PHOSPHORYLATION OF THE PLATELET P47 PHOSPHOPROTEIN IS MEDIATED BY THELIPID PRODUCTS OF PHOSPHOINOSITIDE 3-KINASE

Platelet stimulation by thrombin or the thrombin receptor activating p eptide (TRAP) results in the activation of phosphoinositide 3-kinase a nd the production of the novel polyphosphoinositides phosphatidylinosi tol 3,4-bisphosphate (PtdIns-3,CP2) and phosphatidylinositol 3,4,5-tri sphosphate (PtdIns-3,4,5-P-3). We have shown previously that these lip ids activate calcium-independent protein kinase C (PKC) isoforms in vi tro (Toker, A, Meyer, M., Reddy, K. K, Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PRC in respon se to TRAP is detected by the phosphorylation of the major PKC substra te in platelets, the p47 phosphoprotein, also known as pleckstrin. Her e we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (<1 min) precedes the peak of PtdIns-3,4-P-2 production and is unaffected by c oncentrations of wortmannin (10-100 nw) that block production of this lipid. However prolonged phosphorylation of pleckstrin (>2 min) is inh ibited by wortmannin concentrations that block PtdIns-3,4-P-3 producti on. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC a ctivity. Phosphorylation of pleckstrin could be induced using permeabi lized platelets supplied with exogenous gamma-P-32[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P-3 and dipalmitoyl PtdIns-3,4-P-2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphati dylinositol 4,5-bisphosphate. These results suggest two modes of stimu lating pleckstrin phosphorylation: a rapid activation of PKC (via diac ylglycerol and calcium) followed by a slower activation of calcium-ind ependent PKCs via PtdIns-3,4-P-2.