K-252A INDUCES TYROSINE PHOSPHORYLATION OF THE FOCAL ADHESION KINASE AND NEURITE OUTGROWTH IN HUMAN NEUROBLASTOMA SH-SY5Y CELLS

The protein kinase inhibitor K-252a has been shown to promote choliner gic activity in cultures of rat spinal cord and neuronal survival in c hick dorsal root ganglion cultures. To determine the mechanism by whic h K-252a acts as a neurotrophic factor, we examined the effects of thi s molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinas e C inhibition because downregulation of protein kinase C by long-term treatment with phorbol ester did not prevent K252a-induced tyrosine p hosphorylation. Similarly, the protein kinase C inhibitors H7, GF-1092 03X, and calphostin C did not induce the phosphorylation. We have iden tified one of the phospho-substrates as the pp125 focal adhesion prote in tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integr in interaction. The induction of Fak phosphorylation by K-252a was als o observed in LA-N-5 cells and primary cultures of rat embryonic stria tal cells but not in PC12 cells. The protein kinase C-independent indu ction of tyrosine phosphorylation and the identification of Fak as a s ubstrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathw ay.