Phosphoproteome Dynamics Reveal Novel ERK1/2 MAP Kinase Substrates with Broad Spectrum of Functions

Molecular Systems Biology 9; Article number 669; doi:10.1038/msb.2013.25 Citation: Molecular Systems Biology 9:669 www.molecularsystemsbiology.com

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions

Mathieu Courcelles1,2,6, Christophe Fre´min1,6, Laure Voisin1,6,Se´bastien Lemieux1,3, Sylvain Meloche1,4,* and Pierre Thibault1,2,5,*

1 Institute for Research in Immunology and Cancer, Universite´ de Montreál, Montreal, Quebec, Canada, 2 Department of Biochemistry, Universite´ de Montreál, Montreal, Quebec, Canada, 3 Department of Informatics and Operational Research, Universite´ de Montreál, Montreal, Quebec, Canada, 4 Department of Pharmacology, Universite´ de Montreál, Montreal, Quebec, Canada and 5 Department of Chemistry, Universite´ de Montreál, Montreal, Quebec, Canada 6These authors contributed equally to this work. * Corresponding authors. S Meloche or P Thibault, Institute for Research in Immunology and Cancer, Universite´ de Montreal, P.O. Box 6128, Station Centre-Ville, Montreál, Quebec, Canada H3C 3J7. Tel.: þ 1 514 3436966; Fax.: þ 1 514 3436843; E-mail: [email protected] or Tel.: þ 1 514 3436910; Fax: þ 1 514 3436843; E-mail: [email protected]

Received 22.12.12; accepted 18.4.13

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases. Molecular Systems Biology 9: 669; published online 28 May 2013; doi:10.1038/msb.2013.25 Subject Categories: proteomics; signal transduction Keywords: bioinformatics; cell signaling; MAP kinases; phosphoproteomics; phosphorylation dynamics

Introduction being evaluated clinically for cancer therapy and other indications (http://clinicaltrials.gov). The ubiquitously expressed serine/threonine protein kinases ERK1/2 are multifunctional protein kinases that phospho- extracellular signal-regulated kinase 1 (ERK1) and 2 are rylate target substrates within the minimal consensus motif effector components of the prototypical ERK1/2 mitogen- Ser/Thr–Pro, with a preference for proline at 2 position activated protein (MAP) kinase signaling pathway. ERK1 and (Gonzalez et al, 1991; Songyang et al, 1996). Although ERK2 transduce signals from a wide variety of mitogens, many substrates of ERK1/2 have been reported to date cytokines and differentiation factors to regulate biological (Yoon and Seger, 2006), only a fraction of these have been processes such as cell proliferation, cell-fate specification, adequately validated biochemically. More recent phospho- survival, morphogenesis and immune responses (Pearson proteomics studies have led to the identification of additional et al, 2001). Their activation is catalyzed by the dual-specificity candidate ERK1/2 substrates (Kosako et al, 2009; Old et al, MAP kinase kinases MEK1 and MEK2, which are themselves phosphorylated and activated by upstream MAP kinase kinase 2009; Pan et al, 2009; Carlson et al, 2011), but yielded limited kinases such as Raf (Cobb and Goldsmith, 1995; Cuevas et al, information on the dynamic changes in phosphorylation of the 2007). ERK1 and ERK2 are the only known physiological corresponding substrates. Moreover, very limited overlap of substrates of MEK1/2 (Pearson et al, 2001). Deregulated candidate ERK1/2 substrates was observed between these activity of ERK1/2 is causally associated with neuro–cardio– studies, indicating that these analyses are far from saturation. facial–cutaneous developmental syndromes and with a range Thus, despite the physiological importance of the ERK1/2 of human diseases including cancer, inflammatory disorders MAP kinase pathway and its pharmacological relevance, and neurodegenerative diseases (Schubbert et al, 2007; a comprehensive picture of the substrate repertoire and Lawrence et al, 2008; Tartaglia and Gelb, 2010). Consequently, biochemical reactions regulated by ERK1/2 protein kinases is small molecule inhibitors of the ERK1/2 pathway are currently still lacking.

& 2013 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2013 1 Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

In a systematic effort to identify novel substrates of ERK1/2, phosphorylation events downstream of ERK1/2 kinases, we have capitalized on advances in mass spectrometry (MS) biological triplicates from two populations of rat intestinal instrumentation, quantitative phosphoproteomics and bioin- epithelial cells (IEC-6) were stimulated for 0, 5, 15 or 60 min formatics to measure the dynamic changes in phosphorylation with fetal bovine serum in the presence or absence of of ERK1/2 consensus sequences in response to cell stimulation PD184352. PD184352 is a potent, ATP non-competitive and pharmacological inhibition of MEK1/2 in intestinal inhibitor of MEK1/2 that displays extremely high selectivity epithelial cells. This approach enabled the identification of over a large panel of protein kinases (Bain et al, 2007). When 155 candidate ERK1/2 substrates, of which only 12 proteins used at a low concentration of 2 mM, PD184352 inhibits MEK1/ correspond to previously validated ERK1/2 phosphorylation 2 without affecting the activity of the related MKK5 kinase sites. A subset of these putative substrates was selected and (Mody et al, 2001). Under these conditions, PD184352 almost directly phosphorylated by ERK1 in vitro on the predicted sites, completely inhibits serum-stimulated ERK1/2 activation thereby validating our analysis. Candidate ERK1/2 substrates (Supplementary Figure S1). Proteins from cytosolic and are involved in a broader than appreciated range of pathways nuclear fractions were digested with trypsin and phosphopep- and biological processes. Among the newly identified ERK1/2 tides were enriched on TiO2 microcolumns. Phosphopeptides substrates is the transcriptional factor JunB. We show that were then separated into five different SCX fractions and ERK1 phosphorylates JunB on Ser256 close to the DNA- analyzed by LC-MS/MS on a hybrid LTQ-Orbitrap mass binding domain, resulting in increased DNA-binding affinity spectrometer. Label-free quantitation was used to profile the and transcriptional activity of JunB/c-Fos-activating protein-1 abundance of phosphopeptides that were checked manually (AP-1) complexes. Our findings considerably expand the for accuracy (Marcantonio et al, 2008; Trost et al, 2009). To spectrum of cellular functions under control of the ERK1/2 identify candidate ERK1/2 substrates, additional filtering was MAP kinase pathway. applied to the list of phosphopeptides to select sites within ERK1/2 consensus motifs that display statistically significant changes in abundance upon cell stimulation and MEK1/2 Results inhibition (Figure 1B). We identified 7936 unique phosphorylation sites on 1861 Global proteomic analysis of dynamic proteins, of which two-third represent high-confidence assign- phosphorylation profiles ment with a localization probability of at least 0.75 (Figure 1C We used a quantitative phosphoproteomics approach to and D and Supplementary Table S1). All identifications are measure the dynamics of site-specific phosphorylation available online in ProteoConnections (Courcelles et al, 2011). on a global scale (Figure 1A). To specifically profile The distribution of pS, pT and pY sites was 80, 18, 2%,

IEC-6 cells Serum Cellular Tryptic Phosphopeptides 2D-nano LC stimulation fractionation digestion enrichment TiO2 MS/MS 0 Cytosol Control 5 LTQ- Orbitrap Treated 15 Nucleus MEK1/2 inhibitor 60 (PD184352) n =3 min

Phosphopeptides Label-free quantification Filter for minimal Filter-expected identification with intensity normalization ERK1/2 consensus kinetic profiles

P SP

TP Time Abundance Serum stimulation MEK1/2 inhibition

2500 2% 1.7% 0.3% Proteins 2531 1500 Phosphoproteins 1861 1P 10 100 18% S 6358 17%

Unique peptides 17 876 sites # 500 2P 2177 Unique phosphopeptides 12 529 T 1405

Phosphorylation 0 3P 211 Unique phosphosites 7936 80% Y 173 81% Known phosphosites 1707 020 40 60 80 100 ≥ 4P 37 Localization confidence (%) Figure 1 Experimental workflow and data processing for the identification of candidate ERK1/2 substrates. (A) Experimental workflow for sample processing and MS analysis. (B) Data analysis for the selection of candidate ERK1/2 substrates. (C) Statistics on the number of identified phosphopeptides. (D) Distribution of site-localization confidence data. (E) Distribution of phosphorylated amino acids. (F) Number of phosphorylation sites per peptide.

2 Molecular Systems Biology 2013 & 2013 EMBO and Macmillan Publishers Limited Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

respectively (Figure 1E), and peptides were predominantly Pak1, Pxn, Rai14, Rbm9, Stmn1 and Tpr (Supplementary singly and doubly phosphorylated (Figure 1F), consistent Figure S5). A further 15 proteins have been reported as with previous reports (Pan et al, 2008). We obtained the ERK1/2 substrates but either the phosphorylation site was not dynamic profile of 3015 and 5222 phosphopeptides from the mapped, or a distinct site was identified from these studies cytosol and nuclear fractions, respectively (Supplementary (Supplementary Figure S6). This analysis provided a first level Table S2). The reproducibility of abundance measurements of validation of our approach. We next performed gene from our label-free quantitative procedure was evaluated ontology (GO) analyses on candidate ERK1/2 substrates to to determine a fold-change cutoff above technical and identify functional terms previously reported for these proteins biological variability. The s.d. of these measurements was (Supplementary Table S4, Figure 2B and C). Several proteins 37, and 95% of phosphopeptides showed less than twofold were found with related GO terms: regulation of transcription change across biological replicates (Supplementary Figure S2). (27 proteins, GO:0006355), nucleic acid metabolic process Using a cutoff of twofold change with a P-value of 0.05 (24 proteins, GO:00090304), signal transduction (16 proteins, (two-tailed Student’s t-test), 2510 phosphopeptides displayed GO:0007165), RNA processing (14 proteins, GO:0006396), a significant change in abundance for at least one time cytoskeleton organization (9 proteins, GO:0007010), point of stimulation. We next summed the abundance chromatin organization (8 proteins, GO:0006325), apoptotic changes of the four time points using Slog10(PD184352/ process (6 proteins, GO:0006915) and cell cycle (7 proteins, control) to determine the overall impact of PD184352 GO:0007049). The analysis of cellular components ontology on all quantifiable substrates. A normal distribution showed that ERK1/2 substrates are enriched by 4.5-fold centered on zero was obtained for both cytosolic and nuclear for the term cell junction (14 proteins, GO:0030054) and extracts, indicating that MEK1/2 inhibition resulted in 10.5-fold for nuclear pore (3 proteins, GO:0005643). Interest- an even up- and downregulation of phosphorylated proteins ingly, we also noted a significant enrichment (410-fold) (Supplementary Figure S3). Many phosphopeptides bearing for two related categories associated with nuclear speckles the minimal ERK1/2 consensus motif were downregulated (5 proteins, GO:0016607) and spliceosomes (9 proteins, by PD184352 and thus represent candidate ERK1/2 substrates GO:0005681). Closer inspection of the data indicated that (see below). these two categories comprise members of the SR (serine/ arginine-rich) protein family associated with pre-mRNA splicing. Splicing factors are regulated extensively by phosphorylation (Stamm, 2008), and our results suggest that Phosphoproteome dynamics identify novel ERK1/2 kinases contribute to this regulation. candidate ERK1/2 substrates We used our phosphorylation data to generate a protein- We identified 2296 high-confidence phosphosites on 987 interaction network of the putative ERK1/2 substrates proteins that contained the minimal ERK1/2 consensus using the STRING database that integrates known and motif. Selection of phosphopeptides whose relative predicted interactions from different sources. We combined abundance is regulated positively upon serum stimulation interactions found in rat and their human orthologs, resulting and negatively following MEK1/2 inhibition resulted in in a network of 146 proteins (nodes) and 242 connections a list of 155 candidate ERK1/2 substrates (232 phospho- (edges). This interaction network together with analyses of rylation sites) (Supplementary Table S3 and Figure 2A). functional protein annotations highlighted different Representative stimulation and inhibition temporal profiles subsets of proteins associated with RNA binding, nuclear of regulated phosphopeptides were grouped using fuzzy trafficking, MAPK signaling cell junction membrane, spindle c-means clustering into six groups (arbitrary chosen number) assembly and signaling transcription factors (Figure 3). Inter- to graphically report the general trends of the selected acting proteins identified in this study are highlighted in candidates (Supplementary Figure S4). Of these 232 orange while red circles represent substrates validated in phosphorylation sites, 49 sites (21%) contained the optimal subsequent biochemical assays (see below). Interestingly, we P-X-(S/T)-P ERK1/2 phosphorylation consensus motif. identified several RNA-binding proteins that were regulated in Bioinformatics analyses revealed that 94% of the sites with response to serum activation and inhibition with PD184352, their neighboring proline are conserved in either human or including Hnrnp C, Ddx47 and different Ser/Arg-rich splicing mouse. factors involved in the early steps of spliceosome assembly Binding to docking sites such as the D or DEF domain and pre-mRNA splicing. Several potential substrates modu- confers additional specificity to ERK1/2 kinases (Jacobs et al, lated by ERK1/2 were also involved in cell junction membrane 1999; Tanoue et al, 2000). We found that 22 of the potential signaling such as Mllt4, Tjp1, Tjp2 and Cttn that have ERK1/2 substrates (14%) contain a D domain, whereas one important roles in tight and adherens junctions and in the contains a DEF domain (0.6%) (Supplementary Table S3). In regulation of cell migration. One notable feature of this comparison, 17% (278) and 0.4% (11) of all the phosphopro- network is that out of the 155 candidate ERK1/2 substrates, teins identified in this study have a D or DEF domain, 34 (22%) are directly interconnected in the STRING network. respectively, within 20 amino acids of the phosphorylated site. This high degree of connectivity suggests that ERK1/2 are not This indicates that the putative ERK1/2 substrates are not phosphorylating proteins randomly in the cell but often enriched for these docking domains. regulate members of the same functional pathway or protein The filtered list included 13 previously identified and complexes. biochemically validated ERK1/2 phosphorylation sites present We also noted that changes in the phosphorylation status of on 12 substrates: Bat2, Cttn, Fam129b, Gja1, Hnrnph2, Lima1, several kinases and regulators acting upstream of ERK1/2,

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A Cytosol B 3

Regulation of transcription Purb JunB Ptrf Znf148 Nab1 Prdm2 Rai1 Arid5b Adnp2 2 Signal transduction Rras2 Map2k2 Dab2ip Stxbp4 Sptbn1 Nedd4 Phrf1 ) i RNA processing Ccnl1 Ccnl2 Exosc5 Hnrnpd Pop1 Ddx47 Sfrs2 Ints3 Rnps1

1 Proetin modification process Ptpn12 Pak1 Map2k2 Prpf4b Trip12 Pik3r4 Sptbn1 /DMSO t i Cytoskeleton organization Cdc42ep2 Sipa1l1 Dync1li1 Stmn1 Map1b Chromatin organization Smarca5 Rsf1 Cdyl Setd2 Mcm2 Phf2 Hmg20a 0 Cell cycle Gas2l1 Dync1li1 Mki67 Specc1l Numa1 Cttn

(PD184352 t Apoptotic process Dab2ip Ddx47 Acin1

10 Pxn Stmn1 –1 Pak1 Cell proliferation JunB Ctnnb1 Mki67 log Σ Legend Cell-cell junction organization Gja1 Plekha7 Tjp1 Phosphopeptide Fam129b –2 [pST]P site Translation Rps27 Tpr Candidate ERK1/2 substrate Known ERK1/2 substrate 0 5 10 15 20 25 30 Number of substrates –6 –4 –2 0 2 46 Σ log10(Stimulated t5,15,60 min/Control t0 min)

Nucleus

C Nucleus Cytoplasm

) 2 i Membrane Cytoskeleton

/DMSO t Cell junction i Nucleolus 0 Hnrnph2 Spliceosome

Tpr Chromatin Bat2

(PD184352 t Golgi apparatus

10 Gja1 Rbm9 Nuclear speck –2 log

Σ Rai14 Lima1 Centrosome Nuclear pore Mitochondria –4 Endoplasmic reticulum –6 –4 –2 024 6 0102030405060 70 80 Σ log10(Stimulated t5,15,60 min/Control t0 min) Number of substrates Figure 2 Dynamic changes of protein phosphorylation identify ERK1/2 substrates from cytosolic and nuclear compartments. (A) Two-dimensional representation of quantitative changes in protein phosphorylation following serum stimulation and PD184352 treatment. Each point corresponds to a different phosphopeptide. Candidate ERK1/2 substrates are shown as dark blue circles in the bottom right quadrant and represent phosphopeptides displaying increase in phosphorylation upon serum stimulation and decrease upon PD184352 treatment (two-tailed t-test). Note that a few peptides with (pS/T)-P sites in this quadrant did not have significant abundance change and were not retained as ERK1/2 candidates. Yellow circles correspond to previously known ERK1/2 substrates. (B, C) GO analysis. Candidate ERK1/2 substrates were annotated and classified according to (B) biological process or (C) cellular component GO terms. P is the P-value of Fisher’s exact test and E is the calculated odds ratio for category enrichment. Only E-values above 1 are presented. A subset of proteins belonging to each category is shown. Substrates validated by in vitro kinase assays are shown in red. such as Pak1, Raf1, Map3k1, MEK1, MEK2, Ksr1, Rras2 and Validation of the site-specific phosphorylation of Ywhae (14-3-3 epsilon), were detected in cells treated with candidate substrates by ERK1 in vitro PD184352 (Figure 4). For example, we observed an increase in To further validate the list of candidate ERK1/2 substrates, we the activation loop phosphorylation of MEK1 and MEK2, and selected six unrelated proteins of various functions in Ser621 phosphorylation of Raf1. This observation is (Supplementary Table S5 and Figure 5A) that were purified consistent with the known participation of ERK1/2 in multiple negative feedback regulatory loops in stimulated cells (Ramos, as recombinant GST-fusion proteins from bacteria for sub- 2008; Fritsche-Guenther et al, 2011). Deciphering the complex sequent in vitro kinase assays. In the case of MEK2, the network of signaling events and feedback loops that modulate catalytically inactive GST-MEK2K101E mutant was used to the activity of upstream ERK1/2 regulators upon pharmaco- prevent autophosphorylation of the kinase. All selected logical inhibition of the pathway will be important for proteins were efficiently phosphorylated by recombinant understanding the underlying mechanisms of resistance to active ERK1 in vitro (Figure 5B). In each case, alanine Raf and MEK1/2 inhibitors in the clinic. substitution of the identified phosphorylation residue

4 Molecular Systems Biology 2013 & 2013 EMBO and Macmillan Publishers Limited Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

RNA-binding Nuclear-trafficking proteins Sfrs5 Hnrpr Rae1 nucleoproteins Sfrs4 Sfrs1 Sfrs2 Sfrs11 Sfrs3

Cpsf1 Pom210 Nfx1 Bub1b Sec13 Sfrs6 Nxf1

Ddx23 Hnrnpc Nup153 Nup93 Nup35 Nup50

Tpr Nup107 Nup155 Nupl1 Pabpn1 Rnps1 Nup98 Xpo1 Rangap1 Nup214 Nup54 Nup188

Kif2c Sfrs9 Sf3b3 U2af2 Snrp70 Ncbp1 Sf3a2 Prpf8 Srrm1 Pnn Ranbp2 Nup85 Nup88 Nup37

Cdc20

Ahctf1 Srrm2 Bub3

Dync1li1 Prnp Sf3b2

Ddx47

Snrpd2 Sf3b1 Sf3b4

Incenp

Cpsf7 Ascc3l1

Prpf6 Phf5a

Snrpb2 rpB2

Eftud2 Snrpa Sf3a3

MAPK signaling Cell junctions

Rac1 Membrane signaling Rras2 Lmo7 Nck1

Mllt4 Plekha7 Zyx Rsf1 Src

Limk1 Pak1 Map2k2 Map2k1 Gja4

Gja1 Tjp1 Ctnnb1 Smarca5 Pak2 Fyn Cttn Gja5 Tjp2 Mll2 Lima1 Npm1

Transcription factors Phosphoinositides Microtubules Spindle assembly

Mpak8 Smarca4 Stat3 Cdipt Mtap1a Tubgcp3 Fosb Pik3c2a Nfatc3 Pip5k1c Map1lc3b

Map1b Numa1 Tubgcp2 Batf Junb Fosl1 Pi4k2a Pip5k1b Pik3c2g

Hnrpd Smad3 Fos Lrp8 Pip5k1a Pik3c2b Fosl2 Il2 Nfatc1

Figure 3 Networks of ERK substrates. Identified substrates were overlaid on interaction network from the STRING database (http://string.embl.de). Only high- confidence interaction from experiments or databases were extracted for rat and complemented with human data set. Orange and red circles represent substrates identified and validated in the study, respectively. Major subnetworks concern splicing, nuclear trafficking and nucleoproteins, MAPK signaling, cell junctions and membrane signaling. Binary interactions were removed. Candidate ERK1/2 substrates show a high degree of connectivity (34/155 are interconnected).

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Legend consistent with a functional role (Figure 6B). The regulation of Site phosphorylation Sos1 Grb2 Rac1 AP-1 factors is complex and occurs through multiple mechanisms Σ log (PD184352 treated / control) 10 (Karin et al, 1997). We ﬁrst examined whether Ser256 phospho- –1.5 1.5 rylation could modulate the afﬁnity of JunB for its heterodimeric partner c-Fos. No apparent difference in the binding of JunB Rasd1 Rras2 Pak1 phosphorylation-site mutants to c-Fos was observed in reciprocal Ywhae co-immunoprecipitation experiments (Supplementary Figure S8). Mutations of Ser256 and Thr252 also did not affect the nuclear

MAP3K Raf1 Mos Map3k1 Map3k8 localization of JunB (Supplementary Figure S9). Interestingly, a recent study has suggested that phosphorylation of human JunB

Ksr1 on Ser259 (equivalent to mouse/rat Ser256) by an unidentified kinase primes the phosphorylation of Ser251 and Thr255 (mouse/ MAP2K Mek1 Mek2 rat Thr252) by GSK3b, to create a Fbxw7 phospho-degron that targets JunB for degradation in G2 phase (Perez-Benavente et al, 2012). However, cycloheximide-chase experiments failed to reveal any impact of Ser256 and Thr252 phosphorylation on Erk1 Erk2 MAPK the stability of JunB in exponentially proliferating NIH 3T3 cells (Supplementary Figure S10). Figure 4 Phosphorylation changes in the upstream regulators of ERK1/2 MAP Ser256 is adjacent to the DNA-binding domain of JunB kinases. Identified phosphoproteins (round circles) were mapped on the interaction network from the STRING database. A color gradient is used to (Figure 6B). To assess whether phosphorylation of Ser256 represent the modulation (summed log fold change) of each individual could affect the DNA-binding properties of JunB-containing phosphorylation site following treatment with PD184352. Protein nodes with AP-1 complexes, we measured the binding of JunB/c-Fos multiple phosphorylation sites are represented using a pie chart where each slice heterodimer to TPA-response elements (TREs) and cAMP- corresponds to the abundance change of a unique phosphorylated site. responsive elements (CREs) by electrophoretic mobility shift assay (EMSA). As previously documented (Nakabeppu et al, markedly reduced phosphorylation, confirming the site- 1988), we found that JunB expressed alone binds weakly to the specific assignment. The identity of the in vitro phosphory- TRE, though its affinity for DNA is greatly enhanced by co- lated residue was further confirmed by MS analysis expression of c-Fos (Figure 7 A–D). Inhibition of MEK1/2 (Figure 5C). Additional studies revealed that Numa1 is also activity by PD184352 markedly impaired the binding of AP-1 phosphorylated on Thr2015 by ERK1. Together, these results complexes to their target sequences, suggesting a role of validate the predictive potential of our quantitative phospho- ERK1/2 signaling in regulating the binding of JunB/c-Fos proteomics approach to identify novel ERK1/2 substrates. complexes to DNA (Figure 7A and B). Notably, mutation of Ser256 to alanine in JunB significantly decreased the DNA binding of JunB/c-Fos dimer. As Thr252 is also located in the ERK1/2 phosphorylate JunB on Ser256 in vitro and vicinity of the JunB DNA-binding domain, we further tested in vivo the impact of a Thr-to-Ala substitution, alone or in combina- tion with the Ser-to-Ala mutation at position 256. Whereas the One candidate of interest was JunB, a component of the AP-1 single mutation of Thr252 had a weak effect, the double family of transcription factors (Angel and Karin, 1991). The mutation severely compromised the DNA-binding activity of phosphorylation of Ser256, a residue that lies in a full ERK1/2 the AP-1 complex in a concentration-dependent manner consensus sequence, was markedly downregulated after (Figure 7C and D). Essentially similar results were obtained PD184352 treatment (Figure 5A). In vitro kinase assays using a CRE DNA probe (Supplementary Figure S11). confirmed that ERK1 directly phosphorylates JunB on Ser256 We next evaluated the impact of Ser256 phosphorylation on (Figure 4B and C). Notably, Thr252, which is contained in the the transcriptional activity of JunB complexes using the -73- same tryptic peptide and also lies in a minimal ERK1/2 motif, Col-Luc reporter plasmid. As reported previously (Chiu et al, was not phosphorylated by ERK1 in vitro, highlighting the 1989), expression of JunB alone failed to activate the specificity of these kinases. To determine whether Ser256 of collagenase promoter, while c-Fos slightly increased reporter JunB is phosphorylated by ERK1/2 in vivo, we used a activity (Figure 7E). However, co-expression of JunB with commercial phospho-specific antibody. We first ensured that c-Fos potentiated its transcriptional activity, consistent with the antibody was specific for Ser256 (Supplementary Figure the increase in DNA-binding affinity. This effect was com- S7). Stimulation of cells with the ERK1/2 activator phorbol 12- pletely abolished by treatment with the MEK1/2 inhibitor myristate 13-acetate (PMA) induced the phosphorylation of PD184352 (Figure 7E), arguing for a key role of ERK1/2 endogenous JunB on Ser256, and this signal was abolished by signaling in the transcriptional activation of JunB/c-Fos treatment with PD184352 (Figure 6A). heterodimers. Importantly, alanine substitution of Ser256 and Thr252 impaired the stimulatory effect of JunB on c-Fos transactivation, whereas the single mutants had weaker and ERK1/2 phosphorylation of JunB potentiates DNA more variable effects (Figure 7F). Taken together, these results binding of c-Fos/JunB heterodimers strongly suggest that ERK1/2 phosphorylation of JunB on We next investigated the impact of Ser256 phosphorylation on Ser256 is necessary for the full transcriptional activity of JunB/ JunB regulation. Ser256 is conserved among vertebrates, c-Fos AP-1 complexes.

6 Molecular Systems Biology 2013 & 2013 EMBO and Macmillan Publishers Limited Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

Ddx47 - S9 + ERK1 100 AADEE PDsPTEALQTAA EEEETK 5.0 Mascot score: 65 WT WT S9A 4.8 97 Coomassie 66 50

(intensity) 4.6 10

4.4 97 Intensity (%) Log Autoradiograph 66 4.2 0 0 10 20 30 40 50 60 500 1000 1500 2000 Time (min) m/z

Hmg20a - S105 + ERK1 100 DSN A PKsPLTGYVR 5.5 Mascot score: 42

5.0 WT WT S105A 50 (intensity) Coomassie 10 4.5 66 Intensity (%) Log

4.0 Autoradiograph 66 0 0 10 20 30 40 50 60 200 400 600 800 1000 1200 1400 Time (min) m/z

Map2k2 - S295 100 + ERK1 ELE A SFG R PVV DGADGEPHSVsPR 5.0 Mascot score: 48 4.8

4.6 WT WT S295A 50 (intensity) 4.4 66

10 Coomassie Intensity (%)

Log 4.2 66 Autoradiograph 4.0 0 0 1020304050 60 200400 600 8001000 1200 1400 1600 1800 Time (min) m/z

Numa1 - S1751 + ERK1 2+ 6.4 100 TQ PD GTSVPGEPA sPISQR 6.2 Mascot score: 91 m/z: 1002.45 6.0

(intensity) 5.8 50 10

5.6 WT WT S1757A T2015A S1757A/T2015A Intensity (%) Log 5.4 66 Coomassie 0 0 102030405060 Autoradiograph 200400 600 800 12001000 1400 1600 1800 2000 66 Time (min) m/z

Tjp1 - S390 P O 100 QTPSLPEPKPVYAQVGQ PD VDLPV s PSD GVLPN STHED GILRPSMK 5.0 + ERK1 Mascot score: 68 4.8 4.6 WT WT S390A 50 (intensity) 97 Coomassie 10 4.4 Intensity (%)

Log 4.2 97 Autoradiograph 4.0 0 0 1020304050 60 200400 600 8001000 1200 1400 1600 1800 2000 Time (min) m/z

Junb - S256 + ERK1 6.0 100 DAT PPVsPINMEDQER Mascot score: 74 5.8

5.6

(intensity) 50 WT WT T252A S256A T252A/S256A 10

5.4 66 Coomassie Intensity (%) Log

5.2 66 Autoradiograph 0 0 1020304050 60 200400 600 8001000 1200 1400 1600 1800 Time (min) m/z Figure 5 Validation of ERK1/2 substrates. (A) Phosphorylation kinetic proﬁles of selected ERK1/2 substrates in serum-stimulated cells treated (red) or not (blue) with the MEK1/2 inhibitor PD184352. (B) In vitro kinase assays of wild-type and alanine mutant candidate substrates with recombinant active ERK1. (C) MS/MS analysis of selected ERK1/2 substrates phosphorylated by recombinant active ERK1 in vitro. Source data for this ﬁgure is available on the online supplementary information page.

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PMA PMA+PD DNA-binding 0 30 120 30 120 Time (min) 1 domain 344 mJunB P-S256 270 289293 321 JunB Fos interaction JunB domain (leucine zipper)

P-ERK1 252 256 P-ERK2 Mus musculus PQTVPEARS-----R-DATPPVSPINMEDQERIKVE Homo sapiens PQTVPEARS-----R-DATPPVSPINMEDQERIKVE PQTVPEARS-----R-DATPPVSPINMEDQERIKVE ERK1 Rattus norvegicus PQTVPEARS-----R-DATPPVSPINMEDQERIKVE ERK2 Bos taurus Xenopus laevis PQTVPDTAGVRVDSREGSPAPMSPINMEEQEKIKVE PQTVPDLQS-----S-DGSPPMSPIDMEDQERIKAE α-tub Danio rerio

Figure 6 ERK1/2 phosphorylate JunB on Ser256 in vivo.(A) NIH 3T3 cells were stimulated with PMA in the presence or absence of PD184352. Lysates were analyzed by immunoblotting with a speciﬁc anti-JunB (Ser256) antibody. (B) Schematic representation of mouse JunB structure. Source data for this ﬁgure is available on the online supplementary information page.

Discussion Prpf4b and Pnn, suggests an important and hitherto under- estimated role of ERK1/2 signaling in this process. Several The involvement of the ERK1/2 MAP kinase signaling path- regulators of chromatin organization, such as the high- way in a myriad of cellular processes suggests that many more mobility group protein Hmg20a, the ISWI chromatin remodel- ERK1/2 substrates remain to be discovered. Recent phospho- ing subunit Rsf-1 and the H3K56 methyltransferase Setd2 were proteomics studies of variable scales have used label-free (Old identified. Other potential ERK1/2 substrates are involved in et al, 2009), stable isotope labeling with amino acids in cell cellular junction assembly, mitotic spindle maintenance, or the et al culture (SILAC) (Pan , 2009), two-dimensional-difference DNA damage response. Additional validation efforts will be gel electrophoresis (Kosako et al, 2009) or analog-sensitive required to demonstrate that these proteins are physiological ERK2 kinase (Carlson et al, 2011) technologies to identify substrates of ERK1/2 and to define the biochemical conse- candidate ERK1/2 substrates. Noticeably, a comparative quence of these phosphorylation events. analysis of these studies has revealed very little overlap in In this study, we focused on the regulation of the AP-1 factor potential ERK1/2 substrates (Figure 8). This can be explained JunB. The AP-1 transcription factor is a dimeric basic region- in part by the selection of cell lines with distinct histological leucine zipper (bZIP) complex that comprises members of the origins and by the use of different proteomics approaches. Jun (c-Jun, JunB, JunD), Fos, Atf and Maf protein families However, this also indicates that these analyses have not (Angel and Karin, 1991; Chinenov and Kerppola, 2001). Jun exhaustively mined the repertoire of ERK1/2 substrates, as can form homodimers or heterodimers with other AP-1 factors only a small fraction of them has been identified so far. to recognize different response elements in the enhancers of Here, we have used a robust quantitative phosphoproteo- target genes. Jun proteins have been implicated in a wide mics approach to comprehensively profile dynamic changes in variety of biological functions, including cell proliferation, protein phosphorylation on a global scale. The selection of differentiation, survival, embryonic development and tumor- phosphopeptides with a consensus ERK1/2 phosphorylation igenesis (Jochum et al, 2001; Shaulian and Karin, 2002). The motif that display statistically significant changes in phos- regulation of Jun-containing AP-1 factors is complex and phorylation upon stimulation and inhibition of ERK1/2 occurs at multiple levels: transcription of AP-1 genes, protein signaling enabled the identification of 155 candidate ERK1/2 stability, transcriptional activation by phosphorylation and substrates, of which 128 represent new targets. This represents dimer composition (Karin et al, 1997; Eferl and Wagner, 2003). the largest list of ERK1/2 substrates identified in a single study While the mechanisms of activation of c-Jun have been (Figure 8). Notably, we could associate each substrate extensively studied, much less is known about the regulation identified to a specific dynamic profile of phosphorylation by of JunB. JunB is phosphorylated at Thr102 and Thr104 by the ERK1/2 kinases that will facilitate future studies by other c-Jun N-terminal kinases subfamily of MAP kinases, which investigators to analyze the role of these phosphorylation promotes its transcriptional synergy with c-Maf to activate the events (Supplementary Table 3). Interestingly, these profiles interleukin 4 promoter (Li et al, 1999). It has also been were quite variable depending on the nature of the substrate reported that JunB phosphorylation by cyclin B-Cdk1 on itself (Supplementary Figure S4). In addition to the known Ser23, Thr150 and Ser186 triggers its degradation in mitosis ERK1/2 substrates, we have validated that a selected subset of (Bakiri et al, 2000). A more recent study has proposed that candidates are directly phosphorylated by ERK1 in vitro on the phosphorylation of Ser259 (Ser256 equivalent) primes the assigned site, confirming the predictive value of our data set to phosphorylation of Ser251 and Thr255 to target JunB identify bona fide ERK1/2 substrates. for degradation in G2 by a Fbxw7-dependent mechanism The list of candidate substrates considerably expands the (Perez-Benavente et al, 2012). The kinase responsible for the spectrum of biological functions regulated by the ERK1/2 MAP phosphorylation of Ser256 was not identified in that study. kinase pathway. For example, the identification of several Here, we validated that Ser256 of JunB is a bona fide proteins associated with pre-mRNA splicing, including SR physiological target of ERK1/2. Although we could not family members, heterogeneous nuclear ribonucleoproteins, document any impact of Ser256 phosphorylation on the

8 Molecular Systems Biology 2013 & 2013 EMBO and Macmillan Publishers Limited Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

Flag-c- Flag-c- Fos + Fos + Flag-c-Fos + (1:1) (1:2) HA-JunB pC3 Flag-c-Fos + Ab Flag Flag-c-Fos WT HA-JunB WT + Ab HA HA-JunB – – PD184352 12h PD184352 24h WT TRE probe +++++++–++ Mut. TRE probe –––––––+–– pC3 Flag-c-Fos Flag-c-Fos + Ab Flag Flag-c-Fos WT HA-JunB WT + Ab HA HA-JunB WT HA-JunB S256A HA-JunB T252A HA-JunB T252A/S256A HA-JunB WT HA-JunB S256A HA-JunB T252A HA-JunB T252A/S256A HA-JunB WT TRE probe +++–+++++++++++ Supershift Mut. TRE probe –––+–––––––––––

Supershift

Free probe Free probe

15 25 12 20 9 15 (fold) 6 (fold) 10 3 5 AP1-TRE complex 0 AP1-TRE complex 0 – +– + ++ Flag-c-Fos – + – ++++++++Flag-c-Fos – – + + ++ HA-JunB – – HA-JunB WT WT WT –– – – 12 h 24 h PD184352 S256AT252A S256AT252A

S256A/T252A S256A/T252A 1:1 ratio 1:2 ratio

* 6 8 * 5 7 6 4 5 3 4 (fold) (fold) 2 3 2 1 1 Luciferase activity Luciferase Luciferase activity Luciferase 0 0 – +++ ++ Flag-c-Fos ––+ + + Flag-c-Fos – – – + + + HA-JunB – HA-JunB WT – –+– – PD184352 S256A T252A

S256A/T252A Figure 7 ERK1/2 phosphorylation of JunB on Ser256 promotes cooperative DNA binding of JunB/c-Fos heterodimers. (A, C) HEK 293 cells were transfected with the indicated constructs, and nuclear extracts were analyzed by EMSA using a radiolabeled TRE probe. When indicated, cells were treated with PD184352 for 12 h or 24 h (A). Supershift analyses were performed by incubating nuclear extracts with Flag or HA antibody. Results are representative of three experiments. (B, D) Quantification of EMSA results in A and C by densitometric analysis. Results are normalized to control cells transfected with empty pcDNA3 and represent the mean±s.e.m. of three independent experiments. (E) HEC-1B cells were co-transfected with the indicated constructs and the -73-Col-Luc reporter and pRL-TK control plasmids. After 24 h, the cells were treated or not for 24 h with PD184352. Luciferase activity was measured using the Dual-Luciferase assay. *Po0.05. (F) HEC-1B cells were co-transfected with the indicated JunB constructs and -73-Col-Luc reporter activity was measured as above. Luciferase results are expressed as fold increase over the reporter alone and are representative of at least three independent experiments performed in triplicate. *Po0.05. All of the results are expressed as the mean and the s.e.m. of at least triplicate measurements. Student’s t-test was used to analyze the data. Source data for this figure is available on the online supplementary information page. stability of JunB in cycling cells, we provide strong evidence Taken together, our data provide one of the most comprehen- that Ser256 regulates the cooperative binding of JunB/c-Fos sive phosphoproteomics study to identify ERK1/2 substrates, heterodimers to DNA. Alanine substitution of Ser256 and and represent a unique resource to investigate new areas of Thr252 markedly reduced the binding of JunB/c-Fos hetero- ERK1/2 MAP kinase biology. dimers to TRE and CRE sequences, and prevented the synergistic transactivation of the collagenase promoter by JunB and c-Fos. Accordingly, pharmacological inhibition of Materials and methods ERK1/2 signaling with PD184352 abolished the transcriptional Cell culture activity of JunB/c-Fos heterodimers. Our results add another IEC-6 were grown to confluence in 150 mm Petri dishes, and made layer to the complex and fine regulation of AP-1 activity. quiescent by serum starvation for 24 h. The cells were then pre-treated This contribution presents the first phosphoproteome for 1 h with DMSO (control) or 2 mM PD184352 before stimulation with dynamic study of the ERK1/2 MAP kinase signaling pathway. 10% fetal bovine serum for 0, 5, 15 or 60 min.

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Old et al (2009) Pan et al (2009) Cell line: WW115 (human) Cell line: HeLa (human) Stimulation: B-Raf V600E Stimulation: EGF MEK1/2 inhibitor: U0126 MEK1/2 inhibitor: U0126 Chromatography: anion exchange Phosphopeptides enrichment: TiO2 Chromatography: offline SCX+reverse phase C Mono Q FPLC-reverse phase C18 18 Quantification: label-free Quantification: SILAC 7

2 2 0 0 0 0 18 7 1 2 2 0 3

2 0 5 0 0 0 0 Kosako et al (2009) 1 1 0 0 1 Cell line: NIH3T3 ΔB-Raf:ER (mouse) 0 Stimulation: 4-hydroxy-tamoxifen 1 12 MEK1/2 inhibitor: U0126 Phosphopeptides enrichment: IMAC 128 Quantification: 2D-DIGE 57 gel spot MS analysis

Carlson et al (2011) Courcelles et al Cell line: NIH 3T3-L1 (mouse) Cell line: IEC-6 (rat) Stimulation: EGF Stimulation: fetal bovine serum ATP analog-sensitive ERK2 kinase (in vitro) Kinetic: 0, 5, 15, 60 min Thiophosphate capture MEK1/2 inhibitor: PD184352 Phosphopeptides enrichment: IMAC Phosphopeptides enrichment:TiO2 Chromatography: reverse phase C 18 Chromatography: online SCX-reverse phase C18 Quantification: SILAC Quantification: label-free (3 replicates)

Figure 8 Comparative analysis of phosphoproteomics studies. Comparative analysis of the list of candidate ERK1/2 substrates identiﬁed from four previous phosphoproteomics experiments of variable scale with the present study. The comparison was done at the protein level, as the data set of Kosako et al (2009) did not report the position of the phosphorylated sites.

Reagents, antibodies and plasmids Cellular fractionation and protein extraction PD184352 was a gift from Pfizer. Commercial antibodies were obtained Biological triplicates were generated for MS analysis. Cells (5 108) from the following suppliers: anti-ERK1/2 CT from Upstate Biotech- were washed twice with ice-cold PBS, collected by scrapping, and nology; anti-phospho-ERK1/2(Thr202/Tyr204) and anti-JunB from lysed in lysis buffer (10 mM Tris–HCl, pH 8.4, 140 mM NaCl, 1.5 mM Cell Signaling Technology; anti-phospho-JunB(Ser256) and anti-Hsc70 MgCl2, 0.5% NP-40 (Calbiochem), 1 mM dithiothreitol (DTT), from Santa Cruz Biotechnology; anti-HA from Covance; anti-Flag and protease and phosphatase inhibitor added freshly). After centri- anti-a-tubulin from Sigma. fugation at 1000 g for 3 min at 4 1C, supernatants were transferred pcDNA3-Flag-c-Fos was obtained from Trang Hoang (Universite´ de to separate tubes (cytoplasmic fraction). The pellets were then Montreál). The pGEX-KG-JunB vector was constructed by subcloning resuspended in lysis buffer plus 1/10 of detergent stock (3.3% w/v the mouse JunB sequence from pBabe-JunB (obtained from Philippe sodium deoxycholate, 6.6% Tween 40), vortexed at slow speed, Roux, Universite´ de Montreál) into EcoRI/XhoI sites of pGex-KG vector. incubated on ice for 5 min, and centrifuged at 1000 g for 3 min The pcDNA3-HA-JunB vector was constructed by subcloning mouse at 4 1C. The supernatant was discarded again and the pellet, containing JunB sequence into EcoRI/XhoI sites of pcDNA3-HA. The pGEX-KG- the nuclei, was rinsed with lysis buffer and lysed in extraction DDX47 vector was kindly provided by Takeshi Sekiguchi (Kyushu buffer (20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM University, Japan). The pGEX-KG-Numa1 (amino acids 1605–2016 of EDTA, 25% glycerol) by sonication. Benzonase nuclease HC human protein) plasmid was a kind gift of Nai-Wen Chi (La Jolla, (Novagen) was added to digest nucleic acids and obtain the nuclear California). The pGEX-KG-MEK2(K101E) vector was constructed by fraction. Proteins were precipitated overnight with cold acetone subcloning the human MEK2 sequence into the EcoRI site of pGEX-KG. ( 20 1C) and resuspended in a solution of 1% SDS and 50 mM The kinase-dead K101E mutant of MEK2 was used in the in vitro kinase ammonium bicarbonate. Cytosolic and nuclear protein extracts assays to avoid autophosphorylation of MEK2. The pGEX-KG-Hmg20a were reduced for 20 min at 37 1C with 0.5 mM tris(2-carboxyethyl) vector was constructed by subcloning the human Hmg20a sequence phosphine (TCEP) (Pierce) and then alkylated with 50 mM from pCMV6-XL5-HMG20A (Origene) into XbaI/XhoI sites of pGEX iodoacetamide for 20 min at 37 1C. The excess of iodoacetamide was vector. The pGEX-KG-Tjp1 was constructed by subcloning the first 510 neutralized by the addition of 50 mM DTT. Proteins were quantified amino acids of human Tjp1 from pEGFP-C1-ZO1 (kind gift of Alan with Micro BCA Protein Assay (Pierce), diluted 10 times with Fanning, University of North Carolina) into EcoRI/XhoI sites of pGEX- 50 mM ammonium bicarbonate, and digested with sequencing- KG. pRL-TK and -73 Col-Luc were obtained from J Hiscott (McGill grade trypsin (1:100) (Promega) overnight at 37 1C with agitation. University, Montreal). All mutations and PCR products were verified Tryptic digests were acidified below pH 4.0 with trifluoroacetic acid by DNA sequencing. Sequence of primers used for PCR and details (TFA) to inactivate trypsin and dried in a SpeedVac apparatus about cloning strategies are available upon request. (Thermo).

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Phosphopeptides enrichment and MS This information indicates how similar a profile is compared with the rest of the cluster members. Phosphopeptides (1 mg/replicate) were enriched as described (Thingholm et al, 2006) on home-made TiO2 affinity columns (1.25 mg Titansphere, 5 mm, GL Sciences) and eluted with 30 mlof 1% ammonium hydroxide. Eluates were acidified with 1 mlTFA, Selection of candidate ERK1/2 substrates desalted using 30 mg HLB cartridge (Waters), dried and resuspended in An algorithm, implemented in Perl, was written to select putative 2% acetonitrile (ACN)/0.2% formic acid before analysis. Phospho- ERK1/2 substrates using the following criteria. First, high-confidence peptides were separated online by 2D-nanoLC (Eksigent). Peptides phosphorylation sites (X75%) with the minimal ERK1/2 consensus were first fractionated on a Opti-Guard 1 mm cation SCX column (pS/T)-P motif were selected using ProteoConnections. Second, (Optimize Technologies) using five ammonium acetate salt fractions phosphorylation level must increase after serum stimulation. Phos- (0, 0.25, 0.5, 1 and 2 M) in 2% CAN at pH 3.0. Each fraction was then phopeptides were filtered with a cutoff of Slog (stimulated t / loaded on a reverse-phase precolumn (4 mm length, 360 mm i.d.) and 10 5-60 control t0) X0.3, a value clearly above our biological replica separated on a reverse-phase analytical column (10 cm length, 150 mm measurements. Third, a decrease in phosphorylation-site abundance ˚ i.d.) (Jupiter C18,3mm, 300 A, Phenomenex). Both columns were is observed after treatment with the MEK1/2 inhibitor PD184352. To packed manually. A gradient from 2 to 33% ACN over 53 min followed select downregulated phosphopeptides, a cutoff of Slog10(PD184352 by a gradient from 33 to 60% ACN over 10 min with a flow rate of treated/control) p 0.7 was used, a value 2.5 time above fold change 600 nl/min was used to elute the peptides to the MS system with the found between replicates. Furthermore, at least one time point of the nanoelectrospray source voltage set to 1.7 kV.MS analysis was done on kinetics must show a significant decrease in abundance with a two- a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). MS tailed t-test (P-valuep0.05). Finally, manual inspection of the spectra were acquired with a resolution of 60 000 in FTMS using lock abundance profiles was performed on potential candidates to fix mass. CID MS/MS spectra were acquired in data-dependent mode for potential peak selection errors of the label-free quantification software. the three most abundant multiply charged ions with intensity above 10 000 counts. A dynamic exclusion window was set to 90s. Bioinformatics analyses MS/MS processing for peptide and protein For GO analysis, enrichment and depletion of categories were calculated using odds ratios. Ratios are calculated as: (number of proteins with the identifications GO term in the data set/number of proteins in the data set)/(number of MS/MS spectra peak lists were extracted from Xcalibur raw data files proteins with the GO term in the proteome/number of proteins in the (Thermo Fischer Scientific) and preprocessed using Mascot Distiller proteome). P-values were calculated using Fisher’s exact test. To v2.1.1 (Matrix Science) using the configuration file for low-resolution generate protein interactions network, we first used ProteoConnections MS/MS for the LTQ-Orbitrap. MGF peak lists were searched with Mascot to map the list of candidate ERK1/2 substrates to the STRING 2.1 on a concatenated target/decoy IPI rat database v3.54 (39 928 protein interactions database. We retained only the highest confidence (40.9) sequences) (Kersey et al, 2004) using the following parameters: peptide interactions extracted from experiments and databases found in rat. For mass tolerance±10 p.p.m., fragment mass tolerance±0.5 Da, trypsin all candidates found, we gathered extra interactors one level deeper with two missed cleavages, and the variable modifications carbamido- (white node). Since the rat interactome is not well studied, we chose to methyl (C), deamidation (NQ), oxidation (M), phosphorylation (STY). expand our network by including interactions from human orthologs. All search results were then transferred to ProteoConnections, our in- After converting rat gene identifiers to human, we extracted mapped house bioinformatics platform dedicated to phosphoproteomics analysis interaction from STRING as above. We manually edited the human (Courcelles et al, 2011). Identifications and MS/MS spectra are available network to keep interactions that extended our rat network (connectable online in ProteoConnections (http://www.thibault.iric.ca/proteocon- components) and removed white node (unless needed to connect two nections). From there, a 1% FDR cutoff was applied to peptides assigned subnetworks). Finally, fusion of the rat and the remaining human to proteins with a Po0.05 significance threshold. Phosphorylation-site- network was made using Cytoscape. This software was also used to localization confidence was assigned by ProteoConnections as pre- organize spatially the network for Figure 3. Binary interactions were viously proposed (Olsen et al, 2006). All data files are obtainable at removed to generate the figure. Identified phosphorylation sites were http://www.peptideatlas.org/PASS/PASS00138. compared with Swissprot v15.53 (Boeckmann et al,2003),Phos- pho.ELM v8.2 (Diella et al, 2008) and PhosphositePlus v2.0 (Hornbeck et al, 2004) database to report the fraction of novel sites. ProteoConnec- Data processing for label-free peptide tions was used to indicate the presence of potential docking sequences DEF (motif F-X-[FY]-P) or D domain (motif [KR]2-5-X1-6-[LIV]-X-[LIV]) quantification in the protein sequence of putative ERK1/2 substrates. Peptides detection for all raw files was done using our in-house peptide-detection software. The software retrieves peak intensity value, mass-to-charge ratio, retention time and charge state for each In vitro kinase assays peptide. An intensity threshold of 10 000 counts was selected to detect peptides above the noise level. Detected peptides were then aligned Recombinant GST-taggedcandidate substrates were produced in E. coli with an m/z tolerance of 15 p.p.m., retention time window of ±1 min and purified on glutathione-Sepharose beads. Recombinant proteins and same charge state to get abundance values for all conditions and were incubated in kinase buffer (20 mM Tris–HCl, pH 7.4, 20 mM NaCl, replicates. A median normalization procedure was applied to 10 mM MgCl2, 1 mM DTT) supplemented with 50 mM ATP and 5 mCi 32 1 minimize experimental variability of the peptides population abun- [g- P]ATP for 20 min at 30 C in the presence of 30 ng recombinant dance. Since the peptides population size is not equal between active ERK1 protein (Millipore). The reaction products were analyzed experimental conditions, only reproducibly detected peptides were by SDS-gel electrophoresis, autoradiography and MS/MS. used to calculate the median of samples. Soft clustering of phosphopeptide kinetic profiles was done using fuzzy c-means clustering in MFuzz R package (number of clusters Immunoblot analysis and immunofluorescence centroid c ¼ 6, parameter m ¼ 1.5). The number of clusters was chosen arbitrarily to 6 to show diverse phosphorylation change patterns. microscopy Grouping is done by minimizing the Euclidian distance between the Cell lysis and immunoblot analysis were performed as described phosphopeptide kinetic fold-change profiles with a weighted square previously (Servant et al, 2000). Staining of cells for immunofluores- error function. Fuzzy c-means clustering is a soft-clustering algorithm cence microscopy was performed as described (Julien et al, 2003) and that distinguishes itself from hard-clustering algorithm by providing a the cells were examined on a Zeiss LSM510 confocal microscope. At membership probability value to each member of the clusters. least 150 cells were scored for each coverslip.

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Electrophoretic mobility shift assay Conflict of interest HEK 293 cells were washed once with ice-cold PBS and lysed by The authors declare that they have no conflict of interest. scraping in lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl and 1.5 mM MgCl2) containing 0.1% Nonidet P-40, 0.5 mM DTT, 0.5 mM phenyl- methylsulfonyl fluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin. The nuclei were collected by centrifugation (12 000 g, 5 min, 4 1C) and References washed with lysis buffer without detergent. Nuclear proteins were extracted by suspending the nuclei in ice-cold hypertonic buffer A Angel P, Karin M (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072: (420 mM NaCl, 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT and protease inhibitors) for 10 min on ice. 129–157 After centrifugation (12 000 g, 5 min, 4 1C), the supernatant was Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, transferred to another tube and the nuclear extract was diluted Klevernic I, Arthur JS, Alessi DR, Cohen P (2007) The selectivity of immediately with 1.5 volumes of buffer B (50 mM KCl, 20 mM HEPES, protein kinase inhibitors: a further update. Biochem J 408: 297–315 pH 7.9, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, and protease Bakiri L, Lallemand D, Bossy-Wetzel E, Yaniv M (2000) Cell cycle- inhibitors). The samples were then frozen at 80 1C. dependent variations in c-Jun and JunB phosphorylation: a role in For EMSA, two complementary oligonucleotides containing AP-1 the control of cyclin D1 expression. EMBO J 19: 2056–2068 32 element (TRE or CRE) were end-labeled with [g- P]ATP and purified Boeckmann B, Bairoch A, Apweiler R, Blatter MC, Estreicher A, 32 on a G-25 spin column. The P-labeled oligonucleotide probe was Gasteiger E, Martin MJ, Michoud K, O’Donovan C, Phan I, Pilbout S, incubated in gel shift buffer (4 mM Tris–HCl, pH 7.9, 25 mM KCl, 5 mM Schneider M (2003) The SWISS-PROT protein knowledgebase and MgCl2, 0.4 mM EDTA, 5% glycerol, 0.4 mM DTT) with 10 mg nuclear its supplement TrEMBL in 2003. Nucleic Acids Res 31: 365–370 proteins and 50 mg/ml poly(dI-dC) for 20 min at room temperature. Carlson SM, Chouinard CR, Labadorf A, Lam CJ, Schmelzle K, DNA–protein complexes were separated on 5% native polyacrylamide Fraenkel E, White FM (2011) Large-scale discovery of ERK2 gels, transferred to Whatman 3 MM paper, and exposed to X-ray films. The specificity of binding was assessed using mutant TRE and CRE substrates identifies ERK-mediated transcriptional regulation by oligonucleotides. For supershift experiments, the reaction mixture was ETV3. Sci Signal 4: rs11 pre-incubated for 20 min with 1 mg of antibody against HA (Covance) Chinenov Y, Kerppola TK (2001) Close encounters of many kinds: Fos- or Flag (Sigma). The sequences (50-30) of the oligonucleotides used in Jun interactions that mediate transcription regulatory specificity. the EMSA are: TRE wt (50-TTCCGGCTGACTCATCAAGCG-30); TRE Oncogene 20: 2438–2452 mutant (50-TTCCGGCTGACTTGTCAAGCG-30); CRE wt (50-TTCCGGCT Chiu R, Angel P, Karin M (1989) Jun-B differs in its biological GACGTCATCAAGCG-30); and CRE mutant (50-TTCCGGCTGACGTTGTC properties from, and is a negative regulator of, c-Jun. Cell 59: AAGCG-30). 979–986 Cobb MH, Goldsmith EJ (1995) How MAP kinases are regulated. J Biol Chem 270: 14843–14846 Luciferase reporter assay Courcelles M, Lemieux S, Voisin L, Meloche S, Thibault P (2011) ProteoConnections: a bioinformatics platform to facilitate HEC-1B cells were seeded in 48-well plates and co-transfected by the proteome and phosphoproteome analyses. Proteomics 11: calcium phosphate method with AP-1 expression plasmids (50– 2654–2671 250 ng), 100 ng -73 Col-Luc (collagenase I promoter with single TRE Cuevas BD, Abell AN, Johnson GL (2007) Role of mitogen-activated site) firefly luciferase reporter plasmid and 25 ng Renilla luciferase protein kinase kinase kinases in signal integration. Oncogene 26: internal control pRL-TK. The total amount of transfected DNA was adjusted with pcDNA3. After 48 h, cells were lysed in Passive Lysis 3159–3171 Buffer (Promega) and lysates transferred to 96-well luminometer Diella F, Gould CM, Chica C, Via A, Gibson TJ (2008) Phospho.ELM: a plates. Firefly and Renilla luciferase activities were assayed using the database of phosphorylation sites–update 2008. Nucleic Acids Res Dual-Luciferase Reporter System (Promega) on a MicroBeta 1450 36: D240–D244 luminometer (PerkinElmer). Eferl R, Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 3: 859–868 Fritsche-Guenther R, Witzel F, Sieber A, Herr R, Schmidt N, Braun S, Brummer T, Sers C, Bluthgen N (2011) Strong negative feedback Supplementary information from Erk to Raf confers robustness to MAPK signalling. Mol Syst Supplementary information is available at the Molecular Systems Biol 7: 489 Biology website (www.nature.com/msb). Gonzalez FA, Raden DL, Davis RJ (1991) Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem 266: 22159–22163 Acknowledgements Hornbeck PV, Chabra I, Kornhauser JM, Skrzypek E, Zhang B (2004) PhosphoSite: a bioinformatics resource dedicated to physiological We thank E´ric Bonneil for technical assistance in MS analyses and Ivan protein phosphorylation. Proteomics 4: 1551–1561 Topisirovic for the cellular fractionation protocol. MC is recipient of Jacobs D, Glossip D, Xing H, Muslin AJ, Kornfeld K (1999) Multiple studentships from the Canadian Institutes for Health Research (CIHR), docking sites on substrate proteins form a modular system that BiT program and the Fonds de recherche sur la nature et les mediates recognition by ERK MAP kinase. Genes Dev 13: 163–175 technologies du Que´bec (FQRNT). CF is recipient of fellowships from Jochum W,Passegue E, Wagner EF (2001) AP-1 in mouse development the Cole foundation, the French Association pour la Recherche contre and tumorigenesis. Oncogene 20: 2401–2412 ´ ´ le Cancer (ARC) and the Fonds de la recherche en sante du Quebec Julien C, Coulombe P, Meloche S (2003) Nuclear export of ERK3 by a (FRSQ). PTand SM hold the Canada Research Chairs in Proteomics and CRM1-dependent mechanism regulates its inhibitory action on cell Bioanalytical Spectrometry and Cellular Signaling, respectively. This J Biol Chem work was supported by operating grants from the National Science and cycle progression. 278: 42615–42624 Engineering Research Council (NSERC) to PT, and the CIHR and Karin M, Liu Z, Zandi E (1997) AP-1 function and regulation. Curr Opin Cancer Research Society to SM. IRIC is supported in part by the Cell Biol 9: 240–246 Canadian Center of Excellence in Commercialization and Research, the Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Canada Foundation for Innovation and the FRSQ. Apweiler R (2004) The International Protein Index: an integrated Author Contributions: MC, CF, LV, SM and PT designed research; database for proteomics experiments. Proteomics 4: 1985–1988 MC, CF and LV performed research; MC, CF, LV, SL, SM and PT Kosako H, Yamaguchi N, Aranami C, Ushiyama M, Kose S, Imamoto N, analyzed data; and MC, CF, SM and PT wrote the paper. Taniguchi H, Nishida E, Hattori S (2009) Phosphoproteomics

12 Molecular Systems Biology 2013 & 2013 EMBO and Macmillan Publishers Limited Quantitative phosphoproteomics of ERK1/2 signaling M Courcelles et al

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