Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase.
A novel protein kinase (named PKD) with an NH2-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.
Growth Regulation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
J V
JV
Van Lint
J
J
Sinnett-Smith
E
E
Rozengurt
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation; D007106:Immune Sera; D008168:Lung / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D004075:Diglycerides; D007106:Immune Sera; D010718:Phosphatidylserines; D015240:Phorbol 12,13-Dibutyrate; C042511:1,2-dioleoylphosphatidylserine; C089212:protein kinase D; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C