Multiple Mechanisms for E2F Binding Inhibitionby Phosphorylation of the Retinoblastoma Protein C-terminal Domain

The retinoblastoma proteinC-terminal domain (RbC) is necessary for the tumor suppressor protein's activities in growth suppression and E2F transcription factor inhibition. Cyclin-dependent kinase phosphorylation of RbC contributes to Rb inactivation and weakens the Rb-E2F inhibitory complex. Here we demonstratetwo mechanisms for how RbC phosphorylation inhibits E2F binding. We find that phosphorylation of S788 and S795 weakens the direct association between the N-terminal portion of RbC (RbC N ) and the marked-box domains of E2F and its heterodimerization partner DP. Phosphorylation of these sites and S807/S811 also induces an intramolecular association between RbC and the pocket domain,which overlaps with the site of E2F transactivation domain binding. Areduction in E2F binding affinity occurs with S788/S795 phosphorylation that is additive with the effects of phosphorylation at other sites, and we propose a structural mechanism that explains this additivity. We find that different Rb phosphorylation events have distinct effects on activating E2F family members, which suggests a novel mechanism for how Rb may differentially regulate E2F activities.