Noriyuki Sagata

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition.

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo‐cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product‐regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre‐MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1‐independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.

Nek2B, a novel maternal form of Nek2 kinase, is essential for the assembly or maintenance of centrosomes in early Xenopus embryos.

Nek2, a NIMA‐related kinase, has been postulated to play a role in both the meiotic and mitotic cell cycles in vertebrates. Xenopus has two Nek2 splice variants, Nek2A and Nek2B, which are zygotic and maternal forms, respectively. Here we have examined the role of Nek2B in oocyte meiosis and early embryonic mitosis. Specific inhibition of Nek2B function does not interfere with the oscillation of Cdc2 activity in either the meiotic or mitotic cell cycles; however, it does cause abortive cleavage of early embryos, in which bipolar spindle formation is severely impaired due to fragmentation or dispersal of the centrosomes, to which endogenous Nek2B protein localizes. In contrast, inhibition of Nek2B function does not affect meiotic spindle formation in oocytes, in which functional centrosomes are absent. Thus, strikingly, Nek2B is specifically required for centrosome assembly and/or maintenance (and hence for normal bipolar spindle formation and cleavage) in early Xenopus embryos. Finally, (ectopic) Nek2A but not Nek2B is very labile in cleaving embryos, suggesting that Nek2A cannot replace the centrosomal function of Nek2B in early embryos.

The existence of two distinct Wee1 isoforms in Xenopus: implications for the developmental regulation of the cell cycle.

In eukaryotic cells, the Wee1 protein kinase phosphorylates and inhibits Cdc2, thereby creating an interphase of the cell cycle. In Xenopus, the conventional Wee1 homolog (termed Xe‐Wee1A, or Wee1A for short) is maternally expressed and functions in pregastrula embryos with rapid cell cycles. Here, we have isolated a second, zygotic isoform of Xenopus Wee1, termed Xe‐Wee1B (or Wee1B for short), that is expressed in postgastrula embryos and various adult tissues. When ectopically expressed in immature oocytes, Wee1B inhibits Cdc2 activity and oocyte maturation (or entry into M phase) much more strongly than Wee1A, due to its short C‐terminal regulatory domain. Moreover, ectopic Wee1B, unlike Wee1A, is very labile during meiosis II and cannot accumulate in mature oocytes due to the presence of PEST‐like sequences in its N‐terminal regulatory domain. Finally, when expressed in fertilized eggs, ectopic Wee1B but not Wee1A does affect cell division and impair cell viability in early embryos, due primarily to its very strong kinase activity. These results suggest strongly that the differential expression of Wee1A and Wee1B is crucial for the developmental regulation of the cell cycle in Xenopus.

Overexpression of the cytoplasmic retention signal region of cyclin B2, but not of cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes.

Cyclin B, a regulatory subunit of maturation/M‐phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B‐type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B‐type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N‐termini in Xenopus oocytes; the N‐termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N‐terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B1 N‐terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N‐terminus contained its own CRS sequence, and was able to be rescued by overexpression of full‐length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.

Expression of cell-cycle regulators during Xenopus oogenesis.

In full-grown Xenopus oocytes, cell-cycle regulators and an inactive form of maturation/M phase promoting factor (pre-MPF) are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.