Activation Loop Ser744 and Ser748 in Protein Kinase D Are Transphosphorylated in Vivo Export

@article{citeulike:6029160, abstract = {10.1074/jbc.M101648200 The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) \^{I}¼) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser 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 phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKC\^{I}µ or PKC\^{I}·. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser, revealed that Serphosphorylation accompanies Ser phosphorylation during PKD activation . Ser/Ser phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser and Ser occurs during PKD activation and support the notion of a PKC-PKD phosphorylation cascade.}, author = {Waldron, Richard T. and Rey, Osvaldo and Iglesias, Teresa and Tugal, Tamara and Cantrell, Doreen and Rozengurt, Enrique}, citeulike-article-id = {6029160}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M101648200}, citeulike-linkout-1 = {http://www.jbc.org/content/276/35/32606.abstract}, citeulike-linkout-2 = {http://www.jbc.org/content/276/35/32606.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/11410586}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=11410586}, day = {31}, doi = {10.1074/jbc.M101648200}, journal = {Journal of Biological Chemistry}, keywords = {pkd}, month = {August}, number = {35}, pages = {32606--32615}, posted-at = {2009-10-28 22:00:24}, priority = {2}, title = {Activation Loop Ser744 and Ser748 in Protein Kinase D Are Transphosphorylated in Vivo}, url = {http://dx.doi.org/10.1074/jbc.M101648200}, volume = {276}, year = {2001} }

TY - JOUR ID - citeulike:6029160 L3 - citeulike-article-id:6029160 N2 - 10.1074/jbc.M101648200 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 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 phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCε or PKCη. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser, revealed that Serphosphorylation accompanies Ser phosphorylation during PKD activation . Ser/Ser phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser and Ser occurs during PKD activation and support the notion of a PKC-PKD phosphorylation cascade. IS - 35 JF - Journal of Biological Chemistry EP - 32615 TI - Activation Loop Ser744 and Ser748 in Protein Kinase D Are Transphosphorylated in Vivo VL - 276 SP - 32606 KW - pkd AU - Waldron, Richard AU - Rey, Osvaldo AU - Iglesias, Teresa AU - Tugal, Tamara AU - Cantrell, Doreen AU - Rozengurt, Enrique PY - 2001/08/31/ UR - http://dx.doi.org/10.1074/jbc.M101648200 ER -