Gene Regulation
Phosphorylation-induced Conformational Changes in the Retinoblastoma Protein Inhibit E2F Transactivation Domain Binding*

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Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2FTD) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2FTD binding ∼250-fold and that phosphorylation at Ser608/Ser612 and Thr356/Thr373 is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2FTD binding. We find that phosphorylation at Ser608/Ser612 promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2FTD binding site. We also find that phosphorylation of Thr356/Thr373 inhibits E2FTD binding in a manner that requires the Rb N-terminal domain. Taken together, our results suggest two distinct mechanisms for how phosphorylation of Rb modulates association between E2FTD and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.

Cdk (Cyclin-dependent Kinase)
Cell Cycle
E2F Transcription Factor
Protein Phosphorylation
Retinoblastoma (Rb)

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A Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.

*

This work was supported, in whole or in part, by National Institutes of Health Grants CA132685 (to S. M. R.) and GM68933 (to support the Central California 900-MHz facility), and SS10-RR20939 (to support the University of California Santa Cruz mass spectrometry facility).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.