We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G 2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G 2 . When either a recombinant wild type Cdc25C(WT) or a mutated form of Cdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling egg extracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame the G 2 arrest caused by p42MAPK, driving the extract into M-phase. p90 Rsk a kinase that is the target of p42MAPK, was phosphorylated and activated (pp90 Rsk ) in the G 2 -arrested egg extracts, and was able to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenous Cdc25C in G 2 -arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90 Rsk bound 14-3-3ζ, whereas S287A could not. These data suggest that the link between the p42MAPK signaling pathway and Cdc25C involves the activation of pp90 Rsk and its phosphorylation of Cdc25C at S287, causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90 Rsk phosphorylated-Cdc25C results in a G 2 arrest in a manner similar to the cell cycle delays induced by differentiation signals that occur later in embryonic development.

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 Mphase-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 Mphase even after cyclin destruction, is dependent on the inactivation of p42 MAPK.To date, a large body of research has led to the prevailing view that M-phase promoting factor (MPF, 1 a complex of cyclin B/Cdc2) activation is required for entry into M-phase, and its inactivation is necessary for exit from M-phase (reviewed in Ref. 1). Studies have shown that p42 mitogen-activated protein kinase (p42 MAPK) activation maintains high levels of MPF activity and stabilizes Cyclin B (2-4), leading to the predominant belief that p42 MAPK must sustain M-phase solely by this mechanism.We have previously shown that the activation of p42 MAPK in cycling Xenopus egg extracts by the addition of a constitutively active MAPK kinase (MEK) leads to an arrest of the cell cycle in either G 2 -or M-phase, depending on the timing of p42 MAPK activation (5). If p42 MAPK was activated in cycling egg extracts before entry into M-phase, the cell cycle was arrested in G 2 (4 -7). If p42 MAPK was activated on entry into M-phase, however, Cdc25C phosphatase was hyperphosphorylated (5, 7-9), and nuclear envelope breakdown (NEBD) and chromosome condensation (CC), all markers of M-phase, were sustained (5, 7).Recently, we and others (7, 10) have shown that activation of p42 MAPK by Mos can lead to an M-phase arrest that is maintained even after the inactivation of MPF. We have further shown that MPF levels fall in these extracts due to mitotic cyclin proteolysis (7). Here, we show ...

Previously, we have shown that the addition of a constitutively‐active mitogen‐activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M‐phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M‐phase, or after exit from M‐phase, the extract is arrested in G2. If p42MAPK is activated during entry into M‐phase, the extract is arrested in M‐phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M‐phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M‐phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM‐2. Unexpectedly, in certain M‐phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M‐phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M‐phase‐specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M‐phase, once established, M‐phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.