The 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.02872.001