Studies on proteolysis of protein kinase C with calpain I and II

Earlier reports from this laboratory have shown that protein kinase C (PKC) is cleaved with Ca2(+)-dependent neutral protease (calpain) I or II to produce a catalytically active fragment, and that calpain I, which is active in the micromolar range of Ca2+, may react preferentially with the active form of PKC that is associated with membranes. Subsequently, PKC is shown to exist as a large family of multiple subspecies with subtle individual characteristics. Three types of PKC designated types I, II, and III are purified from rat brain cytosol, which are shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. The aim of the present study was to characterize the proteolysis of each PKC subspecies with calpain I and II. All types of PKC (82 kDa) were converted to two major fragments: a 47-49-kDa catalytic and a 36-kDa regulatory fragments by the cleavage with either calpain I or II. Analysis of the NH2-terminal sequence of the resulting catalytic fragments indicated that both calpain I and II cleaved at one or two specific sites in the variable region (V3) of each PKC molecule of which structure was clearly different among PKC subspecies. From kinetic studies, the cleavage of PKC subspecies with calpain I, and to a lesser extent, with calpain II (active in the millimolar range of Ca2+), was remarkably enhanced by the simultaneous presence of phospholipid and diacylglycerol or phorbol ester, suggesting that the active forms of PKC subspecies were the preferred targets for proteolysis. Whereas, stimulatory abilities of the lipids were variable among PKC subspecies and inactive form of type I PKC was cleaved with calpain I at a significant rate. Quantitative analysis with a fixed amount of calpain under comparable conditions showed that the susceptibilities of the PKC subspecies were distinctly different one another; the relative rates of cleavage of types I, II, and III PKC with calpain I and II were approximately 100:16:2 and 100:48:23, respectively. These results indicated that within the cell various PKC subspecies might be cleaved at different rates under different physiological conditions.