Integrating Old and New Paradigms of G1/S Control

Abstract

The Cdk-Rb-E2F pathway integrates external and internal signals to control progression at the G1/S transition of the mammalian cell cycle. Alterations in this pathway are found in most human cancers, and specific cyclin-dependent kinase Cdk4/6 inhibitors are approved or in clinical trials for the treatment of diverse cancers. In the long-standing paradigm for G1/S control, Cdks inactivate the retinoblastoma tumor suppressor protein (Rb) through phosphorylation, which releases E2F transcription factors to drive cell-cycle progression from G1 to S. However, recent observations in the laboratory and clinic challenge central tenets of the current paradigm and demonstrate that our understanding of the Rb pathway and G1/S control is still incomplete. Here, we integrate these new findings with the previous paradigm to synthesize a current molecular and cellular view of the mammalian G1/S transition. A more complete and accurate understanding of G1/S control will lead to improved therapeutic strategies targeting the cell cycle in cancer.

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Figure 1:. Components of the Cdk-Rb-E2F pathway controlling the G1/S transition.

(A) Simplified model for pathway. (B) Inactive and active states of the key players in the Rb pathway. Cdk4 and Cdk6 (Cdk4/6) have relatively high sequence homology among Cdks. They are inactive as monomers, bound to p16 family proteins, or bound by unphosphorylated p21 and p27 (p21/p27) proteins. Cdk4/6 are activated by association with Cyclin D (CycD) family proteins, but full activity also requires a phosphorylated from of p27 in the complex and phosphorylation on the kinase activation loop. Cdk2 is inactive as a monomer or in complex with p21/p27 family proteins, and it is activated by CycE binding in G1 (or CycA later in the cell cycle) and activation loop phosphorylation. Rb is considered active when hypo-or monophosphorylated; in this state it binds and inhibits E2F. Hyperphosphorylation of Rb leads to its inactivation, dissociation from E2F and subsequent E2F activation. While Rb binds E2F when unphosphorylated during quiescence, it may also be considered inactive in that it cannot perform monophosphorylation-dependent functions.

Figure 2:. Proposed models for Rb inactivation during G1.

(A) The three models differ with respect to whether Cdk4/6 and Cdk2 activate (arrow) or inhibit (cross) Rb and in the timing of their activity relative to the G1/S transition. (B) Roles of Cdk complexes in modulating Rb function. In the canonical Model I, Cdk4/6 partially phosphorylates Rb, resulting in some E2F activity. Transcription of genes such as CycE then activates Cdk2 for full Rb hyperphosphorylation and inactivation for S phase. In Model II, Cdk4/6 monophosphorylates Rb, and the different active Rb species inhibit E2F and form functional protein complexes in G1. Cdk4/6 induces Cdk2 activity through several possible mechanisms, including sequestration of p21/p27 inhibitors and phosphorylation of other targets that may indirectly activate Cdk2. Active Cdk2 then hyperphosphorylates Rb, leading to Rb inactivation and S phase entry. In Model III, Cdk4/6 hyperphosphorylates Rb in G1, which is sufficient for Rb inactivation and S phase entry. Cdk2 activity is required to maintain Rb hyperphosphorylation during S phase.

Figure 3:. APC^Cdh1 and p21/p27 inactivation are required for S phase entry.

S phase activity of Cdk2 is inhibited by p21/p27 and APC^Cdh1, which is a ubiquitin ligase that stimulates CycA degradation. APC^Cdh1 is inactivated by Cdk2 phosphorylation and the protein Emi1. p21/p27 degradation is induced by Cdk2 phosphorylation and the ubiquitin ligase Skp2, which is in turn degraded by APC^Cdh1. Both Emi1 and Skp2 are transcriptional targets of E2F, which places these inactivation events downstream of Rb inactivation. These two connected double negative feedback loops involved in Cdk2 activation are thought to render S phase entry irreversible.

Figure 4:. Cell growth and size signals drive the G1/S transition.

Growth factors trigger the synthesis of CycD and Cdk4/6-dependent activity. Cell growth in G1 dilutes Rb and leads to a decrease in activity of the p38 stress-activated kinase to trigger cell-cycle progression in larger cells.