Mechanism of metaphase arrest by cytostatic factor in vertebrate eggs

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase until correction is made. The proteins Bub1, Mad1, and Mad2 promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome (APC/C) to prevent the ubiquitin-mediated degradation of mitotic regulators. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to cytostatic factor (CSF), a cytoplasmic activity that also inhibits the APC/C but is not regulated by spindle dynamics. Active MAPK is important for the mitotic spindle checkpoint and is required for CSF arrest. This thesis elucidates the mechanism of MAPK-mediated inhibition of the APC/C and CSF arrest. We show that the activity of the MAPK substrate, p90Rsk, is sufficient for activation of Bub1 in vitro , is required for Bub1 activity in vivo, and that the kinase activity of Bub1 is required for the establishment of CSF arrest in egg extracts, thus defining a novel downstream effector of the MAPK/p90 Rsk pathway. Microinjection of Mad1 or Mad2 into early Xenopus embryos caused a metaphase arrest similar to that caused by the MAPK kinase kinase, Mos, or p90Rsk. Moreover, immunodepletion of either Mad1 or Mad2 blocks the ability of Mos to establish CSF arrest in egg extracts. Thus, the spindle checkpoint proteins Mad1 and Mad2 are required for the establishment of CSF arrest in vivo. A Mad2 mutant unable to oligomerize did not cause cell cycle arrest when overexpressed in blastomeres or support Mos-dependent CSF arrest in egg extracts. Therefore, unlike in the spindle checkpoint, Mad2 oligomerization is required for metaphase arrest by CSF. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2, MAPK, or Bub1. This suggests a model whereby CSF arrest by the MAPK pathway is mediated by the Bub1/Mad1/Mad2 proteins in a manner distinct from the spindle checkpoint. In addition to this pathway, this thesis evinces a second independent pathway, initiated by cyclin E/Cdk2, which operates during meiosis II to inhibit the APC/C and help establish CSF arrest. Similar to the MAPK/Bub1/Mad2 pathway, cyclin E/Cdk2 activity is not required for maintenance of CSF arrest once it is established.