Abstract
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 (E2F(TD)) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2F(TD) binding approximately 250-fold and that phosphorylation at Ser(608)/Ser(612) and Thr(356)/Thr(373) is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2F(TD) binding. We find that phosphorylation at Ser(608)/Ser(612) promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2F(TD) binding site. We also find that phosphorylation of Thr(356)/Thr(373) inhibits E2F(TD) 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 E2F(TD) and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.
Highlights
Cells in G1; these observations directly implicate the retinoblastoma protein (Rb)-E2F pathway as an essential control mechanism of the G1-S transition and a critical link between growth factor signaling and cell cycle progression [1, 2]
We show that phosphorylation stimulates an intramolecular interaction between RbPL and the pocket domain that overlaps with the E2F transactivation domain (E2FTD) binding site
RbN Is Required for Phosphorylation-induced Inhibition of E2FTD Binding—To determine the precise sequences and phosphorylation sites within Rb required for inhibition of E2FTD binding, we applied an isothermal titration calorimetry (ITC) assay to quantitatively measure affinities with purified proteins
Summary
Protein Expression and Purification—Human Rb constructs containing a single domain (e.g. RbN or pocket domain) could be expressed with high yields in Escherichia coli. Proteins were purified by Ni2ϩ-nitrilotriacetic acid affinity purification and heparin sulfate chromatography. Rb352–787, Rb380 –787 (wild type and mutants), and RbP⌬PL (Rb380 –787, with 578 – 642 deleted) were expressed in E. coli as glutathione S-transferase fusion proteins. RbPL592– 624, Rb338 –379, Rb55–379, and E2FTD (E2F1, residues 372– 437) were expressed as His fusion proteins in E. coli. Cells were induced for 2– 4 h at 37 °C, and proteins were purified by Ni2ϩ-nitrilotriacetic acid affinity and anion exchange chromatography. Enzymatic Modifications—Rb protein constructs were concentrated to ϳ1–5 mg/ml following purification and phosphorylated in a reaction containing 10 mM MgCl2, 10 mM ATP, 250 mM NaCl, 25 mM Tris (pH 8.0), and 2% Cdk6-CycK or 10% Cdk2-CycA (percentage of mass of the total substrate in the reaction). NMR spectra were processed with NMRPipe and analyzed with NMRViewJ [31, 32]
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