Studies on protein-phosphorylation reactions in isolated chromatin.
The endogenous protein-phosphorylating activity of isolated chromatin was tested. We have found that a group of high-molecular-weight proteins (Mr greater than 50 000) was preferentially phosphorylated when chromatin from mouse ascites cells or from bovine lymphocytes was incubated in the presence of ATP. After disintegration of chromatin by nuclease treatment or by high salt concentration, a larger spectrum of chromatin proteins becomes accessible for phosphorylation by the chromatin-bound protein kinase. Some observations described in this communication may help to partially explain this result. The protein kinase was not found in nucleosomal subunits, indicating a non-random distribution of the enzyme in chromatin. This suggests that enzyme and substrate have to be in close spatial contact for the phosphorylation reaction to occur. Furthermore, we have shown for one protein, histone H1, that phosphorylation sites for the endogenous protein kinase are available on the free but not on the DNA-bound protein, suggesting that phosphate-accepting sites in chromatin proteins may be blocked by protein-DNA or by protein-protein interactions. We also discuss the possibility that chromatin protein kinase occurs in stable complexes with its phosphate-accepting substrates, as has been suggested by the findings of other [Kish, V.M. & Kleinsmith, L.J. (1974) J. Biol. Chem. 249, 750-760].
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D002417:Cattle; D002843:Chromatin / Q000201:enzymology*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004789:Enzyme Activation; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D008836:Micrococcal Nuclease; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D013698:Templates, Genetic
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D004259:DNA-Directed DNA Polymerase; D008836:Micrococcal Nuclease