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Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
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.
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