Phosphoproteomic Analysis Identifies the Tumor Suppressor PDCD4 As a RSK Substrate Negatively Regulated by 14-3-3

Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3

Jacob 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, Université de Montréal, Montreal, QC, Canada H3C 3J7; cDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; dDepartment of Chemistry, Faculty of 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- The 14-3-3 family of pSer/Thr-binding proteins dynamically reg- tions, including cell growth and proliferation. As such, this path- ulates the activity of various client proteins involved in diverse way is frequently deregulated in several types of cancer, including biological processes (11). In response to growth factors, 14-3-3 most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK- proteins orchestrate a complex network of molecular interactions activated protein kinase required for melanoma growth and to achieve well-controlled physiological outputs, such as cell growth proliferation, but relatively little is known about its exact function and proliferation. Many 14-3-3-binding proteins contain sequences and the nature of its substrates. Herein, we used a quantitative that match its general consensus motif, which consists of RSXpS/ phosphoproteomics approach to define the signaling networks pTXP (12). Based on the requirement for an Arg residue at the −3 regulated by RSK in melanoma. To more accurately predict direct position, 14-3-3 client proteins are often phosphorylated by baso- phosphorylation substrates, we defined the RSK consensus phos- philic protein kinases, such as members of the AGC family. phorylation motif and found significant overlap with the binding Quantitative phosphoproteomics has emerged as a powerful consensus of 14-3-3 proteins. We thus characterized the phospho- tool in the elucidation of complex signaling networks. In this dependent 14-3-3 interactome in melanoma cells and found that study, we used quantitative liquid chromatography mass spec- a large proportion of 14-3-3 binding proteins are also potential trometry (LC-MS) to define the RSK phosphoproteome in mel- RSK substrates. Our results show that RSK phosphorylates the anoma cells. We characterized the primary sequence motif spec- tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular ificity of RSK and observed significant overlap with the 14-3-3 localization and interaction with 14-3-3 proteins. We found that binding motif. Characterization of the 14-3-3 interactome in 14-3-3 binding promotes PDCD4 degradation, suggesting an melanoma cells resulted in the identification of a large number important role for RSK in the inactivation of PDCD4 in melanoma. of potential RSK substrates. We characterized the tumor sup- In addition to this tumor suppressor, our results suggest the in- pressor programmed cell death protein 4 (PDCD4) and found – volvement of RSK in a vast array of unexplored biological func- that RSK promotes its degradation in a 14-3-3 dependent manner. tions with relevance in oncogenesis. Together, these results cast insights on the diverse biological functions regulated by RSK in cancer cells. he Ras/MAPK pathway plays a key role in transducing ex- Ttracellular signals to intracellular targets involved in cell Significance growth and proliferation (reviewed in ref. 1). Inappropriate regulation of this pathway leads to a variety of diseases, including The RSK family is a group of Ser/Thr kinases that promotes cell cancer (2). In this pathway, the small GTPase Ras activates the growth and proliferation in response to the Ras/MAPK path- Raf isoforms, which are Ser/Thr kinases frequently mutated in way. Deregulated RSK activity has been associated with dif- human cancers (3). One prominent example is melanoma, which ferent disorders and diseases, such as cancer, but relatively little harbors activating B-Raf mutations (V600E) in a majority of is known regarding the contribution of RSK to tumorigenesis. cases (4). In turn, activated Raf phosphorylates and activates In this study, we describe, to our knowledge, the first global MEK1/2, which themselves phosphorylate and activate the quantitative phosphoproteomic screen to characterize RSK- MAPKs ERK1/2 (5). Once activated, ERK1/2 phosphorylate dependent signaling events in melanoma. Our results show that many substrates, including members of the p90 ribosomal S6 RSK negatively regulates the tumor suppressor PDCD4 by pro- kinase (RSK) family of proteins (6). Although the requirement moting its association to 14-3-3 proteins and subsequent pro- of ERK1/2 signaling in melanoma is well established, relatively teasomal degradation. These findings further implicate RSK as little is known regarding RSK signaling. a promising therapeutic target for the treatment of melanoma The RSK family is composed of four Ser/Thr kinases (RSK1–4) and suggest that RSK plays widespread biological functions that share 73–80% sequence identity and belong to the AGC downstream of the Ras/MAPK pathway. family of basophilic protein kinases (6). The RSK isoforms have 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., and been shown to regulate a number of substrates involved in cell P.P.R. designed research; J.A.G., K.M.G., G.L., E.K., J.T., V.C., and G.R.J. performed re- growth and proliferation, and accordingly, inhibition of their ac- 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. analyzed tivity reduces the proliferation of several cancer cell lines (7, 8). data; and J.A.G. and P.P.R. wrote the paper. Consistent with this, RSK1 and RSK2 were shown to be over- The authors declare no conflict of interest. expressed in breast and prostate cancers (7, 8) and hyperactivated This article is a PNAS Direct Submission. in melanoma (9). Although RSK plays an important role in 1To whom correspondence should be addressed. Email: [email protected]. melanoma (10), relatively little is known about the substrates This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. it regulates. 1073/pnas.1405601111/-/DCSupplemental.

E2918–E2927 | PNAS | Published online July 7, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1405601111 Downloaded by guest on September 27, 2021 Results used an antibody that recognizes the consensus motif (RXXpS/T, PNAS PLUS A Proteomic Strategy for Characterizing the RSK-Dependent where X is any amino acid), which is phosphorylated by AGC Phosphoproteome. To characterize the RSK-dependent phospho- family kinases including RSK (13). We demonstrated that PMA proteome, we devised a comprehensive quantitative MS strategy treatment results in many immunoreactive bands that are se- using pharmacological inhibitors and RNAi (Fig. 1A). As bi- verely reduced when cells are pretreated with either MEK1/2 ological models, we used HEK293 cells treated with the phorbol (PD184352) or RSK (BI-D1870) inhibitors (Fig. 1B). Similarly, ester phorbol-12-myristate-13-acetate (PMA) to acutely stimu- we found that A375 melanoma cells have constitutively high late RSK activity, as well as A375 melanoma cells, which harbor levels of immunoreactive bands, which were also significantly the B-Raf V600E mutation and therefore have constitutively reduced by MEK1/2 or RSK inhibitors. We also depleted RSK1 high RSK activity (10). To optimize these cellular models, we and RSK2 by RNAi, which are the predominantly expressed BIOCHEMISTRY

Fig. 1. Proteomic strategy for the characterization of the RSK-dependent phosphoproteome. (A) Schematic representation of the agonists and pharmacological 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. Protein lysates were resolved by SDS/PAGE and analyzed by immunoblotting with the indicated antibodies. (C) HEK293 and A375 cells were infected with lentiviral shRNA 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 representation of the different conditions analyzed using SILAC and LC-MS/MS. The relative abundance in phosphopeptides was compared between SILAC pairs, which comprised HEK293 and A375 cells treated with MEK1/2 (PD184352) or RSK (BI-D1870) inhibitors, or subjected to a nontarget or RSK1/2 shRNAs.

Galan et al. PNAS | Published online July 7, 2014 | E2919 Downloaded by guest on September 27, 2021 isoforms in both cell types (10). Simultaneous knockdown of and heavy isotope-labeled cells were then combined and digested RSK1/2 resulted in a strong decrease in immunoreactive bands with trypsin and relative changes in protein abundances were compared with cells subjected to a control shRNA (Fig. 1C). measured using MS (Fig. 1D). Both RNAi and inhibitor treat- Together, these data validate our cellular models for further use ments did not globally perturb protein levels, as shown by the in a phosphoproteomic screen. similar distribution of light and heavy proteins across experi- To identify and globally quantify changes in the RSK-dependent mental conditions (Fig. S1). For phosphoproteome analysis, phosphoproteome, we used a stable isotope labeling by amino proteins were digested with trypsin and phosphopeptides were acids in cell culture (SILAC)-based MS approach. Both HEK293 enriched using TiO2 chromatography. Phosphopeptides were 12 14 12 and A375 cells were labeled with either light ( C6 N2-Lys, C6 then further separated using strong cation exchange chroma- 14 13 15 13 15 N4-Arg) or heavy ( C6 N2-Lys, C6 N4-Arg) isotopes of tography and analyzed by LC-MS (Fig. 1D). The relative abun- Lys and Arg and treated with PMA and/or inhibitors as indicated dance of all phosphopeptides was then measured and compared (Fig. 1D). For RNAi experiments, light and heavy isotope- between conditions. labeled HEK293 and A375 cells were infected with lentiviral vectors expressing control or RSK1/2 shRNAs and harvested Global Analysis of the RSK-Dependent Phosphoproteome. In total, following a 3-d selection period in antibiotics. Lysates from light 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. (D and E) Representative MS spectra of light and heavy peptides from Chk1 (S280) and rpS6 (Ser235). (Insets) Representative Western blots using corresponding phosphospecific antibodies. (F and G) MS quantification of the phosphopeptides containing Chk1 Ser280 or rpS6 Ser235 phosphorylation sites. (H) IPA of Gene Ontologies (GO) enriched within down-regulated phosphopeptides.

E2920 | www.pnas.org/cgi/doi/10.1073/pnas.1405601111 Galan et al. Downloaded by guest on September 27, 2021 of two pharmacological inhibitors (PD184352 and BI-D1870) as conditions in both cell lines showed that 168 proteins were PNAS PLUS well as RSK1/2 RNAi in two different cell lines (Fig. 1D). We common to all datasets, from which more than 15 known RSK quantified over 33,329 phosphopeptides (from 10,431 proteins), substrates were identified (Fig. 2 A–C). We show the profile of and the relative changes in abundance of each phosphopeptide four known RSK-regulated phosphorylation sites (GSK3β, were measured in response to RNAi and inhibitor treatments SOS1, rpS6, and Chk1) that were found to be inhibited in re- with a statistical cutoff for SILAC ratios corresponding to 1.5- sponse to MEK1/2 and RSK inhibitors (Fig. 2 D–G and Fig. P < A–C fold ( 0.05) (Dataset S1 and Fig. S2 ). Statistical cut-offs S2D). These results demonstrate the efficiency of our approach were established based on the SILAC ratios of known RSK to enrich in RSK-dependent phosphorylation events and suggest substrates as well as validation by immunoblotting. that our datasets contain many uncharacterized RSK substrates, To increase the probability of identifying direct RSK sub- including PDCD4, Dennd4C, PKN2, and ARHGEF7 (Fig. S2D strates, we reasoned that phosphopeptides containing RSK phos- and Dataset S1). phorylation sites should be similarly affected by MEK1/2 and To characterize the global signature of identified RSK-dependent RSK inhibitors, because both drug treatments result in re- phosphorylation events, we used the Ingenuity Pathway Analysis duced RSK activity (Fig. 1 A and B). Using this approach, we found 695 phosphopeptides (from 688 proteins) in HEK293 platform (IPA). We found enrichments in several cellular and cells that were sensitive to both drug treatments (Fig. 2A and molecular functions, including cell assembly and organization P < P < Fig. S2A). In A375 cells, we found 614 phosphopeptides ( 9.6E-12), cellular function and maintenance ( 9.9E-12), P < H (from 592 proteins) that were sensitive to both drug treatments and cell morphology ( 2.9E-06) (Fig. 2 ). Notably, enrich- (Fig. 2B and Fig. S2B). We applied the same rule to RNAi ments in functions correlated well between cell lines, and refined experiments, where we compared the effect of RSK1/2 silencing analysis revealed enrichments in specific functions, such as or- in HEK293 and A375 cells, and thereby quantified 943 phos- ganization of the cytoskeleton (P < 9.97E-12), organization of phopeptides (from 769 proteins) common to both datasets mitotic spindle (P < 1.77E-09), and cell proliferation (P < 3.16E-07) (Fig. 2C and Fig. S2C). Cross-distribution analysis between (Fig. S2E). BIOCHEMISTRY

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

Galan et al. PNAS | Published online July 7, 2014 | E2921 Downloaded by guest on September 27, 2021 Fig. 4. RSK phosphorylates PDCD4 at S457 and regulates its subcellular localization. (A) HEK293 cells were transfected with PDCD4, serum-starved, and pretreated with PD184352 (10 μM), rapamycin (25 nM), or BI-D1870 (10 μM) for 30 min before PMA (50 ng/mL) stimulation. Phosphorylation was assayed with an 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 RSK1 was 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 were analyzed 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 antibodies to visualize endogenous PDCD4, phalloidin to visualize F-actin, and DAPI to visualize nuclei.

Determination of the RSK Consensus Phosphorylation Motif. Because cent of the preferred phospho-dependent binding motif of 14-3-3 the identified RSK-dependent phosphorylation events may be proteins (12). We observed reduced phosphorylation on peptides directly regulated by RSK or a downstream kinase, we sought to with acidic or hydrophobic residues in the −5 through the −1 distinguish direct substrates from indirect effectors by refining position, indicating that these residues are deterrents to RSK the previously reported RSK consensus phospho-acceptor motif substrate phosphorylation (Fig. 3B). Furthermore, comparing (14). For this, we used a positional scanning peptide library the optimal consensus motif we identified (Fig. 3C) to well- (PSPL) technique in which radiolabeled kinase assays using pu- established RSK substrates revealed excellent concordance (6). rified active RSK1 were performed on a spatially arrayed set of We then used the RSK consensus motif to mine our proteo- peptide mixtures, as described previously (15). From the relative mics data with Scansite (http://scansite.mit.edu) (16). Because of amount of phosphate incorporated into each peptide mixture the observed requirement for an Arg residue at the −3 position, one obtains a quantitative measure of the selectivity for, and we first selected phosphopeptides from all three screens that against, each individual amino acid residue at each position fitted this criterion (Dataset S2), resulting in a total of 500 (Fig. 3A). To visualize the RSK phosphorylation consensus proteins (Fig. 3D). All phosphopeptides were then given a score motif, PSPL signal intensities were translated into probabilities based on how related their sequence was to the optimal RSK and expressed as a matrix of values (Fig. 3B). In agreement with consensus motif. This ranking allowed the classification of po- the previously reported consensus (14), PSPL profiling revealed tential RSK substrates based on two independent scores: con- that RSK is a highly selective kinase that prefers positively cordance to the RSK consensus motif and the amplitude of charged residues at the −5 and −3 positions relative to the the inhibition observed in the phosphoproteomic screens. Both phospho-acceptor site (Fig. 3C). We found a high selectivity variables were given equal weight and a final ranked list was toward Arg over any other basic residues, as well as a preference generated based on the total score for each phosphopeptide for a Ser residue at the −2 position (Fig. 3C), which is reminis- (Dataset S3). Notably, many established RSK substrates were

E2922 | www.pnas.org/cgi/doi/10.1073/pnas.1405601111 Galan et al. Downloaded by guest on September 27, 2021 found to cluster with the highest-ranking phosphopeptides, con- phosphorylating this site, we found that active recombinant PNAS PLUS sistent with the idea that this classification increased the probability RSK1 strongly phosphorylated PDCD4 at Ser457 in vitro of identifying novel RSK substrates, such as the tumorsuppressor (Fig. 4C). protein PDCD4 (Table S1). The protein kinase Akt was previously shown to promote PDCD4 nuclear localization (18). To determine whether RSK RSK Phosphorylates PDCD4 at S457 and Promotes Its Nuclear could similarly regulate PDCD4 subcellular localization, trans- Localization in Melanoma. We first confirmed that PDCD4 phos- fected HEK293 cells were stimulated with PMA and analyzed phorylation was regulated by RSK using the anti-RXXpS/T by immunofluorescence microscopy. Our results show that PMA motif antibody. HEK293 cells were stimulated with PMA, and strongly promotes nuclear accumulation of PDCD4 in HEK293 immunoprecipitated PDCD4 was analyzed for phosphoryla- cells, which is almost completely prevented by pretreatment with tion. Using this method, we found that acute PMA treatment MEK1/2 (PD184352) or RSK (BI-D1870, SL0101) inhibitors strongly stimulated PDCD4 phosphorylation on RXXpS/T (Fig. S3A). We found that Ser457 phosphorylation was respon- consensus sites, which was prevented by treatments with ei- sible for this change in localization, because PMA treatment did ther MEK1/2 or RSK inhibitors (Fig. 4A). The AGC family not affect the localization of the PDCD4 S457A mutant (Fig. member S6K1 was previously shown to promote PDCD4 S3B). Consistent with this, we found that expression of a con- phosphorylation (17), but our data convincingly show that stitutively active form of MEK1 (MEK-DD) was sufficient to inhibition of S6K1 activation (as shown by rpS6 phosphory- promote PDCD4 phosphorylation and its nuclear accumula- lation on Ser240/244) using rapamycin did not prevent tion (Fig. S3 C and D), suggesting that oncogenes within the PDCD4 phosphorylation in response to PMA stimulation Ras/MAPK pathway may similarly regulate PDCD4 localization (Fig. 4A). To identify the exact phosphorylation site(s) regu- in melanoma cells. lated by RSK, we generated different PDCD4 alleles with Ala To determine whether RSK regulates endogenous PDCD4 in substitutions and found that mutation of Ser457 prevented melanoma cells, we analyzed different lines harboring activating most of PDCD4 phosphorylation detected with the anti- mutations in B-Raf (A375, Colo829) or N-Ras (WM852). Al- RXXpS/T motif antibody, which was confirmed using a phos- though PDCD4 protein levels were found to be much lower in two pho-Ser457 antibody (Fig. 4B). Consistent with RSK directly of the three melanoma lines, the ratio of PDCD4 phosphorylation BIOCHEMISTRY

Fig. 5. Identification of PDCD4 as a 14-3-3 binding protein in melanoma. (A) Subtractive fractionation proteomic scheme for enrichment of phospho-dependent 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 identified from A375 melanoma cells. (D) HEK293 cells were transfected with WT PDCD4, serum-starved, and stimulated with PMA (50 ng/mL) for 30 min before being harvested; 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.

Galan et al. PNAS | Published online July 7, 2014 | E2923 Downloaded by guest on September 27, 2021 over total levels was found to be higher in all cell lines compared calization in serum-starved cells (Fig. 4F). Notably, treatment with normal human melanocytes (Fig. 4D). The increased level of cells with MEK1/2 or RSK inhibitors significantly shifted of phosphorylation was found to be sensitive to MEK1/2 and PDCD4 to the cytoplasm, although this was less prominent in re- RSK inhibitors in A375 cells (Fig. 4E), indicating that RSK is sponse to the SL0101 inhibitor. Together, these results confirm required for endogenous PDCD4 phosphorylation in these cells. the identification of PDCD4 as a bona fide RSK substrate and We next assessed the localization of endogenous PDCD4 in highlight the role of RSK in regulating PDCD4 localization A375 cells and found that PDCD4 had a mainly nuclear lo- in 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 14-3-3 isoforms, serum-starved, and stimulated with PMA (50 ng/mL) for 30 min. PDCD4 was immunoprecipitated and 14-3-3 binding was analyzed by immunoblotting. (B) Scansite analysis of PDCD4 sequence for 14-3-3 binding sites, and bar graph representation of PDCD4 Ser76 phosphorylation changes observed in this 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 with PMA (50 ng/mL). The association between 14-3-3β and the different PDCD4 alleles was verified by coimmunoprecipitation. (D) HEK293 cells were transfected with 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 were prepared at each time points and analyzed by immunoblotting. (E) A375 cells were treated with vehicle (DMSO), PD184352 (10 μM), BI-D1870 (10 μM), or SL0101 (50 μM) during a time course of CHX treatment (100 μg/mL). Extracts were prepared at the indicated times and endogenous PDCD4 levels were analyzed by immunoblotting. (F) Densitometric analysis of PDCD4 was performed on CHX time course shown in E and normalized to actin band intensity. The data were then expressed relative to respective controls (t = 0).

E2924 | www.pnas.org/cgi/doi/10.1073/pnas.1405601111 Galan et al. Downloaded by guest on September 27, 2021 A Proteomic Screen Identifies PDCD4 As a 14-3-3 Binding Protein. their association in cells. This finding was confirmed in a GST PNAS PLUS Analysis of the RSK consensus motif revealed a sequence that pull-down assay, which showed that PMA stimulates PDCD4 resembles the reported 14-3-3 binding motif (12). To determine binding to WT 14-3-3 but not the K49E mutant (Fig. 5D). the global involvement of 14-3-3 in the regulation of RSK sub- Phospho-dependent binding was also confirmed using the R18 strates, we characterized the 14-3-3 interactome in A375 mela- inhibitor peptide, which was found to abrogate PDCD4 binding noma cells using affinity purification and MS. We used a pull-down to 14-3-3 (Fig. S4C). To determine whether RSK activity was approach that takes advantage ofamutationin14-3-3(K49E) required for this interaction, we used MEK1/2 and RSK preventing phospho-dependent substrate binding (19) and in- inhibitors and found that both cell treatments disrupted 14-3-3 cluded two subtractive fractionation steps in which nonspecific binding to PDCD4 (Fig. 5E). Taken together, these data reveal (GST alone) and non-phospho-dependent (GST-14-3-3K49E) the functional relationship between RSK and 14-3-3 and vali- interactions were removed (Fig. 5A). A375 cell lysates were date PDCD4 as a RSK-dependent 14-3-3–binding protein. then subjected to 14-3-3 binding chromatography (WT or K49E) RSK Promotes PDCD4 Degradation by Stimulating Its Association to and bound proteins were eluted using MgCl2 and resolved on SDS/PAGE. This method efficiently enriched in proteins 14-3-3. We next asked whether PDCD4 could preferentially bind that are specifically phosphorylated on 14-3-3 binding motifs to specific 14-3-3 isoforms in cells. To address this, we transfected (Fig. 5B). HEK293 cells with five different 14-3-3 isoforms and found that β ζ γ Notably, we identified 340 proteins in the WT 14-3-3 elution PMA stimulation increased PDCD4 binding to 14-3-3 , ,and A from which 66 were also found to be associated to the 14-3-3 (Fig. 6 ). Analysis of the PDCD4 sequence for 14-3-3 binding K49E mutant, resulting in the net identification of 274 poten- sites revealed two potential high-confidence (Ser76 and Ser457) B tial phospho-dependent 14-3-3 client proteins (Fig. 5C and and one low-confidence (Ser67) sites (Fig. 6 ). Notably, Ser76 was also identified in our phosphoproteomic screen as being Dataset S4). Characterization of the identified proteins using B IPA revealed a statistically significant enrichment in the 14-3- regulated by RSK (Fig. 6 ). To determine whether these residues – P < A were responsible for 14-3-3 interaction, HEK293 cells were 3 mediated signaling canonical pathway ( 1.6E-8) (Fig. S4 ). β We also found enrichments in several cellular and molecular cotransfected with 14-3-3 and PDCD4 (WT, S67A, S76A, or functions, including cell growth and proliferation (P < 1.8E-07) S457A), and binding was assessed by coimmunoprecipitation. P < B With this approach, we found that Ser76 and Ser457 (Fig. 6C), and protein synthesis ( 9.8E-06) (Fig. S4 ). Interestingly, we B β found that a large number of 14-3-3 binding proteins were also but not Ser67 (Fig. S5 ), were required for the regulated 14-3-3 interaction. This finding was confirmed using the PDCD4 double BIOCHEMISTRY predicted as RSK substrates in our proteomics screen (54 out of S76/457A mutant, which we found to be completely impaired in 274 proteins, ∼20%). Analysis of these 54 proteins revealed its ability to interact with 14-3-3 in vitro (Fig. S5C) and in cells several known 14-3-3 binding proteins from which two are al- (Fig. 6C). Sequence analysis revealed that both Ser76 and Ser457 ready established RSK substrates (CIC and eIF4B) (Table S2 are conserved in vertebrates (Fig. S5A), suggesting that they play and Dataset S5). important regulatory roles. Importantly, we also identified PDCD4 as a previously un- To determine whether 14-3-3 binding to Ser76/457 regulated identified 14-3-3 binding protein, suggesting that RSK regulates PDCD4 stability, we transfected WT PDCD4 or the S76/457A mutant and analyzed their degradation rates in the presence of cycloheximide. We found that mutation of both phosphorylation sites significantly increased the half-life of PDCD4 (Fig. 6D), suggesting that 14-3-3 promotes PDCD4 degradation. To determine this, we sought to abrogate 14-3-3 binding without dis- rupting PDCD4 phosphorylation using a potent 14-3-3 antagonist termed difopein (20). Notably, we found that disruption of 14-3-3 binding significantly increased the half-life of PDCD4 (Fig. S5 D and E). We next addressed the role of RSK in PDCD4 stability in melanoma cells and found that PDCD4 was rapidly degraded in A375 cells exposed to cycloheximide (Fig. 6E). Notably, we found that treatment of A375 cells with MEK1/2 or RSK inhibitors greatly prevented the degradation of endogenous PDCD4 (Fig. 6 E and F), indicating that RSK negatively regulates PDCD4 in melanoma. Altogether, these results demonstrate that RSK-mediated phosphorylation of PDCD4 promotes its nuclear accumulation and degradation through a mechanism that involves 14-3-3 binding (Fig. 7). Our proteomics results also suggest that many more proteins participate in RSK signaling and their exact functions remain to be fully characterized. Discussion Herein, we describe, to our knowledge, the first global quantitative phosphoproteomic study to identify and quantify RSK- dependent signaling events (Figs. 1 and 2). In addition, we refined the precise RSK phosphorylation consensus motif, high- lighting essential residues that contribute to substrate specificity (Fig. 3). Using this motif, we identified several potential Fig. 7. Schematic representation of the role of RSK in the regulation of the substrates including the tumor suppressor PDCD4 (Fig. 4). We tumor suppressor protein PDCD4. Proposed model whereby the Ras/MAPK found that RSK phosphorylates PDCD4 on Ser457 in melanoma pathway converges on PDCD4 to regulate its nuclear accumulation and cells and thereby promotes its nuclear accumulation. We also proteasomal degradation via a mechanism that involves 14-3-3 proteins. determined the 14-3-3 interactome in melanoma cells and found

Galan et al. PNAS | Published online July 7, 2014 | E2925 Downloaded by guest on September 27, 2021 that a large proportion of predicted RSK substrates interact in other types of cancer characterized by the hyperactivation of with 14-3-3 (Fig. 5). Importantly, we found that RSK regulates the MAPK pathway, such as colon, lung, and thyroid cancer. PDCD4 binding to 14-3-3 by phosphorylating both Ser76 and In conclusion, our study demonstrates the robust and com- Ser457, and functional experiments revealed that 14-3-3 binding prehensive power of our proteomic screen in identifying novel promotes PDCD4 degradation (Fig. 6). In addition to PDCD4, RSK substrates. Our results implicating RSK in the regulation our data revealed that RSK regulates substrates involved in di- of the tumor suppressor PDCD4 also support the possibility that verse biological processes, including functions that have not been RSK is a valuable therapeutic target for the treatment of mela- explored for RSK, such as actin and microtubule dynamics and noma (27). Our proteomics findings also suggest that RSK plays DNA repair. widespread biological functions downstream of the Ras/MAPK The substrate specificity of RSK1 was previously determined pathway and that many more RSK substrates remain to be using a limited set of synthetic peptides (14). Using PSPL, we characterized. have refined this consensus by showing a stricter requirement for Arg at the −5 position (Fig. 3 A and B). This apparent dis- Experimental Procedures crepancy with previous data can be explained by the length of Antibodies. Antibodies targeted against the RXXpS/T motif, ERK1/2 (total, T202/ peptides used in respective assays, as it was shown that a Lys is Y204), GST, rpS6 (total, Ser235/36, Ser240/44), Chk1 (total, Ser280), and RSK tolerated only if there are additional basic residues upstream (S380) were purchased from Cell Signaling Technologies. Anti-RSK1, TSC2, and (at −6 and −7) (14). Because those positions are neutral linker Actin antibodies were purchased from Santa Cruz Biotechnology. The RSK2 residues in the peptide library we have used (Ala at −6, Tyr antibody was purchased from Invitrogen. Anti-Myc, anti-HA, and anti-tubulin at −7), our observation that Arg is preferred to Lys is actually in monoclonal antibodies were purchased from Sigma-Aldrich. The antibody against PDCD4 (Ser457) was purchased from Millipore. All secondary horse- complete agreement with previous data (14). The fact that most radish peroxidase-conjugated antibodies were purchased from Chemicon. RSK substrates have Arg at −5 rather than Lys is logical (6), − because presumably a Lys at 5 would require a cluster of basic DNA Constructs. Human WT PDCD4 was subcloned in frame with a triple HA residues upstream that would be less common. Although we have tag in the pKH3 vector. The plasmid encoding Flag-tagged MEK-DD was only analyzed human RSK1, it is likely that all four RSK iso- described previously (28). The plasmid encoding GST-tagged 14-3-3e was forms have similar substrate specificity when tested in vitro. described previously (29). The plasmid encoding EYFP-Difopein (Dimeric R18 We also found that Ser and Pro residues were preferred at the peptide inhibitor) was described previously (20). The plasmids encoding β, σ, −2 and −1 positions, respectively (Fig. 3B), resulting in a con- γ, ζ, and e 14-3-3 isoforms were purchased from Addgene and described sensus that overlaps significantly with the 14-3-3 binding motif. previously (30). All PDCD4 and 14-3-3 point mutants were generated using For this reason, we have characterized the 14-3-3 interactome the Quikchange methodology (Stratagene). in melanoma cells and identified ∼250 proteins from which ∼20% were also identified in the RSK phosphoproteome Lentiviral Infections for RNA Interference. For shRNA-mediated knockdown, lentiviruses were produced using vectors from the Mission TRC shRNA library. (Fig. 5). Whereas some RSK substrates have previously been HEK293 and A375 were infected in the presence of 4 μg/mL polybrene, and shown to interact with 14-3-3 (e.g., TSC2, Bad, and SOS1) 2 d after viral infection cells were treated and selected with 2 μg/mL puro- (6, 21), our data indicate that 14-3-3 plays a much greater role mycin. shRNA constructs were obtained from Sigma Aldrich (shRSK1, in RSK signaling than previously thought. TRCN470; shRSK2, TRCN537) and validated previously for their on-target Our results indicate that PDCD4 interacts with 14-3-3 in a effects (10). RSK-dependent manner (Figs. 5 and 6). We found that phosphorylation of both Ser76 and Ser457 is required for 14-3-3 Arrayed Positional Scanning Peptide Library. A positional scanning peptide binding, which are sites that are conserved among vertebrate library (PSPL) screening was performed as previously described (31) using species (Fig. S5). Although Ser76 was previously shown to be a set of 200 peptide mixtures with the sequence Y-A-X-X-X-X-X-S/T-X-X-X-X- β located within a SCF TRCP phosphodegron (17), to our knowl- A-G-K-K(biotin), where X is generally an equal molar mixture of the 17 edge this is the first report of a protein kinase regulating this amino acid residues excluding Cys, Ser, and Thr. For each peptide mixture in the set, a single X residue was fixed as one of the 20 unmodified amino residue. We found that 14-3-3 binding promotes PDCD4 deg- acids, phosphothreonine or phosphotyrosine. Aliquots (200 nL) of peptide radation, suggesting that 14-3-3 may facilitate the recruitment of stock solutions (0.6 mM) were transferred to a 1,536-well reaction plate βTRCP the SCF ubiquitin ligase in response to RSK activation. containing 2 μL reaction buffer [50 mM Hepes (pH 7.4), 1 mM EGTA, 1 mM

Binding of 14-3-3 proteins to client proteins can often alter their DTT, 10 mM Mg(OAc)2, and 0.1% Tween 20] in each well. To start the subcellular localization, but in this case we did not find that 14-3-3 reactions, active WT RSK1 (4 ng/μL) and radiolabeled ATP (50 μM, 0.33 μCi/μL participate in the nuclear translocation of PDCD4. Our find- [γ-33P]ATP) were added together to each well, and the plate was sealed and ings demonstrate a molecular mechanism by which the Ras/ incubated at 30 °C for 2 h. Aliquots (200 nL) were then transferred using MAPK pathway and RSK negatively regulates PDCD4 and a pin tool to a streptavidin membrane, which was washed twice with 0.1% thereby likely contributes to melanoma tumorigenesis. SDS in TBS [10 mM Tris (pH 7.5) and 140 mM NaCl), twice with 2 M NaCl, and PDCD4 functions as a tumor suppressor that is lost in many twice with 1% H3PO4 in 2 M NaCl. The membrane was then air-dried and exposed to a phosphor imager screen. Radiolabel incorporation was quan- types of cancer, including lung, colon, and breast cancer, as well tified using QuantityOne software and the data were normalized so that the as glioma (22). One of the most characterized functions of average value for a given position was 1. PDCD4 is as a negative regulator of translation initiation (23). The inhibition of PDCD4 function by RSK is consistent with the Immunoprecipitation and Immunoblotting. Cells were seeded at 2.5 × 106 in fact that RSK was shown to promote cap-dependent translation 10-cm plates, unless otherwise noted. Cell lysates were prepared as pre- (24). Both Akt and S6K1 have previously been shown to regulate viously described (32). Briefly, cells were washed three times with ice-cold μ PDCD4 at Ser67 and Ser457, which results in either decreased PBS and harvested in 600 L lysis buffer [10 mM K3PO4, 1 mM EDTA, 5 mM β protein stability or nuclear translocation, respectively (17, 25). EGTA, 10 mM MgCl2,50mM -glycerophosphate, 0.5% Nonidet P-40, 0.1% Our results indicate that the Ras/MAPK pathway also phos- Brij 35, 0.1% deoxycholic acid, 1 mM sodium orthovanadate (Na3VO4), 1 mM phorylates PDCD4 at a common site (Ser457) but also at Ser76, phenylmethylsulfonyl fluoride, and a Complete Protease Inhibitor Mixture and that RSK seems to be a key regulator of PDCD4 in mela- tablet (Roche)]. For immunoprecipitations, cell lysates were incubated with the indicated antibodies for 2 h, followed by a 1-h incubation with Protein noma (Figs. 4 and 6). Based on the function of PDCD4 as a tu- A-Sepharose CL-4B beads (GE Healthcare). Immunoprecipitates were washed mor suppressor, there has been great interest in identifying small three times in lysis buffer and beads were eluted and boiled in 2× reducing molecules that would act to stabilize PDCD4 in cancer cells (26). sample buffer [5×: 60 mM Tris·HCl (pH 6.8), 25% (vol/vol) glycerol, 2% Our results indicate that RSK inhibitors promote PDCD4 sta- (wt/vol) SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue]. bility in melanoma cells, suggesting that this could be exploited Immunoblotting was performed using a submersible transfer apparatus and

E2926 | www.pnas.org/cgi/doi/10.1073/pnas.1405601111 Galan et al. Downloaded by guest on September 27, 2021 nitrocellulose membranes. Blocking was performed in 5% milk/TBST (0.05% a secondary Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit an- PNAS PLUS Tween-20, 8 mM Tris-Base, 25 mM Tris·HCl, and 154 mM NaCl). Primary tibody (Invitrogen), Texas Red-phalloidin, and DAPI diluted in PBS. Images antibodies were incubated with the membranes in 5% (wt/vol) BSA or 5% were acquired on a Zeiss Axio Imager Z1 wide-field fluorescence microscope milk in TBST and washes were done with TBST. Secondary antibodies con- using a 40× oil immersion objective. jugated to horseradish peroxidase were from Chemicon/Millipore and visualization was done using enhanced chemiluminescence and exposure Protein Stability Assay. The turnover rate of proteins was determined using to X-ray film. cycloheximide (CHX; Sigma Aldrich) to inhibit de novo protein synthesis. HEK293 and A375 cells were treated with CHX (100 μg/mL) with or without Protein Phosphotransferase Assays. For RSK kinase assays, recombinant active PD184352 (10 μM), BI-D1870 (10 μM), or SL0101 (50 μM). After incubation for WT RSK1 (SignalChem) was incubated in the presence or absence of BI-D1870 the indicated time, the cells were harvested and lysed with the lysis buffer μ · (10 M) in kinase buffer [25 mM Tris HCl (pH 7.4), 10 mM MgCl2, and 5 mM described above. Equal amounts of cell lysates were subjected to SDS/PAGE β -glycerophosphate]. Kinase assays were performed with immunopurified and analyzed with the indicated antibodies. Additional information re- full-length HA-tagged PDCD4 as substrate, under linear assay conditions. garding experimental procedures is given in SI Experimental Procedures. Assays were performed for 10 min at 30 °C in kinase buffer supplemented with 5 μCi of [γ-32P]ATP. All samples were subjected to SDS/PAGE followed by 32 ACKNOWLEDGMENTS. We thank all members of our laboratories for their immunoblotting or incorporation of radioactive P label was determined by insightful discussions, as well as Dr. Sylvain Meloche for comments on the autoradiography using a Fuji PhosphorImager with ImageQuant software. manuscript and Marie Cargnello for artwork. This work was supported by grants from the Canadian Cancer Society Research Institute, the Cancer Immunofluoresence Microscopy. For immunofluorescence analyses, HEK293 Research Society, the Canadian Institutes for Health Research (CIHR), and the (transfected with HA-PDCD4) or A375 cells were seeded at 5 × 105 in six-well National Science and Engineering Research Council (to P.P.R.). Work in the plates containing coverslips. Twenty-four hours later, cells were washed laboratories of B.E.T. and B.A.B. was supported by National Institutes of Health (NIH) Grant R01 GM104047 and NIH General Medical Sciences Grant twice in PBS and fixed in 3.7% (vol/vol) formaldehyde for 10 min at room 8P20GM103449, respectively. P.P.R. and P.T., respectively, hold the Canada temperature. Cells were washed twice in PBS and permeabilized for 5 min in Research Chairs in Signal Transduction and Proteomics, and Proteomics and PBS containing 0.2% Triton X-100 and blocked with PBS containing 0.1% Bioanalytical Spectrometry. J.G. holds a Postdoctoral Fellowship from the BSA for 30 min. Cells were incubated for 2 h with indicated anti-HA or anti- CIHR. The Institute for Research in Immunology and Cancer core facilities are PDCD4 antibodies, washed twice with PBS, and incubated for 1 h with supported in part by Le Fonds de Recherche du Québec – Santé.

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