G_q-mediated Akt translocation to the membrane: A novel PIP₃-independent mechanism in platelets

Akt is an important signaling molecule regulating platelet aggregation. Akt is phos-phorylated after translocation to the membrane through G_j signaling pathways by a phosphatidylinositol-3,4,5-trisphosphate(PIP₃)-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared with adenosine 5′-diphosphate in platelets. This study investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor agonists caused translocation of Akt to the membrane rapidly, whereas phosphorylation occurred later. The translocation of Akt was abolished in the presence of a G_q-selective inhibitor or in G_q-deficient murine platelets, indicating that Akt translocation is regulated downstream of G_q pathways. Interestingly, phosphatidylino-sitol 3-kinase (PI3K) inhibitors or P2Y₁₂ antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3K/PIP₃/G_j-independent mechanism. An Akt scaffolding protein, p21-activated kinase (PAK), translocates to the membrane after stimulation with protease-activated receptor agonists in a G_q-dependent manner, with the kinetics of translocation similar to that of Akt. Coimmunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a possible role of PAK in Akt translocation. These results show, for the first time, an important role of the G_q pathway in mediating Akt translocation to the membrane in a novel G_j/PI3K/PIP₃-independent mechanism.