Abstract
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.
Highlights
The retinoblastoma tumor suppressor protein (Rb) functions to regulate multiple critical cellular activities, including the late G1 restriction point, the DNA damage response checkpoint, cell cycle exit, and differentiation (Burkhart and Sage, 2008; Paternot et al, 2010; Henley and Dick, 2012; Johnson and Skotheim, 2013)
We have previously found in kinetic analyses from highly synchronized normal cells and p16-deficient cancer cells that cyclin D:Cdk4/6 is constitutively active throughout early G1 phase at the same time when Rb is repressing E2F target genes (Ezhevsky et al, 1997, 2001; Haberichter et al, 2007)
Our observations demonstrate that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6 complexes, is the functionally active Rb isoform present in early G1 phase
Summary
The retinoblastoma tumor suppressor protein (Rb) functions to regulate multiple critical cellular activities, including the late G1 restriction point, the DNA damage response checkpoint, cell cycle exit, and differentiation (Burkhart and Sage, 2008; Paternot et al, 2010; Henley and Dick, 2012; Johnson and Skotheim, 2013). Given the scientific scrutiny of Rb over the last 25 years, the biochemical identification of the biologically active isoform(s) of Rb required for early G1 phase regulation, DNA damage checkpoint control, cell cycle exit, and differentiation remains unknown. To dissect Rb function and regulation, many early reports utilized supra-physiologic overexpression studies using various cyclins (A, B, D, E) and Cdks (−1, −2, −4, −6) that resulted in Rb inactivation by hyper-phosphorylation associated with an accelerated S-phase entry, and induction of E2F-dependent
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