Studies on the link between cellular signalling and cell cycle control at the G2 checkpoint have shown that, in HeLa cells, epidermal growth factor (EGF) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly inhibit the G2-M transition by preventing the key component of mitosis-promoting factor (MPF), p34cdc2, from expressing protein kinase activity. The kinase activity of active MPF is not inhibited; rather, the conversion of pre-MPF to MPF, i.e., the activating dephosphorylation of p34cdc2, at tyrosine is rapidly blocked (Barth and Kinzel, 1994, Exp. Cell Res. 212:383–388; 1995, J. Cell. Physiol., 162:44–51). The phosphatase responsible, cdc25-C, is activated by phosphorylation in mitotic cells starting at the G2-M transition in an autocatalytic loop with MPF (Hoffmann et al., 1993, EMBO J. 12:53–63). We now show that, concomitant with the prevention of MPF activation, EGF and TPA induced a reduction of the activity of cdc25-C in synchronized cultures. Furthermore, treatment of mitotic HeLa cells with TPA did not influence the kinase activity of MPF but caused a rapid decrease of the specific enzyme activity of cdc25-C, probably due to dephosphorylation of the enzyme, as indicated by reduced binding of monoclonal MPM-2 antibody specific for phosphoepitopes in M phase. Because of its inability to induce signalling during division, EGF failed to influence the activity of cdc25-C in mitotic cells. The scenario in cells late in G2 that are committed to enter mitosis may be as follows: In those cells where the signalling pathways responding to EGF as well as those responding to TPA are still open, cdc25-C is prevented by dephosphorylation from exceeding the threshold level of activity required to initiate the activation of and the autocatalytic feedback loop with p34cdc2 and to enter mitosis. Therefore, cdc25-C appears to represent part of an interface between cellular signalling and cell cycle control in G2 phase. © 1996 Wiley-Liss, Inc.

The mechanism of the transient G2 arrest induced by small doses of ionizing radiation involves the failure to activate the correctly formed pre-mitosis-promoting factor (MPF) complex of cyclin B and p34cdc2 by dephosphorylation at Tyr15 of the latter, as recent studies of other laboratories have indicated. Similar data were obtained with the G2 arrest-inducing agents epidermal growth factor and the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (H. Barth and V. Kinzel, Exp. Cell Res., 212: 383-388, 1994, and H. Barth and V. Kinzel, J. Cell. Physiol., 162: 44-51, 1995). To differentiate the radiation consequences in synchronized HeLa cells from those of 12-0-tetradecanoylphorbol-13-acetate and epidermal growth factor, experiments with a very small dose (1 Gy) have been carried out in cells close to the G2-M border and, for comparison, in mitotic cells. We show that in addition to the failure of p34cdc2 dephosphorylation at Tyr15, radiation with 1 Gy also prevents the activation of the phosphatase cdc25-C, the enzyme catalyzing the MPF activation. In contrast, irradiation of mitotic cells with 1 Gy did not influence that fraction of either MPF or cdc25-C already activated. Moreover, the gain in MPM-2 antigenicity of cdc25-C, usually indicative of an activating phosphorylation, is shown to be prevented by 1 Gy. The data indicate that the initiation of the proposed autocatalytic loop between MPF and cdc25-C becomes interrupted by radiation, but they give no hint at which point.