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Wee1 kinase is a protein that aids in regulating eukaryotic cell divisions (during meiosis II and mitosis) by adequately inhibiting entry into the mitotic phase via the inactivation of Cdk1.(1) Along with the growth and activities occurring within the cell during the cell cycle,the chromosomes of a cell are duplicated to consist of two chromatids each. These reparations should be completed and checked for errors to continue into the mitotic phase(2) – hence, Wee1 is utilized to steady the cell until deemed ready to go forth and divide. Then the Wee1 activity renders control to post-translational modification mechanisms of phosphorylation and ubiquitination that down-regulates/ degrades Wee1.(6)

When the Wee1 gene within a cell expresses a surplus of this kinase protein, the cell is delayed within the Growth 2 Phase and results in the cell becoming abnormally large. Comparatively, when the cell has an insufficient expression of Wee1, the transition occurs pre-maturely with the emerging cell being relatively small with a higher chance of malfunction. (4) Therefore, it is observed that the level of expression can be related to health issues (For example, tumour development has been linked to over-expression and DNA damage, as in aging, has been linked to under-expression of Wee1) Treatment for cancer often involves administration of small-molecule inhibitors that interacts with Wee1; ultimately interfering with the function of Wee1 to induce apoptosis in the malignant cells.(5) There are two known homologues of the WEE1 gene in humans (WEE1A and WEE1B) and they code for WEE1A kinase and Wee1B kinase, respectively. Wee1A is discussed in depth in the next section but it is worth noting that the main difference involves WEE1B only being expressed in oocytes for modulating meiotic arrest. The exact structure is currently (2017) still unknown. (3)

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The WEE 1 gene can be located on chromosome 11 and includes 19 809 bases. Containing 1 protein kinase domain, the gene encodes for a nuclear protein (A tyrosine Kinase.) The protein expressed by the WEE1A homologue catalyzes the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase and appears to coordinate the transition between DNA replication and mitosis by protecting the nucleus from cytoplasmically activated CDC2 kinase. It also acts as a negative regulator of entry into mitosis (G2 to M transition) by protecting the nucleus from cytoplasmically activated cyclin B1-complexed CDK1 before the onset of mitosis and, in turn, does this by mediating phosphorylation of CDK1 on Tyr-15. It is known to specifically phosphorylate and inactivate cyclin B1-complexed CDK1 - reaching a maximum during G2 phase and a minimum as cells enter M phase. Phosphorylation of cyclin B1-CDK1 occurs, exclusively on Tyr-15, and phosphorylation of monomeric CDK1 does not occur. Its activity increases during S and G2 phases and then decreases at M phase as it is hyper phosphorylated. A correlated decrease in protein level occurs at M/G1 phase; probably due to its degradation.

Wee1 Structure
Wee1A protein kinase consists of 646 amino acids. Its secondary structure contains 14 alpha helices and 9 beta strands[1].

Wee1 Post-Translational Modifications
Wee1A activity is regulated through post-translational modification, more specifically phosphorylation and ubiquitination. Phosphorylation downregulates Wee1A protein kinase function. Phosphorylation at Thr-239 is the strongest regulator of Wee1A kinase activity[6]. Phosphorylation signals the βTrCP-complex which ubiquinates and degrades Wee1A protein kinase. The following table shows the sites of phosphorylation and the enzyme that performs the action: When dephosphorylation occurs Wee1A kinase activity is upregulated again. Dephosphorylation of Thr-239 is mediated by CTDP1[1]. The following diagram shows the primary structure of Wee1A with is Most important sites of Post-transtlational modification[6].

The wee box contains Thr-239. The Cyclin A/CDK2 complex binds to the RxL1 site and downregulates Wee1A action by preventing Crm1 binding and sequential export of Wee1A out of the nucleus. Crm1 uses the RxL1 site as its binding site[6]. Wee1A protein kinase also takes part in post-translational modification of other structures, specifically through phosphorylating them. It downregulates the function of CDK1 by phosphorylating its Tyrosine residue at position 15. This prevents entry into the mitotic phase of cell division[7].

The Essence - Within Cell:
The cell cycle is an extraordinary but convoluted process. Consequently, eukaryotic cells have evolved regulatory mechanisms to minimize complications - such as the G2 checkpoint within which Wee1 negatively regulates the G2/M transition. (7) Wee1 is one of two inhibitory kinases that enable the restraint of a cell within the second growth phase. This is a precautionary step of delay. It allows for a pause within which to detect and repair cell damage/ irregularities before transitioning into the mitotic phase (as errors cannot be reversed once the sister-chromatids have separated in the cell cycle.) The third kinase contributing to the control in delay of the G2/M transition is a stimulatory kinase which counteracts the inhibitory actions. This combination of active and negative regulation results in another function: the release into the mitotic phase oozes with energy for a rapid launch into the actively dividing M phase. (1)

The Context -Within regulating system:
The network of kinases described above steer the activation of Cdk1-cyclin B1. Cdk1, is key for the commencement of mitosis, with versatile regulatory functions and a range of targets throughout the cell cycle (as its presence remains fairly consistent.) For its specific role in G2/M transition, it forms the complex with Cyclin B1 and is shuttled into the nucleus (by Importin b) and then swiftly transported back into the cytoplasm (by Crm1.) The stimulatory kinase mentioned above, CAK, waits in the nucleus to phosphorylates Cdk1 on T 161 and induce a conformational change in the protein’s tertiary structure – exposing active binding sites for substrates.

Wee1 is also located in the nucleus (as it is a nuclear protein) but it catalyzes an inhibitory phosphorylation reaction in Cdk1 on Y 15. , alongside the exposed binding site – ensuring the complex is inactive and thereby forming a non-competitive antagonistic relationship within the regulating system.. Myt1 (associated with the Golgi apparatus and ER) ensures that the complex remains inactive when it is flung back into the cytoplasm; creating the tension needed for the energetic initiation of the M phase. (1)

The focus: As Wee1 Kinase:
Wee1 kinase phosphorylates a specific Tyrosine amino acid on the Cdk1-cyclin B1 complex (Y 15 on the Cdk1 component) to maintain the complex in an inactive state and inhibit the initiation of the G2/M transition. (7) Thereby, it contributes to a necessary delay at the G2 checkpoint in the cell cycle for inspection and repairs to cells before the division of sister-chromatids (either in meiosis II or mitosis). (1) Wee1 activity is found not to be increased by unreplicated DNA (such as when the Cell cycle is halted before synthesis can take place in S-phase) but is found to be heavily repressed as soon as the M phase commences. This lead to the hypothesis that the activation of the Cdk1-cyclin B1 complex is due to a negative regulatory mechanism acting on Wee1 - causing the sudden decrease in levels at the G2/M transition point in the cell cycle after its highest peak in the G2 phase. Thus far, studies have concurred and indicate that Wee1 is reactivated during the performance of tests that exclude protein phosphatase inhibitors. (7,8) and prevents its hyperphosphorylation at M phase initiation. An accumulation of Scientific research, such as in the above elaboration, has revealed that (under normal cell circumstances) a fairly predictable pattern of Wee1 Kinase activity with supporting arguments still undergoing scrutinization. During the M and G1 phases, the Wee1 kinase levels decrease in relation to the protein’s degradation. This plateaus and, as transcription presumably increases, the gradient rises again in association with the elevation of Wee1 quantity and activity during the progression of S phase and G2 phase. Here, it peaks and then there is a sudden drop in Wee1 levels and the downwards inclination continues with degradation until the cycle repeats itself. (8)

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Wee1 kinase is a significant regulator of the G2/M checkpoint. This has implications on the cell cycle, such as adding a negative phosphorylation on CDK1 (Tyr15) according to Magnussen et al.¹, which will inactivate the CDK1/cyclin B complex and halt the cell cycle. Such unscheduled tyrosine phosphorylation can play a role in disease – for example cancer².

The degree of expression of Wee1 kinases, can vary in several types of tumours, despite being “potential therapeutic targets”². A hyper-expression of Wee1 is seen in osteosarcoma, glioblastomas and breast cancers. Up-regulation of Wee1 is associated with ulceration, thicker tumours and reduced relapse-free survival. Hypo-expressions, however, are seen in ‘non-small-cell lung cancer’ and are associated with a higher repetition rate. According to these results, in the research done by Hunter T.², the high levels of Wee1 that is observed, protect the cells against DNA damage and cell death. In cells where double-stranded DNA damage occurred, it was seen that Wee1 was absent, and that may have been the cause of the damage.

Wee1 mutants can also have a major effect on other diseases. According to Anda S. et al.³, “Wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage.” DNA damage will contribute to ageing, which can play a role in disease either indirectly with apoptosis, or directly with cell dysfunction.

One can therefore see the link that Wee1 has to disease is quite significant. It can play a role in cancer development and expression, as well as diseases related to ageing of cells and cellular dysfunction.

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WEE1 plays an important role in the phosphorylation and inactivation of CDK1, as discussed above, arresting the G2 checkpoint in the cell cycle to give the cell the opportunity to respond to DNA damage.1 Inhibiting this function has been proven critical in cancer patients that require undifferentiated cells to be destroyed before division instead of entering the final mitotic phases of the cell cycle. Small-molecule inhibitors are used as research tools to disrupt protein interactions and render its target molecule temporarily, or permanently, dysfunctional. Two of such inhibitors discussed will be AZD-1775 and MK-1775.

AZD-1775
The AZD-1775 inhibitor selectively targets the WEE1 protein at the G2 checkpoint. At this phase, the inhibition of WEE1 activity precludes the phosphorylation of CDC2.2 Without activated CDC2 it cannot bind to cyclin, which impairs the cyclin-dependent kinases (CDK) that is needed for the transition between the G2-M phase.2 Their absence therefore invalidates the G2 DNA damage checkpoint. The subsequent cell can now no longer enter mitosis. Although cell growth is unhindered, metaphase of the cell cycle will not commence.2 This will lead to apoptosis during the treatment of DNA damaging chemotherapeutic agents.1-2 In normal human cells p53 is a gene that plays a critical regulatory role at the G1 checkpoint, patients with a deficient/mutated p53 (e.g. Cancer patients) rely solely on the G2 checkpoint for DNA damage repair.2 The annulment of G2-M renders tumour cells more susceptible to antineoplastic agents and augment their cytotoxicity, also, ultimately leading to apoptosis.2

MK-1775
MK-1775 is a potent derivative of pyrazolo-pyrimidine that selectively inhibits the WEE1 gene. Studies demonstrated a direct inhibition of CDK1 substrates based on the concentrations of phosphorylated tyrosine-15 on CDC2. Under normal circumstances CDK1 activity is halted, during G2 and early mitosis, by WEE1 for the cell to undergo chromatid separation and nuclear envelope reconstruction.1 CDK1 is reactivated after successful DNA repair as is restores regular cell cycle progression. In the event of mutation the prolonged G2-M arrest will trigger apoptosis as this metaphase-promoting factor accumulates.3 WEE1 inhibition does not give the cell the opportunity to pause at the G2 checkpoint, resulting in premature mitotic entry.1 CDK1 activity continues undisrupted and the cell progresses with metaphase during mitosis. Any mutation or DNA damage will not be repaired, enter the cell cycle at G2 and be divided along with other chromosomes. Without detection this mutation will continue to enter the cell cycle and replicate without the presence of WEE1 and either disrupt protein function or result in a chromosomal mutation.3