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 inhibitio n of conventional isoforms of protein kinase C (cPKC) in the development of cleft palate (CP) in the offspring of female mice treated with the mycotox in, secalonic acid D (SAD). These experiments were aimed at assessing wheth er SAD inhibits commercially available pure cPKC (PKC alpha, -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 mu M by di rect extrapolation and 2.7 to 4 mu M by logarithmic regression). The loss o f inhibitory effect of SAD upon removal of the regulatory domain of PKC bet a II, the most predominant cPKC in the palate, suggested that the inhibitio n was mediated by the regulatory subunit. Kinetic analysis suggested a lack of competitive interaction for SAD with the binding sites for Ca2+ and pho sphatidyl serine (PS). Antibody directed against residues 19-32 of the pseu dosubstrate region of PKC beta II, however, competitively reversed the inhi bition of PKC beta II by SAD, suggesting that the pseudosubstrate is the si te of interaction of SAD. Further, SAD inhibited the cleavage of the pseudo substrate from PKC beta II by the endoproteinase Arg-C. The fact that the a ctivity of Arg-C itself was not inhibited by SAD suggests that SAD interfer es with the preceding step involving the cofactor-induced release of the ps eudosubstrate from the active site of PKC beta II, a novel mechanism. (C) 2 000 Academic Press.