Differential PKC-dependent and -independent PKD activation by G protein ? subunits of the Gq family: selective stimulation of PKD Ser??? autophosphorylation by G?q.

ABSTRACT: 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 G?q-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 G? and stimulated with aluminum fluoride (AlF4?). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser???, a prominent and rapid PKC transphosphorylation site, and Ser???, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4? potently induced PKD activation and Ser??? and Ser??? phosphorylation, in the presence of cotransfected G?q, G?11, G?14 or G?15. 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 G?11-, G?14-, and G?15-mediated enhancement of Ser??? phosphorylation induced by AlF4?, and largely abolished Ser??? phosphorylation. In contrast, Ser??? phosphorylation was almost completely intact, and Ser??? phosphorylation was significantly activated in cells cotransfected with G?q. Importantly, the differential Ser??? phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of G?q but not G?11. Taken together, our results suggest that G?q, but not the closely related G?11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser???.

SUBMITTER: Waldron RT 

PROVIDER: S-EPMC3286641 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature