SEQUESTRATION OF A G-PROTEIN BETA-GAMMA SUBUNIT OR ADP-RIBOSYLATION OF RHO CAN INHIBIT THROMBIN-INDUCED ACTIVATION OF PLATELET PHOSPHOINOSITIDE 3-KINASES

Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytos keletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-bin ding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTP gamma S requires a functional sma ll G protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta gamma subunits of heterotrimeric G-prote ins (G beta gamma). We now report that the activated PI 3-K that is as sociated with CSK can be inhibited by a recombinant protein containing the G beta gamma-binding pleckstrin homology domain of beta-adrenergi c receptor kinase 1 (beta ARK-PH). Inhibition is blocked by G beta gam ma. PI 3-K in nonactivated platelet CSK is activated by GTP gamma S bu t unaffected by beta ARK-PH or G beta gamma, Western blots indicate th at activated platelet CSK contains a novel 110-kDa PI 3-K(gamma) that has been shown to be stimulated by G beta gamma and to lack binding si tes for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunopre cipitates obtained via p85 subunit-directed antibodies can be activate d by GTP gamma S but not by G beta gamma. PI 3-K that is stimulatable by G beta gamma remains soluble, as does PI 3-K(gamma), and is unaffec ted by Rho, In contrast, ADP-ribosylation of Rho present in p85 immuno precipitates is inhibitory, Further, activation of PI 3-K in permeabil ized platelets exposed to thrombin or GTP gamma S is inhibited by beta ARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and G beta gamma each, respectively, contributes to the activatio n of different PI 3-Ks (p85-containing heterodimer and PI3-K (gamma)) in thrombin-stimulated platelets.