G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon.
Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.
Rey
Osvaldo
O
Department of Medicine, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, David Geffen School of Medicine, University of California-Los Angeles, CA 90095, USA.
Reeve
Joseph R
JR
Zhukova
Elena
E
Sinnett-Smith
James
J
Rozengurt
Enrique
E
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013347:Subcellular Fractions; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; C497256:Prkce protein, mouse; C089212:protein kinase D; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon