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.
Nakagami
H
H
Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Japan.
Sekine
M
M
Murakami
H
H
Shinmyo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins; D056741:Cyclin D; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D017343:Genes, Plant; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
D018797:Cell Cycle Proteins; D056741:Cyclin D; D016213:Cyclins; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D010940:Plant Proteins; C107938:RRB1 protein, Zea mays; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases