EMI1 switches from being a substrate to an inhibitor of APC/CCDH1 to start the cell cycle

Abstract

Mammalian cells integrate mitogen and stress signaling prior to the end of G1 phase to decide whether or not to enter the cell cycle1–4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program, and inactivate an E3 ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/CCdh1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but cannot after inactivation of APC/CCdh1, arguing that APC/CCdh1 inactivation is the point-of-no-return for cell cycle entry3. While rapid inactivation of APC/CCdh1 requires early mitotic inhibitor 1 (Emi1)3,5, the molecular mechanism controlling this cell cycle commitment step is unknown. Here we show that cell cycle commitment is mediated by an Emi1-APC/CCdh1 dual-negative feedback switch, in which Emi1 is both a substrate and an inhibitor of APC/CCdh1. The inactivation switch triggers a transition between a state with low Emi1 levels and high APC/CCdh1 activity during G1 to a state with high Emi1 levels and low APC/CCdh1 activity during S and G2. Cell-based analysis, in vitro reconstitution, and modeling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Together, our study argues that mammalian cells commit to the cell cycle by increasing CDK2 activity and Emi1 mRNA expression to trigger a one-way APC/CCdh1 inactivation switch mediated by Emi1 transitioning from a substrate to an inhibitor of APC/CCdh1.

Bistable switches as integrators and actuators during cell cycle progression

“…Inactivation of APC/C Cdh1 generally occurs late in G1 and is induced by cyclin E/CDK2 complexes, suggesting that engagement of the Rb-E2F switch should be required prior to the APC/C Cdh1 switch. Indeed, Cappell et al demonstrated that APC/C Cdh1 inactivation was observed in the vast majority of cells only after prior engagement of the Rb-E2F switch and within 1 h before S phase initiation [75]. Consistent with the idea that this mechanism confers a temporal order to the switches, E2F-dependent expression of Emi1 [77] was also shown to play a crucial role in the kinetics of APC/C Cdh1 inactivation and was required to make the switch irreversible, thus preventing reactivation of APC/C Cdh1 during S phase.…”

Preparation for DNA replication: the key to a successful S phase

“…In addition, E2F‐stimulated transcription drives production of the APC/C inhibitor protein EMI1 whose levels rise through the G1/S transition (reviewed in ). EMI1 is subsequently stabilized by a variety of ubiquitination and phosphorylation events near the G1/S transition . Once APC/C is inactivated, DNA replication (i.e., S phase) is destined to begin.…”

A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

“…3e), we hypothesized that, in the presence of CDK4/6 inhibitor, full Rb phosphorylation and inactivation may occur only after APC/C CDH1 inactivation. This is possible since APC/C CDH1 inactivation can be mediated by cyclin E-CDK2 phosphorylation on CDH1 even in the absence of EMI1 12,13 . This hypothesis is further strengthened by the finding that APC/C CDH1 inactivates at approximately the same level of cyclin E/A-CDK activity in the alternate and canonical pathway (Extended Data Fig.…”

“…Previous studies of the canonical pathway showed that APC/C CDH1 inactivation can be initiated either by E2F-mediated activation of cyclin E-CDK2 or expression of the APC/C CDH1 inhibitor EMI1 12,13 . However, irreversible APC/C CDH1 inactivation requires a double negative feedback between EMI1 and APC/C CDH1 (Fig.…”

“…Furthermore, once APC/C CDH1 inactivation is initiated, cell-cycle entry becomes irreversible in respect to various types of stress, reflecting an underlying G1 commitment point (Fig. 1a, top) [11][12][13] .…”