G₂ Phase - End of G₂/Entry Into Mitosis

End of G₂/Entry Into Mitosis

See also: maturation promoting factor, biochemical switches in the cell cycle

Mitotic entry is determined by a threshold level of active cyclin B1/CDK1 complex. In vertebrates, there are five cyclin B isoforms (B1, B2, B3, B4, and B5), but specific role of each of these isoforms in regulating mitotic entry is still unclear. It is known that cyclin B1 can compensate for loss of both cyclin B2 (and vice versa in Drosophila). Cyclin B1/CDK1 activity is regulated both spatially and temporally during G₂ phase to ensure proper entry into mitosis.

Cyclin B1 transcription begins at the end of S phase after DNA replication. Its promoter contains consensus binding sequences for a number of transcription factors, including p53, p21, Ets, Ap-1, NF-Y, c-Myc, TFE3, and USF. Cyclin B1 accumulates in the cytoplasm throughout G₂, where it binds to and activates CDK1’s kinase activity. CDK1 activity is modulated primarily through regulation of its inhibitory phosphorylation sites at Thr14 and Tyr15. Wee1 and Myt1 phosphorylate these two residues, with Wee1 acting on the Tyr15 site and Myt1 acting predominantly on the Thr14 site. However, Myt1 has a separate inhibitory effect on CDK1; it can also sequester CDK1 in the cytoplasm via interaction with Myt1’s C-terminal domain. CDK1 is dephosphorylated primarily through the actions of Cdc25, which can dephosphorylate both the Thr14 and Tyr15 residues of CDK1. There are three isoforms of Cdc25 (A, B, and C) in mammalian cells, all of which have been shown to have roles in regulation of G₂ phase.

CDK1, in turn, phosphorylates and modulates the activity of Wee1 and the Cdc25 isoforms A and C. Specifically, CDK1 phosphorylation inhibits Wee1 kinase activity, activates Cdc25C phosphatase activity, and stabilizes Cdc25A. Thus, CDK1 forms a positive feedback loop with Cdc25 and a double negative feedback loop with Wee1 (essentially a net positive feedback loop). These loops encode a hysteretic bistable switch in CDK1 activity relative to Cyclin B1 levels. It is thought that this hysteretic behavior ensures that cells commit to mitosis even if cyclin B1 levels falter.

In mammals, cyclin B1/CDK1 translocation to the nucleus is activated by phosphorylation of five serine sites on cyclin B1’s cytoplasmic retention site (CRS): S116, S26, S128, S133, and S147. In Xenopus laevis, Cyclin B1 contains four analogous CRS serine phosphorylation sites (S94, S96, S101, and S113) indicating that this mechanism is highly conserved. Nuclear export is also inactivated by phosphorylation of Cyclin B1’s Nuclear export signal (NES). The regulators of these phosphorylation sites are still largely unknown but several factors have been identified, including extracellular signal-regulated kinases (ERKs), PLK1, and CDK1 itself. Upon reaching some threshold level of phosphorylation, translocation of cyclin B1/CDK1 to the nucleus is extremely rapid. Once in the nucleus, Cyclin B1/CDK1 phosphorylates many targets in preparation for mitosis, including Histone H1, nuclear lamins, centrosomal proteins, and Microtubule Associated Proteins (MAPs).

Recently, evidence has emerged suggesting a more important role for cyclin A2/CDK complexes in regulating entry into mitosis. Cyclin A2/CDK2 activity begins in early S phase and increases during G₂. Cdc25B has been shown to dephosphorylate Tyr15 on CDK2 in early-to-mid G₂ in a manner similar to the aforementioned CDK1 mechanism. Downregulation of cyclin A2 in U2OS cells increases Wee1 activity and lowers Plk1 and Cdc25C activity. However, cyclin A2/CDK complexes do not function strictly as activators of cyclin B1/CDK1 in G₂, as CDK2 has been shown to be required for activation of the p53-independent G₂ checkpoint activity, perhaps through a stabilizing phosphorylation on Cdc6. CDK2-/- cells also have aberrantly high levels of Cdc25A. Cyclin A2/CDK1 has also been shown to mediate proteosomal destruction of Cdc25B. These pathways are often deregulated in cancer.