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Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

ABSTRACT: Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.

SUBMITTER: Xue L

PROVIDER: S-EPMC3326509 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

Xue Liang L Wang Wen-Horng WH Iliuk Anton A Hu Lianghai L Galan Jacob A JA Yu Shuai S Hans Michael M Geahlen Robert L RL Tao W Andy WA

Proceedings of the National Academy of Sciences of the United States of America 20120326 15

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of ...[more]

PMID: 22451900

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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

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

Publications

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

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