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 func- tions 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 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. 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.