Poster: Cell Cycle & Cytokinesis

Abs # 1093: Functional Analysis of CDK Inhibitors from Arabidopsis thaliana

Presenter:	Nakai, Tomohiro , to-nakai@bs.aist-nara.ac.jp
Authors	Nakai, Tomohiro  (A)   Sekine, Masami  (A)   Shinmyo, Atsuhiko  (A)
Affiliations:	(A): Nara Institute of Science and Technology

In all eukaryotes, the cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK activity is controlled by various regulatory factors during the cell cycle and CDK inhibitors play a major role in controlling the G1/S transition through the control of CDK activity. Based on the complete Arabidopsis genome analysis, seven genes have been identified as CDK inhibitor. These genes were designated Kip-related proteins (KRPs) or Interactor of Cdc2 kinase (ICK). The KRP/ICK genes encode proteins sharing sequence homology with an animal CDK inhibitor p27^Kip1. In this study, we have examined the function of KRP protein with respect to the interaction with Cyclin/CDK. Using in vitro transcription/translation system, we have prepared all KRP proteins. With the exception of KRP7, all KRPs interact with Cyclin D2/CDKA and Cyclin D4/CDKB complexes but none interact with Cyclin D2, Cyclin D4, CDKA and CDKB protein alone in an in vitro binding assay. We have purified recombinant proteins, KRP2, 6 and 7 from E. coli expressing these proteins and examined their inhibition activities on histone H1 kinase activity of Cyclin D2/CDKA complex that is purified from insect cells. Increasing amounts of KRP2 and 6 proteins in kinase reactions resulted in greater reduction of kinase activity. Surprisingly, relatively less reduction of kinase activity was also observed by adding KRP7, while KRP7 did not stably interact with Cyclin/CDK complexes. Additionally, inhibition activities of other KRPs have been observed on histone H1 kinase assays by using tag-fusion proteins. Together with binding and inhibition properties of KRP proteins, binding ability is thought to correlate with inhibition property and we will discuss the mechanism of interaction between KRPs and Cyclin/CDK complexes.