V-CRK, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NCR-mediated neunte outgrowth in pheochromocytoma (PC12) cells (Hempstead et al. Mol. Cell Biol. 14;1964-1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 hr of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 80 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation; naive undifferentialed v-Crk expressing PCI 2 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EOF to serum and NGF deprived v-Crk expressing cells, which also causes neunte outgrowth, promoted complete and longterm survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkAexpressing PC12 cells exhibited a Gl arrest when incubated for two weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC 12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited downregulation in MAP kinase and JNK activities while in native cells, these activities increased within 6-8 hours after NGF deprivation. Thus, unlike with differentiation, constituitive activation of MAP kinase may not be required for v-Crk-induced cell survival.