Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13.
Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.
Yuan
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Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
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D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D008954:Models, Biological; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018004:Receptors, Bombesin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D017607:Aluminum Compounds; D018076:DNA, Complementary; D007211:Indoles; D008164:Luminescent Proteins; D015778:Minor Histocompatibility Antigens; D011518:Proto-Oncogene Proteins; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D000409:Alanine; D005459:Fluorides; C064758:Ro 31-8220; C032311:aluminum fluoride