{"gene":"MASTL","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2009,"finding":"Greatwall kinase (Gwl/MASTL) is activated during M phase by Cdk1/cyclin B (MPF), and once activated, promotes inactivation of PP2A/B55delta phosphatase — the major 'antimitotic' phosphatase directed against CDK phosphosites — independently of continued MPF activity. Removal of PP2A/B55delta rescues the inability of Gwl-depleted Xenopus egg extracts to enter M phase.","method":"Xenopus egg extract depletion/add-back experiments, in vitro phosphatase activity assays, biochemical reconstitution","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution in Xenopus extracts with depletion and rescue experiments, multiple orthogonal biochemical methods, foundational mechanistic study","pmids":["19793917"],"is_preprint":false},{"year":2010,"finding":"Human MASTL (the ortholog of Greatwall) localizes to the nucleus in interphase and re-localizes in part to centrosomes during mitosis when it is active. MASTL RNAi causes G2 delay, slow chromosome condensation, anaphase sister chromatid separation failure, cytokinesis failure, and incomplete cyclin B1 destruction. MASTL supports phosphorylation of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C.","method":"RNAi knockdown in human cells, immunofluorescence localization, live-cell imaging, immunoblotting of mitotic phosphoproteins","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal localization by immunofluorescence tied to functional loss-of-function phenotypes, multiple orthogonal readouts in a single focused study","pmids":["20818157"],"is_preprint":false},{"year":2010,"finding":"In vivo genetic studies in mice demonstrate that Mastl (Greatwall) is required for mitotic exit downstream of Cdk1; Cdc20-null cells can exit mitosis upon inactivation of both Cdk1 and Mastl, and this mitotic exit depends on PP2A phosphatase complexes containing B55α or B55δ regulatory subunits.","method":"Genetically engineered mouse models, conditional knockout, in vivo tumor regression assays","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with defined molecular pathway, replicated across multiple cell types and tumor models","pmids":["21156286"],"is_preprint":false},{"year":2014,"finding":"Mastl is required in mouse oocytes for timely activation of APC/C to allow meiosis I exit and for the rapid rise of Cdk1 activity needed for entry into meiosis II. Mastl-null oocytes reach metaphase I normally but show delayed anaphase I onset and fail to enter meiosis II, reassembling a nuclear structure with decondensed chromatin instead.","method":"Oocyte-specific conditional Mastl knockout mouse model, live imaging, immunoblotting","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined cellular and molecular phenotype, in vivo mammalian model","pmids":["25246615"],"is_preprint":false},{"year":2015,"finding":"MASTL is essential for Cdc20-independent recruitment of cyclin B1 to the mitotic APC/C in prometaphase. This MASTL-directed binding critically supports efficient cyclin B1 destruction after spindle checkpoint release. MASTL RNAi causes anaphase bridges, attributed to insufficient cyclin B1 destruction.","method":"RNAi knockdown, co-immunoprecipitation of cyclin B1 with APC/C, cell biology/immunofluorescence","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and RNAi with specific molecular phenotype, single lab, single study","pmids":["25750436"],"is_preprint":false},{"year":2016,"finding":"PP1 is associated with MASTL and is capable of partially dephosphorylating and deactivating MASTL during mitotic exit. Small decreases in Cdk1 activity during metaphase are sufficient to initiate PP1 reactivation, which in turn partially deactivates MASTL to release inhibition of PP2A, creating a feedback loop that drives complete MASTL deactivation and switch-like phosphatase activation during mitotic exit.","method":"Co-immunoprecipitation of PP1 with MASTL, phosphoprotein analysis, mathematical modeling, kinase/phosphatase activity assays in human cells","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, activity assays, and mathematical modeling as orthogonal methods, single lab","pmids":["26872783"],"is_preprint":false},{"year":2016,"finding":"MASTL is critical for timing of mitotic entry after DNA damage checkpoint recovery, acting through ARPP19/ENSA to inhibit PP2A. Constitutively active MASTL promotes CDK1(Tyr15) dephosphorylation and accelerates mitotic re-entry. Downregulation of MASTL or ARPP19/ENSA delays mitotic re-entry after DNA damage and causes premature APC/C activation, causing cells to proceed from G2 directly to G1 skipping mitosis.","method":"RNAi knockdown, expression of constitutively active MASTL, cell cycle analysis, immunoblotting for CDK1-Tyr15 and APC/C substrates","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular pathway placement, two orthogonal methods, single lab","pmids":["26923777"],"is_preprint":false},{"year":2017,"finding":"The proliferative function of MASTL in breast cancer cells requires its kinase activity and the presence of PP2A-B55 complexes; genetic ablation of MASTL (CRISPR/Cas9) impairs proliferation of a subset of breast cancer cells and shows therapeutic effect in vivo.","method":"CRISPR/Cas9 knockout, RNAi, kinase-dead mutant rescue, in vivo xenograft tumor regression","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutant and PP2A-B55 dependency tested, in vivo validation, single lab","pmids":["29229993"],"is_preprint":false},{"year":2018,"finding":"In platelets, thrombocytopenia-associated MASTL mutation leads to aberrant platelet activation characterized by hyperstabilized pseudopods and actin polymerization defects mimicking PP2A inhibition. Multiple components of the actin cytoskeleton show abnormal hyperphosphorylation. These defects are rescued in vitro and in vivo by inhibiting upstream kinases PKA, PKC, or AMPK, revealing an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells via PP2A-B55.","method":"Conditional megakaryocyte/platelet Mastl knockout mice, knock-in thrombocytopenia-associated mutation mouse model, phosphoproteomics (mass spectrometry), pharmacological rescue in vivo","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mouse genetic models, phosphoproteomics, in vivo pharmacological rescue, multiple orthogonal methods","pmids":["30252678"],"is_preprint":false},{"year":2018,"finding":"RSK (ribosomal S6 kinase) phosphorylates MASTL at Thr297 (conserved only among mammalian MASTLs) after oocyte activation, sustaining CDK1-MASTL-ENSA activity and PP2A suppression despite absence of cyclin B, thereby delaying pronuclear formation. This RSK-MASTL pathway is required for mammalian-specific prolonged meiotic exit and faithful paternal pronucleus formation.","method":"Mouse oocyte live imaging, phosphomutant constructs, kinase inhibitor experiments, immunoblotting for MASTL and ENSA phosphorylation","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — identification of specific phosphorylation site on MASTL by RSK using phosphomutant rescue and kinase inhibition, with functional consequence in live oocytes","pmids":["30293837"],"is_preprint":false},{"year":2019,"finding":"The non-conserved middle region (NCMR) of MASTL is crucial for target specificity and activity. Key phosphorylation sites for MASTL activation were determined to arise from autophosphorylation versus exogenous kinases. Hydrogen/deuterium exchange data show the C-lobe forms a stable structure while the N-lobe is dynamic; truncated MASTL constructs retaining a cryptic C-lobe in the NCMR display catalytic activity with different substrate targets.","method":"Phosphoproteomic assay with MASTL constructs, hydrogen/deuterium exchange mass spectrometry, in vitro kinase assays with truncation and point mutants","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural HDX-MS analysis combined with in vitro kinase assays and phosphoproteomics, multiple orthogonal methods in single study","pmids":["31852836"],"is_preprint":false},{"year":2020,"finding":"AKT phosphorylates MASTL at residue T299, which plays a critical role in MASTL activation. AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which in turn promotes mitotic progression through PP2A inhibition. AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting/silencing MASTL.","method":"In vitro kinase assay, phospho-site mutant constructs (T299A), immunoblotting, MASTL knockdown/inhibition in colorectal cancer cell lines","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay identifying specific phospho-site combined with phosphomutant and loss-of-function cell-based experiments, single lab","pmids":["32123010"],"is_preprint":false},{"year":2020,"finding":"MASTL promotes cell contractility and breast cancer cell motility/invasion through a kinase-independent mechanism. MASTL associates with MRTF-A (myocardin-related transcription factor A) and increases its nuclear retention and SRF transcriptional activity, regulating genes including GEF-H1, TPM4, VCL, and MYH10. Kinase-dead MASTL is sufficient to regulate cell spreading and MRTF-A/SRF transcriptional activity.","method":"Co-immunoprecipitation of MASTL with MRTF-A, kinase-dead MASTL expression, transcriptome and proteome profiling, nuclear fractionation, RNAi knockdown with invasion/migration assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP establishing direct MASTL-MRTF-A interaction, kinase-dead mutant proving kinase-independent mechanism, transcriptome/proteome profiling, multiple orthogonal methods","pmids":["32311005"],"is_preprint":false},{"year":2022,"finding":"MASTL/Greatwall sustains mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10, thereby inhibiting PI3K-AKT activity via the feedback loop downstream of mTORC1. Genetic depletion of MASTL results in inefficient feedback and AKT hyperactivity. These defects are rescued by expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, limiting PP2A/B55-dependent dephosphorylation of IRS1 and GRB10.","method":"Genetic MASTL depletion (mouse and cell line), phosphomimetic ENSA/ARPP19 rescue, in vitro mTORC1 kinase assay on MASTL, phospho-IRS1/GRB10 immunoblotting, in vivo glucose tolerance tests in mice","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay identifying mTORC1 as MASTL kinase, genetic rescue with phosphomimetic substrates, in vivo metabolic phenotype, multiple orthogonal methods","pmids":["36354735"],"is_preprint":false},{"year":2022,"finding":"Phosphorylation of the MASTL C-tail turn motif at S861 (mouse) is dispensable for kinase function in intact mammalian cells, as shown by complementation of tamoxifen-inducible conditional Mastl knockout mouse embryonic fibroblasts with S861A mutant. Activation loop phosphorylations (T193 and T206) remain required.","method":"Tamoxifen-inducible conditional Mastl knockout MEFs, genetic complementation with phosphosite mutants, computational molecular docking","journal":"Journal of biomolecular structure & dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean genetic complementation in mammalian cells for specific phospho-site, single lab, single study; computational docking supportive but not Tier 1","pmids":["36270968"],"is_preprint":false},{"year":2022,"finding":"A SILAC-based whole-cell lysate in vitro kinase screen identified 59 phospho-sites on 67 proteins as potential MASTL substrates beyond ARPP19/ENSA. Subsequent in vitro kinase assays suggested MASTL may phosphorylate hnRNPM, YB1, and TUBA1C under certain in vitro conditions.","method":"SILAC in vitro kinase screen with cell lysates, in vitro kinase assays for candidate substrates","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vitro screen with partial follow-up kinase assays, substrates not validated in cells, single lab, single method for each candidate","pmids":["35732702"],"is_preprint":false},{"year":2023,"finding":"E6AP is the E3 ubiquitin ligase that mediates MASTL protein degradation under normal conditions. Upon DNA damage, ATM phosphorylates E6AP at Ser-218, causing E6AP dissociation from MASTL, stabilizing MASTL protein levels, and enabling timely cell cycle recovery from the DNA damage checkpoint. E6AP depletion reduced DNA damage signaling and promoted checkpoint recovery in a MASTL-dependent manner.","method":"E3 ligase identification by co-immunoprecipitation and mass spectrometry, E6AP depletion, ATM inhibition, phospho-site mutant of E6AP (S218A), cell cycle recovery assays, ubiquitination assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP/MS identification of E3 ligase, phospho-site mutant validation, epistasis experiment (MASTL-dependent rescue), multiple orthogonal methods in peer-reviewed study","pmids":["37672026"],"is_preprint":false},{"year":2017,"finding":"E2F8 transcription factor directly activates MASTL expression by binding to the MASTL promoter, as validated by western blot, dual luciferase assay, and ChIP-qPCR. E2F8 overexpression shortens cisplatin-induced G2/M arrest by promoting MASTL-mediated mitotic progression, an effect cancelled by MASTL inhibition.","method":"Dual luciferase promoter assay, ChIP-qPCR, western blot, MASTL inhibition rescue experiment","journal":"Biomedicine & pharmacotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (luciferase, ChIP-qPCR, rescue experiment) establishing transcriptional regulation, single lab","pmids":["28605876"],"is_preprint":false},{"year":2003,"finding":"A missense mutation in FLJ14813 (MASTL), E167D (G-to-C at nucleotide 565), segregates perfectly with autosomal dominant thrombocytopenia in a kindred of 51 family members and is absent from 94 unaffected controls, identifying MASTL as a candidate disease gene for THC2 thrombocytopenia.","method":"Genetic linkage/segregation analysis, sequencing of family cohort","journal":"Human heredity","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — genetic segregation in large kindred establishes disease association, but no direct functional mechanism of the mutation shown in this paper","pmids":["12890928"],"is_preprint":false},{"year":2009,"finding":"In vivo morpholino knockdown of MASTL in zebrafish results in deficiency of circulating thrombocytes and decreased expression of thrombopoietin receptor c-mpl and CD41/GpIIb. Both wild-type and E167D mutant MASTL localize to the nucleus when transiently expressed in baby hamster kidney cells.","method":"Zebrafish morpholino knockdown, northern blot, transient expression with localization by fluorescence in BHK cells","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — in vivo morpholino knockdown with specific thrombocyte phenotype and molecular markers, plus direct localization experiment; single lab","pmids":["19460416"],"is_preprint":false},{"year":2025,"finding":"In Xenopus oocytes, loss of Greatwall (Gwl/MASTL) and subsequent hyperactivation of PP2A-B55 prevents phosphorylation of Wee1/Myt1 and the APC/C complex, blocking APC/C activation. This impairs cyclin degradation and partial CDK1 inactivation required at the meiosis I–meiosis II transition, prevents mos-MAPK-Rsk1/2 pathway activation due to insufficient Mos accumulation, and blocks Erp1 degradation, revealing a feedback loop between APC/C and Erp1.","method":"Gwl depletion from Xenopus oocytes, immunoblotting for APC/C substrates, cyclin B levels, Wee1/Myt1 phosphorylation, Mos/MAPK pathway components","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion experiments in Xenopus oocyte system with multiple molecular readouts; preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"MASTL facilitates phosphorylation of Y-box binding protein-1 (YBX1); phosphorylated YBX1 transcriptionally activates PAK4, identifying the MASTL/YBX1/PAK4 axis as a downstream effector pathway. STK24, a stress-responsive kinase, can activate MASTL in HCC under lenvatinib exposure. Disruption of this axis by MASTL inhibition restores HCC sensitivity to lenvatinib.","method":"Co-immunoprecipitation and mass spectrometry identifying YBX1 as MASTL-associated protein, ChIP-qPCR for YBX1-PAK4 transcriptional regulation, phosphorylation analysis for STK24-MASTL interaction","journal":"Hepatology (Baltimore, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS, ChIP-qPCR, and phosphorylation analysis as orthogonal methods; single lab","pmids":["40456026"],"is_preprint":false},{"year":2017,"finding":"MASTL is required for anaphase entry in proliferating primordial germ cells (PGCs); Mastl-null PGCs enter mitosis normally but fail to proceed beyond a metaphase-like stage, and subsequent cell death eliminates female germ cells. Simultaneous deletion of Ppp2r1a (PP2A α subunit) rescues the mitotic progression defect in Mastl-null PGCs, placing PP2A downstream of Mastl in this pathway.","method":"PGC-specific conditional Mastl knockout mouse model, genetic epistasis with Ppp2r1a deletion, immunofluorescence, cell counting","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO in vivo with genetic epistasis (PP2A rescue) establishing pathway position, in vivo mammalian model","pmids":["28224044"],"is_preprint":false},{"year":2021,"finding":"MASTL promotes pancreatic cancer progression by modulating EGFR protein stability and kinase signaling; MASTL loss reduces EGFR protein levels and downstream signaling, and combined targeting of MASTL with gemcitabine shows increased cancer cell killing.","method":"Loss- and gain-of-function studies in PC cell lines, murine PC models (KC and KPC), immunoblotting for EGFR stability, pharmacological combination experiments","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with mechanistic molecular readout (EGFR stability), in vivo models, single lab","pmids":["34331012"],"is_preprint":false}],"current_model":"MASTL (Greatwall kinase) is a serine/threonine kinase activated during mitotic entry by Cdk1/cyclin B and by AKT (at T299) and RSK (at T297 in mammals); once active, it phosphorylates ENSA and ARPP19, which then inhibit PP2A/B55 phosphatase complexes, thereby maintaining Cdk1 substrate phosphorylation and the mitotic state. PP1 dephosphorylates and deactivates MASTL during mitotic exit to create a bistable switch. MASTL also acts kinase-independently by associating with MRTF-A to promote nuclear retention and SRF-dependent transcription of actomyosin contractility genes, and its protein stability is regulated by the E3 ubiquitin ligase E6AP, which is dissociated from MASTL upon ATM-mediated phosphorylation during DNA damage to enable checkpoint recovery. Beyond mitosis, the MASTL-PP2A/B55 axis controls mTORC1-S6K1 feedback phosphorylation of IRS1 and GRB10 to restrain PI3K-AKT activity, and MASTL regulates EGFR stability and Wnt/β-catenin signaling in cancer contexts."},"narrative":{"mechanistic_narrative":"MASTL (Greatwall kinase) is a serine/threonine kinase that enforces the mitotic and meiotic state by suppressing the principal anti-mitotic phosphatase PP2A/B55, thereby preserving Cdk1-substrate phosphorylation [PMID:19793917, PMID:21156286]. Activated during M phase by Cdk1/cyclin B, MASTL drives inactivation of PP2A/B55delta independently of continued MPF activity, and depleting this phosphatase rescues the mitotic-entry failure of Greatwall-depleted extracts [PMID:19793917]; in mammalian cells and mice this axis is required for both mitotic entry and exit, with PP2A complexes bearing B55alpha/B55delta acting genetically downstream of MASTL [PMID:21156286, PMID:28224044]. MASTL phosphorylates ENSA/ARPP19, which in turn inhibit PP2A/B55 to sustain CDK1 activity and time mitotic re-entry after DNA-damage checkpoint recovery [PMID:26923777]. The switch is made bistable by PP1, which associates with MASTL and partially dephosphorylates and deactivates it at mitotic exit, releasing PP2A inhibition [PMID:26872783]. MASTL also licenses Cdc20-independent recruitment of cyclin B1 to the APC/C and supports efficient cyclin B1 destruction [PMID:25750436], and in oocytes and primordial germ cells it is required for timely APC/C activation and progression through the meiosis I–II transition [PMID:25246615, PMID:28224044]. Its activity is tuned by multiple upstream kinases—AKT (T299), RSK (T297/Thr297), and mTORC1—and by autophosphorylation, with the non-conserved middle region and activation-loop sites governing specificity and activity [PMID:30293837, PMID:31852836, PMID:32123010, PMID:36354735]; mTORC1-directed phosphorylation routes MASTL into a metabolic feedback loop that sustains S6K1-dependent phosphorylation of IRS1 and GRB10 to restrain PI3K-AKT signaling [PMID:36354735]. MASTL protein levels are set by the E3 ligase E6AP, which is dissociated upon ATM-mediated phosphorylation during DNA damage to stabilize MASTL and enable checkpoint recovery [PMID:37672026], and MASTL transcription is driven by E2F8 [PMID:28605876]. Beyond catalysis, MASTL acts kinase-independently by binding MRTF-A to promote its nuclear retention and SRF-dependent transcription of actomyosin contractility genes, driving breast cancer cell motility and invasion [PMID:32311005]. In cancer contexts MASTL supports proliferation through PP2A-B55, modulates EGFR stability, and operates a YBX1/PAK4 axis linked to lenvatinib resistance [PMID:29229993, PMID:34331012, PMID:40456026]. A recurrent missense mutation (E167D) in MASTL co-segregates with autosomal dominant THC2 thrombocytopenia [PMID:12890928], consistent with a non-mitotic role for MASTL in platelet actin-cytoskeletal dynamics via PP2A-B55 [PMID:30252678].","teleology":[{"year":2009,"claim":"Established the core mechanistic logic that Greatwall/MASTL maintains mitosis not by direct substrate phosphorylation alone but by suppressing the anti-mitotic phosphatase PP2A/B55.","evidence":"Xenopus egg extract depletion/add-back with in vitro phosphatase assays and biochemical reconstitution","pmids":["19793917"],"confidence":"High","gaps":["Did not identify the molecular intermediary linking MASTL to PP2A/B55 inhibition","Mechanism shown in extract, not yet in intact mammalian cells"]},{"year":2010,"claim":"Defined the human MASTL loss-of-function phenotype and localization, establishing it as a Cdk1-downstream effector required across multiple mitotic transitions.","evidence":"RNAi knockdown, immunofluorescence, live-cell imaging and phosphoprotein immunoblotting in human cells","pmids":["20818157"],"confidence":"High","gaps":["Centrosomal relocalization function not mechanistically defined","Direct substrates not identified"]},{"year":2010,"claim":"Provided in vivo genetic proof that MASTL acts through B55alpha/B55delta-containing PP2A for mitotic exit, anchoring the pathway in mammalian physiology.","evidence":"Conditional knockout mouse models with genetic epistasis and in vivo tumor regression assays","pmids":["21156286"],"confidence":"High","gaps":["Did not resolve the bistability/switch behavior of the system"]},{"year":2014,"claim":"Extended MASTL function to meiosis, showing it is needed for timely APC/C activation and the CDK1 rise driving the meiosis I-II transition.","evidence":"Oocyte-specific conditional Mastl knockout mouse, live imaging and immunoblotting","pmids":["25246615"],"confidence":"High","gaps":["Mechanism of APC/C activation timing by MASTL not fully resolved at molecular level"]},{"year":2015,"claim":"Linked MASTL to APC/C function directly by showing it is required for Cdc20-independent cyclin B1 recruitment and efficient destruction.","evidence":"RNAi with co-immunoprecipitation of cyclin B1 with APC/C in human cells","pmids":["25750436"],"confidence":"Medium","gaps":["Single-lab Co-IP without reciprocal validation","Whether the cyclin B1-APC/C effect is kinase-dependent not resolved"]},{"year":2016,"claim":"Identified PP1 as the deactivating phosphatase for MASTL, explaining the switch-like, bistable transition of phosphatase activity at mitotic exit.","evidence":"Co-IP of PP1 with MASTL, activity assays and mathematical modeling in human cells","pmids":["26872783"],"confidence":"Medium","gaps":["Direct PP1-MASTL dephosphorylation sites not fully mapped","Single lab"]},{"year":2016,"claim":"Placed MASTL in DNA-damage checkpoint recovery, showing it times mitotic re-entry through ARPP19/ENSA-mediated PP2A inhibition.","evidence":"RNAi and constitutively active MASTL with cell cycle analysis and CDK1-Tyr15/APC/C immunoblotting","pmids":["26923777"],"confidence":"Medium","gaps":["Upstream signal coupling DNA damage to MASTL not defined here","Single lab"]},{"year":2017,"claim":"Demonstrated that MASTL's pro-proliferative role in cancer depends on its kinase activity and on PP2A-B55, validating it as a therapeutic target.","evidence":"CRISPR/Cas9 knockout, kinase-dead rescue and in vivo xenograft regression in breast cancer cells","pmids":["29229993"],"confidence":"Medium","gaps":["Which cancer subsets are dependent not predicted by a marker","Single lab"]},{"year":2017,"claim":"Identified E2F8 as a direct transcriptional activator of MASTL coupling it to cell-cycle gene-expression control and chemoresistance.","evidence":"Dual luciferase promoter assay, ChIP-qPCR and MASTL-inhibition rescue","pmids":["28605876"],"confidence":"Medium","gaps":["Single lab","Broader transcriptional network controlling MASTL not mapped"]},{"year":2017,"claim":"Showed MASTL is required for anaphase entry in primordial germ cells, with PP2A genetically downstream, reinforcing the conserved MASTL-PP2A axis in germline mitosis.","evidence":"PGC-specific conditional knockout mouse with Ppp2r1a epistasis and immunofluorescence","pmids":["28224044"],"confidence":"High","gaps":["Cause of female-specific germ cell death downstream not fully defined"]},{"year":2018,"claim":"Revealed a non-mitotic role for MASTL in postmitotic platelets, controlling actin-cytoskeletal dynamics through PP2A-B55 and linking it mechanistically to thrombocytopenia.","evidence":"Conditional and knock-in mutant mice, phosphoproteomics and in vivo pharmacological rescue","pmids":["30252678"],"confidence":"High","gaps":["Direct cytoskeletal substrates of the MASTL-PP2A axis in platelets not individually validated"]},{"year":2018,"claim":"Identified RSK phosphorylation of MASTL at Thr297 as a mammalian-specific mechanism sustaining MASTL-ENSA activity during prolonged meiotic exit independent of cyclin B.","evidence":"Mouse oocyte live imaging, phosphomutant constructs and kinase inhibition","pmids":["30293837"],"confidence":"High","gaps":["Structural basis of T297-dependent activation not resolved"]},{"year":2019,"claim":"Resolved structural and regulatory determinants of MASTL activity, distinguishing autophosphorylation from exogenous activating phosphorylations and assigning specificity to the non-conserved middle region.","evidence":"HDX mass spectrometry, in vitro kinase assays with truncation/point mutants and phosphoproteomics","pmids":["31852836"],"confidence":"High","gaps":["No full-length crystal/cryo-EM structure","In-cell relevance of cryptic C-lobe activity not established"]},{"year":2020,"claim":"Identified AKT phosphorylation at T299 as an activating input linking growth-factor signaling to MASTL-driven mitotic progression.","evidence":"In vitro kinase assay, T299A phosphomutant and loss-of-function in colorectal cancer cells","pmids":["32123010"],"confidence":"Medium","gaps":["Single lab","Interplay of AKT and Cdk1 inputs on MASTL not quantitatively resolved"]},{"year":2020,"claim":"Uncovered a kinase-independent function of MASTL, binding MRTF-A to drive its nuclear retention and SRF-dependent contractility gene expression underlying cancer cell invasion.","evidence":"Co-IP, kinase-dead mutant, transcriptome/proteome profiling and invasion assays in breast cancer cells","pmids":["32311005"],"confidence":"High","gaps":["Direct vs indirect nature of MASTL-MRTF-A interaction not crystallographically defined"]},{"year":2021,"claim":"Connected MASTL to receptor tyrosine kinase signaling in cancer by showing it modulates EGFR protein stability and downstream signaling.","evidence":"Loss/gain-of-function in pancreatic cancer cells and KC/KPC mouse models with EGFR immunoblotting","pmids":["34331012"],"confidence":"Medium","gaps":["Molecular mechanism of EGFR stabilization not defined","Single lab"]},{"year":2022,"claim":"Extended the MASTL-PP2A/B55 axis into metabolic signaling, showing mTORC1-phosphorylated MASTL sustains S6K1-dependent IRS1/GRB10 phosphorylation to restrain PI3K-AKT.","evidence":"Genetic depletion, phosphomimetic ENSA/ARPP19 rescue, in vitro mTORC1 kinase assay and in vivo glucose tolerance tests","pmids":["36354735"],"confidence":"High","gaps":["Tissue-specific contribution to systemic metabolism not fully mapped"]},{"year":2022,"claim":"Refined the activation-site requirements of MASTL, showing the C-tail turn motif S861 is dispensable while activation-loop sites T193/T206 remain required.","evidence":"Inducible conditional Mastl knockout MEFs complemented with phosphosite mutants plus docking","pmids":["36270968"],"confidence":"Medium","gaps":["Docking-based structural inferences not experimentally validated","Single lab"]},{"year":2022,"claim":"Broadened the candidate MASTL substrate repertoire beyond ARPP19/ENSA through an unbiased in vitro screen.","evidence":"SILAC in vitro kinase screen of cell lysates with follow-up kinase assays for candidates","pmids":["35732702"],"confidence":"Low","gaps":["Candidate substrates (hnRNPM, YB1, TUBA1C) not validated in cells","Single in vitro method per candidate"]},{"year":2023,"claim":"Identified the E3 ligase E6AP controlling MASTL turnover and showed ATM-dependent E6AP phosphorylation stabilizes MASTL to enable checkpoint recovery.","evidence":"Co-IP/MS E3 ligase identification, E6AP S218A mutant, ATM inhibition and ubiquitination/recovery assays","pmids":["37672026"],"confidence":"High","gaps":["Ubiquitination site(s) on MASTL not mapped"]},{"year":2025,"claim":"Defined a MASTL/YBX1/PAK4 effector axis and an STK24 input in hepatocellular carcinoma linking MASTL to drug resistance.","evidence":"Co-IP/MS, ChIP-qPCR and phosphorylation analysis in HCC cells","pmids":["40456026"],"confidence":"Medium","gaps":["Direct YBX1 phosphosite not validated","Single lab"]},{"year":2025,"claim":"Dissected how loss of Greatwall and PP2A-B55 hyperactivation blocks the meiosis I-II transition by preventing Wee1/Myt1 and APC/C phosphorylation, revealing an APC/C-Erp1 feedback loop.","evidence":"Gwl depletion from Xenopus oocytes with immunoblotting of APC/C substrates and Mos/MAPK components (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Generality to mammalian meiosis not shown"]},{"year":null,"claim":"The physiological substrate repertoire of MASTL beyond ARPP19/ENSA, and the structural basis integrating its multiple activating inputs, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No validated in-cell substrates beyond ARPP19/ENSA","No full-length structure of MASTL","Mechanism of kinase-independent MRTF-A binding undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,10,11]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,9,13]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,19]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,4,5,6]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[3,9,22]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,13,23]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[6,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[16]}],"complexes":[],"partners":["ENSA","ARPP19","PP2A/B55","PP1","MRTF-A","E6AP","APC/C","YBX1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96GX5","full_name":"Serine/threonine-protein kinase greatwall","aliases":["Microtubule-associated serine/threonine-protein kinase-like","MAST-L"],"length_aa":879,"mass_kda":97.3,"function":"Serine/threonine kinase that plays a key role in M phase by acting as a regulator of mitosis entry and maintenance (PubMed:19680222). Acts by promoting the inactivation of protein phosphatase 2A (PP2A) during M phase: does not directly inhibit PP2A but acts by mediating phosphorylation and subsequent activation of ARPP19 and ENSA at 'Ser-62' and 'Ser-67', respectively (PubMed:38123684). ARPP19 and ENSA are phosphatase inhibitors that specifically inhibit the PPP2R2D (PR55-delta) subunit of PP2A. Inactivation of PP2A during M phase is essential to keep cyclin-B1-CDK1 activity high (PubMed:20818157). Following DNA damage, it is also involved in checkpoint recovery by being inhibited. Phosphorylates histone protein in vitro; however such activity is unsure in vivo. May be involved in megakaryocyte differentiation","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Nucleus; Cleavage furrow","url":"https://www.uniprot.org/uniprotkb/Q96GX5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MASTL","classification":"Common Essential","n_dependent_lines":1185,"n_total_lines":1208,"dependency_fraction":0.9809602649006622},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MASTL","total_profiled":1310},"omim":[{"mim_id":"618638","title":"HECT DOMAIN E3 UBIQUITIN PROTEIN LIGASE 3; HECTD3","url":"https://www.omim.org/entry/618638"},{"mim_id":"618002","title":"MICROTUBULE-ASSOCIATED SERINE/THREONINE KINASE 4; MAST4","url":"https://www.omim.org/entry/618002"},{"mim_id":"613992","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B, DELTA; PPP2R2D","url":"https://www.omim.org/entry/613992"},{"mim_id":"609135","title":"APLASTIC ANEMIA","url":"https://www.omim.org/entry/609135"},{"mim_id":"608221","title":"MICROTUBULE-ASSOCIATED SERINE/THREONINE KINASE-LIKE; MASTL","url":"https://www.omim.org/entry/608221"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MASTL"},"hgnc":{"alias_symbol":["FLJ14813","THC2","Gwl"],"prev_symbol":[]},"alphafold":{"accession":"Q96GX5","domains":[{"cath_id":"3.30.200.20","chopping":"30-113_199-222","consensus_level":"medium","plddt":84.7495,"start":30,"end":222},{"cath_id":"1.10.510.10","chopping":"116-178_741-870","consensus_level":"medium","plddt":91.6731,"start":116,"end":870}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96GX5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96GX5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96GX5-F1-predicted_aligned_error_v6.png","plddt_mean":54.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MASTL","jax_strain_url":"https://www.jax.org/strain/search?query=MASTL"},"sequence":{"accession":"Q96GX5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96GX5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96GX5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96GX5"}},"corpus_meta":[{"pmid":"21156286","id":"PMC_21156286","title":"Targeting 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Removal of PP2A/B55delta rescues the inability of Gwl-depleted Xenopus egg extracts to enter M phase.\",\n      \"method\": \"Xenopus egg extract depletion/add-back experiments, in vitro phosphatase activity assays, biochemical reconstitution\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution in Xenopus extracts with depletion and rescue experiments, multiple orthogonal biochemical methods, foundational mechanistic study\",\n      \"pmids\": [\"19793917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human MASTL (the ortholog of Greatwall) localizes to the nucleus in interphase and re-localizes in part to centrosomes during mitosis when it is active. MASTL RNAi causes G2 delay, slow chromosome condensation, anaphase sister chromatid separation failure, cytokinesis failure, and incomplete cyclin B1 destruction. MASTL supports phosphorylation of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C.\",\n      \"method\": \"RNAi knockdown in human cells, immunofluorescence localization, live-cell imaging, immunoblotting of mitotic phosphoproteins\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal localization by immunofluorescence tied to functional loss-of-function phenotypes, multiple orthogonal readouts in a single focused study\",\n      \"pmids\": [\"20818157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In vivo genetic studies in mice demonstrate that Mastl (Greatwall) is required for mitotic exit downstream of Cdk1; Cdc20-null cells can exit mitosis upon inactivation of both Cdk1 and Mastl, and this mitotic exit depends on PP2A phosphatase complexes containing B55α or B55δ regulatory subunits.\",\n      \"method\": \"Genetically engineered mouse models, conditional knockout, in vivo tumor regression assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with defined molecular pathway, replicated across multiple cell types and tumor models\",\n      \"pmids\": [\"21156286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mastl is required in mouse oocytes for timely activation of APC/C to allow meiosis I exit and for the rapid rise of Cdk1 activity needed for entry into meiosis II. Mastl-null oocytes reach metaphase I normally but show delayed anaphase I onset and fail to enter meiosis II, reassembling a nuclear structure with decondensed chromatin instead.\",\n      \"method\": \"Oocyte-specific conditional Mastl knockout mouse model, live imaging, immunoblotting\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined cellular and molecular phenotype, in vivo mammalian model\",\n      \"pmids\": [\"25246615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MASTL is essential for Cdc20-independent recruitment of cyclin B1 to the mitotic APC/C in prometaphase. This MASTL-directed binding critically supports efficient cyclin B1 destruction after spindle checkpoint release. MASTL RNAi causes anaphase bridges, attributed to insufficient cyclin B1 destruction.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation of cyclin B1 with APC/C, cell biology/immunofluorescence\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and RNAi with specific molecular phenotype, single lab, single study\",\n      \"pmids\": [\"25750436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PP1 is associated with MASTL and is capable of partially dephosphorylating and deactivating MASTL during mitotic exit. Small decreases in Cdk1 activity during metaphase are sufficient to initiate PP1 reactivation, which in turn partially deactivates MASTL to release inhibition of PP2A, creating a feedback loop that drives complete MASTL deactivation and switch-like phosphatase activation during mitotic exit.\",\n      \"method\": \"Co-immunoprecipitation of PP1 with MASTL, phosphoprotein analysis, mathematical modeling, kinase/phosphatase activity assays in human cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, activity assays, and mathematical modeling as orthogonal methods, single lab\",\n      \"pmids\": [\"26872783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MASTL is critical for timing of mitotic entry after DNA damage checkpoint recovery, acting through ARPP19/ENSA to inhibit PP2A. Constitutively active MASTL promotes CDK1(Tyr15) dephosphorylation and accelerates mitotic re-entry. Downregulation of MASTL or ARPP19/ENSA delays mitotic re-entry after DNA damage and causes premature APC/C activation, causing cells to proceed from G2 directly to G1 skipping mitosis.\",\n      \"method\": \"RNAi knockdown, expression of constitutively active MASTL, cell cycle analysis, immunoblotting for CDK1-Tyr15 and APC/C substrates\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular pathway placement, two orthogonal methods, single lab\",\n      \"pmids\": [\"26923777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The proliferative function of MASTL in breast cancer cells requires its kinase activity and the presence of PP2A-B55 complexes; genetic ablation of MASTL (CRISPR/Cas9) impairs proliferation of a subset of breast cancer cells and shows therapeutic effect in vivo.\",\n      \"method\": \"CRISPR/Cas9 knockout, RNAi, kinase-dead mutant rescue, in vivo xenograft tumor regression\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutant and PP2A-B55 dependency tested, in vivo validation, single lab\",\n      \"pmids\": [\"29229993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In platelets, thrombocytopenia-associated MASTL mutation leads to aberrant platelet activation characterized by hyperstabilized pseudopods and actin polymerization defects mimicking PP2A inhibition. Multiple components of the actin cytoskeleton show abnormal hyperphosphorylation. These defects are rescued in vitro and in vivo by inhibiting upstream kinases PKA, PKC, or AMPK, revealing an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells via PP2A-B55.\",\n      \"method\": \"Conditional megakaryocyte/platelet Mastl knockout mice, knock-in thrombocytopenia-associated mutation mouse model, phosphoproteomics (mass spectrometry), pharmacological rescue in vivo\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mouse genetic models, phosphoproteomics, in vivo pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"30252678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RSK (ribosomal S6 kinase) phosphorylates MASTL at Thr297 (conserved only among mammalian MASTLs) after oocyte activation, sustaining CDK1-MASTL-ENSA activity and PP2A suppression despite absence of cyclin B, thereby delaying pronuclear formation. This RSK-MASTL pathway is required for mammalian-specific prolonged meiotic exit and faithful paternal pronucleus formation.\",\n      \"method\": \"Mouse oocyte live imaging, phosphomutant constructs, kinase inhibitor experiments, immunoblotting for MASTL and ENSA phosphorylation\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — identification of specific phosphorylation site on MASTL by RSK using phosphomutant rescue and kinase inhibition, with functional consequence in live oocytes\",\n      \"pmids\": [\"30293837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The non-conserved middle region (NCMR) of MASTL is crucial for target specificity and activity. Key phosphorylation sites for MASTL activation were determined to arise from autophosphorylation versus exogenous kinases. Hydrogen/deuterium exchange data show the C-lobe forms a stable structure while the N-lobe is dynamic; truncated MASTL constructs retaining a cryptic C-lobe in the NCMR display catalytic activity with different substrate targets.\",\n      \"method\": \"Phosphoproteomic assay with MASTL constructs, hydrogen/deuterium exchange mass spectrometry, in vitro kinase assays with truncation and point mutants\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural HDX-MS analysis combined with in vitro kinase assays and phosphoproteomics, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31852836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AKT phosphorylates MASTL at residue T299, which plays a critical role in MASTL activation. AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which in turn promotes mitotic progression through PP2A inhibition. AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting/silencing MASTL.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutant constructs (T299A), immunoblotting, MASTL knockdown/inhibition in colorectal cancer cell lines\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay identifying specific phospho-site combined with phosphomutant and loss-of-function cell-based experiments, single lab\",\n      \"pmids\": [\"32123010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MASTL promotes cell contractility and breast cancer cell motility/invasion through a kinase-independent mechanism. MASTL associates with MRTF-A (myocardin-related transcription factor A) and increases its nuclear retention and SRF transcriptional activity, regulating genes including GEF-H1, TPM4, VCL, and MYH10. Kinase-dead MASTL is sufficient to regulate cell spreading and MRTF-A/SRF transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation of MASTL with MRTF-A, kinase-dead MASTL expression, transcriptome and proteome profiling, nuclear fractionation, RNAi knockdown with invasion/migration assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP establishing direct MASTL-MRTF-A interaction, kinase-dead mutant proving kinase-independent mechanism, transcriptome/proteome profiling, multiple orthogonal methods\",\n      \"pmids\": [\"32311005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MASTL/Greatwall sustains mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10, thereby inhibiting PI3K-AKT activity via the feedback loop downstream of mTORC1. Genetic depletion of MASTL results in inefficient feedback and AKT hyperactivity. These defects are rescued by expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, limiting PP2A/B55-dependent dephosphorylation of IRS1 and GRB10.\",\n      \"method\": \"Genetic MASTL depletion (mouse and cell line), phosphomimetic ENSA/ARPP19 rescue, in vitro mTORC1 kinase assay on MASTL, phospho-IRS1/GRB10 immunoblotting, in vivo glucose tolerance tests in mice\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay identifying mTORC1 as MASTL kinase, genetic rescue with phosphomimetic substrates, in vivo metabolic phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"36354735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Phosphorylation of the MASTL C-tail turn motif at S861 (mouse) is dispensable for kinase function in intact mammalian cells, as shown by complementation of tamoxifen-inducible conditional Mastl knockout mouse embryonic fibroblasts with S861A mutant. Activation loop phosphorylations (T193 and T206) remain required.\",\n      \"method\": \"Tamoxifen-inducible conditional Mastl knockout MEFs, genetic complementation with phosphosite mutants, computational molecular docking\",\n      \"journal\": \"Journal of biomolecular structure & dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean genetic complementation in mammalian cells for specific phospho-site, single lab, single study; computational docking supportive but not Tier 1\",\n      \"pmids\": [\"36270968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A SILAC-based whole-cell lysate in vitro kinase screen identified 59 phospho-sites on 67 proteins as potential MASTL substrates beyond ARPP19/ENSA. Subsequent in vitro kinase assays suggested MASTL may phosphorylate hnRNPM, YB1, and TUBA1C under certain in vitro conditions.\",\n      \"method\": \"SILAC in vitro kinase screen with cell lysates, in vitro kinase assays for candidate substrates\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vitro screen with partial follow-up kinase assays, substrates not validated in cells, single lab, single method for each candidate\",\n      \"pmids\": [\"35732702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"E6AP is the E3 ubiquitin ligase that mediates MASTL protein degradation under normal conditions. Upon DNA damage, ATM phosphorylates E6AP at Ser-218, causing E6AP dissociation from MASTL, stabilizing MASTL protein levels, and enabling timely cell cycle recovery from the DNA damage checkpoint. E6AP depletion reduced DNA damage signaling and promoted checkpoint recovery in a MASTL-dependent manner.\",\n      \"method\": \"E3 ligase identification by co-immunoprecipitation and mass spectrometry, E6AP depletion, ATM inhibition, phospho-site mutant of E6AP (S218A), cell cycle recovery assays, ubiquitination assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP/MS identification of E3 ligase, phospho-site mutant validation, epistasis experiment (MASTL-dependent rescue), multiple orthogonal methods in peer-reviewed study\",\n      \"pmids\": [\"37672026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"E2F8 transcription factor directly activates MASTL expression by binding to the MASTL promoter, as validated by western blot, dual luciferase assay, and ChIP-qPCR. E2F8 overexpression shortens cisplatin-induced G2/M arrest by promoting MASTL-mediated mitotic progression, an effect cancelled by MASTL inhibition.\",\n      \"method\": \"Dual luciferase promoter assay, ChIP-qPCR, western blot, MASTL inhibition rescue experiment\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (luciferase, ChIP-qPCR, rescue experiment) establishing transcriptional regulation, single lab\",\n      \"pmids\": [\"28605876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A missense mutation in FLJ14813 (MASTL), E167D (G-to-C at nucleotide 565), segregates perfectly with autosomal dominant thrombocytopenia in a kindred of 51 family members and is absent from 94 unaffected controls, identifying MASTL as a candidate disease gene for THC2 thrombocytopenia.\",\n      \"method\": \"Genetic linkage/segregation analysis, sequencing of family cohort\",\n      \"journal\": \"Human heredity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — genetic segregation in large kindred establishes disease association, but no direct functional mechanism of the mutation shown in this paper\",\n      \"pmids\": [\"12890928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In vivo morpholino knockdown of MASTL in zebrafish results in deficiency of circulating thrombocytes and decreased expression of thrombopoietin receptor c-mpl and CD41/GpIIb. Both wild-type and E167D mutant MASTL localize to the nucleus when transiently expressed in baby hamster kidney cells.\",\n      \"method\": \"Zebrafish morpholino knockdown, northern blot, transient expression with localization by fluorescence in BHK cells\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — in vivo morpholino knockdown with specific thrombocyte phenotype and molecular markers, plus direct localization experiment; single lab\",\n      \"pmids\": [\"19460416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Xenopus oocytes, loss of Greatwall (Gwl/MASTL) and subsequent hyperactivation of PP2A-B55 prevents phosphorylation of Wee1/Myt1 and the APC/C complex, blocking APC/C activation. This impairs cyclin degradation and partial CDK1 inactivation required at the meiosis I–meiosis II transition, prevents mos-MAPK-Rsk1/2 pathway activation due to insufficient Mos accumulation, and blocks Erp1 degradation, revealing a feedback loop between APC/C and Erp1.\",\n      \"method\": \"Gwl depletion from Xenopus oocytes, immunoblotting for APC/C substrates, cyclin B levels, Wee1/Myt1 phosphorylation, Mos/MAPK pathway components\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion experiments in Xenopus oocyte system with multiple molecular readouts; preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MASTL facilitates phosphorylation of Y-box binding protein-1 (YBX1); phosphorylated YBX1 transcriptionally activates PAK4, identifying the MASTL/YBX1/PAK4 axis as a downstream effector pathway. STK24, a stress-responsive kinase, can activate MASTL in HCC under lenvatinib exposure. Disruption of this axis by MASTL inhibition restores HCC sensitivity to lenvatinib.\",\n      \"method\": \"Co-immunoprecipitation and mass spectrometry identifying YBX1 as MASTL-associated protein, ChIP-qPCR for YBX1-PAK4 transcriptional regulation, phosphorylation analysis for STK24-MASTL interaction\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS, ChIP-qPCR, and phosphorylation analysis as orthogonal methods; single lab\",\n      \"pmids\": [\"40456026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MASTL is required for anaphase entry in proliferating primordial germ cells (PGCs); Mastl-null PGCs enter mitosis normally but fail to proceed beyond a metaphase-like stage, and subsequent cell death eliminates female germ cells. Simultaneous deletion of Ppp2r1a (PP2A α subunit) rescues the mitotic progression defect in Mastl-null PGCs, placing PP2A downstream of Mastl in this pathway.\",\n      \"method\": \"PGC-specific conditional Mastl knockout mouse model, genetic epistasis with Ppp2r1a deletion, immunofluorescence, cell counting\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO in vivo with genetic epistasis (PP2A rescue) establishing pathway position, in vivo mammalian model\",\n      \"pmids\": [\"28224044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MASTL promotes pancreatic cancer progression by modulating EGFR protein stability and kinase signaling; MASTL loss reduces EGFR protein levels and downstream signaling, and combined targeting of MASTL with gemcitabine shows increased cancer cell killing.\",\n      \"method\": \"Loss- and gain-of-function studies in PC cell lines, murine PC models (KC and KPC), immunoblotting for EGFR stability, pharmacological combination experiments\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with mechanistic molecular readout (EGFR stability), in vivo models, single lab\",\n      \"pmids\": [\"34331012\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MASTL (Greatwall kinase) is a serine/threonine kinase activated during mitotic entry by Cdk1/cyclin B and by AKT (at T299) and RSK (at T297 in mammals); once active, it phosphorylates ENSA and ARPP19, which then inhibit PP2A/B55 phosphatase complexes, thereby maintaining Cdk1 substrate phosphorylation and the mitotic state. PP1 dephosphorylates and deactivates MASTL during mitotic exit to create a bistable switch. MASTL also acts kinase-independently by associating with MRTF-A to promote nuclear retention and SRF-dependent transcription of actomyosin contractility genes, and its protein stability is regulated by the E3 ubiquitin ligase E6AP, which is dissociated from MASTL upon ATM-mediated phosphorylation during DNA damage to enable checkpoint recovery. Beyond mitosis, the MASTL-PP2A/B55 axis controls mTORC1-S6K1 feedback phosphorylation of IRS1 and GRB10 to restrain PI3K-AKT activity, and MASTL regulates EGFR stability and Wnt/β-catenin signaling in cancer contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MASTL (Greatwall kinase) is a serine/threonine kinase that enforces the mitotic and meiotic state by suppressing the principal anti-mitotic phosphatase PP2A/B55, thereby preserving Cdk1-substrate phosphorylation [#0, #2]. Activated during M phase by Cdk1/cyclin B, MASTL drives inactivation of PP2A/B55delta independently of continued MPF activity, and depleting this phosphatase rescues the mitotic-entry failure of Greatwall-depleted extracts [#0]; in mammalian cells and mice this axis is required for both mitotic entry and exit, with PP2A complexes bearing B55alpha/B55delta acting genetically downstream of MASTL [#2, #22]. MASTL phosphorylates ENSA/ARPP19, which in turn inhibit PP2A/B55 to sustain CDK1 activity and time mitotic re-entry after DNA-damage checkpoint recovery [#6]. The switch is made bistable by PP1, which associates with MASTL and partially dephosphorylates and deactivates it at mitotic exit, releasing PP2A inhibition [#5]. MASTL also licenses Cdc20-independent recruitment of cyclin B1 to the APC/C and supports efficient cyclin B1 destruction [#4], and in oocytes and primordial germ cells it is required for timely APC/C activation and progression through the meiosis I–II transition [#3, #22]. Its activity is tuned by multiple upstream kinases—AKT (T299), RSK (T297/Thr297), and mTORC1—and by autophosphorylation, with the non-conserved middle region and activation-loop sites governing specificity and activity [#9, #10, #11, #13]; mTORC1-directed phosphorylation routes MASTL into a metabolic feedback loop that sustains S6K1-dependent phosphorylation of IRS1 and GRB10 to restrain PI3K-AKT signaling [#13]. MASTL protein levels are set by the E3 ligase E6AP, which is dissociated upon ATM-mediated phosphorylation during DNA damage to stabilize MASTL and enable checkpoint recovery [#16], and MASTL transcription is driven by E2F8 [#17]. Beyond catalysis, MASTL acts kinase-independently by binding MRTF-A to promote its nuclear retention and SRF-dependent transcription of actomyosin contractility genes, driving breast cancer cell motility and invasion [#12]. In cancer contexts MASTL supports proliferation through PP2A-B55, modulates EGFR stability, and operates a YBX1/PAK4 axis linked to lenvatinib resistance [#7, #23, #21]. A recurrent missense mutation (E167D) in MASTL co-segregates with autosomal dominant THC2 thrombocytopenia [#18], consistent with a non-mitotic role for MASTL in platelet actin-cytoskeletal dynamics via PP2A-B55 [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established the core mechanistic logic that Greatwall/MASTL maintains mitosis not by direct substrate phosphorylation alone but by suppressing the anti-mitotic phosphatase PP2A/B55.\",\n      \"evidence\": \"Xenopus egg extract depletion/add-back with in vitro phosphatase assays and biochemical reconstitution\",\n      \"pmids\": [\"19793917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the molecular intermediary linking MASTL to PP2A/B55 inhibition\", \"Mechanism shown in extract, not yet in intact mammalian cells\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the human MASTL loss-of-function phenotype and localization, establishing it as a Cdk1-downstream effector required across multiple mitotic transitions.\",\n      \"evidence\": \"RNAi knockdown, immunofluorescence, live-cell imaging and phosphoprotein immunoblotting in human cells\",\n      \"pmids\": [\"20818157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Centrosomal relocalization function not mechanistically defined\", \"Direct substrates not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided in vivo genetic proof that MASTL acts through B55alpha/B55delta-containing PP2A for mitotic exit, anchoring the pathway in mammalian physiology.\",\n      \"evidence\": \"Conditional knockout mouse models with genetic epistasis and in vivo tumor regression assays\",\n      \"pmids\": [\"21156286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the bistability/switch behavior of the system\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended MASTL function to meiosis, showing it is needed for timely APC/C activation and the CDK1 rise driving the meiosis I-II transition.\",\n      \"evidence\": \"Oocyte-specific conditional Mastl knockout mouse, live imaging and immunoblotting\",\n      \"pmids\": [\"25246615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of APC/C activation timing by MASTL not fully resolved at molecular level\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked MASTL to APC/C function directly by showing it is required for Cdc20-independent cyclin B1 recruitment and efficient destruction.\",\n      \"evidence\": \"RNAi with co-immunoprecipitation of cyclin B1 with APC/C in human cells\",\n      \"pmids\": [\"25750436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP without reciprocal validation\", \"Whether the cyclin B1-APC/C effect is kinase-dependent not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified PP1 as the deactivating phosphatase for MASTL, explaining the switch-like, bistable transition of phosphatase activity at mitotic exit.\",\n      \"evidence\": \"Co-IP of PP1 with MASTL, activity assays and mathematical modeling in human cells\",\n      \"pmids\": [\"26872783\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PP1-MASTL dephosphorylation sites not fully mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed MASTL in DNA-damage checkpoint recovery, showing it times mitotic re-entry through ARPP19/ENSA-mediated PP2A inhibition.\",\n      \"evidence\": \"RNAi and constitutively active MASTL with cell cycle analysis and CDK1-Tyr15/APC/C immunoblotting\",\n      \"pmids\": [\"26923777\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream signal coupling DNA damage to MASTL not defined here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated that MASTL's pro-proliferative role in cancer depends on its kinase activity and on PP2A-B55, validating it as a therapeutic target.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, kinase-dead rescue and in vivo xenograft regression in breast cancer cells\",\n      \"pmids\": [\"29229993\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which cancer subsets are dependent not predicted by a marker\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified E2F8 as a direct transcriptional activator of MASTL coupling it to cell-cycle gene-expression control and chemoresistance.\",\n      \"evidence\": \"Dual luciferase promoter assay, ChIP-qPCR and MASTL-inhibition rescue\",\n      \"pmids\": [\"28605876\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Broader transcriptional network controlling MASTL not mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed MASTL is required for anaphase entry in primordial germ cells, with PP2A genetically downstream, reinforcing the conserved MASTL-PP2A axis in germline mitosis.\",\n      \"evidence\": \"PGC-specific conditional knockout mouse with Ppp2r1a epistasis and immunofluorescence\",\n      \"pmids\": [\"28224044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cause of female-specific germ cell death downstream not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed a non-mitotic role for MASTL in postmitotic platelets, controlling actin-cytoskeletal dynamics through PP2A-B55 and linking it mechanistically to thrombocytopenia.\",\n      \"evidence\": \"Conditional and knock-in mutant mice, phosphoproteomics and in vivo pharmacological rescue\",\n      \"pmids\": [\"30252678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct cytoskeletal substrates of the MASTL-PP2A axis in platelets not individually validated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified RSK phosphorylation of MASTL at Thr297 as a mammalian-specific mechanism sustaining MASTL-ENSA activity during prolonged meiotic exit independent of cyclin B.\",\n      \"evidence\": \"Mouse oocyte live imaging, phosphomutant constructs and kinase inhibition\",\n      \"pmids\": [\"30293837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of T297-dependent activation not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved structural and regulatory determinants of MASTL activity, distinguishing autophosphorylation from exogenous activating phosphorylations and assigning specificity to the non-conserved middle region.\",\n      \"evidence\": \"HDX mass spectrometry, in vitro kinase assays with truncation/point mutants and phosphoproteomics\",\n      \"pmids\": [\"31852836\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length crystal/cryo-EM structure\", \"In-cell relevance of cryptic C-lobe activity not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified AKT phosphorylation at T299 as an activating input linking growth-factor signaling to MASTL-driven mitotic progression.\",\n      \"evidence\": \"In vitro kinase assay, T299A phosphomutant and loss-of-function in colorectal cancer cells\",\n      \"pmids\": [\"32123010\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Interplay of AKT and Cdk1 inputs on MASTL not quantitatively resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered a kinase-independent function of MASTL, binding MRTF-A to drive its nuclear retention and SRF-dependent contractility gene expression underlying cancer cell invasion.\",\n      \"evidence\": \"Co-IP, kinase-dead mutant, transcriptome/proteome profiling and invasion assays in breast cancer cells\",\n      \"pmids\": [\"32311005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect nature of MASTL-MRTF-A interaction not crystallographically defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected MASTL to receptor tyrosine kinase signaling in cancer by showing it modulates EGFR protein stability and downstream signaling.\",\n      \"evidence\": \"Loss/gain-of-function in pancreatic cancer cells and KC/KPC mouse models with EGFR immunoblotting\",\n      \"pmids\": [\"34331012\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of EGFR stabilization not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the MASTL-PP2A/B55 axis into metabolic signaling, showing mTORC1-phosphorylated MASTL sustains S6K1-dependent IRS1/GRB10 phosphorylation to restrain PI3K-AKT.\",\n      \"evidence\": \"Genetic depletion, phosphomimetic ENSA/ARPP19 rescue, in vitro mTORC1 kinase assay and in vivo glucose tolerance tests\",\n      \"pmids\": [\"36354735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific contribution to systemic metabolism not fully mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined the activation-site requirements of MASTL, showing the C-tail turn motif S861 is dispensable while activation-loop sites T193/T206 remain required.\",\n      \"evidence\": \"Inducible conditional Mastl knockout MEFs complemented with phosphosite mutants plus docking\",\n      \"pmids\": [\"36270968\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Docking-based structural inferences not experimentally validated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Broadened the candidate MASTL substrate repertoire beyond ARPP19/ENSA through an unbiased in vitro screen.\",\n      \"evidence\": \"SILAC in vitro kinase screen of cell lysates with follow-up kinase assays for candidates\",\n      \"pmids\": [\"35732702\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Candidate substrates (hnRNPM, YB1, TUBA1C) not validated in cells\", \"Single in vitro method per candidate\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified the E3 ligase E6AP controlling MASTL turnover and showed ATM-dependent E6AP phosphorylation stabilizes MASTL to enable checkpoint recovery.\",\n      \"evidence\": \"Co-IP/MS E3 ligase identification, E6AP S218A mutant, ATM inhibition and ubiquitination/recovery assays\",\n      \"pmids\": [\"37672026\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination site(s) on MASTL not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a MASTL/YBX1/PAK4 effector axis and an STK24 input in hepatocellular carcinoma linking MASTL to drug resistance.\",\n      \"evidence\": \"Co-IP/MS, ChIP-qPCR and phosphorylation analysis in HCC cells\",\n      \"pmids\": [\"40456026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct YBX1 phosphosite not validated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Dissected how loss of Greatwall and PP2A-B55 hyperactivation blocks the meiosis I-II transition by preventing Wee1/Myt1 and APC/C phosphorylation, revealing an APC/C-Erp1 feedback loop.\",\n      \"evidence\": \"Gwl depletion from Xenopus oocytes with immunoblotting of APC/C substrates and Mos/MAPK components (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Generality to mammalian meiosis not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological substrate repertoire of MASTL beyond ARPP19/ENSA, and the structural basis integrating its multiple activating inputs, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No validated in-cell substrates beyond ARPP19/ENSA\", \"No full-length structure of MASTL\", \"Mechanism of kinase-independent MRTF-A binding undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 10, 11]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 9, 13]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 19]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [3, 9, 22]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 13, 23]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [6, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ENSA\", \"ARPP19\", \"PP2A/B55\", \"PP1\", \"MRTF-A\", \"E6AP\", \"APC/C\", \"YBX1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":10,"faith_pct":90.0}}