title: Effects of the putatively oncogenic protein kinase Calpha D294G mutation on enzymatic activity and cell growth and its occurrence in human thyroid neoplasias.
abstract: A point mutation of protein kinase Calpha (PKCalpha) has been described in pituitary adenomas and in follicular adenomas and thyroid carcinomas. The mutation results in an exchange of aspartic acid into a glycine of the amino acid 294 of PKCalpha, which is located adjacent to the Ca (2+)-binding hinge region and has been proposed as an activation inhibitor. To investigate its biochemical sequelae, we constructed the mutated enzyme and expressed it in human embryonic kidney cells (HEK). The K M of the purified enzyme for Ca (2+) and its K M for the substrate MBP 4 - 14 was not altered by the mutation. Translocation of PKCalpha to HEK cell membranes upon activation was not changed and the mutant potently inhibited cell proliferation upon 5-fold stable overexpression in HEK cells. Thus, loss of function in mutated PKCalpha was excluded. A screen for the mutation using a restriction assay with a sensitivity of at least 8 % for the mutated DNA did not show any mutation in 11 carcinoma and 13 adenomatous thyroid samples. We conclude that the A294G mutation of PKCalpha does not detectably affect its biochemical properties in vitro or in vivo, and is at least rare in thyroid neoplasias, in Germany.
authors.affiliation: German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Assert
authors.forename: R
authors.initials: R
authors.lastname: Kötter
authors.forename: U
authors.initials: U
authors.lastname: Schiemann
authors.forename: P
authors.initials: P
authors.lastname: Goretzki
authors.forename: A F H
authors.initials: AF
authors.lastname: Pfeiffer
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004622:Embryo, Mammalian; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013964:Thyroid Neoplasms / Q000201:enzymology* / Q000473:pathology*; D014162:Transfection
chemical_list: D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium