Entry into mitosis is triggered by the activation of cyclin‐dependent kinase 1 (Cdk1). This simple reaction rapidly and irreversibly sets the cell up for division. Even though the core step in triggering mitosis is so simple, the regulation of this cellular switch is highly complex, involving a large number of interconnected signalling cascades. We do have a detailed knowledge of most of the components of this network, but only a poor understanding of how they work together to create a precise and robust system that ensures that mitosis is triggered at the right time and in an orderly fashion. In this review, we will give an overview of the literature that describes the Cdk1 activation network and then address questions relating to the systems biology of this switch. How is the timing of the trigger controlled? How is mitosis insulated from interphase? What determines the sequence of events, following the initial trigger of Cdk1 activation? Which elements ensure robustness in the timing and execution of the switch? How has this system been adapted to the high levels of replication stress in cancer cells?

Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3–5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell’s progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation. 3 Main TextCdk1 phosphorylates over 1000 proteins 1,2 within the brief 20-30 minute window of mitotic entry, triggering centrosome separation and chromosome condensation in prophase, followed by nuclear envelope breakdown (NEBD) and mitotic spindle formation in prometaphase, and the alignment of bi-oriented sister chromatids at the metaphase plate 3 . Binding of a Cyclin partner is critical for allosteric activation of CDKs. Two families of mitotic Cyclins, termed A and B, work with Cdk1 to orchestrate mitotic entry in higher eukaryotes 4,5 . Despite the central importance of these proteins for cell cycle control, the functional specialisation of mammalian A and B-type Cyclins remains unclear. Following the depletion of maternal pools of early embryonic Cyclin A1 and B3, somatic mammalian cells express one A-type Cyclin, A2, and two B-type cyclins, B1 and B2. Genetic depletion in mice suggests an essential role for Cyclin A2 for development, but not for embryonic fibroblast proliferation 6 . Depletion of Human Cyclin A2 by siRNA delays mitotic entry, and this is further enhanced by co-depletion of Cyclin B1 7-9 . Likewise, work in mammalian cell extracts documented how Cyclin A synergises with Cyclin B to control the mitotic entry threshold at the level for Cdk1 activation 10 . A mechanism involving Plk1 activation has been suggested 11,12 , although an essential role of Plk1 in the G2/M transition remains contentious 13 . Likewise, work in mammalian cell extracts documented how Cyclin A synergises with Cyclin B to control the mitotic entry threshold at the level for Cdk1 activation 10 . A confounding factor in the genetic analysis of Cyclin A2 has been its dual role in S-phase and mitosis 14 , making it difficult to directly investigate G2 specific defects. Murine Cyclin B1 is essential for development 15 and critical for mitotic entry in early mouse embryos 16 . Conversely, mice lacking Cyclin B2 live healthily, without apparent defects 15 . These results stand in stark contrast to observations from experiments involving siRNA depletion of B-type Cyclins in human cancer cell lines that show surprisingly mild mitotic entry defects 9,1...