Exit from mitosis

The Xenopus system has proven instrumental in determining the mechanism controlling exit from mitosis at the metaphase/anaphase transition. Studies in this area have relied heavily on extracts prepared from fully mature oocytes/ unfertilized eggs that are arrested at metaphase of the second meiotic division. Upon Ca2+ addition, anaphase is initiated and the extract enters the first embryonic cell cycle to replicate DNA. The activity responsible for metaphase arrest was discovered by Masui at the same time as MPF (Masui & Markert l97l), and given the name cytostatic factor (CSF). CSF has never been purified and therefore its molecular composition is unknown. However, an extensive series of studies show that an active MAPK pathway is necessary for CSF arrest, which is usually monitored by assaying components for cleavage arrest at metaphase in early embryos. Thus CSF arrest can be achieved by expression of Mos, a MAP kinase kinase kinase synthesized during maturation, by constitutively active MAP kinase kinase or by thiophosphorylated, phosphatase-resistant MAP kinase (Sagata 1997 for review). Because activated MAP kinase is localized at kinetochores in mitosis (Zecevic et al 1998) and certain kinetochore-based microtubule motors are altered in CSF-arrested spindles (Duesbery et al 1997), it is generally believed that CSF is located on or acts at the kinetochore. CSF-dependent metaphase arrest is fundamentally different from the spindle assembly/kinetochore attachment checkpoint in the somatic cell cycle, since in the former an intact metaphase spindle is present whereas in the latter microtubules are depolymerized and spindles are absent.

Recently the target of MAP kinase responsible for CSF arrest has been identified as the protein kinase p90Rsk. Rsk was originally identified, purified and cloned as the kinase responsible for phosphorylation of ribosomal protein S6 during maturation (Erikson & Maller 1986). Subsequently, it was found to be activated by phosphorylation, and identification in this laboratory of MAP kinase as the upstream activating kinase defined the initial features of the MAP kinase cascade (Sturgill et al 1988). Although Rsk deactivated by protein phosphatases can be reactivated by MAP kinase, recombinant Rsk cannot be activated by MAP kinase in vitro. The dilemma was resolved recently by studies that identified PDK1 as an additional kinase that is required for Rsk activation (Jensen et al 1999). With this information and a variety of deletion mutants, Gross et al (1999) generated a constitutively active form of Rsk that requires only PDK1 phosphorylation for activation. This enzyme was found to cause CSF arrest at metaphase when injected into blastomeres (Gross et al 1999), and depletion of Rsk from egg extracts depleted CSF activity, which could be restored by re-addition of Rsk (Bhatt & Ferrell 1999). Importantly, Rsk produced this CSF arrest without activation of feedback loops that activate endogenous MAP kinase or Rsk. These results indicate that Rsk is the only substrate ofMAP kinase needed to mediate CSF arrest (Fig. 2).

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