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MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit
K Takenaka1, Y Gotoh, E Nishida
1Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Japan.
Abstract:
In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.
Insights
Mitogen-activated protein kinase (MAPK) is not essential for cell cycle progression in Xenopus egg extracts. However, MAPK is crucial for the spindle assembly checkpoint, ensuring proper mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Early embryonic cell cycles in Xenopus laevis involve complex regulatory networks.
- Maturation promoting factor (MPF) and MAP kinase (MAPK) pathways are key regulators of the M phase.
- The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis.
Purpose of the Study:
- To investigate the role of MAP kinase (MAPK) in Xenopus laevis egg cell cycle extracts.
- To determine if MAPK is required for normal M phase entry and exit.
- To elucidate the involvement of MAPK in the spindle assembly checkpoint (SAC).
Main Methods:
- Depletion of MAP kinase (MAPK) from Xenopus egg extracts using specific antibodies.
- Monitoring of maturation promoting factor (MPF) activity in MAPK-depleted and mock-treated extracts.
- Assessment of the spindle assembly checkpoint (SAC) function in MAPK-depleted extracts with and without recombinant MAPK.
- Induction of mitotic arrest by activating MAPK in the absence of specific cellular cues.
Main Results:
- MAP kinase (MAPK) depletion did not affect the periodic activation and deactivation of maturation promoting factor (MPF), indicating MAPK is dispensable for M phase entry and exit.
- The spindle assembly checkpoint (SAC) failed to operate in MAPK-depleted extracts.
- Recombinant MAPK addition restored SAC function in depleted extracts.
- Strong activation of MAPK alone was sufficient to induce mitotic arrest in vitro.
Conclusions:
- Classical MAP kinase (MAPK) is essential for the spindle assembly checkpoint (SAC) in Xenopus cell cycle extracts.
- MAPK activation alone can trigger mitotic arrest, highlighting its sufficiency in regulating cell cycle progression.
- These findings clarify the distinct roles of MAPK in M phase regulation and checkpoint control.