Novel mechanism of protein kinase C inhibition involving the pseudosubstrate region by secalonic acid D in vitro.
Evidence from studies in mice suggests a mechanistic role for the inhibition of conventional isoforms of protein kinase C (cPKC) in the development of cleft palate (CP) in the offspring of female mice treated with the mycotoxin, secalonic acid D (SAD). These experiments were aimed at assessing whether SAD inhibits commercially available pure cPKC (PKCalpha, -beta, -gamma) and at identifying the mechanism of such an inhibition in vitro. Secalonic acid D inhibited the three isozymes similarly (IC50 of 5 to 6.2 microM by direct extrapolation and 2.7 to 4 microM by logarithmic regression). The loss of inhibitory effect of SAD upon removal of the regulatory domain of PKCbetaII, the most predominant cPKC in the palate, suggested that the inhibition was mediated by the regulatory subunit. Kinetic analysis suggested a lack of competitive interaction for SAD with the binding sites for Ca(2+) and phosphatidyl serine (PS). Antibody directed against residues 19-32 of the pseudosubstrate region of PKCbetaII, however, competitively reversed the inhibition of PKCbetaII by SAD, suggesting that the pseudosubstrate is the site of interaction of SAD. Further, SAD inhibited the cleavage of the pseudosubstrate from PKCbetaII by the endoproteinase Arg-C. The fact that the activity of Arg-C itself was not inhibited by SAD suggests that SAD interferes with the preceding step involving the cofactor-induced release of the pseudosubstrate from the active site of PKCbetaII, a novel mechanism.
Balasubramanian
G
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Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri, 65211, USA.
Reddy
C S
CS
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000633:toxicity; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D064546:Protein Kinase C beta; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D014966:Xanthenes / Q000633:toxicity*; D044004:Xanthones
D007527:Isoenzymes; D009183:Mycotoxins; D010718:Phosphatidylserines; D014966:Xanthenes; D044004:Xanthones; C009218:secalonic acid; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; D002118:Calcium