Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells

The mechanism by which nitric oxide (NO) protects from apoptosis is a matte r of debate. We have shown previously that phosphorylation of tyrosine resi dues participates in the protection from apoptosis in insulin-producing RIN m5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the acti vation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-depri ved cells to 10 muM DETA/NO results in protection from degradation of the a nti-apoptotic protein Bcl-2, together with a reduction of cytochrome c rele ase from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activati on of guanylate cyclase and PKG. DETA/NO was also able to induce autophosph orylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c -Src kinase inhibitor PPI abrogated the actions of DETA/NO on cGMP formatio n, PKG activation, caspase activation, cytochrome c release from mitochondr ia, and Bcl-2 phosphorylation and degradation in serum-deprived cells, We t hus propose that activation of c-Src is an early step in the chain of event s that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria. (C) 2001 Elsevier Science Inc. All rights reserved.