ARTICLE

FURTHER CHARACTERIZATION OF THE F1-HISTONE PHOSPHOKINASE OF METAPHASE-ARRESTED ANIMAL CELLS

¹ From the Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014

Exponentially growing Chinese hamster cells are found to contain two major phosphokinase activities with specificity for the phosphorylation of F1 (lysine-rich) histone. These two activities, designated KI and KII, were extracted with 0.35 M NaCl and fractionated in 0.2 M NaCl by Sephadex G-200 gel filtration. KI, which is similar to the ubiquitous cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphokinase, differs from KII by several criteria. KII is mol wt 90,000, cAMP independent, rapidly turned over in vivo, low K_m for ATP, and phosphorylates F1 histone at several unique sites. Comparative examination of metaphase-arrested (M) and counterpart interphase (I) cells for these two activities reveals that KII is responsible for the overall high activity in M-arrested cells. Pulse labeling of cells with ³²P during traverse of the G₂-M phase of the cell cycle reveals an in vivo tryptic-phosphopeptide pattern in whole unfractionated F1 which is unique to M cells. Seven major phosphopeptides derived by in vitro phosphorylation of F1 with the KII enzyme correspond to these M cell-specific phosphorylation sites observed in vivo. It is suggested that KII activity predominates during the G₂-M transition and that F1 is its natural in vivo substrate.

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