Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II.
A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.
D047388:Casein Kinases; D002467:Cell Nucleus / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology
D009698:Nucleoproteins; D010761:Phosphorus Radioisotopes; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D006493:Heparin; D011794:Quercetin; D011494:Protein Kinases; D047388:Casein Kinases