Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding.
Since S-nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by nitric oxide (NO), and since protein kinase C (PKC) has critical thiol residues which influence its kinase activity, we have determined whether NO could regulate this enzyme. Initial studies were carried out with purified PKC and the NO-generating agent S-nitrosocysteine. This agent decreased phosphotransferase activity of PKC in a Ca(2+)- and oxygen-dependent manner with an IC50 of 75 microM. Phorbol ester binding was affected partially only at higher concentrations (> 100 microM) of S-nitrosocysteine. This inactivation of PKC was blocked by the NO scavenger oxyhemoglobin or reversed by dithiothreitol. It is likely that NO initially induced an S-nitrosylation of vicinal thiols, which were then oxidized to form an intramolecular disulfide. Other NO-generating agents such as S-nitroso-N-acetylpenicillamine and sodium nitroprusside, as well as authentic NO gas, induced similar types of PKC modifications. In intact B16 melanoma cells treated with S-nitrosocysteine a rapid decrease in PKC activity in both cytosol and membrane was observed. Unlike in experiments with purified PKC, in intact cells treated with S-nitrosocysteine the phorbol ester binding also decreased to a rate equal to that of PKC activity. These modifications were readily reversed by treating the homogenates with dithiothreitol in test tubes or by removing the NO-generating source from intact cells. To determine whether the limited amounts of NO generated within the intact cells could induce this type of PKC modification, the macrophage cell line IC-21 was treated with lipopolysacharide and Ca2+ ionophore A23187 to induce the NO production. With an increase in generation of NO (3-12-h period) in these cells, a parallel and irreversible decrease in PKC activity and phorbol ester binding was observed. A specific inhibitor for NO synthase, NG-monomethyl-L-arginine, inhibited both the production of NO and PKC inactivation. In experiments using purified enzyme or intact cells there was no decrease in cAMP-dependent protein kinase activity. Conceivably, NO production for limited time induces a reversible inactivation of PKC due to the formation of a disulfide bridge(s), whereas the chronic production of NO could induce irreversible inactivation of PKC. The reversible or irreversible inactivations of PKC may in part influence NO-mediated cytoprotective or cytotoxic actions, respectively.
Gopalakrishna
R
R
Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles 90033.
Chen
Z H
ZH
Gundimeda
U
U
D000818:Animals; D002461:Cell Line, Transformed; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D026403:S-Nitrosothiols
D008070:Lipopolysaccharides; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D015240:Phorbol 12,13-Dibutyrate; C021315:S-nitrosocysteine; D011493:Protein Kinase C; D003545:Cysteine