Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3
Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 13, 2014 (received for review April 9, 2014)
Significance
The RSK family is a group of Ser/Thr kinases that promotes cell growth and proliferation in response to the Ras/MAPK pathway. Deregulated RSK activity has been associated with different disorders and diseases, such as cancer, but relatively little is known regarding the contribution of RSK to tumorigenesis. In this study, we describe, to our knowledge, the first global quantitative phosphoproteomic screen to characterize RSK-dependent signaling events in melanoma. Our results show that RSK negatively regulates the tumor suppressor PDCD4 by promoting its association to 14-3-3 proteins and subsequent proteasomal degradation. These findings further implicate RSK as a promising therapeutic target for the treatment of melanoma and suggest that RSK plays widespread biological functions downstream of the Ras/MAPK pathway.
Abstract
The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis.
Acknowledgments
We thank all members of our laboratories for their insightful discussions, as well as Dr. Sylvain Meloche for comments on the manuscript and Marie Cargnello for artwork. This work was supported by grants from the Canadian Cancer Society Research Institute, the Cancer Research Society, the Canadian Institutes for Health Research (CIHR), and the National Science and Engineering Research Council (to P.P.R.). Work in the 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 8P20GM103449, respectively. P.P.R. and P.T., respectively, hold the Canada Research Chairs in Signal Transduction and Proteomics, and Proteomics and Bioanalytical Spectrometry. J.G. holds a Postdoctoral Fellowship from the CIHR. The Institute for Research in Immunology and Cancer core facilities are supported in part by Le Fonds de Recherche du Québec – Santé.
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Published online: July 7, 2014
Published in issue: July 22, 2014
Acknowledgments
We thank all members of our laboratories for their insightful discussions, as well as Dr. Sylvain Meloche for comments on the manuscript and Marie Cargnello for artwork. This work was supported by grants from the Canadian Cancer Society Research Institute, the Cancer Research Society, the Canadian Institutes for Health Research (CIHR), and the National Science and Engineering Research Council (to P.P.R.). Work in the 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 8P20GM103449, respectively. P.P.R. and P.T., respectively, hold the Canada Research Chairs in Signal Transduction and Proteomics, and Proteomics and Bioanalytical Spectrometry. J.G. holds a Postdoctoral Fellowship from the CIHR. The Institute for Research in Immunology and Cancer core facilities are supported in part by Le Fonds de Recherche du Québec – Santé.
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3, Proc. Natl. Acad. Sci. U.S.A.
111 (29) E2918-E2927,
https://doi.org/10.1073/pnas.1405601111
(2014).
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