Nitric Oxide (NO) Induces Nitration of Protein Kinase C (PKC ), Facilitating PKC Translocation via Enhanced PKC -RACK2 Interactions

Activation of protein kinase C (PKC) by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKC by NO remain largely unknown. 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. In this investigation, we demonstrate that NO donors promote translocation and activation of PKC in an NOand peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKC in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKC in vivo in the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKC and its receptor for activated C kinase, RACK2, illustrated a peroxynitrite-dependent increase in PKC -RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assaybased protein-protein interaction assay, PKC proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKC by NO donors, namely nitration of PKC , facilitates its interaction with RACK2 and promotes translocation and activation of PKC . These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway.