Activation of hepatocyte protein kinase C by redox-cycling quinones.
The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
G E
GE
Kass
S K
SK
Duddy
S
S
Orrenius
D000818:Animals; D016227:Benzoquinones; D002469:Cell Separation; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014812:Vitamin K / Q000494:pharmacology
D016227:Benzoquinones; D011809:Quinones; D013438:Sulfhydryl Compounds; D014812:Vitamin K; D011493:Protein Kinase C; C034257:duroquinone