alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression

In Chinese hamster embryonic fibroblasts (IIC9 cells) alpha-thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G(1) phase of the cell cycle. We investigated in IIC9 cells, the role of beta-arrestin1 in alpha-thrombin signaling to these pathways. alpha-Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative beta-arrestin1 (beta-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of beta-arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a beta-arrestin1 mutant, which impairs binding to c-Src (beta-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents alpha-thrombin-stimulated Akt phosphorylation. Expression of beta- arrestin1(V53D) does not prevent G(1) progression, as its expression has no effect on (3)H]thymidine incorporation into DNA. In agreement with the ineffectiveness of beta-arrestin1(V53D) to block G(1) progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of beta-arrestin1(V53D). Thus in IIC9 cells, alpha-thrombin activates rapid beta-arrestin1-dependent and sustained beta-arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the beta-arrestin1-independent PI 3-kinase/Akt pathway prevents G(1) progression, inhibition of the beta-arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.