Oxidant-induced S-gutathiolation inactivates protein kinase C-alpha (PKC-alpha): A potential mechanism of PKC isozyme regulation

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxy gen species (ROS) through as yet undefined oxidative modifications of the i sozyme structure. We previously reported that Cys-containing, Arg-rich pept ide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC mi ght be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide li nkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutat hiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examin e whether PKC could be inactivated by S-glutathiolation, we used the thiol- specific oxidant diamide because its oxidant activity is restricted to indu ction of disulfide bridge formation. Diamide weakly inactivated purified re combinant cPKC-alpha, and this was markedly potentiated to nearly full inac tivation by 100 mu M GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH we re both fully reversed by DTT. Likewise, GSH markedly potentiated diamide i nactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, is an element of, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-a inactivation was associated with S-glutathiolati on of the isozyme, cPKC-alpha S-glutathiolation was demonstrated by the DTT -reversible incorporation of [S-35]GSH into the isozyme structure and by an associated change in the migration position of cPKC-a in nonreducing SDS-P AGE. Diamide treatment of NM3T3 cells likewise induced potent, DTT-reversib le inactivation of cPKC-alpha: in association with [S-35] S-thiolation of t he isozyme. Taken together, the results indicate that PKC isozymes can be o xidatively inactivated by S-thiolation reactions involving endogenous thiol s such as GSH.