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Wee1 kinase is a protein that aids in regulating eukaryotic cell cycles leading up to divisions in meiosis and mitosis. This involves activation of cyclin/CDK Complexes (when CDKi activity is ebbed) to enhance cell progression or adequately inhibiting entry into the next phase (via impeding CDC25 activity) when in excess during replication stress.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. The preparations for this should be completed and checked for errors to continue into the mitotic phase2 – hence, Wee1 is utilized to steady the cell in systems such as the RPA–ATR pathway. Then the Wee1 activity renders control to post-translational modification mechanisms of phosphorylation and ubiquitination that down-regulates/ degrades Wee1.3 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 Wee 1 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

Gene Wee1

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The wee1 gene belongs to the protein-kinase superfamily and can be located on chromosome 11.6 It contains 19, 809 bases. The gene encodes for a tyrosine-kinase nuclear protein, which catalyzes the inhibitory tyrosine phosphorylation of CDC2 / Cyclin B kinase. It is also known to coordinate DNA replication and mitosis transitions, as it protects the nucleus from cytoplasmically activated CDC2 kinase.6 The wee1 gene is known to be a negative regulator of entry into mitosis, by mediating the phosphorylation of CDK1 on Tyr-15 and, therefore, protecting the nucleus from cytoplasmically activated cyclin B/CDK1 complex before the start of mitosis.7 Phosphorylation of the cyclin B1-CDK1 occurs exclusively on Tyr-15. The gene 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.7 The activity of the gene increases during the S and G2 phases of the cell cycle and decreases during the M phase, when hyper phosphorylation occurs. A correlated decrease in protein level occurs at M/G1 phase, which can be attributed to the degradation of the chromosome gene.7-8

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  Chromosome 11

Wee1 Structure and Post-Translational Modifications

Wee1 Structure

Wee1A protein kinase consists of 646 amino acids. Its secondary structure contains 14 alpha helices and 9 beta strands9.

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  An outline of the secondary structure of Wee1A protein kinase. Indicating where the helices and strands are positioned according to the amino acid sequence (UniProtKB, 2017).

Caption2Wee1A protein kinase structure[3]

As stated above, it forms part of the protein kinase superfamily, Ser/Thr protein kinase family and Wee1 subfamily. The family name indicates that Wee1A is selectively phosphorylated at Serine and Threonine residues. Chain A is a 289 amino acid residue that forms part of the Wee1A protein kinase (residue 291-575). It contains several sequence and structural domains10.One of the domains that it contains is the protein kinase domain (residue 299-569)9. This domain forms the catalytic subunit of protein kinases, where a phosphate is cleaved from ATP and transferred to another protein11. It also contains phosphorylase kinase domain 1 and transferase (phosphotransferase) domain 1, which are structural domains of two superfamilies. These structural domains form functional units.

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  The protein kinase catalytic domain[5].

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 activity12. 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:

Site of phosphorylation	Enzyme
Ser-642	BRSK1 and BRSK2 [1]
Ser-53	PLK1[1]
Ser-123	Cyclin B/CDK1 complex[1]
Thr-239 (Wee Box)	Cyclin B/CDK1 [6] or Cyclin A/CDK2 complexes

When dephosphorylation occurs Wee1A kinase activity is upregulated again. Dephosphorylation of Thr-239 is mediated by CTDP1.9 The following diagram shows the primary structure of Wee1A with is Most important sites of Post-transtlational modification12.

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  Wee1A: 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 site12. 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 division13.

Wee1 Function and Role in Cell Signalling

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 overactive Wee1 negatively regulates the G2/M transition when acting as an effector activated by the CHK1 transducer. A lesser concentration of activated Wee1, in contrast, is used to positively regulate cell progression during transitions such as from G1 to S phase by activating Cyclin A/CDK2 or Cyclin E/CDK2 complexes.14 Wee1 and Myt1 can act as inhibitory kinases that enable the restraint of a cell within the second growth phase. 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.) A third kinase involved in controlling the G2/M transition is a stimulatory kinase (CAK1) which counteracts these 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. Activation of Wee1 via the CHK1 transducer causes inhibition of the cyclin A/CDK2 complex via deterring the expression of CDC25 (necessary for the activation of the complex) so that it is repressed from allowing the expression of FoxM1 will produce cyclin B and commence the second growth phase. 1

The Context -Within regulating system:

The network of kinases described above help steer the activation of cyclin B1/CDK1. 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:

Inhibitory phosphorylation by Wee1 during the Cell Cycle

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. 14 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.14-15 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.15

Wee1 Role in Disease

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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.16 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 cancer.5

The degree of expression of Wee1 kinases, can vary in several types of tumours, despite being “potential therapeutic targets”5. 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.5, 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.17, “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.

Wee1 in Relation to The Effect of Small-Molecule Inhibitors on The Cell Cycle

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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.3 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 CDC2.18 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.18 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.18 This will lead to apoptosis during the treatment of DNA damaging chemotherapeutic agents.3,18 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.18 The annulment of G2-M renders tumour cells more susceptible to antineoplastic agents and augment their cytotoxicity, also, ultimately leading to apoptosis.18 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.3 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.19 WEE1 inhibition does not give the cell the opportunity to pause at the G2 checkpoint, resulting in premature mitotic entry.3 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.19

Inhibitor	Phase Affected	Cell Growth	Cell Death	Consequence
AZD-1775	G2	√	√	Apoptosis
MK-1775	G2/early metaphase	√	X	Dysfunction/Mutation