Narasimha, Anil MKaulich, ManuelShapiro, Gary SChoi, Yoon JSicinski, PiotrDowdy, Steven F2014-08-132014Narasimha, Anil M, Manuel Kaulich, Gary S Shapiro, Yoon J Choi, Piotr Sicinski, and Steven F Dowdy. 2014. “Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.” eLife 3 (1): e02872. doi:10.7554/eLife.02872. http://dx.doi.org/10.7554/eLife.02872.2050-084Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12717606The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase. DOI: http://dx.doi.org/10.7554/eLife.02872.001en-UScell cycleRb tumor suppressorcyclin D:Cdk4mono-phosphorylated Rbp16G1 phasehumanJournal Article2014-08-1310.7554/eLife.02872