Partial displacement of histone H1 from chromatin is required before it can be phosphorylated by mitotic H1 kinase in vitro.
The massive nonselective and reversible phosphorylation of histone H1 during mitosis is a universal phenomenon among eukaryotes. The growth-associated kinase responsible for this phosphorylation is identical to the maturation promoting factor, a key regulator of the cell cycle. Here we showed that growth-associated kinase, isolated from mitotic HeLa cells which were capable of phosphorylating HeLa H1 in vitro with high activity and mostly at the same sites phosphorylated during mitosis in vivo (assayed by two-dimensional analysis of tryptic phosphopeptides), did not significantly phosphorylate chromatin-bound or nuclear H1 in vitro. Its inability to phosphorylate chromatin-bound H1 did not change when the amount of kinase was increased or the incubation was prolonged. The resistance of chromatin-bound H1 to phosphorylation did not result from chromatin aggregation. Rapid phosphorylation of H1 in vitro, as well as in a nuclear system, was restored when NaCl concentrations were raised above 200 mM where H1:DNA interactions are weakened. At 300 mM NaCl, chromatin-bound H1 was phosphorylated in a subset of the sites observed for free H1 phosphorylated in vitro. These results suggest that active displacement of H1 from chromatin DNA may take place before H1 can be fully phosphorylated during mitosis.
Jerzmanowski
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Cole
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D001345:Autoradiography; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015636:Magnesium Chloride / Q000378:metabolism; D008938:Mitosis; D010449:Peptide Mapping; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002843:Chromatin; D006657:Histones; D015636:Magnesium Chloride; D012965:Sodium Chloride; D004247:DNA; D006656:Protamine Kinase; D014357:Trypsin