FUNCTIONAL ANALYSIS OF CDK INHIBITOR FROM ARABIDOPSIS THALIANA

拟南芥CDK抑制剂的功能分析

基本信息

批准号：
13640648
负责人：
SEKINE Masami
金额：
$ 2.24万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The development of plants depends largely on the cell division activity that is simultaneously controlled by the inherited program of ontogenesis and by environmental conditions. Eukaryotic cells undergo the sequential series of events when they divide, and the proper progression of cell division through the cell cycle requires the coordinated activation of serine/threonine protein kinases called cyclin-dependent kinases (CDKs). The CDKs bind to regulatory subunit, cyclins, which drive progression through the cell-division cycle. CDK activity is controlled by a variety of mechanisms, including binding to cyclins and phosphorylation by the CDK-activating kinase. In addition, a family of low-molecular-weight proteins, designated CDK inhibitors (CKIs), inhibits CDK activity by physically association with the monomer of CDK or the cyclin/CDK complexes. In mammals, two different CKI families can be distinguished on the basis of their mode of action and sequence similarity: the INK4 and the Kip/Cip families. The Kip/Cip family comprises three gene products: p21^<cip1>, p27^<kip1>, and p57^<kip2>. These CKIs bind to the cyclin/CDK complexes. In plants, seven genes, designated Kip-related proteins (KRPs), have been found in Arabidopsis and these genes share sequence similarity with the mammalian p27^<kip1>.In this study, the biochemical analysis of KRP proteins was conducted to characterize their functions. With the exception of KRP7, all KRPs interact with cyclin/CDK complexes but none interact with cyclin and CDK protein alone using in vitro binding assay. We have purified three recombinant KRP proteins from E. coli. These proteins have inhibitory property on the kinase activity of Arabidopsis CyclinD2/CDKA complex, which is purified from insect cells, in a different dose-dependent manner.

植物的发展在很大程度上取决于由个体生成和环境条件同时控制的细胞分裂活性。真核细胞分裂时会经历一系列事件，并且细胞分裂通过细胞周期的正常进展需要丝氨酸/苏氨酸蛋白激酶的协调激活，称为Cyclin依赖性激酶（CDKS）。 CDK与调节亚基，细胞周期蛋白结合，该亚基蛋白可以通过细胞分割周期驱动进展。 CDK活性由多种机制控制，包括与细胞周期蛋白结合和CDK激活激酶的磷酸化。此外，指定的CDK抑制剂（CKIS）的低分子量蛋白家族通过与CDK或细胞周期蛋白/CDK复合物的物理相关性抑制CDK活性。在哺乳动物中，可以根据其作用方式和序列相似性来区分两个不同的CKI家族：Ink4和KIP/CIP家族。 KIP/CIP家族包括三个基因产品：p21^<cip1>，p27^<kip1>和p57^<kip2>。这些CKI与细胞周期蛋白/CDK复合物结合。在植物中，已经在拟南芥中发现了七个指定为KIP相关蛋白（KRP）的基因，并且这些基因与哺乳动物p27^<kip1>具有序列相似性。在这项研究中，KRP蛋白的生化分析被进行表征，以表征它们功能。除KRP7外，所有KRP都与细胞周期蛋白/CDK复合物相互作用，但没有使用体外结合测定法与细胞周期蛋白和CDK蛋白相互作用。我们已经从大肠杆菌中纯化了三种重组KRP蛋白。这些蛋白质对拟南芥cyclind2/cdka复合物的激酶活性具有抑制性能，该蛋白质以不同的剂量依赖性方式从昆虫细胞中纯化。