Abstract

The indole carbazole staurosporine is an extraordinarily potent antiproliferative agent that inhibits the growth of cultured mammalian cells at concentrations of less than 1 nM. The antiproliferative activity of staurosporine is attributed to its potent inhibition of diverse protein kinases, but the mechanism of staurosporine inhibition has not been elucidated for any protein kinase. Protein kinase C (PKC) is a family of Ca(2+)- and phosphatidylserine-dependent protein kinases that are activated in vivo by the second messenger diacylglycerol. A fully active, Ca(2+)- and phosphatidylserine-independent, catalytic fragment of PKC that contains only the catalytic domain of the enzyme can be produced by limited proteolysis. Previous studies indicated that staurosporine inhibits PKC by binding its catalytic domain. In this study, we define the kinetics of inhibition by staurosporine of a catalytic fragment of rat brain PKC-gamma and of a catalytic fragment generated from a rat brain PKC-alpha/PKC-beta mixture. Our kinetic results provide evidence that staurosporine inhibits PKC by binding to a site of the catalytic domain other than the ATP substrate and protein substrate binding sites. Staurosporine inhibition appears to entail binding at a conserved site in the catalytic domain of PKC, because staurosporine inhibited rat brain PKC-alpha, PKC-beta, and PKC-gamma, as well as the catalytic fragments of PKC-beta and PKC-gamma, with similar protencies. The kinetics of inhibition of the catalytic fragment of PKC-gamma were uncompetitive with respect to histone III-S, providing evidence that the binding of histone III-S at the active site of the catalytic fragment precedes the binding of staurosporine to the enzyme. Taken in the context of previous mechanistic studies of PKC-catalyzed histone III-S phosphorylation, these results provide evidence that staurosporine binds to a complex of PKC, MgATP, and histone III-S, thereby forming a complex that cannot break down to products. In addition, the inhibitory kinetics observed when the ATP concentration was varied provided evidence that staurosporine reduces the affinity of MgATP for the catalytic fragment of PKC-gamma. Thus, the kinetics of inhibition of the catalytic fragment of PKC-gamma by staurosporine provide evidence that staurosporine inhibits PKC by a mixed mechanism.