Staurosporine up-regulates the expression of phorbol dibutyrate binding sites in human platelets

Abstract

Tumor-promoting phorbol esters bind to and activate protein kinase C (PKC). Staurosporine, a potent PKC inhibitor, interferes with PKC catalytic activity without altering phorbol ester binding sites in cell-free systems. We found that, unlike cell-free systems, treatment of intact platelets with staurosporine enhances the expression of phorbol 12,13-dibutyrate (PDBu) binding sites. Incubation of platelets at 37° with staurosporine (25 nM to 1 μM and 2 nM tritiated PDBu ([ 3H]PDBu) increased the amount of [ 3H]PDBu specifically bound to intact platelets by approximately 10 to 200% of control values. This effect was rapid and plateaued after 10 min of cell treatment. Scatchard analysis of the data showed that staurosporine (500 nM) significantly increased the total binding capacity B max from 42.9 ± 15.4 × 10 3 to 78 ± 7.3 × 10 3 sites per platelet and reduced the apparent dissociation constant value K d from 30.8 ± 8.6 nM to 9.4 ± 3.4 nM. Enhanced PDBu binding capacity and affinity were also observed with human mononuclear and polymorphonuclear leukocytes. Fractionation of staurosporine-treated platelets showed an increased binding capacity of the particulate fraction (102%) and decreased binding capacity of the soluble fraction (60%) compared to controls, with no change in the affinity of PDBu binding to these fractions. Chelation of internal calcium with BAPTA did not significantly attenuate the staurosporine-mediated rise in PBDu binding but prevented the platelet-activating factor-induced response, indicating that cytosolic calcium does not play an important role in these Staurosporine effects. These results show that, in addition to interfering with PKC protein-phosphorylating activity, staurosporine enhances PDBu binding affinity and capacity in intact platelets. This latter effect appears to be due to translocation of soluble PDBu binding sites, presumably PKC units.