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= G1/S Phase Transition State =

The G1/S checkpoint was first characterized in 1971, and is the scientific term used to describe the mechanisms within the cell that allow it to determine if conditions are appropriate to proceed to DNA synthesis. In the presence of growth hormones, the various inhibitory pathways that prevent synthesis will be inhibited, allowing synthesis to proceed. In the absence of growth hormone, or when DNA damage is detected, the inhibitory elements of the checkpoint such as the CDK inhibitors become active, preventing the cell from proceeding to synthesis.

History/Discovery
One of the first papers that discussed the possibility of a restriction point was published in 1971. In this paper, Temin showed that certain populations of chicken cells required incubation with serum in order to proceed through the cell cycle, while other populations of cells entered S phase without exposure to serum. This observation was linked to the existence of a point in the cell cycle at which cells were committed to enter mitosis despite the external environment. Expanding on this research, the restriction point was first identified as the “R-point” in 1974. It was proposed that the “R-point” is the section of the G1 phase in many different species when the switch to an active growth phase occurs. It was suggested that progression past this point is dependent on sufficient build up of specific proteins in the cell.

Mechanism
A brief visual depiction of the proteins involved in the G1/S checkpoint can be found here. []

Initiation
G1 phase progression is influenced by growth factors, however, after the interaction of RB and cyclin/CDK complexes occurs, the cell loses its dependence on growth factors.

In the presence of the appropriate growth factors the AKT pathway is triggered. When the AKT pathway is active, it inhibits the family of forkhead transcription factors (FoxO). FoxO has antiproliferative and pro-apoptotic effects. FoxO induces transcription of genes involved in cell cycle arrest, such as cyclin-dependent kinase inhibitors (CKIs), p21, p27KIP1 and p15INK4B. FOXO is also able to repress transcription of cyclin D. In the event that protein kinase B phosphorylates FoxO, FoxO translocates to the cytoplasm which prevents transcription of its target genes. FoxO then promotes cell survival and proliferation. By inhibiting the activity of FoxO the AKT pathway allows cell proliferation.

Regulation in response to damage
In the presence of TGF-ß, Smad3 is activated which suppresses cell progress before the G1/S phase checkpoint. In the event, where DNA damage is detected, the ATM/ATR pathway induces a rapid cascade. Depending on the type of DNA damage, ATM phosphorylates Chk2 kinases, while ATR phosphorylates Chk1 kinases. The main portion of this cascade is when Chk2/Chk1 triggers phosphorylation of the Cdc25A phosphatase, which causes Cdc25A ubquitination then demolition by the proteasome. Also from DNA damage, the ATM/ATR pathway activation can cause the phosphorylation of p53. Phosphorylation stabilizes the p53 protein in the cell nucleus, which causes induction of the p21 CDK inhibitor. p21 is able to bind to cyclin E-CDK2, which inhibits the S phase promoter allowing for a G1 arrest. The inhibition of CDK2 also allows for dephosphorylation of Rb and thus inhibition of the E2F.

Transduction
The CDK inhibitors are a major class of proteins involved in regulating the transition of the G1/S checkpoint. They are typically the effectors activated in response to damage. They have become increasingly well characterized having been tested in various biochemical assays, and recent attempts have been made to create a mathematical model of the mechanisms involved in the regulation of the restriction point. In the absence of growth factor, FoxO1/3 have been demonstrated to primarily activate three proteins: p15INK4b, p27KIP1, and p21CIP1. When activated by FoxO1/3, p27, p21, and p15 inhibit cyclin-dependent kinase 2 and Cdk4/6 activity.

In the presence of activated AKT, the transcription of p15, p27 and p21 is down regulated and existing proteins are slowly deactivated, modulating the formation of CDK4/cyclin D complexes and CDK2/cyclin E complexes. Recent literature on the G1/S checkpoint suggests that CDK complex formation and activity may be modulated by a variety of other CDK inhibitors that are not controlled by FoxO, including p16, p18, p19 and p57. These FoxO independent CDK inhibitors are regulated by extracellular signals such as hormones, or by intracellular signals such as those indicative of DNA damage. The various inhibitors are typically divided into two categories: the INK/ARF CDK inhibitors which target CDK4 and 6, and the KIP/CIP CDK inhibitors which mainly target CDK2 and promote the formation of the CDK4 cyclin D complex. Another important protein involved in the regulation of CDKs is Cdc25A, which has been found to activate CDK in response to the Myc triggered activation of Stat3.

Response
For the cell to progress from G phase to S phase, it is regulated by cyclins and CDKs. Cyclin D will interact with CDK4 and CDK6. In the nucleus CDK4 and cyclin D requires CKIs to form the cyclin D/CDK4 complex. p21 has a separate binding site for cyclin D and CDK and it has been found that the binding site will promote cyclin D and CDK4 to form into a complex. Along with promoting the assembly of cyclin D/CDK4, p21 is involved in kinase acitiviy in the RB pathway. The cyclin D/CDK4 complex becomes active when phosphorylated by CDK-activating kinase, CAK. Cyclin D/CDK4/6 will phosphorylate pocket protein pRB. When pRB is phosphorylated, it unbinds to E2F, which frees E2F. E2F is a transcription factor, which controls genes that allow the cell to proceed to S phase. Some of these genes are cyclin E and CDK2. Cyclin E and CDK2 appears near the end of G1 phase. It is essential for the cell to move into S phase. Cyclin E has promoter region where E2F is able to bind to transcribe cyclin E. Cyclin E is the activator of CDK2. Unlike cyclin D where the main objective is to phosphorylate Rb, cyclin E/CDK2 phosphorylate more than one substrate that is important to move into S phase. This is indicated by experiments where the Cyclin E/CDK2 are rate limiting in cells with Rb pathway and without Rb pathway. Some of the other substrates that cyclin E and CDK2 phosphorylate are H1 histone and CDK inhibitor p27KIP1. Cyclin E/CDK2 reinforces CDK4 to phosphorylate Rb and is involved with the destruction of p27KIP1.

In addition to phosphorylation, CDK4 and CDK6 are found to inactivate the CDK2 inhibitors CIP/KIP of p21 and p27 by sequestering these CKIs. Cyclin E depends on cyclin D and CDK4/6 sequestration of CIP/KIP. CDK2 will become active which then reinforce CDK4 activating the pRB protein. CDK2 also triggers the destruction of p27kip1.

Role
The G1/S checkpoint is crucial for controlling the cell cycle, and ensuring division of cells occur only when the cell is prepared for synthesis, with no damage or mutations, and in the presence of the appropriate extracellular signals. Because of its role in regulating cell proliferation, mutations to various proteins are oncogenes, and mutations can result in various human diseases including cancer.

Human Disease
One of the main features of cancer is uncontrolled cell growth and the division of cells without the presence of proper growth factors. These features are connected to disruptions in control over the cell cycle including problems with the G1/S phase transition state. The connection between cancer and disruptions in the G1/S phase transition state was first discussed in the literature in 1974 in reference to gene mutations. Disruptions in the form of gene mutations involved in the transition state are the most common cell cycle change that occurs with human cancers. Specifically, mutations in the p53 tumor suppressor gene are the most common change that is observed. The next most frequent mutation involves the p16 gene which inhibits CDK 4/6. Additional evidence of the role of disruptions of the G1/S phase transition phase in cancer occurrence are that DNA tumor virus oncoproteins have a role in preventing the restriction point from occurring. If for example Ras is mutated, then S phase can be activated without growth factors being present as Ras can mimic the signals normally sent by these factors. The inactivation of p53 and RB tumor suppressor proteins eventually occur with these mutations meaning the cell can transition through the G1 checkpoint to the S phase.

Current Research
A potential conflict in the literature occurs over whether the “restriction point” and G1/S transition state are always referring to the same actual point in the cell cycle. It has been suggested that there are actually two separate points that are commonly referred to as the restriction point. One of these points is growth factor dependent while the other step is nutritionally dependent. The nutritionally dependent step is observed to occur later in the G1 phase than the growth factor dependent step. This second nutritionally dependent stage is thought to be genetically similar to the START step in the  yeast cell cycle. Divisions have been drawn between these two checkpoints in terms of the CDKs and cyclins that are involved. Cyclin D is linked to the regulation of the true restriction point that is growth factor dependent. In contrast, cyclin E is linked to the regulation of the later occurring nutrient dependent stage.