Phosphoproteomics Analyses to Identify the Candidate Substrates and Signaling Intermediates of the Non-Receptor Tyrosine Kinase, SRMS
SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylaton sites) is a non-receptor tyrosine kinase that belongs to the BRK family kinases (BFKs) and is evolutionarily related to the Src family kinases (SFKs). Like SFKs and BFKs, the SRMS protein comprises of two domains involved in protein-protein interactions, namely, the Src-homology 3 domain (SH3) and Src-homology 2 domain (SH2) and one catalytic kinase domain. Unlike members of the BFKs and SFKs, the biochemical and cellular role of SRMS is poorly understood primarily due to the lack of information on the substrates and signaling intermediates regulated by the kinase. Previous biochemical studies have shown that wild type SRMS is enzymatically active and leads to the tyrosine-phosphorylation of several proteins, when expressed exogenously in mammalian cells. These tyrosine-phosphorylated proteins represent the candidate cellular substrates of SRMS which are largely unknown. Further, previous studies have determined that the SRMS protein displays a characteristic punctate cytoplasmic localization pattern in mammalian cells. These SRMS cytoplasmic puncta are uncharacterized and may provide insights into the biochemical and cellular role of the kinase. Here, we utilized mass spectrometry-based quantitative label-free phosphoproteomics to (a) identify the candidate SRMS cellular substrates and (b) candidate signaling intermediates regulated by SRMS, in HEK293 cells expressing ectopic SRMS. Specifically, using a phosphotyrosine enrichment strategy we identified 663 candidate SRMS substrates and consensus substrate-motifs of SRMS. We used customized peptide arrays and performed the high-throughput validation of a subset of the identified candidate SRMS substrates. Further, we independently validated Vimentin and Sam68 as bonafide SRMS substrates. Next, using Titanium dioxide (TiO2)-based phosphopeptide enrichment columns, we identified multiple signaling intermediates of SRMS. Functional gene enrichment analyses revealed several common and unique cellular processes regulated by the candidate SRMS substrates and signaling intermediates. Overall, these studies led to the identification of a significant number of novel and biologically relevant SRMS candidate substrates and signaling intermediates, which mapped to a number of cellular and biological processes primarily involved in cell cycle regulation, apoptosis, RNA processing, DNA repair and protein synthesis. These findings provide an important resource for future mechanistic studies to investigate the cellular and physiological functions of the SRMS. Studies towards characterizing the SRMS cytoplasmic puncta showed that the SRMS punctate structures do not colocalize with some of the major cellular organelles investigated, such as the mitochondria, endoplasmic reticulum, golgi bodies and lysosomes. However, studies investigating the involvement of the SRMS domains in puncta-localization revealed that the SRMS SH2 domain partly regulates this localization pattern. These results highlight the potential role of the SRMS SH2 domain in the localization of SRMS to these cytoplasmic sites and lay important groundwork for future characterization studies.
SRMS, PTK70, substrates, BRK, FRK, Src, phosphoproteomics, mass spectrometry, PTK6, PTK5, non-receptor tyrosine kinase
Doctor of Philosophy (Ph.D.)