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 matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F 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 activation 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-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation 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 PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.
Tejedo
J R
JR
Laboratory of Biochemistry of the Immune System, Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, University of Sevilla, Avenida Sanchez Pizjuan, 4, 41009, Seville, Spain.
Ramírez
R
R
Cahuana
G M
GM
Rincón
P
P
Sobrino
F
F
Bedoya
F J
FJ
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D006152:Cyclic GMP / Q000096:biosynthesis; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D006162:Guanylate Cyclase / Q000502:physiology; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D014226:Triazenes / Q000494:pharmacology
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D016895:Culture Media, Serum-Free; D020030:Nitric Oxide Donors; D014226:Triazenes; D009569:Nitric Oxide; D016392:Proto-Oncogene Proteins pp60(c-src); D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP