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Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin
1Department of Cell Biology, University of Alberta, Edmonton, Canada.
Abstract:
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.
Insights
Mitogen-activated protein kinase kinase (MEK) activation in Xenopus egg extracts drives cell cycle arrest. Mos protein addition also activates p42MAPK, influencing G2 or M-phase arrest timing and maintenance.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitogen-activated protein kinase kinase (MAPKK, also known as MEK) activation triggers the p42MAPK pathway.
- This pathway can induce cell cycle arrest at G2 or M-phase depending on activation timing.
Purpose of the Study:
- To investigate the effects of recombinant Mos protein on cell cycle progression in Xenopus egg extracts.
- To determine if Mos protein mimics MEK's effects on p42MAPK activation and cell cycle arrest.
- To elucidate the role of the p42MAPK pathway in maintaining M-phase independently of cyclin B/Cdc2 (MPF).
Main Methods:
- Addition of constitutively-active MEK or recombinant Mos protein to cycling Xenopus egg extracts.
- Analysis of cell cycle stage by nuclear morphology and microtubule arrays.
- Immunoblotting using monoclonal antibody MPM-2 to detect M-phase specific phosphorylations.
- Assay of histone H1 kinase activity and Cyclin B2 levels.
Main Results:
- Mos protein addition induced G2 arrest when added early and M-phase arrest when added later, similar to MEK.
- M-phase arrest was characterized by condensed chromosomes, mitotic microtubule arrays, and sustained MPM-2 phosphoproteins.
- In some M-phase arrested extracts, Cyclin B2 was degraded, yet M-phase was maintained with low maturation promoting factor (MPF) activity.
Conclusions:
- The p42MAPK pathway, activated by Mos, can induce and maintain M-phase arrest in Xenopus egg extracts.
- While MPF is crucial for M-phase entry, the p42MAPK pathway can sustain M-phase post-proteolysis of mitotic cyclins.