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

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.

SUBMITTER: Galan JA

PROVIDER: S-EPMC4115529 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Galan Jacob A JA Geraghty Kathryn M KM Lavoie Geneviève G Kanshin Evgeny E Tcherkezian Joseph J Calabrese Viviane V Jeschke Grace R GR Turk Benjamin E BE Ballif Bryan A BA Blenis John J Thibault Pierre P Roux Philippe P PP

Proceedings of the National Academy of Sciences of the United States of America 20140707 29

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 sig ...[more]

PMID: 25002506

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Project description:Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. The Cancer Genome Atlas (TCGA) data showed that down regulation of DLC1 is not due to methylation or mutations. To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we stably overexpressed DLC1-full-length (DLC1-FL) in T-47D breast cancer cells; this inhibited cell colony formation significantly in vitro compared to its control counterpart. Label-free global proteomic and TiO2 phosphopeptide enrichment assays (ProteomeXchange identifier PXD008220) showed that 205 and 122 phosphopeptides were unique to DLC1-FL cells and T-47D-control cells, respectively, whereas 6,726 were quantified by phosphoproteomics analysis in both conditions. The top three significant clusters of differentially phosphopeptides identified by DAVID pathway analysis represent cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Phosphoproteomics analysis documented an inverse relation between DLC1 expression and several phosphopeptides including epithelial cell transforming sequence 2 (ECT2). Decreased phosphorylation of ECT2 at the residue T359, critical for its active conformational change, was validated by western blot. In addition, the ECT2 T359-containing phosphopeptide was detected in both basal and luminal patient-derived breast cancers breast cancer phosphoproteomics data on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay portal. Together, for the first time, this implicates ECT2 phosphorylation in breast cancer, which has been proposed as a therapeutic target in lung cancer. In conclusion, this data suggests that low expression of DLC1 is associated with poor prognosis. Targeting ECT2 phosphopeptides could provide a promising mechanism for controlling poor prognosis seen in DLC1low ER+ breast cancer.

Dataset Information

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

Publications

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

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