Phosphorylation of c-Crk II on the negative regulatory Tyr(222) mediates nerve growth factor-induced cell spreading and morphogenesis

The Crk family of adaptor proteins participate in diverse signaling pathway s that regulate growth factor-induced proliferation, anchorage-dependent DN A synthesis, and cytoskeletal reorganization, important for cell adhesion a nd motility, Using kidney epithelial 293T cells for transient co-transfecti on studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-recep tor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II pho sphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperph osphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tye(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphoryl ation, followed by a transient initial dissociation of the c-Crk II paxilli n complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cel lular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-C rk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas prev ious studies have shown that Crk SH2 binding to paxillin is critical for ce ll adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulati ng turnover of multiprotein complexes, a critical aspect of cytoskeletal pl asticity and actin dynamics.