Activation of CDK-activating kinase is dependent on interaction with H-type cyclins in plants

cDNAs encoding cyclin H homologs were isolated from poplar (Populus tremula x tremuloides) and rice (Oryza sativa) plants, and were designated Pt;cycH ;1 and Os;cycH;1, respectively. The deduced amino-acid sequences showed 40- 60% similarity to human cyclin H and Schizosaccharomyces pombe Mcs2, with h igher similarity in the cyclin box region. While Pt;cycH;1 and Os;cycH;1 we re expressed in all tissues examined, the transcripts accumulated abundantl y in dividing cells. Expression of Os;cycH;1 was abundant in the S-phase in partially synchronized suspension cells, and was induced by submergence in internodes of deepwater rice. A yeast two-hybrid assay demonstrated that b oth Pt;CycH;1 and Os;CycH;1 were able to interact with rice R2 kinase, whic h is structurally and functionally similar to cyclin-dependent kinase (CDK) -activating kinase (CAK) of vertebrates. Moreover, an in vitro pull-down as say showed that Os;CycH;1 specifically bound to R2 but not to other rice CD Ks. When R2 was expressed in budding yeast CAK mutant, the suppression acti vity in terms of temperature-sensitivity was enhanced by co-expression with Os;cycH;1. Furthermore, in vitro kinase assay indicated that the kinase ac tivities of R2 on CDKs and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II were markedly elevated by binding to Os;CycH; 1. Our results suggest that cyclin H is a regulatory subunit of CAK, which positively controls CDK- and CTD-kinase activities in plant cells.