Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4.
Phosphorylation of target proteins by cyclin D1-Cdk4 requires both substrate docking and kinase activity. In addition to the ability of cyclin D1-Cdk4 to catalyze the phosphorylation of consensus sites within the primary amino acid sequence of a substrate, maximum catalytic activity requires the enzyme complex to anchor at a site remote from the phospho-acceptor site. A novel Cdk4 docking motif has been defined within a stretch of 19 amino acids from the C-terminal domain of the Rb protein that are essential for Cdk4 binding. Mutation or deletion of the docking motif prevents Cdk4-dependent phosphorylation of full-length Rb protein or C-terminal Rb fragments in vitro and in cells, while a peptide encompassing the Cdk4 docking motif specifically inhibits Cdk4-dependent phosphorylation of Rb. Cyclin D1-Cdk4 can overcome the growth-suppressive activity of Rb in both cell cycle progression and colony formation assays; however, while mutants of Rb in which the Cdk4 docking site has been either deleted or mutated retain growth suppressor activity, they are resistant to inactivation by cyclin D1-Cdk4. Finally, binding of Cdk4 to its docking site can inhibit cleavage of exogenous and endogenous Rb in response to distinct apoptotic signals. The Cdk4 docking motif in Rb gives insight into the mechanism by which enzyme specificity is ensured and highlights a role for Cdk4 docking in maintaining the Rb protein in a form that favors cell survival rather than apoptosis.
Wallace
Maura
M
CRUK Laboratories, University of Dundee Medical School, Dundee DD1 9SY, United Kingdom.
Ball
Kathryn L
KL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases