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.
Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk–cyclin complexes, including Cdk1–cyclin A, Cdk1–cyclin B, and Cdk2–cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk–cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism
In early animal development, cell proliferation and differentiation are tightly linked and coordinated. It is important, therefore, to know how the cell cycle is controlled during early development. Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell-cycle progression. In Xenopus laevis, three isoforms of cdc25 have been identified, viz. cdc25A, cdc25B and cdc25C. In this study, we isolated a cDNA encoding a novel Xenopus Cdc25 phosphatase (named cdc25D). We investigated the temporal and spatial expression patterns of the four cdc25 isoforms during early Xenopus development, using RT-PCR and whole-mount in situ hybridization. cdc25A and cdc25C were expressed both maternally and zygotically, whereas cdc25B and cdc25D were expressed zygotically. Both cdc25A and cdc25C were expressed mainly in prospective neural regions, whereas cdc25B was expressed preferentially in the central nervous system (CNS), such as the spinal cord and the brain. Interestingly, cdc25D was expressed in the epidermal ectoderm of the late-neurula embryo, and in the liver diverticulum endoderm of the mid-tailbud embryo. In agreement with the spatial expression patterns in whole embryos, inhibition of bone morphogenetic protein (BMP), a crucial step for neural induction, induced an upregulation of cdc25B, but a downregulation of cdc25D in animal cap assays. These results indicate that different cdc25 isoforms are differently expressed and play different roles during early Xenopus development. KEY WORDS: cell cycle, Xenopus, cdc25, cdc25D, cell proliferationIn early animal development, cell-cycle progression and exit from the cell cycle must be precisely controlled, since aberrant cell proliferation results in malformation or hyperplasia. It is important, therefore, to know how the cell cycle is controlled during development. Despite the importance of cell-cycle control in embryogenesis, however, relatively less attention has been paid to the expression patterns of cell-cycle regulators than to those of other factors that are involved in patterning and differentiation. Since amphibian Xenopus laevis is one of the most intensely investigated model animals in both developmental biology and cell-cycle control at the molecular level, this animal is highly suitable for studies on the cell-cycle control during embryogenesis.Over the last three decades, numerous studies have contributed to the understanding of the core mechanisms underlying cell-cycle control. Especially, extensive studies in yeast and mammalian cultured cells have identified many essential regulators that govern Int. J. Dev. Biol. 55: [627][628][629][630][631][632]
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