Stimulation of ascites tumor RNA polymerase II by protein kinase

Abstract

The activity of purified RNA polymerase II from Novikoff ascites [rat hepatoma] tumor cells is stimulated 5 to 7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzes the incorporation of .gamma.-32P 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 CM cellulose, phosphocellulose and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylylimidodiphosphate (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 casein or phosvitin but does not catalyze the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic AMP or cyclic GMP over a concentration range of 10-6-10-4 M. Protein kinase activity is not inhibited by the regulatory subunit of rabbit muscle protein kinase or the heat-stable inhibitor of cyclic 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.