PHORBOL ESTER TPA RAPIDLY PREVENTS ACTIVATION OF P34(CDC2) HISTONE H1KINASE AND CONCOMITANTLY THE TRANSITION FROM G2 PHASE TO MITOSIS IN SYNCHRONIZED HELA-CELLS

HeLa cells in G2 phase are temporarily inhibited and prevented from en tering mitosis by treatment with the phorbol ester TPA (12-O-tetradeca noylphorbol-13-acetate), whereas cells in mitosis are refractory to TP A and divide. In this study the possibility was tested that TPA may in terfere with the regulatory cycle of MPF (mitosis promoting factor), t he rate-limiting protein kinase for cell division. MPF, consisting of the catalytic subunit p34(cdc2) and the regulatory subunit Cyclin B, i s known to be activated at the transition from G2 phase to mitosis thr ough dephosphorylation at Tyr(15) and to become inactivated after meta phase by proteolysis. Treatment of HeLa cells (synchronized around the G2-M transition) with TPA (10(-7) M) has now been shown to induce an overall decrease of the histone H1 kinase activity associated with ant i-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 b y TPA. Moreover, the p34(cdc2)/Cyclin B complex formation was also una ffected. However, p34(cdc2) from cultures treated with TPA was more in tensely stained by anti-phosphotyrosine antibodies than that of contro l cells, indicating that TPA treatment probably prevented the tyrosine dephosphorylation required for expression of the histone H1 kinase ac tivity of the complex. The results indicate that TPA treatment of HeLa cultures rapidly stops the G2-M transition because it very rapidly pr events the p34(cdc2)/Cyclin B complex in G2 cells from developing hist one H1 kinase activity. (C) 1994 Academic Press, Inc.