Cyclin D activates the Rb tumor suppressor by mono-phosphorylation

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.DOI: http://dx.doi.org/10.7554/eLife.02872.001

RB1: a prototype tumor suppressor and an enigma

“…Remarkably, the single phosphorylation event can be found at many, perhaps all, of the 14 known sites for CDK phosphorylation. Narasimha et al (2014) show that monophosphorylated protein is the predominant form of pRB in contact-inhibited cells and cells arrested by DNA damage, situations in which pRB is known to be active (Fig. 3A).…”

“…This selectivity suggests that the various perturbations of this "pathway" are not identical but that specific mutations have different consequences in different contexts. Data indicating that pRB retains some degree of E2F regulation in INK4A mutant cells or when phosphorylated by cyclin D-dependent kinases (Haberichter et al 2007;Narasimha et al 2014) and evidence that hyperphosphorylated pRB interacts with the mTORC2 complex and attenuates Akt activation (Zhang et al 2016) support the view that the inactivation of pRB by phosphorylation is not functionally equivalent to the mutation of the RB1 gene.…”

“…In both cases, the structural changes driven by individual phosphorylation events alter specific binding domains but do not compromise the overall integrity of the protein and leave other binding surfaces intact. These observations are especially significant when combined with experiments that used isoelectric focusing gels to assess the number of phosphorylation events on endogenous pRB (Narasimha et al 2014). Narasimha et al (2014) reported that the "hypophosphorylated" form of pRB isolated from asynchronously dividing cells in tissue culture is entirely composed of monophosphorylated protein.…”

“…These observations are especially significant when combined with experiments that used isoelectric focusing gels to assess the number of phosphorylation events on endogenous pRB (Narasimha et al 2014). Narasimha et al (2014) reported that the "hypophosphorylated" form of pRB isolated from asynchronously dividing cells in tissue culture is entirely composed of monophosphorylated protein. Remarkably, the single phosphorylation event can be found at many, perhaps all, of the 14 known sites for CDK phosphorylation.…”

Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium

“…Recently it is proposed that Cyclin D-CDK4/6 catalyzes mono-phosphorylation of Rb to activate early G1 functions of multiple, mono-phospho Rb isoforms and to prevent cell cycle exit to G0, regulated in part by un-phosphorylated Rb. 17 Importantly, Cyclin D-CDK4/6 complexes can activate Cyclin E-CDK2 indirectly by sequestering G1-phase p21/p27-type Cyclin-dependent Kinase Inhibitors (CKIs), which inhibit Cyclin E-CDK2 activity.…”