Activation of protein kinase D by signaling through the alpha subunit of the heterotrimeric G protein G(q).
Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active alpha subunit of G(q) (Galpha(q)Q209L) exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with phorbol 12,13-dibutyrate. In contrast, transient transfection of COS-7 cells with activated Galpha(12)Q229L or Galpha(13)Q226L neither promoted PKD activation nor interfered with the increase of PKD activity induced by phorbol 12,13-dibutyrate. The addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(q) induced a marked increase in PKD activity, which was comparable with that induced by expression of Galpha(q)Q209L. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride. Expression of a COOH-terminal fragment of Galpha(q) that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either Galpha(q) or bombesin was completely prevented by mutation of Ser(744) and Ser(748) to Ala in the kinase activation loop of PKD. Our results show that Galpha(q) activation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous Galpha(q) mediates PKD activation in response to acute bombesin receptor stimulation.
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Department of Medicine, School of Medicine, the CURE Digestive Diseases Research Center, and Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
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D000409:Alanine / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001839:Bombesin / Q000494:pharmacology; D019556:COS Cells; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007211:Indoles / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D018004:Receptors, Bombesin / Q000819:agonists; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D017607:Aluminum Compounds; D004791:Enzyme Inhibitors; D007211:Indoles; D008164:Luminescent Proteins; D008301:Maleimides; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C070515:bisindolylmaleimide I; D000409:Alanine; D001839:Bombesin; D005459:Fluorides; C064758:Ro 31-8220; C032311:aluminum fluoride