Molecular Mechanisms for Phosphorylation Driven Dissociation of Rb-E2F Complexes

Abstract

The Retinoblastoma Protein (Rb) functions as a negative regulator of cell growth in part by physically sequestering and repressing the transactivation activity of E2F. It is well established that phosphorylation of Rb by Cyclin Dependent Kinases disrupts binding between Rb and E2F, however it is unknown which of the 15 CDK consensus phosphorylation sites on Rb are required to disrupt the interaction between the pocket domain of Rb and the transactivation domain of E2F (E2FTD). In this work, we use calorimetric assays to reveal that phosphorylation at S608/S612 and T356/T373 are together sufficient to reduce the affinity of E2FTD for Rb pocket 250-fold, the same as fully-phosphorylated Rb. Nuclear Magnetic Resonance is used to identify the phosphorylation-dependent conformational changes that directly inhibit E2FTD binding. Specifically, we have found that phosphorylation of S608/S612 promotes intramolecular binding between the flexible pocket linker and the pocket domain of Rb, while phosphorylation at T356/T373 enhances binding between the N-terminal domain and pocket domain of Rb. Taken together, our results reveal two novel mechanisms for how phosphorylation of Rb modulates binding between E2FTD and Rb pocket, and we describe for the first time a function for the N-terminal domain in the inactivation of Rb.