G2phase,Gap 2 phase,orGrowth 2 phase,is the third subphase ofinterphasein thecell cycledirectly precedingmitosis.It follows the successful completion ofS phase,during which the cell’sDNAisreplicated.G2phase ends with the onset ofprophase,the first phase of mitosis in which the cell’schromatincondenses intochromosomes.
G2phase is a period of rapid cell growth andprotein synthesisduring which the cell prepares itself for mitosis. Curiously, G2phase is not a necessary part of the cell cycle, as some cell types (particularly youngXenopusembryos[1]and somecancers[2]) proceed directly from DNA replication to mitosis. Though much is known about thegenetic networkwhich regulates G2 phase and subsequent entry into mitosis, there is still much to be discovered concerning its significance and regulation, particularly in regards to cancer. One hypothesis is that the growth in G2phase is regulated as a method of cell size control. Fission yeast (Schizosaccharomyces pombe) has been previously shown to employ such a mechanism, viaCdr2-mediated spatial regulation ofWee1activity.[3]Though Wee1 is a fairly conserved negative regulator of mitotic entry, no general mechanism of cell size control in G2 has yet been elucidated.
Biochemically, the end of G2phase occurs when a threshold level of activecyclin B1/CDK1complex, also known asMaturation promoting factor(MPF) has been reached.[4]The activity of this complex is tightly regulated during G2.In particular, the G2checkpoint arrests cells in G2in response to DNA damage through inhibitory regulation of CDK1.
Homologous recombinational repair
editDuring mitoticS phase,DNA replicationproduces two nearly identicalsister chromatids.DNA double-strand breaks that arise after replication has progressed or during the G2 phase can berepairedbefore cell division occurs (M-phase of thecell cycle). Thus, during the G2 phase, double-strand breaks in one sister chromatid may be repaired byhomologous recombinationalrepair using the other intact sister chromatid as template.[5]
End of G2/entry into mitosis
editThis sectionneeds additional citations forverification.(December 2023) |
Mitotic entry is determined by a threshold level of active cyclin-B1/CDK1 complex, also known as cyclin-B1/Cdc2 or thematuration promoting factor(MPF). Active cyclin-B1/CDK1 triggers irreversible actions in early mitosis, includingcentrosomeseparation,nuclear envelopebreakdown, andspindleassembly. In vertebrates, there are five cyclin Bisoforms(B1,B2,B3,B4,andB5), but the 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 inDrosophila).[6]Saccharomyces cerevisiaecontains six B-type cyclins (Clb1-6), with Clb2 being the most essential for function. In both vertebrates and S. cerevisiae, it is speculated that the presence of multiple B-type cyclins allows different cyclins to regulate different portions of the G2/M transition while also making the transitionrobustto perturbations.[7]
Subsequent discussions will focus on the spatial and temporal activation of cyclin B1/CDK in mammalian cells, but similar pathways are applicable in both other metazoans and in S. cerevisiae.
Cyclin B1 synthesis and degradation
editCyclin B1 levels are suppressed throughout G1 and S phases by theanaphase-promoting complex(APC), an E3 ubiquitin ligase which targets cyclin B1 for proteolysis. Transcription begins at the end of S phase after DNA replication, in response to phosphorylation of transcription factors such asNF-Y,FoxM1andB-Mybby upstream G1 and G1/S cyclin-CDK complexes.[8]
Regulation of cyclin-B1/CDK1 activity
editIncreased levels of cyclin B1 cause rising levels of cyclin B1-CDK1 complexes throughout G2, but the complex remains inactive prior to the G2/M transition due to inhibitory phosphorylation by the Wee1 and Myt1 kinases. Wee1 is localized primarily to the nucleus and acts on the Tyr15 site, while Myt1 is localized to the outer surface of the ER and acts predominantly on the Thr14 site.
The effects of Wee1 and Myt1 are counteracted by phosphatases in the cdc25 family, which remove the inhibitory phosphates on CDK1 and thus convert the cyclin B1-CDK1 complex to its fully activated form, MPF.
Active cyclinB1-CDK1 phosphorylates and modulates the activity of Wee1 and the Cdc25 isoforms A and C. Specifically, CDK1 phosphorylation inhibits Wee1 kinase activity, activates Cdc25Cphosphataseactivity via activating the intermediate kinasePLK1,and stabilizes Cdc25A. Thus, CDK1 forms apositive feedbackloop with Cdc25 and a doublenegative feedbackloop with Wee1 (essentially a net positive feedback loop).
Positive feedback and switch-like activation
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These positive feedback loops encode ahystereticbistableswitch in CDK1 activity relative to cyclin B1 levels (see figure). This switch is characterized by two distinct stable equilibria over a bistable region of cyclin B1 concentrations. One equilibrium corresponds to interphase and is characterized by inactivity of Cyclin-B1/CDK1 and Cdc25, and a high level of Wee1 and Myt1 activity. The other equilibrium corresponds to M-phase and is characterized by high activity of Cyclin-B1/CDK1 and Cdc25, and low Wee1 and Myt1 activity. Within the range of bistability, a cell’s state depends upon whether it was previously in interphase or M-phase: the threshold concentration for entering M-phase is higher than the minimum concentration that will sustain M-phase activity once a cell has already exited interphase.
Scientists have both theoretically and empirically validated the bistable nature of the G2/M transition. TheNovak-Tyson modelshows that the differential equations modelling the cyclin-B/CDK1-cdc25-Wee1-Myt1 feedback loop admit two stable equilibria over a range of cyclin-B concentrations.[9]Experimentally, bistability has been validated by blocking endogenous cyclin B1 synthesis and titrating interphase and M-phase cells with varying concentrations of non-degradable cyclin B1. These experiments show that the threshold concentration for entering M-phase is higher than the threshold for exiting M-phase: nuclear envelope break-down occurs between 32-40 nm cyclin-B1 for cells exiting interphase, while the nucleus remains disintegrated at concentrations above 16-24 nm in cells already in M-phase.[10]
This bistable, hysteretic switch is physiologically necessary for at least three reasons.[11]First, the G2/M transition signals the initiation of several events, such as chromosome condensation and nuclear envelope breakdown, that markedly change the morphology of the cell and are only viable in dividing cells. It is therefore essential that cyclin-B1/CDK1 activation occurs in a switch-like manner; that is, cells should rapidly settle into a discrete M-phase state after the transition, and should not persist in a continuum of intermediate states (e.g., with a partially decomposed nuclear envelope). This requirement is satisfied by the sharpdiscontinuityseparating the interphase and M-phase equilibrium levels of CDK1 activity; as the cyclin-B concentration increases beyond the activation threshold, the cell rapidly switches to the M-phase equilibrium.
Secondly, it is also vital that the G2/M transition occur unidirectionally, or only once per cell cycle Biological systems are inherentlynoisy,and small fluctuations in cyclin B1 concentrations near the threshold for the G2/M transition should not cause the cell to switch back and forth between interphase and M-phase states. This is ensured by the bistable nature of the switch: after the cell transitions to the M-phase state, small decreases in the concentration of cyclin B do not cause the cell to switch back to interphase.
Finally, the continuation of the cell cycle requires persisting oscillations in cyclin-B/CDK1 activity as the cell and its descendants transition in and out of M-phase. Negative feedback provides one essential element of this long-term oscillation: cyclin-B/CDK activates APC/C, which causes degradation of cyclin-B from metaphase onwards, restoring CDK1 to its inactive state. However, simple negative feedback loops lead todamped oscillationsthat eventually settle on a steady state. Kinetic models show that negative feedback loops coupled with bistable positive feedback motifs can lead to persistent, non-damped oscillations (seerelaxation oscillator) of the kind required for long-term cell cycling.
Positive feedback
editThe positive feedback loop mentioned above, in which cyclin-B1/CDK1 promotes its own activation by inhibiting Wee1 and Myst1 and activating cdc25, does not inherently include a “trigger” mechanism to initiate the feedback loop. Recently, evidence has emerged suggesting a more important role forcyclin A2/CDK complexes in regulating the initiation of this switch. Cyclin A2/CDK2activity begins in early S phase and increases during G2.Cdc25B has been shown to dephosphorylate Tyr15 on CDK2 in early-to-mid G2in a manner similar to the aforementioned CDK1 mechanism. Downregulation of cyclin A2 in U2OS cells delays cyclin-B1/CDK1 activation by increasing Wee1 activity and lowering 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 G2checkpoint activity, perhaps through a stabilizing phosphorylation onCdc6.CDK2-/- cells also have aberrantly high levels of Cdc25A. Cyclin A2/CDK1 has also been shown to mediate proteasomal destruction of Cdc25B. These pathways are often deregulated in cancer.[7]
Spatial regulation
editIn addition to the bistable and hysteretic aspects of cyclin B1-CDK1 activation, regulation of subcellular protein localization also contributes to the G2/M transition. Inactive cyclin B1-CDK1 accumulates in the cytoplasm, begins to be activated by cytoplasmic cdc25, and then is rapidly sequestered into the nucleus during prophase (as it is further activated). In mammals, cyclin B1/CDK1 translocation to thenucleusis activated by phosphorylation of fiveserinesites on cyclin B1's cytoplasmic retention site (CRS): S116, S26, S128, S133, and S147. InXenopus 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'snuclear export signal(NES). The regulators of these phosphorylation sites are still largely unknown but several factors have been identified, includingextracellular 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, includinghistone H1,nuclear lamins,centrosomal proteins,andmicrotubule associated proteins (MAPs).
The subcellular localization of cdc25 also shifts from the cytosol to the nucleus during prophase. This is accomplished via removal of nuclear localization sequence (NLS)-obscuring phosphates and phosphorylation of the nuclear export signal. It is thought that the simultaneous transport of cdc25 and cyclin-B1/CDK1 into the nucleus amplify the switch-like nature of the transition by increasing the effective concentrations of the proteins.[7]
G2/M DNA damage arrest
editCells respond toDNA damageor incompletely replicated chromosomes in G2 phase by delaying the G2/M transition so as to prevent attempts to segregate damaged chromosomes. DNA damage is detected by the kinasesATMandATR,which activateChk1,an inhibitory kinase of Cdc25. Chk1 inhibits Cdc25 activity both directly and by promoting its exclusion from the nucleus.[7]The net effect is an increase in the threshold of cyclin B1 required to initiate the hysteretic transition to M-phase, effectively stalling the cell in G2 until the damage is repaired by mechanisms such as homology-directed repair (see above).[4]
Long-term maintenance of the G2 arrest is also mediated byp53,which is stabilized in response to DNA damage. CDK1 is directly inhibited by three transcriptional targets of p53:p21,Gadd45,and14-3-3σ.Inactive Cyclin B1/CDK1 is sequestered in the nucleus by p21,[12]while active Cyclin B1/CDK1 complexes are sequestered in the cytoplasm by 14-3-3σ.[13]Gadd45 disrupts the binding of Cyclin B1 and CDK1 through direct interaction with CDK1. P53 also directly transcriptionally represses CDK1.[13][14]
Medical relevance
editMutations in several genes involved in the G2/M transition are implicated in many cancers. Overexpression of both cyclin B and CDK1, oftentimes downstream of loss oftumor suppressorssuch as p53, can cause an increase in cell proliferation.[7]Experimental approaches to mitigate these changes include both pharmacological inhibition of CDK1 and downregulation of cyclin B1 expression (e.g., viasiRNA).[15][16]
Other attempts to modulate the G2/M transition for chemotherapy applications have focused on the DNA damage checkpoint. Pharmacologically bypassing the G2/M checkpoint via inhibition of Chk1 has been shown to enhance cytotoxicity of other chemotherapy drugs. Bypassing the checkpoint leads to the rapid accumulation of deleterious mutations, which is thought to drive the cancerous cells intoapoptosis.Conversely, attempts to prolong the G2/M arrest have also been shown to enhance the cytotoxicity of drugs likedoxorubicin.These approaches remain in clinical and pre-clinical phases of research.[17]
References
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- ^Liskay RM (April 1977)."Absence of a measurable G2 phase in two Chinese hamster cell lines".Proceedings of the National Academy of Sciences of the United States of America.74(4): 1622–5.Bibcode:1977PNAS...74.1622L.doi:10.1073/pnas.74.4.1622.PMC430843.PMID266201.)
- ^Moseley JB, Mayeux A, Paoletti A, Nurse P (June 2009). "A spatial gradient coordinates cell size and mitotic entry in fission yeast".Nature.459(7248): 857–60.Bibcode:2009Natur.459..857M.doi:10.1038/nature08074.PMID19474789.S2CID4330336.
- ^abSha W, Moore J, Chen K, Lassaletta AD, Yi CS,Tyson JJ,Sible JC (February 2003)."Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts".Proceedings of the National Academy of Sciences of the United States of America.100(3): 975–80.Bibcode:2003PNAS..100..975S.doi:10.1073/pnas.0235349100.PMC298711.PMID12509509.
- ^Burgoyne PS, Mahadevaiah SK, Turner JM (October 2007). "The management of DNA double-strand breaks in mitotic G2, and in mammalian meiosis viewed from a mitotic G2 perspective".BioEssays.29(10): 974–86.doi:10.1002/bies.20639.PMID17876782.S2CID36778078.
- ^Porter LA, Donoghue DJ (2003). "Cyclin B1 and CDK1: nuclear localization and upstream regulators".Progress in Cell Cycle Research.5:335–47.PMID14593728.
- ^abcdeMorgan, David Owen, 1958- (2007).The cell cycle: principles of control.New Science Press.ISBN978-0-19-920610-0.OCLC70173205.
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- ^Novak B, Tyson JJ (December 1993). "Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos".Journal of Cell Science.106(4): 1153–68.doi:10.1242/jcs.106.4.1153.PMID8126097.
- ^Sha W, Moore J, Chen K, Lassaletta AD, Yi CS, Tyson JJ, Sible JC (February 2003)."Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts".Proceedings of the National Academy of Sciences of the United States of America.100(3): 975–80.Bibcode:2003PNAS..100..975S.doi:10.1073/pnas.0235349100.PMC298711.PMID12509509.
- ^Pomerening JR, Sontag ED, Ferrell JE (April 2003). "Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2".Nature Cell Biology.5(4): 346–51.doi:10.1038/ncb954.PMID12629549.S2CID11047458.
- ^Charrier-Savournin FB, Château MT, Gire V, Sedivy J, Piette J, Dulic V (September 2004)."p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress".Molecular Biology of the Cell.15(9): 3965–76.doi:10.1091/mbc.E03-12-0871.PMC515331.PMID15181148.
- ^abTaylor WR, Stark GR (April 2001). "Regulation of the G2/M transition by p53".Oncogene.20(15): 1803–15.doi:10.1038/sj.onc.1204252.PMID11313928.S2CID9543421.
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- ^Asghar U, Witkiewicz AK, Turner NC, Knudsen ES (February 2015)."The history and future of targeting cyclin-dependent kinases in cancer therapy".Nature Reviews. Drug Discovery.14(2): 130–46.doi:10.1038/nrd4504.PMC4480421.PMID25633797.
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