title: Oxidants induce phosphorylation of ribosomal protein S6.
abstract: We have investigated the phosphorylation of the ribosomal S6 protein which may be on the pathway of mitogenic stimulation in response to oxidants. Mouse epidermal cells JB6 (clone 41) were exposed to active oxygen generated extracellularly by glucose/glucose oxidase (producing H2O2) or xanthine oxidase (producing H2O2 plus superoxide) or active oxygen produced intracellularly by the metabolism of menadione (producing mostly superoxide). All three sources of active oxygen induced rapidly a protein kinase activity which phosphorylated S6 in cellular extracts prepared in the presence of the phosphatase inhibitor beta-glycerophosphate. Maximal activity was reached within 15 min of exposure, and phosphorylation occurred specifically at serine residues. Strong activation of the protein kinase activity was also observed by diamide which selectively oxidizes SH functions. The following observations characterize the reaction: 1) Extracellular addition of catalase but not Cu,Zn-superoxide dismutase was inhibitory, implicating H2O2 rather than superoxide as the active species. 2) Exposure of JB6 cells to reagent H2O2 or H2O2 released by glucose/glucose oxidase resulted in a measurable increase in intracellular free Ca2+. 3) The intracellular Ca2+ complexer quin 2 suppressed the reaction. 4) The calmodulin antagonist trifluoperazine prevented the activation of the protein kinase. 5) Exposure of cells to Mn2+ and La3+, which stimulate calmodulin-dependent activities, potently increased the S6 kinase activity of the cell extracts. 6) Desalted extracts strictly required the addition of Mg2+ and their activity was inhibited by Mn2+. In contrast, the phosphorylation of a 95-kDa protein was strongly stimulated by Mn2+. 7) For several agonists, i.e. active oxygen, phorbol 12-myristate 13-acetate, and serum, tryptic peptide analysis yielded the same phosphopeptides, suggesting that a common S6 kinase is involved in these reactions. From these data we propose that oxidants induce an increase in intracellular free Ca2+ which activates a Ca2+/calmodulin-dependent protein kinase and, as a consequence, an S6 kinase.
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002460:Cell Line; D003958:Diamide / Q000494:pharmacology; D004817:Epidermis; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D014812:Vitamin K / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D010748:Phosphopeptides; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D003958:Diamide; D014812:Vitamin K; D000001:Calcimycin; D002118:Calcium