Phosphorylation of histones 1 and 3 and nonhistone high mobility group 14 by an endogenous kinase in HeLa metaphase chromosomes.
We have developed a simple in vitro system for studying phosphorylation in isolated HeLa metaphase chromosomes which utilizes the endogenous protein kinase and phosphoprotein phosphatase activities in the chromosomes. Because the isolated chromosomes retain the specificity of phosphorylation seen in vivo, this system offers unique possibilities for studying the properties and regulation of the kinase and phosphatase by adding exogenous substances and observing their effects. It should also be useful for studying the sites of phosphorylation, since proteins can be more easily labeled to high specific activity with 32P in this system than in vivo. The pattern of proteins phosphorylated in isolated metaphase chromosomes appears to be nearly identical with the pattern found in vivo. Among the histones (H) only H1 and H3 are phosphorylated, but several nonhistone proteins, including high mobility group (HMG) 14, are also phosphorylated. Since HMG 14 has been implicated as a structural protein of actively transcribing chromatin, our results suggest that phosphorylation of chromatin proteins may be involved in the shutoff of transcription during mitosis. Tryptic peptide maps and analysis of the phosphorylated amino acids indicate that H1A, H1B, HMG 14, and H3 are phosphorylated at the same sites in vitro in metaphase chromosomes as in mitotic cells in vivo. The major site of phosphorylation of histone H3, both in vivo and in vitro, has been identified as serine 10. HMG 14 is phosphorylated both at serine and threonine residues.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D002877:Chromosomes, Human / Q000201:enzymology*; D006367:HeLa Cells / Q000201:enzymology; D006609:High Mobility Group Proteins; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008677:Metaphase; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; D006609:High Mobility Group Proteins; D006657:Histones; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D006656:Protamine Kinase; D002118:Calcium