Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.

In vertebrate unfertilized eggs, metaphase arrest in Meiosis II is mediated by an activity known as cytostatic factor (CSF). CSF arrest is dependent upon Mos-dependent activation of the MAPK/Rsk pathway, and Rsk activates the spindle checkpoint kinase Bub1, leading to inhibition of the anaphase-promoting complex (APC), an E3 ubiquitin ligase required for the metaphase/anaphase transition. However, it is not known whether Bub1 is required for the establishment of CSF arrest or whether other pathways also contribute. Here, we show that immunodepletion of Bub1 from egg extracts blocks the ability of Mos to establish CSF arrest, and arrest can be restored by the addition of wild-type, but not kinase-dead, Bub1. The appearance of CSF arrest at Meiosis II may result from coexpression of cyclin E/Cdk2 with the MAPK/Bub1 pathway. Cyclin E/Cdk2 was able to cause metaphase arrest in egg extracts even in the absence of Mos and could also inhibit cyclin B degradation in oocytes when expressed at anaphase of Meiosis I. Once it has been established, metaphase arrest can be maintained in the absence of MAPK, Bub1, or cyclin E/Cdk2 activity. Both pathways are independent of each other, but each appears to block activation of the APC, which is required for cyclin B degradation and the metaphase/anaphase transition.

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.

Polo-like kinases comprise a family of evolutionarily conserved serine/threonine protein kinases that play multiple roles in cell cycle regulation. In addition to the N-terminal catalytic domain, polo-like kinases have one or two highly conserved C-terminal non-catalytic regions, termed polo boxes. These motifs are required for targeting these kinases to subcellular mitotic structures. Here we report that kinase-dead Xenopus pololike kinase (Plx1NA) functions as a competitor of endogenous Plx1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G 2 -M transition in vivo. However, kinase-dead Plx1 with a point mutation in the polo box region (Plx1NAWF) did not have inhibitory effects. The ability of Plx1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of Plx1NA but not Plx1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-Plx1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant Plx1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for Plx1 function in both the G 2 -M and the metaphase/anaphase transitions during the cell cycle.

In Xenopus development the mid-blastula transition (MBT) marks a dramatic change in response of the embryo to ionizing radiation. Whereas inhibition of cyclin D1-Cdk4 and cyclin A2-Cdk2 by p27Xic1 has been linked to cell cycle arrest and prevention of apoptosis in embryos irradiated post-MBT, distinct roles for these complexes during apoptosis are evident in embryos irradiated pre-MBT. Cyclin A2 is cleaved by caspases to generate a truncated complex termed ⌬N-cyclin A2-Cdk2, which is kinase active, not inhibited by p27Xic1 , and not sensitive to degradation by the ubiquitin-mediated proteasome pathway. Moreover, ⌬N-cyclin A2-Cdk2 has an expanded substrate specificity and can phosphorylate histone H2B at Ser-32, which may facilitate DNA cleavage. Consistent with a role for cyclin A2 in apoptosis, the addition of ⌬N-cyclin A2-Cdk2, but not fulllength cyclin A2-Cdk2, to Xenopus egg extracts triggers apoptotic DNA fragmentation even when caspases are not activated. Similarly, cyclin D1 is targeted by caspases, and the generated product exhibits higher affinity for p27Xic1 , leading to reduced phosphorylation of the retinoblastoma protein (pRB) during apoptosis. These data suggest that caspase cleavage of both cyclin D1-Cdk4 and cyclin A2-Cdk2 promotes specific apoptotic events in embryos undergoing apoptosis in response to ionizing radiation.