Iterative tyrosine phosphorylation controls non-canonical domain utilization in Crk

Crk, the prototypical member of a class of SH2 and SH3 domain-containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-SH3N signaling. Here, we show using LC-MS/MS and by generating phosphospecific antibodies that, iteratively with Y221, the Crk SH3C is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. While phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind Polyproline Type II (PPII) ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix based bio-informatics approaches, and unbiased MS, we found that the phosphoSH3C binds several SH2 domain-containing proteins, including specific non-receptor tyrosine kinases - Abl via pY251 and Csk via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.