Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-04, Vol.109 (15), p.5615-5620
• Main Authors: Xue, Liang, Wang, Wen-Horng, Iliuk, Anton, Hu, Lianghai, Galan, Jacob A, Yu, Shuai, Hans, Michael, Geahlen, Robert L, Tao, W. Andy
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Antibodies; B-Lymphocytes - enzymology; Biological Sciences; Breast cancer; breast neoplasms; Breast Neoplasms - enzymology; cell division; Cell lines; Centrosome - enzymology; Datasets; Enzyme Assays - methods; Epithelial cells; Epithelial Cells - enzymology; Female; Humans; Intracellular Signaling Peptides and Proteins - metabolism; Kinases; Mass spectrometers; Mass spectrometry; Mass spectroscopy; Molecular Sequence Data; Peptides; Phosphoproteins - metabolism; Phosphorylation; Physical Sciences; protein kinases; Protein Kinases - metabolism; Protein-Tyrosine Kinases - metabolism; Proteomics; Proteomics - methods; Reproducibility of Results; spleen; Substrate Specificity; Substrates; Syk Kinase; tyrosine
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1119418109

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.