title: Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts.
abstract: Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.
authors.affiliation: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hoshi
authors.forename: E
authors.initials: E
authors.lastname: Nishida
authors.forename: H
authors.initials: H
authors.lastname: Sakai
mesh_terms: D001769:Blood; D002118:Calcium / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D006133:Growth Substances / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008168:Lung / Q000166:cytology; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D010748:Phosphopeptides / Q000032:analysis; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002364:Caseins; D006133:Growth Substances; D006657:Histones; D008869:Microtubule-Associated Proteins; D010703:Phorbol Esters; D010748:Phosphopeptides; D010982:Platelet-Derived Growth Factor; D047428:Protein Kinase Inhibitors; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium