Phosphopleckstrin inhibits G beta gamma-activable platelet phosphatidylinositol-4,5-bisphosphate 3-kinase

Pleckstrin, the prototypic protein containing two copies of the pleckstrin homology domain, is a prominent substrate of protein kinase C in platelets and neutrophils. Both cell types have p85 subunit-containing phosphoinositide 3-kinase (p85/PI3K) and non-p85-containing PI3K (PI3K gamma) that is activated by beta gamma subunits of heterotrimeric GTP-binding proteins. We have shown that a PI3K product, phosphatidylinositol (PI) 3,4,5-trisphosphate, promotes pleckstrin phosphorylation in platelets. Since pleckstrin homology domains are thought to interact with G beta gamma heterodimers and/or PI(4,5)P-2, we have examined the effects of recombinant pleckstrins on platelet PI3K gamma and p85/PI3K activities. Depending upon its phosphorylation/charged state, pleckstrin inhibits PI3K gamma, but not p85/PI3K. Pleckstrin-mediated inhibition of PI3K gamma is overcome by excess G beta gamma and is restricted to PI(4,5)P-2 as substrate, i.e. pleckstrin does not inhibit phosphorylation of PI(4)P or PI. Consistent with this, activation of protein kinase C by exposure of platelets to beta-phorbol diester (to increase endogenous pleckstrin phosphorylation) prior to platelet lysis causes inhibition of G beta gamma-stimulatable PI3K activity only with respect to PI(4,5)P-2 substrate. This phosphopleckstrin-mediated inhibition is overcome by increasing concentrations of G beta gamma. We propose that phosphorylation of pleckstrin may constitute an important inhibitory mechanism for PI3K gamma-mediated cell signaling.