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A Code of Mono-phosphorylation Modulates the Function of RB.

ABSTRACT: Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.

SUBMITTER: Sanidas I

PROVIDER: S-EPMC6424368 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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A Code of Mono-phosphorylation Modulates the Function of RB.

Sanidas Ioannis I Morris Robert R Fella Katerina A KA Rumde Purva H PH Boukhali Myriam M Tai Eric C EC Ting David T DT Lawrence Michael S MS Haas Wilhelm W Dyson Nicholas J NJ

Molecular cell 20190130 5

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional speci ...[more]

PMID: 30711375

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Project description:The data were acquired by performing multiplexed proteomics with TMT10-plex reagents using both and Orbitrap Fusion and an Orbitrap Lumos mass spectrometer and applying an SPS-supported LC-MS2/MS3 method. Samples were anti-FLAG immunoprecipitates of RB phospho-isoforms from human RPE1 cells. Samples were labeled with the specific TMT reagents as follows: Sample pool 1: 126, Bridge; 127n, RB wild-type (replicate 1); 127c, RB_DELTA_cdk (replicate 1); 128n, FLAG-RB wild-type (replicate 1); 128c, FLAG-RB_DELTA_cdk (replicate 1); 129n, FLAG-RB_DELTA_cdk + S230 (replicate 1); 129c, FLAG-RB_delta_cdk + S249 (replicate 1); 130n, ,FLAG-RB_delta_cdk + T252 (replicate 1); 131, Bridge. Sample pool 2: 126, Bridge; 127n, RB wild-type (replicate 2); 127c, RB_delta_cdk (replicate 2); 128n, FLAG-RB wild-type (replicate 2); 128c, FLAG-RB_delta_cdk (replicate 2); 129n, FLAG-RB_delta_cdk + S230 (replicate 2); 129c, FLAG-RB_delta_cdk + S249 (replicate 2), 130n, ,FLAG-RB_delta_cdk + T252 (replicate 2), 131, Bridge. Sample pool 3: 126, Bridge; 127n, RB wild-type (replicate 3); 127c, RB_delta_cdk (replicate 3); 128n, FLAG-RB wild-type (replicate 3); 128c, FLAG-RB_delta_cdk (replicate 3); 129n, FLAG-RB_delta_cdk + S230 (replicate 3); 129c, FLAG-RB_delta_cdk + S249 (replicate 3); 130n, FLAG-RB_delta_cdk + T252 (replicate 3); 131, Bridge. Sample pool 4: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 1); 127c, FLAG-RB_delta_cdk + T373 (replicate 1); 128n, FLAG-RB_delta_cdk + S608 (replicate 1); 128c, FLAG-RB_delta_cdk + S612 (replicate 1); 129n, FLAG-RB_delta_cdk + S780 (replicate 1); 129c, FLAG-RB_delta_cdk + S788 (replicate 1); 130n, FLAG-RB_delta_cdk + S795 (replicate 1); 131, Bridge. Sample pool 5: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 2); 127c, FLAG-RB_delta_cdk + T373 (replicate 2); 128n, FLAG-RB_delta_cdk + S608 (replicate 2); 128c, FLAG-RB_delta_cdk + S612 (replicate 2); 129n, FLAG-RB_delta_cdk + S780 (replicate 2); 129c, FLAG-RB_delta_cdk + S788 (replicate 2); 130n, FLAG-RB_delta_cdk + S795 (replicate 2); 131, Bridge. Sample pool 6: 126, Bridge; 127n, FLAG-RB_delta_cdk + T356 (replicate 3); 127c, FLAG-RB_delta_cdk + T373 (replicate 3); 128n, FLAG-RB_delta_cdk + S608 (replicate 3); 128c, FLAG-RB_delta_cdk + S612 (replicate 3); 129n, FLAG-RB_delta_cdk + S780 (replicate 3); 129c, FLAG-RB_delta_cdk + S788 (replicate 3); 130n, FLAG-RB_delta_cdk + S795 (replicate 3); 131, Bridge. Sample pool 7: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 1); 127c, FLAG-RB_delta_cdk + S811 (replicate 1); 128n, FLAG-RB_delta_cdk + T821 (replicate 1); 128c, FLAG-RB_delta_cdk + T826 (replicate 1); 131, Bridge. Sample pool 8: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 2); 127c, FLAG-RB_delta_cdk + S811 (replicate 2); 128n, FLAG-RB_delta_cdk + T821 (replicate 2); 128c, FLAG-RB_delta_cdk + T826 (replicate 2); 131, Bridge. Sample pool 9: 126, Bridge; 127n, FLAG-RB_delta_cdk + S807 (replicate 3); 127c, FLAG-RB_delta_cdk + S811 (replicate 3); 128n, FLAG-RB_delta_cdk + T821 (replicate 3); 128c, FLAG-RB_delta_cdk + T826 (replicate 3); 131, Bridge.

Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

Dataset Information

A Code of Mono-phosphorylation Modulates the Function of RB.

Publications

A Code of Mono-phosphorylation Modulates the Function of RB.

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