Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP.
Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma / Q000201:enzymology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D000596:Amino Acids; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D003994:Bucladesine; D012319:RNA Polymerase II; D007545:Isoproterenol