Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid

Abstract

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Gαq/11 (Gαq/11 KO cells) or Gα12/13 (Gα12/13 KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Gα12/13 KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Gα12/13 KO cells in autophosphorylation activity) achieved at 3 μM. In contrast, LPA did not induce any significant increase in PKD activity in Gαq/11 KO cells. However, LPA induced a significantly increased PKD activity when Gαq/11 KO cells were transfected with Gαq. LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Gα12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Gα12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi, and G12 and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.