Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction

By using cycling Xenopus egg extracts, we have previously found that if mitogen-activated protein kinase (p42 MAPK) is activated on entry into mitosis (M-phase), the extract is arrested with condensed chromosomes and spindle microtubules. Here we show that these arrested extracts have high levels of M-phase promoting factor (MPF, Cyclin B/Cdc2) activity, stabilized levels of Cyclin B, and sustained M-phase-specific phosphorylations. We also examined the role of p42 MAPK in DNA damage checkpoint-arrested extracts that were induced to enter M-phase by the addition of Cdc25C protein. In these extracts, Cdc25C protein triggers the abrupt, premature activation of MPF and entry into M-phase. MPF activity then drops suddenly due to Cyclin B proteolysis, just as p42 MAPK is activated. Unexpectedly, however, M-phase is sustained, as judged by maintenance of M-phase-specific phosphorylations and condensed chromosomes. To determine if this M-phase arrest depended on p42 MAPK activation, we added PD98059 (PD), an inhibitor of p42 MAPK activation, to egg extracts with exogenous Cdc25. Both untreated and PD-treated extracts entered M-phase simultaneously, with a sharp peak of MPF activity. However, only PD-treated extracts subsequently exited from M-phase and entered interphase. In PD-treated extracts, p42 MAPK was not activated, and the transition to interphase was accompanied by the formation of decondensed nuclei and the disappearance of M-phase-specific phosphorylation of proteins. These results show that although entry into M-phase requires the activation of MPF, exit from M-phase even after cyclin destruction, is dependent on the inactivation of p42 MAPK.