Regulation of cell cycle transition by a cyclin-dependent kinase in trypanosomes

细胞周期蛋白依赖性蛋白激酶(CDK)是真核生物细胞周期的基本调节因子，在细胞周期中发挥重要作用。 通过与细胞周期蛋白结合激活，其丰度在细胞周期中发生变化。一个周期和多个周期 CDKs在酵母细胞周期调控中起作用，而多个周期蛋白和多个CDKs控制细胞周期 动物的进化。锥虫似乎使用了一种截然不同的细胞周期控制系统 不同于真菌和动物。一个类似PHO80的细胞周期蛋白Cyc2和一个CDK相关的激酶CRK1控制着G1/S 而B型细胞周期蛋白、细胞周期蛋白6和另一种CDK相关的激酶CRK3控制着G2/M的转变。 然而，CRK1如何在细胞周期进程中发挥作用仍不清楚，主要是因为它的下游 目标尚不清楚。目前的建议旨在了解G1/S细胞的潜在机制 细胞周期转换及其在不同生命周期形式间细胞周期调控的分子基础 布氏毛虫T.brucei在这项建议的目标1中，我们计划应用化学遗传学方法来鉴定CRK1底物 从前循环和血流的形式及CRK1调节的细胞通路的比较分析 在这两种形式之间。目标2是了解细胞周期依赖蛋白的潜在机制 翻译启动，重点是CRK1对两个翻译启动因素的调节。目标3是 通过研究CRK1对DNA的调控来剖析CRK1在DNA复制启动中的作用 CDC45-McM2-7-GINS复合体中的复制因子。通过分子，细胞生物学，化学遗传， 和生化方法，我们在这项提案中的总体目标是了解CRK1的机械作用 在G1/S对照中通过调节DNA复制解旋酶和在G1特异性蛋白翻译中 通过调节翻译启动因素来启动翻译。这些研究的结果不仅将 极大地促进了我们对锥虫细胞周期转变机制的理解，但也 可以确认CRK1及其锥虫特异性下游通路为抗锥虫的药物靶点 化疗。

Cyclin-dependent kinases (CDKs) are the fundamental regulators of the cell cycle in eukaryotes, which are activated by binding to cyclins whose abundance changes during the cell cycle. A single cyclin and multiple CDKs function in yeast cell cycle control, whereas multiple cyclins and multiple CDKs control the cell cycle progression in animals. Trypanosomes appear to employ a cell cycle control system that is strikingly different from that in fungi and animals. A PHO80-like cyclin, CYC2, and a CDK-related kinase, CRK1, control the G1/S transition, whereas a B-type cyclin, CYC6, and another CDK-related kinase, CRK3, govern the G2/M transition. However, how CRK1 exert their roles in cell cycle progression remains elusive, mainly because its downstream targets are not known. The current proposal aims to understand the mechanisms underlying the G1/S cell cycle transition and the molecular basis for the distinct cell cycle regulation between different life cycle forms of T. brucei. In aim 1 of this proposal, we plan to apply chemical genetic approach to identify CRK1 substrates from the procyclic and bloodstream forms and to comparatively analyze the CRK1-regulated cellular pathways between the two forms. Aim 2 is to understand the mechanisms underlying the cell cycle-dependent protein translation initiation, with a focus on the regulation of two translation initiation factors by CRK1. Aim 3 is to dissect the role of CRK1 in DNA replication initiation by investigating CRK1-mediated regulation of the DNA replication factors in the Cdc45-Mcm2-7-GINS complex. Through molecular, cell biological, chemical genetic, and biochemical approaches, our overall goal in this proposal is to understand the mechanistic roles of CRK1 in the G1/S control through regulating the DNA replicative helicase and in G1-specific protein translation initiation through regulating translation initiation factors. The outcomes from these studies not only will significantly advance our understanding of the mechanisms of cell cycle transitions in trypanosomes, but also could validate CRK1 and its trypanosome-specific downstream pathways as drug targets for anti-trypanosome chemotherapy.