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).
Maturation promoting factor (MPF), a complex of cyclin-dependent kinase 1 and cyclin B, drives oocyte maturation in all animals. Mechanisms to block MPF activation in developing oocytes must exist to prevent precocious cell cycle progression prior to oocyte maturation and fertilization. This study sought to determine the developmental consequences of precociously activating MPF in oocytes prior to fertilization. Whereas depletion of Myt1 in Xenopus oocytes causes nuclear envelope breakdown in vitro, depletion of the Myt1 ortholog WEE-1.3 in C. elegans hermaphrodites causes precocious oocyte maturation in vivo. Although such oocytes are ovulated, they are fertilization incompetent. Novel phenotypes have been observed in these precociously maturing oocytes, such as chromosome coalescence, aberrant meiotic spindle organization, and the expression of a meiosis II post-fertilization marker. Furthermore, co-depletion studies of CDK-1 and WEE-1.3 demonstrate that WEE-1.3 is dispensable in the absence of CDK-1, suggesting that CDK-1 is a major target of WEE-1.3 in C. elegans oocytes (Burrows, 2006).
Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggest that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control (Mueller, 1995).
M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which are maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. The C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. These results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B (Palmer, 1998).
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).
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).
During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. When cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented from being activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, acquisition of autoamplification competence does not require the Mos/MAPK pathway (Karaiskou, 2004).
Thus, Plx1 protein, crucial for the function of the auto-amplification feedback loop in full-grown oocytes is not expressed in small oocytes. Both Cdc25 and Myt1 are direct substrates of Plk1 during M phase. The results indicate that overexpression of Plk1 in stage IV oocytes authorizes cyclin B1 to form active complexes with Cdc2. This observation shows that in oocytes, Plk1 participates in the formation of an active MPF trigger by downregulating Myt1. Moreover, it indicates that progesterone unresponsiveness of small oocytes depends on a stable activity of Myt1 kinase, because of Plx1 absence. Although Plk1 expression prevents Tyr15 phosphorylation of Cdc2 after cyclin B overexpression, Cdc25 is not fully activated. This shows that full activation of Cdc25 requires a further regulatory mechanism. Indeed, Xenopus Cdc25 can be negatively regulated through Ser287 phosphorylation by several kinases, including Chk1 and PKA. Cdc25C, which is phosphorylated on Ser287 in Xenopus prophase oocytes, is dephosphorylated by type 1 phosphatase (PP1) at GVBD. Since the PP1 inhibitor I prevents meiotic maturation, PP1 could participate in the regulation of the MPF autoamplification loop by catalyzing the removal of the inhibitory Ser287 phosphate, and could therefore be involved in the regulation of Cdc25 during oogenesis (Karaiskou, 2004).
In competent oocytes, Plx1 action on Cdc25 is antagonized by an okadaic acid-sensitive phosphatase, involving PP2A activity. This explains why the auto-amplification mechanism can be artificially activated by okadaic acid. However, okadaic acid is unable to promote Cdc2 activation in small incompetent oocytes, showing that the loop implying Cdc2, Cdc25, Plx1 and PP2A is not functional in growing oocytes. The most probable explanation for this defect is the absence of Plx1 in stage IV oocytes. Indeed, it has been shown, both in vivo and in vitro, that expression of Plk1 is sufficient to restore the activation of MPF in response to okadaic acid in incompetent oocytes. Plx1 is therefore the missing factor explaining why the auto-amplification of MPF is defective in small oocytes (Karaiskou, 2004).
Altogether, these experiments show that the incompetence of small oocytes to resume meiosis is ensured by the absence of Plx1 resulting in a double negative control on MPF activation. (1) The formation of active complexes between Cdc2 and newly synthesized cyclins is prevented by a sustained activity of Myt1 that escapes downregulation by Plx1. (2) Cdc25 activation that is normally achieved through a feedback loop involving Plx1 is also prevented. Further investigation will be necessary to discover (1) how Plx1 expression is controlled by cell size at the end of oogenesis; (2) how PP2A controls Cdc25 activity in small oocytes, and (3) how the initial steps of the progesterone transduction pathway connect to MPF regulators, allowing the female germ cell to resume meiosis when oocyte growth is completed (Karaiskou, 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 (Inoue, 2005 and references therein).
In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. This study provides evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, this study shows that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, it was demonstrated that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. A model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop (Gaffré, 2011).
This study focused on the role and regulation of Myt1, the member of the Wee1 family of inhibitory kinases that is expressed in Xenopus prophase I oocytes. The Wee1 family of kinases comprises Wee1, which is present in all eukaryotes, and Myt1, which is restricted to metazoans. Wee1 is a nuclear kinase, phosphorylating Cdc2 on Tyr15, whereas Myt1 possesses a dual Thr14/Tyr15 Cdc2-phosphorylating activity and associates with cell membranes. In Xenopus oocytes only Myt1 is detectable at the protein level during the last stages of growth and the early steps of meiosis reinitiation. The two forms of Xenopus Wee1 (Wee1A and Wee1B) are not expressed in the prophase I-arrested oocytes. Wee1A becomes detectable only after completion of the first meiotic division. Myt1 kinase has been implicated in both male and female gametogenesis in various animals, either alone, as is the case in C. elegans, Drosophila, starfish and Xenopus, or in concert with Wee1, as in mouse oocytes. In starfish, Myt1 has been clearly demonstrated to be involved both in the G2 arrest of oocytes and in meiosis re-entry, which involves downregulation of Myt1 activity by Akt phosphorylation. Whether Myt1 also plays an essential role in Xenopus oocyte meiotic resumption remains unclear (Gaffré, 2011).
The main aim of this study was to determine the contribution of Myt1 to the prophase arrest of fully grown Xenopus oocytes and to meiosis re-entry. A crucial issue was to determine which kinases are responsible for the initial phosphorylation and downregulation of Myt1 activity following progesterone treatment. As well as Cdc2, candidate kinases activated at around the same time include Plx1 (the Xenopus homolog of Drosophila Polo kinase) and members of the MAPK cascade, including Mos and p90Rsk kinase (Gaffré, 2011).
This study found that Myt1 function is required to maintain prophase I arrest in fully grown oocytes, such that experimental Myt1 inhibition promoted meiosis re-entry. At the onset of maturation, the inactivating phosphorylation of Myt1 was found to precede the activating phosphorylation of Cdc25 and to be mediated principally by Cdc2 itself in association with newly synthesized Cyclins, rather than by the Mos/MAPK cascade or Plx1. A model is proposed in which the significant upregulation of Cyclin B synthesis following progesterone stimulation produces a small population of active Cdc2-Cyclin B, which is responsible for early Myt1 phosphorylation and inhibition, ahead of full Cdc25 activation and thus entry into the auto-amplification loop. In Xenopus, the change in the balance between Cyclin B synthesis and Myt1 activity following hormone stimulation is therefore a key feature of meiotic re-entry (Gaffré, 2011).
Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. This study reports the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain; its deletion results in the localization of Myt1Hu primarily to the nucleus (Liu, 1997).
Activation of the Cdc2.cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. This study examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. Human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreases during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2. cyclin B1 is capable of phosphorylating Myt1 in vitro, but this phosphorylation does not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2.cyclin complexes (Booher, 1997).
The Myt1 protein kinase functions to negatively regulate Cdc2-cyclin B complexes by phosphorylating Cdc2 on threonine 14 and tyrosine 15. Throughout interphase, human Myt1 localizes to the endoplasmic reticulum and Golgi complex, whereas Cdc2-cyclin B1 complexes shuttle between the nucleus and the cytoplasm. Overproduction of either kinase-active or kinase-inactive forms of Myt1 blocks the nuclear-cytoplasmic shuttling of cyclin B1 and causes cells to delay in the G2 phase of the cell cycle. The COOH-terminal 63 amino acids of Myt1 serve as a Cdc2-cyclin B1 interaction domain. Myt1 mutants lacking this domain no longer bind cyclin B1 and do not efficiently phosphorylate Cdc2-cyclin B1 complexes in vitro. In addition, cells overproducing mutant forms of Myt1 lacking the interaction domain exhibit normal trafficking of cyclin B1 and unperturbed cell cycle progression. These results suggest that the docking of Cdc2-cyclin B1 complexes to the COOH terminus of Myt1 facilitates the phosphorylation of Cdc2 by Myt1, overproduction of Myt1 perturbs cell cycle progression by sequestering Cdc2-cyclin B1 complexes in the cytoplasm (Liu, 1999).
Activation of Cdc2, is the universal event controlling the onset of mitosis. In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory phosphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevents catastrophic premature entry into mitosis. The function of Myt1 was defined by overexpression studies in both S. pombe and a human osteosarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevents entry into mitosis in both cell types; however, Myt1 catalytic activity is not essential for the cell cycle delay observed with human cells. Myt1 expression is restricted to proliferating cells. Furthermore, no major decline was detected in Myt1 protein abundance prior to the entry into mitosis, which coincides with the loss of Myt1 activity. Mitotic phosphoepitopes, recognized by the monoclonal antibody MPM-2, have been localized to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, is able to associate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevents its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduces its ability to phosphorylate Cdc2 in vitro. The C-terminal domain of Myt1 is required for recruitment of Cdc2, and it is inferred that this domain lies in the cytoplasm because it can interact with and is phosphorylated by Cdc2. In conclusion, it is proposed that Myt1 can negatively regulate Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of which require the C-terminal domain: (1) Myt1 can bind and sequester Cdc2/cyclin B1 in the cytoplasm preventing entry into the nucleus, and (2) it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibiting its catalytic activity (Wells, 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).
The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. The interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles was studied in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, are unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 is required for cyclin B-Cdc2 activation, the action of Plk1 is mediated primarily through suppression of Myt1 rather than through activation of Cdc25. It is proposed that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1 (Okano-Uchida, 2003).
Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase, is crucially involved in multiple events during the M phase. A consensus phosphorylation sequence for Plk1 has been identified by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position -2 are important for optimal phosphorylation by Plk1. Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) serves as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, does not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displays its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1 (Nakajima, 2003).
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