Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells.
A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was cAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles 32P per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its Km was 30 microM. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnCl2 and 4- to 9-fold by deoxymononucleotides. The nuclei of HeLa cells contained 64% of the total kinase of which 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a cAMP-independent protein kinase which is similar to glycogen synthase kinase.
D002051:Burkitt Lymphoma / Q000201:enzymology*; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors; D006801:Humans; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification; D009711:Nucleotides / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000302:isolation & purification*
D009363:Neoplasm Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D006006:Glycogen Synthase; D011494:Protein Kinases