J. Cell Sci. Suppl. 12, 99-116 (1989) Printed in Great Britain @ The Company ofBiologists Limited 1989 99 Temporal regulation of cdc2 mitotic kinase activity and cyclin degradation in cell-free extracts of Xenopus eggs MARIE-ANNE FELIX1, JONATHAN PINES2, TIM HUNT3 and ERIC KARSENTI1 1European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstr. 1, 6900 Heidelberg, FRG 2The Salk Institute, P.O. Box 858800, San Diego, California 92138-9216, USA 3 University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW, England Summary In cleaving Xenopus eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases. During mitosis a number of proteins show increased phosphorylation due to the activation of a histone H I kinase, the homologue of the cdc2+ gene product of the yeast Schizosaccharomyces pombe. We have studied the regulation of the activity of this enzyme in cell-free extracts of Xenopus eggs. In extracts of activated eggs incubated at 22°C, histone H I kinase activity shows two peaks of activation and disappearance. Activation occurs in two stages. The first stage requires protein synthesis, whereas the second does not. The second stage of activation involves post-translational activation of the kinase. Kinase activity rises to a peak and then abruptly disappears. Added sea urchin cyclin is degraded at the time of disappearance of kinase activity. The oscillation in kinase activity is then repeated, usually with lower amplitude. Post- translational activation of the kinase requires a membrane- containing particulate cellular component, whose role has yet to be defined. The kinase can still be activated in the presence of EDTA or in the presence of the ATP analogue, 6-dimethylaminopurine, which implies that phosphorylation of the kinase complex is not required for activation. Under these conditions, however, the kinase activity does not show its normal sudden disappearance, and added cyclin is perfectly stable. These observations are consistent with the idea that post- translational activation of the kinase involves protein phosphatase activity, whereas switching off the kinase requires an AT P- Mg2+- dependent reaction, perhaps due to protein phosphorylation. Introduction During the first few hours following fertilization of Xenopus laevis eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases, without any significant G; and G 2 phases (Newport and Kirschner, 1982; Signoret and Lefresne, 1971). The rapid alternation between two cytoplasmic states, interphase and mitosis, has been interpreted in terms of a ‘master oscillator’ that controls the various structural and metabolic events which characterize S and M phases (Kirschner et al. 1985). ' The molecular nature of this oscillator is beginning to be defined. The level of overall protein phosphorylation increases in M phase in all eukaryotes (Ajiro et al. 1981; Bradbury et al. 1973; Capony et al. 1986; Dorée et al. 1983; Karsenti et al. 1987; Mailer et al. 1977; Meijer et al. 1982; Peaucellier et al. 1984). This is correlated Key words: cdc2+ , cyclin, Xenopus eggs, protein kinase.