Activation of Protein Kinase D by Signaling through Rho and the α Subunit of the Heterotrimeric G Protein G13
Journal of Biological Chemistry
Protein kinase D (PKD/PKC) 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 G␣ 13 also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum
... m fluoride-stimulated G␣ 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 G␣ 13 . PKD activation in response to G␣ 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 G␣ 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 G␣ 13 also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that G␣ 13 contributes to PKD activation through a Rho-and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both G␣ q and G␣ 13 in response to bombesin receptor stimulation. Protein kinase C (PKC), 1 a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways regulating a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation (1, 2). Molecular cloning has demonstrated the presence of multiple PKC isoforms (2-5), i.e. conventional PKCs (␣, ␤ 1 , ␤ 2 , and ␥), novel PKCs (␦, ⑀, , and ), and atypical PKCs ( and ), all of which possess a highly conserved catalytic domain.