Wee1 Homolog Research Articles

PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with cyclin-dependent kinase 4/6 (CDK4/6) inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of patient-derived xenografts (PDXs) from patients with 22 ER+ breast cancer demonstrated that protein kinase, membrane-associated tyrosine/threonine one (PKMYT1), a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX-derived organoids and PDXs, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Trypanosoma cruzi DNA Polymerase β Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).

DNA polymerase β plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase β, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase β have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase β is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase β, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase β as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase β, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi.

IDENTIFICATION OF WEE GENES IN DICTYOSTELIUM DISCOIDEUM

Cell cycle is a basic cellular mechanism that allows for cellular growth and development, regeneration or repair, and wear of tissues or even regeneration of the whole organism in certain cases. A failure during the cell cycle process leads to various dire consequences. Various proteins and kinases play a vital role in the regulation of the cell cycle to meet cellular demands and needs. Amongst the kinases, Wee1 is a key regulator of cell cycle progression which inhibits mitotic entry by phosphorylating the tyrosine residue on the M-phase-inducing kinase, Cdk1, at the G2/M transition. In Dictyostelium discoideum, wee1 genes that regulate the cell cycle progression at the G2/M transition have not been identified and studied yet. Our findings support the existence of the Wee1 homolog in D. discoideum, which could play a role in cell division at G2/M.

Antisense long non‑coding RNA WEE2‑AS1 regulates human vascular endothelial cell viability via cell cycleG2/M transition in arteriosclerosis obliterans.

Long non-coding RNAs (lncRNAs) affect atherosclerosis by regulating the physiological and pathological processes of endothelial cells; however, the role of lncRNA WEE2 antisense RNA 1 (WEE2-AS1) in arteriosclerosis obliterans (ASO) is not completely understood. The present study aimed to explore the function of lncRNA WEE2-AS1 in human vascular endothelial cells. The results indicated that lncRNA WEE2-AS1 was significantly elevated in plasma and artery tissue samples of patients with ASO compared with healthy controls. The fluorescence in situ hybridization results suggested that lncRNA WEE2-AS1 was expressed in the cytoplasm and nuclei of primary human umbilical vein endothelial cells (HUVECs). The Cell Counting Kit-8 assay results suggested that lncRNA WEE2-AS1 knockdown significantly promoted HUVEC viability, whereas lncRNA WEE2-AS1 overexpression inhibited HUVEC viability compared with the negative control groups. Furthermore, analysis of the cell cycle by flow cytometry indicated that lncRNA WEE2-AS1 knockdown significantly decreased the proportion of cells in the G0/G1 phase and significantly increased the proportion of cells in the G2/M phase compared with the negative control group. However, lncRNA WEE2-AS1 overexpression had no significant effect on cell cycle distribution compared with the negative control group. The western blotting results indicated that lncRNA WEE2-AS1 knockdown significantly reduced the expression levels of phosphorylated cyclin dependent kinase 1, WEE1 homolog 2 and myelin transcription factor 1, but increased the expression level of cell division cycle 25B compared with the negative control group. lncRNA WEE2-AS1 overexpression displayed the opposite effect on protein expression. Collectively, the present study suggested that lncRNA WEE2-AS1 was significantly upregulated in ASO and may serve a role in regulating human vascular endothelial cell viability. Further investigation into lncRNA WEE2-AS1 may broaden the current understanding of the molecular mechanism underlying ASO, and aid with the identification of specific probes and precise targeted drugs for the diagnosis and treatment of ASO.

Novel WEE2 gene variants identified in patients with fertilization failure and female infertility

To determine whether variants in the WEE2 (WEE1 homolog 2, also known as WEE1B) gene, which has been known to function in the formation of pronuclei during fertilization, contribute to fertilization failure. Case-control genetic study. University hospital. Ninety infertile women with repeated cycles of pronucleus formation failure undergoing invitro fertilization and/or intracytoplasmic sperm injection treatment as well as 200 fertile control women. Genomic DNA was extracted from the peripheral blood. The whole exons of WEE2 were amplified by means of polymerase chain reaction and then Sanger sequencing was performed. Variants analysis of WEE2 gene. We identified five subjects that were subjected to homozygous or compound-heterozygous variants of WEE2: case 1 (from a consanguineous family) with homozygous frameshift variant: c.293_294insCTGAGACACCAGCCCAACC (p.Pro98Pro fsX2); case 2 with homozygous missense variant: c.1576T>G (p.Tyr526Asp); and three cases with compound-heterozygous variants: case 3: c.991C>A (p.His331Asn) and c.1304_1307delCCAA (p.Thr435Met fsX31); case 4: c.341_342 del AA (p.Lys114Asn fsX20) and c.864G>C (p.Gln288His); and case 5: c.1A>G (p.0?) and c.1261G>A (p.Gly421Arg). Besides c.1576T>G (from case 2) and c.864G>C (from case 4), which have been previously reported as rare single nucleotide polymorphisms (SNPs), the other six variants were novel and predicted by software to be deleterious. The parental genotypes of case 1 and case 2 indicated that the detected homozygous variants were inherited in an autosomal recessive mode. All of the detected variants were absent from the control cohort. Novel variants found in WEE2, which is autosomal-recessive inherited, may be related to recurrent pronucleus formation failure and female infertility.

The interaction between RACK1 and WEE1 regulates the growth of gastric cancer cell line HGC27

Receptor of activated C Kinase 1 (RACK1) is an essential scaffold and anchoring protein, which serves an important role in multiple tumorigenesis signaling pathways. The present study aimed to investigate the expression of RACK1 in gastric cancer (GC), and its association with the occurrence and development of GC. In addition, the effect and mechanism of RACK1 overexpression on the growth, and proliferation of GC cells was examined. Firstly, the protein expression of RACK1 was detected in 70 cases of GC tissues and 30 cases of noncancerous tissues using immunohistochemical staining, and the association between clinical and pathological features of GC was analyzed. Secondly, the mRNA and protein expression of RACK1 was determined in the poorly-differentiated human gastric cancer cell line HGC27 and gastric epithelial cell line GES-1. The growth of HGC27 cells following the upregulation of RACK1 was detected using MTT method. Subsequently, the interaction and co-location between RACK1, and WEE1 homolog (S. pombe) (WEE1) in HGC27 cells was confirmed using co-immunoprecipitation and indirect immunofluorescence. The expression level of RACK1 in GC was significantly lower compared with that in pericarcinous tissues (P<0.05). The protein level of RACK1 expression correlated with tumor node metastasis stage, tumor differentiation and lymph node metastasis. The mRNA and protein levels of RACK1 in HGC27 cells were significantly reduced, and overexpressed RACK1 downregulated WEE1 protein expression, thus inhibiting the growth of HGC27 cells. Co-immunoprecipitation and immunofluorescence confirmed that RACK1, and WEE1 interacted and co-located in the cytoplasm of HGC27 cells. Therefore, the abnormal expression of RACK1 in GC tissues was identified to be involved in the occurrence and development of GC. Overexpression of RACK1 was able to inhibit the growth of HGC27 cells. The current study suggests that low expression of RACK1 is an important indicator of poor prognosis of GC. RACK1 and WEE1 interact to regulate the growth of HGC27 cells.

Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth.

BackgroundThe successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments.ResultsWe used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9) and BMP binding endothelial regulator (BMPER) was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11), bone morphogenetic protein 15 (BMP15) and WEE1 homolog 2 (S. pombe)(WEE2) which play critical roles in follicular development but other biomarkers are also likely to play significant roles in this process.ConclusionsTo our knowledge, this is the first in vivo spatio-temporal exploration of transcriptomes derived from early follicles in sheep.

Cdk1 Modulation Ensures the Coordination of Cell-Cycle Events during the Switch from Meiotic Prophase to Mitosis

Budding yeast cells that enter the developmental path of meiosis do not commit to finishing meiosis until after prophase I and the realization of such meiosis-specific events as pairing of homologous chromosomes and initiation of recombination. If the meiosis-inducing signal is withdrawn prior to commitment, cells exit meiosis and return to mitosis. The timing of this transition poses a singular problem for maintaining genome integrity. Cells in meiotic prophase have already replicated their DNA, but they have not undergone the morphological changes intrinsic to mitosis, including budding. Successful re-entry into mitosis requires that these cells bud but not rereplicate their DNA, reversing the normal order of mitosis. This study focuses on the cellular mechanisms that permit this dramatically altered order of cell-cycle events. By developing a microfluidics assay to monitor individual cells, we show that the successful transition from meiotic prophase to mitosis requires the modulation of Cdk1 activity to coordinate cell-cycle events. The S. cerevisiae Wee1 homolog Swe1 prevents the formation of multinucleate cells by restraining M phase CDK activity to allow bud formation prior to nuclear division. The remaining S phase CDK activity promotes bud formation and prevents origin licensing so that DNA cannot rereplicate between bud formation and nuclear division. Once a bud has formed, M phase CDK drives cells through a normal mitotic division. Our study uncovers the essential requirement of Swe1 to modulate CDK activity to coordinate cell-cycle events and maintain genome integrity during the transition from meiotic prophase to mitosis.

Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos

The WEE1 kinase family has been shown to be the major kinase family responsible for phosphorylating Tyr 15 on cyclin-dependent kinase 1 (CDK1). WEE1 homolog 2 (WEE2, also known as WEE1B) was first identified in Xenopus laevis and more recently in humans and mice, and is responsible for phosphorylating the CDK1 inhibitory site and maintaining meiotic arrest in oocytes. However, the mechanism by which WEE1B is regulated in one-cell stage mouse embryos remains to be elucidated. In the present study, we examined the role of WEE1B-Ser 15 in G2/M transition of one-cell stage mouse embryos. WEE1B-Ser 15 was phosphorylated during the G1 and S phases, whereas Ser 15 was dephosphorylated during G2 and M phases in vivo. Overexpression of the phosphor-mimic Wee1B-S15D mutant delayed the re-entry of embryos into mitosis more efficiently than Wee1B-wild type (Wee1B-WT) by direct phosphorylation of CDK1-Tyr 15. The results of the present study suggested that WEE1B acts as a direct downstream substrate of protein kinase A (PKA) and that Ser 15 of WEE1B is a potential PKA phosphorylation target in the G2/M transition of mouse embryos. In addition, WEE1B inhibits mitosis by negatively regulating M phase promoting factor activity in one-cell stage mouse embryos.

Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces cerevisiae

Mitotic cyclin-dependent kinase (CDK) is activated by Cdc25 phosphatase through dephosphorylation at the Wee1-mediated phosphorylation site. In Saccharomyces cerevisiae, regulation of Mih1 (Cdc25 homologue) remains unclear because inactivation/degradation of Swe1 (Wee1 homologue) is the main trigger for G2/M transition. By deleting all mitotic cyclins except Clb2, a strain was created where Mih1 became essential for mitotic entry at high temperatures. Using this novel assay, the essential domain of Mih1 was identified and Mih1 regulation was characterized. Mih1(3E1D) with phosphomimetic substitutions of four putative PKC target residues in Mih1 had a reduced complementation activity, whereas Mih1(4A) with those nonphosphorylatable substitutions was active. The band pattern of Mih1 by SDS-PAGE was similar to that of Mih1(4A) only after inactivation of Pkc1 in a pkc1(ts) mutant. Over-expression of GFP-tagged Mih1 or GFP-Mih1(4A) accumulated as dot-like structures in the nucleus, whereas GFP-Mih1(3E1D) was localized in the cytoplasm. Over-expression of an active form of Pkc1 excluded GFP-Mih1 from the nucleus, but had minimal effect on GFP-Mih1(4A) localization. Furthermore, addition of ectopic nuclear localization signal to the Mih1(3E1D) sequence recovered complementation activity and nuclear localization. These results suggest that Mih1 is negatively regulated by Pkc1-mediated phosphorylation, which excludes it from the nucleus under certain conditions.

Quantitative Proteomics Reveals that miR-155 Regulates the PI3K-AKT Pathway in Diffuse Large B-Cell Lymphoma

The aberrant expression of microRNA-155 (miR-155), which has emerged as having a significant impact on the biological characteristics of lymphocytes, plays important roles in B-cell malignancies, such as diffuse large B-cell lymphoma (DLBCL). DLBCL is the most common non-Hodgkin's lymphoma in the adult population, accounting for approximately 40% of newly diagnosed non-Hodgkin's lymphoma cases globally. To determine the specific function of miR-155, a quantitative proteomics approach was applied to examine the inhibitory effects of miR-155 on protein synthesis in DLBCL cells. PIK3R1 (p85α), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, was identified as a direct target of miR-155. A luciferase reporter was repressed through the direct interaction of miR-155 and the p85α 3'-untranslated region, and overexpression of miR-155 down-regulated both the transcription and translation of p85α. The PI3K-AKT signaling pathway was highly activated by the sustained overexpression of miR-155 in DHL16 cells, whereas knockdown of miR-155 in OCI-Ly3 cells diminished AKT activity. Taken together, our results reveal a novel target involved in miR-155 biological characteristics and provide a molecular link between the overexpression of miR-155 and the activation of PI3K-AKT in DLBCL.

Expressional Difference of RHEB, HDAC1, and WEE1 Proteins in the Stromal Tumors of the Breast and Their Significance in Tumorigenesis

BackgroundFibroadenoma (FA) and phyllodes tumor (PT) are stromal tumors of breast and are histologically similar. There are no established differences in tumorigenesis and oncogene expression among them. Ras homolog enriched in brain (RHEB) plays an important role in cell growth and cell-cycle control, histone deacetylase 1 (HDAC1) is an important factor in breast tumor progression and prognosis, and WEE1 homolog (WEE1) functions as a tumor suppressor. No studies on the expressional differences of these proteins in FA and PT have been reported to date.MethodsThe expression of these proteins in FA, PT, and normal breast was compared. We used 102 cases of FA and 25 cases of benign PT.ResultsIn epithelial cells, the expression of RHEB, HDAC1, and WEE1 was lowest in PT, higher in FA, and most enhanced in normal breast. In addition, the expression of RHEB and HDAC1 was higher in the stromal cells of PT than in FA and normal breast.ConclusionsBoth epithelial and stromal cells of FA and PT express these proteins, which indicates that epithelial cells play an important role in the development of stromal tumors. In addition, the expressional differences of these proteins may be associated with the tumorigenesis of breast stromal tumors.

WEE2 Is an Oocyte-Specific Meiosis Inhibitor in Rhesus Macaque Monkeys1

WEE1 homolog 2 (WEE2, also known as WEE1B) is a newly identified member of the WEE kinase family that is conserved from yeast to humans. The aim of the present study was to determine the spatiotemporal expression pattern and the function of WEE2 during oocyte maturation in a nonhuman primate species, the rhesus macaque. Among 11 macaque tissues examined, WEE2 transcript is predominantly expressed in the ovary and only weakly detectable in the testis. Within the ovary, WEE2 mRNA is exclusively localized in the oocyte and appears to accumulate during folliculogenesis, reaching the highest level in preovulatory follicles. Microinjection of a full-length WEE2-GFP (green fluorescent protein) fusion mRNA indicates a specific nuclear localization of WEE2 protein in both growing and fully grown germinal vesicle (GV)-intact oocytes. Taking the long double-stranded RNA-mediated RNA interference approach, we found that down-regulation of WEE2 led to meiotic resumption in a subset of GV oocytes even in the presence of a phosphodiesterase 3 inhibitor. On the other hand, overexpression of WEE2 delays the reentry of oocytes into meiosis in both mice and monkeys. These findings suggest that WEE2 is a conserved oocyte-specific meiosis inhibitor that functions downstream of cAMP in nonhuman primates.

Hydroquinone, a Benzene Metabolite, Induces Hog1-dependent Stress Response Signaling and Causes Aneuploidy in Saccharomyces cerevisiae

Previously, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae by arresting the cell cycle at the G2/M transition as a result of the activation of the Hog1 (p38 MAPK homolog)-Swe1 (Wee1 homolog) pathway. In this experiment, we examined the aneuploidy forming effects of hydroquinone, a benzene metabolite, since both phenyl hydroquinone and hydroquinone are Ames-test negative carcinogens and share similar molecular structures. As was seen in phenyl hydroquinone, hydroquinone induced aneuploidy in yeast by delaying the cell cycle at the G2/M transition. Deficiencies in SWE1 and HOG1 abolished the hydroquinone-induced delay at the G2/M transition and aneuploidy formation. Furthermore, Hog1 was phosphorylated by hydroquinone, which may stabilize Swe1. These data indicate that the hydroquinone-induced G2/M transition checkpoint, which is activated by the Hog1-Swe1 pathway, plays a role in the formation of aneuploidy.

Phenyl hydroquinone, an Ames test‐negative carcinogen, induces Hog1‐dependent stress response signaling

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G(1) and G(2)/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G(2)/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G(2)/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroquinone-induced G(2)/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.

AgSwe1p Regulates Mitosis in Response to Morphogenesis and Nutrients in MultinucleatedAshbya gossypiiCells

Nuclei in the filamentous, multinucleated fungus Ashbya gossypii divide asynchronously. We have investigated what internal and external signals spatially direct mitosis within these hyphal cells. Mitoses are most common near cortical septin rings found at growing tips and branchpoints. In septin mutants, mitoses are no longer concentrated at branchpoints, suggesting that the septin rings function to locally promote mitosis near new branches. Similarly, cells lacking AgSwe1p kinase (a Wee1 homologue), AgHsl1p (a Nim1-related kinase), and AgMih1p phosphatase (the Cdc25 homologue that likely counteracts AgSwe1p activity) also have mitoses distributed randomly in the hyphae as opposed to at branchpoints. Surprisingly, however, no phosphorylation of the CDK tyrosine 18 residue, the conserved substrate of Swe1p kinases, was detected in normally growing cells. In contrast, abundant CDK tyrosine phosphorylation was apparent in starving cells, resulting in diminished nuclear density. This starvation-induced CDK phosphorylation is AgSwe1p dependent, and overexpressed AgSwe1p is sufficient to delay nuclei even in rich nutrient conditions. In starving cells lacking septins or AgSwe1p negative regulators, the nuclear density is further diminished compared with wild type. We have generated a model in which AgSwe1p may regulate mitosis in response to cell intrinsic morphogenesis cues and external nutrient availability in multinucleated cells.

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7–Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

A WEE1 homologue from Arabidopsis thaliana

Little is known about the genes that regulate cyclinB-Cdc2 complexes at the G2/M transition of the plant cell cycle although in yeast and animals cdc25 and wee1 are central regulators of cdc2. Here we describe the isolation, by reverse transcription polymerase chain reaction (RT-PCR), of a WEE1 cDNA (AtWEE1) in Arabidopsis thaliana (L.) Heynh. Semi-quantitative RT-PCR showed that AtWEE1 expression was confined to actively dividing regions of the plant. The overexpression of AtWEE1 in fission yeast (Schizosaccharomyces pombe) caused cells to arrest, and to grow but not divide, resulting in very elongated cells. Our data provide evidence for a functional WEE1 in A. thaliana.

The existence of two distinct Wee1 isoforms in Xenopus: implications for the developmental regulation of the cell cycle.

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. Here, we have isolated a second, zygotic isoform of Xenopus Wee1, termed Xe-Wee1B (or Wee1B for short), 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.

Activation of the Mitogen-activated Protein Kinase ERK1 during Meiotic Progression of Mouse Pachytene Spermatocytes

Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G(2)/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G(2)/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.