Phosphoproteomics analysis identifies novel candidate substrates of the non-receptor tyrosine kinase, SRMS

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) or PTK 70 (Protein tyrosine kinase 70) is a non-receptor tyrosine kinase that belongs to the BRK family kinases (BFKs). The gene encoding SRMS was first discovered in 1994. Yet to date less is known about the cellular role of SRMS primarily due to 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 1259 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 represented various biological processes typically linked to cell growth and RNA metabolism. Analyses of the sequence surrounding the phospho-sites in these proteins led us to identify novel candidate SRMS consensus substrate motifs. We utilized customized high-throughput peptide arrays to validate a subset of the candidate SRMS substrates and motifs identified in our MS-based analyses. Finally, we independently validated Vimentin and Sam68, as bonafide SRMS substrates through in vitro and in vivo assays. Overall, our study led to the identification of a significant number of novel and biologically relevant SRMS candidate substrates, which suggests the involvement of the kinase in a vast array of unexplored cellular functions.