Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid.
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 Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha(12/13) 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 Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm. In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells. However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q). 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 Galpha(12/13) 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 G(q) is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells. We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.
Yuan
Jingzhen
J
Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
Slice
Lee W
LW
Gu
Jennifer
J
Rozengurt
Enrique
E
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D008246:Lysophospholipids; C089212:protein kinase D; D011493:Protein Kinase C; D020962:Heterotrimeric GTP-Binding Proteins