Death of a Dogma: Cyclin D Activates Rb by Mono-phosphorylation

Abstract

The current textbook dogma of G₁ cell cycle progression proposes that cyclin D-Cdk4/Cdk6 inactivates the pRb tumor suppressor during early G₁ phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors that specifically turn on the cyclin E gene, which then activate Cdk2 and complete pRb’s inactivation by hyper-phosphorylation at the restriction point. However, this model has remained largely untested from a biochemical standpoint for more than 20 years. Moreover, the biologically active form(s) of pRb present during early G₁ phase is uncharacterized, and a precise understanding of a potential “pRb phospho-code,” regulating association with individual pRb partners, remains elusive. Recently, using quantitative 2D isoelectric focusing (2D IEF) to directly count phosphates on pRb, we have shown pRb to be exclusively mono-phosphorylated in early G₁ phase by cyclin D-Cdk4/Cdk6 complexes acting to modify only one of each of the 14 Cdk phosphorylation sites per individual pRb molecule. Mono-phosphorylated pRb is functionally active in early G1 phase and binds to and represses E2Fs as well as the E1a oncoprotein. Biologically, cells undergoing a DNA damage response activate cyclin D-Cdk4/Cdk6 to generate mono-phosphorylated pRb to regulate global transcription, whereas un-phosphorylated pRb is inactive during a DNA damage response. At the late G₁ restriction point, activation of cyclin E-Cdk2 complexes inactivates pRb by quantum hyper-phosphorylation. These observations fundamentally change our understanding of G₁ cell cycle progression and show that mono-phosphorylated pRb, generated by cyclin D-Cdk4/Cdk6, is the biologically active pRb isoform in early G₁ phase that represses E2F transcription and that cyclin E-Cdk2 is the pRb inactivating kinase. These findings raise clear questions about the role of cyclin D1 as an oncogene and about the cellular state elicited by drugs targeting cyclin D1’s partner kinases, Cdk4 and Cdk6. Herein, we summarize the findings leading to this paradigm shift and offer a hypothesis reconciling the apparently contradictory roles of cyclin D-Cdk4/Cdk6 as pRb activator and as a common oncogenic driver.