Activation of protein kinase D by signaling through the α subunit of the heterotrimeric G protein Gq

Protein kinase D (PKD/PKC mu) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active alpha subunit of G(q) (G alpha(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 G alpha(12)Q229L or G alpha(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 cotransfected with PKD and wild type G alpha(q) induced a marked increase in PKD activity, which was comparable with that induced by expression of G alpha(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 G alpha(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 G alpha(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 G alpha(q) activation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous G alpha(q) mediates PKD activation in response to acute bombesin receptor stimulation.