FUNCTIONAL ANALYSIS OF CDK INHIBITOR FROM ARABIDOPSIS THALIANA

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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.