Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro

The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro. Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.