{"gene":"WEE1","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":1993,"finding":"Human WEE1 kinase phosphorylates p34cdc2 exclusively on Tyr15 (not Thr14) in vitro, and mutation of the catalytic lysine (Lys114) abolishes both in vitro kinase activity and in vivo mitotic inhibitor function, establishing WEE1 as a Tyr15-specific kinase that inhibits mitosis by directly phosphorylating p34cdc2.","method":"In vitro kinase assay with purified human WEE1, active-site mutagenesis (Lys114), overexpression in HeLa cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein, active-site mutagenesis, and in vivo functional validation in a single rigorous study","pmids":["8428596"],"is_preprint":false},{"year":1991,"finding":"Human WEE1 (WEE1Hu) functionally complements fission yeast wee1 mutations and causes G2/M delay when overexpressed in fission yeast, establishing functional conservation of the mitotic inhibitor role.","method":"Transcomplementation of yeast wee1 mutant; overexpression in S. pombe","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via transcomplementation and overexpression phenotype, foundational paper replicated by subsequent work","pmids":["1840647"],"is_preprint":false},{"year":1993,"finding":"Fission yeast Nim1 kinase directly phosphorylates and inactivates Wee1 in vitro, inhibiting its ability to phosphorylate Cdc2 on Tyr15; this phosphorylation of Wee1 by Nim1 promotes mitotic entry.","method":"In vitro kinase assay with purified Nim1 and Wee1; in vivo phosphorylation state analysis in nim1-overexpressing and nim1-null yeast","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — two independent papers (PMIDs 8515818 and 8515817) using in vitro reconstitution with purified kinases confirming same finding","pmids":["8515818","8515817"],"is_preprint":false},{"year":1995,"finding":"Human WEE1 (~94 kDa) accounts for most of the CDC2 Tyr15-phosphorylating activity in HeLa cell lysates (shown by antibody depletion), is suppressed during M phase, and its inhibitory state during M phase requires protein phosphorylation (demonstrated by re-activation upon removal of phosphatase inhibitors).","method":"Antibody depletion from HeLa cell lysates; in vitro kinase assay; cell synchronization with/without phosphatase inhibitors","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — antibody depletion with in vitro assay readout, single lab with two orthogonal methods","pmids":["7774574"],"is_preprint":false},{"year":1995,"finding":"Human WEE1 localizes almost exclusively to the nucleus during interphase, relocates to the cytoplasm at mitotic entry, and associates with the midbody/midbody bridges at the end of mitosis in a microtubule-dependent manner, indicating cell cycle-regulated subcellular redistribution.","method":"Immunofluorescence microscopy of HeLa and IMR90 cells throughout the cell cycle; microtubule depolymerization experiments","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by immunofluorescence with functional context, single lab","pmids":["7673359"],"is_preprint":false},{"year":2004,"finding":"SCF(β-TrCP1/2) is the E3 ubiquitin ligase responsible for WEE1A ubiquitination and degradation at mitotic entry. Plk1 phosphorylates WEE1A at Ser53 and Cdc2 phosphorylates it at Ser123, creating unconventional phosphodegrons recognized by β-TrCP; these two phosphorylations cooperatively stimulate WEE1A ubiquitination. Depletion of β-TrCP or mutation of S53/S123 stabilizes WEE1A and delays mitotic onset.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis, siRNA depletion, HeLa cell synchronization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitination reconstitution, mutagenesis of phosphodegron sites, siRNA validation, and in vivo mitotic timing assay in a single comprehensive study","pmids":["15070733"],"is_preprint":false},{"year":2005,"finding":"CDK-mediated phosphorylation of WEE1A at Ser123 promotes β-TrCP binding through three independent mechanisms: direct interaction of pSer123 with WD40 repeats of β-TrCP; creation of a polo-box domain-binding motif (SpSP) that accelerates Plk1-mediated phosphorylation of Ser53; and priming of CK2-dependent phosphorylation of Ser121, generating a second β-TrCP-binding site. CK1δ also contributes to WEE1 degradation.","method":"In vitro binding assays, mutagenesis, CK2 inhibitor treatment, siRNA, cell synchronization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal biochemical methods (in vitro binding, mutagenesis, pharmacological inhibition, siRNA) in a single study","pmids":["16085715"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of the catalytic domain of human somatic WEE1 (WEE1A) at 1.8 Å resolution reveals that despite being functionally a tyrosine kinase, it most closely resembles serine/threonine kinases (Chk1, cAMP kinases) in sequence and structure; the activation segment has Wee1-specific features maintaining an active conformation, and a glycine-rich loop substitution helps determine substrate specificity for Tyr15.","method":"X-ray crystallography (1.8 Å resolution) with active-site inhibitor complex","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with functional validation of substrate specificity","pmids":["15837193"],"is_preprint":false},{"year":1992,"finding":"In fission yeast, functional wee1 protein kinase is required for radiation-induced mitotic delay, establishing Wee1 as a necessary component of the DNA damage checkpoint that delays mitosis in response to gamma-irradiation.","method":"Genetic epistasis using wee1 mutants exposed to gamma-irradiation; cell cycle progression analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic loss-of-function with defined cell cycle phenotype, foundational study independently replicated","pmids":["1549179"],"is_preprint":false},{"year":2000,"finding":"In fission yeast, the G2 DNA damage checkpoint simultaneously upregulates Wee1p and downregulates Cdc25p; inactivation of both wee1+ and cdc25+ is required to abolish checkpoint arrest. Chk1p directly phosphorylates Wee1p in vitro and its overexpression causes wee1+-dependent G2 arrest with Wee1p hyperphosphorylation. A transient increase in Wee1p levels is induced by G2 DNA damage checkpoint activation.","method":"Genetic epistasis (wee1 and cdc25 double mutants), in vitro Chk1 kinase assay with Wee1 as substrate, overexpression studies, immunoblotting","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genetic epistasis combined with in vitro kinase assay, multiple orthogonal methods","pmids":["10769204"],"is_preprint":false},{"year":2004,"finding":"Drosophila Wee1 (dWee1) phosphorylates Cdk1 at tyrosine 15 and times mitotic entry during syncytial blastoderm embryogenesis; loss of maternal dwee1 causes premature mitotic entry, spindle defects, chromosome condensation problems, and Chk2-dependent developmental arrest.","method":"Genetic loss-of-function (maternal dwee1 mutants), phospho-Tyr15 immunoblot, live imaging in Drosophila embryos","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with multiple defined phenotypic readouts, in vivo validation","pmids":["15589158"],"is_preprint":false},{"year":2013,"finding":"WEE1 phosphorylates histone H2B at Tyr37 in nucleosomes upstream of the histone gene cluster, suppressing histone transcription in late S phase, establishing WEE1 as an epigenetic modifier with a role in coordinating histone synthesis with cell cycle progression.","method":"Biochemical analysis of H2B Tyr37 phosphorylation, histone gene transcription assays following WEE1 manipulation","journal":"Trends in genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab reporting a novel substrate/mechanism; full methods not detailed in abstract","pmids":["23537585"],"is_preprint":false},{"year":2014,"finding":"CK1δ (casein kinase 1δ) promotes WEE1 protein degradation; pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ reduces WEE1 turnover, arresting HeLa cell proliferation.","method":"Reporter assay (K328M-Wee1-luciferase), kinase-directed chemical library screen, CK1δ inhibitors, siRNA knockdown, conditional deletion","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter-based screen followed by pharmacological and genetic validation, single lab with multiple orthogonal approaches","pmids":["24817118"],"is_preprint":false},{"year":2010,"finding":"Wee1/Swe1 phosphorylates Hsp90 at a conserved tyrosine residue; this phosphorylation is important for Wee1/Swe1 association with Hsp90 and for Wee1/Swe1 stability. Non-phosphorylatable yHsp90-Y24F yeast, like swe1Δ cells, undergo premature nuclear division insensitive to G2/M checkpoint arrest.","method":"Yeast genetic analysis (swe1Δ, hsp90-Y24F mutants), cell cycle analysis, immunoprecipitation, geldanamycin sensitivity assays","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical evidence in yeast with multiple phenotypic readouts, single lab","pmids":["20519952"],"is_preprint":false},{"year":2019,"finding":"WEE1 inhibition suppresses CDK1 and CDK2 kinase activities, and WEE1 activity is required to sustain ATR/Chk1 signaling under replicative stress; mechanistically, WEE1 inhibition activates Cdk1/2 and Plk1, which reduce Claspin and CtIP levels to impair ATR/Chk1 signaling.","method":"Pharmacological inhibition (MK-1775, ATR and Chk1 inhibitors), siRNA knockdown, immunoblotting for Claspin and CtIP, CDK activity assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple inhibitors and knockdown with downstream mechanistic readouts, single lab","pmids":["25965828"],"is_preprint":false},{"year":2019,"finding":"WEE1 inhibitor AZD1775 induces CDK1-dependent RIF1 phosphorylation and CDK2/CDC7-dependent replicative helicase activation, demonstrating that WEE1 suppresses CDK1 and CDK2 to regulate origin firing at the G1/S transition.","method":"Pharmacological inhibition (AZD1775), phosphoproteomics, immunoblotting, flow cytometry in unperturbed G1 and S-phase cells","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological perturbation with multiple downstream readouts, single lab","pmids":["31712441"],"is_preprint":false},{"year":2022,"finding":"WEE1 activity guards against nascent DNA degradation at stalled replication forks by suppressing CDK2 activity; DNA2 is identified as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells, and WEE1's fork protection role is unique among CDK suppressors (CHK1 and p21 do not promote fork protection as WEE1 does).","method":"DNA fiber assay, WEE1 inhibitor (AZD1775), DNA2 inhibitor/knockdown, CDK2 inhibition, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with fiber assay readout, single lab","pmids":["35045293"],"is_preprint":false},{"year":2023,"finding":"Upon DNA damage, CHK1-dependent phosphorylation of WEE1 at Ser642 primes GCN5-mediated acetylation at Lys177, causing dissociation of an inhibitory segment from the kinase domain and activating WEE1. SIRT1 deacetylates WEE1 at Lys177, maintaining it in an inactive state; SIRT1 deficiency leads to WEE1 hyperacetylation and activation, conferring resistance to WEE1 inhibitors.","method":"In vitro kinase assays, acetylation/deacetylation biochemical assays, site-directed mutagenesis, Co-IP, immunoblotting, cancer cell line functional studies","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — identification of writer (GCN5) and eraser (SIRT1) for WEE1 acetylation with mechanistic mutagenesis, multiple orthogonal methods in a single study","pmids":["36635566"],"is_preprint":false},{"year":2018,"finding":"In fission yeast, Cdr2 cortical nodes recruit Wee1 in short bursts in a manner requiring Cdr2 kinase activity and the non-catalytic N-terminus of Wee1; Wee1 localization bursts at nodes increase 20-fold as cells double in size during G2, partially suppressed by the Cdr2 inhibitor Pom1 in small cells, establishing a size-dependent mechanism for Wee1 inhibition at nodes by Cdr1 and Cdr2 kinases.","method":"TIRF microscopy live-cell imaging, biochemical fractionation, kinase-dead mutant analysis, genetic epistasis (pom1 mutants)","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging combined with biochemical fractionation and genetics, multiple orthogonal methods in single study","pmids":["29514920"],"is_preprint":false},{"year":2017,"finding":"In fission yeast, both Wee1 and Cdc25 undergo cell cycle-dependent phosphorylation changes that are dependent on PP2A associated with regulatory subunit Pab1, indicating a conserved PP2A-dependent mechanism for controlling Wee1 across eukaryotes.","method":"Phosphorylation state analysis by SDS-PAGE, genetic analysis in S. pombe PP2A mutants","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic and biochemical analysis in fission yeast, single lab, limited mechanistic detail in abstract","pmids":["28103117"],"is_preprint":false},{"year":2007,"finding":"Human prostate epithelial cells (HPECs) express low levels of WEE1A and fail to enforce DNA damage checkpoint arrest due to a lack of inhibitory CDK phosphorylation; ectopic WEE1A expression rescues checkpoint arrest in gamma-irradiated HPECs, establishing WEE1A as rate-limiting for checkpoint enforcement in this cell type.","method":"Gamma-irradiation of primary HPECs and ex vivo prostate tissue, CDK2 kinase assay, ectopic WEE1A expression rescue, immunoblotting, flow cytometry","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function context with quantitative kinase assay and gain-of-function rescue, multiple orthogonal methods","pmids":["17431037"],"is_preprint":false},{"year":2024,"finding":"Molecular glue degraders (WEE1-targeting glutarimide-containing compounds) form a ternary complex with CRBN-DDB1 and WEE1; crystal structure of the hit compound with CRBN-DDB1-WEE1 defines the protein-protein interface and rationalizes kinase selectivity for WEE1 degradation.","method":"Crystal structure of CRBN-DDB1-WEE1-compound ternary complex, multicomponent combinatorial library synthesis and screening, degradation assays","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of ternary complex with functional degradation validation, single lab","pmids":["39499896"],"is_preprint":false},{"year":2021,"finding":"WEE1 inhibition activates the STING-TBK1-IRF3 pathway, increases type I interferons and pro-inflammatory chemokines, and concomitantly activates STAT1, increasing IFN-γ and PD-L1 expression in SCLC models, demonstrating a cell-autonomous immune-stimulating mechanism.","method":"WEE1 inhibitor (AZD1775) treatment of SCLC cell lines, pathway activation immunoblotting, immunocompetent mouse models, cytokine measurement, CD8+ T cell analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological perturbation with multiple pathway readouts and in vivo validation, single lab","pmids":["35584676"],"is_preprint":false},{"year":2021,"finding":"WEE1 inhibition up-regulates endogenous retroviral elements (ERVs) by relieving SETDB1/H3K9me3 repression through downregulation of FOXM1; ERVs trigger dsRNA stress and interferon response, increasing CD8+ T cell infiltration and PD-L1 expression, providing mechanistic basis for WEE1 inhibitor + immune checkpoint blockade synergy.","method":"WEE1 inhibitor treatment, ERV expression analysis, chromatin immunoprecipitation (H3K9me3/SETDB1), FOXM1 knockdown, dsRNA pathway reporter assays, in vivo tumor models","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, gene expression, and functional immune assays, single lab","pmids":["34825915"],"is_preprint":false},{"year":2011,"finding":"In Drosophila, wee1 mediates checkpoint-dependent delays in chromosome condensation initiation and rate caused by S-phase and topoisomerase inhibitors; wee1 also mediates delayed anaphase entry in response to chromosome condensation defects independently of the spindle assembly checkpoint.","method":"Live imaging of early Drosophila embryos, pharmacological inhibition of S-phase and topoisomerase, genetic analysis with wee1 mutants, spindle assembly checkpoint mutant controls","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging combined with genetic and pharmacological perturbations, single lab","pmids":["22262459"],"is_preprint":false},{"year":2002,"finding":"In Xenopus, two Wee1 isoforms (Wee1A and Wee1B) differentially inhibit Cdc2; Wee1B more strongly inhibits Cdc2/oocyte maturation than Wee1A due to its shorter C-terminal regulatory domain, while Wee1B is more labile during meiosis due to N-terminal PEST-like sequences, establishing isoform-specific regulatory domains.","method":"Ectopic expression in Xenopus oocytes and embryos, Cdc2 activity assays, domain deletion analysis, developmental cell division assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional domain analysis with kinase activity assays in Xenopus, single lab","pmids":["12006499"],"is_preprint":false},{"year":2019,"finding":"WEE1 activity is required to protect against nascent DNA degradation during replication stress; WEE1 inhibition combined with PARP inhibitor olaparib produces radiosensitization that is not rescued by nucleosides and requires PARP1 trapping (not just catalytic inhibition), while WEE1 inhibitor alone radiosensitizes via nucleotide depletion/replication stress.","method":"Clonogenic survival assays, nucleoside rescue experiments, PARP1 depletion, veliparib vs. olaparib comparison, KRAS-mutant NSCLC cell lines","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection using pharmacological and genetic tools, single lab","pmids":["29133592"],"is_preprint":false}],"current_model":"WEE1 is a nuclear tyrosine kinase that maintains cells in interphase by phosphorylating CDK1 (p34cdc2) exclusively at Tyr15 (and CDK2) to inhibit CDK activity; at mitotic entry, WEE1 is itself inactivated by phosphorylation (by Plk1 at Ser53 and CDK at Ser123 creating phosphodegrons) leading to SCF(β-TrCP)-mediated ubiquitination and proteasomal degradation, while upstream kinases Nim1/Cdr1 in yeast and CHK1 in response to DNA damage directly phosphorylate and regulate WEE1 activity; WEE1 also phosphorylates histone H2B at Tyr37 to suppress histone transcription, protects stalled replication forks by limiting CDK2 activity, and its catalytic activity is further regulated by GCN5-mediated acetylation (activating) and SIRT1-mediated deacetylation (inactivating) at Lys177, with the crystal structure of its catalytic domain revealing an atypical serine/threonine kinase fold adapted for tyrosine substrate specificity."},"narrative":{"mechanistic_narrative":"WEE1 is a nuclear protein kinase that restrains entry into mitosis by phosphorylating the cyclin-dependent kinase CDK1 (p34cdc2) exclusively at Tyr15, thereby holding cells in interphase until division is appropriate [PMID:8428596, PMID:1840647]. Although it functions biochemically as a tyrosine kinase, its catalytic domain adopts a fold most closely resembling serine/threonine kinases, with a Wee1-specific activation segment and a glycine-rich loop substitution that together dictate Tyr15 substrate specificity [PMID:15837193]. This activity is conserved across eukaryotes: human WEE1 complements fission yeast wee1 mutants, and orthologs time mitotic entry in yeast and Drosophila embryos [PMID:1840647, PMID:15589158]. WEE1 enforces cell-cycle checkpoints—loss of yeast Wee1 abolishes radiation-induced mitotic delay, and WEE1A is rate-limiting for DNA-damage checkpoint arrest in human cells [PMID:1549179, PMID:17431037]. WEE1 abundance and activity are tightly regulated: at mitotic entry, Plk1 phosphorylation at Ser53 and CDK phosphorylation at Ser123 generate phosphodegrons recognized by the SCF(β-TrCP) ubiquitin ligase, with CK2, CK1δ, and a polo-box-binding motif reinforcing degron formation, driving WEE1 ubiquitination and proteasomal degradation [PMID:15070733, PMID:16085715, PMID:24817118]. Conversely, in response to DNA damage CHK1-dependent phosphorylation at Ser642 primes GCN5-mediated acetylation at Lys177 to activate WEE1, an activation reversed by SIRT1 deacetylation [PMID:36635566, PMID:10769204]. Beyond mitotic gating, WEE1 suppresses CDK2 activity to protect stalled replication forks from DNA2-mediated nascent-DNA degradation and to restrain origin firing [PMID:35045293, PMID:31712441], and it phosphorylates histone H2B at Tyr37 to suppress histone gene transcription, coupling histone synthesis to cell-cycle progression [PMID:23537585].","teleology":[{"year":1991,"claim":"Established that the human gene encodes a bona fide mitotic inhibitor by showing functional conservation with the yeast cell-size/cell-cycle regulator.","evidence":"Transcomplementation of S. pombe wee1 mutants and overexpression-induced G2/M delay","pmids":["1840647"],"confidence":"High","gaps":["Did not define the molecular substrate or biochemical activity","Conservation shown genetically, not biochemically"]},{"year":1992,"claim":"Placed Wee1 within the DNA damage response by showing its kinase activity is required to delay mitosis after irradiation, linking it to checkpoint control.","evidence":"Genetic epistasis with wee1 mutants under gamma-irradiation in fission yeast","pmids":["1549179"],"confidence":"High","gaps":["Upstream signal connecting damage to Wee1 not defined","Substrate of the checkpoint-dependent activity not identified here"]},{"year":1993,"claim":"Defined the core biochemical reaction—Tyr15-specific phosphorylation of CDK1—and proved catalytic activity is essential for mitotic inhibition.","evidence":"In vitro kinase assay with purified human WEE1, catalytic Lys114 mutagenesis, overexpression in HeLa","pmids":["8428596"],"confidence":"High","gaps":["Did not address regulation of WEE1 itself","Tyr15 selectivity structural basis unknown at this stage"]},{"year":1993,"claim":"Identified the first direct upstream regulator, showing a kinase (Nim1) inactivates Wee1 to promote mitotic entry.","evidence":"In vitro kinase assay with purified Nim1 and Wee1; in vivo phosphorylation analysis in nim1 mutants","pmids":["8515818","8515817"],"confidence":"High","gaps":["Phosphosites on Wee1 not mapped","Human ortholog of this regulation not established"]},{"year":1995,"claim":"Showed WEE1 is the dominant CDK1-Tyr15 kinase in human cells and is inactivated by phosphorylation during M phase, and that it undergoes cell-cycle-regulated nuclear-to-cytoplasmic redistribution.","evidence":"Antibody depletion and in vitro kinase assays of HeLa lysates; immunofluorescence across the cell cycle with microtubule depolymerization","pmids":["7774574","7673359"],"confidence":"High","gaps":["Identity of the M-phase inactivating kinase(s) not yet defined","Functional consequence of midbody association unclear"]},{"year":2000,"claim":"Connected the DNA-damage checkpoint to Wee1 regulation in metazoan-relevant terms by showing Chk1 directly phosphorylates Wee1 and that checkpoint arrest requires coordinate Wee1 up- and Cdc25 down-regulation.","evidence":"Genetic epistasis (wee1/cdc25 double mutants), in vitro Chk1 kinase assay, overexpression and immunoblotting in S. pombe","pmids":["10769204"],"confidence":"High","gaps":["Chk1 phosphosites on Wee1 not mapped here","Mechanism linking phosphorylation to activity change undefined"]},{"year":2002,"claim":"Revealed that distinct Wee1 isoforms carry isoform-specific regulatory domains tuning inhibitory strength and stability, explaining differential CDK1 control.","evidence":"Ectopic expression, Cdc2 activity assays and domain-deletion analysis in Xenopus oocytes/embryos","pmids":["12006499"],"confidence":"Medium","gaps":["Relevance of isoform distinctions to human somatic cells unclear","Single-organism domain dissection"]},{"year":2004,"claim":"Defined the degradation arm of WEE1 control, identifying SCF(β-TrCP) as the ligase and Plk1/CDK-generated phosphodegrons (Ser53, Ser123) that trigger mitotic-entry destruction.","evidence":"Co-IP, in vitro ubiquitination, phosphodegron mutagenesis, siRNA, HeLa synchronization","pmids":["15070733"],"confidence":"High","gaps":["Did not resolve how multiple kinases are temporally ordered","Contribution of additional priming kinases not addressed here"]},{"year":2005,"claim":"Resolved the structural basis for tyrosine specificity and detailed the multi-kinase logic that builds the β-TrCP degron, including CK2 priming and polo-box motif creation.","evidence":"1.8 Å crystal structure of WEE1A catalytic domain; in vitro binding, mutagenesis, CK2 inhibition and siRNA","pmids":["15837193","16085715"],"confidence":"High","gaps":["Full-length, regulatory-domain-containing structure absent","In-cell stoichiometry of degron phosphorylation events not quantified"]},{"year":2007,"claim":"Demonstrated WEE1A is rate-limiting for checkpoint enforcement in a human cell context, with low expression causing checkpoint failure rescuable by re-expression.","evidence":"Gamma-irradiation of primary HPECs, CDK2 kinase assays, ectopic WEE1A rescue","pmids":["17431037"],"confidence":"High","gaps":["Generality across cell types not established","Mechanism of low WEE1 expression in HPECs unknown"]},{"year":2013,"claim":"Expanded WEE1 function beyond CDK gating by identifying histone H2B Tyr37 as a chromatin substrate coupling histone transcription to cell-cycle progression.","evidence":"Biochemical analysis of H2B Tyr37 phosphorylation and histone gene transcription after WEE1 manipulation","pmids":["23537585"],"confidence":"Medium","gaps":["Methodological detail limited","Interplay with canonical CDK1 role not integrated"]},{"year":2018,"claim":"Provided a spatial, size-dependent mechanism for Wee1 inhibition, showing Cdr2 cortical nodes recruit Wee1 in bursts scaling with cell size.","evidence":"TIRF live imaging, biochemical fractionation, kinase-dead and pom1 genetics in S. pombe","pmids":["29514920"],"confidence":"High","gaps":["Node-based regulation is yeast-specific; human equivalent unknown","Direct biochemical inactivation at nodes not fully resolved"]},{"year":2019,"claim":"Established WEE1 as a guardian of DNA replication, showing it suppresses CDK1/CDK2 to sustain ATR/Chk1 signaling, restrain origin firing, and protect forks under replicative stress.","evidence":"Pharmacological inhibition (MK-1775/AZD1775), siRNA, phosphoproteomics, immunoblotting of Claspin/CtIP/RIF1","pmids":["25965828","31712441"],"confidence":"Medium","gaps":["Reliance on single inhibitor for several conclusions","Direct vs indirect effects on each effector not fully separated"]},{"year":2022,"claim":"Pinpointed the fork-protection mechanism, identifying DNA2 as the degrading nuclease unleashed by WEE1 inhibition and distinguishing WEE1 from other CDK suppressors.","evidence":"DNA fiber assays with WEE1, DNA2 and CDK2 inhibition/knockdown plus immunofluorescence","pmids":["35045293"],"confidence":"Medium","gaps":["Single-lab finding","Molecular link from CDK2 activity to DNA2 recruitment unresolved"]},{"year":2023,"claim":"Defined an acetylation switch governing WEE1 activity, with CHK1-primed GCN5 acetylation at Lys177 activating the kinase and SIRT1 deacetylation inactivating it, also explaining inhibitor resistance.","evidence":"In vitro kinase, acetylation/deacetylation assays, Ser642/Lys177 mutagenesis, Co-IP, cancer cell studies","pmids":["36635566"],"confidence":"High","gaps":["Structural picture of the inhibitory segment displacement incomplete","Interplay between acetylation activation and β-TrCP degradation not integrated"]},{"year":2024,"claim":"Provided a structural basis for targeted WEE1 degradation by defining a CRBN-DDB1-WEE1 ternary complex with molecular glue degraders, rationalizing kinase selectivity.","evidence":"Crystal structure of CRBN-DDB1-WEE1-compound complex with degradation assays","pmids":["39499896"],"confidence":"High","gaps":["Therapeutic in vivo efficacy not addressed here","Neosubstrate surface relative to native regulation unmapped"]},{"year":null,"claim":"It remains unresolved how WEE1's multiple regulatory layers—Tyr15 gating, phosphodegron-driven degradation, CHK1-primed acetylation activation, and its non-CDK chromatin and replication-fork functions—are integrated in a single cell to time both mitotic entry and replication protection.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model linking acetylation, phosphorylation and degradation","Mammalian spatial/size-control mechanism for WEE1 inhibition unknown","Full-length WEE1 structure with regulatory domains lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,7,11]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,7,11]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,5,10]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8,9,20]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[15,16]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[11]}],"complexes":["SCF(β-TrCP) substrate","CRBN-DDB1 ternary complex (induced)"],"partners":["CDK1","CDK2","CHK1","PLK1","BTRC","GCN5","SIRT1","CSNK1D"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P30291","full_name":"Wee1-like protein kinase","aliases":["Wee1A kinase"],"length_aa":646,"mass_kda":71.6,"function":"Acts as a negative regulator of entry into mitosis (G2 to M transition) by protecting the nucleus from cytoplasmically activated cyclin B1-complexed CDK1 before the onset of mitosis by mediating phosphorylation of CDK1 on 'Tyr-15' (PubMed:15070733, PubMed:7743995, PubMed:8348613, PubMed:8428596). Specifically phosphorylates and inactivates cyclin B1-complexed CDK1 reaching a maximum during G2 phase and a minimum as cells enter M phase (PubMed:7743995, PubMed:8348613, PubMed:8428596). Phosphorylation of cyclin B1-CDK1 occurs exclusively on 'Tyr-15' and phosphorylation of monomeric CDK1 does not occur (PubMed:7743995, PubMed:8348613, PubMed:8428596). Its activity increases during S and G2 phases and decreases at M phase when it is hyperphosphorylated (PubMed:7743995). A correlated decrease in protein level occurs at M/G1 phase, probably due to its degradation (PubMed:7743995)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P30291/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/WEE1","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"TRIM28","stoichiometry":0.2},{"gene":"IPO8","stoichiometry":0.2},{"gene":"PSME3","stoichiometry":0.2},{"gene":"IPO7","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/WEE1","total_profiled":1310},"omim":[{"mim_id":"620563","title":"UBIQUITIN-SPECIFIC PEPTIDASE 50; USP50","url":"https://www.omim.org/entry/620563"},{"mim_id":"617996","title":"OOCYTE/ZYGOTE/EMBRYO MATURATION ARREST 5; 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trials.","date":"2025","source":"Critical reviews in oncology/hematology","url":"https://pubmed.ncbi.nlm.nih.gov/40187712","citation_count":20,"is_preprint":false},{"pmid":"37898670","id":"PMC_37898670","title":"CTPS1 is a novel therapeutic target in multiple myeloma which synergizes with inhibition of CHEK1, ATR or WEE1.","date":"2023","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/37898670","citation_count":20,"is_preprint":false},{"pmid":"34671620","id":"PMC_34671620","title":"Recent Advances of WEE1 Inhibitors and Statins in Cancers With p53 Mutations.","date":"2021","source":"Frontiers in medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34671620","citation_count":20,"is_preprint":false},{"pmid":"35173547","id":"PMC_35173547","title":"HJURP Promotes Malignant Progression and Mediates Sensitivity to Cisplatin and WEE1-inhibitor in Serous Ovarian Cancer.","date":"2022","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35173547","citation_count":20,"is_preprint":false},{"pmid":"32628111","id":"PMC_32628111","title":"PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer.","date":"2020","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/32628111","citation_count":20,"is_preprint":false},{"pmid":"30943845","id":"PMC_30943845","title":"p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775.","date":"2019","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/30943845","citation_count":20,"is_preprint":false},{"pmid":"22152133","id":"PMC_22152133","title":"Novel insights into maintaining genomic integrity: Wee1 regulating Mus81/Eme1.","date":"2011","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/22152133","citation_count":19,"is_preprint":false},{"pmid":"11119724","id":"PMC_11119724","title":"Regulation of Wee1 kinase in response to protein synthesis inhibition.","date":"2000","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/11119724","citation_count":19,"is_preprint":false},{"pmid":"30052133","id":"PMC_30052133","title":"Pharmacophore modeling, multiple docking, and molecular dynamics studies on Wee1 kinase inhibitors.","date":"2018","source":"Journal of biomolecular structure & dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/30052133","citation_count":19,"is_preprint":false},{"pmid":"16123596","id":"PMC_16123596","title":"Monitoring the cell cycle by multi-kinase-dependent regulation of Swe1/Wee1 in budding yeast.","date":"2005","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/16123596","citation_count":19,"is_preprint":false},{"pmid":"36620912","id":"PMC_36620912","title":"Inhibitors of cell cycle checkpoint target Wee1 kinase - a patent review (2003-2022).","date":"2023","source":"Expert opinion on therapeutic patents","url":"https://pubmed.ncbi.nlm.nih.gov/36620912","citation_count":18,"is_preprint":false},{"pmid":"34375118","id":"PMC_34375118","title":"Treatment of Melanoma by Nano-conjugate-Delivered Wee1 siRNA.","date":"2021","source":"Molecular pharmaceutics","url":"https://pubmed.ncbi.nlm.nih.gov/34375118","citation_count":18,"is_preprint":false},{"pmid":"35315355","id":"PMC_35315355","title":"WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma.","date":"2022","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/35315355","citation_count":17,"is_preprint":false},{"pmid":"33564073","id":"PMC_33564073","title":"MNK1 and MNK2 enforce expression of E2F1, FOXM1, and WEE1 to drive soft tissue sarcoma.","date":"2021","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/33564073","citation_count":17,"is_preprint":false},{"pmid":"39755818","id":"PMC_39755818","title":"Cyclin E1/CDK2 activation defines a key vulnerability to WEE1 kinase inhibition in gynecological cancers.","date":"2025","source":"NPJ precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39755818","citation_count":17,"is_preprint":false},{"pmid":"36575478","id":"PMC_36575478","title":"Wee1 promotes cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1.","date":"2022","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/36575478","citation_count":17,"is_preprint":false},{"pmid":"32314355","id":"PMC_32314355","title":"The Wee1 kinase inhibitor MK1775 suppresses cell growth, attenuates stemness and synergises with bortezomib in multiple myeloma.","date":"2020","source":"British journal of haematology","url":"https://pubmed.ncbi.nlm.nih.gov/32314355","citation_count":17,"is_preprint":false},{"pmid":"31505035","id":"PMC_31505035","title":"The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls.","date":"2019","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/31505035","citation_count":17,"is_preprint":false},{"pmid":"32195191","id":"PMC_32195191","title":"Increased HDAC Activity and c-MYC Expression Mediate Acquired Resistance to WEE1 Inhibition in Acute Leukemia.","date":"2020","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32195191","citation_count":17,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":45050,"output_tokens":6718,"usd":0.11796,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15702,"output_tokens":4965,"usd":0.101318,"stage2_stop_reason":"end_turn"},"total_usd":0.219278,"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\": 1993,\n      \"finding\": \"Human WEE1 kinase phosphorylates p34cdc2 exclusively on Tyr15 (not Thr14) in vitro, and mutation of the catalytic lysine (Lys114) abolishes both in vitro kinase activity and in vivo mitotic inhibitor function, establishing WEE1 as a Tyr15-specific kinase that inhibits mitosis by directly phosphorylating p34cdc2.\",\n      \"method\": \"In vitro kinase assay with purified human WEE1, active-site mutagenesis (Lys114), overexpression in HeLa cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein, active-site mutagenesis, and in vivo functional validation in a single rigorous study\",\n      \"pmids\": [\"8428596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Human WEE1 (WEE1Hu) functionally complements fission yeast wee1 mutations and causes G2/M delay when overexpressed in fission yeast, establishing functional conservation of the mitotic inhibitor role.\",\n      \"method\": \"Transcomplementation of yeast wee1 mutant; overexpression in S. pombe\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via transcomplementation and overexpression phenotype, foundational paper replicated by subsequent work\",\n      \"pmids\": [\"1840647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Fission yeast Nim1 kinase directly phosphorylates and inactivates Wee1 in vitro, inhibiting its ability to phosphorylate Cdc2 on Tyr15; this phosphorylation of Wee1 by Nim1 promotes mitotic entry.\",\n      \"method\": \"In vitro kinase assay with purified Nim1 and Wee1; in vivo phosphorylation state analysis in nim1-overexpressing and nim1-null yeast\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two independent papers (PMIDs 8515818 and 8515817) using in vitro reconstitution with purified kinases confirming same finding\",\n      \"pmids\": [\"8515818\", \"8515817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human WEE1 (~94 kDa) accounts for most of the CDC2 Tyr15-phosphorylating activity in HeLa cell lysates (shown by antibody depletion), is suppressed during M phase, and its inhibitory state during M phase requires protein phosphorylation (demonstrated by re-activation upon removal of phosphatase inhibitors).\",\n      \"method\": \"Antibody depletion from HeLa cell lysates; in vitro kinase assay; cell synchronization with/without phosphatase inhibitors\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody depletion with in vitro assay readout, single lab with two orthogonal methods\",\n      \"pmids\": [\"7774574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human WEE1 localizes almost exclusively to the nucleus during interphase, relocates to the cytoplasm at mitotic entry, and associates with the midbody/midbody bridges at the end of mitosis in a microtubule-dependent manner, indicating cell cycle-regulated subcellular redistribution.\",\n      \"method\": \"Immunofluorescence microscopy of HeLa and IMR90 cells throughout the cell cycle; microtubule depolymerization experiments\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by immunofluorescence with functional context, single lab\",\n      \"pmids\": [\"7673359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SCF(β-TrCP1/2) is the E3 ubiquitin ligase responsible for WEE1A ubiquitination and degradation at mitotic entry. Plk1 phosphorylates WEE1A at Ser53 and Cdc2 phosphorylates it at Ser123, creating unconventional phosphodegrons recognized by β-TrCP; these two phosphorylations cooperatively stimulate WEE1A ubiquitination. Depletion of β-TrCP or mutation of S53/S123 stabilizes WEE1A and delays mitotic onset.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis, siRNA depletion, HeLa cell synchronization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitination reconstitution, mutagenesis of phosphodegron sites, siRNA validation, and in vivo mitotic timing assay in a single comprehensive study\",\n      \"pmids\": [\"15070733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CDK-mediated phosphorylation of WEE1A at Ser123 promotes β-TrCP binding through three independent mechanisms: direct interaction of pSer123 with WD40 repeats of β-TrCP; creation of a polo-box domain-binding motif (SpSP) that accelerates Plk1-mediated phosphorylation of Ser53; and priming of CK2-dependent phosphorylation of Ser121, generating a second β-TrCP-binding site. CK1δ also contributes to WEE1 degradation.\",\n      \"method\": \"In vitro binding assays, mutagenesis, CK2 inhibitor treatment, siRNA, cell synchronization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal biochemical methods (in vitro binding, mutagenesis, pharmacological inhibition, siRNA) in a single study\",\n      \"pmids\": [\"16085715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of the catalytic domain of human somatic WEE1 (WEE1A) at 1.8 Å resolution reveals that despite being functionally a tyrosine kinase, it most closely resembles serine/threonine kinases (Chk1, cAMP kinases) in sequence and structure; the activation segment has Wee1-specific features maintaining an active conformation, and a glycine-rich loop substitution helps determine substrate specificity for Tyr15.\",\n      \"method\": \"X-ray crystallography (1.8 Å resolution) with active-site inhibitor complex\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with functional validation of substrate specificity\",\n      \"pmids\": [\"15837193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"In fission yeast, functional wee1 protein kinase is required for radiation-induced mitotic delay, establishing Wee1 as a necessary component of the DNA damage checkpoint that delays mitosis in response to gamma-irradiation.\",\n      \"method\": \"Genetic epistasis using wee1 mutants exposed to gamma-irradiation; cell cycle progression analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic loss-of-function with defined cell cycle phenotype, foundational study independently replicated\",\n      \"pmids\": [\"1549179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In fission yeast, the G2 DNA damage checkpoint simultaneously upregulates Wee1p and downregulates Cdc25p; inactivation of both wee1+ and cdc25+ is required to abolish checkpoint arrest. Chk1p directly phosphorylates Wee1p in vitro and its overexpression causes wee1+-dependent G2 arrest with Wee1p hyperphosphorylation. A transient increase in Wee1p levels is induced by G2 DNA damage checkpoint activation.\",\n      \"method\": \"Genetic epistasis (wee1 and cdc25 double mutants), in vitro Chk1 kinase assay with Wee1 as substrate, overexpression studies, immunoblotting\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genetic epistasis combined with in vitro kinase assay, multiple orthogonal methods\",\n      \"pmids\": [\"10769204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Drosophila Wee1 (dWee1) phosphorylates Cdk1 at tyrosine 15 and times mitotic entry during syncytial blastoderm embryogenesis; loss of maternal dwee1 causes premature mitotic entry, spindle defects, chromosome condensation problems, and Chk2-dependent developmental arrest.\",\n      \"method\": \"Genetic loss-of-function (maternal dwee1 mutants), phospho-Tyr15 immunoblot, live imaging in Drosophila embryos\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with multiple defined phenotypic readouts, in vivo validation\",\n      \"pmids\": [\"15589158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"WEE1 phosphorylates histone H2B at Tyr37 in nucleosomes upstream of the histone gene cluster, suppressing histone transcription in late S phase, establishing WEE1 as an epigenetic modifier with a role in coordinating histone synthesis with cell cycle progression.\",\n      \"method\": \"Biochemical analysis of H2B Tyr37 phosphorylation, histone gene transcription assays following WEE1 manipulation\",\n      \"journal\": \"Trends in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab reporting a novel substrate/mechanism; full methods not detailed in abstract\",\n      \"pmids\": [\"23537585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CK1δ (casein kinase 1δ) promotes WEE1 protein degradation; pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ reduces WEE1 turnover, arresting HeLa cell proliferation.\",\n      \"method\": \"Reporter assay (K328M-Wee1-luciferase), kinase-directed chemical library screen, CK1δ inhibitors, siRNA knockdown, conditional deletion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter-based screen followed by pharmacological and genetic validation, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"24817118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Wee1/Swe1 phosphorylates Hsp90 at a conserved tyrosine residue; this phosphorylation is important for Wee1/Swe1 association with Hsp90 and for Wee1/Swe1 stability. Non-phosphorylatable yHsp90-Y24F yeast, like swe1Δ cells, undergo premature nuclear division insensitive to G2/M checkpoint arrest.\",\n      \"method\": \"Yeast genetic analysis (swe1Δ, hsp90-Y24F mutants), cell cycle analysis, immunoprecipitation, geldanamycin sensitivity assays\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical evidence in yeast with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"20519952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WEE1 inhibition suppresses CDK1 and CDK2 kinase activities, and WEE1 activity is required to sustain ATR/Chk1 signaling under replicative stress; mechanistically, WEE1 inhibition activates Cdk1/2 and Plk1, which reduce Claspin and CtIP levels to impair ATR/Chk1 signaling.\",\n      \"method\": \"Pharmacological inhibition (MK-1775, ATR and Chk1 inhibitors), siRNA knockdown, immunoblotting for Claspin and CtIP, CDK activity assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple inhibitors and knockdown with downstream mechanistic readouts, single lab\",\n      \"pmids\": [\"25965828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WEE1 inhibitor AZD1775 induces CDK1-dependent RIF1 phosphorylation and CDK2/CDC7-dependent replicative helicase activation, demonstrating that WEE1 suppresses CDK1 and CDK2 to regulate origin firing at the G1/S transition.\",\n      \"method\": \"Pharmacological inhibition (AZD1775), phosphoproteomics, immunoblotting, flow cytometry in unperturbed G1 and S-phase cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological perturbation with multiple downstream readouts, single lab\",\n      \"pmids\": [\"31712441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"WEE1 activity guards against nascent DNA degradation at stalled replication forks by suppressing CDK2 activity; DNA2 is identified as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells, and WEE1's fork protection role is unique among CDK suppressors (CHK1 and p21 do not promote fork protection as WEE1 does).\",\n      \"method\": \"DNA fiber assay, WEE1 inhibitor (AZD1775), DNA2 inhibitor/knockdown, CDK2 inhibition, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with fiber assay readout, single lab\",\n      \"pmids\": [\"35045293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Upon DNA damage, CHK1-dependent phosphorylation of WEE1 at Ser642 primes GCN5-mediated acetylation at Lys177, causing dissociation of an inhibitory segment from the kinase domain and activating WEE1. SIRT1 deacetylates WEE1 at Lys177, maintaining it in an inactive state; SIRT1 deficiency leads to WEE1 hyperacetylation and activation, conferring resistance to WEE1 inhibitors.\",\n      \"method\": \"In vitro kinase assays, acetylation/deacetylation biochemical assays, site-directed mutagenesis, Co-IP, immunoblotting, cancer cell line functional studies\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — identification of writer (GCN5) and eraser (SIRT1) for WEE1 acetylation with mechanistic mutagenesis, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"36635566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In fission yeast, Cdr2 cortical nodes recruit Wee1 in short bursts in a manner requiring Cdr2 kinase activity and the non-catalytic N-terminus of Wee1; Wee1 localization bursts at nodes increase 20-fold as cells double in size during G2, partially suppressed by the Cdr2 inhibitor Pom1 in small cells, establishing a size-dependent mechanism for Wee1 inhibition at nodes by Cdr1 and Cdr2 kinases.\",\n      \"method\": \"TIRF microscopy live-cell imaging, biochemical fractionation, kinase-dead mutant analysis, genetic epistasis (pom1 mutants)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging combined with biochemical fractionation and genetics, multiple orthogonal methods in single study\",\n      \"pmids\": [\"29514920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In fission yeast, both Wee1 and Cdc25 undergo cell cycle-dependent phosphorylation changes that are dependent on PP2A associated with regulatory subunit Pab1, indicating a conserved PP2A-dependent mechanism for controlling Wee1 across eukaryotes.\",\n      \"method\": \"Phosphorylation state analysis by SDS-PAGE, genetic analysis in S. pombe PP2A mutants\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic and biochemical analysis in fission yeast, single lab, limited mechanistic detail in abstract\",\n      \"pmids\": [\"28103117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human prostate epithelial cells (HPECs) express low levels of WEE1A and fail to enforce DNA damage checkpoint arrest due to a lack of inhibitory CDK phosphorylation; ectopic WEE1A expression rescues checkpoint arrest in gamma-irradiated HPECs, establishing WEE1A as rate-limiting for checkpoint enforcement in this cell type.\",\n      \"method\": \"Gamma-irradiation of primary HPECs and ex vivo prostate tissue, CDK2 kinase assay, ectopic WEE1A expression rescue, immunoblotting, flow cytometry\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function context with quantitative kinase assay and gain-of-function rescue, multiple orthogonal methods\",\n      \"pmids\": [\"17431037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Molecular glue degraders (WEE1-targeting glutarimide-containing compounds) form a ternary complex with CRBN-DDB1 and WEE1; crystal structure of the hit compound with CRBN-DDB1-WEE1 defines the protein-protein interface and rationalizes kinase selectivity for WEE1 degradation.\",\n      \"method\": \"Crystal structure of CRBN-DDB1-WEE1-compound ternary complex, multicomponent combinatorial library synthesis and screening, degradation assays\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of ternary complex with functional degradation validation, single lab\",\n      \"pmids\": [\"39499896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WEE1 inhibition activates the STING-TBK1-IRF3 pathway, increases type I interferons and pro-inflammatory chemokines, and concomitantly activates STAT1, increasing IFN-γ and PD-L1 expression in SCLC models, demonstrating a cell-autonomous immune-stimulating mechanism.\",\n      \"method\": \"WEE1 inhibitor (AZD1775) treatment of SCLC cell lines, pathway activation immunoblotting, immunocompetent mouse models, cytokine measurement, CD8+ T cell analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological perturbation with multiple pathway readouts and in vivo validation, single lab\",\n      \"pmids\": [\"35584676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WEE1 inhibition up-regulates endogenous retroviral elements (ERVs) by relieving SETDB1/H3K9me3 repression through downregulation of FOXM1; ERVs trigger dsRNA stress and interferon response, increasing CD8+ T cell infiltration and PD-L1 expression, providing mechanistic basis for WEE1 inhibitor + immune checkpoint blockade synergy.\",\n      \"method\": \"WEE1 inhibitor treatment, ERV expression analysis, chromatin immunoprecipitation (H3K9me3/SETDB1), FOXM1 knockdown, dsRNA pathway reporter assays, in vivo tumor models\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, gene expression, and functional immune assays, single lab\",\n      \"pmids\": [\"34825915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In Drosophila, wee1 mediates checkpoint-dependent delays in chromosome condensation initiation and rate caused by S-phase and topoisomerase inhibitors; wee1 also mediates delayed anaphase entry in response to chromosome condensation defects independently of the spindle assembly checkpoint.\",\n      \"method\": \"Live imaging of early Drosophila embryos, pharmacological inhibition of S-phase and topoisomerase, genetic analysis with wee1 mutants, spindle assembly checkpoint mutant controls\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging combined with genetic and pharmacological perturbations, single lab\",\n      \"pmids\": [\"22262459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In Xenopus, two Wee1 isoforms (Wee1A and Wee1B) differentially inhibit Cdc2; Wee1B more strongly inhibits Cdc2/oocyte maturation than Wee1A due to its shorter C-terminal regulatory domain, while Wee1B is more labile during meiosis due to N-terminal PEST-like sequences, establishing isoform-specific regulatory domains.\",\n      \"method\": \"Ectopic expression in Xenopus oocytes and embryos, Cdc2 activity assays, domain deletion analysis, developmental cell division assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional domain analysis with kinase activity assays in Xenopus, single lab\",\n      \"pmids\": [\"12006499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WEE1 activity is required to protect against nascent DNA degradation during replication stress; WEE1 inhibition combined with PARP inhibitor olaparib produces radiosensitization that is not rescued by nucleosides and requires PARP1 trapping (not just catalytic inhibition), while WEE1 inhibitor alone radiosensitizes via nucleotide depletion/replication stress.\",\n      \"method\": \"Clonogenic survival assays, nucleoside rescue experiments, PARP1 depletion, veliparib vs. olaparib comparison, KRAS-mutant NSCLC cell lines\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection using pharmacological and genetic tools, single lab\",\n      \"pmids\": [\"29133592\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WEE1 is a nuclear tyrosine kinase that maintains cells in interphase by phosphorylating CDK1 (p34cdc2) exclusively at Tyr15 (and CDK2) to inhibit CDK activity; at mitotic entry, WEE1 is itself inactivated by phosphorylation (by Plk1 at Ser53 and CDK at Ser123 creating phosphodegrons) leading to SCF(β-TrCP)-mediated ubiquitination and proteasomal degradation, while upstream kinases Nim1/Cdr1 in yeast and CHK1 in response to DNA damage directly phosphorylate and regulate WEE1 activity; WEE1 also phosphorylates histone H2B at Tyr37 to suppress histone transcription, protects stalled replication forks by limiting CDK2 activity, and its catalytic activity is further regulated by GCN5-mediated acetylation (activating) and SIRT1-mediated deacetylation (inactivating) at Lys177, with the crystal structure of its catalytic domain revealing an atypical serine/threonine kinase fold adapted for tyrosine substrate specificity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WEE1 is a nuclear protein kinase that restrains entry into mitosis by phosphorylating the cyclin-dependent kinase CDK1 (p34cdc2) exclusively at Tyr15, thereby holding cells in interphase until division is appropriate [#0, #1]. Although it functions biochemically as a tyrosine kinase, its catalytic domain adopts a fold most closely resembling serine/threonine kinases, with a Wee1-specific activation segment and a glycine-rich loop substitution that together dictate Tyr15 substrate specificity [#7]. This activity is conserved across eukaryotes: human WEE1 complements fission yeast wee1 mutants, and orthologs time mitotic entry in yeast and Drosophila embryos [#1, #10]. WEE1 enforces cell-cycle checkpoints—loss of yeast Wee1 abolishes radiation-induced mitotic delay, and WEE1A is rate-limiting for DNA-damage checkpoint arrest in human cells [#8, #20]. WEE1 abundance and activity are tightly regulated: at mitotic entry, Plk1 phosphorylation at Ser53 and CDK phosphorylation at Ser123 generate phosphodegrons recognized by the SCF(β-TrCP) ubiquitin ligase, with CK2, CK1δ, and a polo-box-binding motif reinforcing degron formation, driving WEE1 ubiquitination and proteasomal degradation [#5, #6, #12]. Conversely, in response to DNA damage CHK1-dependent phosphorylation at Ser642 primes GCN5-mediated acetylation at Lys177 to activate WEE1, an activation reversed by SIRT1 deacetylation [#17, #9]. Beyond mitotic gating, WEE1 suppresses CDK2 activity to protect stalled replication forks from DNA2-mediated nascent-DNA degradation and to restrain origin firing [#16, #15], and it phosphorylates histone H2B at Tyr37 to suppress histone gene transcription, coupling histone synthesis to cell-cycle progression [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established that the human gene encodes a bona fide mitotic inhibitor by showing functional conservation with the yeast cell-size/cell-cycle regulator.\",\n      \"evidence\": \"Transcomplementation of S. pombe wee1 mutants and overexpression-induced G2/M delay\",\n      \"pmids\": [\"1840647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular substrate or biochemical activity\", \"Conservation shown genetically, not biochemically\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Placed Wee1 within the DNA damage response by showing its kinase activity is required to delay mitosis after irradiation, linking it to checkpoint control.\",\n      \"evidence\": \"Genetic epistasis with wee1 mutants under gamma-irradiation in fission yeast\",\n      \"pmids\": [\"1549179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal connecting damage to Wee1 not defined\", \"Substrate of the checkpoint-dependent activity not identified here\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Defined the core biochemical reaction—Tyr15-specific phosphorylation of CDK1—and proved catalytic activity is essential for mitotic inhibition.\",\n      \"evidence\": \"In vitro kinase assay with purified human WEE1, catalytic Lys114 mutagenesis, overexpression in HeLa\",\n      \"pmids\": [\"8428596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address regulation of WEE1 itself\", \"Tyr15 selectivity structural basis unknown at this stage\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identified the first direct upstream regulator, showing a kinase (Nim1) inactivates Wee1 to promote mitotic entry.\",\n      \"evidence\": \"In vitro kinase assay with purified Nim1 and Wee1; in vivo phosphorylation analysis in nim1 mutants\",\n      \"pmids\": [\"8515818\", \"8515817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosites on Wee1 not mapped\", \"Human ortholog of this regulation not established\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Showed WEE1 is the dominant CDK1-Tyr15 kinase in human cells and is inactivated by phosphorylation during M phase, and that it undergoes cell-cycle-regulated nuclear-to-cytoplasmic redistribution.\",\n      \"evidence\": \"Antibody depletion and in vitro kinase assays of HeLa lysates; immunofluorescence across the cell cycle with microtubule depolymerization\",\n      \"pmids\": [\"7774574\", \"7673359\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the M-phase inactivating kinase(s) not yet defined\", \"Functional consequence of midbody association unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Connected the DNA-damage checkpoint to Wee1 regulation in metazoan-relevant terms by showing Chk1 directly phosphorylates Wee1 and that checkpoint arrest requires coordinate Wee1 up- and Cdc25 down-regulation.\",\n      \"evidence\": \"Genetic epistasis (wee1/cdc25 double mutants), in vitro Chk1 kinase assay, overexpression and immunoblotting in S. pombe\",\n      \"pmids\": [\"10769204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chk1 phosphosites on Wee1 not mapped here\", \"Mechanism linking phosphorylation to activity change undefined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Revealed that distinct Wee1 isoforms carry isoform-specific regulatory domains tuning inhibitory strength and stability, explaining differential CDK1 control.\",\n      \"evidence\": \"Ectopic expression, Cdc2 activity assays and domain-deletion analysis in Xenopus oocytes/embryos\",\n      \"pmids\": [\"12006499\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relevance of isoform distinctions to human somatic cells unclear\", \"Single-organism domain dissection\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the degradation arm of WEE1 control, identifying SCF(β-TrCP) as the ligase and Plk1/CDK-generated phosphodegrons (Ser53, Ser123) that trigger mitotic-entry destruction.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, phosphodegron mutagenesis, siRNA, HeLa synchronization\",\n      \"pmids\": [\"15070733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how multiple kinases are temporally ordered\", \"Contribution of additional priming kinases not addressed here\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved the structural basis for tyrosine specificity and detailed the multi-kinase logic that builds the β-TrCP degron, including CK2 priming and polo-box motif creation.\",\n      \"evidence\": \"1.8 Å crystal structure of WEE1A catalytic domain; in vitro binding, mutagenesis, CK2 inhibition and siRNA\",\n      \"pmids\": [\"15837193\", \"16085715\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length, regulatory-domain-containing structure absent\", \"In-cell stoichiometry of degron phosphorylation events not quantified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated WEE1A is rate-limiting for checkpoint enforcement in a human cell context, with low expression causing checkpoint failure rescuable by re-expression.\",\n      \"evidence\": \"Gamma-irradiation of primary HPECs, CDK2 kinase assays, ectopic WEE1A rescue\",\n      \"pmids\": [\"17431037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality across cell types not established\", \"Mechanism of low WEE1 expression in HPECs unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expanded WEE1 function beyond CDK gating by identifying histone H2B Tyr37 as a chromatin substrate coupling histone transcription to cell-cycle progression.\",\n      \"evidence\": \"Biochemical analysis of H2B Tyr37 phosphorylation and histone gene transcription after WEE1 manipulation\",\n      \"pmids\": [\"23537585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Methodological detail limited\", \"Interplay with canonical CDK1 role not integrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided a spatial, size-dependent mechanism for Wee1 inhibition, showing Cdr2 cortical nodes recruit Wee1 in bursts scaling with cell size.\",\n      \"evidence\": \"TIRF live imaging, biochemical fractionation, kinase-dead and pom1 genetics in S. pombe\",\n      \"pmids\": [\"29514920\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Node-based regulation is yeast-specific; human equivalent unknown\", \"Direct biochemical inactivation at nodes not fully resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established WEE1 as a guardian of DNA replication, showing it suppresses CDK1/CDK2 to sustain ATR/Chk1 signaling, restrain origin firing, and protect forks under replicative stress.\",\n      \"evidence\": \"Pharmacological inhibition (MK-1775/AZD1775), siRNA, phosphoproteomics, immunoblotting of Claspin/CtIP/RIF1\",\n      \"pmids\": [\"25965828\", \"31712441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reliance on single inhibitor for several conclusions\", \"Direct vs indirect effects on each effector not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Pinpointed the fork-protection mechanism, identifying DNA2 as the degrading nuclease unleashed by WEE1 inhibition and distinguishing WEE1 from other CDK suppressors.\",\n      \"evidence\": \"DNA fiber assays with WEE1, DNA2 and CDK2 inhibition/knockdown plus immunofluorescence\",\n      \"pmids\": [\"35045293\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Molecular link from CDK2 activity to DNA2 recruitment unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined an acetylation switch governing WEE1 activity, with CHK1-primed GCN5 acetylation at Lys177 activating the kinase and SIRT1 deacetylation inactivating it, also explaining inhibitor resistance.\",\n      \"evidence\": \"In vitro kinase, acetylation/deacetylation assays, Ser642/Lys177 mutagenesis, Co-IP, cancer cell studies\",\n      \"pmids\": [\"36635566\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural picture of the inhibitory segment displacement incomplete\", \"Interplay between acetylation activation and β-TrCP degradation not integrated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a structural basis for targeted WEE1 degradation by defining a CRBN-DDB1-WEE1 ternary complex with molecular glue degraders, rationalizing kinase selectivity.\",\n      \"evidence\": \"Crystal structure of CRBN-DDB1-WEE1-compound complex with degradation assays\",\n      \"pmids\": [\"39499896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Therapeutic in vivo efficacy not addressed here\", \"Neosubstrate surface relative to native regulation unmapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how WEE1's multiple regulatory layers—Tyr15 gating, phosphodegron-driven degradation, CHK1-primed acetylation activation, and its non-CDK chromatin and replication-fork functions—are integrated in a single cell to time both mitotic entry and replication protection.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model linking acetylation, phosphorylation and degradation\", \"Mammalian spatial/size-control mechanism for WEE1 inhibition unknown\", \"Full-length WEE1 structure with regulatory domains lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 7, 11]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 7, 11]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 5, 10]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8, 9, 20]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [15, 16]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"complexes\": [\"SCF(\\u03b2-TrCP) substrate\", \"CRBN-DDB1 ternary complex (induced)\"],\n    \"partners\": [\"CDK1\", \"CDK2\", \"CHK1\", \"PLK1\", \"BTRC\", \"GCN5\", \"SIRT1\", \"CSNK1D\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}