Stimulation of ascites tumor RNA polymerase II by protein kinase.
The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008113:Liver Neoplasms; D008274:Magnesium / Q000494:pharmacology; D009374:Neoplasms, Experimental / Q000201:enzymology; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification / Q000494:pharmacology*
D000643:Ammonium Chloride; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D000645:Ammonium Sulfate