The in vitro phosphorylation of chromatin by the catalytic subunit of cAMP-dependent protein kinase.

When a mixture of DNA-free core histones (H) from calf thymus is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, phosphate is incorporated primarily into H2B and, to a lesser extent, into H3 and H4. In contrast, when the phosphorylation of the DNA-free histones is compared to the phosphorylation of histones in long chromatin or in nucleosome core, only one of the core histones, H3, is phosphorylated. The site of modification of H3 has been identified as serine 10, which is located in the highly charged basic NH2-terminal region of the molecule. The other sites of phosphorylation in H2B nd H4 are completely masked when the histones are complexed with DNA. It is only when the nucleosome core structure is perturbed that these additional sites become accessible to the kinase. If long chromatin which contains H1 is phosphorylated, histone 1 is also phosphorylated by catalytic subunit at serine 38. However, the addition of excess H1 to H1-depleted long chromatin inhibits the phosphorylation of H3. In addition to the histones, the high mobility group (HMG) protein, HMG 14, was also found to be a good substrate for the kinase. Phosphorylation of HMG 17 in comparison was much less.