Activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ in protein kinase D are transphosphorylated in vivo

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) μ) activity has become controversial. 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 PKD transfected in COS-7 and HEK 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 lymphocytes and Rat-1 fibroblasts, indicated that Ser ⁷⁴⁸ phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser⁷⁴⁸ phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCε of PKCη. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser⁷⁴⁴, revealed that Ser⁷⁴⁴ phosphorylation accompanies Ser⁷⁴⁸ phosphorylation during PKD activation in vivo. Ser⁷⁴⁴/Ser⁷⁴⁸ phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser⁷⁴⁴ and Ser⁷⁴⁸ occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.