Dataset Information

Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

ABSTRACT: Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways.

SUBMITTER: Petrone A

PROVIDER: S-EPMC4937516 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

Petrone Adam A Adamo Mark E ME Cheng Chao C Kettenbach Arminja N AN

Molecular & cellular proteomics : MCP 20160501 7

Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in m ...[more]

PMID: 27134283

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Project description:Protein phosphorylation is a crucial regulatory mechanism that controls many aspects of cellular signaling. Casein kinase 2 (CK2), a constitutively expressed and active kinase, plays key roles in an array of cellular events including transcription and translation, ribosome biogenesis, cell cycle progression, and apoptosis. CK2 is implicated in cancerous transformation and is a therapeutic target in anti-cancer therapy. The specific and selective CK2 ATP competitive inhibitor, CX-4945 (silmitaseratib), is currently in phase 2 clinical trials. While many substrates and interactors of CK2 have been identified, less is known about CK2 substrates in mitosis. In the present work, we utilize CX-4945 and quantitative phosphoproteomics to inhibit CK2 activity in mitotically arrested HeLa cells and determine candidate CK2 substrates. We identify 330 phosphorylation sites on 202 proteins as significantly decreased in abundance upon inhibition of CK2 activity. Motif analysis of decreased sites reveals a linear kinase motif with aspartic and glutamic amino acids downstream of the phosphorylated residues, which is consistent with known substrate preferences for CK2. To validate specific candidate CK2 substrates, we perform in vitro kinase assays using purified components. Furthermore, we identified CK2 interacting proteins by affinity purification-mass spectrometry (AP-MS). To investigate the biological processes regulated by CK2 in mitosis, we perform network analysis and identify an enrichment of proteins involved in chromosome condensation, chromatin organization, and RNA processing. We demonstrate that overexpression of CK2 in HeLa cells affects proper chromosome condensation. Previously, we found that phosphoprotein phosphatase 6 (PP6), but not phosphoprotein phosphatase 2A (PP2A), opposes CK2 phosphorylation of the condensin I complex, which is essential for chromosome condensation. Here, we extend this observation and demonstrate that PP6 opposition of CK2 is a more general cellular regulatory mechanism.

Dataset Information

Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

Publications

Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

Similar Datasets

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