Enzymology of Mitosis Promoting Factor (MPF)

在真核细胞中，Cdc2-cyclin B复合物是诱导有丝分裂的关键调控激酶。在脊椎动物中，Cdc2在间期通过抑制其Thr-14和tir -15残基的磷酸化而受到负调控。双特异性磷酸酶Cdc25C通过去磷酸化这些残基在G2/M过渡时激活Cdc2-cyclin B。如果基因组没有被正确复制或受到损伤，进入有丝分裂就会受到抑制。这些调节机制分别被称为DNA复制和DNA损伤检查点，它们的部分功能是阻断Cdc25C的作用。在脊椎动物中，在DNA复制检查点反应中Cdc25C的下调涉及蛋白激酶Chk1的磷酸化。例如，在蛙卵提取物中，Chk1的爪蟾同源物（Xchk1）磷酸化Ser-287上的Cdc25C。Xenopus Atr （Xatr）激活Xchk1以响应DNA复制阻断。此外，Xchk1的Xatr依赖性磷酸化需要一种名为Claspin的新蛋白。我们建议对包含Cdc25、Xchk1、Claspin和Xatr的调控通路进行全面的研究。将追求以下实验目标。1. Cdc25的控制。我们将进一步研究Cdc25被Xck1下调的机制。2. Xchk1的调控。我们将进一步分析控制Xchk1作用的机制。3. Claspin的功能解剖。研究将进一步阐明Claspin的生化功能。Claspin-Echk1通路新组分的鉴定。将使用各种搜索器来确定该网络中的其他监管机构。

In eukaryotic cells, the Cdc2-cyclin B complex is a key regulatory kinase that induces mitosis. In vertebrates, Cdc2 is negatively regulated during interphase by inhibitory phosphorylation on its Thr-14 and Tyr-15 residues. The dual-specificity phosphatase Cdc25C activates Cdc2-cyclin B at the G2/M transition by dephosphorylating these residues. The entry into mitosis is suppressed if the genome has not been replicated properly or has suffered damage. These regulatory mechanisms, which are referred to as the DNA replication and DNA damage checkpoints, respectively, function in part by blocking the action of Cdc25C. In vertebrates, the down-regulation of Cdc25C during a DNA replication checkpoint response involves phosphorylation by the protein kinase Chk1. In frog egg extracts, for example, the Xenopus homologue of Chk1 (Xchk1) phosphorylates Cdc25C on Ser-287. The activation on Xchk1 in response to DNA replication blocks is carried out by Xenopus Atr (Xatr), Furthermore, the Xatr-dependent phosphorylation of Xchk1 requires a novel protein named Claspin. A comprehensive study of the regulatory pathway containing Cdc25, Xchk1, Claspin, and Xatr is proposed. The following experimental objectives will be pursued. 1. Control of Cdc25. Additional studies on the mechanism by which Cdc25 is down-regulated by Xck1 will be conducted. 2. Regulation of Xchk1. The mechanisms which control the action of Xchk1 will be analyzed further. 3. Functional dissection of Claspin. Studies will be carried out to elucidate the biochemical function of Claspin. 4. Identification of Novel Components in the Claspin-Echk1 Pathway. Various searchers will be employed to identify additional regulators in this network.