Activation loop Ser(744) and Ser(748) in protein kinase D are transphosphorylated in vivo

The importance of activation loop phosphorylation in the regulation of prot ein kinase D (PKD/protein kinase C (PKC) mu) activity has become controvers ial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length P KD transfected in COS-7 and EIEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphoc ytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was abs ent from unstimulated cells. In contrast, dramatic increases in Ser(748) ph osphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKC epsilo n or PKC eta. Western analysis using a second phosphospecific antibody, whi ch primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(7 44) phosphorylation accompanies Ser(748) phosphorylation during PKD activat ion in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD act ivity, indicative of transphosphorylation. Our results provide new experime ntal evidence indicating that activation loop phosphorylation at Ser(744) a nd Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.