G2 Phase
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G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of
interphase Interphase is the portion of the cell cycle that is not accompanied by visible changes under the microscope, and includes the G1, S and G2 phases. During interphase, the cell grows (G1), replicates its DNA (S) and prepares for mitosis (G2). A c ...
in the
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
directly preceding
mitosis In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is mainta ...
. It follows the successful completion of
S phase S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during ...
, during which the cell’s DNA is replicated. G2 phase ends with the onset of
prophase Prophase () is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin ret ...
, the first phase of mitosis in which the cell’s
chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
condenses into
chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
s. G2 phase is a period of rapid cell growth and
protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside Cell (biology), cells, homeostasis, balancing the loss of cellular proteins (via Proteolysis, degradation or Protein targeting, export) through the product ...
during which the cell prepares itself for mitosis. Curiously, G2 phase is not a necessary part of the cell cycle, as some cell types (particularly young ''
Xenopus ''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-known ...
'' embryos and some
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
s)) proceed directly from DNA replication to mitosis. Though much is known about the genetic network which 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 G2 phase is regulated as a method of cell size control. Fission yeast (''
Schizosaccharomyces pombe ''Schizosaccharomyces pombe'', also called "fission yeast", is a species of yeast used in traditional brewing and as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Cells typically meas ...
'') has been previously shown to employ such a mechanism, via Cdr2-mediated spatial regulation of
Wee1 Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast ''Schizosaccharomyces pombe'' (''S. pombe'')Wee1has a molecular mass of 96 kDa and is a key regulator of cell cycle progression. It influences ...
activity. 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 G2 phase occurs when a threshold level of active
cyclin B1 G2/mitotic-specific cyclin-B1 is a protein that in humans is encoded by the ''CCNB1'' gene. Function Cyclin B1 is a regulatory protein involved in mitosis. The gene product complexes with p34 (Cdk1) to form the maturation-promoting factor (MPF) ...
/
CDK1 Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine protein kinase, and is a key player in cell cycle regulation. It has been highly studied in t ...
complex, also known as
Maturation promoting factor Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor) is the cyclin-Cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF ...
(MPF) has been reached. The activity of this complex is tightly regulated during G2. In particular, the G2 checkpoint arrests cells in G2 in response to DNA damage through inhibitory regulation of CDK1.


Homologous recombinational repair

During mitotic
S phase S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during ...
,
DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
produces two nearly identical
sister chromatids A sister chromatid refers to the identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together by a common centromere. In other words, a sister chromatid may also be said to be 'one-half' of the dup ...
. DNA double-strand breaks that arise after replication has progressed or during the G2 phase can be repaired before cell division occurs (M-phase of the
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
). Thus, during the G2 phase, double-strand breaks in one sister chromatid may be repaired by
homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
al repair using the other intact sister chromatid as template.


End of G2/entry into mitosis

Mitotic entry is determined by a threshold level of active cyclin-B1/CDK1 complex, also known as cyclin-B1/Cdc2 or the
maturation promoting factor Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor) is the cyclin-Cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF ...
(MPF). Active cyclin-B1/CDK1 triggers irreversible actions in early mitosis, including
centrosome In cell biology, the centrosome (Latin centrum 'center' + Greek sōma 'body') (archaically cytocentre) is an organelle that serves as the main microtubule organizing center (MTOC) of the animal cell, as well as a regulator of cell-cycle prog ...
separation, nuclear envelope breakdown, and
spindle Spindle may refer to: Textiles and manufacturing * Spindle (textiles), a straight spike to spin fibers into yarn * Spindle (tool), a rotating axis of a machine tool Biology * Common spindle and other species of shrubs and trees in genus ''Euony ...
assembly. In vertebrates, there are five cyclin B
isoform A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isof ...
s ( B1, B2, B3, B4, and B5), 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 in ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
''). ''Saccharomyces cerevisiae'' contains 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 transition
robust Robustness is the property of being strong and healthy in constitution. When it is transposed into a system, it refers to the ability of tolerating perturbations that might affect the system’s functional body. In the same line ''robustness'' ca ...
to perturbations. 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

Cyclin B1 levels are suppressed throughout G1 and S phases by the
anaphase-promoting complex Anaphase-promoting complex (also called the cyclosome or APC/C) is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cull ...
(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 as NF-Y,
FoxM1 Forkhead box protein M1 is a protein that in humans is encoded by the ''FOXM1'' gene. The protein encoded by this gene is a member of the FOX family of transcription factors. Its potential as a target for future cancer treatments led to it being ...
and B-Myb by upstream G1 and G1/S cyclin-CDK complexes.


Regulation of cyclin-B1/CDK1 activity

Increased 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. Wee1is 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 Cdc25C
phosphatase In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid Ester, monoester into a phosphate ion and an Alcohol (chemistry), alcohol. Because a phosphatase enzyme catalysis, catalyzes the hydrolysis of its Substrate ...
activity via activating the intermediate kinase
PLK1 Serine/threonine-protein kinase PLK1, also known as polo-like kinase 1 (PLK-1) or serine/threonine-protein kinase 13 (STPK13), is an enzyme that in humans is encoded by the ''PLK1'' ( polo-like kinase 1) gene. Structure PLK1 consists of 603 am ...
, and stabilizes Cdc25A. Thus, CDK1 forms a
positive feedback Positive feedback (exacerbating feedback, self-reinforcing feedback) is a process that occurs in a feedback loop which exacerbates the effects of a small disturbance. That is, the effects of a perturbation on a system include an increase in the ...
loop with Cdc25 and a double negative feedback loop with Wee1 (essentially a net positive feedback loop).


Positive feedback and switch-like activation

These positive feedback loops encode a
hysteretic Hysteresis is the dependence of the state of a system on its history. For example, a magnet may have more than one possible magnetic moment in a given magnetic field, depending on how the field changed in the past. Plots of a single component of ...
bistable switch 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. The Novak-Tyson model shows 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. 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. This bistable, hysteretic switch is physiologically necessary for at least three reasons. 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 sharp discontinuity separating 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 inherently noisy, 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 to damped oscillations that 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 (see
relaxation oscillator In electronics a relaxation oscillator is a nonlinear electronic oscillator circuit that produces a nonsinusoidal repetitive output signal, such as a triangle wave or square wave. on Peter Millet'Tubebookswebsite The circuit consists of a feedba ...
) of the kind required for long-term cell cycling.


Positive feedback

The 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 for
cyclin A2 Cyclin-A2 is a protein that in humans is encoded by the ''CCNA2'' gene. It is one of the two types of cyclin A: cyclin A1 is expressed during meiosis and embryogenesis while cyclin A2 is expressed in dividing somatic cells. Function Cyclin A2 ...
/CDK complexes in regulating the initiation of this switch. Cyclin A2/
CDK2 Cyclin-dependent kinase 2, also known as cell division protein kinase 2, or Cdk2, is an enzyme that in humans is encoded by the ''CDK2'' gene. The protein encoded by this gene is a member of the cyclin-dependent kinase family of Ser/Thr protein ...
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 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 G2 checkpoint activity, perhaps through a stabilizing phosphorylation on
Cdc6 Cell division control protein 6 homolog is a protein that in humans is encoded by the ''CDC6'' gene. The protein encoded by this gene is highly similar to Saccharomyces cerevisiae Cdc6, a protein essential for the initiation of DNA replication. ...
. 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.


Spatial regulation

In 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 the
nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom * Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucl ...
is activated by phosphorylation of five
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
sites on cyclin B1's cytoplasmic retention site (CRS): S116, S26, S128, S133, and S147. In ''
Xenopus laevis The African clawed frog (''Xenopus laevis'', also known as the xenopus, African clawed toad, African claw-toed frog or the ''platanna'') is a species of African aquatic frog of the family Pipidae. Its name is derived from the three short claws ...
'', 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 A nuclear export signal (NES) is a short target peptide containing 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. It has the opposite ...
(NES). The regulators of these phosphorylation sites are still largely unknown but several factors have been identified, including
extracellular signal-regulated kinases In molecular biology, extracellular signal-regulated kinases (ERKs) or classical MAP kinases are widely expressed protein kinase intracellular signalling molecules that are involved in functions including the regulation of meiosis, mitosis, and p ...
(ERKs),
PLK1 Serine/threonine-protein kinase PLK1, also known as polo-like kinase 1 (PLK-1) or serine/threonine-protein kinase 13 (STPK13), is an enzyme that in humans is encoded by the ''PLK1'' ( polo-like kinase 1) gene. Structure PLK1 consists of 603 am ...
, 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 Histone H1 is one of the five main histone protein families which are components of chromatin in eukaryotic cells. Though highly conserved, it is nevertheless the most variable histone in sequence across species. Structure Metazoan H1 prote ...
, nuclear lamins, centrosomal proteins, and microtubule 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.


G2/M DNA damage arrest

Cells respond to
DNA damage DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...
or 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 kinases ATM and
ATR ATR may refer to: Medicine * Acute transfusion reaction * Ataxia telangiectasia and Rad3 related, a protein involved in DNA damage repair Science and mathematics * Advanced Test Reactor, nuclear research reactor at the Idaho National Laboratory, ...
, which activate
Chk1 Checkpoint kinase 1, commonly referred to as Chk1, is a serine/threonine-specific protein kinase that, in humans, is encoded by the ''CHEK1'' gene. Chk1 coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chk ...
, an inhibitory kinase of Cdc25. Chk1 inhibits Cdc25 activity both directly and by promoting its exclusion from the nucleus. 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). Long-term maintenance of the G2 arrest is also mediated by
p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...
, which is stabilized in response to DNA damage. CDK1 is directly inhibited by three transcriptional targets of p53:
p21 p21Cip1 (alternatively p21Waf1), also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a cyclin-dependent kinase inhibitor (CKI) that is capable of inhibiting all cyclin/CDK complexes, though is primarily associated ...
, Gadd45, and 14-3-3σ. Inactive Cyclin B1/CDK1 is sequestered in the nucleus by p21, while active Cyclin B1/CDK1 complexes are sequestered in the cytoplasm by 14-3-3σ. Gadd45 disrupts the binding of Cyclin B1 and CDK1 through direct interaction with CDK1. P53 also directly transcriptionally represses CDK1.


Medical relevance

Mutations 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 of tumor suppressors such as p53, can cause an increase in cell proliferation. Experimental approaches to mitigate these changes include both pharmacological inhibition of CDK1 and downregulation of cyclin B1 expression (e.g., via
siRNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating ...
). 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 into
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
. Conversely, attempts to prolong the G2/M arrest have also been shown to enhance the cytotoxicity of drugs like
doxorubicin Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used toge ...
. These approaches remain in clinical and pre-clinical phases of research.


References

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