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 protein s that have been implicated in regulating the TrkA receptor tyrosine k inase and potentiating Nerve Growth Factor (NGF)-mediated neurite outg rowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Bio l. 14:1964-1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic ce ll death elicited by NGF-withdrawal in native, v-Crk, and TrkA-express ing PC12 cells. While more than 50% of native PC12 cells underwent apo ptosis within 48 h of NGF withdrawal, the V-Crk and TrkA-expressing ce lls were much more resistant to apoptosis under these conditions, wher eby approximately 70 and 95%, respectively, of the cells were alive. T he ability of v-Crk to delay apoptosis required prior NGF-dependent di fferentiation, since naive undifferentiated v-Crk expressing PC12 cell s or cells that express v-Crk mutants that are defective in NGF signal ing were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk ex pressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotro phic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a recepto r tyrosine kinase under conditions of growth factor deprivation is ess ential for preventing apoptosis. However, unlike native PC12 cells, ne ither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when i ncubated for 2 weeks in NGF. Thus, v-Crk and TrkA may rotect NGF depri ved PC12 by preventing cell cycle arrest and hence an aborted entry in to a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6-8 h after NGF de privation. Thus, unlike v-Crk-mediated augmentation of differentiation , sustained activation of MAP kinase may not be required for v-Crk-ind uced cell survival.