Phosphoproteomics Analysis Identifies Novel Candidate Substrates of the Nonreceptor Tyrosine Kinase, S rc- r elated Kinase Lacking C-terminal Regulatory Tyrosine and N-terminal M yristoylation S ites (SRMS)

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites), also known as PTK 70 (Protein tyrosine kinase 70), is a non-receptor tyrosine kinase that belongs to the BRK family of kinases (BFKs). To date less is known about the cellular role of SRMS primarily because of the unidentified substrates or signaling intermediates regulated by the kinase. In this study, we used phosphotyrosine antibody-based immunoaffinity purification in large-scale label-free quantitative phosphoproteomics to identify novel candidate substrates of SRMS. Our analyses led to the identification of 1258 tyrosine-phosphorylated Peptides which mapped to 663 phosphoproteins, exclusively from SRMS-expressing cells. DOK1, a previously characterized SRMS substrate, was also identified in our analyses. Functional enrichment analyses revealed that the candidate SRMS substrates were enriched in various biological processes including protein ubiquitination, mitotic cell cycle, energy metabolism and RNA processing, as well as Wnt and TNF signaling. Analyses of the sequence surrounding the phospho-sites in these proteins revealed novel candidate SRMS consensus substrate motifs. We utilized customized high-throughput peptide arrays to validate a subset of the candidate SRMS substrates identified in our MS-based analyses. Finally, we independently validated Vimentin and Sam68, as bona fide SRMS substrates through in vitro and in vivo assays. Overall, our study identified a number of novel and biologically relevant SRMS candidate substrates, which suggests the involvement of the kinase in a vast array of unexplored cellular functions.