Yeast Wee1

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2 (Sia, 1998).

The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G2 activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p (Drosophila homolog: Grapes) is the endpoint of this checkpoint pathway. Overexpression of Chk1p causes a wee1(+)-dependent G2 arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggest that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14-3-3 proteins to cause the cytoplasmic retention of the 14-3-3/Cdc25p complexes. However, the G2 DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1(+) and cdc25(+) abolishes checkpoint control. Activation of the G2 DNA damage checkpoint induces a transient increase in Wee1p levels. It is concluded that the G2 DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability (Raleigh, 2000).

Development of a multicellular organism requires that mitosis and morphogenesis be coordinated. These processes must also be synchronized during the growth of unicellular organisms. In the yeast Saccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud. Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form results in the formation of multinucleate cells in budding yeast. Immunofluorescence analysis of these multinulceate cells shows that mitosis and bud formation are no longer linked. Swe1 (Wee1 of budding yeast) is required for coupling mitosis to bud formation during a perturbed cell cycle. When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk. In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation. Affinity purification, two-hybrid analysis, and mutant characterization results suggest that Cdc5 and Swe1 interact. From these results, it is concluded that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function. These results also suggest that Cdc5 may be a negative regulator of Swe1 (Bartholomew, 2001).

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay (Harrison, 2001).

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, have been identified as Hsp90 substrates and the kinase domain has been shown to be sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function (Goes, 2001).

The G2 DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases. However, the in vivo role of these kinases in checkpoint regulation has been unclear. In the fission yeast Schizosaccharomyces pombe, Mik1 is a target of both checkpoints and the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints. Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance. In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint (Rhind, 2001).

In eukaryotic cells, the Wee1 protein kinase phosphorylates and inhibits Cdc2, thereby creating an interphase of the cell cycle. In Xenopus, the conventional Wee1 homolog (termed Xe-Wee1A, or Wee1A for short) is maternally expressed and functions in pregastrula embryos with rapid cell cycles. A second, zygotic isoform of Xenopus Wee1, termed Xe-Wee1B (or Wee1B for short) has been isolated, that is expressed in postgastrula embryos and various adult tissues. When ectopically expressed in immature oocytes, Wee1B inhibits Cdc2 activity and oocyte maturation (or entry into M phase) much more strongly than Wee1A, due to its short C-terminal regulatory domain. Moreover, ectopic Wee1B, unlike Wee1A, is very labile during meiosis II and cannot accumulate in mature oocytes due to the presence of PEST-like sequences in its N-terminal regulatory domain. Finally, when expressed in fertilized eggs, ectopic Wee1B but not Wee1A does affect cell division and impair cell viability in early embryos, due primarily to its very strong kinase activity. These results suggest strongly that the differential expression of Wee1A and Wee1B is crucial for the developmental regulation of the cell cycle in Xenopus (Okamoto, 2002).

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylates Swe1 and this modification serves as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. It is proposed that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events (Asano, 2005).

This study delineates how Swe1 regulation is orchestrated by multiple components as cells progress through the cell cycle. Cla4-dependent septin filament formation early in the cell cycle permits assembly of a platform consisting of Hsl1 (Nim1-related kinase) and its adaptor Hsl7, a critical step that is required for the recruitment of Clb2-Cdc28-phosphorylated Swe1 and Cdc5 later in the cell cycle. Phosphorylated Swe1 further promotes Cdc5 localization to the platform by providing a docking site for the polo-box domain of Cdc5. The data show that both the Hsl1-Hsl7 platform and the primed Swe1 are two crucial elements for Cdc5-dependent Swe1 hyperphosphorylation and subsequent degradation at the bud neck. This coordinated, multistep, Swe1 regulation clearly provides a means to monitor the completion of earlier cell cycle events and to effectively bring about Swe1 destruction at the time of mitotic entry. Once unleashed from the Swe1-imposed G2 delay, Clb-Cdc28 can induce mitotic entry unimpeded (Asano, 2005).

The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Upregulation of Chk1 by mutation or overexpression bypasses the requirement for upstream regulators or DNA damage to promote a G2 cell cycle arrest. Fission yeast were screened for mutations that rendered cells resistant to overexpressed chk1+. A mutation was identified in tra1, which encodes one of two homologs of transformation/transcription domain-associated protein (TRRAP), an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed chk1+, suggesting this phenotype is due to a HAT activity, although expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing chk1+ and accumulate inactive Wee1 protein. The changed division response (Cdr) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and it was shown that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation (Calonge, 2010).

Wee1 homologs in invertebrates

Regulatory phosphorylation of the Cdc2p kinase by Wee1p-type kinases prevents eukaryotic cells from entering mitosis or meiosis at an inappropriate time. The canonical Wee1p kinase is a soluble protein that functions in the eukaryotic nucleus. All metazoa also have a membrane-associated Wee1p-like kinase named Myt1, and the first genetic characterization of this less well-studied kinase is described. The Caenorhabditis elegans Myt1 ortholog is encoded by the wee-1.3 gene, and six dominant missense mutants prevent primary spermatocytes from entering M phase but do not affect either oocyte meiosis or any mitotic division. These six dominant wee-1.3(gf) mutations are located in a four amino acid region near the C terminus and they cause self-sterility of hermaphrodites. Second-site intragenic suppressor mutations in wee-1.3(gf) restore self-fertility to these dominant sterile hermaphrodites, permitting genetic dissection of this kinase. Ten intragenic wee-1.3 suppressor mutations were recovered and they form an allelic series that includes semi-dominant, hypomorphic and null mutations. These mutants reveal that WEE-1.3 protein is required for embryonic development, germline proliferation and initiation of meiosis during spermatogenesis. This suggests that a novel, sperm-specific pathway negatively regulates WEE-1.3 to allow the G2/M transition of male meiosis I, and that dominant wee-1.3 mutants prevent this negative regulation (Lamitina, 2002).

In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B-Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases, exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. In oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B-Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator (Okumura, 2002).

Protein interactions of vertebrate Wee1

In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s). Five of the eight positive clones identified encode 14-3-3beta. In vivo binding assay in 293 cells has shown that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta. The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta. The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection. Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase. Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability (Wang, 2000).

The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggest that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuates the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1 (Lee, 2001).

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. The adaptor protein Crk is required for apoptotic signaling in egg extracts. Moreover, removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevents apoptosis. The relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. A specific interaction has been demonstrated between tyrosine-phosphorylated Wee1 and the Crk SH2 domain: recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerates apoptosis in egg extracts, and this acceleration is largely dependent on the presence of endogenous Crk protein. Since other Cdk inhibitors, such as roscovitine and Myt1, do not act like Wee1 to accelerate apoptosis, it is proposed that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator (Smith, 2000).

The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. The C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. A mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells (Smith, 2002).

Wee kinases in Xenopus

The Wee kinases block entry into mitosis by phosphorylating and inhibiting the activity of the mitotic cyclin-dependent kinase, Cdk1. The various Xenopus Wee kinases have unique temporal and spatial patterns of expression during development. A new Wee1-like kinase, Xenopus Wee2, has been isolated and characterized. By both in vivo and in vitro tests, Xenopus Wee2 functions as a Wee1-like kinase. The previously isolated Wee1-like kinase, Xenopus Wee1, is expressed only as maternal gene product. In contrast, Xenopus Wee2 is predominantly a zygotic gene product, while the third Wee kinase, Xenopus Myt1, is both a maternal and zygotic gene product. Concurrent with the changing levels of these Cdk inhibitory kinases, the pattern of embryonic cell division becomes asynchronous and spatially restricted in the Xenopus embryo. Interestingly, once zygotic transcription begins, Xenopus Wee2 is expressed in regions of the embryo that are devoid of mitotic cells, such as the involuting mesoderm. In contrast, Xenopus Myt1 is expressed in regions of the embryo that have high levels of proliferation, such as the developing neural tissues. The existence of multiple Wee kinases may help explain how distinct patterns of cell division arise and are regulated during development (Leise, 2002).

Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. The contribution has been investigated of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, it is shown that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. Overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis (Murakami, 2004).

Modulation of the cell cycle appears to play an important role in both Xenopus and Drosophila embryogenesis. Prior to gastrulation, both organisms undergo a burst of rapid cell divisions followed by a gradual expansion of the cell cycle. Zygotic cell cycle components are synthesized after the MBT and previous studies have indicated that zygotic proteins do play a role in regulating Cdc2 activity during gastrulation. In Drosophila, cell cycle inhibition is observed at the ventral furrow, a region somewhat analogous to the Xenopus blastopore, and this inhibition is achieved by the removal of a zygotic activator of Cdc2. Specifically, the spatially restricted expression of the Tribbles protein results in the degradation of the String/Cdc25C phosphatase in cells surrounding the ventral furrow. In Xenopus, the zone of non-mitotic cells in the mid-late gastrula is identical to the area of zygotic Wee1B/Wee2 RNA expression, suggesting that zygotic expression of a Cdc2 inhibitor, Wee1B/Wee2, might play an analogous role in frog embryogenesis. Interestingly, the expansion of the cell cycle after the MBT (and during gastrulation) is regulated by zygotic components in Drosophila, but is regulated by maternally derived components in Xenopus. In Xenopus, the maternally regulated program of cell cycle expansion has been implicated in the onset of zygotic transcription, cytoplasmic blebbing and pseudopod formation (at the MBT), but has not been previously implicated in the coordinated tissue morphogenesis that takes place during gastrulation. This study demonstrates that the maternal Wee1 protein contributes to the cell cycle downregulation that occurs during Xenopus gastrulation. The findings also indicate that the maternally directed program of cell cycle control, rather than simply facilitating the transcription of zygotic components, plays a direct role in morphogenesis (Murakami, 2004).

The requirement of cell cycle regulation for the coordinated cell movements of gastrulation is another shared feature of Drosophila and Xenopus embryogenesis. In flies, the Tribbles-mediated degradation of Cdc25C permits the invagination of mesodermal cells at the ventral furrow, one of the earliest events of gastrulation. Similarly, in this study, cell cycle inhibition mediated by Wee1 was found to be important for epiboly, involution and convergent-extension, all of which are major morphogenetic processes that contribute to normal Xenopus gastrulation. Thus, although flies and frogs may use different molecular components to regulate the embryonic cell cycle, it appears that in both organisms the inhibition of cell division is essential for the complex morphogenetic movements required for gastrulation (Murakami, 2004).

Wee1 is upregulated by tyrosine autophosphorylation following the MBT and at gastrulation. This upregulation appears to be required for Wee1 function in early gastrula embryos given that neither kinase-inactive Wee1 or a Wee1 protein containing mutations in the tyrosine phosphorylation sites are able to rescue the defects produced by MO-Wee1-depletion. These findings are consistent with previous observations that upregulation of Wee1 activity by tyrosine autophosphorylation is critical for Wee1 function in the first mitotic cell cycle. Taken together, these studies indicate that the maternal Wee1 protein functions at distinct developmental points to coordinate cell cycle progression with events that control the organization of the embryonic body plan. Moreover, this work contributes to a growing body of evidence that cell cycle regulation is likely to be crucial for a wide variety of morphogenetic processes. Wee1 is a primary cell cycle target of the budding morphogenesis checkpoint in S. cerevisiae, and in mammalian cells, there is evidence that inhibition of cell proliferation is necessary for cell migration. Collectively, these studies suggest that 'morphogenesis' checkpoints, which coordinate cell shape changes and movement with cell proliferation, will be crucial for normal development and organogenesis, and may also play an important role in the balance between deregulated cell proliferation and metastasis (Murakami, 2004).

During the meiotic cell cycle in Xenopus oocytes, p90rsk, the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1/Wee1. However, p90rsk is inactivated after fertilization due to the degradation of Mos. The Polo-like kinase Plx1, instead of p90rsk, interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90rsk-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus (Inoue, 2005).

Although the interruption of the Plx1-Myt1 interaction by T478A mutation largely impaired the Plx1 phosphorylation and inhibition of Myt1, it seemed to have a relatively small (although significant) effect on early embryonic cell divisions. This is presumably due, however, to the dramatic increase in levels of the Myt1/Wee1-antagonizing Cdc25A phosphatase during this period. In somatic cells, however, the level of Cdc25A is considerably lower; therefore, the interruption of the Plx1-Myt1 interaction in somatic cells might have a significantly larger effect on cell divisions. In human somatic cells, Myt1 localizes to the endoplasmic reticulum and Golgi complex. Therefore, the binding of Plk1/Plx1 or the Myt1 substrate Cdc2-cyclin B to Myt1 could also be responsible, at least in part, for their known localization and function at the Golgi complex. Indeed, in Drosophila embryos, Myt1 has been implicated in Golgi fragmentation (Cornwell, 2002), a mitotic event involving both Cdc2 and Plk1 (Lin, 2000; Sutterlin, 2001; Inoue, 2005 and references therein).

Structure of mammalian Wee kinase

Phosphorylation is critical to regulation of the eukaryotic cell cycle. Entry to mitosis is triggered by the cyclin-dependent kinase CDK1 (Cdc2), which is inactivated during the preceding S and G2 phases by phosphorylation of T14 and Y15. Two homologous kinases, Wee1, which phosphorylates Y15, and Myt1, which phosphorylates both T14 and Y15, mediate this inactivation. The crystal structure of the catalytic domain of human somatic Wee1 (Wee1A) complexed with an active-site inhibitor has been determined at 1.8Å resolution. Although Wee1A is functionally a tyrosine kinase, in sequence and structure it most closely resembles serine/threonine kinases such as Chk1 and cAMP kinases. The crystal structure shows that although the catalytic site closely resembles that of other protein kinases, the activation segment contains Wee1-specific features that maintain it in an active conformation and, together with a key substitution in its glycine-rich loop, help determine its substrate specificity (Squire, 2005).

Regulation of expression of mammalian Wee1

Wee1 kinase activity is necessary for the control of the first embryonic cell cycle following the fertilization of meiotically mature Xenopus oocytes. Wee1 mRNA is present in immature oocytes, but Wee1 protein does not accumulate in immature oocytes or during the early stages of progesterone-stimulated maturation. This delay in Wee1 translation is critical since premature Wee1 protein accumulation has been shown to inhibit oocyte maturation. Evidence is provided that Wee1 protein accumulation is regulated at the level of mRNA translation. This translational control is directed by sequences within the Wee1 mRNA 3'-untranslated region (3' UTR). Specifically, cytoplasmic polyadenylation element (CPE) sequences within the Wee1 3' UTR are necessary for full translational repression in immature oocytes. The data further indicate that while CPE-independent mechanisms may regulate the levels of Wee1 protein accumulation during progesterone-stimulated oocyte maturation, the timing of Wee1 mRNA translational induction is directed through a CPE-dependent mechanism (Charlesworth, 2000).

M-phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. The wee1 gene promoter was isolated and found to contain one AP-1 binding motif. The promoter is directly activated by the immediate early gene product c-Fos at cellular G(1)/S phase. In antigen-specific Th1 cells stimulated by antigen, transactivation of the c-fos and wee1 kinase genes occurs sequentially at G(1)/S, and the substrate of wee1 kinase, cdc2-Tyr15, is subsequently phosphorylated at late G(1)/S. Under prolonged expression of the c-fos gene, however, the amount of wee1 kinase is increased and its target cdc2 molecule is constitutively phosphorylated on its tyrosine residue; Th1 cells go into aberrant mitosis. Thus, an immediate early gene product, c-Fos/AP-1, directly transactivates the wee1 kinase gene at G(1)/S. The transient increase in c-fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G(1)/S phase of the cell cycle (Kawasaki, 2001).

The breast cancer tumor-suppressor gene, BRCA1, encodes a protein with a BRCT domain -- a motif that is found in many proteins that are implicated in DNA damage response and in genome stability. Phosphorylation of BRCA1 by the DNA damage-response proteins ATM, ATR and hCds1/Chk2 (Drosophila homolog: loki) changes in response to DNA damage and at replication-block checkpoints. Although cells that lack BRCA1 have an abnormal response to DNA damage, the exact role of BRCA1 in this process has remained unclear. BRCA1 is shown in this study to be essential for activating the Chk1 kinase that regulates DNA damage-induced G2/M arrest. Thus, BRCA1 controls the expression, phosphorylation and cellular localization of Cdc25C and Cdc2/cyclin B kinase-proteins that are crucial for the G2/M transition. BRCA1 regulates the expression of both Wee1 kinase, an inhibitor of Cdc2/cyclin B kinase, and the 14-3-3 family of proteins that sequesters phosphorylated Cdc25C and Cdc2/cyclin B kinase in the cytoplasm. It is concluded that BRCA1 regulates key effectors that control the G2/M checkpoint and is therefore involved in regulating the onset of mitosis (Yarden, 2002).

Signaling upstream of Wee1

Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. The mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest has been investigated using Xenopus egg extracts as a model system. Wee1-depleted extracts are unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restores their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK phosphorylates recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK results in a modest (approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts have demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, evidence is presented that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts results in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully (Walter, 2000).

The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress. Chfr is a ubiquitin ligase. When transfected into HEK293T cells, Myc-Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, it has been demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2-M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis (Kang, 2002).

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation. What might be the additional Mos target? Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF (MPF is a complex of cyclin B and Cdc2 kinase). Wee family member Myt1 is likely to be the Mos target in this process, since Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. It is proposed that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes (Peter, 2002).

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. A G1 substrate of the APC has been identified that has been termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit (Ayad, 2003).

Substrate competition as a source of ultrasensitivity in the inactivation of Wee1

The mitotic regulators Wee1 and Cdk1 can inactivate each other through inhibitory phosphorylations. This double-negative feedback loop is part of a bistable trigger that makes the transition into mitosis abrupt and decisive. To generate a bistable response, some component of a double-negative feedback loop must exhibit an ultrasensitive response to its upstream regulator. This study experimentally demonstrates that Wee1 exhibits a highly ultrasensitive response to Cdk1. Several mechanisms can, in principle, give rise to ultrasensitivity, including zero-order effects, multisite phosphorylation, and competition mechanisms. It was found that the ultrasensitivity in the inactivation of Wee1 arises mainly through two competition mechanisms: competition between two sets of phosphorylation sites in Wee1 and between Wee1 and other high-affinity Cdk1 targets. Based on these findings, it was possible to reconstitute a highly ultrasensitive Wee1 response with purified components. Competition provides a simple way of generating the equivalent of a highly cooperative allosteric response (Kim, 2007).

The mitotic regulator cyclin B-Cdk1 is controlled by a system of two double-negative feedback loops and a positive feedback loop. Active Cdk1 brings about the inactivation of the nuclear kinase Wee1 and the cytoplasmic, membrane-associated kinase Myt1, which, when active, can inactivate Cdk1 through phosphorylation of Thr 14 and/or Tyr 15. In addition, active Cdk1 brings about the activation of Cdc25, which can then dephosphorylate the sites phosphorylated by Wee1 and Myt1 (Kim, 2007 and references therein).

Under the proper circumstances, positive and double-negative feedback loops can exhibit bistability. This means that the system can adopt either of two alternative steady states in response to a constant stimulus and can toggle between these states in response to small changes in stimulus. Recent experimental work has demonstrated that the Cdk1/Wee1/Myt1/Cdc25 system is, in fact, bistable and can toggle between a stable interphase state, with Cdk1 and Cdc25 inactive and Wee1 and Myt1 active, and a stable mitotic state with Cdk1 and Cdc25 active and Wee1 and Myt1 inactive (Kim, 2007 and references therein).

A bistable signaling system must include a positive feedback loop, a double-negative feedback loop, or the equivalent, although sometimes this feedback loop may be difficult to appreciate. However, the presence of positive or double-negative feedback does not guarantee that a system will be bistable; the shapes of the steady-state stimulus/response curves for the individual legs of the loop are important as well. It is also important that some component of the loop exhibit a sigmoidal, ultrasensitive steady-state response -- that is, a nonlinear response resembling that of a cooperative enzyme -- rather than a hyperbolic, Michaelian response. For the particular case of a two-component positive or double-negative feedback, it is easy to show that simple Michaelian response functions do not support bistability. This is also true for an arbitrarily large positive feedback loop, provided that the components of the loop constitute a strongly monotone system. Given that the Cdk1/Wee1/Myt1/Cdc25 system is bistable, it seemed plausible that some component of the system would exhibit a highly ultrasensitive steady-state response to its upstream regulator (Kim, 2007 and references therein).

Ultrasensitivity is not only important for allowing positive feedback loops to generate bistable responses, but also for effective signal propagation down cascades. With Michaelian steady-state responses, an n-fold change in stimulus always yields a less than n-fold change in response. After several levels in a signaling cascade, the loss in signal contrast can be severe. Ultrasensitivity can help restore the original contrast and can also amplify it, converting graded inputs into more abrupt and switch-like outputs. In addition, ultrasensitivity is required for generating oscillations in negative feedback loops of certain lengths. Thus, ultrasensitivity may be important in a wide range of signaling contexts (Kim, 2007 and references therein).

Several examples of ultrasensitive responses have been documented experimentally. The earliest were the phosphorylation of phosphorylase and isocitrate dehydrogenase in vitro, where the observed sigmoidal responses could be largely attributed to zero-order ultrasensitivity, a phenomenon that occurs when the kinase and/or phosphatase that regulate the steady-state level of substrate phosphorylation are operating near saturation. More recent examples have included the activation of MEK and p42 MAPK by Mos in Xenopus oocyte extracts, AMP kinase activation in INS-1 cells, and JNK activation in several cell types. Thus, ultrasensitivity is a recurring motif in cell signaling (Kim, 2007 and references therein).

A variety of plausible mechanisms have been proposed to account for the ultrasensitivity observed in these systems. For example, the ultrasensitive response of MEK to Mos may be generated by competition between CK2β and MEK for access to Mos, and the response of p42 MAPK to MEK may be generated by nonprocessive multisite phosphorylation. However, these postulated mechanisms remain largely untested (Kim, 2007 and references therein).

This study examined whether the inactivation of Wee1 by Cdk1 in Xenopus egg extracts is Michaelian or ultrasensitive. It was found that the inactivation of Wee1, as assessed by the steady-state phosphorylation of one critical residue (Thr 150), is highly ultrasensitive, with an apparent Hill coefficient of 3.5. Several plausible mechanisms were tested to account for the observed ultrasensitivity. It was found that some of the ultrasensitivity is intrinsic to the core Wee1/cyclin B-Cdk1/Wee1 phosphatase system, with the main mechanism for intrinsic ultrasensitivity being competition between two sets of phosphorylation sites in Wee1 for access to Cdk1. In addition, much of the observed ultrasensitivity is extrinsic to the core system. The main mechanism of extrinsic ultrasensitivity appears to be competition between Wee1 and other high-affinity substrates for access to Cdk1. As proof of principle for the idea of competition as a source of ultrasensitivity, the highly ultrasensitive, switch-like inhibition of Wee1 by Cdk1 was reconstituted in vitro with purified recombinant components (Kim, 2007).

Wee kinase and meiosis

Meiotic cells undergo two successive divisions without an intervening S phase. However, the mechanism of S-phase omission between the two meiotic divisions is largely unknown. Wee1, a universal mitotic inhibitor, is absent in immature (but not mature) Xenopus oocytes, being down-regulated specifically during oogenesis; this down-regulation is most likely due to a translational repression. Even the modest ectopic expression of Wee1 in immature (meiosis I) oocytes can induce interphase nucleus reformation and DNA replication just after meiosis I. Thus, the presence of Wee1 during meiosis I converts the meiotic cell cycle into a mitotic-like cell cycle having an S phase. In contrast, Myt1, a Wee1-related kinase, is present and directly involved in G2 arrest of immature oocytes, but its ectopic expression has little effect on the meiotic cell cycle. These results strongly indicate that the absence of Wee1 in meiosis I ensures the meiotic cell cycle in Xenopus oocytes. It is suggested that absence of Wee1 may be a well-conserved mechanism for omitting interphase or S phase between the two meiotic divisions (Nakajo, 2000).

In most species, the meiotic cell cycle is arrested at the transition between prophase and metaphase through unclear somatic signals. Activation of the Cdc2-kinase component of maturation promoting factor (MPF) triggers germinal vesicle breakdown after the luteinizing hormone (LH) surge and reentry into the meiotic cell cycle. Although high levels of cAMP and activation of protein kinase A (PKA) play a critical role in maintaining an inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. Using a small-pool expression-screening strategy, several putative PKA substrates have been identified from a mouse oocyte cDNA library. One of these clones encodes a Wee1-like kinase that prevents progesterone-induced oocyte maturation when expressed in Xenopus oocytes. Unlike the widely expressed Wee1 and Myt1, mWee1B mRNA and its protein are expressed only in oocytes, and mRNA downregulation by RNAi injection in vitro or transgenic overexpression of RNAi in vivo causes a leaky meiotic arrest. Ser15 residue of mWee1B is the major PKA phosphorylation site in vitro, and the inhibitory effects of the kinase are enhanced when this residue is phosphorylated. Thus, mWee1B is a key MPF inhibitory kinase in mouse oocytes, functions downstream of PKA, and is required for maintaining meiotic arrest (Han, 2005).

A mechanism for controlled breakage of under-replicated chromosomes during mitosis

While DNA replication and mitosis occur in a sequential manner, precisely how cells maintain their temporal separation and order remains elusive. This study unveils a double-negative feedback loop between replication intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4 (see Drosophila Mus81 and Mus312), which renders DNA replication and mitosis mutually exclusive. MUS81 nuclease is constitutively active throughout the cell cycle (see Drosophila cell cycle) but requires association with SLX4 for efficient substrate targeting. To preclude toxic processing of replicating chromosomes, WEE1 (see Drosophila Wee1) kinase restrains CDK1 (see Drosophila Cdk1) and PLK1 (see Drosophila polo)-mediated MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers widespread nucleolytic breakage of replication intermediates, halting DNA replication and leading to chromosome pulverization. Unexpectedly, premature entry into mitosis-licensed by unrestrained CDK1 activity during S phase-requires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation (Duda, 2016).

wee: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | References

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