Phosphoproteomic analysis identifies the tumorsuppressor PDCD4 as a RSK substrate negativelyregulated by 14-3-3Jacob A. Galana,b, Kathryn M. Geraghtyc, Geneviève Lavoiea, Evgeny Kanshina,d, Joseph Tcherkeziana,b,Viviane Calabresea,b, Grace R. Jeschkee, Benjamin E. Turke, Bryan A. Balliff, John Blenisc, Pierre Thibaulta,d,and Philippe P. Rouxa,b,1

aInstitute for Research in Immunology and Cancer, Montreal, QC, Canada H3C 3J7; bDepartment of Pathology and Cell Biology, Faculty of Medicine, Universitéde Montréal, Montreal, QC, Canada H3C 3J7; cDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; dDepartment of Chemistry, Facultyof Arts and Science, Université de Montréal, Montreal, QC, Canada H3C 3J7; eDepartment of Pharmacology, Yale University School of Medicine, New Haven,CT 06510; and fDepartment of Biology, University of Vermont, Burlington, VT 05405

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 13, 2014 (received for review April 9, 2014)

The Ras/MAPK signaling cascade regulates various biological func-tions, including cell growth and proliferation. As such, this path-way is frequently deregulated in several types of cancer, includingmost cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth andproliferation, but relatively little is known about its exact functionand the nature of its substrates. Herein, we used a quantitativephosphoproteomics approach to define the signaling networksregulated by RSK in melanoma. To more accurately predict directphosphorylation substrates, we defined the RSK consensus phos-phorylation motif and found significant overlap with the bindingconsensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found thata large proportion of 14-3-3 binding proteins are also potentialRSK substrates. Our results show that RSK phosphorylates thetumor suppressor PDCD4 (programmed cell death protein 4) ontwo serine residues (Ser76 and Ser457) that regulate its subcellularlocalization and interaction with 14-3-3 proteins. We found that14-3-3 binding promotes PDCD4 degradation, suggesting animportant role for RSK in the inactivation of PDCD4 in melanoma.In addition to this tumor suppressor, our results suggest the in-volvement of RSK in a vast array of unexplored biological func-tions with relevance in oncogenesis.

The Ras/MAPK pathway plays a key role in transducing ex-tracellular signals to intracellular targets involved in cell

growth and proliferation (reviewed in ref. 1). Inappropriateregulation of this pathway leads to a variety of diseases, includingcancer (2). In this pathway, the small GTPase Ras activates theRaf isoforms, which are Ser/Thr kinases frequently mutated inhuman cancers (3). One prominent example is melanoma, whichharbors activating B-Raf mutations (V600E) in a majority ofcases (4). In turn, activated Raf phosphorylates and activatesMEK1/2, which themselves phosphorylate and activate theMAPKs ERK1/2 (5). Once activated, ERK1/2 phosphorylatemany substrates, including members of the p90 ribosomal S6kinase (RSK) family of proteins (6). Although the requirementof ERK1/2 signaling in melanoma is well established, relativelylittle is known regarding RSK signaling.The RSK family is composed of four Ser/Thr kinases (RSK1–4)

that share 73–80% sequence identity and belong to the AGCfamily of basophilic protein kinases (6). The RSK isoforms havebeen shown to regulate a number of substrates involved in cellgrowth and proliferation, and accordingly, inhibition of their ac-tivity reduces the proliferation of several cancer cell lines (7, 8).Consistent with this, RSK1 and RSK2 were shown to be over-expressed in breast and prostate cancers (7, 8) and hyperactivatedin melanoma (9). Although RSK plays an important role inmelanoma (10), relatively little is known about the substratesit regulates.

The 14-3-3 family of pSer/Thr-binding proteins dynamically reg-ulates the activity of various client proteins involved in diversebiological processes (11). In response to growth factors, 14-3-3proteins orchestrate a complex network of molecular interactionsto achieve well-controlled physiological outputs, such as cell growthand proliferation. Many 14-3-3-binding proteins contain sequencesthat match its general consensus motif, which consists of RSXpS/pTXP (12). Based on the requirement for an Arg residue at the −3position, 14-3-3 client proteins are often phosphorylated by baso-philic protein kinases, such as members of the AGC family.Quantitative phosphoproteomics has emerged as a powerful

tool in the elucidation of complex signaling networks. In thisstudy, we used quantitative liquid chromatography mass spec-trometry (LC-MS) to define the RSK phosphoproteome in mel-anoma cells. We characterized the primary sequence motif spec-ificity of RSK and observed significant overlap with the 14-3-3binding motif. Characterization of the 14-3-3 interactome inmelanoma cells resulted in the identification of a large numberof potential RSK substrates. We characterized the tumor sup-pressor programmed cell death protein 4 (PDCD4) and foundthat RSK promotes its degradation in a 14-3-3–dependent manner.Together, these results cast insights on the diverse biological func-tions regulated by RSK in cancer cells.

Significance

The RSK family is a group of Ser/Thr kinases that promotes cellgrowth and proliferation in response to the Ras/MAPK path-way. Deregulated RSK activity has been associated with dif-ferent disorders and diseases, such as cancer, but relatively littleis known regarding the contribution of RSK to tumorigenesis.In this study, we describe, to our knowledge, the first globalquantitative phosphoproteomic screen to characterize RSK-dependent signaling events in melanoma. Our results show thatRSK negatively regulates the tumor suppressor PDCD4 by pro-moting its association to 14-3-3 proteins and subsequent pro-teasomal degradation. These findings further implicate RSK asa promising therapeutic target for the treatment of melanomaand suggest that RSK plays widespread biological functionsdownstream of the Ras/MAPK pathway.

Author contributions: J.A.G., K.M.G., G.L., E.K., J.T., V.C., G.R.J., B.E.T., B.A.B., J.B., P.T., andP.P.R. designed research; J.A.G., K.M.G., G.L., E.K., J.T., V.C., and G.R.J. performed re-search; J.A.G., K.M.G., G.L., E.K., J.T., G.R.J., B.E.T., B.A.B., J.B., P.T., and P.P.R. analyzeddata; and J.A.G. and P.P.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: philippe.roux@umontreal.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405601111/-/DCSupplemental.

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ResultsA Proteomic Strategy for Characterizing the RSK-DependentPhosphoproteome. To characterize the RSK-dependent phospho-proteome, we devised a comprehensive quantitative MS strategyusing pharmacological inhibitors and RNAi (Fig. 1A). As bi-ological models, we used HEK293 cells treated with the phorbolester phorbol-12-myristate-13-acetate (PMA) to acutely stimu-late RSK activity, as well as A375 melanoma cells, which harborthe B-Raf V600E mutation and therefore have constitutivelyhigh RSK activity (10). To optimize these cellular models, we

used an antibody that recognizes the consensus motif (RXXpS/T,where X is any amino acid), which is phosphorylated by AGCfamily kinases including RSK (13). We demonstrated that PMAtreatment results in many immunoreactive bands that are se-verely reduced when cells are pretreated with either MEK1/2(PD184352) or RSK (BI-D1870) inhibitors (Fig. 1B). Similarly,we found that A375 melanoma cells have constitutively highlevels of immunoreactive bands, which were also significantlyreduced by MEK1/2 or RSK inhibitors. We also depleted RSK1and RSK2 by RNAi, which are the predominantly expressed

Fig. 1. Proteomic strategy for the characterization of the RSK-dependent phosphoproteome. (A) Schematic representation of the agonists and pharma-cological inhibitors used in this study. (B) HEK293 and A375 cells were serum-starved for 24 h before incubation with PD184352 (10 μM) or BI-D1870 (10 μM)for 30 min in HEK293 cells and 2 h in A375 cells, respectively. HEK293 cells were stimulated with PMA (50 ng/mL) for 30 min or left unstimulated. Proteinlysates were resolved by SDS/PAGE and analyzed by immunoblotting with the indicated antibodies. (C) HEK293 and A375 cells were infected with lentiviralshRNA constructs targeted against a scrambled sequence (Scr) or RSK1/2. After selection, cells were serum-starved and stimulated with either PMA (50 ng/mL)or left unstimulated. Protein lysates were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (D) Schematic representationof the different conditions analyzed using SILAC and LC-MS/MS. The relative abundance in phosphopeptides was compared between SILAC pairs, whichcomprised HEK293 and A375 cells treated with MEK1/2 (PD184352) or RSK (BI-D1870) inhibitors, or subjected to a nontarget or RSK1/2 shRNAs.

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isoforms in both cell types (10). Simultaneous knockdown ofRSK1/2 resulted in a strong decrease in immunoreactive bandscompared with cells subjected to a control shRNA (Fig. 1C).Together, these data validate our cellular models for further usein a phosphoproteomic screen.To identify and globally quantify changes in the RSK-dependent

phosphoproteome, we used a stable isotope labeling by aminoacids in cell culture (SILAC)-based MS approach. Both HEK293and A375 cells were labeled with either light (12C6

14N2-Lys,12C6

14N4-Arg) or heavy (13C615N2-Lys,

13C615N4-Arg) isotopes of

Lys and Arg and treated with PMA and/or inhibitors as indicated(Fig. 1D). For RNAi experiments, light and heavy isotope-labeled HEK293 and A375 cells were infected with lentiviralvectors expressing control or RSK1/2 shRNAs and harvestedfollowing a 3-d selection period in antibiotics. Lysates from light

and heavy isotope-labeled cells were then combined and digestedwith trypsin and relative changes in protein abundances weremeasured using MS (Fig. 1D). Both RNAi and inhibitor treat-ments did not globally perturb protein levels, as shown by thesimilar distribution of light and heavy proteins across experi-mental conditions (Fig. S1). For phosphoproteome analysis,proteins were digested with trypsin and phosphopeptides wereenriched using TiO2 chromatography. Phosphopeptides werethen further separated using strong cation exchange chroma-tography and analyzed by LC-MS (Fig. 1D). The relative abun-dance of all phosphopeptides was then measured and comparedbetween conditions.

Global Analysis of the RSK-Dependent Phosphoproteome. In total,we analyzed six different SILAC pairs by comparing the effects

Fig. 2. Characterization of the RSK-dependent phosphoproteome. (A–C) Log2 ratios of phosphopeptides identified comparing MEK1/2 (PD184352) and RSK(BI-D1870) inhibition, or RSK1/2 depletion by RNAi in HEK293 and A375 cells. Log2 ratios below −1.5-fold were considered as significantly down-regulated. (Dand E) Representative MS spectra of light and heavy peptides from Chk1 (S280) and rpS6 (Ser235). (Insets) Representative Western blots using correspondingphosphospecific antibodies. (F and G) MS quantification of the phosphopeptides containing Chk1 Ser280 or rpS6 Ser235 phosphorylation sites. (H) IPA of GeneOntologies (GO) enriched within down-regulated phosphopeptides.

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of two pharmacological inhibitors (PD184352 and BI-D1870) aswell as RSK1/2 RNAi in two different cell lines (Fig. 1D). Wequantified over 33,329 phosphopeptides (from 10,431 proteins),and the relative changes in abundance of each phosphopeptidewere measured in response to RNAi and inhibitor treatmentswith a statistical cutoff for SILAC ratios corresponding to 1.5-fold (P < 0.05) (Dataset S1 and Fig. S2 A–C). Statistical cut-offswere established based on the SILAC ratios of known RSKsubstrates as well as validation by immunoblotting.To increase the probability of identifying direct RSK sub-

strates, we reasoned that phosphopeptides containing RSK phos-phorylation sites should be similarly affected by MEK1/2 andRSK inhibitors, because both drug treatments result in re-duced RSK activity (Fig. 1 A and B). Using this approach, wefound 695 phosphopeptides (from 688 proteins) in HEK293cells that were sensitive to both drug treatments (Fig. 2A andFig. S2A). In A375 cells, we found 614 phosphopeptides(from 592 proteins) that were sensitive to both drug treatments(Fig. 2B and Fig. S2B). We applied the same rule to RNAiexperiments, where we compared the effect of RSK1/2 silencingin HEK293 and A375 cells, and thereby quantified 943 phos-phopeptides (from 769 proteins) common to both datasets(Fig. 2C and Fig. S2C). Cross-distribution analysis between

conditions in both cell lines showed that 168 proteins werecommon to all datasets, from which more than 15 known RSKsubstrates were identified (Fig. 2 A–C). We show the profile offour known RSK-regulated phosphorylation sites (GSK3β,SOS1, rpS6, and Chk1) that were found to be inhibited in re-sponse to MEK1/2 and RSK inhibitors (Fig. 2 D–G and Fig.S2D). These results demonstrate the efficiency of our approachto enrich in RSK-dependent phosphorylation events and suggestthat our datasets contain many uncharacterized RSK substrates,including PDCD4, Dennd4C, PKN2, and ARHGEF7 (Fig. S2Dand Dataset S1).To characterize the global signature of identified RSK-dependent

phosphorylation events, we used the Ingenuity Pathway Analysisplatform (IPA). We found enrichments in several cellular andmolecular functions, including cell assembly and organization(P < 9.6E-12), cellular function and maintenance (P < 9.9E-12),and cell morphology (P < 2.9E-06) (Fig. 2H). Notably, enrich-ments in functions correlated well between cell lines, and refinedanalysis revealed enrichments in specific functions, such as or-ganization of the cytoskeleton (P < 9.97E-12), organization ofmitotic spindle (P < 1.77E-09), and cell proliferation (P < 3.16E-07)(Fig. S2E).

Fig. 3. Peptide library profiling of the optimal substrate motif for RSK. (A) A spatially arrayed PSPL was subjected to in vitro phosphorylation with activeRSK1 and radiolabeled ATP. Aliquots of each reaction were spotted onto a membrane and exposed to a phosphor storage screen. (B) Matrix of intensitiesderived from results shown in A. (C) Web logo representation of the RSK consensus phosphorylation motif. (D) Schematic representation of our globalproteomic data from all experimental conditions. The data highlight the number of peptides and proteins affected by the MEK1/2 and RSK inhibitors, as wellas the RSK1/2 RNAi. The proportions of phosphopeptides that fit the RSK consensus motif are indicated.

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Determination of the RSK Consensus Phosphorylation Motif. Becausethe identified RSK-dependent phosphorylation events may bedirectly regulated by RSK or a downstream kinase, we sought todistinguish direct substrates from indirect effectors by refiningthe previously reported RSK consensus phospho-acceptor motif(14). For this, we used a positional scanning peptide library(PSPL) technique in which radiolabeled kinase assays using pu-rified active RSK1 were performed on a spatially arrayed set ofpeptide mixtures, as described previously (15). From the relativeamount of phosphate incorporated into each peptide mixtureone obtains a quantitative measure of the selectivity for, andagainst, each individual amino acid residue at each position(Fig. 3A). To visualize the RSK phosphorylation consensusmotif, PSPL signal intensities were translated into probabilitiesand expressed as a matrix of values (Fig. 3B). In agreement withthe previously reported consensus (14), PSPL profiling revealedthat RSK is a highly selective kinase that prefers positivelycharged residues at the −5 and −3 positions relative to thephospho-acceptor site (Fig. 3C). We found a high selectivitytoward Arg over any other basic residues, as well as a preferencefor a Ser residue at the −2 position (Fig. 3C), which is reminis-

cent of the preferred phospho-dependent binding motif of 14-3-3proteins (12). We observed reduced phosphorylation on peptideswith acidic or hydrophobic residues in the −5 through the −1position, indicating that these residues are deterrents to RSKsubstrate phosphorylation (Fig. 3B). Furthermore, comparingthe optimal consensus motif we identified (Fig. 3C) to well-established RSK substrates revealed excellent concordance (6).We then used the RSK consensus motif to mine our proteo-

mics data with Scansite (http://scansite.mit.edu) (16). Because ofthe observed requirement for an Arg residue at the −3 position,we first selected phosphopeptides from all three screens thatfitted this criterion (Dataset S2), resulting in a total of 500proteins (Fig. 3D). All phosphopeptides were then given a scorebased on how related their sequence was to the optimal RSKconsensus motif. This ranking allowed the classification of po-tential RSK substrates based on two independent scores: con-cordance to the RSK consensus motif and the amplitude ofthe inhibition observed in the phosphoproteomic screens. Bothvariables were given equal weight and a final ranked list wasgenerated based on the total score for each phosphopeptide(Dataset S3). Notably, many established RSK substrates were

Fig. 4. RSK phosphorylates PDCD4 at S457 and regulates its subcellular localization. (A) HEK293 cells were transfected with PDCD4, serum-starved, andpretreated with PD184352 (10 μM), rapamycin (25 nM), or BI-D1870 (10 μM) for 30 min before PMA (50 ng/mL) stimulation. Phosphorylation was assayed withan anti-RXXpS/T motif antibody. (B) HEK293 cells were transfected with WT PDCD4 or the S67A and S457A mutants, serum-starved, and stimulated with PMA(50 ng/mL) for 30 min. Phosphorylation was assayed by immunoblotting using the phospho-Ser457 and anti-RXXpS/T motif antibodies. (C) Recombinant RSK1was incubated with immunopurified PDCD4 in a kinase reaction with [γ-32P]ATP. The resulting samples were subjected to SDS/PAGE and the gel auto-radiographed. In parallel, samples were immunoblotted with phospho-Ser457 antibodies. (D) Normal human melanocytes and three melanoma cell lines wereanalyzed for PDCD4 levels and phosphorylation. (E) The phosphorylation status of PDCD4 at Ser457 was analyzed in A375 cells treated with PD184352 (10 μM)or BI-D1870 (10 μM) for 1 h. (F) A375 cells treated as in E were imaged using immunofluorescence microscopy. Cells were stained with anti-PDCD4 antibodiesto visualize endogenous PDCD4, phalloidin to visualize F-actin, and DAPI to visualize nuclei.

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found to cluster with the highest-ranking phosphopeptides, con-sistent with the idea that this classification increased the probabilityof identifying novel RSK substrates, such as the tumorsuppressorprotein PDCD4 (Table S1).

RSK Phosphorylates PDCD4 at S457 and Promotes Its NuclearLocalization in Melanoma. We first confirmed that PDCD4 phos-phorylation was regulated by RSK using the anti-RXXpS/Tmotif antibody. HEK293 cells were stimulated with PMA, andimmunoprecipitated PDCD4 was analyzed for phosphoryla-tion. Using this method, we found that acute PMA treatmentstrongly stimulated PDCD4 phosphorylation on RXXpS/Tconsensus sites, which was prevented by treatments with ei-ther MEK1/2 or RSK inhibitors (Fig. 4A). The AGC familymember S6K1 was previously shown to promote PDCD4phosphorylation (17), but our data convincingly show thatinhibition of S6K1 activation (as shown by rpS6 phosphory-lation on Ser240/244) using rapamycin did not preventPDCD4 phosphorylation in response to PMA stimulation(Fig. 4A). To identify the exact phosphorylation site(s) regu-lated by RSK, we generated different PDCD4 alleles with Alasubstitutions and found that mutation of Ser457 preventedmost of PDCD4 phosphorylation detected with the anti-RXXpS/T motif antibody, which was confirmed using a phos-pho-Ser457 antibody (Fig. 4B). Consistent with RSK directly

phosphorylating this site, we found that active recombinantRSK1 strongly phosphorylated PDCD4 at Ser457 in vitro(Fig. 4C).The protein kinase Akt was previously shown to promote

PDCD4 nuclear localization (18). To determine whether RSKcould similarly regulate PDCD4 subcellular localization, trans-fected HEK293 cells were stimulated with PMA and analyzedby immunofluorescence microscopy. Our results show that PMAstrongly promotes nuclear accumulation of PDCD4 in HEK293cells, which is almost completely prevented by pretreatment withMEK1/2 (PD184352) or RSK (BI-D1870, SL0101) inhibitors(Fig. S3A). We found that Ser457 phosphorylation was respon-sible for this change in localization, because PMA treatment didnot affect the localization of the PDCD4 S457A mutant (Fig.S3B). Consistent with this, we found that expression of a con-stitutively active form of MEK1 (MEK-DD) was sufficient topromote PDCD4 phosphorylation and its nuclear accumula-tion (Fig. S3 C and D), suggesting that oncogenes within theRas/MAPK pathway may similarly regulate PDCD4 localizationin melanoma cells.To determine whether RSK regulates endogenous PDCD4 in

melanoma cells, we analyzed different lines harboring activatingmutations in B-Raf (A375, Colo829) or N-Ras (WM852). Al-though PDCD4 protein levels were found to be much lower in twoof the three melanoma lines, the ratio of PDCD4 phosphorylation

Fig. 5. Identification of PDCD4 as a 14-3-3 binding protein in melanoma. (A) Subtractive fractionation proteomic scheme for enrichment of phospho-de-pendent 14-3-3 binding proteins. (B) Eluates were resolved by SDS/PAGE and gels stained with Coomassie or subjected to immunoblotting using the 14-3-3–binding motif antibody. (C) Comparisons of proteomic datasets between predicted RSK substrates (from Dataset S4) and 14-3-3 interacting proteins identifiedfrom A375 melanoma cells. (D) HEK293 cells were transfected with WT PDCD4, serum-starved, and stimulated with PMA (50 ng/mL) for 30 min before beingharvested; 14-3-3 binding was analyzed in a pull-down assay. (E) HEK293 cells were transfected with WT PDCD4, serum-starved, pretreated with PD184352(10 μM) or BI-D1870 (10 μM) followed by PMA (50 ng/mL) stimulation for 30 min. PDCD4 interaction to GST-14-3-3 was assessed as in D.

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over total levels was found to be higher in all cell lines comparedwith normal human melanocytes (Fig. 4D). The increased levelof phosphorylation was found to be sensitive to MEK1/2 andRSK inhibitors in A375 cells (Fig. 4E), indicating that RSK isrequired for endogenous PDCD4 phosphorylation in these cells.We next assessed the localization of endogenous PDCD4 inA375 cells and found that PDCD4 had a mainly nuclear lo-

calization in serum-starved cells (Fig. 4F). Notably, treatmentof cells with MEK1/2 or RSK inhibitors significantly shiftedPDCD4 to the cytoplasm, although this was less prominent in re-sponse to the SL0101 inhibitor. Together, these results confirmthe identification of PDCD4 as a bona fide RSK substrate andhighlight the role of RSK in regulating PDCD4 localizationin melanoma.

Fig. 6. RSK mediates site-specific 14-3-3 binding to PDCD4 and thereby promotes its degradation. (A) HEK293 cells were transfected with different 14-3-3isoforms, serum-starved, and stimulated with PMA (50 ng/mL) for 30 min. PDCD4 was immunoprecipitated and 14-3-3 binding was analyzed by immuno-blotting. (B) Scansite analysis of PDCD4 sequence for 14-3-3 binding sites, and bar graph representation of PDCD4 Ser76 phosphorylation changes observed inthis study. (C) HEK293 cells were cotransfected with 14-3-3β and WT PDCD4 or the S76A, S457A, and S76/457A mutants, serum-starved, and stimulated withPMA (50 ng/mL). The association between 14-3-3β and the different PDCD4 alleles was verified by coimmunoprecipitation. (D) HEK293 cells were transfectedwith WT PDCD4 or the double phosphorylation mutant S76/457A and treated with PMA (50 ng/mL) during a CHX (100 μg/mL) time course. Extracts wereprepared at each time points and analyzed by immunoblotting. (E) A375 cells were treated with vehicle (DMSO), PD184352 (10 μM), BI-D1870 (10 μM), orSL0101 (50 μM) during a time course of CHX treatment (100 μg/mL). Extracts were prepared at the indicated times and endogenous PDCD4 levels wereanalyzed by immunoblotting. (F) Densitometric analysis of PDCD4 was performed on CHX time course shown in E and normalized to actin band intensity. Thedata were then expressed relative to respective controls (t = 0).

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A Proteomic Screen Identifies PDCD4 As a 14-3-3 Binding Protein.Analysis of the RSK consensus motif revealed a sequence thatresembles the reported 14-3-3 binding motif (12). To determinethe global involvement of 14-3-3 in the regulation of RSK sub-strates, we characterized the 14-3-3 interactome in A375 mela-noma cells using affinity purification and MS. We used a pull-downapproach that takes advantage of a mutation in 14-3-3 (K49E)preventing phospho-dependent substrate binding (19) and in-cluded two subtractive fractionation steps in which nonspecific(GST alone) and non-phospho-dependent (GST-14-3-3K49E)interactions were removed (Fig. 5A). A375 cell lysates werethen subjected to 14-3-3 binding chromatography (WT or K49E)and bound proteins were eluted using MgCl2 and resolvedon SDS/PAGE. This method efficiently enriched in proteinsthat are specifically phosphorylated on 14-3-3 binding motifs(Fig. 5B).Notably, we identified 340 proteins in the WT 14-3-3 elution

from which 66 were also found to be associated to the 14-3-3K49E mutant, resulting in the net identification of 274 poten-tial phospho-dependent 14-3-3 client proteins (Fig. 5C andDataset S4). Characterization of the identified proteins usingIPA revealed a statistically significant enrichment in the 14-3-3–mediated signaling canonical pathway (P < 1.6E-8) (Fig. S4A).We also found enrichments in several cellular and molecularfunctions, including cell growth and proliferation (P < 1.8E-07)and protein synthesis (P < 9.8E-06) (Fig. S4B). Interestingly, wefound that a large number of 14-3-3 binding proteins were alsopredicted as RSK substrates in our proteomics screen (54 out of274 proteins, ∼20%). Analysis of these 54 proteins revealedseveral known 14-3-3 binding proteins from which two are al-ready established RSK substrates (CIC and eIF4B) (Table S2and Dataset S5).Importantly, we also identified PDCD4 as a previously un-

identified 14-3-3 binding protein, suggesting that RSK regulates

their association in cells. This finding was confirmed in a GSTpull-down assay, which showed that PMA stimulates PDCD4binding to WT 14-3-3 but not the K49E mutant (Fig. 5D).Phospho-dependent binding was also confirmed using the R18inhibitor peptide, which was found to abrogate PDCD4 bindingto 14-3-3 (Fig. S4C). To determine whether RSK activity wasrequired for this interaction, we used MEK1/2 and RSKinhibitors and found that both cell treatments disrupted 14-3-3binding to PDCD4 (Fig. 5E). Taken together, these data revealthe functional relationship between RSK and 14-3-3 and vali-date PDCD4 as a RSK-dependent 14-3-3–binding protein.

RSK Promotes PDCD4 Degradation by Stimulating Its Association to14-3-3. We next asked whether PDCD4 could preferentially bindto specific 14-3-3 isoforms in cells. To address this, we transfectedHEK293 cells with five different 14-3-3 isoforms and found thatPMA stimulation increased PDCD4 binding to 14-3-3 β, ζ, and γ(Fig. 6A). Analysis of the PDCD4 sequence for 14-3-3 bindingsites revealed two potential high-confidence (Ser76 and Ser457)and one low-confidence (Ser67) sites (Fig. 6B). Notably, Ser76was also identified in our phosphoproteomic screen as beingregulated by RSK (Fig. 6B). To determine whether these residueswere responsible for 14-3-3 interaction, HEK293 cells werecotransfected with 14-3-3β and PDCD4 (WT, S67A, S76A, orS457A), and binding was assessed by coimmunoprecipitation.With this approach, we found that Ser76 and Ser457 (Fig. 6C),but not Ser67 (Fig. S5B), were required for the regulated 14-3-3βinteraction. This finding was confirmed using the PDCD4 doubleS76/457A mutant, which we found to be completely impaired inits ability to interact with 14-3-3 in vitro (Fig. S5C) and in cells(Fig. 6C). Sequence analysis revealed that both Ser76 and Ser457are conserved in vertebrates (Fig. S5A), suggesting that they playimportant regulatory roles.To determine whether 14-3-3 binding to Ser76/457 regulated

PDCD4 stability, we transfected WT PDCD4 or the S76/457Amutant and analyzed their degradation rates in the presence ofcycloheximide. We found that mutation of both phosphorylationsites significantly increased the half-life of PDCD4 (Fig. 6D),suggesting that 14-3-3 promotes PDCD4 degradation. To de-termine this, we sought to abrogate 14-3-3 binding without dis-rupting PDCD4 phosphorylation using a potent 14-3-3 antagonisttermed difopein (20). Notably, we found that disruption of 14-3-3binding significantly increased the half-life of PDCD4 (Fig. S5 Dand E). We next addressed the role of RSK in PDCD4 stabilityin melanoma cells and found that PDCD4 was rapidly degradedin A375 cells exposed to cycloheximide (Fig. 6E). Notably, wefound that treatment of A375 cells with MEK1/2 or RSK inhib-itors greatly prevented the degradation of endogenous PDCD4(Fig. 6 E and F), indicating that RSK negatively regulates PDCD4in melanoma.Altogether, these results demonstrate that RSK-mediated

phosphorylation of PDCD4 promotes its nuclear accumulationand degradation through a mechanism that involves 14-3-3 bind-ing (Fig. 7). Our proteomics results also suggest that many moreproteins participate in RSK signaling and their exact functionsremain to be fully characterized.

DiscussionHerein, we describe, to our knowledge, the first global quan-titative phosphoproteomic study to identify and quantify RSK-dependent signaling events (Figs. 1 and 2). In addition, werefined the precise RSK phosphorylation consensus motif, high-lighting essential residues that contribute to substrate speci-ficity (Fig. 3). Using this motif, we identified several potentialsubstrates including the tumor suppressor PDCD4 (Fig. 4). Wefound that RSK phosphorylates PDCD4 on Ser457 in melanomacells and thereby promotes its nuclear accumulation. We alsodetermined the 14-3-3 interactome in melanoma cells and found

Fig. 7. Schematic representation of the role of RSK in the regulation of thetumor suppressor protein PDCD4. Proposed model whereby the Ras/MAPKpathway converges on PDCD4 to regulate its nuclear accumulation andproteasomal degradation via a mechanism that involves 14-3-3 proteins.

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that a large proportion of predicted RSK substrates interactwith 14-3-3 (Fig. 5). Importantly, we found that RSK regulatesPDCD4 binding to 14-3-3 by phosphorylating both Ser76 andSer457, and functional experiments revealed that 14-3-3 bindingpromotes PDCD4 degradation (Fig. 6). In addition to PDCD4,our data revealed that RSK regulates substrates involved in di-verse biological processes, including functions that have not beenexplored for RSK, such as actin and microtubule dynamics andDNA repair.The substrate specificity of RSK1 was previously determined

using a limited set of synthetic peptides (14). Using PSPL, wehave refined this consensus by showing a stricter requirementfor Arg at the −5 position (Fig. 3 A and B). This apparent dis-crepancy with previous data can be explained by the length ofpeptides used in respective assays, as it was shown that a Lys istolerated only if there are additional basic residues upstream(at −6 and −7) (14). Because those positions are neutral linkerresidues in the peptide library we have used (Ala at −6, Tyrat −7), our observation that Arg is preferred to Lys is actually incomplete agreement with previous data (14). The fact that mostRSK substrates have Arg at −5 rather than Lys is logical (6),because presumably a Lys at −5 would require a cluster of basicresidues upstream that would be less common. Although we haveonly analyzed human RSK1, it is likely that all four RSK iso-forms have similar substrate specificity when tested in vitro.We also found that Ser and Pro residues were preferred at the−2 and −1 positions, respectively (Fig. 3B), resulting in a con-sensus that overlaps significantly with the 14-3-3 binding motif.For this reason, we have characterized the 14-3-3 interactomein melanoma cells and identified ∼250 proteins from which∼20% were also identified in the RSK phosphoproteome(Fig. 5). Whereas some RSK substrates have previously beenshown to interact with 14-3-3 (e.g., TSC2, Bad, and SOS1)(6, 21), our data indicate that 14-3-3 plays a much greater rolein RSK signaling than previously thought.Our results indicate that PDCD4 interacts with 14-3-3 in a

RSK-dependent manner (Figs. 5 and 6). We found that phos-phorylation of both Ser76 and Ser457 is required for 14-3-3binding, which are sites that are conserved among vertebratespecies (Fig. S5). Although Ser76 was previously shown to belocated within a SCFβTRCP phosphodegron (17), to our knowl-edge this is the first report of a protein kinase regulating thisresidue. We found that 14-3-3 binding promotes PDCD4 deg-radation, suggesting that 14-3-3 may facilitate the recruitment ofthe SCFβTRCP ubiquitin ligase in response to RSK activation.Binding of 14-3-3 proteins to client proteins can often alter theirsubcellular localization, but in this case we did not find that 14-3-3participate in the nuclear translocation of PDCD4. Our find-ings demonstrate a molecular mechanism by which the Ras/MAPK pathway and RSK negatively regulates PDCD4 andthereby likely contributes to melanoma tumorigenesis.PDCD4 functions as a tumor suppressor that is lost in many

types of cancer, including lung, colon, and breast cancer, as wellas glioma (22). One of the most characterized functions ofPDCD4 is as a negative regulator of translation initiation (23).The inhibition of PDCD4 function by RSK is consistent with thefact that RSK was shown to promote cap-dependent translation(24). Both Akt and S6K1 have previously been shown to regulatePDCD4 at Ser67 and Ser457, which results in either decreasedprotein stability or nuclear translocation, respectively (17, 25).Our results indicate that the Ras/MAPK pathway also phos-phorylates PDCD4 at a common site (Ser457) but also at Ser76,and that RSK seems to be a key regulator of PDCD4 in mela-noma (Figs. 4 and 6). Based on the function of PDCD4 as a tu-mor suppressor, there has been great interest in identifying smallmolecules that would act to stabilize PDCD4 in cancer cells (26).Our results indicate that RSK inhibitors promote PDCD4 sta-bility in melanoma cells, suggesting that this could be exploited

in other types of cancer characterized by the hyperactivation ofthe MAPK pathway, such as colon, lung, and thyroid cancer.In conclusion, our study demonstrates the robust and com-

prehensive power of our proteomic screen in identifying novelRSK substrates. Our results implicating RSK in the regulationof the tumor suppressor PDCD4 also support the possibility thatRSK is a valuable therapeutic target for the treatment of mela-noma (27). Our proteomics findings also suggest that RSK playswidespread biological functions downstream of the Ras/MAPKpathway and that many more RSK substrates remain to becharacterized.

Experimental ProceduresAntibodies. Antibodies targeted against the RXXpS/Tmotif, ERK1/2 (total, T202/Y204), GST, rpS6 (total, Ser235/36, Ser240/44), Chk1 (total, Ser280), and RSK(S380) were purchased from Cell Signaling Technologies. Anti-RSK1, TSC2, andActin antibodies were purchased from Santa Cruz Biotechnology. The RSK2antibody was purchased from Invitrogen. Anti-Myc, anti-HA, and anti-tubulinmonoclonal antibodies were purchased from Sigma-Aldrich. The antibodyagainst PDCD4 (Ser457) was purchased from Millipore. All secondary horse-radish peroxidase-conjugated antibodies were purchased from Chemicon.

DNA Constructs. Human WT PDCD4 was subcloned in frame with a triple HAtag in the pKH3 vector. The plasmid encoding Flag-tagged MEK-DD wasdescribed previously (28). The plasmid encoding GST-tagged 14-3-3e wasdescribed previously (29). The plasmid encoding EYFP-Difopein (Dimeric R18peptide inhibitor) was described previously (20). The plasmids encoding β, σ,γ, ζ, and e 14-3-3 isoforms were purchased from Addgene and describedpreviously (30). All PDCD4 and 14-3-3 point mutants were generated usingthe Quikchange methodology (Stratagene).

Lentiviral Infections for RNA Interference. For shRNA-mediated knockdown,lentiviruses were produced using vectors from theMission TRC shRNA library.HEK293 and A375 were infected in the presence of 4 μg/mL polybrene, and2 d after viral infection cells were treated and selected with 2 μg/mL puro-mycin. shRNA constructs were obtained from Sigma Aldrich (shRSK1,TRCN470; shRSK2, TRCN537) and validated previously for their on-targeteffects (10).

Arrayed Positional Scanning Peptide Library. A positional scanning peptidelibrary (PSPL) screening was performed as previously described (31) usinga set of 200 peptide mixtures with the sequence Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K(biotin), where X is generally an equal molar mixture of the 17amino acid residues excluding Cys, Ser, and Thr. For each peptide mixture inthe set, a single X residue was fixed as one of the 20 unmodified aminoacids, phosphothreonine or phosphotyrosine. Aliquots (200 nL) of peptidestock solutions (0.6 mM) were transferred to a 1,536-well reaction platecontaining 2 μL reaction buffer [50 mM Hepes (pH 7.4), 1 mM EGTA, 1 mMDTT, 10 mM Mg(OAc)2, and 0.1% Tween 20] in each well. To start thereactions, active WT RSK1 (4 ng/μL) and radiolabeled ATP (50 μM, 0.33 μCi/μL[γ-33P]ATP) were added together to each well, and the plate was sealed andincubated at 30 °C for 2 h. Aliquots (200 nL) were then transferred usinga pin tool to a streptavidin membrane, which was washed twice with 0.1%SDS in TBS [10 mM Tris (pH 7.5) and 140 mM NaCl), twice with 2 M NaCl, andtwice with 1% H3PO4 in 2 M NaCl. The membrane was then air-dried andexposed to a phosphor imager screen. Radiolabel incorporation was quan-tified using QuantityOne software and the data were normalized so that theaverage value for a given position was 1.

Immunoprecipitation and Immunoblotting. Cells were seeded at 2.5 × 106 in10-cm plates, unless otherwise noted. Cell lysates were prepared as pre-viously described (32). Briefly, cells were washed three times with ice-coldPBS and harvested in 600 μL lysis buffer [10 mM K3PO4, 1 mM EDTA, 5 mMEGTA, 10 mM MgCl2, 50 mM β-glycerophosphate, 0.5% Nonidet P-40, 0.1%Brij 35, 0.1% deoxycholic acid, 1 mM sodium orthovanadate (Na3VO4), 1 mMphenylmethylsulfonyl fluoride, and a Complete Protease Inhibitor Mixturetablet (Roche)]. For immunoprecipitations, cell lysates were incubated withthe indicated antibodies for 2 h, followed by a 1-h incubation with ProteinA-Sepharose CL-4B beads (GE Healthcare). Immunoprecipitates were washedthree times in lysis buffer and beads were eluted and boiled in 2× reducingsample buffer [5×: 60 mM Tris·HCl (pH 6.8), 25% (vol/vol) glycerol, 2%(wt/vol) SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue].Immunoblotting was performed using a submersible transfer apparatus and

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nitrocellulose membranes. Blocking was performed in 5% milk/TBST (0.05%Tween-20, 8 mM Tris-Base, 25 mM Tris·HCl, and 154 mM NaCl). Primaryantibodies were incubated with the membranes in 5% (wt/vol) BSA or 5%milk in TBST and washes were done with TBST. Secondary antibodies con-jugated to horseradish peroxidase were from Chemicon/Millipore andvisualization was done using enhanced chemiluminescence and exposureto X-ray film.

Protein Phosphotransferase Assays. For RSK kinase assays, recombinant activeWT RSK1 (SignalChem) was incubated in the presence or absence of BI-D1870(10 μM) in kinase buffer [25 mM Tris·HCl (pH 7.4), 10 mM MgCl2, and 5 mMβ-glycerophosphate]. Kinase assays were performed with immunopurifiedfull-length HA-tagged PDCD4 as substrate, under linear assay conditions.Assays were performed for 10 min at 30 °C in kinase buffer supplementedwith 5 μCi of [γ-32P]ATP. All samples were subjected to SDS/PAGE followed byimmunoblotting or incorporation of radioactive 32P label was determined byautoradiography using a Fuji PhosphorImager with ImageQuant software.

Immunofluoresence Microscopy. For immunofluorescence analyses, HEK293(transfected with HA-PDCD4) or A375 cells were seeded at 5 × 105 in six-wellplates containing coverslips. Twenty-four hours later, cells were washedtwice in PBS and fixed in 3.7% (vol/vol) formaldehyde for 10 min at roomtemperature. Cells were washed twice in PBS and permeabilized for 5 min inPBS containing 0.2% Triton X-100 and blocked with PBS containing 0.1%BSA for 30 min. Cells were incubated for 2 h with indicated anti-HA or anti-PDCD4 antibodies, washed twice with PBS, and incubated for 1 h with

a secondary Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit an-tibody (Invitrogen), Texas Red-phalloidin, and DAPI diluted in PBS. Imageswere acquired on a Zeiss Axio Imager Z1 wide-field fluorescence microscopeusing a 40× oil immersion objective.

Protein Stability Assay. The turnover rate of proteins was determined usingcycloheximide (CHX; Sigma Aldrich) to inhibit de novo protein synthesis.HEK293 and A375 cells were treated with CHX (100 μg/mL) with or withoutPD184352 (10 μM), BI-D1870 (10 μM), or SL0101 (50 μM). After incubation forthe indicated time, the cells were harvested and lysed with the lysis bufferdescribed above. Equal amounts of cell lysates were subjected to SDS/PAGEand analyzed with the indicated antibodies. Additional information re-garding experimental procedures is given in SI Experimental Procedures.

ACKNOWLEDGMENTS. We thank all members of our laboratories for theirinsightful discussions, as well as Dr. Sylvain Meloche for comments on themanuscript and Marie Cargnello for artwork. This work was supported bygrants from the Canadian Cancer Society Research Institute, the CancerResearch Society, the Canadian Institutes for Health Research (CIHR), and theNational Science and Engineering Research Council (to P.P.R.). Work in thelaboratories of B.E.T. and B.A.B. was supported by National Institutes ofHealth (NIH) Grant R01 GM104047 and NIH General Medical Sciences Grant8P20GM103449, respectively. P.P.R. and P.T., respectively, hold the CanadaResearch Chairs in Signal Transduction and Proteomics, and Proteomics andBioanalytical Spectrometry. J.G. holds a Postdoctoral Fellowship from theCIHR. The Institute for Research in Immunology and Cancer core facilities aresupported in part by Le Fonds de Recherche du Québec – Santé.

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