DIFFERENTIAL INHIBITION OF PROTEIN-KINASE-C ISOZYMES BY UCN-01, A STAUROSPORINE ANALOG

UCN-01 (7-hydroxystaurosporine) has been demonstrated to be a potent i nhibitor of tumor cell growth both in cell culture and with in vivo xe nograft models. The ability of UCN-01 to inhibit the kinase activity o f recombinant protein kinase C (PKC) isozymes alpha, beta, gamma, delt a, epsilon, and zeta was characterized using an in vitro kinase assay. Two distinct groups of isozymes could be defined on the basis of rela tive potency of kinase inhibition. UCN-01 was 15-20-fold more potent f or inhibition of the Ca2+-dependent isozymes, compared with the Ca2+-i ndependent isozymes. In contrast, UCN-02 (the diastereomer of UCN-01) and staurosporine exhibited less ability to discriminate between Ca2+- dependent and -independent isozymes. PKC-zeta was not inhibited by UCN 01, UCN-02, or staurosporine. IC50 values for UCN-01 inhibition of the Ca2+-dependent PKC-alpha, -beta, and -gamma were 29, 34, and 30 nM, r espectively, and for the Ca2+-independent PKC-delta and -epsilon were 530 and 590 nM, respectively. IC50 values for staurosporine inhibition of the isozymes alpha, beta, and gamma were 58, 65, and 49 nM, respec tively, and for the isozymes delta and epsilon were 325 and 160 nM, re spectively. UCN-02 was significantly less potent for the inhibition of PKC-alpha, -beta, -gamma, -delta, and -epsilon (IC50 values of 530, 7 00, 385, 2800, and 1200 nM, respectively). An analysis of the inhibiti on by UCN-01 and staurosporine of the kinase activity of PKC-alpha and -delta indicated mixed inhibition kinetics. Increasing the ATP concen tration resulted in decreased potency, as shown by increased IC50 valu es. In contrast, increasing the peptide substrate concentration result ed in increased potency, as shown by decreased IC50 values. Increasing concentrations of myelin basic protein as a PKC-alpha or -delta subst rate also caused increased potency of inhibition by UCN-01. Because of the competitive nature of inhibition with respect to ATP and the unco mpetitive nature with respect to substrate, the concentrations of thes e substrates can have dramatically different effects on the degree of inhibition observed. These data also suggest that UCN-01 may be an imp ortant tool for the dissection of PKC isozyme contributions to signal transduction pathways.