Phorbol Ester TPA Rapidly Prevents Activation of p34 Histone H1 Kinase and Concomitantly the Transition from G2 Phase to Mitosis in Synchronized HeLa Cells

HeLa cells in G2 phase are temporarily inhibited and prevented from entering mitosis by treatment with the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate), whereas cells in mitosis are refractory to TPA and divide. In this study the possibility was tested that TPA may interfere with the regulatory cycle of MPF (mitosis promoting factor), the rate-limiting protein kinase for cell division. MPF, consisting of the catalytic subunit p34^cdc2 and the regulatory subunit Cyclin B, is known to be activated at the transition from G2 phase to mitosis through dephosphorylation at Tyr¹⁵ and to become inactivated after metaphase by proteolysis. Treatment of HeLa cells (synchronized around the G2-M transition) with TPA (10^-7M) has now been shown to induce an overall decrease of the histone H1 kinase activity associated with anti-p34^cdc2 immunoprecipitates after about 20 to 30 min. In metaphase cells, the histone H1 kinase activity of p34^cdc2 was shown to remain unaffected by TPA treatment. In cultures enriched in G2 cells neither the amount of p34^cdc2 protein nor that of Cyclin B was influenced by TPA. Moreover, the p34^cdc2/Cyclin B complex formation was also unaffected. However, p34^cdc2 from cultures treated with TPA was more intensely stained by anti-phosphotyrosine antibodies than that of control cells, indicating that TPA treatment probably prevented the tyrosine dephosphorylation required for expression of the histone H1 kinase activity of the complex. The results indicate that TPA treatment of HeLa cultures rapidly stops the G2-M transition because it very rapidly prevents the p34^cdc2/Cyclin B complex in G2 cells from developing histone H1 kinase activity.