A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN's seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding.
Keywords: C-terminal Src kinase; CSK; CUB; Cas; Crk-associated substrate; CrkCT10 regulator of kinase Crk-Like; CrkL; DCBLD2; ESDN; Functional phosphoproteomics; GRB2-associated-binding protein 1; GST; Gab1; IRS-1; LC; MS; Mass spectrometry; PCR; Phosphorylation; SFK; SH2; SILAC; Signal transduction; Src family kinase; Src family tyrosine kinase; WCE; complement C1r/C1s Uegf Bmp1; discoidin CUB and LCCL domain containing 2; endothelial and smooth muscle cell-derived neuropilin-like protein; glutathione stransferase; insulin receptor substrate-1; liquid chromatography; mass spectrometry; polymerase chain reaction; stable-isotope labeling by amino acids in cell culture; whole cell extract.
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