Phosphosecretome profiling and kinase-substrate interaction screening reveal key extracellular c-Src-regulated pathways

Secreted protein kinases are responsible for the phosphorylation of a vast number of extracellular phosphoproteins. The tyrosine kinase c-Src is secreted through the vesicular trafficking pathway, however, mechanistic insights into extracellular substrate specificity and potential therapeutic opportunities in cancer remain unexplored. Here, we show that the SH4/Unique region of c-Src is required for its secretion. Limited proteolysis-mass spectrometry together with mutagenesis further confirmed predictions of in-silico molecular docking of active c-Src structure with its bona fide substrate TIMP2, that the Src homology 3 (SH3) domain is critical for kinase interaction with the P31xxP34 motif in the amino domain of TIMP2. Interestingly, comprehensive unbiased phosphoproteomic studies revealed an enrichment of PxxP containing extracellular substrates as potential targets of secreted c-Src. Cell-based studies using custom anti-c-Src antibodies against the c-Src SH3-TIMP2 interacting interface revealed that blocking secreted Src not only disrupts kinase-substrate interaction but also inhibits prostate cancer cell proliferation. Together, our data provide insights into the intricate role of secreted c-Src in extracellular phosphorylation.