Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro.
cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D002868:Chromosomal Proteins, Non-Histone; D007527:Isoenzymes; D008345:Manganese; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium