Oxidant-induced S-glutathiolation 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 oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.
Ward
N E
NE
Departments of Cancer Biology and Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Stewart
J R
JR
Ioannides
C G
CG
O'Brian
C A
CA
D000818:Animals; D001921:Brain / Q000201:enzymology; D003958:Diamide / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004357:Drug Synergism; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000378:metabolism*; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D003958:Diamide; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D005978:Glutathione; D004229:Dithiothreitol