distinct from the mainly cytoplasmic structures of myc-Sept7 (Fig. 37B). Interestingly, co-expression

82 Results of BE-ERK3 induces co-localization of myc-Sept7, indicating that MK5 and Sept7 can simultaneously bind to ERK3. This could be a mechanism how ERK3 is targeting MK5 to Sept7-containing filamentous cytoplasmic structures. Analyzing the direct interaction of all three proteins in a ternary complex, GST- MK5 pull-down of GFP-Sept7 in the absence and presence of His-ERK3 was performed by Stefanie Schumacher (Fig. 38). As a positive control, GFP-ERK3 was added, which was efficiently enriched in the GST-MK5 pull-down fraction. Alone, GFP-Sept7 did not bind to GST-MK5 and was only co-purified in the pull-down in the presence of BE-ERK3. This experiment

Fig. 38. MK5 interaction with Sept7 is bridged by supports the notion that ERK3 acts as an ERK3-binding. GST pull-down assay with GST-MK5 was performed either with GFP-Sept7, GFP-ERK3, or adaptor or a docking platform for Sept7 GFP-Sept7 + BE-ERK3. and MK5. Another experiment focused on endogenous Sept7 structures in dKO and dRes MEF cells. Compared to ectopically expressed Sept7, the endogenous Sept7 was not exclusively found in filamentous structures. Especially in ERK3/MK5 dKO cells Sept7 was localized to granular complexes in the cytosol, whereas the rescue of ERK3/MK5-proteins resulted in re- distribution of endogenous Sept7 to a more filamentous structure (Fig. 39A). Septin filaments were partially strongly connected to the actin filaments, especially at intercellular contact points, which were more frequently observed in ERK3/MK5 dRes MEFs than in dKO cells. Because of the high cell-to-cell diversity of Sept7 structures, the quantification of Sept7 organization was not possible. To get a general view of Sept7 organization, the solubility of Sept7-containing structures in dKO and dRes cells was analyzed. Two buffers systems were utilized for this issue. On one hand, a permeabilization buffer in combination with low speed centrifugation and, on the other hand, harsher detergent lysis combined with ultracentrifugation was used to clearly separate granular soluble fractions from higher molecular filamentous Sept7 structures. Comparing dKO with rescued cells, a minor increase of insoluble Sept7, pelleted by ultracentrifugation, could be observed in dRes MEFs lysates (Fig. 39B).

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A

B

Fig. 39. Impaired cellular septin organization due to knockout of ERK3 and MK5 in MEF cells. A) Cells were seeded on glass cover slips, fixed with 4% PFA, and stained with polyclonal anti-Sept7 antibody. F-actin staining was performed using Alexa647-conjugated phalloidin. B) The solubility of Sept7-containing structures was analyzed using a permeabilization buffer and low speed centrifugation or a detergent lysis followed by ultracentrifugation to separate high molecular filamentous from soluble granular Sept7 structures.

To analyze the role of Sept7 in cells knockdown studies have been performed using siRNA for the human Sept7 transiently transfected in HeLa cells. Scrambled siRNA was used as a control. The knockdown of Sept7 could be confirmed by western blotting and immunofluorescence (Fig. 40A and B). Microscopic analysis revealed morphological differences, which were also detected after scrambled siRNA transfection. Since it has been reported that Sept7 can influence cytokinesis (Kremer et al., 2007), growth of HeLa cells depending on Sept7 knockdown was analyzed. A reduction in growth could be detected

84 Results within the first three days after transfection with Sept7 siRNA (Fig. 39C). The same effect could be observed after transfection with scrambled siRNA, even though the growth inhibition was not as high. Microscopic observation of transfected cells revealed that the lower values detected in WST-1 assay were caused by an increased cell death.

A B

C

Fig. 40. Effects of Sept7 gene silencing with siRNA in HeLa cells. A) Immunofluorescent staining of Sept7 in HeLa cells, 72 h post transfection with siRNAs, showed strong reduction of the fluorescence signal using confocal microscope. B) Western blotting of HeLa cell lysates from day 1 till day 5 after transfection with siRNA using anti-Sept7 and anti-GAPDH antibodies. C) Differences in growth of untransfected, scrambled siRNA transfected or Sept7 siRNA transfected HeLa cells. Cell viability was measured in triplicates using the WST-1 standard procedure.

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4.4 The neuronal function of the ERK3/MK5-signaling module

4.4.1 Regulation of septin organization via Borg proteins

Using in vitro kinase assay with recombinant Sept7ΔN and Sept7ΔC, no phosphorylation by recombinant MK5 or ERK3 could be detected (Fig. 41). Truncated mutants of Sept7 were used, because recombinant full-length Sept7 could not be purified from E. coli. The in vitro activation of MK5 and ERK3 was approved by investigation of their known substrate phosphorylation of the small heat shock protein beta-1 (Hsp1b; also known as Hsp25) and myelin basic protein (MBP), respectively.

Fig. 41. Sept7 is not phosphorylated by ERK3 or MK5. Since expression of full-length Sept7 in E. coli was not successful, recombinant protein of His-tagged N- and C-terminal deletion mutants of Sept7 were expressed and applied to an in vitro kinase assay. As positive controls for kinase activity of ERK3 and MK5 their known substrates myelin basic protein (MBP) and small heat shock protein beta-1 (Hspb1, also known as Hsp25) were used, respectively.

Thus, Sept7 is not phosphorylated by MK5 or ERK3, there might be other substrates of these kinases, which can control septin filament organization. A know septin2/6/7-specific regulator of filament assembly is Borg3, which similarly to Borg1 and 2 and binds to the septin GTPase domain (Joberty et al., 2001, Sheffield et al., 2003). Most probably, ERK3 and MK5 can regulate septin structures via phosphorylation of Borg proteins. Recombinant GST-tagged Borg1-3 were expressed in E. coli and used as substrates for ERK3 and MK5 in an in vitro kinase assay (Fig. 42). Recombinant baculovirus-expressed 6xHis-ERK3 possesses intrinsic activity and can efficiently phosphorylate Borg1, Borg2 and to a lesser extend also Borg3. As a positive control for the ERK3 kinase activity, MBP was employed. Using p38-activated GST-MK5 in the kinase assay, Borg2 could be phosphorylated by MK5 in vitro with

86 Results comparable efficiency to that of its in vitro substrate Hsp25. Borg3 is also phosphorylated by MK5 to a somewhat lower extent. The degree of isoform-specific Borg protein phosphorylation also correlated with the number of putative phosphorylation sites for ERK3 (xSPx; Borg1: 7x, Borg2: 3x, Borg3: 1x) and MK5 ([FVIL]xRxxS; Borg1: 0x, Borg2: 1x, Borg3: 0x), respectively. Phosphorylation of Borg3 by MK5 might occur at a threonine-residue.

Fig. 42 In vitro phosphorylation of Borg1, 2, and 3 by ERK3 and MK5. Recombinant GST-tagged Borg1, Borg2, and Borg3 were expressed in E. coli and used for in vitro kinase assay with Sf9- expressed, active His-ERK3 or with E. coli expressed, p38-activated GST-MK5. Incorporated phosphate from γ-33P-ATP was detected by phospho-imaging. As positive control the known ERK3 substrate myelin basic protein (MBP) and the known MK5 substrate heat shock protein beta-1 (Hspb1, also known as Hsp25) were used.

4.4.2 Overexpression-dependent phenotype in ex vivo primary neurons

Kal7 and Sept7 are both involved in dendritic spine formation. Sept7 was found in postsynaptic density fractions of the brain (Peng et al., 2004) and is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons. Interestingly, overexpression of Sept7 increased dendrite branching to a certain degree, and, more importantly, knockdown of Sept7 led to a greater proportion of immature dendritic protrusions and impaired dendritic branching (Tada et al., 2007, Xie et al., 2007a). Kal7 also stimulates spine formation and maturation (Penzes et al., 2003). It is regulated via phosphorylation by CaMKII, a protein kinase of the same family as MK5, and as a result of phosphorylation-dependent translocation from dendrite shafts into nearby spines, it induces phosphorylation of the Rac-target PAK in spines (Xie et al., 2007a). PAKs have recently identified as activators of the ERK3/MK5-module (De la Mota-Peynado et al., 2011, Deleris

87 Results et al., 2011). Taken together, these results strongly suggest a complex role of the ERK3/MK5-signaling module in regulation of dendritic spine formation. For direct analysis of the effect of the ERK3/MK5-signaling module on Sept7-dependent neuronal morphogenesis co-overexpression studies in mouse hippocampal neurons were performed. Neurons were isolated from embryonic day 18 BALB/c mice (E18.5) and cultivated on coated glass coverslips. After 5 days of in vitro culture (5DIV) cells were transfected with pcDNA6/BioEase-ERK3, pcDNA3.1-HA-MK5, or pRSK5-myc-Sept7 together with pEGFP to enable selection of transfected neurons. Neuronal morphology of transfected cells were analyzed three days post transfection (5+3DIV) in regard to dendrite number and branching. Neuronal cultures were fixed with 4% PFA and GFP-positive neurons were visualized using confocal microscopy. The overall complexity of statistically relevant, selected neurons transfected with pEGFP alone (CTRL) or with myc-Sept7/pEGFP without (Sept7) or together with BE-ERK3 and HA-MK5 (ERK3/MK5/Sept7) was analyzed and is shown in Fig. 43A. While Sept7 alone does not significantly increase neuronal complexity, co-expression of ERK3/MK5 leads to obviously more complex neurons possessing more dendrites with apparently more branches. The dendritic complexity was subjected to Sholl analysis. Sholl analysis counts the number of intersections made by dendrites with concentric circles of increasing distance from the cell body, reflecting both dendrite number and intersections and represents a very sensitive and objective measurement of complex branching. Performing Sholl analysis on distinct types of transfected neurons revealed that expression of Sept7, MK5, ERK3 or ERK3/MK5 together with the EGFP construct does not lead to an increase in the Sholl index (Fig. 43B). However, MK5 and ERK3 lead to a highly significant increase of counted intersections of the transfected neurons in all distances from the soma when co-expressed with Sept7. Since Sept7 has also been described to alter dendritic spine morphology (Xie et al., 2007a), spine number in the differently transfected neurons were qualitatively analyzed. The number of spines displays a significant increase only in ERK3/MK5/Sept7 expressing dendrites (Fig. 43C). Determination of spine morphology at later stages of neuronal differentiation was also performed by transfecting primary neurons at DIV10 and inspecting morphology at DIV15 (Fig. 43D). In cells, overexpressing Sept7 alone, dendritic complexity and spine formation is rather low and did not differ significantly from control cells. Again, overexpression of Sept7 together with ERK3 and MK5 (ERK3/MK5/Sept7) leads to a dramatically increased dendritic complexity. In addition, spine formation is significantly increased in number and size in these cells (inserts in Fig. 43D).

88 Results

A

B C

D

Fig. 43. Sept7-dependent stimulation of dendritic outgrowth and increased spine number by overexpression of the ERK3/MK5-complex in primary hippocampal neurons. Primary neurons were isolated at embryonic day 18. Transfection was performed with BE-tagged ERK3, HA-tagged MK5, myc-tagged Sept7 or their combinations together with pEGFP at DIV5 (A-C) or DIV10 (D). 72 (A- C) or 120 (D) hours post transfection cells were fixed and analyzed using confocal microscopy. A) Representative images of statistical relevant neurons overexpressing GFP (CTRL), Sept7 or ERK3, MK5 and Sept7 together (scale bar 50 µm). B) Sholl analysis of neurons differently transfected with the constructs coding for the indicated proteins and EGFP. C) Qualitative analysis of dendritic spine morphology from the experiment in B). D) Representative images of primary neurons expressing EGFP and Sept7 or ERK3/MK5/Sept7, respectively (scale bar 50 µm). Inserts display spine morphology (at DIV15). 89 Results

4.4.3 In vivo spinal phenotype in hippocampal neurons of MK5-deficient mice

To elucidate the role of the ERK3/MK5-module in spine formation in vivo, hippocampal sections of brains of adult MK5-deficient and WT animals were visualized by Golgi stain, a vital staining, which decorates a limited number of neurons at random in their entirety of soma and dendrites (Heimrich and Frotscher, 1991). Spine number was determined by confocal microscopy and image analysis (experiments were performed in the laboratory of Prof. Dr. Britsch, Ulm University, Germany). Typical stains for WT and MK5 knockout (Shi et al., 2003) brains are shown in Fig. 44A. Image analysis reveals a significant lower number of dendritic spines in this region in the MK5-deficient animals (Fig. 44B). For an independent verification of these results, MK5-deficient mice were crossed with Thy1- YFP transgenic mice that express YFP at high levels in motor and sensory neurons, and subsets of central neurons (Feng et al., 2000). In these mice, pyramidal neurons are selectively labeled in the hippocampus and, hence, this line provides a strong and specific "Golgi-like" vital marker for this neuronal subset, with minimal labeling of nearby axons. Typical YFP stained dendrites of these mice (MK5) compared with “wild type” mice carrying only the YFP transgene (WT) are shown in Fig. 44C. Image analysis demonstrated a significance of these differences (Fig. 44D). These results are in line with the Golgi stain and confirm a role of the ERK3/MK5-module for spine formation in vivo.

A B

C D

Fig. 44. In vivo determination of spine numbers for MK5-deficiency in mice. A) To visualize hippocampal neurons Golgi stain was performed with 150 µm vibratom sections of three WT and MK5-/- mice each. Representative images are illustrated. B) Statistical analysis of spine number from three animals of each genotype. Number of dendrites analyzed n=78 (WT) and n=95 (MK5 knockout). C) Analysis of denditic spines from “WT” and MK5-/- animals expressing neuron-specific Thy1-YFP reporter protein. D) Statistical analysis of spine number from three animals of each genotype. Number of dendrites analyzed n=131 (WT) and n=162 (MK5 knockout).

90 Discussion

5 Discussion

5.1 Functional aspects of ERK3/MK5-mediated signaling in MEF cells

5.1.1 The implication in cell cycle progression

The performed experiments demonstrated that targeted deletion of MK5 and ERK3 gene compromised growth rates of isolated mouse embryonic fibroblasts (Fig. 10A). The growth delay could also be observed in SV40-derived large T antigen immortalized MEFs (Fig. 10B). These results indicate a functional role of ERK3 and/or MK5 in the regulation of cell growth and proliferation. Interestingly, protein expression of both kinases was enhanced in highly proliferative tumor-derived cell lines, whereas the protein levels in fully differentiated cells of adult mouse tissues were close to the detection limit (Fig. 8A/B). On the other hand, ERK3 mRNA has been detected in almost all adult mouse tissue except for heart, liver, and spleen (Turgeon et al., 2000), whereas analysis of ERK3 protein showed detectable amounts in heart and thymus (Fig. 8B), which might result from distinct tissue-specific degradation levels of ERK3. This supports the model of ERK3 functional regulation on protein level (Coulombe et al., 2003, Coulombe et al., 2004).

Cell cycle analysis demonstrated that ERK3 is enriched in G1 phase (Coulombe et al., 2003), thus, stable, ectopically expressed ERK3 seems to block S phase entry in NIH 3T3 fibroblasts (Coulombe et al., 2003). ERK3 has been shown to be enriched during differentiation of PC-12 and C2C12 cells (Coulombe et al., 2003), whereas treatment of PC- 12 cells with NGF for 48 h did not influence ERK3 protein levels (Fig. 13B). The mechanism of S-phase blockade was not identified. There is evidence that nucleocytoplasmic shuttling is necessary for ERK3’s ability to block S-phase entry (Julien et al., 2003). Interestingly, by analysis of ERK3 phosphorylation, it has been demonstrated that ERK3 is phosphorylated during pro-metaphase of mitosis at multiple sites within the C-terminus (Ser684, Ser688, Thr698 and Ser705) (Tanguay et al., 2010). Phosphorylation was abolished at the transition to G1 phase (Tanguay et al., 2010). Stimulation with nocodazole, which inhibits the formation of the spindle apparatus and retains cells in mitosis, leads to stabilization of the C-terminal phosphorylation (Tanguay et al., 2010). Stably transfected PC-12 cells treated with nocodazole showed higher levels of GFP-ERK3 (Fig.17). ERK3 phosphorylation seems to be dual-regulated via Cdk1 and Cdc14A/B (Hansen et al., 2008, Tanguay et al., 2010). Unfortunately, the described phospho-shift could not be reproduced with our cell lines. The biological function of this C-terminal phosphorylation remains elusive. It has not been shown, whether ERK3 activity is influenced by these phosphorylations. ERK3 might have different

91 Discussion functions during cell cycle progression. On the one hand, it can act as a factor promoting G1 arrest and, on the other hand, it is involved in metaphase-anaphase transition during mitosis. A regulatory function of ERK3 during mitosis has been shown in HT-29 colon cancer cells using a lentiviral-based shRNA genetic screen (Moffat et al., 2006). In this high-throughput screen, ERK3 knockdown resulted in a significant increase in the mitotic index. Within another interesting study, it has been established that ERK3 is essential for spindle stability and for metaphase-anaphase transition in mouse oocyte meiosis (Li et al., 2010). This model can bring substantial benefits for ongoing studies on ERK3-related mechanisms in cell cycle progression. Probably, a septin-dependent regulation of ERK3 by interaction with Sept7 might have functional relevance. In yeast, contribution of septin filament in cell cycle control has already been demonstrated (Barral et al., 1999). Overall, there is certain evidence that ERK3 is involved in cell cycle control, whereas underlying mechanisms remain unknown.

An implication of MK5 in cell cycle progression has not been described, whereas regulatory function in cell cycle control could be shown for its nearest homolog MK2. Under stress stimuli, MK2 phosphorylates Cdc25B/C phosphatases, which are involved in triggering mitosis entry by dephosphorylation of cyclinB-bound Cdc2 (Manke et al., 2005). In MEF cells, single deletion of the MK5 gene leads to reduced cell growth (Fig. 10) MK2 can regulate p53- mediated cell cycle arrest via phosphorylation of HDM2 (human MDM2 homolog) (Weber et al., 2005). Interestingly, a previous study on Ras-induced tumor senescence suggests direct phosphorylation of p53 at Ser37 by MK5, which promotes p53 activity and subsequent cell cycle arrest (Sun et al., 2007). The proline-directed site at Ser37 is more common for MAPK- and CDK-mediated phosphorylation, whereas the role of p38, responsible for MK5-activation, or ERK3 was not examined in this study. In contrast, it was possible to show interaction of MK5 with the Mdm2-binding protein (Mtbp), which could be another possible pathway of p53 regulation by MK5 (section 4.3.6.3). The Mtbp-binding to the E3 ligase Mdm2 induces its stabilization and the continuous ubiquitination and degradation of p53. MK5 could serve as a molecular switch, which could enhance p53-mediated signaling by direct phosphorylation and probably disruption of negative feedback loops via interaction with Mtbp.

5.1.2 The impact on cell migration by alteration of the actin filament

The main MK substrate, involved in the regulation of cell migration by controlling the organization of the actin filament, is the heat shock protein beta-1 (Hspb1, also known as Hsp25). The Hspb1 can form complex aggregates, which serve as chaperones, for instance,

92 Discussion after heat shock (Lambert et al., 1999). MK2 is able to phosphorylate Hspb1 in response to p38-activation, which prevents Hspb1-complex formation. MK2 serves as the main Hspb1 phosphorylating kinase upon stress. MK5 is able to phosphorylate Hspb1 to a certain extent in vitro (Fig. 9), whereas in vivo relevance is arguable, since it has been shown, that stress- induced phosphorylation of Hspb1 is entirely abolished in MK2/3 knockout MEFs.

Differences in actin filament structures have been observed in MEF cells lacking ERK3 and MK5 (Fig. 12). The dKO cells were characterized by less organized F-actin structures compared to dRes cells. Staining of endogenous Sept7 in dKO cells also revealed altered septin filaments (Fig. 39). Septin structures are cooperating with the actin filament (Weirich et al., 2008). Septin filaments are usually found at the inner plasmamembrane. In dKO MEFs, enrichment of actin and septin structures at cell-to-cell contact points was considerably reduced (Fig. 39). There is evidence that MK5 can phosphorylate the focal adhesion kinase (FAK), which is involved in cellular adhesion and spreading processes (Yoshizuka and colleagues, unpublished). In breast cancer cells, it has been reported that the lack of FAK caused impaired tumor invasion by reducing cell mobility (Chan et al., 2009). The loss of cell- cell contact and cell communication could probably influence cell growth and motility.

Less is known about effects of ERK3 kinase activity on actin filament organization, whereas in several publications describe a PKA-dependent F-actin regulation by MK5 (Gerits et al., 2007a, Kostenko et al., 2011b). It has been assumed that MK2 and MK5 are not cooperating downstream to p38 in stress-induced Hspb1 phosphorylation, since MK5 is more likely to be activated by PKA-mediated S115 phosphorylation in a p38-independent manner. However, stimulation experiments with forskolin using HEK293 cells did not influence activity or T182- phosphorylation of ectopically expressed GST-MK5.

Recent data suggested activation of ERK3 downstream to Cdc42 GTPases, which are global signaling molecules involved in the organization of cytoskeletal structures (De la Mota- Peynado et al., 2011, Deleris et al., 2011). Cdc42 belongs to the family of Rho GTPases, which are cellular signal amplifier downstream of a variety of activated receptors. The ERK3/MK5-signaling module might serve as additional pathway in mammalian cells, which connects septin structures with the actin filament. Since ERK3/MK5-deletion do not lead to complete disruption of the F-actin, it is more likely that further signaling pathways are essential in the regulation of the actin filament organization.

93 Discussion

5.2 ERK3 acting as sensor for environmental changes

5.2.1 ERK3 protein levels are sensitive to extracellular pH changes

The cellular protein level of ERK3 is known to be regulated by the 26S proteasome. It has been demonstrated that ERK3 is degraded after ubiquitination of its free N-terminal amino group, resulting in an estimated half life of about 30 min (Coulombe et al., 2003, Coulombe et al., 2004). Only ERK7 has also been shown to be regulated by proteasomal degradation (Kuo et al., 2004), while other MAPKs are stable proteins, whose functions are controlled by phosphorylation and subcellular localization (English et al., 1999). Interestingly, ERK3 and ERK7 share a long C-terminal extension, which particular function is not fully understood. Deletion of C-terminal parts leads to changes in their subcellular localization (Abe et al., 1999, Bind et al., 2004). In proliferating cells, ERK3 is constitutively degraded, independent of the activation-loop phosphorylation or kinase activity. Whereas, it has been shown that the protein levels are upregulated during differentiation of PC-12 and C2C12 cells (Coulombe et al., 2003).

A strong accumulation of ERK3 could be observed in PC-12 cells after three hours of NGF- stimulation (Fig. 13A). During the first hour of NGF-treatment, ERK3 protein completely disappeared, whereas, after three hours, the protein level was rescued. Another experiment revealed that this temporal loss of ERK3 is independently to NGF-stimulation (Fig. 13B). It could be shown that alkalinization of DMEM, induced by the absence of 5% CO2, was strongly affecting stability of ERK3 (Fig. 14B). Acidification of the medium (pH 6.4) leads to increased ERK3 levels, whereas alkaline-induced fast degradation occurred via proteasomal degradation (Fig. 14A). By the use of pH-stable medium and known proteasome inhibitors, MG132 and bortezomib, ERK3 protein levels stayed the same (Fig. 14A/B). Analysis of the MEF cells, generated in our research group, revealed that pH-dependent regulation of ERK3 is not cell-type specific for PC-12 cells (Fig.15). Ectopically expressed GFP-ERK3 is fast degraded due to alkaline pH, whereas the GFP-signal was not completely abolished (Fig. 16B/C). Appearance of several GFP-specific or ERK3-specific fragments could be observed by western blotting of the stimulated stably transfected cells. This might indicate that upon degradation of GFP-ERK3 specific fragments with altered stability emerged. It has been reported by Bind & colleagues that GFP-ERK3 can be cleaved within the C-terminus by an unknown caspase, which induced nuclear localization of truncated GFP-ERK3 fragments in HeLa cells. Thus, this cleavage of GFP-ERK3 occurred independently of the proteasome. Two possible caspase-like cleavage sites, located at amino acids 388–398 (DVTDEEEVQVD) and amino acids 543–546 (DVVD) have been identified. Deletion of these

94 Discussion acidic caspase-like binding regions result in loss of specific GFP-ERK3 fragments in western blot and diminishing nuclear localization of GFP-ERK3 signal (Bind et al., 2004). In regard to pH-dependent regulation of ERK3, these charged acidic motifs might undergo conformational changes, due to altered pH, which can influence (GFP-) ERK3 stability.

The molecular structure and the resulting protein function are dependent on the pH of the surrounding milieu and protonation-deprotonation events regulating biological properties by changing surface charge of biomolecules (Casey et al., 2010). An accumulation of GFP- ERK3 at the Golgi (Bind et al., 2004) might be a result of the more acidic pH (6-6.7) milieu in this cellular compartment. Within the nucleus protein stability is reduced by the slightly alkaline pH condition (7.2) (Casey et al., 2010). It has been further shown that extracellular acidic pH can stabilize GFP-ERK3 protein in PC-12 cells leading to significantly increased GFP-signal after 4 hours, whereas alkaline-induced degradation of GFP-ERK3 has not been detected for GFP or an instable d2GFP (Fig. 16 B/C).

5.2.2 Possible mechanisms of the pH-dependent ERK3 degradation

The intracellular pH is mostly acidic and differs within the distinct subcellular compartments. Anion and cation channels, present on the plasmamembrane and cytosolic vesicles, are pivotal regulators of the intracellular pH. Metabolic processes, such as generation of ATP by glycolysis and oxidative phosphorylation, lead to constitutive intracellular acidification, which must be compensated by other cellular mechanisms. Activity of Na+-H+ exchangers (NHEs) leads to an increase of intracellular pH (Casey et al., 2010). For instance, it has been demonstrated that p38α is able to phosphorylate and activate NHE1, which causes increased intracellular pH in the murine pro-B-cell line FL5.12A (Khaled et al., 2001).

Interestingly, another type of ion exchangers has been identified within a MK5 yeast two- hybrid screen (Fig. 25; Tab. 10). Several C-terminal fragments of the regulatory subunit beta- 1 (Atp1b1) of Na+-K+ transporting ATPase have been identified as MK5 positive prey clones and interactions have been confirmed by pull-down assay of ectopically expressed proteins in HEK293 cells (Fig. 34). Transmembraneous Na+-K+ ATPases consist of an active and a regulatory subunit. For each subunit, four isoforms are known, which vary in their tissue- specific protein expression (Skou, 2004, Pestov et al., 2011). No putative MK5 phosphorylation motif could be found within Atp1b1 amino acid sequence, whereas the four distinct active subunits display a high number of proline-directed phosphorylation sites.

95 Discussion

Especially, the Atp1a2 contains six SP-sites. These sites are preferentially phosphorylated by many kinases including MAPKs such as ERK3. Newborn mice, deficient in Atp1a2 gene, have less dilated lungs (Moseley et al., 2003, Ikeda et al., 2004) as it has been demonstrated for ERK3 knockout mice (Klinger et al., 2009). The Atp1a2-/- phenotype could be explained by disruption of neuronal activity in these mice. Probably, the ERK3/MK5-signaling module might cooperate in regulation of neuronal Na+-K+ ATPases activity. Emerging electrostatic changes at the plasmamembrane, which also occur by pH changes, might interfere with ERK3 protein stability and/or activity. The pH-dependent regulation of ERK3 could play an important role in the ATPase-activation process. In contrast, inhibition of Na+-K+ ATPases by ouabain could not prevent alkaline-induced degradation of ERK3 in PC-12 cells stably transfected with GFP-ERK3, whereas inhibition of ATP synthesis by antimycin A lead to stable GFP-ERK3 protein levels (Fig. 18).

Furthermore, inhibition of alkaline-induced degradation of GFP-ERK3 could be detected upon co-stimulation with cobalt chloride (Fig. 17, Fig. 18), which is commonly used as a hypoxia-mimicking agent. Cobalt chloride (CoCl2) has been shown to induce expression of the alpha subunit of hypoxia-inducible factor 1 (HIF1α) (Goldberg et al., 1988, Huang et al.,

2003). The CoCl2 is also known to activate conventional MAPKs pathways in PC-12 cells

(Lan et al., 2011). A possible activation of ERK3 in response to CoCl2 might stabilize the protein, since interaction of ERK3 with MK5 also stabilizes ERK3 protein levels (Fig. 15).

Both, phosphorylation and dephosphorylation events seem to be necessary in regulation of cellular ERK3 protein levels. By the use of several protein kinase or phosphatase inhibitors, it has been demonstrated that GFP-ERK3 protein degradation can be completely blocked by treatment with staurosporine, okadaic acid, and ortho-vanadate (Fig. 18, Fig. 19, and, Fig. 22). These substances broadly inhibit serine/threonine protein kinases, serine/threonine protein phosphatases and protein tyrosine phosphatases, respectively. Interestingly, vanadate has also been described as a potent inhibitor of Na+-K+ ATPase (Cantley et al., 1977). The mode of ERK3 stabilization upon treatment with okadaic acid might not caused by serine/threonine protein phosphatase inhibition, since no effect could be shown in response to other comparable compounds, such as microcystine-LR and calyculin A, targeting Ser/Thr protein phosphatases (Fig. 19). In this regard, an off-target effect of okadaic acid seems to be more likely. Overall, there is profound evidence that phosphorylation and protein stability of ERK3 are strongly connected.

96 Discussion

5.3 Interacting partners of the ERK3/MK5-signaling module

5.3.1 In vitro activation of the ERK3/MK5-signaling module

Hsp25 phosphorylation by GST-tagged MK5 can be increased by co-incubation with His- tagged ERK3 or GST-tagged p38α in an in vitro kinase assay experiment (Fig. 9, Fig. 41). Recombinant GST-tagged p38α strongly induced MK5-activity, whereas Hsp25 phosphorylation in the presence of Sf9-expressed ERK3 was not as high as with p38α- activated MK5. So far, it is not clear whether ERK3 activation loop phosphorylation at S189 is necessary to promote MK5-activation (Perander et al., 2008b). MK5-activation upon interaction with ERK3 might occur in response to ERK3-activation and S189-phosphorylation by an additional kinase (Deleris et al., 2008). MK5 interaction with inactivated ERK3 kinase has been shown to be more transient. Activation loop phosphorylation at S189 increases the binding affinity and promotes T182 phosphorylation of MK5 accompanied by phosphorylation of several other ERK3 C-terminal sites (Deleris et al., 2008).

A kinase screen of one hundred distinct serine/threonine protein kinases was performed to identify the possible protein ‘kinase X’, which might phosphorylate ERK3 at S189 and might increase ERK3-dependent induction of MK5-activity. Analysis of MK5-dependent Z’-Lyte substrate phosphorylation revealed a significant increase of ERK3-induced MK5 activity in case of co-stimulation with p21-activated kinase 1/3 (PAK1/3), microtubule affinity-regulated kinase 1/3 (MARK1/3), PDZ-binding kinase (PBK), and testis-specific kinase 2 (TSK2) (Fig. 21). Control experiments could show direct phosphorylation of the MK-specific substrate peptide by PAKs and TSK2. MARK1/3 and PBK seem to have an effect on MK5-dependent substrate phosphorylation independent of ERK3. Unfortunately, significant increase of MK5 substrate phosphorylation, in the presence of ERK3 together with one of the positive kinase, was not observed (Fig. 21).

Recently, p21-activated kinases have been reconsidered as ERK3 activation loop phosphorylating enzymes (De la Mota-Peynado et al., 2011, Deleris et al., 2011). Deleris et al. identified PAKs as ERK3 kinases using mass spectrometric analysis of proteins, which have been co-purified with His-ERK3 (1-365 aa) kinase dead mutant. Alternatively, ERK3 has been described as PAK substrate using peptide microarray approach (De la Mota- Peynado et al., 2011). Induction of ERK3 phosphorylation in response to overexpression of active Rac1 could be disrupted by RNA silencing of PAK1, 2, and 3 (Deleris et al., 2011). Activation of ERK3 by PAKs results in increased phosphorylation of Hsp27 by MK5, showing that activity of MK5, upon interaction with S189-phosphorylated ERK3, is increased. In vitro kinase assay with recombinant PAKs showed phosphorylation of ERK3 by group I PAKs

97 Discussion

(isoform 1-3) (Fig. 22). Interestingly, inhibition of PAKs by the inhibitory peptide PAK18 did not influence the alkaline-induced degradation of GFP-ERK3 stably transfected in PC-12 cells (Fig. 22).

The family of microtubule affinity-regulated kinases (MARKs) is a known regulator of tau proteins. The multiple phosphorylations of tau promote neuron-specific assembly of the microtubule filaments, which is necessary for neuronal differentiation, dendrite growth, and spine formation (Chen et al., 2006, Thies and Mandelkow, 2007). Since activation of ERK3 by MARK could not be shown, functional cooperation in neuronal development should be considered.

The PDZ-binding kinase (PBK; also known as T-LAK cell-originated protein kinase, TOPK) has been shown to act as MAPKK, which can activate p38 in proliferating tumor cells in response to insulin-like growth factor stimulation (Ayllon and O'Connor, 2007). Further studies revealed functional participation in PI3K-PTEN-AKT pathway regulating cell migration of lung cancer cells (Shih et al., 2011) PDZ-interaction with p53 leads to altered transcriptional activity (Hu et al., 2010). The testis-specific kinase 2 is conserved in mammals, whereas no relevant physiological role has been described, so far. Functional correlation to ERK3 could not be found.

5.3.2 The absence of ERK3 and MK5 lead to changes in the phosphoproteome

Changes in the phosphoproteome have been analyzed in MK5-deficient adult tissues (1) and immortalized MEF cells lacking ERK3 and MK5 (2) using kinase-specific substrate antibodies for the phospho-motif of PKA (1, 2), PKB (2), PKC (1, 2), PKD (2), CDK (1, 2), ATM/ATR (1, 2), and MAPK (2). Evidence of substrate phosphorylation by MK5 can be given by analysis of MK5-related basophilic motifs of the AGC family kinases PKA, PKB, PKC, and PKD. The LXRXXS/T-motif of PKD is similar to MK5 phosphorylation site. The proline-directed phosphorylation-motifs of CDKs and MAPKs are indicators for potential ERK3 phospho-sites. Changes in the phosphoprotein pattern might suggest participation of ERK3 and MK5.

In tissues, differences in the obtained phosphoprotein pattern could be observed in brain, heart, spleen, and thymus (Tab. 9, Fig. 23). A promising phospho-band was obtained after staining with CDK substrate phospho-motif antibody. The identified protein migrated with a molecular mass of 25 kDa and phosphorylation was lost in MK5-deficient lysates of the brain and thymus (Fig. 23). Probably, direct phosphorylation by ERK3, which has been shown to be reduced in MK5 knockout tissue (Schumacher et al., 2004), caused this decrease in

98 Discussion phosphorylation of this 25 kDa protein. ERK3 protein expression analysis revealed detectable amounts in brain (Fig. 8A), heart and thymus (Fig. 8B), which supports the idea of specific functions in these tissues. Mapping of the found phosphoprotein is not possible, because of the high number of potential CDK-substrates. Another possible substrate, migrating at a molecular weight of 22 kDa, has been recognized in the heart using the PKC motif antibody. Phosphorylation was not present in MK5-deficient heart, suggesting a potential substrate of MK5 or at least MK5/(ERK3)-dependent regulation of PKC-mediated substrate phosphorylation. It has already been described that the ERK3/MK5-signaling module might have functions in the hypertrophic heart (Dingar et al., 2010).

General induction of phosphorylation events by cell-stimulation with the PKC-activating agent PMA and tyrosine protein phosphatase inhibition by pervanadate increased levels of phosphoproteins detectable in MEF lysates. Comparing dKO MEFs and dRes cells, differences in the phosphoprotein pattern have been observed using phospho-motif PKA, PKB, PKC, and PKD antibodies. Stimulation-dependent phosphorylation of substrates, migrating with a molecular mass of 80 kDa (PKA), 160 kDa (PKB), 35 kDa (PKC), and 50 kDa (PKD) have been recognized in stimulated ERK3/MK5-rescued MEFs (Fig. 24). Since these motifs are basophilic and comparable to that of MK5, direct MK5-dependent phosphorylation could be considered, but further analysis is required to prove the relevance and identity of the appearing phosphoproteins.

5.3.3 Evaluation of the biological relevance of the novel MK5-interacting proteins

Septin-8

Interaction of MK5 with the GTP-binding protein septin-8 (Sept8) was most interesting, since several prey clones containing different parts of Sept8 cDNA have been identified within two independently performed yeast two-hybrid screens using a brain-specific mouse cDNA library (Fig. 25, Tab. 10). Binding of MK5 to Sept8 could be confirmed by pull-down and co- localization studies (Fig. 28). Interaction of Sept8 with MK5 might be involved in general regulation of the septin filaments. The septins have been identified as temperature-sensitive mutants involved in cytokinesis (Hartwell, 1971). The name arose from the Latin word septum. For instance, a septum is generated between two daughter cells at the end of the process of division in living cells. Septin filaments are also present as structural diffusion barrier responsible for septation of fungal hyphae (Sudbery, 2001).

99 Discussion

In mammals, there are 14 known septin isoforms showing multiple splice variants which are characterized by tissue-specific distribution (Hall et al., 2005). Septins are involved in many cellular processes such as cytokinesis, vesicular transport and vesicle fusion and they act as important regulators of other cytoskeletal systems, including microfilaments and microtubules (Kinoshita, 2003). Septin structures are found during developmental processes like neurogenesis, spermatogenesis, and as supporting structure for epithelial cilia (Barral, 2010). Septins are known to form heteropolymeric structures consisting of a core unit of three distinct isoforms. Many different combinations are possible, whereas the crystal structure of Sept2/6/7-complex has already been solved (Sirajuddin et al., 2007).

Septin 8, together with Sept6, Sept10, Sept11 and Sept14, is classified as group II septin, which is constituted by septins with a long C-terminus. MK5 seems to prefer interaction with Sept8 splice variant carrying an additional C-terminal extension of 55 amino acids (Fig. 26). Sept8 has been described as an important factor involved in exocytosis by activated-platelets (Blaser et al., 2004). Another study revealed a neuron-specific function in exocytosis by mediating the binding of vesicle-associated membrane protein 2 to synaptophysin (Ito et al., 2009). Overexpression of Sept8 in HEK293 cell leads to a cytosolic vesicle-like distribution (Fig. 28), whereas filamentous structures could not be found. Only by additional over- expression of Sept7, which normally forms cytosolic homo-polymers, the integration of Sept8 into the septin filament could be observed (Fig. 29). The binding of MK5 to Sept8 might influence the subcellular localization of the kinase in neurons, since Sept8 has been reported to be involved in neuronal exocytic transport. There is substantial evidence that MK5 can interfere with septin filament via Sept8-binding and might play a functional role in the organization of septin filaments. Further discussion on the putative neuronal functions of the ERK3/MK5-signaling module will follow in section 5.4.

Kalirin-7

A cDNA clone coding for a fragment of 353 amino acids of the Rho guanine nucleotide exchange factor kalirin-7 (Kal7) has been identified within a yeast two-hybrid screen using a brain cDNA library (Fig. 25, Tab. 10). Specific interaction has been confirmed by pull-down studies with recombinant Kal7-fragments and co-localization studies in HEK293 cells (Fig. 30). Kinase assay with these fragments demonstrated that Kal7 is an in vitro substrate of MK5, which is phosphorylated at Ser487 (Fig. 30).

100 Discussion

Multiple kalirin splice variants exist, whereas Kal7 is the major Kalrn gene product, which is expressed in the brain. In neurons, the multi-domain spanning protein (190kDa) is predominantly localized at the postsynaptic density (PSD) via binding of PSD-95 through its PDZ-binding motif (Penzes et al., 2000). GEF-activity of Kal7 strongly depends on multiple phosphorylation events. For instance, it is known that phosphorylation at Thr1590 by Cdk5 increases the catalytic GEF-activity (Xin et al., 2008). Similar effects have been observed upon phosphorylation of Thr95 by CaMKII (Xie et al., 2007b). Deletion of both phosphorylation sites caused altered spine morphology. Analysis of immunoprecipitated Kal7 protein from mouse brain lysates showed about 22 distinct phosphorylated sites, including the MK5 putative site at Ser487 (Kiraly et al., 2011). The S487 is located within an SH3- domain, which is necessary for interaction with longer kalirin isoforms Kal9 and Kal12 (Schiller et al., 2006). MK5-mediated phosphorylation could play an important role in the regulation of Rho GEF-activity.

Mice with a deletion of a Kal7-unique exon in their kalrn gene display a significant decrease in dendritic spine density in the hippocampus, altered long-term potentiation (LTP), and focal learning impairments (Ma et al., 2008). The effect of Kal7 activity on spine formation is mainly due to changes in actin filament organization through activation of EphB-Kal7-Rac1- PAK-dependent pathway (Penzes et al., 2003). A functional role for Kal7 has been described in the early onset of coronary artery disease (Wang et al., 2007), schizophrenia (Kushima et al., 2010), and Alzheimer’s disease (Youn et al., 2007). MK5 might regulate Kal7-activity in neurons, which causes altered spine morphology. This could explain the reduced number of dendritic spines found in the hippocampal neurons of MK5-deficient mice (Fig. 43).

Mdm2-binding protein (Mtbp)

The MK5-interaction with the Mdm2-binding protein (Mtbp) has been identified in a parallel screen using a normalized mouse cDNA library. The use of a normalized universal mouse cDNA has the advantage that rare genes with low transcript number were artificially amplified, increasing the possibility of finding rare interactions. MK5-Mtbp interaction has been approved in pull-down studies with ectopically expressed proteins in HEK293 cell lysates (Fig. 32A/B). Overexpressed MK5 could also be purified using recombinant Mtbp immobilized to a sepharose matrix (Fig. 32C). Both proteins are exclusively localized in the nucleus, when overexpressed in HEK293 cells, and the presence of ectopically expressed Mtbp seems to reduce ERK3-dependent nuclear export of MK5 (Fig. 33).

101 Discussion

Mtbp has been described by the group of Mark. T. Boyd (Liverpool, United Kingdom) as Mdm2-interacting protein, which inhibits auto-ubiquitination of Mdm2 leading to protein stabilization. As a result, ubiquitination of Mdm2-substrate p53 is upregulated. Overexpression of Mtbp has been shown to negatively affect cell growth, which seems to be independent of Mdm2-stabilization (Boyd et al., 2000a). Mtbp is also present in humans (designated as MTBP) and the related gene is found on chromosome 8 (Boyd et al., 2000b). So far, no clear physiological function has been described. First studies suggest that tumor metastasis is increased in heterozygous mice, whereas homozygous disruption of the Mtbp gene promotes early embryonic lethality (Iwakuma et al., 2008). In heterozygous mice decreased Mtbp has been demonstrated to inhibit myc-induced B-cell proliferation and lymphomagenesis, suggesting Mtbp function as an important factor in myc-induced tumorigenesis (Odvody et al., 2010). Interestingly, MK5 has also been demonstrated to be involved in the regulation of Myc expression (Kress et al., 2011).

Recently, it has been published that Mtbp is crucial for proper mitotic progression in HeLa cells. The protein is found at the kinetochores, where it might interfere with the Mad1/Mad2 complex during pro-metaphase regulating chromosome segregation. Down-modulation of Mtbp results in abnormal aneuploidy, decreased cell proliferation, senescence, and cell death (Agarwal et al., 2011). Interestingly, ERK3 was also found at the mitotic spindle in mouse oocytes (Li et al., 2010). In the pro-metaphase ERK3 protein is enriched (Tanguay et al., 2010), whereas Mtbp has been found to be rapidly degraded during mitosis (Agarwal et al., 2011). Activation of MK5 by ERK3 might cause phosphorylation of Mtbp, including two putative MK5 phosphorylation sites. Thus, phosphorylation of Mtbp might trigger its cleavage and induce transition to anaphase. The research on Mtbp function is still at early stages, but possible implication in regulation of p53-function via a MK5-Mtbp dependent pathway could not be excluded, whereas further investigations have to be performed.

Sodium/potassium-transporting ATPase subunit beta-1 (Atp1b1)

Substantial evidence of MK5-interaction with the regulatory subunit beta-1 (Atp1b1) of the sodium/potassium-transporting ATPase (Na+-K+ ATPase) is provided in the current study, since several distinct cDNA fragments could be identified in two independent yeast two- hybrid screens. Interaction has been confirmed by pull-down studies in HEK293 cells (Fig. 34), whereas MK2 was also shown to interact with the Atp1b1 fragment in yeast and mammalian cells.

102 Discussion

The Na+-K+ ATPase has initially been described in 1957 by Jens Christian Skou (Skou, 1957). In 1997, he received the Nobel Prize in Chemistry for the first discovery of an ion- transporting enzyme. The Na+-K+ ATPase moves three Na+ ions out of, and two K+ ions into the cell, for each ATP that is hydrolyzed (Morth et al., 2011). Na+-K+ ATPases are known to be involved in the generation of resting membrane potential in neurons (Skou, 1957), maintenance of cell volume (Wehner and Tinel, 2000), regulating secondary transport via retaining sodium gradient (Wright et al., 2011), and as signaling transducer, activating, among others, MAPKs pathways (Lei and Ingbar, 2011). Expression of the atp1b1 gene has been shown to be under control of the transcription factors SP1 and CREB (Matlhagela et al., 2005). It has also been demonstrated that MK5 mRNA is increased after stimulation with forskolin (Gerits et al., 2009). The MK5 promoter of the human, mouse, and rat gene contains a CRE-binding site, indicating similar regulation of MK5 and Atp1b1 gene expression. The interaction of the MK5/ERK3-complex, which seems to have an impact on neuronal morphology, might also influence neuronal function via interaction with Atp1b1.

Other potential MK5-interacting proteins

Interactions with other identified yeast two-hybrid clones still have to be confirmed in mammalian cells (Tab. 10). The doublecortin-like kinase 1 (Dclk1) is a less characterized kinase, which might have a function in neurogenesis. Dclk1 and Dclk2 have been demonstrated to be important for spindle formation during M-phase in neuronal cells and might control the fate of neuronal progenitors during cortical neurogenesis (Shu et al., 2006). Interaction of MK5 and Dclk1 is probable compelling for neuronal function of the ERK3/MK5- complex and will be discussed later (section 5.4). Another microtubule-interacting protein, identified in the screen, is the microtubule-associated protein 1a (Mtap1a), which is important for linking microtubules to other cytoskeletal elements. For example, it has been reported that Mtap1a can bind to Tiam1 (Takefuji et al., 2007), which belongs to the family of Rac GEFs such as kalirin.

Another possible MK5-interacting protein, the cell division cycle and apoptosis regulator protein 1 (Ccar1, Carp1), is a known E3 ligase that targets apical caspases and p53, promoting DNA damage-induced degradation of p53 independently to Mdm2 (Yang and El- Deiry, 2007, Yang et al., 2007). Ccar1 could be another target of MK5, besides Mtbp, to regulate the stability and transcriptional activity of p53.

103 Discussion

Interaction of MK5 with the small nuclear ribonucleoprotein 48 (snRNP48) component of the splicosome and the keratin-like protein 222 (Krt222) cannot be evaluated, since no functions have yet been described. MK5-interaction with the carboxypeptidase E (Cpe) fails to give any direct functional correlation, except for its neuronal expression. The Cpe has been demonstrated to catalyze the C-terminal cleavage of neuropeptide, such as prohormones. The enzyme is involved in secretion of bioactive peptides such as neurotrophins, in need for proper synaptic development and neuroprotection (Thomas and Davies, 2005).

5.3.4 Evaluation of the biological relevance of the novel ERK3-interacting proteins

By using the ras recruitment system, GEF Cdc25, the ubiquitin-like protein 3 (Ubl3), the MAP kinase phosphatase 6, cop9, and septin-7 have been identified as potential ERK3-interacting partners (Tab. 11). In following pull-down studies, only interaction with Cdc25, Ubl3, and septin-7 could be confirmed. The Cdc25 can activate yeast ras independent to the bait protein and has been regarded as false positive candidate (Fig. 35). ERK3 interaction with Ubl3 might indicate a posttranslational modification of ERK3. In contrast to the N-terminal ubiquitination of ERK3 regulating its degradation, ubiquitination by Ubls are normally not affecting protein cleavage. Modification by Ubl-binding rather induces conformational changes of the targets affecting the affinity for ligands or other interacting molecules and alter substrate localization (Hochstrasser, 2009). Ubl3 was identified in 1999 (Chadwick et al., 1999), and so far, no cellular substrate or underlying mechanism of substrate-targeting has been established.

Interaction of ERK3 with the structural protein Sept7 has been approved by pull-down assays and co-localization studies (Fig. 36, Fig. 37, and Fig. 38). Interaction studies with deletion mutants of Sept7 and ERK3 revealed that the C-terminal coiled coil (CC) domain of Sept7 binds to ERK3 within amino acid 358 and 481 (Fig. 36D). The Sept7 interaction site seems to be conserved in ERK4, since binding has also been confirmed by pull-down (S. Kant, unpublished). The Sept7 interacting site does not overlap with the MK5-binding site in ERK3 (FHIEDEVDDIL, 332-342 aa). Simultaneous binding of MK5 and Sept7 to ERK3 could be observed by pull-down (Fig. 38). In an overexpression system, ERK3 serves as a bridge connecting MK5 to Sept7-containing filaments (Fig. 39), leading to the hypothesis that the ERK3/MK5-signaling module is recruited to septin structure and might be important for septin-related functions.

104 Discussion

As already described, septins are implicated in developmental processes like neurogenesis, spermatogenesis, and as supporting structure for epithelial cilia (Barral, 2010). Functional classification of the single isoforms proved to be difficult, since septin filaments are heteropolymers with a high variety of possible isoform-specific combinations. The Sept7 has been shown to favorably interact with septin 1, 4, 6, 8, 9, 10, and 11, whereas interaction with non-septin protein gave no evidence of a functional role (Nakahira et al., 2010). A neuron-specific function of Sept7 in the regulation of dendritic branching and spine formation has been described (Tada et al., 2007, Xie et al., 2007a). Involvement of septins in neuronal development and differentiation is one of the most prominent functions in mammals, and numerous investigations have been performed. The following section will connect possible neuronal implication of the ERK3/MK5-signaling module with known mechanisms associated with septins.

5.4 The ERK3/MK5-signaling module affects neuronal morphology by reorganizing septin filaments

The first implication of a neuron-specific function of mammalian septins was reported in 1998. Four distinct mammalian septins, including Sept7, have been found to interact with the rat brain sec6/8b complex, which is required for vesicle trafficking and relocalization of proteins to the plasma membrane of neurons (Hsu et al., 1998). Another study focused on targeting of area-specific protein expression of these four mammalian septin isoforms (CDCrel-1 [Sept5], Septin6, CDC10 [Sept7], and H5 [Sept4]) in the brain. A broad functional role of the related septin filaments depending on their isoform-specific composition has been proposed (Kinoshita et al., 2000). Regarding to the growing number of identified mammalian septins, the list of neuron-specific functions of distinct isoforms has been extended. For instance, Sept5 in complex with Sept7 also Sept2 (Nedd5) has been shown to be necessary for neurotransmitter release and deletion of Sept5 gene leads to impaired neuron function (Peng et al., 2002). The Sept8 isoform has been demonstrated to be involved in exocytosis by targeting the vesicle-associated membrane protein 2 (Ito et al., 2009). Interaction of Sept8 with MK5 might relocalize the kinase to the septin filament at distinct subcellular regions of the neuron. Currently, septins are though to have two major functions in nerve cells. They serve as important regulators in exocytic vesicle transport and can act as structural skeleton in close relation to other filaments, which is indispensable for neuronal differentiation.

105 Discussion

A functional role of septin filaments in differentiation, affecting morphology, has firstly been described in PC-12 cells. The expression of a GTPase dead Sept2 resulted in altered neurite outgrowth in PC-12 cells upon NGF-treatment (Peng et al., 2002). Similar function in neuronal differentiation, regulating dendrite branching and spine formation in primary hippocampal neurons, has initially been reported for Sept7 (Tada et al., 2007, Xie et al., 2007a) and later for Sept6 (Cho et al., 2011). Recently, Sept14 together with Sept4 have been shown to be important for neuronal migration during the development of the cerebral cortex (Shinoda et al., 2010).

Characterization of presynaptic septin complexes in mammalian hippocampal neurons showed a main contribution by Sept3, Sept5, Sept6, Sept7 and Sept11 (Tsang et al., 2011). Most of the studies described a primary role of Sept7 in neuronal development, since its down-regulation by siRNA clearly affected neuronal morphology (Tada et al., 2007, Xie et al., 2007a). Using transfected primary neurons, it has been demonstrated that the Sept7- overexpression-dependent phenotype was enhanced by additional overexpression of ERK3 and MK5. Simultaneous expression leads to increased complexity in dendrite branching and a higher number of spines (Fig. 43). Further analysis of neuronal morphology also suggested an increase of dendrite thickness upon overexpression (Fig. 43D). In contrast, targeted deletion of other isoforms, such as Sept3, revealed no obvious phenotypic changes in developing neurons (Fujishima et al., 2007), which might be due to compensation by other isoforms.

Regarding to the organization of septin filaments, Sept7 dimer-formation is mainly due to interaction of their GTPase domain (G interface), which is also true for Sept2 dimers (Sirajuddin et al., 2007). A second type of septin-septin-interaction, mostly found in heteromers, is due to binding of their N- and C-terminal extensions (NC interface). Structural and biochemical analysis showed that the Sept7 homodimers are highly flexible structures compared to Sept2 homodimers. The binding affinity of two distinct Sept7 monomers strongly depends on GTP-binding that induced conformational changes, which is not required to Sept2 dimers (Sirajuddin et al., 2007, Zent et al., 2011). This might indicate a possible mechanism in the assembly and disassembly of septin filaments by regulated GDP/GTP binding to accelerate Sept7 dimerization or dissociation.

Organization of septin structures has been reported to be regulated by Cdc42 effector proteins also know as binders of Rho GTPases (Borgs) (Joberty et al., 2001, Sheffield et al., 2003). Interaction of Cdc42 and Borg proteins strongly influences the stability of septin filaments. Phosphorylation of Borg proteins by ERK3 and MK5 (Fig. 42) might stabilize septin structures and induce filament formation. Additionally, Cdc42 has been demonstrated to activate ERK3 kinase via PAKs, which might be required for local regulation of Borg activity

106 Discussion at Sept7-containing filamentous structures. It has already been reported that activation of Cdc42 upon EphB receptor activation regulates dendritic spine development (Irie and Yamaguchi, 2002). Another small Rho GTPase, which is activated downstream of the EphB receptor, is Rac1 (Penzes et al., 2003). Interestingly, MK5 interacts and in vitro phosphorylates Kal7 (Fig. 30), which represents the Rho GEF of Rac1 GTPase. Phosphorylation of Kal7 by MK5 could serve as a feed-forward control of this activation process by stimulating GEF-activity for Rac1. Deletion of Kal7 leads to a strong reduction of dendritic spines in cultured hippocampal neurons (Xie et al., 2007b). So far, it is not clear whether Sept7-dependent morphological changes require the presence of ERK3 and MK5 or the presence of other factors. Activation of Rac1/Cdc42 is also affecting the actin filament. Impaired dendritic branching and altered spine morphology required multiple factors changing organization of the neuroskeleton, including actin-, septin-, and microtubule filaments. Septin filaments are known to cooperate with actin- and microtubule skeleton. Several models of crosstalk have been established (Saarikangas and Barral, 2011). Recruitment of MK5 to septin filaments might promote cooperation with the actin filament due to Kal7-binding, moreover a potential interaction with microtubule filament might occur by MK5-mediated regulation of Dclk1 and Mtap1a.

The assembly of the septin ring at the bud neck of yeast is controlled by the activity of the Cdc42-like small GTPases Rga1 and Rga2, which subsequently activate Cla4, a p21- activated protein kinase analog in yeast. Cla4 is able to phosphorylate septins (especially Cdc10; Cdc3) resulting in its GTP-binding and subsequent formation of heteromeric ring structures (Versele and Thorner, 2004) (Fig. 45A). The reported data implicate that the ERK3/MK5-complex regulates septin-dependent processes at the neck of dendritic spines and neuronal branch points in a manner similar to the molecular mechanisms identified in yeast (Fig. 45B).

In the brain, septins are involved in neuronal disorders and associate with several neurological diseases including Alzheimer’s disease (Takehashi et al., 2004), Parkinson’s disease (Ihara et al., 2003, Shehadeh et al., 2009) and schizophrenia (Barr et al., 2004, Pennington et al., 2008). Importantly, the results of the in vitro overexpression studies are complemented by the evidence of impaired spine formation of hippocampal neurons in vivo in mice lacking MK5 (Fig. 44), which also displaying reduced levels of ERK3 (Shi et al., 2003, Schumacher et al., 2004). A transgenic mouse model expressing a constitutive active MK5 mutant (L337A) is characterized by an impaired cognitive functions, showing gender-specific less anxious behavior in the elevated plus maze (EPM) test (Gerits et al., 2007b). ERK3 and MK5 knockout mice display poorly understood perinatal and embryonic lethality, respectively (Schumacher et al., 2004, Klinger et al., 2009).

107 Discussion

ERK3 knockout mice lethality is due to acute respiratory failure within the first minutes after birth, although these animals display normal lung morphology. One may speculate that respiratory rhythm-generating neurons residing within the hindbrain could be impaired in their functional cooperation caused by reduced ability to form dendrites and spines. A similar scenario in other specific brain regions could also explain the enigmatic embryonic lethality seen in the MK5- deficient mice when backcrossed to the C57/B6J strain, without showing any morphological and histological abnormalities (Schumacher et al., 2004). The observed lower neuronal complexity found in the hippocampus of MK5-deficient mice could cause problems in generation of long-term memory and, which result in disoriented behavior and impaired place navigation (Morris et al., 1982), as occasionally observed by our group in this strain (A. Kotlyarov and T. Fig. 45. Comparison of the molecular mechanisms regulating septin assembly in yeast and mammalian Yakovleva, unpublished). This work neurons. A) In yeast, septin ring formation is regulated by the Cdc42-like small GTPase analogs Rga1 and Rga2, describes a Sept7-dependent leading to subsequent activation of the protein kinase Cla4. Cla4 directly phosphorylates the septin7 analog Cdc10 regulation of neuronal morphogenesis resulting in formation of heteromergic complexes. Small GTPase activity also regulates the actin filaments, which by the ERK3/MK5-signaling module cooperate with the septins. B) Hypothetical scheme of the ERK3/MK5-signaling module regulating mammalian septin and might explain the pleiotropic assembly at dendritic protrusions. The ERK3/MK5/Sept7- phenotypes of the ERK3 and MK5 signaling complex is activated by Cdc42 and Rac GTPase via p21-activated protein kinases (PAKs). Septin heteromeric knockout models. The protein functions structures are generated via ERK3/MK5/Sept7-mediated recruitment and phosphorylation of Borg proteins. The MK5 could also be of relevance to other substrate Kalirin-7, a Rho GEF and known activator of Rac GTPases further contributes to PAK activation and actin septin-dependent processes in filament formation. mammals.

108 Conclusions and perspectives

6 Conclusions and perspective

This doctoral thesis dealt with the characterization of the physiological function of the ERK3/MK5-signaling module. The identification of novel interaction partners of ERK3 and MK5 by extensive yeast-based library screens was the primary aim of this study.

The tissue-specific determination of MK5 protein expression revealed highest abundance in thymus, brain, kidney, lung and spleen, whereas ERK3 protein could only be detected in the thymus and the heart. High levels of ERK3 and MK5 protein expression have also been detected in selected neuroblastoma cell lines. In HEK293 cell lysates, ectopically expressed MK5 can be strongly activated by co-expressed ERK3. Their interaction has been confirmed on endogenous level. Analysis of primary isolated MEFs lacking ERK3 and MK5 showed impairment of cell growth, migration and organization of the actin filament.

In PC-12 cells, endogenous ERK3 protein and ectopically expressed GFP-ERK3 is degraded in response to alkalinization of the culture medium. The pH-dependent ERK3-cleavage can be inhibited by proteasome inhibitors, cobalt chloride, antimycin A, staurosporine, okadaic acid and ortho-vanadate. The mechanisms of distinct inhibitions remain unknown, but future experiments should focus on activation site phosphorylation-dependent stabilization or destabilization of ERK3.

An in vitro screen of Ser/Thr protein kinases revealed an increased activity of the ERK3/MK5-kinases in the presence of microtubule affinity-regulated kinase 1/3, p21- activated kinase 1/3, PDZ-binding kinase, and the testis-specific kinase 2. From control experiments, induction of substrate phosphorylation by MK5 due to kinase X-dependent phosphorylation of ERK3 could not be shown for any of these kinases. Using another kinase assay approach it could be verified that phosphorylation of ERK3 is increased in the presence of group I PAKs (isoform 1-3).

Phosphoproteome analysis in MK5-deficient tissues using phospho-(Ser/Thr) kinase substrate antibodies revealed a potential substrate of ERK3, which migrates with a molecular mass of 25 kDa. The phosphoprotein was specifically found in lysates of the brain and the thymus. Related investigation of ERK3/MK5-deficient MEF cells showed the absence of PMA or pervanadate-inducible phosphorylation of substrate proteins migrating with a molecular mass of 35, 50, 75, and 170 kDa as possible substrates for MK5. Further efforts have to be made to identify these proteins and to approve the phosphorylation by ERK3 or MK5.

In MK5 yeast two-hybrid screens, interactions with Septin 8, sodium/potassium transporting ATPase subunit beta 1, Rho nucleotide exchange factor kalirin 7 (Kal7), and the Mdm2-

109 Conclusions and perspectives binding protein have been identified and confirmed by pull-down assays and co-localization studies. Interestingly, using the ras recruitment system, an interaction of ERK3 with another septin isoforms Sept7 has been found. Analysis of the endogenous Sept7 distribution in MEF cells revealed a less organized septin filament in ERK3/MK5-deficient cells compared to ERK3/MK5-rescued MEFs. In vitro kinase assay showed no phosphorylation of Sept7 by MK5 or ERK3, whereas both kinases can phosphorylate the Borg proteins, which are known regulator of septin filament organization.

In neurons, the overexpression of Sept7 lead to higher complexity in dendrite branching and spine formation and could be significantly increased by additional expression of ERK3 and MK5. Interestingly, hippocampal neurons of MK5-deficient mice showed a decrease in spine number, which suggests the in vivo relevance of the ERK3/MK5-dependent neuronal functions regulating spine formation. Future experiments should address the MK5/ERK3- dependent reorganization of septin filaments in neurons. The use of kinase dead mutants, as well as monospecific inhibitors for MK5, could be of substantial benefit. It would be also interesting to analyze, whether hippocampal neurons of MK5-deficient mice display functional deficits. The MK5-dependent phosphorylation of Kal7, which has been shown in vitro, could have relevance in the regulation of neuronal spine dynamics. Further experiments have to be done to show the importance of S487 phosphorylation of Kal7, for instance, by showing the influence on GEF-activity of Kal7 in the activation of Rac1.

It is tempting to speculate that the neuronal function of the ERK3/MK5-signaling module explains the pleiotropic phenotypes of the ERK3 and MK5 knockout mice. The corresponding protein functions of ERK3 and MK5 should be further analyzed in other septin-dependent processes in mammals.

110 References

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125

8 Appendix

8.1 List of tables and figures

Tab. 1. Mouse strains

Tab. 2. Cell lines

Tab. 3. Plasmids

Tab. 4. Oligonucleotides

Tab. 5. Antibodies

Tab. 6. Further chemicals

Tab. 7. Laboratory Equipment

Tab. 8. Kits

Tab. 9. Phospho-motif antibody staining pattern of PKA, PKC, CDK, and ATM/ATR kinase of MK5-deficient compared to wild type tissues showed additional (A) or missing (B) phosphoprotein-bands.

Tab. 10. Positive cDNA prey clones identified in three distinct yeast-two-hybrid screens using MK5 as bait.

Tab. 11. Overview of positive clones obtained from yeast based screening following the ras recruitment system (RRS).

Fig. 1. Cascades of conventional MAP kinase activation.

Fig. 2. Overview of biological processes involving ERK1/2-mediated substrate phosphorylation.

Fig. 3. The p38 MAP kinases are activated in response to inflammatory cytokines, growth factors and environmental stress.

Fig. 4. Phylogenetic tree of the human kinome.

Fig. 5. Structural analysis of ERK3-related fragments.

Fig. 6. Phylogenetic tree of MAPK-activated protein kinases (MAPKAPKs).

Fig. 7. Substrate targets of ribosomal-S6-kinases 1-4 (RSK1–4), the mitogen- and stress- activated-kinases 1 and 2 (MSK1, 2), and MAPK-interacting kinases 1 and 2 (MNK1, 2).

Fig. 8. ERK3 and MK5 protein expression in selected cell lines (A) and adult mouse tissues (A/B).

Fig. 9. Interaction of ERK3 and MK5 induced activation of MK5 and cytosolic relocalization of both kinases.

Fig. 10. Mouse embryonic fibroblasts (MEF) deficient in MK5 and ERK3 (dKO) showed impaired proliferation and decreased sensitivity to glucose starvation.

Fig. 11. ERK3/MK5 targeted deletion affects cell migration of immortalized MEFs.

Fig. 12. The actin filament is less organized in immortalized ERK3/MK5-deficient MEFs.

Fig. 13. ERK3 protein level is highly regulated in PC-12 cells.

Fig. 14. ERK3 degradation in response to environmental changes can be blocked by proteasomal inhibitors.

Fig. 15. ERK3 protein levels are positively and negatively regulated through acidification and alkalinization of the culture medium.

Fig. 16. In stably transfected PC-12 cells GFP-ERK3 is degraded in response to alkaline extracellular pH.

Fig. 17. GFP-ERK3 protein cleavage can be influenced by bortezomib, nocodazole, PMA, and cobalt chloride.

Fig. 18. The pH-dependent cleavage of GFP-ERK3 can be blocked by cobalt chloride, antimycin A, and staurosporine.

Fig. 19. High dosage of okadaic acid and broad inhibition of tyrosine phosphatase can block degradation of GFP-ERK3.

Fig. 20. In presence of ERK3, MK5-mediated substrate phosphorylation is significantly increased upon additional incubation with MARK1/3, PAK1/3, PBK, or TSK2 significantly increased MK-substrate phosphorylation.

Fig. 21. Verification of potential ERK3/MK5-complex activating kinases.

Fig. 22. Phosphorylation of ERK3 is increased in presence of PAK1, 2, and 3, whereas inhibition of PAKs do not influence ERK3 stability.

Fig. 23. Analysis of substrate phospho-motif pattern of PKA, PKC, CDK, and ATM/ATR in MK5-deficient murine adult tissues.

Fig. 24. PKA, PKB (Akt), PKC, and PKD substrate phospho-motif antibody detection in immortalized dKO MEFs (1) compared to dRes MEFs (2).

Fig. 25. Reconstitution of MK5-bait interacting prey-vectors together with the empty, MK2-, or MK5-bait vector in yeast.

Fig. 26. MK5 interacts with C-terminal part of Septin8 (Sept8).

Fig. 27. MK5 interacts with Sept8 fragments in HEK293 in yeast and cell lysates.

Fig. 28. Septin 8 (Sept8) is partially co-localized with MK5.

Fig. 29. Sept7 and Sept8 generated strong cytosolic filamentous structures.

Fig. 30. Kal7 as in vitro substrate of MK5.

Fig. 31. Co-localization studies revealed that Kal7 interacts with MK5.

Fig. 32. The Mdm2-binding protein (Mtbp) interacts with MK5 in vitro.

Fig. 33. Mtbp and MK5 are exclusively nuclear localized.

Fig. 34. The sodium/potassium-transporting ATPase subunit beta 1 (Atp1b1) can be precipitated by MK5 in pull-down assay.

Fig. 35. GST-ERK3 pull-down with GFP-tagged fragments of RRS-derived positive clones.

Fig. 36. Pull-down studies with deletion mutants of Sept7 and ERK3.

Fig. 37. ERK3 acts as a docking platform for MK5 and Sept7.

Fig. 38. MK5 interaction with Sept7 is bridged by ERK3-binding.

Fig. 39. Impaired cellular septin organization due to knockout of ERK3 and MK5 in MEF cells.

Fig. 40. Effects of Sept7 gene silencing with siRNA in HeLa cells.

Fig. 41. Sept7 is not phosphorylated by ERK3 or MK5.

Fig. 42. In vitro phosphorylation of Borg1, 2, and 3 by ERK3 and MK5.

Fig. 43. Sept7-dependent stimulation of dendritic outgrowth and increased spine number by overexpression of the ERK3/MK5-complex in primary hippocampal neurons.

Fig. 44. Determination of spine numbers for MK5-deficiency in mice in vivo.

Fig. 45. Comparison of the molecular mechanisms regulating septin assembly in yeast and mammalian neurons.

8.2 List of recombinant kinases (Proqinase GmbH, Freiburg)

ACV-R1 0372- human, C-terminal fragment, GST-HIS fusion ACVR1 ACTRI; ACVRLK2; ALK2; SKR1 NM_001105 145-509 TKL 0000-1 protein, expressed in Sf9 cells AKT1 0132- human, C-terminal fragment, activated, GST-HIS AKT1 PKB; RAC; PRKBA; RAC-ALPHA NM_005163 106-480 AGC 0000-2 fusion protein, expressed in Sf9 cells AKT2 0276- human, C-terminal fragment, activated, GST-HIS AKT2 PRKBB; PKBBETA; RAC-BETA NM_001626 107-481 AGC 0000-2 fusion protein, expressed in Sf9 cells AKT3 0436- human, C-terminal fragment, GST-HIS fusion AKT3 PKBG; PRKBG; STK-2; RAC- NM_005465 106-479 AGC 0000-2 protein, activated, expressed in Sf9 cells gamma; RAC-PK-gamma AMPK-alpha1 fl 0289- human, full length, GST-HIS fusion protein, PRKAA1 MGC33776; MGC57364 NM_006251 1-550 CAMK 0000-1 expressed in Sf9 cells AMPK-alpha1 tr 0383- human, N-terminal fragment, GST-HIS fusion PRKAA1 MGC33776; MGC57364 NM_006251 1-312 CAMK 0000-1 protein, expressed in Sf9 cells ARK5 0300- human, full length, GST-HIS fusion protein, NUAK1 KIAA0537 NM_014840 1-661 CAMK 0000-1 expressed in Sf9 cells Aurora-A 0166- human, full length, GST-HIS fusion protein, STK6 AIK; ARK1; AURA; BTAK; STK15; NM_003600 1-403 Other 0000-1 expressed in Sf9 cells MGC34538 Aurora-B 0190- human, full-length, GST-HIS fusion protein, AURKB AIK2; AIM1; ARK2; AurB; IPL1; NM_004217 2-344 Other 0000-1 expressed in Sf9 cells STK5; AIM-1; STK12 Aurora-C 0315- human, full length, GST-HIS fusion protein, AURKC AIE2; AIK3; STK13 AB017332 1-275 Other 0000-1 expressed in Sf9 cells B-RAF wt 0403- human, C-terminal fragment, GST-HIS fusion BRAF B-Raf; B-raf-1; BRAF1; RAFB1 NM_004333 417-766 TKL 0000-1 protein, expressed in Sf9 cells B-RAF VE 0404- human, C-terminal fragment, mutationally BRAF B-Raf; B-raf-1; BRAF1; RAFB1 NM_004333 417-766 TKL 0000-1 activated (V600E), GST-HIS fusion protein, V600E expressed in Sf9 cells CDC42BPB 0382- human, N-terminal fragment, GST-HIS fusion CDC42BPB MRCK-beta; MRCKb NM_006035 1-472 AGC 0000-1 protein, expressed in Sf9 cells CDK1/CycB1 0134- human, full length, GST-HIS fusion proteins, CDC2 p34 NM_001786 1-297/1-433 CMGC 0135-1 expressed in Sf9 cells CDK1/CycE 0134- human, full length, GST-HIS fusion proteins, CDC2 p34 NM_001786 1-297/1-395 CMGC 0055-1 expressed in Sf9 cells CDK2/CycA 0050- human, full length, GST-HIS fusion proteins, CDK2 p33(CDK2) NM_001798 1-298/1-432 CMGC 0054-1 expressed in Sf9 cells CDK2/CycE 0050- human, full length, GST-HIS fusion proteins, CDK2 p33(CDK2) NM_001798 1-298/1-395 CMGC 0055-1 expressed in Sf9 cells CDK3/CycE 0212- human, full length, GST-HIS fusion proteins, CDK3 n/a NM_001258 1-305/1-395 CMGC 0055-1 expressed in Sf9 cells CDK4/CycD1 0142- human, full length, GST-HIS fusion proteins, CDK4 CMM3; PSK-J3; MGC14458 NM_000075 4-303/4-295 CMGC 0143-1 expressed in Sf9 cells CDK4/CycD3 0142- human, full length, CDK4: GST-HIS fusion CDK4 CMM3; PSK-J3; MGC14458 NM_000075 4-303/1-293 CMGC

0373-1 protein, CycD3: HIS-tagged protein, expressed in Sf9 cells CDK5/p25NCK 0356- human, full length, CDK5: GST-HIS fusion CDK5 PSSALRE NM_004935 1-292/99-307 CMGC 0389-1 protein, p25NCK: HIS-tagged protein, expressed in Sf9 cells CDK5/p35NCK 0356- human, full length, CDK5: GST-HIS fusion CDK5 PSSALRE NM_004935 1-292/1-307 CMGC 0355-1 protein, p35NCK: HIS-tagged protein, expressed in Sf9 cells CDK6/CycD1 0051- human, full length, GST-HIS fusion proteins, CDK6 PLSTIRE; MGC59692 NM_001259 1-326/4-295 CMGC 0143-1 expressed in Sf9 cells CDK7/CycH/MAT1 0366- human, full length, CDK7: GST-HIS fusion CDK7 CAK1; CDKN7; STK1; p39MO15 NM_001799 1-346/1- CMGC 0360-4 protein, CycH/MAT1: HIS-tagged proteins, 323/1-309 expressed in Sf9 cells CDK8/CycC 0376- human, full length, CDK8: GST-HIS fusion CDK8 K35 NM_001260 1-463/1-283 CMGC 0390-1 protein, CycC: HIS-tagged protein, expressed in Sf9 cells CDK9/CycT 0371- human, full length, CDK9: GST-HIS fusion CDK9 C-2k; CDC2L4K35; PITALRE; TAK NM_001261 1-372/1-726 CMGC 0345-1 protein, CycT: HIS-tagged protein, expressed in Sf9 cells CHK1 0282- human, full length, GST-HIS fusion protein, CHEK1 n/a NM_001274 1-476 CAMK 0000-1 expressed in Sf9 cells CK1-alpha1 0406- human, full length, GST-HIS fusion protein, CSNK1A1 CK1; HLCDGP1; PRO2975 DQ082865 1-365 CK1 0000-1 expressed in Sf9 cells CK2-alpha1 0124- human, full length, GST-HIS fusion protein, CSNK2A1 CKII; CK2A1 NM_001895 1-391 Other 0000-1 expressed in Sf9 cells CK2-alpha2 0412- human, full length, GST-HIS fusion protein, CSNK2A2 n/a NM_001896 1-350 Other 0000-1 expressed in Sf9 cells CLK1 0447- human, full length, GST-HIS fusion protein, CLK1 CLK; CLK/STY NM_004071 1-484 CMGC 0000-1 expressed in Sf9 cells COT 0180- human, internal fragment, GST-HIS fusion MAP3K8 EST; ESTF; TPL2; Tpl-2; c-COT NM_005204 30-397 STE 0000-1 protein, expressed in Sf9 cells DAPK1 0332- human, N-terminal fragment, GST-HIS fusion DAPK1 n/a NM_004938 1-363 CAMK 0000-1 protein, expressed in Sf9 cells ERK2 0634- human, full length, activated, untagged, MAPK1 ERK, ERT1, MAPK2, P42MAPK, NM_002745 1-360 CMGC 0000-7 expressed in E.coli PRKM1, PRKM2, p38, p40, p41 GSK3-beta 0310- human, full length,GST-HIS fusion protein, GSK3B n/a L33801 1-420 CMGC 0000-1 expressed in Sf9 cells HRI 0444- human, full length, GST-HIS fusion protein, EIF2AK1 HCR NM_014413.2 1-630 Other 0000-1 expressed in Sf9 cells IKK-beta 0258- human, full length, GST-HIS fusion protein, IKBKB IKK2; IKKB; NFKBIKB AF029684 1-756 Other 0000-1 expressed in Sf9 cells IKK-epsilon 0320- human, full length, GST-HIS fusion protein, IKBKE IKKE; IKKI; IKK-i; KIAA0151 NM_014002 1-716 Other

0000-1 expressed in Sf9 cells IRAK4 0268- human, C-terminal fragment, GST-HIS fusion IRAK4 REN64; NY-REN-64 AF445802.1 104-460 TKL 0000-1 protein, expressed in Sf9 cells JNK1 0419- human, full length, HIS-tagged protein, expressed MAPK8 JNK; JNK1A2; JNK21B1/2; NM_002750 1-385 CMGC 0000-1 in Sf9 cells PRKM8; SAPK1 JNK3 0214- human, full length, GST-HIS fusion protein, MAPK10 JNK3A; PRKM10; p493F12; NM_002753 1-422 CMGC 0000-1 expressed in Sf9 cells FLJ12099; p54bSAPK MAPKAPK3 0633- human, full length, activated, untagged, MAPKAPK3 3PK; MAPKAP; MAPKAP3 NM_004653 1-382 CAMK 0000-1 expressed in E.coli MARK1 0432- human, full length, GST-HIS fusion protein, MARK1 KIAA1477 NM_018650 1-780 CAMK 0000-1 expressed in Sf9 cells MARK3 0433- human, full length, GST-HIS fusion protein, MARK3 CTAK1; KP78; PAR1A NM_002376.4 1-713 CAMK 0000-1 expressed in Sf9 cells MEK1 wt 0550- human, full length, activated, untagged, MAP2K1 MAPKK1, MKK1, PRKMK1 NM_002755 1-393 STE 0000-3 expressed in Sf9 cells MKK6 SDTD 0396- human, full length, mutationally activated (S207D, MAP2K6 MAPKK6; MEK6; MKK6; PRKMK6; NM_002758 1-334 STE 0000-1 T211D), GST-HIS fusion protein, expressed in SAPKK3 S207T211D Sf9 cells MST4 0286- human, full length, GST-HIS fusion protein, MST4 MASK NM_016542 1-416 STE 0000-1 expressed in Sf9 cells MYLK2 0445- human, full length, GST-HIS fusion protein, MYLK2 KMLC; MLCK; skMLCK NM_033118 1-596 CAMK 0000-1 expressed in Sf9 cells NEK2 0290- human, full length, GST-HIS fusion protein, NEK2 NLK1; HsPK21; HsPK 21 NM_002497 1-445 Other 0000-1 expressed in Sf9 cells NEK6 0209- human, C-terminal fragment, GST-HIS fusion NEK6 SID6-1512 NM_014397 8-313 Other 0000-1 protein, expressed in Sf9 cells NIK 0344- human, full length, GST-HIS fusion protein, MAP3K14 FTDCR1B; HS; HSNIK NM_003954 1-947 STE 0000-1 expressed in Sf9 cells NLK 0253- human, full length, GST-HIS fusion protein, NLK n/a NM_016231 1-515 CMGC 0000-1 expressed in Sf9 cells p38-alpha 0443- human, full length, activated, untagged, MAPK14 p38-alpha NM_001315 1-360 CMGC 0000-3 expressed in E.coli p38-beta 0455- human, full length, activated, untagged, MAPK11 p38-beta NM_002751 1-364 CMGC 0000-3 expressed in E.coli PAK1 0357- human, full length, GST-HIS fusion protein, PAK1 PAKalpha NM_002576 1-545 STE 0000-1 expressed in Sf9 cells PAK2 0304- human, C-terminal fragment, GST-HIS fusion PAK2 PAK65; PAKgamma NM_002577 3-524 STE 0000-1 protein, expressed in Sf9 cells PAK3 0422- human, full length, GST-HIS fusion protein, PAK3 CDKN1A; MRX30; OPHN3; NM_002578 1-544 STE 0000-1 expressed in Sf9 cells PAK3beta; bPAK; hPAK3 PAK4 0365- human, full length, GST-HIS fusion protein, PAK4 n/a NM_005884 1-591 STE 0000-1 expressed in Sf9 cells

PAK6 0423- human, full length, GST-HIS fusion protein, PAK6 PAK5 NM_020168 1-681 STE 0000-1 expressed in Sf9 cells PAK7 0410- human, full length, GST-HIS fusion protein, PAK7 KIAA1264; MGC26232 NM_020341 1-719 STE 0000-1 expressed in Sf9 cells PBK 0185- human, full length, GST-HIS fusion protein, PBK TOPK; SPK; Nori-3; FLJ14385 NM_018492 1-322 Other 0000-2 expressed in Sf9 cells PCTAIRE1 0189- human, C-terminal fragment, GST-HIS fusion PCTK1 PCTAIRE; PCTGAIRE NM_006201 107-496 CMGC 0000-1 protein, expressed in Sf9 cells S153A PDK1 0353- human, N-terminal fragment, GST-HIS fusion PDPK1 PkB kinase; MGC20087; NM_002610 1-460 AGC 0000-1 protein, expressed in Sf9 cells MGC35290; PRO0461 PIM1 0186- human, full length, GST-HIS fusion protein, PIM1 PIM NM_002648 1-313 CAMK 0000-1 expressed in Sf9 cells PIM2 0223- human, full length, GST-HIS fusion protein, PIM2 n/a NM_006875 1-311 CAMK 0000-1 expressed in Sf9 cells PKC-alpha 0222- human, full length, GST-HIS fusion protein, PRKCA PKCA; PRKACA; PKC-alpha NM_002737 1-672 AGC 0000-1 expressed in Sf9 cells PKC-beta1 0067- human, full length, GST fusion protein, expressed PRKCB1 PKCB; PRKCB; PRKCB2; NM_002738 1-671 AGC 0000-1 in Sf9 cells MGC41878; PKC-beta PKC-beta2 0116- human, full length, GST fusion protein, expressed PRKCB1 PKCB; PRKCB; PRKCB2; X07109 1-673 AGC 0000-1 in Sf9 cells MGC41878; PKC-beta PKC-delta 0232- human, full length, GST-HIS fusion protein, PRKCD MAY1; MGC49908; nPKC-delta NM_006254 1-675 AGC 0000-1 expressed in Sf9 cells PKC-epsilon 0203- human, full length, GST-HIS fusion protein, PRKCE PKCE; nPKC-epsilon NM_005400 1-737 AGC 0000-1 expressed in Sf9 cells PKC-eta 0420- human, full length, GST fusion protein, expressed PRKCH PKC-L, PKCL, PRKCL, nPKC-eta NM_006255.2 1-683 AGC 0000-1 in Sf9 cells PKC-gamma 0204- human, full length, GST-HIS fusion protein, PRKCG PKCC; PKCG; SCA14; MGC57564 NM_002739 1-697 AGC 0000-1 expressed in Sf9 cells PKC-iota 0114- human, full length, GST-HIS fusion protein, PRKCI PKCI; DXS1179E; MGC26534; NM_002740 1-587 AGC 0000-1 expressed in Sf9 cells nPKC-iota PKC-mu 0115- human, full length, GST-HIS fusion protein, PRKD1 PKD; PKCM; PKC-MU NM_002742 1-912 AGC 0000-1 expressed in Sf9 cells PKC-theta 0113- human, full length, GST-HIS fusion protein, PRKCQ PRKCT; nPKC-theta NM_006257 1-706 AGC 0000-1 expressed in Sf9 cells PKC-zeta 0117- human, C-terminal fragment, GST-HIS fusion PRKCZ PKC2 NM_002744 6-584 AGC 0000-1 protein, expressed in Sf9 cells PLK1 0183- human, full length, GST-HIS fusion protein, PLK1 PLK; STPK13 NM_005030 1-603 Other 0000-1 expressed in Sf9 cells PLK3 0437- human, full length, GST-HIS fusion protein, PLK3 CNK; FNK; PRK U56998.1 1-607 Other 0000-1 expressed in Sf9 cells PRK1 0207- human, full length, GST-HIS fusion protein, PKN1 DBK; PKN; PAK1; PRK1; NM_002741 1-942 AGC 0000-1 expressed in Sf9 cells PRKCL1; MGC46204

ROCK2 0347- human, internal fragment, GST-HIS fusion ROCK2 KIAA0619; ROC2 NM_004850 6-553 AGC 0000-1 protein, expressed in Sf9 cells S6K 0318- human, N-terminal fragment, activated, GST-HIS RPS6KB1 S6K1; STK14A NM_003161 1-400 AGC 0000-2 fusion protein, expressed in Sf9 cells SAK 0306- human, full length, GST-HIS fusion protein, PLK4 SAK; STK18 NM_014264 1-970 Other 0000-1 expressed in Sf9 cells SGK1 0199- human, full length, activated, GST fusion protein, SGK n/a NM_005627 1-431 AGC 0000-2 expressed in Sf9 cells SGK3 0198- human, full length, activated, GST fusion protein, SGKL CISK; SGK2; SGK3 NM_013257.2 1-496 AGC 0000-2 expressed in Sf9 cells SNARK 0414- human, full length, GST-HIS fusion protein, NUAK2 DKFZp434J037 NM_030952 1-628 CAMK 0000-1 expressed in Sf9 cells SNK 0277- human, full length, GST-HIS fusion protein, PLK2 SNK NM_006622 1-685 Other 0000-1 expressed in Sf9 cells SRPK1 0434- human, full length, GST-HIS fusion protein, SRPK1 SFRSK1 NM_003137 1-655 CMGC 0000-1 expressed in Sf9 cells SRPK2 0381- human, full length, GST-HIS fusion protein, SRPK2 SFRSK2 NM_182692 1-688 CMGC 0000-1 expressed in Sf9 cells TGFB-R1 0397- human, C-terminal fragment, GST-HIS fusion TGFBR1 ALK5; ACVRLK4 NM_004612 200-503 TK 0000-1 protein, expressed in Sf9 cells TSF1 0187- human, full length, GST-HIS fusion protein, STK16 KRCT; MPSK; PKL12 NM_003691 1-305 Other 0000-1 expressed in Sf9 cells TSK2 0220- human, full length, GST-HIS fusion protein, TSSK2 DGS-G; SPOGA2; FLJ38613 NM_053006 1-358 CAMK 0000-1 expressed in Sf9 cells TTK 0284- human, full length,GST-HIS fusion protein, TTK ESK; MPS1L1; PYT NM_003318 1-857 other 0000-1 expressed in Sf9 cells VRK1 0302- human, full length, GST-HIS fusion protein, VRK1 n/a NM_003384 1-396 CK1 0000-1 expressed in Sf9 cells

MEKK2 PR-071219 0.8 mg/ml; 12 µl; HIS-tagged; not yet fully tested !! MEKK3 PR-071106 0.1 mg/ml; 100 µl; GST-tagged; not yet fully tested !! MKK7 PR-071220 0.15 mg/ml; 60 µl; tagless; not yet fully tested !! MKK4 PR-070711 0.8 mg/ml; 12 µl; HIS-tagged; not yet fully tested !! MEK5 PR-070522 0.2 mg/ml; 50 µl; GST-tagged; not yet fully tested !! ERK5 PR-071017 0.3 mg/ml; 30 µl; tagless; not yet fully tested !! MAPKAPK5 PR-071218 0.14 mg/ml; 70 µl; tagless; not yet fully tested !! MAPKAPK2 PR-071121 0.12 mg/ml; 80 µl; tagless; not yet fully tested !!

8.3 Erklärung

Hiermit erkläre ich, dass ich die Dissertation mit dem Titel „Regulation and Function of ERK3/MK5-mediated Signaling“ selbstständig verfasst habe. Alle benutzten Hilfsmittel sowie evtl. zur Hilfeleistung herangezogene Institutionen sind vollständig angegeben.

Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten oder anderer Personen in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Ich habe die Dissertation in der Arbeitsgruppe Gaestel / Kotlyarov im Institut für Physiologische Chemie an der Medizinischen Hochschule Hannover angefertigt.

Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen ähnlichen Zweck zur Beurteilung eingereicht.

Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig und der Wahrheit entsprechend gemacht habe.

Hannover, den

(Unterschhrift)

Name: Frank Brand

8.4 Danksagung

Herrn Prof. Dr. Matthias Gaestel danke ich für die Möglichkeit diese Arbeit in seinem Labor anfertigen zu können, seiner steten Diskussionsbereitschaft und im Besonderen für die große Unterstützung bei dem Verfassen des Manuskripts für eine mögliche Veröffentlichung meiner Daten.

Bei Herrn Prof. Dr. Ernst Ungewickell möchte ich mich ganz herzlich für die übernommene Zweitbetreuung bedanken.

Herrn Dr. Alexey Kotlyarov möchte ich danken, für die stets kritische Betrachtung der erhaltenen Ergebnisse sowie zahlreichen Ideen für weitere Experimente.

Dr. Shashi Kant und Stefanie Schuhmacher möchte ich danken für die geleistete Vorarbeit, ohne die dieses Thema nicht zu Stande gekommen wäre.

Prof. Dr. Stefan Britsch, Dr. Ruth Simon und Jacqueline Andratschke möchte ich für die Zusammenarbeit bei den neuronalen Untersuchungen der MK5-knockout-Tiere sowie für die schöne Zeit, die ich in Ulm hatte, vielmals bedanken.

Allen anderen ehemaligen und derzeitigen Mitarbeitern der Arbeitsgruppe, Anastassiia, Christopher, Dorothee, Inga, Jessica, Juri, Manoj, Mohammad, Natascha und Tatiana, möchte ich für die zahlreichen Diskussionen, die angenehme Atmosphäre und emotionale Stütze danken.

Auch bei den restlichen Mitarbeitern der Physiologischen Chemie möchte ich mich für die hervorragende Arbeitsatmosphäre und die Arbeitsgruppen-übergreifenden Hilfe danken.

Natürlich möchte ich mich auch bei meiner Familie und bei all meinen Freunden bedanken, die mir bei der Entscheidung zur Promotion bei Seite standen und mir den nötigen Rückhalt gaben.

Veröffentlichung:

Brand F, Klutz AM, Jacobson KA, Fredholm BB, Schulte G. Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC. Eur J Pharmacol. 2008 Aug 20;590(1-3):36-42

Brand F, Schumacher S, Kant S, Menon MB, Simon R, Britsch S, Gaestel M, and Kotlyarov A. The ERK3-MK5 signalling complex regulates septin function and dendrite morphology. Manuscript submitted.

Hannover, den

(Unterschhrift)

Name: Frank Brand