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) mu) 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 Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.
Waldron
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RT
Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, and Molecular Biology Institute, UCLA School of Medicine and UCLA-CURE Digestive Diseases Research Center, Los Angeles, California 90095, USA.
Rey
O
O
Iglesias
T
T
Tugal
T
T
Cantrell
D
D
Rozengurt
E
E
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D014162:Transfection
D000906:Antibodies; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C