Enzymology of Mitosis Promoting Factor (MPF)

在真核细胞中，Cdc 2-细胞周期蛋白B复合物是诱导有丝分裂的关键调节激酶。在脊椎动物中，Cdc 2在间期通过其Thr-14和Tyr-15残基上的抑制性磷酸化而负调控。双特异性磷酸酶Cdc 25 C通过使这些残基去磷酸化而在G2/M转换时激活Cdc 2-细胞周期蛋白B。如果基因组没有被正确复制或受到损伤，则进入有丝分裂受到抑制。这些调节机制，分别被称为DNA复制和DNA损伤检查点，部分通过阻断Cdc 25 C的作用发挥作用。在脊椎动物中，Cdc 25 C在DNA复制检查点反应期间的下调涉及蛋白激酶Chk 1的磷酸化。例如，在青蛙卵提取物中，Chk 1（Xchk 1）的爪蟾同源物磷酸化Ser-287上的Cdc 25 C。Xchk 1的激活是由非洲爪蟾Atr（Xatr）响应DNA复制阻断而进行的。此外，Xatr依赖的Xchk 1磷酸化需要一种名为Claspin的新蛋白。提出了一个全面的研究的调节途径，包括Cdc 25，Xchk 1，Claspin，和Xatr。将追求以下实验目标。1.控制Cdc 25。将对Xck 1下调Cdc 25的机制进行进一步研究。2. Xchk 1的规则。控制Xchk 1作用的机制将进一步分析。3. Claspin的功能解剖。将进行研究以阐明Claspin的生化功能。4. Claspin-Echk 1通路中新组分的鉴定。将雇用各种搜索人员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.