Enzymology of Mitosis Promoting Factor (MPF)

在真核细胞中，CDC2-Cyclin B复合体是诱导有丝分裂的关键调节蛋白。在脊椎动物中，Cdc2在间期通过抑制其Thr-14和Tyr-15残基的磷酸化而受到负调控。双特异性磷酸酶CDc25C通过使这些残基去磷酸化来激活G2/M转换过程中的CDc2-Cyclin B。如果基因组没有被正确复制或受到破坏，进入有丝分裂的过程就会受到抑制。这些调控机制分别被称为DNA复制检查点和DNA损伤检查点，部分通过阻止CDC25C的作用发挥作用。在脊椎动物中，在DNA复制检查点反应过程中，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(Xchk1)的非洲爪哇同源物使Ser-287上的CDc25C磷酸化。Xchk1在DNA复制受阻后的激活是由非洲爪蛙ATR(XatR)完成的，此外，XatR依赖的Xchk1的磷酸化需要一种新的蛋白质Claspin。建议对包含CDC25、Xchk1、Claspin和XatR的调控通路进行全面研究。将实现以下实验目标。1.CDC25的控制。此外，还将对Xck 1下调CDC25的机制进行进一步研究。2.对Xchk1的调控。控制Xchk1作用的机制将进一步分析。3.对Claspin的功能剖析。将进行研究，以阐明claspin的生化功能。4.鉴定Claspin-Echk1通路中的新成分。将雇用各种搜索者来确定这一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.