Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser⁷⁴⁸ autophosphorylation by Gαq.
Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4⁻). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser⁷⁴⁴, a prominent and rapid PKC transphosphorylation site, and Ser⁷⁴⁸, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4⁻ potently induced PKD activation and Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser⁷⁴⁸ phosphorylation induced by AlF4⁻, and largely abolished Ser⁷⁴⁴ phosphorylation. In contrast, Ser⁷⁴⁸ phosphorylation was almost completely intact, and Ser⁷⁴⁴ phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser⁷⁴⁸ phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser⁷⁴⁸.
David Geffen School of Medicine, Department of Medicine, Division of Digestive Diseases, CURE: Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles, USA. rwaldronster@gmail.com
Richard T
RT
Waldron
Giulio
G
Innamorati
M Eugenia
ME
Torres-Marquez
James
J
Sinnett-Smith
Enrique
E
Rozengurt
D016475:3T3 Cells; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection
D017607:Aluminum Compounds; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D044385:GTP-Binding Protein alpha Subunits; C062258:Pasteurella multocida toxin; D010703:Phorbol Esters; D011480:Protease Inhibitors; D043562:Receptors, G-Protein-Coupled; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C; D005459:Fluorides; C032311:aluminum fluoride