Molecular mechanisms mediating nitric oxide-induced activation of protein kinase C epsilon.

摘要

Activation of protein kinase C (PKC) epsilon by either endogenously or exogenously produced nitric oxide (NO) has been implicated in the development of cardioprotection. However, the molecular mechanisms underlying the activation of PKCepsilon by NO remain largely unknown. Phosphorylation of PKC at the activation loop by PDK-1 and membrane targeting of PKC, which is promoted by anchoring proteins, are two important mechanisms that regulate the enzyme's activity. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. We demonstrate that the NO donor SNAP increases the particulate expression and activity of PKCepsilon in cardiomyocytes in an NO- and peroxynitrite-dependent fashion. SNAP induces peroxynitrite-mediated tyrosine nitration of PKCepsilon in cardiomyocytes in vitro, and nitrotyrosines were also detected on PKCepsilon in vivo in the hearts of rabbits administered a protective dose of the NO donor DETA/NO, as determined by Western immunoblotting. Co-immunoprecipitation of PKCepsilon and its specific anchoring protein RACK2 showed a peroxynitrite-dependent increase in PKCepsilon-RACK2 interactions in SNAP-treated cardiomyocytes. Moreover, recombinant PKCepsilon treated with the peroxynitrite donor SIN-1 exhibited increased binding to recombinant RACK2 protein, as determined by ELISA. Our data demonstrate that post-translational modification, namely nitration, of PKCepsilon by NO donors facilitates its interaction with RACK2, thereby promoting translocation and activation of PKCepsilon. These findings offer a plausible novel mechanism of NO-mediated activation of PKC signaling pathway.