Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.
The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.
Gopalakrishna
R
R
Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles 90033.
Anderson
W B
WB
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008934:Mitogens / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002273:Carcinogens; D008934:Mitogens; D010718:Phosphatidylserines; D010743:Phospholipids; D013438:Sulfhydryl Compounds; D010504:Periodic Acid; D015240:Phorbol 12,13-Dibutyrate; C009288:metaperiodate; D011493:Protein Kinase C; D002118:Calcium; D004229:Dithiothreitol