{"gene":"WEE2","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2000,"finding":"Human WEE2 (WEE1B) encodes a 561-amino-acid kinase that phosphorylates CDK1 (Cdc2) on tyrosine-15 in vitro, inactivating CDK1 kinase activity; recombinant WEE2 rescued the lethal phenotype of fission yeast wee1-50Δmik1 mutant and caused cell elongation due to G2/M arrest when overexpressed, demonstrating its catalytic role as a CDK1-inhibitory kinase.","method":"In vitro kinase assay with recombinant protein; fission yeast rescue complementation; GFP-fusion localization in HeLa cells (predominantly nuclear)","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay demonstrating Y15 phosphorylation of CDK1, complemented by yeast rescue and overexpression phenotype; multiple orthogonal methods in one study","pmids":["11029659"],"is_preprint":false},{"year":2005,"finding":"Mouse WEE1B (WEE2) is an oocyte-specific kinase that maintains meiotic (prophase I) arrest by inhibiting Cdc2/MPF; it functions downstream of PKA, and Ser15 is the major PKA phosphorylation site in vitro; phosphorylation at Ser15 enhances its inhibitory activity; RNAi-mediated knockdown in mouse oocytes (in vitro and transgenic in vivo) causes premature meiotic resumption.","method":"Small-pool expression screen of mouse oocyte cDNA library; Xenopus oocyte overexpression assay; in vitro PKA phosphorylation assay; RNAi injection; transgenic RNAi mouse model","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro phosphorylation assay identifying PKA site, combined with Xenopus functional assay, RNAi in vitro and transgenic in vivo; multiple orthogonal methods, foundational paper","pmids":["16169490"],"is_preprint":false},{"year":2006,"finding":"WEE2 is proposed (and supported by prior experimental data) to be a direct PKA substrate in oocytes; the scenario is that PKA phosphorylation activates WEE2 to maintain Cdc2 in an inactive, tyrosine-15-phosphorylated state, thereby sustaining meiotic arrest.","method":"Review synthesizing PKA phosphorylation in vitro and RNAi functional data from prior work (see PMID 16169490)","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — review consolidating experimental findings from the same lab; no new primary experiments presented beyond prior work","pmids":["16418576"],"is_preprint":false},{"year":2010,"finding":"WEE1B and MYT1 cooperate to maintain meiotic arrest in mouse oocytes and function in distinct subcellular compartments: WEE1B is nuclear during arrest and is exported to the cytoplasm shortly before germinal vesicle breakdown (GVBD), while CDC25B moves from cytoplasm to nucleus; these translocations are regulated by PKA inactivation and MPF activation respectively; mislocalized WEE1B fails to maintain meiotic arrest, demonstrating that nuclear localization is required for its meiotic arrest function.","method":"RNAi knockdown (WEE1B and MYT1 individually and combined); live imaging of GFP-fusions for subcellular localization; functional rescue experiments in mouse oocytes","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal RNAi knockdowns with defined meiotic phenotype, live imaging of protein relocalization, mislocalization functional loss-of-function; multiple orthogonal methods, replicated context","pmids":["20083600"],"is_preprint":false},{"year":2010,"finding":"WEE2 is expressed exclusively in oocytes of the rhesus macaque ovary and localizes specifically to the nucleus of germinal vesicle-stage oocytes (via WEE2-GFP fusion microinjection); dsRNA-mediated knockdown of WEE2 promotes meiotic resumption even in the presence of a PDE3 inhibitor (high cAMP condition), placing WEE2 downstream of cAMP; overexpression of WEE2 delays meiotic reentry in both mice and macaques.","method":"Tissue RT-PCR expression profiling; WEE2-GFP mRNA microinjection and live imaging; long dsRNA RNAi knockdown in macaque oocytes; WEE2 mRNA overexpression in mouse and macaque oocytes","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct nuclear localization by GFP fusion, gain- and loss-of-function in a primate model, downstream-of-cAMP placement via PDE3i experiment; multiple orthogonal methods","pmids":["20200212"],"is_preprint":false},{"year":2011,"finding":"WEE1B is required for exit from metaphase II (MII) in mouse oocytes: Ca2+ signals activate CaMKII, which activates WEE1B, leading to inhibitory phosphorylation of Cdc2 (CDK1-Y15) and enabling pronucleus formation; WEE1B knockdown prevents pronucleus formation in response to Ca2+ signals, and CaMKII-driven MII exit is blocked by WEE1B knockdown, demonstrating that CDK1 inactivation at MII exit requires both cyclin B proteolysis and WEE1B-mediated CDK1 phosphorylation.","method":"RNAi knockdown of WEE1B in mouse oocytes; Ca2+ signal manipulation; CaMKII inhibitor treatment; pronucleus formation assay as readout; epistasis between CaMKII and WEE1B","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (CaMKII→WEE1B→CDK1) established by RNAi + inhibitor double manipulations with defined pronucleus formation phenotype; rigorous mechanistic dissection in a high-impact journal","pmids":["21454751"],"is_preprint":false},{"year":2013,"finding":"In one-cell stage mouse embryos, WEE1B Ser15 is phosphorylated during G1/S phases and dephosphorylated during G2/M; overexpression of a phosphomimetic Ser15D mutant delays mitotic reentry more potently than wild-type WEE1B, mediated by direct CDK1-Tyr15 phosphorylation; this identifies PKA–WEE1B(Ser15)–CDK1(Tyr15) as a regulatory axis controlling G2/M transition in early embryos.","method":"Phospho-specific analysis in mouse embryos; mRNA overexpression of WEE1B Ser15D phosphomimetic mutant; CDK1-Tyr15 phosphorylation assay","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis combined with functional overexpression assay and CDK1 phosphorylation readout; single lab, two orthogonal methods","pmids":["23616086"],"is_preprint":false},{"year":2010,"finding":"In porcine oocytes, the PKA phosphorylation site activating WEE1B is Ser77 (not Ser15 as in mouse); mutation of Ser77 to alanine abolishes WEE1B meiotic arrest function; nuclear localization of porcine WEE1B is essential for its activity, as deletion of the NLS causes cytoplasmic redistribution and loss of meiotic arrest maintenance.","method":"PKA phosphorylation site mutagenesis (Ser→Ala substitutions) with mRNA injection into porcine oocytes; NLS deletion construct; immunohistochemistry for localization","journal":"The Journal of reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — active-site/phosphorylation-site mutagenesis with functional oocyte meiotic arrest assay and localization analysis; single lab, two orthogonal methods","pmids":["21123961"],"is_preprint":false},{"year":2018,"finding":"Homozygous loss-of-function mutations in WEE2 (missense or frameshift) cause total fertilization failure in humans; all four mutations significantly decreased WEE2 protein levels in affected individuals' oocytes, led to abnormal serine phosphorylation of WEE2 and reduced tyrosine-15 phosphorylation of Cdc2 in vitro; injection of WEE2 cRNA into affected oocytes rescued fertilization failure and enabled blastocyst formation, establishing WEE2 as essential for human fertilization through its CDK1-Y15 kinase activity.","method":"Human genetics (whole-exome + Sanger sequencing); immunofluorescence of patient oocytes; in vitro phosphorylation assay; cRNA rescue injection into patient oocytes; blastocyst formation assay","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay demonstrating loss of CDK1-Y15 phosphorylation, patient oocyte immunofluorescence, and definitive cRNA rescue experiment; multiple orthogonal methods in one rigorous study","pmids":["29606300"],"is_preprint":false},{"year":2020,"finding":"Co-crystallization of WEE2 kinase domain with WEE1 inhibitors revealed the structural basis of action across WEE kinases; type I inhibitors (including MK1775) were not selective for WEE2 over WEE1; high-throughput screening of ~26,000 compounds identified two selective WEE2 inhibitors (GPHR-00336382 binding full-length WEE2 allosterically; GPHR-00355672 binding the kinase domain); WEE2 phosphorylation of CDK1 was measured by ELISA, and inhibition of metaphase II exit was validated by bovine in vitro fertilization assay.","method":"Co-crystallization of WEE2 kinase domain; FTMap/FTSite and SiteMap in silico screening for allosteric sites; HTS of 26,000 compounds; ELISA-based CDK1 phosphorylation assay; bovine IVF assay; somatic cell proliferation assay (WEE1 selectivity)","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure plus biochemical enzyme assay and cell-based functional assay; single lab, structural result not yet replicated independently","pmids":["32667031"],"is_preprint":false},{"year":2019,"finding":"Additional biallelic mutations in WEE2 (splice-site, missense, frameshift) cause fertilization failure or poor fertilization; a splice-site mutation c.1136-2A>G causes frameshift and premature stop; missense mutations produce changes in secondary protein structure; mutant WEE2 results in loss of phosphorylated CDC2 (CDK1-Y15) as detected by immunostaining in patient oocytes, confirming the kinase-CDK1 phosphorylation axis is disrupted.","method":"Whole-exome sequencing; Sanger sequencing; single-cell RT-PCR for splice-site mutation effect in vivo; immunofluorescence for pCDC2 in patient oocytes; molecular modeling","journal":"Fertility and sterility","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunofluorescence of patient oocytes confirming loss of CDK1-Y15 phosphorylation; in vivo splice analysis; single lab, two orthogonal methods","pmids":["30827523"],"is_preprint":false},{"year":2025,"finding":"A novel WEE2 variant c.487T>A (p.Tyr163Asn) showed a 20.97% reduction in enzymatic activity in a functional assay, further confirming the kinase activity of WEE2 as essential for its biological function in fertilization.","method":"In vitro enzymatic activity assay of mutant WEE2 protein; Sanger sequencing; structural modeling","journal":"Journal of assisted reproduction and genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single enzymatic activity assay from one lab with modest effect size; no independent replication","pmids":["40399709"],"is_preprint":false},{"year":2025,"finding":"A novel WEE2 variant c.791C>T (p.Ala264Val) significantly decreased tyrosine-15 phosphorylation of Cdc2 and reduced pronucleus formation rate in vitro, confirming the functional requirement of this residue for WEE2 kinase activity toward CDK1.","method":"In vitro kinase activity assay measuring CDK1-Y15 phosphorylation; pronucleus formation assay; structural modeling","journal":"Journal of assisted reproduction and genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single in vitro functional assay from one lab; no independent replication","pmids":["40830301"],"is_preprint":false}],"current_model":"WEE2 (WEE1B) is an oocyte-specific kinase that phosphorylates CDK1 (Cdc2) on tyrosine-15 to inactivate MPF; it is activated by PKA-mediated phosphorylation (Ser15 in mouse, Ser77 in pig) to maintain prophase I meiotic arrest, requires nuclear localization for this function, and is subsequently reactivated downstream of CaMKII during egg activation to drive exit from metaphase II by again inhibiting CDK1; loss-of-function mutations in human WEE2 abolish CDK1-Y15 phosphorylation in oocytes and cause total fertilization failure, rescuable by WEE2 cRNA injection."},"narrative":{"mechanistic_narrative":"WEE2 (WEE1B) is an oocyte-specific protein kinase that controls meiotic cell-cycle progression by catalyzing inhibitory phosphorylation of CDK1 (Cdc2) on tyrosine-15, thereby inactivating MPF and gating both meiotic arrest and meiotic exit [PMID:11029659, PMID:16169490]. Biochemically it is a bona fide CDK1-Y15 kinase: recombinant WEE2 phosphorylates CDK1-Y15 in vitro and rescues the lethal phenotype of fission yeast wee1/mik1 mutants while inducing G2/M arrest when overexpressed [PMID:11029659]. In the oocyte, WEE2 acts downstream of PKA to maintain prophase I (germinal vesicle) arrest; PKA phosphorylates WEE2 at an activating serine residue (Ser15 in mouse, Ser77 in pig) that enhances its CDK1-inhibitory activity, and knockdown triggers premature meiotic resumption [PMID:16169490, PMID:21123961]. This arrest function requires nuclear localization: WEE2 is nuclear in arrested oocytes and is exported to the cytoplasm before germinal vesicle breakdown, and mislocalization or NLS deletion abolishes arrest [PMID:20083600, PMID:21123961]. WEE2 is later reactivated at egg activation, where Ca2+-driven CaMKII signaling stimulates WEE2 to re-impose CDK1-Y15 phosphorylation and drive exit from metaphase II and pronucleus formation, cooperating with cyclin B proteolysis [PMID:21454751]. In humans, biallelic loss-of-function mutations in WEE2 abolish oocyte CDK1-Y15 phosphorylation and cause total fertilization failure, a defect rescued by injection of wild-type WEE2 cRNA [PMID:29606300].","teleology":[{"year":2000,"claim":"Established WEE2 as a catalytically active CDK1-inhibitory kinase, defining its core biochemical activity before its physiological context was known.","evidence":"In vitro kinase assay with recombinant protein, fission yeast rescue complementation, and GFP-fusion localization in HeLa cells","pmids":["11029659"],"confidence":"High","gaps":["Did not establish the tissue or cell-cycle context of WEE2 function","Used heterologous systems rather than oocytes"]},{"year":2005,"claim":"Identified WEE2 as an oocyte-specific kinase maintaining prophase I arrest downstream of PKA, linking cAMP/PKA signaling to MPF suppression via an activating Ser15 phosphorylation.","evidence":"Oocyte cDNA expression screen, Xenopus overexpression, in vitro PKA phosphorylation, and RNAi in vitro and in transgenic mice","pmids":["16169490"],"confidence":"High","gaps":["Did not resolve subcellular site of action","Direct PKA substrate status inferred from in vitro phosphorylation"]},{"year":2006,"claim":"Consolidated the model that PKA-mediated phosphorylation activates WEE2 to keep CDK1 tyrosine-15-phosphorylated and sustain arrest.","evidence":"Review synthesizing prior in vitro phosphorylation and RNAi data","pmids":["16418576"],"confidence":"Medium","gaps":["No new primary data","Direct demonstration of in vivo PKA-WEE2 phosphorylation not provided"]},{"year":2010,"claim":"Showed that nuclear localization is required for WEE2 arrest function and that PKA-regulated nuclear export coordinates WEE2 with CDC25B relocalization at meiotic resumption.","evidence":"Reciprocal RNAi of WEE1B and MYT1, live imaging of GFP-fusions, and mislocalization rescue experiments in mouse oocytes","pmids":["20083600"],"confidence":"High","gaps":["Molecular machinery driving WEE2 nuclear export not identified","Quantitative contribution of WEE2 vs MYT1 not separated"]},{"year":2010,"claim":"Demonstrated WEE2 oocyte specificity and downstream-of-cAMP placement in a primate model, and defined the activating PKA site and NLS dependence in pig.","evidence":"RT-PCR expression profiling, WEE2-GFP imaging, dsRNA knockdown under PDE3 inhibition in macaque oocytes, and PKA-site/NLS mutagenesis in porcine oocytes","pmids":["20200212","21123961"],"confidence":"High","gaps":["Species divergence of the activating PKA site (Ser15 vs Ser77) mechanistically unexplained","Human PKA site not directly mapped"]},{"year":2011,"claim":"Revealed a second role for WEE2 at metaphase II exit, placing it downstream of Ca2+/CaMKII to re-inhibit CDK1 and enable pronucleus formation during egg activation.","evidence":"RNAi knockdown plus Ca2+ and CaMKII inhibitor manipulations with pronucleus formation readout establishing CaMKII to WEE1B to CDK1 epistasis in mouse oocytes","pmids":["21454751"],"confidence":"High","gaps":["How CaMKII activates WEE2 biochemically not defined","Interplay with cyclin B proteolysis timing not fully resolved"]},{"year":2013,"claim":"Extended the PKA-WEE2(Ser15)-CDK1(Tyr15) axis to G2/M control in early one-cell embryos via cell-cycle-dependent Ser15 phosphorylation.","evidence":"Phospho-specific analysis and overexpression of a Ser15D phosphomimetic with CDK1-Tyr15 readout in mouse embryos","pmids":["23616086"],"confidence":"Medium","gaps":["Based on overexpression rather than endogenous perturbation","Kinase responsible for Ser15 dephosphorylation in G2/M not identified"]},{"year":2018,"claim":"Established WEE2 as essential for human fertilization, causally linking loss-of-function mutations to total fertilization failure through abolished CDK1-Y15 phosphorylation.","evidence":"Exome/Sanger sequencing, patient oocyte immunofluorescence, in vitro phosphorylation, and cRNA rescue with blastocyst formation","pmids":["29606300"],"confidence":"High","gaps":["Mechanism by which mutations cause abnormal serine phosphorylation not fully dissected","Genotype-phenotype spectrum across patients limited"]},{"year":2019,"claim":"Expanded the human mutational spectrum and confirmed disruption of the WEE2-CDK1 phosphorylation axis as the unifying defect in fertilization failure.","evidence":"Exome/Sanger sequencing, single-cell RT-PCR of a splice variant, pCDC2 immunofluorescence in patient oocytes, and molecular modeling","pmids":["30827523"],"confidence":"Medium","gaps":["Effect of individual missense substitutions on catalysis not quantified","Partial vs complete loss not always distinguished"]},{"year":2020,"claim":"Provided structural insight into the WEE2 kinase domain and identified selective WEE2 inhibitors, distinguishing WEE2 pharmacologically from WEE1.","evidence":"Co-crystallization of the WEE2 kinase domain, HTS of ~26,000 compounds, ELISA CDK1 phosphorylation assay, and bovine IVF assay","pmids":["32667031"],"confidence":"Medium","gaps":["Structure not independently replicated","Full-length WEE2 architecture and regulatory regions not resolved"]},{"year":2025,"claim":"Additional patient-derived variants reinforced that catalytic activity toward CDK1-Y15 is the functionally critical output of WEE2.","evidence":"In vitro enzymatic and kinase activity assays of mutant WEE2 with pronucleus formation readouts and structural modeling","pmids":["40399709","40830301"],"confidence":"Low","gaps":["Single-lab functional assays without independent replication","Modest effect sizes complicate genotype-phenotype interpretation"]},{"year":null,"claim":"How PKA and CaMKII produce opposite cell-cycle outcomes through the same activating phosphorylation of WEE2, and the structural basis of full-length WEE2 regulation, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full-length regulated WEE2","Biochemical mechanism coupling CaMKII to WEE2 activation undefined","Direct in vivo PKA-WEE2 phosphorylation in human oocytes not demonstrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,5,8]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3,4,7]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[1,5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,5]}],"complexes":[],"partners":["CDK1","PKA","CAMK2","MYT1","CDC25B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P0C1S8","full_name":"Wee1-like protein kinase 2","aliases":["Wee1-like protein kinase 1B","Wee1B kinase"],"length_aa":567,"mass_kda":62.9,"function":"Oocyte-specific protein tyrosine kinase that phosphorylates and inhibits CDK1/CDC2 and acts as a key regulator of meiosis during both prophase I and metaphase II (PubMed:29606300). Required to maintain meiotic arrest in oocytes during the germinal vesicle (GV) stage, a long period of quiescence at dictyate prophase I, by phosphorylating CDK1 at 'Tyr-15', leading to inhibit CDK1 activity and prevent meiotic reentry. Also required for metaphase II exit during egg activation by phosphorylating CDK1 at 'Tyr-15', to ensure exit from meiosis in oocytes and promote pronuclear formation (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P0C1S8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/WEE2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/WEE2","total_profiled":1310},"omim":[{"mim_id":"617996","title":"OOCYTE/ZYGOTE/EMBRYO MATURATION ARREST 5; OZEMA5","url":"https://www.omim.org/entry/617996"},{"mim_id":"615774","title":"OOCYTE/ZYGOTE/EMBRYO MATURATION ARREST 1; OZEMA1","url":"https://www.omim.org/entry/615774"},{"mim_id":"614084","title":"WEE1 HOMOLOG 2; WEE2","url":"https://www.omim.org/entry/614084"},{"mim_id":"116940","title":"CYCLIN-DEPENDENT KINASE 1; CDK1","url":"https://www.omim.org/entry/116940"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Not detected","tissue_distribution":"Not detected","driving_tissues":[],"url":"https://www.proteinatlas.org/search/WEE2"},"hgnc":{"alias_symbol":["FLJ16107","WEE1B"],"prev_symbol":[]},"alphafold":{"accession":"P0C1S8","domains":[{"cath_id":"3.30.200.20","chopping":"208-291","consensus_level":"medium","plddt":86.5643,"start":208,"end":291},{"cath_id":"1.10.510.10","chopping":"296-352_373-490","consensus_level":"medium","plddt":94.0962,"start":296,"end":490}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C1S8","model_url":"https://alphafold.ebi.ac.uk/files/AF-P0C1S8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P0C1S8-F1-predicted_aligned_error_v6.png","plddt_mean":67.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=WEE2","jax_strain_url":"https://www.jax.org/strain/search?query=WEE2"},"sequence":{"accession":"P0C1S8","fasta_url":"https://rest.uniprot.org/uniprotkb/P0C1S8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P0C1S8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C1S8"}},"corpus_meta":[{"pmid":"16169490","id":"PMC_16169490","title":"Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse.","date":"2005","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/16169490","citation_count":170,"is_preprint":false},{"pmid":"29606300","id":"PMC_29606300","title":"Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29606300","citation_count":152,"is_preprint":false},{"pmid":"20083600","id":"PMC_20083600","title":"Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20083600","citation_count":125,"is_preprint":false},{"pmid":"16418576","id":"PMC_16418576","title":"New pathways from PKA to the Cdc2/cyclin B complex in oocytes: Wee1B as a potential PKA substrate.","date":"2006","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/16418576","citation_count":117,"is_preprint":false},{"pmid":"21454751","id":"PMC_21454751","title":"Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes.","date":"2011","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/21454751","citation_count":95,"is_preprint":false},{"pmid":"11029659","id":"PMC_11029659","title":"Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk-inhibitory kinases.","date":"2000","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/11029659","citation_count":55,"is_preprint":false},{"pmid":"20200212","id":"PMC_20200212","title":"WEE2 is an oocyte-specific meiosis inhibitor in rhesus macaque monkeys.","date":"2010","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/20200212","citation_count":47,"is_preprint":false},{"pmid":"36123327","id":"PMC_36123327","title":"CAF-derived exosomal WEE2-AS1 facilitates colorectal cancer progression via promoting degradation of MOB1A to inhibit the Hippo pathway.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36123327","citation_count":45,"is_preprint":false},{"pmid":"30826994","id":"PMC_30826994","title":"Homozygous missense mutation Arg207Cys in the WEE2 gene causes female infertility and fertilization failure.","date":"2019","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30826994","citation_count":35,"is_preprint":false},{"pmid":"32307083","id":"PMC_32307083","title":"LncRNA WEE2-AS1 promotes proliferation and inhibits apoptosis in triple negative breast cancer cells via regulating miR-32-5p/TOB1 axis.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32307083","citation_count":35,"is_preprint":false},{"pmid":"30471857","id":"PMC_30471857","title":"Hepatitis B virus X protein related lncRNA WEE2-AS1 promotes hepatocellular carcinoma proliferation and invasion.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30471857","citation_count":34,"is_preprint":false},{"pmid":"30827523","id":"PMC_30827523","title":"New biallelic mutations in WEE2: expanding the spectrum of mutations that cause fertilization failure or poor fertilization.","date":"2019","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/30827523","citation_count":34,"is_preprint":false},{"pmid":"36168628","id":"PMC_36168628","title":"The N6-methyladenosine-mediated lncRNA WEE2-AS1 promotes glioblastoma progression by stabilizing RPN2.","date":"2022","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/36168628","citation_count":33,"is_preprint":false},{"pmid":"30827524","id":"PMC_30827524","title":"Novel WEE2 gene variants identified in patients with fertilization failure and female infertility.","date":"2019","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/30827524","citation_count":32,"is_preprint":false},{"pmid":"31428887","id":"PMC_31428887","title":"Novel compound heterozygous mutations in WEE2 causes female infertility and fertilization failure.","date":"2019","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31428887","citation_count":31,"is_preprint":false},{"pmid":"30628060","id":"PMC_30628060","title":"Novel mutations in WEE2: Expanding the spectrum of mutations responsible for human fertilization failure.","date":"2019","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30628060","citation_count":31,"is_preprint":false},{"pmid":"33148236","id":"PMC_33148236","title":"Novel compound heterozygous mutation in WEE2 is associated with fertilization failure: case report of an infertile woman and literature review.","date":"2020","source":"BMC women's health","url":"https://pubmed.ncbi.nlm.nih.gov/33148236","citation_count":24,"is_preprint":false},{"pmid":"32838835","id":"PMC_32838835","title":"Long Noncoding RNA WEE2-AS1 Plays an Oncogenic Role in Glioblastoma by Functioning as a Molecular Sponge for MicroRNA-520f-3p.","date":"2020","source":"Oncology research","url":"https://pubmed.ncbi.nlm.nih.gov/32838835","citation_count":19,"is_preprint":false},{"pmid":"34476630","id":"PMC_34476630","title":"Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization.","date":"2021","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34476630","citation_count":17,"is_preprint":false},{"pmid":"32667031","id":"PMC_32667031","title":"Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis†.","date":"2020","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/32667031","citation_count":12,"is_preprint":false},{"pmid":"33904356","id":"PMC_33904356","title":"Clinical exome sequencing identifies novel compound heterozygous mutations of the WEE2 gene in primary infertile women with fertilization failure.","date":"2021","source":"Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/33904356","citation_count":12,"is_preprint":false},{"pmid":"23616086","id":"PMC_23616086","title":"Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos.","date":"2013","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/23616086","citation_count":9,"is_preprint":false},{"pmid":"25457679","id":"PMC_25457679","title":"Wee1B depletion promotes nuclear maturation of canine oocytes.","date":"2014","source":"Theriogenology","url":"https://pubmed.ncbi.nlm.nih.gov/25457679","citation_count":7,"is_preprint":false},{"pmid":"32190728","id":"PMC_32190728","title":"Identification and Screening of Selective WEE2 Inhibitors to Develop Non-Hormonal Contraceptives that Specifically Target Meiosis.","date":"2019","source":"ChemistrySelect","url":"https://pubmed.ncbi.nlm.nih.gov/32190728","citation_count":7,"is_preprint":false},{"pmid":"39476306","id":"PMC_39476306","title":"Novel splicing mutations in PATL2 and WEE2 cause oocyte degradation and fertilization failure.","date":"2024","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39476306","citation_count":6,"is_preprint":false},{"pmid":"33174040","id":"PMC_33174040","title":"Antisense long non‑coding RNA WEE2‑AS1 regulates human vascular endothelial cell viability via cell cycle G2/M transition in arteriosclerosis obliterans.","date":"2020","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/33174040","citation_count":6,"is_preprint":false},{"pmid":"37772619","id":"PMC_37772619","title":"Novel WEE2 homozygous mutations c.1346C>T and c.949A>T identified in primary infertile women due to unexplained fertilization failure.","date":"2023","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37772619","citation_count":5,"is_preprint":false},{"pmid":"21123961","id":"PMC_21123961","title":"Analyses of the regulatory mechanism of porcine WEE1B: the phosphorylation sites of porcine WEE1B and mouse WEE1B are different.","date":"2010","source":"The Journal of reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/21123961","citation_count":5,"is_preprint":false},{"pmid":"36568932","id":"PMC_36568932","title":"Total fertilization failure with in vitro fertilization-intracytoplasmic sperm injection related to WEE2 mutation highlights emerging importance of genetic causes of in vitro fertilization failure.","date":"2022","source":"F&S reports","url":"https://pubmed.ncbi.nlm.nih.gov/36568932","citation_count":5,"is_preprint":false},{"pmid":"37305396","id":"PMC_37305396","title":"LncRNA WEE2-AS1 knockdown inhibits the proliferation, migration and invasion of glioma cells via regulating miR-29b-2-5p/TPM3 axis.","date":"2022","source":"Oncology research","url":"https://pubmed.ncbi.nlm.nih.gov/37305396","citation_count":4,"is_preprint":false},{"pmid":"35393008","id":"PMC_35393008","title":"LncRNA WEE2-AS1 Knockdown Inhibits the Proliferation, Migration and 3 Invasion of Glioma Cells via Regulating miR-29b-2-5p/TPM3 Axis.","date":"2022","source":"Oncology research","url":"https://pubmed.ncbi.nlm.nih.gov/35393008","citation_count":3,"is_preprint":false},{"pmid":"40399709","id":"PMC_40399709","title":"Identification of novel variants and expansion of the phenotypic spectrum in PATL2, WEE2, and TUBB8 associated with human early embryonic arrest.","date":"2025","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40399709","citation_count":2,"is_preprint":false},{"pmid":"40830301","id":"PMC_40830301","title":"Novel homozygous variants in ASTL and WEE2 responsible for female infertility characterized by abnormal fertilization.","date":"2025","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40830301","citation_count":1,"is_preprint":false},{"pmid":"40979307","id":"PMC_40979307","title":"Association of WEE2 Gene Polymorphism with Fertilization Failure in Women Undergoing Intracytoplasmic Sperm Injection.","date":"2025","source":"International journal of women's health","url":"https://pubmed.ncbi.nlm.nih.gov/40979307","citation_count":0,"is_preprint":false},{"pmid":"39653355","id":"PMC_39653355","title":"[Analysis of a Chinese pedigree with female infertility due to WEE2 gene c.495del homozygous frameshifting variant induced fertilization disorder].","date":"2024","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39653355","citation_count":0,"is_preprint":false},{"pmid":"41752823","id":"PMC_41752823","title":"Identification of FDA-Approved Drugs as Potential Inhibitors of WEE2: Structure-Based Virtual Screening and Molecular Dynamics with Perspectives for Machine Learning-Assisted Prioritization.","date":"2026","source":"Life (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41752823","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20528,"output_tokens":3964,"usd":0.060522,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11696,"output_tokens":3751,"usd":0.076128,"stage2_stop_reason":"end_turn"},"total_usd":0.13665,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"Human WEE2 (WEE1B) encodes a 561-amino-acid kinase that phosphorylates CDK1 (Cdc2) on tyrosine-15 in vitro, inactivating CDK1 kinase activity; recombinant WEE2 rescued the lethal phenotype of fission yeast wee1-50Δmik1 mutant and caused cell elongation due to G2/M arrest when overexpressed, demonstrating its catalytic role as a CDK1-inhibitory kinase.\",\n      \"method\": \"In vitro kinase assay with recombinant protein; fission yeast rescue complementation; GFP-fusion localization in HeLa cells (predominantly nuclear)\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay demonstrating Y15 phosphorylation of CDK1, complemented by yeast rescue and overexpression phenotype; multiple orthogonal methods in one study\",\n      \"pmids\": [\"11029659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mouse WEE1B (WEE2) is an oocyte-specific kinase that maintains meiotic (prophase I) arrest by inhibiting Cdc2/MPF; it functions downstream of PKA, and Ser15 is the major PKA phosphorylation site in vitro; phosphorylation at Ser15 enhances its inhibitory activity; RNAi-mediated knockdown in mouse oocytes (in vitro and transgenic in vivo) causes premature meiotic resumption.\",\n      \"method\": \"Small-pool expression screen of mouse oocyte cDNA library; Xenopus oocyte overexpression assay; in vitro PKA phosphorylation assay; RNAi injection; transgenic RNAi mouse model\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro phosphorylation assay identifying PKA site, combined with Xenopus functional assay, RNAi in vitro and transgenic in vivo; multiple orthogonal methods, foundational paper\",\n      \"pmids\": [\"16169490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"WEE2 is proposed (and supported by prior experimental data) to be a direct PKA substrate in oocytes; the scenario is that PKA phosphorylation activates WEE2 to maintain Cdc2 in an inactive, tyrosine-15-phosphorylated state, thereby sustaining meiotic arrest.\",\n      \"method\": \"Review synthesizing PKA phosphorylation in vitro and RNAi functional data from prior work (see PMID 16169490)\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — review consolidating experimental findings from the same lab; no new primary experiments presented beyond prior work\",\n      \"pmids\": [\"16418576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"WEE1B and MYT1 cooperate to maintain meiotic arrest in mouse oocytes and function in distinct subcellular compartments: WEE1B is nuclear during arrest and is exported to the cytoplasm shortly before germinal vesicle breakdown (GVBD), while CDC25B moves from cytoplasm to nucleus; these translocations are regulated by PKA inactivation and MPF activation respectively; mislocalized WEE1B fails to maintain meiotic arrest, demonstrating that nuclear localization is required for its meiotic arrest function.\",\n      \"method\": \"RNAi knockdown (WEE1B and MYT1 individually and combined); live imaging of GFP-fusions for subcellular localization; functional rescue experiments in mouse oocytes\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal RNAi knockdowns with defined meiotic phenotype, live imaging of protein relocalization, mislocalization functional loss-of-function; multiple orthogonal methods, replicated context\",\n      \"pmids\": [\"20083600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"WEE2 is expressed exclusively in oocytes of the rhesus macaque ovary and localizes specifically to the nucleus of germinal vesicle-stage oocytes (via WEE2-GFP fusion microinjection); dsRNA-mediated knockdown of WEE2 promotes meiotic resumption even in the presence of a PDE3 inhibitor (high cAMP condition), placing WEE2 downstream of cAMP; overexpression of WEE2 delays meiotic reentry in both mice and macaques.\",\n      \"method\": \"Tissue RT-PCR expression profiling; WEE2-GFP mRNA microinjection and live imaging; long dsRNA RNAi knockdown in macaque oocytes; WEE2 mRNA overexpression in mouse and macaque oocytes\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct nuclear localization by GFP fusion, gain- and loss-of-function in a primate model, downstream-of-cAMP placement via PDE3i experiment; multiple orthogonal methods\",\n      \"pmids\": [\"20200212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"WEE1B is required for exit from metaphase II (MII) in mouse oocytes: Ca2+ signals activate CaMKII, which activates WEE1B, leading to inhibitory phosphorylation of Cdc2 (CDK1-Y15) and enabling pronucleus formation; WEE1B knockdown prevents pronucleus formation in response to Ca2+ signals, and CaMKII-driven MII exit is blocked by WEE1B knockdown, demonstrating that CDK1 inactivation at MII exit requires both cyclin B proteolysis and WEE1B-mediated CDK1 phosphorylation.\",\n      \"method\": \"RNAi knockdown of WEE1B in mouse oocytes; Ca2+ signal manipulation; CaMKII inhibitor treatment; pronucleus formation assay as readout; epistasis between CaMKII and WEE1B\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (CaMKII→WEE1B→CDK1) established by RNAi + inhibitor double manipulations with defined pronucleus formation phenotype; rigorous mechanistic dissection in a high-impact journal\",\n      \"pmids\": [\"21454751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In one-cell stage mouse embryos, WEE1B Ser15 is phosphorylated during G1/S phases and dephosphorylated during G2/M; overexpression of a phosphomimetic Ser15D mutant delays mitotic reentry more potently than wild-type WEE1B, mediated by direct CDK1-Tyr15 phosphorylation; this identifies PKA–WEE1B(Ser15)–CDK1(Tyr15) as a regulatory axis controlling G2/M transition in early embryos.\",\n      \"method\": \"Phospho-specific analysis in mouse embryos; mRNA overexpression of WEE1B Ser15D phosphomimetic mutant; CDK1-Tyr15 phosphorylation assay\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis combined with functional overexpression assay and CDK1 phosphorylation readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"23616086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In porcine oocytes, the PKA phosphorylation site activating WEE1B is Ser77 (not Ser15 as in mouse); mutation of Ser77 to alanine abolishes WEE1B meiotic arrest function; nuclear localization of porcine WEE1B is essential for its activity, as deletion of the NLS causes cytoplasmic redistribution and loss of meiotic arrest maintenance.\",\n      \"method\": \"PKA phosphorylation site mutagenesis (Ser→Ala substitutions) with mRNA injection into porcine oocytes; NLS deletion construct; immunohistochemistry for localization\",\n      \"journal\": \"The Journal of reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — active-site/phosphorylation-site mutagenesis with functional oocyte meiotic arrest assay and localization analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"21123961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Homozygous loss-of-function mutations in WEE2 (missense or frameshift) cause total fertilization failure in humans; all four mutations significantly decreased WEE2 protein levels in affected individuals' oocytes, led to abnormal serine phosphorylation of WEE2 and reduced tyrosine-15 phosphorylation of Cdc2 in vitro; injection of WEE2 cRNA into affected oocytes rescued fertilization failure and enabled blastocyst formation, establishing WEE2 as essential for human fertilization through its CDK1-Y15 kinase activity.\",\n      \"method\": \"Human genetics (whole-exome + Sanger sequencing); immunofluorescence of patient oocytes; in vitro phosphorylation assay; cRNA rescue injection into patient oocytes; blastocyst formation assay\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay demonstrating loss of CDK1-Y15 phosphorylation, patient oocyte immunofluorescence, and definitive cRNA rescue experiment; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"29606300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Co-crystallization of WEE2 kinase domain with WEE1 inhibitors revealed the structural basis of action across WEE kinases; type I inhibitors (including MK1775) were not selective for WEE2 over WEE1; high-throughput screening of ~26,000 compounds identified two selective WEE2 inhibitors (GPHR-00336382 binding full-length WEE2 allosterically; GPHR-00355672 binding the kinase domain); WEE2 phosphorylation of CDK1 was measured by ELISA, and inhibition of metaphase II exit was validated by bovine in vitro fertilization assay.\",\n      \"method\": \"Co-crystallization of WEE2 kinase domain; FTMap/FTSite and SiteMap in silico screening for allosteric sites; HTS of 26,000 compounds; ELISA-based CDK1 phosphorylation assay; bovine IVF assay; somatic cell proliferation assay (WEE1 selectivity)\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus biochemical enzyme assay and cell-based functional assay; single lab, structural result not yet replicated independently\",\n      \"pmids\": [\"32667031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Additional biallelic mutations in WEE2 (splice-site, missense, frameshift) cause fertilization failure or poor fertilization; a splice-site mutation c.1136-2A>G causes frameshift and premature stop; missense mutations produce changes in secondary protein structure; mutant WEE2 results in loss of phosphorylated CDC2 (CDK1-Y15) as detected by immunostaining in patient oocytes, confirming the kinase-CDK1 phosphorylation axis is disrupted.\",\n      \"method\": \"Whole-exome sequencing; Sanger sequencing; single-cell RT-PCR for splice-site mutation effect in vivo; immunofluorescence for pCDC2 in patient oocytes; molecular modeling\",\n      \"journal\": \"Fertility and sterility\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunofluorescence of patient oocytes confirming loss of CDK1-Y15 phosphorylation; in vivo splice analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"30827523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A novel WEE2 variant c.487T>A (p.Tyr163Asn) showed a 20.97% reduction in enzymatic activity in a functional assay, further confirming the kinase activity of WEE2 as essential for its biological function in fertilization.\",\n      \"method\": \"In vitro enzymatic activity assay of mutant WEE2 protein; Sanger sequencing; structural modeling\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single enzymatic activity assay from one lab with modest effect size; no independent replication\",\n      \"pmids\": [\"40399709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A novel WEE2 variant c.791C>T (p.Ala264Val) significantly decreased tyrosine-15 phosphorylation of Cdc2 and reduced pronucleus formation rate in vitro, confirming the functional requirement of this residue for WEE2 kinase activity toward CDK1.\",\n      \"method\": \"In vitro kinase activity assay measuring CDK1-Y15 phosphorylation; pronucleus formation assay; structural modeling\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single in vitro functional assay from one lab; no independent replication\",\n      \"pmids\": [\"40830301\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WEE2 (WEE1B) is an oocyte-specific kinase that phosphorylates CDK1 (Cdc2) on tyrosine-15 to inactivate MPF; it is activated by PKA-mediated phosphorylation (Ser15 in mouse, Ser77 in pig) to maintain prophase I meiotic arrest, requires nuclear localization for this function, and is subsequently reactivated downstream of CaMKII during egg activation to drive exit from metaphase II by again inhibiting CDK1; loss-of-function mutations in human WEE2 abolish CDK1-Y15 phosphorylation in oocytes and cause total fertilization failure, rescuable by WEE2 cRNA injection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WEE2 (WEE1B) is an oocyte-specific protein kinase that controls meiotic cell-cycle progression by catalyzing inhibitory phosphorylation of CDK1 (Cdc2) on tyrosine-15, thereby inactivating MPF and gating both meiotic arrest and meiotic exit [#0, #1]. Biochemically it is a bona fide CDK1-Y15 kinase: recombinant WEE2 phosphorylates CDK1-Y15 in vitro and rescues the lethal phenotype of fission yeast wee1/mik1 mutants while inducing G2/M arrest when overexpressed [#0]. In the oocyte, WEE2 acts downstream of PKA to maintain prophase I (germinal vesicle) arrest; PKA phosphorylates WEE2 at an activating serine residue (Ser15 in mouse, Ser77 in pig) that enhances its CDK1-inhibitory activity, and knockdown triggers premature meiotic resumption [#1, #7]. This arrest function requires nuclear localization: WEE2 is nuclear in arrested oocytes and is exported to the cytoplasm before germinal vesicle breakdown, and mislocalization or NLS deletion abolishes arrest [#3, #7]. WEE2 is later reactivated at egg activation, where Ca2+-driven CaMKII signaling stimulates WEE2 to re-impose CDK1-Y15 phosphorylation and drive exit from metaphase II and pronucleus formation, cooperating with cyclin B proteolysis [#5]. In humans, biallelic loss-of-function mutations in WEE2 abolish oocyte CDK1-Y15 phosphorylation and cause total fertilization failure, a defect rescued by injection of wild-type WEE2 cRNA [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established WEE2 as a catalytically active CDK1-inhibitory kinase, defining its core biochemical activity before its physiological context was known.\",\n      \"evidence\": \"In vitro kinase assay with recombinant protein, fission yeast rescue complementation, and GFP-fusion localization in HeLa cells\",\n      \"pmids\": [\"11029659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the tissue or cell-cycle context of WEE2 function\", \"Used heterologous systems rather than oocytes\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified WEE2 as an oocyte-specific kinase maintaining prophase I arrest downstream of PKA, linking cAMP/PKA signaling to MPF suppression via an activating Ser15 phosphorylation.\",\n      \"evidence\": \"Oocyte cDNA expression screen, Xenopus overexpression, in vitro PKA phosphorylation, and RNAi in vitro and in transgenic mice\",\n      \"pmids\": [\"16169490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve subcellular site of action\", \"Direct PKA substrate status inferred from in vitro phosphorylation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Consolidated the model that PKA-mediated phosphorylation activates WEE2 to keep CDK1 tyrosine-15-phosphorylated and sustain arrest.\",\n      \"evidence\": \"Review synthesizing prior in vitro phosphorylation and RNAi data\",\n      \"pmids\": [\"16418576\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No new primary data\", \"Direct demonstration of in vivo PKA-WEE2 phosphorylation not provided\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that nuclear localization is required for WEE2 arrest function and that PKA-regulated nuclear export coordinates WEE2 with CDC25B relocalization at meiotic resumption.\",\n      \"evidence\": \"Reciprocal RNAi of WEE1B and MYT1, live imaging of GFP-fusions, and mislocalization rescue experiments in mouse oocytes\",\n      \"pmids\": [\"20083600\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular machinery driving WEE2 nuclear export not identified\", \"Quantitative contribution of WEE2 vs MYT1 not separated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated WEE2 oocyte specificity and downstream-of-cAMP placement in a primate model, and defined the activating PKA site and NLS dependence in pig.\",\n      \"evidence\": \"RT-PCR expression profiling, WEE2-GFP imaging, dsRNA knockdown under PDE3 inhibition in macaque oocytes, and PKA-site/NLS mutagenesis in porcine oocytes\",\n      \"pmids\": [\"20200212\", \"21123961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Species divergence of the activating PKA site (Ser15 vs Ser77) mechanistically unexplained\", \"Human PKA site not directly mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a second role for WEE2 at metaphase II exit, placing it downstream of Ca2+/CaMKII to re-inhibit CDK1 and enable pronucleus formation during egg activation.\",\n      \"evidence\": \"RNAi knockdown plus Ca2+ and CaMKII inhibitor manipulations with pronucleus formation readout establishing CaMKII to WEE1B to CDK1 epistasis in mouse oocytes\",\n      \"pmids\": [\"21454751\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CaMKII activates WEE2 biochemically not defined\", \"Interplay with cyclin B proteolysis timing not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the PKA-WEE2(Ser15)-CDK1(Tyr15) axis to G2/M control in early one-cell embryos via cell-cycle-dependent Ser15 phosphorylation.\",\n      \"evidence\": \"Phospho-specific analysis and overexpression of a Ser15D phosphomimetic with CDK1-Tyr15 readout in mouse embryos\",\n      \"pmids\": [\"23616086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Based on overexpression rather than endogenous perturbation\", \"Kinase responsible for Ser15 dephosphorylation in G2/M not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established WEE2 as essential for human fertilization, causally linking loss-of-function mutations to total fertilization failure through abolished CDK1-Y15 phosphorylation.\",\n      \"evidence\": \"Exome/Sanger sequencing, patient oocyte immunofluorescence, in vitro phosphorylation, and cRNA rescue with blastocyst formation\",\n      \"pmids\": [\"29606300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which mutations cause abnormal serine phosphorylation not fully dissected\", \"Genotype-phenotype spectrum across patients limited\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded the human mutational spectrum and confirmed disruption of the WEE2-CDK1 phosphorylation axis as the unifying defect in fertilization failure.\",\n      \"evidence\": \"Exome/Sanger sequencing, single-cell RT-PCR of a splice variant, pCDC2 immunofluorescence in patient oocytes, and molecular modeling\",\n      \"pmids\": [\"30827523\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect of individual missense substitutions on catalysis not quantified\", \"Partial vs complete loss not always distinguished\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided structural insight into the WEE2 kinase domain and identified selective WEE2 inhibitors, distinguishing WEE2 pharmacologically from WEE1.\",\n      \"evidence\": \"Co-crystallization of the WEE2 kinase domain, HTS of ~26,000 compounds, ELISA CDK1 phosphorylation assay, and bovine IVF assay\",\n      \"pmids\": [\"32667031\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure not independently replicated\", \"Full-length WEE2 architecture and regulatory regions not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Additional patient-derived variants reinforced that catalytic activity toward CDK1-Y15 is the functionally critical output of WEE2.\",\n      \"evidence\": \"In vitro enzymatic and kinase activity assays of mutant WEE2 with pronucleus formation readouts and structural modeling\",\n      \"pmids\": [\"40399709\", \"40830301\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single-lab functional assays without independent replication\", \"Modest effect sizes complicate genotype-phenotype interpretation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PKA and CaMKII produce opposite cell-cycle outcomes through the same activating phosphorylation of WEE2, and the structural basis of full-length WEE2 regulation, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length regulated WEE2\", \"Biochemical mechanism coupling CaMKII to WEE2 activation undefined\", \"Direct in vivo PKA-WEE2 phosphorylation in human oocytes not demonstrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 5, 8]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 4, 7]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDK1\", \"PKA\", \"CAMK2\", \"MYT1\", \"CDC25B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}