Cancer-preventive selenocompounds induce a specific redox modification of cysteine-rich regions in Ca(2+)-dependent isoenzymes of protein kinase C.
Since protein kinase C (PKC) serves as a receptor for phorbol ester type tumor promoters and oxidants and has unique redox-active cysteine-rich regions, we have determined whether various chemopreventive selenocompounds could affect this enzyme. At lower concentrations, selenite decreased the kinase activity (IC50 = 0.5 microM), while at higher concentrations it decreased phorbol ester binding. However, when the catalytic and regulatory domains of PKC were separated by proteolysis, the catalytic domain retained its sensitivity to selenite, while the regulatory domain lost its sensitivity. Cysteine residues were quantitated in PKC modified with selenite by using 5,5'-dithiobis(2-nitrobenzoic acid) and also by using 2-nitro-5-thiosulfobenzoic acid after sulfitolysis. At lower concentrations, selenite induced a modification of four cysteine residues resulting in the formation of two disulfides, while at higher concentrations it induced a modification of seven to eight cysteine residues resulting in the formation of three to four disulfides. Contrary to selenite, selenocystine and selenodiglutathione (GSSeSG) readily inactivated the kinase activity, but not the phorbol ester binding. These two agents induced a two-stage modification of PKC; a limited modification at low concentrations leads to a loss of affinity for ATP, while an excessive modification at high concentrations leads to a loss of Vmax. Selenocystine and GSSeSG were 100,000-fold more potent than GSSG in inactivating PKC. The isoenzymes alpha, beta, and gamma exhibited an identical susceptibility to these selenocompounds. These results suggested that the cysteine residues present within the catalytic domain of these isoenzymes, although apart in the sequence, may be clustered in the tertiary structure to react with selenite, as well as may be in close proximity to some of the cysteines in the regulatory domain. Selenite did not affect protein kinase A, whereas GSSeSG and selenocystine inactivated the catalytic subunit after dissociation from the regulatory subunit at concentrations 100- and 800-fold, respectively, higher than that required for PKC inactivation. All three selenocompounds did not affect the activities of phosphorylase kinase and protein phosphatase 2A. Taken together, these results suggest that the accessible redox-active cysteine residues present in the PKC catalytic domain can react with certain specificity with redox-active selenocompounds such as selenite, selenocystine, and GSSeSG relative to other protein kinases tested.
Gopalakrishna
R
R
Department of Cell and Neurobiology, School of Medicine, University of Southern California, Los Angeles 90033, USA. rgopalak@hsc.usc.edu
Gundimeda
U
U
Chen
Z H
ZH
D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003545:Cysteine; D003553:Cystine / Q000031:analogs & derivatives / Q000494:pharmacology; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009579:Nitrobenzoates; D016566:Organoselenium Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D054648:Protein Phosphatase 2; D051381:Rats; D018038:Sodium Selenite / Q000494:pharmacology
D016588:Anticarcinogenic Agents; D004220:Disulfides; D007527:Isoenzymes; D009579:Nitrobenzoates; D016566:Organoselenium Compounds; C009226:selenocystine; C017710:selenodiglutathione; D003553:Cystine; C041724:2-nitro-5-thiosulfobenzoic acid; D004228:Dithionitrobenzoic Acid; C497260:Prkcd protein, rat; D010764:Phosphorylase Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D005978:Glutathione; D018038:Sodium Selenite; D003545:Cysteine; D002118:Calcium; D004229:Dithiothreitol