Cyclin A triggers Mitosis either via Greatwall or Cyclin B

Abstract

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...

Triggering mitosis

“…Moreover, even codepletion of cyclins B1 and B2 is not sufficient to block mitotic entry in HeLa and RPE‐1 cells, suggesting that cyclin A can compensate to some extent for the mitotic entry function of cyclin B . We have recently revisited this question using degron tags, a more efficient and precise depletion approach compared to siRNA . This study supports the idea that cyclin A activity in G2 phase is essential to trigger mitosis.…”

“…Accordingly, cyclin B is known to be recruited to the kinetochore and has recently been shown to contribute to spindle assembly checkpoint signalling by phosphorylating Mps1 . A large subset of the cyclin B‐specific substrates that we have identified using degron‐mediated depletion is indeed associated with the centromere/kinetochore, supporting the importance of this targeted localisation of cyclin B . Nevertheless, we also identified cyclin B‐specific substrates that are distributed all over the chromosomes, such as Ki67, and proteins at the cell cortex.…”

“…A body of evidence suggests that Cdk1 in complex with cyclin A constitutes the trigger of mitotic entry . Our recent work using degron tags provided further evidence that this is, indeed, a G2‐specific function of cyclin A . The action of this trigger is twofold: Firstly, it sets in motion the feedback loops that lead to ultrasensitive and bistable Cdk1 Try15 dephosphorylation by Cdc25 activation and Wee1 inhibition , and secondly, it triggers inactivation of PP2A:B55 via activation of Greatwall and phosphorylation of Ensa/ARPP19 that also follows the dynamic of a bistable system .…”