{"gene":"TJP2","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":1994,"finding":"TJP2/ZO-2 was identified as a tight junction-associated peripheral membrane protein that co-immunoprecipitates with ZO-1 from MDCK cell extracts, establishing it as a member of the MAGUK protein family containing guanylate kinase-like and other conserved domains, localized exclusively to epithelial tight junctions (not fascia adherens).","method":"Co-immunoprecipitation, immunofluorescence, cDNA cloning and sequence analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with localization, foundational paper replicated widely","pmids":["8132716"],"is_preprint":false},{"year":1996,"finding":"ZO-2 contains three PDZ domains, an SH3 domain, and a guanylate kinase-like domain, and undergoes alternative splicing producing isoforms differing in a 36-amino acid C-terminal region; the domain architecture indicates multiple protein-protein interaction capacities.","method":"cDNA cloning, sequence analysis of multiple ZO-2 cDNA clones","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — complete cDNA sequence with structural domain mapping, foundational","pmids":["8824195"],"is_preprint":false},{"year":1999,"finding":"ZO-2 directly binds to the COOH-terminal YV sequence of claudins-1 through -8 via its PDZ1 domain in vitro; ZO-2 is recruited to claudin-based networks through both PDZ1/claudin-COOH and PDZ2/ZO-1-PDZ2 interactions.","method":"In vitro binding assays, transfection of claudins into L fibroblasts, co-localization in epithelial cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding with domain mapping, replicated across multiple claudins and in multiple cell systems","pmids":["10601346"],"is_preprint":false},{"year":1999,"finding":"ZO-2 directly interacts with F-actin in vitro (cosedimentation assay) and directly binds both ZO-1 and occludin; in vivo, ZO-1 and ZO-2 exist primarily as independent ZO-1·ZO-2 complexes rather than a trimeric ZO-1·ZO-2·ZO-3 complex.","method":"Actin cosedimentation, in vitro binding assays with recombinant proteins, immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro binding plus in vivo immunoprecipitation with orthogonal methods","pmids":["10575001"],"is_preprint":false},{"year":1999,"finding":"Cingulin interacts with ZO-2 via an NH2-terminal fragment (residues 1-378) in pull-down assays from epithelial and insect cell lysates; ZO-1 and ZO-2 immunoprecipitates contain cingulin, confirming in vivo interaction.","method":"Pull-down assays, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus in vivo co-IP, replicated","pmids":["10613913"],"is_preprint":false},{"year":2000,"finding":"Protein 4.1R isoforms (135 and 150 kDa) specifically interact with ZO-2; the interaction domains were mapped to exons 19-21 of 4.1R and residues 1054-1118 of ZO-2, providing a molecular link between tight junctions and the actin/spectrin cytoskeleton.","method":"Yeast two-hybrid, immunocolocalization, immunoprecipitation, in vitro binding studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including domain mapping and in vivo co-IP","pmids":["10874042"],"is_preprint":false},{"year":2001,"finding":"Adenovirus type 9 E4-ORF1 oncoprotein binds ZO-2 via a C-terminal PDZ-binding motif interacting with the first PDZ domain of ZO-2; this interaction causes aberrant cytoplasmic sequestration of ZO-2. Overexpression of wild-type ZO-2 (but not a mutant lacking PDZ2/3) interfered with E4-ORF1-induced focus formation, implicating ZO-2 as a candidate tumor suppressor.","method":"Co-immunoprecipitation, immunofluorescence, focus formation assays, domain-deletion mutants","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — domain-specific binding mapped by mutagenesis plus functional rescue assay","pmids":["11598001"],"is_preprint":false},{"year":2002,"finding":"ZO-2 localizes to the nucleus of sparse epithelial cells in clusters that partially co-localize with splicing factor SC35; nuclear staining diminishes at confluence and can be induced by impairing cell-cell contacts or mechanical injury. ZO-2 nuclear translocation is mediated by the actin cytoskeleton and involves shuttling of a pre-existing pool rather than newly synthesized protein. Nuclear export is sensitive to leptomycin B.","method":"Immunofluorescence, live-cell imaging, leptomycin B treatment, calcium switch assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — direct localization with functional inhibitor experiments, multiple conditions tested","pmids":["11855865"],"is_preprint":false},{"year":2002,"finding":"Nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B) via its PDZ-1 domain; this association was confirmed by yeast two-hybrid, co-immunoprecipitation, and co-localization in nuclei of epithelial and endothelial cells. ZO-1 does not associate with SAF-B, indicating non-redundant nuclear functions.","method":"Yeast two-hybrid, co-immunoprecipitation, co-localization by confocal microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — domain-mapped interaction confirmed by orthogonal methods","pmids":["12403786"],"is_preprint":false},{"year":2003,"finding":"A TJP2 mutation in the first PDZ domain (found in Amish families with familial hypercholanemia) reduces PDZ domain stability and ligand binding in vitro, and is associated with morphological changes in hepatic tight junctions.","method":"In vitro PDZ domain stability/binding assays, patient genetic analysis, liver histology","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1 — in vitro functional domain assay with structural consequence in patient tissue","pmids":["12704386"],"is_preprint":false},{"year":2003,"finding":"Tyrosine phosphorylation of the C-terminal tail of occludin (by c-Src in vitro) significantly reduces binding of ZO-2 (as well as ZO-1 and ZO-3) to occludin, but does not affect occludin-F-actin binding.","method":"In vitro phosphorylation (c-Src), GST pull-down binding assays","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with defined substrate and binding consequence","pmids":["12604349"],"is_preprint":false},{"year":2004,"finding":"ZO-2 associates with Jun, Fos and C/EBP transcription factors both at the nucleus and at the TJ region of epithelial cells; this association downregulates AP-1-dependent gene transcription in a dose-dependent manner, with both amino and carboxyl domains of ZO-2 capable of inhibiting transcription.","method":"GST pull-down, gel shift analysis (EMSA), co-immunoprecipitation, reporter gene (CAT) assays, immunolocalization","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing interaction and functional consequence","pmids":["14720506"],"is_preprint":false},{"year":2004,"finding":"Nuclear ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B1 and actin. Multiple NLS signals in the amino region mediate nuclear import; only the second of two putative NES sequences is functional (confirmed by ovalbumin-coupled peptide nuclear injection assay). The NLS region also regulates AP-1-dependent transcription.","method":"Nuclear fractionation, co-immunoprecipitation, NLS deletion analysis, nuclear export assay with ovalbumin-coupled peptides","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches with domain-level resolution","pmids":["15194440"],"is_preprint":false},{"year":2004,"finding":"ARVCF interacts with ZO-2 (and ZO-1) via a C-terminal PDZ-binding motif; the PDZ domains of ZO-2 can mediate nuclear localization of ARVCF, establishing a PDZ-domain-dependent mechanism for nuclear targeting.","method":"Co-immunoprecipitation, localization experiments, domain deletion analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional localization, single lab","pmids":["15456900"],"is_preprint":false},{"year":2005,"finding":"hScrib directly interacts with ZO-2 via two PDZ domains of hScrib and the C-terminal PDZ-binding motif of ZO-2; a point mutation in the LRR of hScrib that delocalizes it from the plasma membrane also abolishes ZO-2 interaction.","method":"Direct interaction assay, co-localization, mutagenesis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 — single pulldown/co-localization, domain mapped by mutagenesis","pmids":["15975580"],"is_preprint":false},{"year":2006,"finding":"ZO-2 (along with ZO-1) is required for tight junction strand formation and claudin polymerization; double knockdown/knockout of ZO-1 and ZO-2 in epithelial cells abolishes TJ formation. ZO-1 and ZO-2 independently determine the site of claudin polymerization, requiring dimerization and recruitment to the lateral membrane.","method":"Homologous recombination knockout + siRNA knockdown, exogenous ZO-1/ZO-2 rescue, forced membrane dimerization","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with clean KO/KD and rescue, definitive functional readout","pmids":["16923393"],"is_preprint":false},{"year":2006,"finding":"ZO-2 has four functional nuclear export signals (NES-0, NES-1, NES-2, NES-3); NES-0 and NES-3 are directly functional (confirmed by nuclear injection assay with ovalbumin-coupled peptides); NES-1 becomes functional upon phosphorylation at Ser369; mutation of any single NES is sufficient to cause nuclear accumulation of full-length ZO-2.","method":"Nuclear export assay with microinjection of NES-ovalbumin conjugates, leptomycin B sensitivity, transfection of NES mutants","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 — in vivo nuclear export assay with defined peptides and mutagenesis","pmids":["16920099"],"is_preprint":false},{"year":2006,"finding":"EcN (probiotic E. coli Nissle 1917) restores epithelial barrier integrity in EPEC-infected cells by enhancing ZO-2 expression and redistributing ZO-2 to cell boundaries, a process mediated by silencing of PKCζ.","method":"DNA microarray, immunofluorescence, Western blotting, PKC inhibitors, TEER measurement","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods but mechanistic link is correlative/inhibitor-based","pmids":["17087734"],"is_preprint":false},{"year":2007,"finding":"ZO-2 downregulates cyclin D1 transcription via an E box in the cyclin D1 promoter by interacting with c-Myc; the complex also recruits HDAC1 to the E box, and HDAC activity is required for ZO-2-mediated repression. ZO-2 and c-Myc co-immunoprecipitate.","method":"Reporter gene (CAT) assays, deletion analysis, EMSA, ChIP, co-immunoprecipitation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including ChIP confirming in vivo complex at the promoter","pmids":["17881732"],"is_preprint":false},{"year":2007,"finding":"ZO-2 silencing in MDCK cells increases paracellular permeability (gate function), disrupts fence function (non-polarized E-cadherin distribution), decreases occludin and E-cadherin expression in mature monolayers, delays arrival of ZO-1 and occludin to the plasma membrane during calcium switch, and causes atypical monolayer architecture with widened intercellular spaces.","method":"siRNA knockdown, TEER measurement, dextran permeability assay, immunofluorescence, calcium switch assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — clean KD with multiple defined phenotypic readouts","pmids":["17374535"],"is_preprint":false},{"year":2008,"finding":"ZO-2 knockout mice die shortly after implantation due to arrest in early gastrulation; ZO-2-deficient embryos show decreased proliferation at E6.5, increased apoptosis at E7.5, altered apical junctional complex architecture, and increased paracellular permeability. ZO-3 knockout mice have no obvious phenotype.","method":"Gene knockout, embryo histology, permeability tracer assay, immunostaining, cell proliferation/apoptosis assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with defined developmental and barrier phenotype","pmids":["18172007"],"is_preprint":false},{"year":2008,"finding":"ZO-1 and ZO-2 are required for integration of myosin-2 into the zonula adherens (ZA); in ZO1(ko)/2(kd) cells, myosin-2 fails to integrate into ZA, and rescue by full-length ZO-1 or ZO-2 (or ZO-1 lacking PDZ1/2 but not PDZ1/2/3) restores myosin-2 integration. ZO-1/2-dependent RhoA/ROCK signaling spatiotemporally regulates ZA establishment.","method":"Knockout/knockdown, domain-deletion rescue, FRET RhoA activity assay, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — epistasis via KO/KD with domain-resolution rescue and FRET-based signaling readout","pmids":["18596233"],"is_preprint":false},{"year":2010,"finding":"ZO-2 forms a complex with YAP2 via the first PDZ domain of ZO-2 binding the PDZ-binding motif of YAP2; endogenous ZO-2 and YAP2 co-localize in the nucleus. ZO-2 facilitates nuclear localization and pro-apoptotic function of YAP2 in a PDZ-domain-dependent manner.","method":"Co-immunoprecipitation, co-localization, domain-deletion analysis, apoptosis assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — domain-mapped interaction with functional consequence (apoptosis), multiple methods","pmids":["20868367"],"is_preprint":false},{"year":2010,"finding":"Genomic duplication of TJP2 leads to overexpression of ZO-2 protein; this overexpression decreases phosphorylation of GSK-3β and alters expression of apoptosis-regulating genes, causing progressive hearing loss (DFNA51) via increased susceptibility to apoptosis of inner ear cells.","method":"Genomic sequencing, family analysis, RT-PCR, Western blot, phosphorylation assays","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — human genetic gain-of-function with downstream phosphorylation readout, but correlative for mechanistic link","pmids":["20602916"],"is_preprint":false},{"year":2013,"finding":"JAM-A associates directly with ZO-2 (and indirectly with afadin); this complex, along with PDZ-GEF1, activates the small GTPase Rap2c to regulate epithelial barrier function and apical cytoskeleton contraction via RhoA and nonmuscle myosin phosphorylation.","method":"Direct binding assay, co-immunoprecipitation, siRNA knockdown, permeability assay, RhoA activity assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — direct binding plus signaling pathway epistasis with multiple orthogonal readouts","pmids":["23885123"],"is_preprint":false},{"year":2013,"finding":"SNX27 interacts with ZO-2 via the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2; when tight junctions are disrupted by calcium chelation, ZO-2 transiently localizes to SNX27-positive early endosomes. Depletion of SNX27 decreases ZO-2 (but not ZO-1) mobility at junctions and increases junctional permeability.","method":"Proteomics, co-immunoprecipitation, co-localization, FRAP, permeability assay, siRNA knockdown","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — proteomic identification plus domain-mapped interaction with functional trafficking and barrier readouts","pmids":["23826934"],"is_preprint":false},{"year":2014,"finding":"Protein-truncating mutations in TJP2 cause failure of ZO-2 protein localization and disruption of tight junction structure, leading to severe cholestatic liver disease (PFIC4).","method":"Patient genetic analysis, protein localization studies in patient tissue, electron microscopy of tight junctions","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — human genetic loss-of-function with protein localization and ultrastructural phenotype","pmids":["24614073"],"is_preprint":false},{"year":2016,"finding":"ZO-2 is SUMOylated; it associates with SUMO E2 enzyme Ubc9 and deconjugating proteases SENP1/SENP3; lysine 730 in the GuK domain is a SUMOylation site. Mutation of K730 (mimicking constitutive SUMOylation) retains ZO-2 in cytoplasm and abolishes its inhibitory effect on GSK3β activity and β-catenin/TCF-4-mediated transcription; ZO-2 directly binds GSK3β and forms a complex with β-catenin.","method":"Co-immunoprecipitation, Ubc9 fusion-directed SUMOylation, site-directed mutagenesis, reporter gene assays","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 1 — direct SUMOylation assay with site mutagenesis and functional signaling readout","pmids":["27604867"],"is_preprint":false},{"year":2016,"finding":"ZO-2 silencing in renal epithelial MDCK cells induces cell hypertrophy by: (1) prolonging G1 phase via increased cyclin D1; (2) increasing protein synthesis via nuclear accumulation of YAP leading to reduced PTEN expression, activation of Akt/mTOR/S6K1. In vivo, compensatory renal hypertrophy after uninephrectomy is accompanied by decreased ZO-2 and nuclear YAP.","method":"siRNA knockdown, cell cycle analysis, flow cytometry, reporter assays, mTOR pathway inhibitors, in vivo uninephrectomy model","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — in vitro KD with defined pathway epistasis confirmed in vivo","pmids":["27009203"],"is_preprint":false},{"year":2018,"finding":"The organophosphate pesticide methamidophos covalently binds to ZO-2 at serine, tyrosine, and lysine residues (identified by mass spectrometry), inducing ZO-2 phosphorylation and reducing ZO-2/occludin interaction. Covalent modification at a lysine ubiquitination site (K) interferes with ZO-2 degradation and TJ sealing, demonstrated by transfection with a ZO-2 mutant at a MET target lysine residue.","method":"Mass spectrometry, co-immunoprecipitation, site-directed mutagenesis, transfection, permeability assay","journal":"Toxicology and applied pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — mass spectrometry identification with mutagenesis functional validation, single lab","pmids":["30291936"],"is_preprint":false},{"year":2019,"finding":"Activation of the Ca2+-sensing receptor triggers PKC/WNK4 signaling, leading to ZO-2 phosphorylation and concentration at tight junctions. In low calcium, ZO-2 is protected from degradation by association with 14-3-3ζ and 14-3-3σ proteins; upon Ca2+ restoration, ZO-2/14-3-3 complexes move to cell borders and dissociate (14-3-3 is proteasomally degraded; ZO-2 integrates into TJs). The unique region 2 of ZO-2 and S261 within an NLS are critical for 14-3-3 interaction and nuclear import.","method":"Co-immunoprecipitation, kinase inhibitors, calcium switch assay, domain mutagenesis, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — signaling pathway epistasis with domain mutagenesis and functional localization readout","pmids":["31318316"],"is_preprint":false},{"year":2021,"finding":"Liver-specific deletion of Tjp2 in mice causes lower Cldn1 protein levels, dilated canaliculi, reduced microvilli density, aberrant radixin and BSEP distribution, mild progressive cholestasis, and lower expression of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10; a cholic acid diet causes severe cholestasis and liver necrosis in Tjp2-deficient but not control mice.","method":"Conditional knockout in hepatocytes/cholangiocytes, biochemical analyses, electron microscopy, immunostaining, fluorescein-dextran permeability","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with multiple defined molecular and functional readouts","pmids":["33465371"],"is_preprint":false},{"year":2021,"finding":"Nuclear ZO-2 facilitates TEAD entry into the nucleus; ZO-2 and TEAD interact in the cytoplasm (confirmed by proximity ligation, immunoprecipitation, pull-down); inhibition of nPKCδ promotes ZO-2/TEAD cytoplasmic interaction and co-importation. Nuclear exit of ZO-2/TEAD is enhanced by nPKCε-mediated activation of a ZO-2 NES.","method":"Proximity ligation assay, co-immunoprecipitation, GST pull-down, siRNA knockdown, kinase inhibitors, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal interaction assays with defined kinase-dependent mechanism","pmids":["34010016"],"is_preprint":false},{"year":2021,"finding":"ZO-2 functions as a scaffold for the Hippo pathway by associating with LATS1; ZO-2 silencing reduces LATS kinase activity and leads to nuclear accumulation of YAP. In liver steatosis, ZO-2 is silenced and this correlates with diminished LATS activity; metformin (AMPK activator blocking JNK) restores ZO-2 and claudin-1 expression in steatotic liver.","method":"Co-immunoprecipitation (ZO-2/LATS1), siRNA knockdown, kinase activity assays, in vivo obese rat model, immunofluorescence","journal":"Tissue barriers","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP for scaffold function, supported by in vivo model","pmids":["34689705"],"is_preprint":false},{"year":2022,"finding":"ZO-2 is polyubiquitinated at K759 and K992 (K48-linked, targeting for proteasomal degradation), confirmed by mutation of these sites reducing ubiquitination and extending ZO-2 half-life. K730 (SUMOylation site) mutation increases ubiquitination and decreases half-life. Mutation of any of these lysines reduces TJ sealing.","method":"Co-immunoprecipitation with ubiquitin, TUBES (tandem ubiquitin-binding entities), site-directed mutagenesis, half-life assay (CHX chase), TEER measurement","journal":"Cells","confidence":"High","confidence_rationale":"Tier 1 — direct ubiquitination assay with site mutagenesis and functional consequence","pmids":["36291162"],"is_preprint":false},{"year":2022,"finding":"In the mouse liver, Tjp2 negatively regulates Yap and Wwtr1/Taz protein expression; hepatocyte-specific (but not cholangiocyte-specific) Tjp2 deletion leads to DDC-diet-induced hepatocyte-to-cholangiocyte transdifferentiation in a Yap/Taz-dependent manner.","method":"Conditional knockout (hepatocyte- vs cholangiocyte-specific), immunostaining, Yap/Taz protein level analysis, dietary challenge","journal":"NPJ Regenerative medicine","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with mechanistic pathway epistasis (Yap/Taz dependence)","pmids":["36151109"],"is_preprint":false},{"year":2023,"finding":"ZNF582 upregulates TJP2 protein expression; increased TJP2 then binds ERK2, promotes ERK2 protein expression, and suppresses ERK2 phosphorylation, thereby inhibiting ccRCC growth and metastasis.","method":"TMT quantitative proteomics, co-immunoprecipitation, Western blot, orthotopic tumor models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — proteomics-identified interaction confirmed by Co-IP with in vivo functional readout, single lab","pmids":["36966163"],"is_preprint":false},{"year":2023,"finding":"ZO-2 is required for p190A RhoGAP to activate LATS kinases and the Hippo pathway; interaction of p190A with ZO-2 is dependent on RasGAP. Both RasGAP and ZO-2 are necessary for p190A to promote mesenchymal-to-epithelial transition and contact inhibition of proliferation.","method":"Co-immunoprecipitation, siRNA knockdown, LATS kinase activity assays, tumor growth assays, reporter assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — interaction identified with functional epistasis across multiple assays","pmids":["37995182"],"is_preprint":false},{"year":2024,"finding":"ZO-2 acts as a scaffold to promote LATS1/YAP interaction: ZO-2 brings LATS1 (via SH3 domain) and YAP (via PDZ domain) together, enabling LATS1-dependent phosphorylation and cytoplasmic retention/inactivation of YAP, maintaining Hippo pathway activation and contact inhibition of proliferation.","method":"Co-immunoprecipitation, domain-deletion analysis, LATS1 kinase assays, YAP phosphorylation assays","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 — mechanistic reconstitution of scaffolding with defined domains and kinase assay","pmids":["39462647"],"is_preprint":false},{"year":2024,"finding":"Absence of ZO-2 reduces apical membrane rigidity, inhibits γ-actin and JAM-A recruitment to cell borders, facilitates p114RhoGEF and afadin accumulation at junctions, and increases mechanical tension at TJs (measured by FRET). ZO-2 KD cells show impaired responses to substrate stiffness and topography, with increased YAP and Snail nuclear accumulation.","method":"Atomic force microscopy, FRET tension probes, immunofluorescence, siRNA knockdown, in silico binding stability analysis","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1-2 — direct biophysical measurement (AFM, FRET) with genetic KD and mechanistic pathway readouts","pmids":["38473701"],"is_preprint":false},{"year":2025,"finding":"ZO-2 colocalizes with CEP164 at the distal appendage of the mother centriole and is present at mitotic spindle poles, the basal body of primary cilia, and spermatozoa tails. ZO-2 depletion alters centriolar protein levels (CEP164, centriolin, CEP135), inhibits astral and mitotic spindle microtubule growth, increases NuMA and decreases KIF14/TPX2/p-Aurora at spindle poles, reduces mitotic spindle length, and blocks primary cilia development. KIF14, NuMA, and p-Aurora co-immunoprecipitate with ZO-2; NuMA and Aurora-A bind distinct ZO-2 segments.","method":"Immunofluorescence, co-immunoprecipitation, siRNA knockdown, domain binding assays","journal":"Cell and tissue research","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization with functional KD phenotypes, single lab with multiple readouts","pmids":["40728639"],"is_preprint":false},{"year":2025,"finding":"ZO-2 is identified as a novel NTCP-binding protein by immunopurification/LC-MS/MS; ZO-2 knockdown or knockout reduces NTCP at the cell surface, decreasing HBV attachment and infection. HBV surface element preS1 dissociates NTCP from ZO-2 and promotes formation of NTCP-preS1-actin complexes that are internalized; actin polymerization is required for preS1 internalization and HBV infection.","method":"Immunopurification + LC-MS/MS, siRNA knockdown/knockout, HBV infection assay, co-immunoprecipitation, actin inhibitor (latrunculin A)","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 — proteomic identification confirmed by KO functional assay with mechanistic dissection of receptor trafficking","pmids":["41870046"],"is_preprint":false},{"year":2025,"finding":"c-Abl directly binds to and phosphorylates the C-terminus of ZO-2; c-Abl also stimulates JAK1 activity, which subsequently phosphorylates the N-terminus of ZO-2. By RNAi knockdown/rescue, c-Abl regulates cellular morphology and migration through ZO-2 phosphorylation.","method":"In vitro kinase assay, co-immunoprecipitation, RNAi knockdown/rescue, traction force microscopy","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with defined substrate plus functional rescue experiments","pmids":["41259016"],"is_preprint":false}],"current_model":"TJP2/ZO-2 is a MAGUK scaffold protein that localizes to epithelial tight junctions and, in sparse/subconfluent cells, to the nucleus; at tight junctions it directly binds claudins (via PDZ1), occludin, ZO-1, cingulin, JAM-A, and the actin cytoskeleton to organize the paracellular barrier and drive claudin polymerization together with ZO-1; in the nucleus it associates with transcription factors (AP-1/Jun/Fos/C/EBP, c-Myc, TEAD) and the SAF-B chromatin protein to suppress proliferative gene expression (cyclin D1, Wnt/β-catenin targets); it acts as a scaffold for the Hippo pathway by recruiting LATS1 and YAP via its SH3 and PDZ domains to promote YAP phosphorylation and cytoplasmic retention, thereby enforcing contact inhibition; its intracellular traffic between TJ, cytoplasm, and nucleus is controlled by multiple NLS/NES sequences, SUMOylation at K730, K48-polyubiquitination at K759/K992 for proteasomal turnover, interaction with 14-3-3 proteins in low-calcium conditions, and phosphorylation cascades including PKC/WNK4 and c-Abl/JAK1; loss-of-function mutations cause progressive cholestatic liver disease (PFIC4) by disrupting TJ localization of ZO-2, reducing claudin-1 and bile acid transporter (BSEP/ABCB11) expression, and deregulating YAP/Taz-mediated hepatocyte transdifferentiation."},"narrative":{"teleology":[{"year":1994,"claim":"Identifying ZO-2 as a ZO-1-interacting MAGUK protein at tight junctions established the existence of a multi-protein scaffold at the cytoplasmic face of TJs beyond ZO-1 alone.","evidence":"Co-immunoprecipitation from MDCK cells with immunofluorescence localization","pmids":["8132716"],"confidence":"High","gaps":["Binding domains not yet mapped","Functional role at TJ unknown"]},{"year":1996,"claim":"Determination of the full domain architecture (three PDZ, SH3, GuK domains) revealed the multi-interaction capacity of ZO-2 and explained how it could serve as a hub scaffold.","evidence":"cDNA cloning and sequence analysis with alternative splice isoform characterization","pmids":["8824195"],"confidence":"High","gaps":["Ligands for each domain not identified","No functional assays for individual domains"]},{"year":1999,"claim":"Demonstration that ZO-2 directly binds claudins (via PDZ1), occludin, ZO-1, cingulin, and F-actin defined the molecular connections through which ZO-2 links transmembrane TJ proteins to the cytoskeleton.","evidence":"In vitro binding/cosedimentation assays, co-immunoprecipitation, and transfection of claudins into fibroblasts","pmids":["10601346","10575001","10613913"],"confidence":"High","gaps":["Whether ZO-2 is sufficient for TJ strand formation unknown","Regulation of these interactions uncharacterized"]},{"year":2002,"claim":"Discovery that ZO-2 shuttles to the nucleus in subconfluent cells and interacts with SAF-B via PDZ1 revealed a non-redundant nuclear role distinct from ZO-1, linking TJ disassembly to changes in nuclear function.","evidence":"Immunofluorescence with leptomycin B and calcium switch; yeast two-hybrid and co-IP for SAF-B interaction","pmids":["11855865","12403786"],"confidence":"High","gaps":["Transcriptional targets of nuclear ZO-2 unknown","NLS/NES sequences not yet mapped"]},{"year":2003,"claim":"A PDZ1 mutation causing familial hypercholanemia linked ZO-2 to human liver disease and showed that PDZ1 integrity is essential for hepatic TJ function.","evidence":"Patient genetic analysis with in vitro PDZ domain stability/binding assays and liver histology","pmids":["12704386"],"confidence":"High","gaps":["Mechanism of cholestasis not resolved","Whether complete loss of function causes more severe disease unknown"]},{"year":2004,"claim":"Identification of ZO-2 as a transcriptional repressor of AP-1 targets through association with Jun/Fos/C/EBP, and mapping of multiple NLS/NES sequences, established how ZO-2 nuclear shuttling translates into gene regulation.","evidence":"GST pull-down, EMSA, reporter assays, NLS deletion analysis, nuclear export peptide assays","pmids":["14720506","15194440"],"confidence":"High","gaps":["Genome-wide transcriptional targets not defined","Phosphorylation-dependent regulation of NES not yet shown"]},{"year":2006,"claim":"Double knockout/knockdown of ZO-1 and ZO-2 abolished TJ strand formation, proving these two proteins are the essential and sufficient scaffolds for claudin polymerization; four NES sequences with phosphorylation-dependent activation were mapped.","evidence":"Genetic epistasis (KO/KD/rescue in EpH4 cells); NES-ovalbumin injection assays with NES mutants","pmids":["16923393","16920099"],"confidence":"High","gaps":["Relative contributions of ZO-1 vs ZO-2 not fully separated","In vivo confirmation of NES phosphoregulation lacking"]},{"year":2007,"claim":"ZO-2 was shown to repress cyclin D1 transcription through c-Myc/HDAC1 recruitment to the E-box, and ZO-2 knockdown increased paracellular permeability and disrupted fence function, connecting nuclear and junctional roles to cell cycle control and polarity.","evidence":"ChIP, reporter assays, co-IP for c-Myc; siRNA KD in MDCK with TEER, dextran permeability, and calcium switch","pmids":["17881732","17374535"],"confidence":"High","gaps":["Whether cyclin D1 repression is the main growth-inhibitory mechanism unknown","Redundancy with ZO-1 at TJs not fully resolved"]},{"year":2008,"claim":"ZO-2 knockout embryos died at gastrulation with proliferation defects, apoptosis, and barrier failure, proving ZO-2 is essential for early mammalian development and non-redundant with ZO-1 in vivo.","evidence":"Gene knockout mice with embryo histology, permeability tracer, proliferation/apoptosis assays","pmids":["18172007"],"confidence":"High","gaps":["Cell-type-specific requirements in later development unknown","Molecular basis of embryonic lethality not dissected"]},{"year":2010,"claim":"ZO-2 was found to interact with YAP2 via PDZ1 and facilitate its nuclear localization and pro-apoptotic activity, establishing ZO-2 as a Hippo pathway component; separately, TJP2 duplication causing overexpression was linked to progressive hearing loss (DFNA51).","evidence":"Co-IP, domain-deletion analysis, apoptosis assays (YAP2); family genomic analysis with phosphorylation readouts (DFNA51)","pmids":["20868367","20602916"],"confidence":"High","gaps":["Whether ZO-2 scaffolds LATS-YAP phosphorylation not yet tested","DFNA51 mechanism remains correlative"]},{"year":2014,"claim":"Protein-truncating TJP2 mutations were shown to cause severe progressive familial intrahepatic cholestasis (PFIC4) with TJ ultrastructural disruption, establishing complete ZO-2 loss as a cause of Mendelian liver disease.","evidence":"Patient genetic analysis with immunolocalization and electron microscopy of hepatic TJs","pmids":["24614073"],"confidence":"High","gaps":["Downstream molecular pathology (claudin, transporter levels) not fully characterized at this point","No animal model recapitulating PFIC4"]},{"year":2016,"claim":"SUMOylation at K730 was identified as a switch controlling ZO-2 subcellular localization and its ability to inhibit β-catenin/Wnt signaling; ZO-2 silencing was shown to cause cell hypertrophy via nuclear YAP/Akt/mTOR activation, integrating post-translational control with growth regulation.","evidence":"Ubc9-directed SUMOylation, site mutagenesis, reporter assays; siRNA KD with cell cycle/mTOR pathway analysis and in vivo uninephrectomy model","pmids":["27604867","27009203"],"confidence":"High","gaps":["SUMO ligase identity unknown","Whether SUMOylation and ubiquitination are coordinated not tested"]},{"year":2019,"claim":"Ca2+-sensing receptor/PKC/WNK4 signaling was found to drive ZO-2 phosphorylation and TJ recruitment, while 14-3-3 proteins protect ZO-2 from degradation in low calcium, revealing how extracellular calcium signals control ZO-2 trafficking between cytoplasm and TJs.","evidence":"Co-IP, kinase inhibitors, calcium switch, domain mutagenesis including S261 in NLS","pmids":["31318316"],"confidence":"High","gaps":["Direct WNK4 phosphorylation sites on ZO-2 not mapped","14-3-3 binding stoichiometry and specificity not resolved"]},{"year":2021,"claim":"Liver-specific Tjp2 deletion in mice recapitulated key PFIC4 features including reduced claudin-1, BSEP/Abcb11, and canalicular defects, while nuclear ZO-2 was shown to co-import TEAD via PKCδ/ε-regulated shuttling, expanding ZO-2's nuclear transcriptional partnerships.","evidence":"Hepatocyte-specific conditional KO with EM, biochemistry, and dietary challenge; proximity ligation and kinase inhibitors for TEAD interaction","pmids":["33465371","34010016"],"confidence":"High","gaps":["Whether TEAD nuclear import is the key driver of cholestasis phenotype untested","ZO-2-LATS scaffolding mechanism not yet reconstituted"]},{"year":2022,"claim":"K48-linked polyubiquitination at K759 and K992 was shown to target ZO-2 for proteasomal degradation, and interplay with K730 SUMOylation was demonstrated, establishing a post-translational code controlling ZO-2 turnover and TJ sealing; hepatocyte-specific Tjp2 loss was shown to derepress Yap/Taz and enable hepatocyte-to-cholangiocyte transdifferentiation.","evidence":"TUBES ubiquitin capture, site-directed mutagenesis, CHX chase, TEER; hepatocyte- vs cholangiocyte-specific KO with dietary DDC challenge","pmids":["36291162","36151109"],"confidence":"High","gaps":["E3 ubiquitin ligase identity unknown","Full ubiquitin/SUMO crosstalk mechanism not resolved"]},{"year":2023,"claim":"ZO-2 was identified as essential for p190A RhoGAP-mediated LATS activation and as an ERK2-binding protein suppressing ERK phosphorylation, broadening ZO-2's scaffolding role to RhoGAP-Hippo and MAPK signaling.","evidence":"Co-IP, siRNA KD, LATS kinase activity assays, tumor growth assays (p190A); TMT proteomics, co-IP, orthotopic tumor models (ERK2)","pmids":["37995182","36966163"],"confidence":"High","gaps":["Direct binding interface of p190A-ZO-2 not mapped","ERK2 interaction confirmed in single lab only"]},{"year":2024,"claim":"The scaffolding mechanism for Hippo signaling was reconstituted: ZO-2 bridges LATS1 via SH3 and YAP via PDZ domains to enable LATS1-dependent YAP phosphorylation; biophysically, ZO-2 loss reduces apical rigidity and increases TJ mechanical tension.","evidence":"Domain-deletion co-IP with LATS1 kinase/YAP phosphorylation assays; AFM and FRET tension probes in KD cells","pmids":["39462647","38473701"],"confidence":"High","gaps":["Structural basis of SH3-LATS1 and PDZ-YAP interfaces not determined","How mechanical tension feedback integrates with Hippo signaling unknown"]},{"year":2025,"claim":"ZO-2 was localized to centrosomes and the basal body of primary cilia and shown to regulate mitotic spindle organization, and was independently identified as a stabilizer of the bile acid transporter NTCP at the cell surface required for HBV entry; c-Abl/JAK1 were identified as kinases that directly phosphorylate ZO-2 to control cell morphology and migration.","evidence":"Co-IP with spindle/centrosomal proteins and siRNA KD (cilia/spindle); LC-MS/MS immunopurification with KO functional HBV assay (NTCP); in vitro kinase assays with RNAi rescue (c-Abl/JAK1)","pmids":["40728639","41870046","41259016"],"confidence":"High","gaps":["Centrosomal localization confirmed in single lab only","Whether NTCP stabilization contributes to PFIC4 pathology untested","JAK1 phosphorylation sites on ZO-2 not mapped"]},{"year":null,"claim":"Key unresolved questions include the identity of the E3 ubiquitin ligase(s) targeting ZO-2, the structural basis for ZO-2's scaffolding of LATS1-YAP, the genome-wide transcriptional program controlled by nuclear ZO-2, and how ZO-2's centrosomal and ciliary functions relate to its established junctional and nuclear roles.","evidence":"","pmids":[],"confidence":"Low","gaps":["E3 ligase unknown","No high-resolution structure of ZO-2 scaffold complexes","No genome-wide ChIP-seq or nuclear transcriptomics for ZO-2","Centrosome/cilia role not independently replicated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,15,22,33,37,38]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[11,18,27,32]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,5,39]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[15,21]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,8,11,12,18,32]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,15,19,30]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,5,39,40]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[40]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[40]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[27,30,32]}],"pathway":[{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,2,15,19,26]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[22,28,33,37,38]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[11,18,27,32]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[18,28,40]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,26,31,35]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[27,34]}],"complexes":["ZO-1/ZO-2 heterodimer","Tight junction plaque complex"],"partners":["TJP1","OCLN","CLDN1","CGN","F11R","LATS1","YAP1","ABL1"],"other_free_text":[]},"mechanistic_narrative":"TJP2/ZO-2 is a MAGUK family scaffold protein that organizes epithelial tight junctions, suppresses proliferative gene expression in the nucleus, and enforces contact inhibition through the Hippo pathway. At tight junctions, ZO-2 binds claudins via its PDZ1 domain, occludin, ZO-1, cingulin, JAM-A, and F-actin, and together with ZO-1 is essential for claudin polymerization into TJ strands and paracellular barrier formation; its absence causes loss of barrier integrity, reduced apical membrane rigidity, and in the liver leads to progressive cholestatic disease (PFIC4) with diminished claudin-1 and BSEP expression [PMID:10601346, PMID:16923393, PMID:24614073, PMID:33465371]. In the nucleus, ZO-2 represses AP-1- and β-catenin/TCF-dependent transcription by associating with Jun/Fos, c-Myc, and SAF-B, downregulates cyclin D1 via c-Myc/HDAC1 recruitment, and acts as a Hippo pathway scaffold that bridges LATS1 (via its SH3 domain) and YAP (via its PDZ domain) to promote YAP phosphorylation and cytoplasmic retention [PMID:14720506, PMID:17881732, PMID:39462647, PMID:12403786]. ZO-2 subcellular distribution is controlled by multiple NLS/NES sequences regulated by PKC, c-Abl/JAK1 phosphorylation, SUMOylation at K730, K48-linked polyubiquitination at K759/K992 for proteasomal turnover, and calcium-dependent 14-3-3 association [PMID:16920099, PMID:31318316, PMID:36291162, PMID:27604867, PMID:41259016]."},"prefetch_data":{"uniprot":{"accession":"Q9UDY2","full_name":"Tight junction protein 2","aliases":["Tight junction protein ZO-2","Zona occludens protein 2","Zonula occludens protein 2"],"length_aa":1190,"mass_kda":134.0,"function":"Plays a role in tight junctions and adherens junctions (By similarity). Acts as a positive regulator of RANKL-induced osteoclast differentiation, potentially via mediating downstream transcriptional activity (By similarity)","subcellular_location":"Cell junction, adherens junction; Cell membrane; Cell junction, tight junction; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UDY2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TJP2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000119139","cell_line_id":"CID000737","localizations":[{"compartment":"cell_contact","grade":3},{"compartment":"cytoplasmic","grade":1}],"interactors":[{"gene":"TJP1","stoichiometry":10.0},{"gene":"BCKDK","stoichiometry":4.0},{"gene":"CALD1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"MAGI1","stoichiometry":0.2},{"gene":"PGAM5","stoichiometry":0.2},{"gene":"BIN3","stoichiometry":0.2},{"gene":"KIF14","stoichiometry":0.2},{"gene":"GTF2H5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000737","total_profiled":1310},"omim":[{"mim_id":"619658","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 7, WITH OR WITHOUT HEARING LOSS; PFIC7","url":"https://www.omim.org/entry/619658"},{"mim_id":"619232","title":"HYPERCHOLANEMIA, FAMILIAL 3; FHCA3","url":"https://www.omim.org/entry/619232"},{"mim_id":"617677","title":"ZO2-ASSOCIATED SPECKLE PROTEIN","url":"https://www.omim.org/entry/617677"},{"mim_id":"617431","title":"UBIQUITIN-SPECIFIC PEPTIDASE 53; USP53","url":"https://www.omim.org/entry/617431"},{"mim_id":"615878","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 4; PFIC4","url":"https://www.omim.org/entry/615878"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Plasma membrane","reliability":"Enhanced"},{"location":"Cell Junctions","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TJP2"},"hgnc":{"alias_symbol":["ZO-2","X104","ZO2"],"prev_symbol":["DFNA51"]},"alphafold":{"accession":"Q9UDY2","domains":[{"cath_id":"2.30.42.10","chopping":"26-124","consensus_level":"high","plddt":86.1002,"start":26,"end":124},{"cath_id":"2.30.42.10","chopping":"307-384","consensus_level":"high","plddt":86.9264,"start":307,"end":384},{"cath_id":"2.30.42.10","chopping":"501-673","consensus_level":"medium","plddt":90.8371,"start":501,"end":673},{"cath_id":"3.40.50.300","chopping":"727-890_897-919","consensus_level":"high","plddt":84.7832,"start":727,"end":919}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UDY2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UDY2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UDY2-F1-predicted_aligned_error_v6.png","plddt_mean":63.03},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TJP2","jax_strain_url":"https://www.jax.org/strain/search?query=TJP2"},"sequence":{"accession":"Q9UDY2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UDY2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UDY2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UDY2"}},"corpus_meta":[{"pmid":"10601346","id":"PMC_10601346","title":"Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10601346","citation_count":919,"is_preprint":false},{"pmid":"16923393","id":"PMC_16923393","title":"ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16923393","citation_count":656,"is_preprint":false},{"pmid":"8132716","id":"PMC_8132716","title":"Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.","date":"1994","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/8132716","citation_count":396,"is_preprint":false},{"pmid":"10575001","id":"PMC_10575001","title":"Protein interactions at the tight junction. 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co-immunoprecipitates with ZO-1 from MDCK cell extracts, establishing it as a member of the MAGUK protein family containing guanylate kinase-like and other conserved domains, localized exclusively to epithelial tight junctions (not fascia adherens).\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, cDNA cloning and sequence analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with localization, foundational paper replicated widely\",\n      \"pmids\": [\"8132716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ZO-2 contains three PDZ domains, an SH3 domain, and a guanylate kinase-like domain, and undergoes alternative splicing producing isoforms differing in a 36-amino acid C-terminal region; the domain architecture indicates multiple protein-protein interaction capacities.\",\n      \"method\": \"cDNA cloning, sequence analysis of multiple ZO-2 cDNA clones\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete cDNA sequence with structural domain mapping, foundational\",\n      \"pmids\": [\"8824195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ZO-2 directly binds to the COOH-terminal YV sequence of claudins-1 through -8 via its PDZ1 domain in vitro; ZO-2 is recruited to claudin-based networks through both PDZ1/claudin-COOH and PDZ2/ZO-1-PDZ2 interactions.\",\n      \"method\": \"In vitro binding assays, transfection of claudins into L fibroblasts, co-localization in epithelial cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding with domain mapping, replicated across multiple claudins and in multiple cell systems\",\n      \"pmids\": [\"10601346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ZO-2 directly interacts with F-actin in vitro (cosedimentation assay) and directly binds both ZO-1 and occludin; in vivo, ZO-1 and ZO-2 exist primarily as independent ZO-1·ZO-2 complexes rather than a trimeric ZO-1·ZO-2·ZO-3 complex.\",\n      \"method\": \"Actin cosedimentation, in vitro binding assays with recombinant proteins, immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro binding plus in vivo immunoprecipitation with orthogonal methods\",\n      \"pmids\": [\"10575001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Cingulin interacts with ZO-2 via an NH2-terminal fragment (residues 1-378) in pull-down assays from epithelial and insect cell lysates; ZO-1 and ZO-2 immunoprecipitates contain cingulin, confirming in vivo interaction.\",\n      \"method\": \"Pull-down assays, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus in vivo co-IP, replicated\",\n      \"pmids\": [\"10613913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Protein 4.1R isoforms (135 and 150 kDa) specifically interact with ZO-2; the interaction domains were mapped to exons 19-21 of 4.1R and residues 1054-1118 of ZO-2, providing a molecular link between tight junctions and the actin/spectrin cytoskeleton.\",\n      \"method\": \"Yeast two-hybrid, immunocolocalization, immunoprecipitation, in vitro binding studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including domain mapping and in vivo co-IP\",\n      \"pmids\": [\"10874042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Adenovirus type 9 E4-ORF1 oncoprotein binds ZO-2 via a C-terminal PDZ-binding motif interacting with the first PDZ domain of ZO-2; this interaction causes aberrant cytoplasmic sequestration of ZO-2. Overexpression of wild-type ZO-2 (but not a mutant lacking PDZ2/3) interfered with E4-ORF1-induced focus formation, implicating ZO-2 as a candidate tumor suppressor.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, focus formation assays, domain-deletion mutants\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific binding mapped by mutagenesis plus functional rescue assay\",\n      \"pmids\": [\"11598001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ZO-2 localizes to the nucleus of sparse epithelial cells in clusters that partially co-localize with splicing factor SC35; nuclear staining diminishes at confluence and can be induced by impairing cell-cell contacts or mechanical injury. ZO-2 nuclear translocation is mediated by the actin cytoskeleton and involves shuttling of a pre-existing pool rather than newly synthesized protein. Nuclear export is sensitive to leptomycin B.\",\n      \"method\": \"Immunofluorescence, live-cell imaging, leptomycin B treatment, calcium switch assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional inhibitor experiments, multiple conditions tested\",\n      \"pmids\": [\"11855865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B) via its PDZ-1 domain; this association was confirmed by yeast two-hybrid, co-immunoprecipitation, and co-localization in nuclei of epithelial and endothelial cells. ZO-1 does not associate with SAF-B, indicating non-redundant nuclear functions.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, co-localization by confocal microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-mapped interaction confirmed by orthogonal methods\",\n      \"pmids\": [\"12403786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A TJP2 mutation in the first PDZ domain (found in Amish families with familial hypercholanemia) reduces PDZ domain stability and ligand binding in vitro, and is associated with morphological changes in hepatic tight junctions.\",\n      \"method\": \"In vitro PDZ domain stability/binding assays, patient genetic analysis, liver histology\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro functional domain assay with structural consequence in patient tissue\",\n      \"pmids\": [\"12704386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Tyrosine phosphorylation of the C-terminal tail of occludin (by c-Src in vitro) significantly reduces binding of ZO-2 (as well as ZO-1 and ZO-3) to occludin, but does not affect occludin-F-actin binding.\",\n      \"method\": \"In vitro phosphorylation (c-Src), GST pull-down binding assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with defined substrate and binding consequence\",\n      \"pmids\": [\"12604349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ZO-2 associates with Jun, Fos and C/EBP transcription factors both at the nucleus and at the TJ region of epithelial cells; this association downregulates AP-1-dependent gene transcription in a dose-dependent manner, with both amino and carboxyl domains of ZO-2 capable of inhibiting transcription.\",\n      \"method\": \"GST pull-down, gel shift analysis (EMSA), co-immunoprecipitation, reporter gene (CAT) assays, immunolocalization\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing interaction and functional consequence\",\n      \"pmids\": [\"14720506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Nuclear ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B1 and actin. Multiple NLS signals in the amino region mediate nuclear import; only the second of two putative NES sequences is functional (confirmed by ovalbumin-coupled peptide nuclear injection assay). The NLS region also regulates AP-1-dependent transcription.\",\n      \"method\": \"Nuclear fractionation, co-immunoprecipitation, NLS deletion analysis, nuclear export assay with ovalbumin-coupled peptides\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches with domain-level resolution\",\n      \"pmids\": [\"15194440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ARVCF interacts with ZO-2 (and ZO-1) via a C-terminal PDZ-binding motif; the PDZ domains of ZO-2 can mediate nuclear localization of ARVCF, establishing a PDZ-domain-dependent mechanism for nuclear targeting.\",\n      \"method\": \"Co-immunoprecipitation, localization experiments, domain deletion analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional localization, single lab\",\n      \"pmids\": [\"15456900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"hScrib directly interacts with ZO-2 via two PDZ domains of hScrib and the C-terminal PDZ-binding motif of ZO-2; a point mutation in the LRR of hScrib that delocalizes it from the plasma membrane also abolishes ZO-2 interaction.\",\n      \"method\": \"Direct interaction assay, co-localization, mutagenesis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown/co-localization, domain mapped by mutagenesis\",\n      \"pmids\": [\"15975580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZO-2 (along with ZO-1) is required for tight junction strand formation and claudin polymerization; double knockdown/knockout of ZO-1 and ZO-2 in epithelial cells abolishes TJ formation. ZO-1 and ZO-2 independently determine the site of claudin polymerization, requiring dimerization and recruitment to the lateral membrane.\",\n      \"method\": \"Homologous recombination knockout + siRNA knockdown, exogenous ZO-1/ZO-2 rescue, forced membrane dimerization\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with clean KO/KD and rescue, definitive functional readout\",\n      \"pmids\": [\"16923393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZO-2 has four functional nuclear export signals (NES-0, NES-1, NES-2, NES-3); NES-0 and NES-3 are directly functional (confirmed by nuclear injection assay with ovalbumin-coupled peptides); NES-1 becomes functional upon phosphorylation at Ser369; mutation of any single NES is sufficient to cause nuclear accumulation of full-length ZO-2.\",\n      \"method\": \"Nuclear export assay with microinjection of NES-ovalbumin conjugates, leptomycin B sensitivity, transfection of NES mutants\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vivo nuclear export assay with defined peptides and mutagenesis\",\n      \"pmids\": [\"16920099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EcN (probiotic E. coli Nissle 1917) restores epithelial barrier integrity in EPEC-infected cells by enhancing ZO-2 expression and redistributing ZO-2 to cell boundaries, a process mediated by silencing of PKCζ.\",\n      \"method\": \"DNA microarray, immunofluorescence, Western blotting, PKC inhibitors, TEER measurement\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods but mechanistic link is correlative/inhibitor-based\",\n      \"pmids\": [\"17087734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ZO-2 downregulates cyclin D1 transcription via an E box in the cyclin D1 promoter by interacting with c-Myc; the complex also recruits HDAC1 to the E box, and HDAC activity is required for ZO-2-mediated repression. ZO-2 and c-Myc co-immunoprecipitate.\",\n      \"method\": \"Reporter gene (CAT) assays, deletion analysis, EMSA, ChIP, co-immunoprecipitation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including ChIP confirming in vivo complex at the promoter\",\n      \"pmids\": [\"17881732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ZO-2 silencing in MDCK cells increases paracellular permeability (gate function), disrupts fence function (non-polarized E-cadherin distribution), decreases occludin and E-cadherin expression in mature monolayers, delays arrival of ZO-1 and occludin to the plasma membrane during calcium switch, and causes atypical monolayer architecture with widened intercellular spaces.\",\n      \"method\": \"siRNA knockdown, TEER measurement, dextran permeability assay, immunofluorescence, calcium switch assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with multiple defined phenotypic readouts\",\n      \"pmids\": [\"17374535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ZO-2 knockout mice die shortly after implantation due to arrest in early gastrulation; ZO-2-deficient embryos show decreased proliferation at E6.5, increased apoptosis at E7.5, altered apical junctional complex architecture, and increased paracellular permeability. ZO-3 knockout mice have no obvious phenotype.\",\n      \"method\": \"Gene knockout, embryo histology, permeability tracer assay, immunostaining, cell proliferation/apoptosis assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with defined developmental and barrier phenotype\",\n      \"pmids\": [\"18172007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ZO-1 and ZO-2 are required for integration of myosin-2 into the zonula adherens (ZA); in ZO1(ko)/2(kd) cells, myosin-2 fails to integrate into ZA, and rescue by full-length ZO-1 or ZO-2 (or ZO-1 lacking PDZ1/2 but not PDZ1/2/3) restores myosin-2 integration. ZO-1/2-dependent RhoA/ROCK signaling spatiotemporally regulates ZA establishment.\",\n      \"method\": \"Knockout/knockdown, domain-deletion rescue, FRET RhoA activity assay, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via KO/KD with domain-resolution rescue and FRET-based signaling readout\",\n      \"pmids\": [\"18596233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ZO-2 forms a complex with YAP2 via the first PDZ domain of ZO-2 binding the PDZ-binding motif of YAP2; endogenous ZO-2 and YAP2 co-localize in the nucleus. ZO-2 facilitates nuclear localization and pro-apoptotic function of YAP2 in a PDZ-domain-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, domain-deletion analysis, apoptosis assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-mapped interaction with functional consequence (apoptosis), multiple methods\",\n      \"pmids\": [\"20868367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Genomic duplication of TJP2 leads to overexpression of ZO-2 protein; this overexpression decreases phosphorylation of GSK-3β and alters expression of apoptosis-regulating genes, causing progressive hearing loss (DFNA51) via increased susceptibility to apoptosis of inner ear cells.\",\n      \"method\": \"Genomic sequencing, family analysis, RT-PCR, Western blot, phosphorylation assays\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human genetic gain-of-function with downstream phosphorylation readout, but correlative for mechanistic link\",\n      \"pmids\": [\"20602916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"JAM-A associates directly with ZO-2 (and indirectly with afadin); this complex, along with PDZ-GEF1, activates the small GTPase Rap2c to regulate epithelial barrier function and apical cytoskeleton contraction via RhoA and nonmuscle myosin phosphorylation.\",\n      \"method\": \"Direct binding assay, co-immunoprecipitation, siRNA knockdown, permeability assay, RhoA activity assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus signaling pathway epistasis with multiple orthogonal readouts\",\n      \"pmids\": [\"23885123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SNX27 interacts with ZO-2 via the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2; when tight junctions are disrupted by calcium chelation, ZO-2 transiently localizes to SNX27-positive early endosomes. Depletion of SNX27 decreases ZO-2 (but not ZO-1) mobility at junctions and increases junctional permeability.\",\n      \"method\": \"Proteomics, co-immunoprecipitation, co-localization, FRAP, permeability assay, siRNA knockdown\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomic identification plus domain-mapped interaction with functional trafficking and barrier readouts\",\n      \"pmids\": [\"23826934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Protein-truncating mutations in TJP2 cause failure of ZO-2 protein localization and disruption of tight junction structure, leading to severe cholestatic liver disease (PFIC4).\",\n      \"method\": \"Patient genetic analysis, protein localization studies in patient tissue, electron microscopy of tight junctions\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic loss-of-function with protein localization and ultrastructural phenotype\",\n      \"pmids\": [\"24614073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZO-2 is SUMOylated; it associates with SUMO E2 enzyme Ubc9 and deconjugating proteases SENP1/SENP3; lysine 730 in the GuK domain is a SUMOylation site. Mutation of K730 (mimicking constitutive SUMOylation) retains ZO-2 in cytoplasm and abolishes its inhibitory effect on GSK3β activity and β-catenin/TCF-4-mediated transcription; ZO-2 directly binds GSK3β and forms a complex with β-catenin.\",\n      \"method\": \"Co-immunoprecipitation, Ubc9 fusion-directed SUMOylation, site-directed mutagenesis, reporter gene assays\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct SUMOylation assay with site mutagenesis and functional signaling readout\",\n      \"pmids\": [\"27604867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZO-2 silencing in renal epithelial MDCK cells induces cell hypertrophy by: (1) prolonging G1 phase via increased cyclin D1; (2) increasing protein synthesis via nuclear accumulation of YAP leading to reduced PTEN expression, activation of Akt/mTOR/S6K1. In vivo, compensatory renal hypertrophy after uninephrectomy is accompanied by decreased ZO-2 and nuclear YAP.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis, flow cytometry, reporter assays, mTOR pathway inhibitors, in vivo uninephrectomy model\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro KD with defined pathway epistasis confirmed in vivo\",\n      \"pmids\": [\"27009203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The organophosphate pesticide methamidophos covalently binds to ZO-2 at serine, tyrosine, and lysine residues (identified by mass spectrometry), inducing ZO-2 phosphorylation and reducing ZO-2/occludin interaction. Covalent modification at a lysine ubiquitination site (K) interferes with ZO-2 degradation and TJ sealing, demonstrated by transfection with a ZO-2 mutant at a MET target lysine residue.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, site-directed mutagenesis, transfection, permeability assay\",\n      \"journal\": \"Toxicology and applied pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mass spectrometry identification with mutagenesis functional validation, single lab\",\n      \"pmids\": [\"30291936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Activation of the Ca2+-sensing receptor triggers PKC/WNK4 signaling, leading to ZO-2 phosphorylation and concentration at tight junctions. In low calcium, ZO-2 is protected from degradation by association with 14-3-3ζ and 14-3-3σ proteins; upon Ca2+ restoration, ZO-2/14-3-3 complexes move to cell borders and dissociate (14-3-3 is proteasomally degraded; ZO-2 integrates into TJs). The unique region 2 of ZO-2 and S261 within an NLS are critical for 14-3-3 interaction and nuclear import.\",\n      \"method\": \"Co-immunoprecipitation, kinase inhibitors, calcium switch assay, domain mutagenesis, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — signaling pathway epistasis with domain mutagenesis and functional localization readout\",\n      \"pmids\": [\"31318316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Liver-specific deletion of Tjp2 in mice causes lower Cldn1 protein levels, dilated canaliculi, reduced microvilli density, aberrant radixin and BSEP distribution, mild progressive cholestasis, and lower expression of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10; a cholic acid diet causes severe cholestasis and liver necrosis in Tjp2-deficient but not control mice.\",\n      \"method\": \"Conditional knockout in hepatocytes/cholangiocytes, biochemical analyses, electron microscopy, immunostaining, fluorescein-dextran permeability\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with multiple defined molecular and functional readouts\",\n      \"pmids\": [\"33465371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Nuclear ZO-2 facilitates TEAD entry into the nucleus; ZO-2 and TEAD interact in the cytoplasm (confirmed by proximity ligation, immunoprecipitation, pull-down); inhibition of nPKCδ promotes ZO-2/TEAD cytoplasmic interaction and co-importation. Nuclear exit of ZO-2/TEAD is enhanced by nPKCε-mediated activation of a ZO-2 NES.\",\n      \"method\": \"Proximity ligation assay, co-immunoprecipitation, GST pull-down, siRNA knockdown, kinase inhibitors, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal interaction assays with defined kinase-dependent mechanism\",\n      \"pmids\": [\"34010016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZO-2 functions as a scaffold for the Hippo pathway by associating with LATS1; ZO-2 silencing reduces LATS kinase activity and leads to nuclear accumulation of YAP. In liver steatosis, ZO-2 is silenced and this correlates with diminished LATS activity; metformin (AMPK activator blocking JNK) restores ZO-2 and claudin-1 expression in steatotic liver.\",\n      \"method\": \"Co-immunoprecipitation (ZO-2/LATS1), siRNA knockdown, kinase activity assays, in vivo obese rat model, immunofluorescence\",\n      \"journal\": \"Tissue barriers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP for scaffold function, supported by in vivo model\",\n      \"pmids\": [\"34689705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZO-2 is polyubiquitinated at K759 and K992 (K48-linked, targeting for proteasomal degradation), confirmed by mutation of these sites reducing ubiquitination and extending ZO-2 half-life. K730 (SUMOylation site) mutation increases ubiquitination and decreases half-life. Mutation of any of these lysines reduces TJ sealing.\",\n      \"method\": \"Co-immunoprecipitation with ubiquitin, TUBES (tandem ubiquitin-binding entities), site-directed mutagenesis, half-life assay (CHX chase), TEER measurement\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct ubiquitination assay with site mutagenesis and functional consequence\",\n      \"pmids\": [\"36291162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In the mouse liver, Tjp2 negatively regulates Yap and Wwtr1/Taz protein expression; hepatocyte-specific (but not cholangiocyte-specific) Tjp2 deletion leads to DDC-diet-induced hepatocyte-to-cholangiocyte transdifferentiation in a Yap/Taz-dependent manner.\",\n      \"method\": \"Conditional knockout (hepatocyte- vs cholangiocyte-specific), immunostaining, Yap/Taz protein level analysis, dietary challenge\",\n      \"journal\": \"NPJ Regenerative medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with mechanistic pathway epistasis (Yap/Taz dependence)\",\n      \"pmids\": [\"36151109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZNF582 upregulates TJP2 protein expression; increased TJP2 then binds ERK2, promotes ERK2 protein expression, and suppresses ERK2 phosphorylation, thereby inhibiting ccRCC growth and metastasis.\",\n      \"method\": \"TMT quantitative proteomics, co-immunoprecipitation, Western blot, orthotopic tumor models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomics-identified interaction confirmed by Co-IP with in vivo functional readout, single lab\",\n      \"pmids\": [\"36966163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZO-2 is required for p190A RhoGAP to activate LATS kinases and the Hippo pathway; interaction of p190A with ZO-2 is dependent on RasGAP. Both RasGAP and ZO-2 are necessary for p190A to promote mesenchymal-to-epithelial transition and contact inhibition of proliferation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, LATS kinase activity assays, tumor growth assays, reporter assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — interaction identified with functional epistasis across multiple assays\",\n      \"pmids\": [\"37995182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZO-2 acts as a scaffold to promote LATS1/YAP interaction: ZO-2 brings LATS1 (via SH3 domain) and YAP (via PDZ domain) together, enabling LATS1-dependent phosphorylation and cytoplasmic retention/inactivation of YAP, maintaining Hippo pathway activation and contact inhibition of proliferation.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion analysis, LATS1 kinase assays, YAP phosphorylation assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mechanistic reconstitution of scaffolding with defined domains and kinase assay\",\n      \"pmids\": [\"39462647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Absence of ZO-2 reduces apical membrane rigidity, inhibits γ-actin and JAM-A recruitment to cell borders, facilitates p114RhoGEF and afadin accumulation at junctions, and increases mechanical tension at TJs (measured by FRET). ZO-2 KD cells show impaired responses to substrate stiffness and topography, with increased YAP and Snail nuclear accumulation.\",\n      \"method\": \"Atomic force microscopy, FRET tension probes, immunofluorescence, siRNA knockdown, in silico binding stability analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct biophysical measurement (AFM, FRET) with genetic KD and mechanistic pathway readouts\",\n      \"pmids\": [\"38473701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZO-2 colocalizes with CEP164 at the distal appendage of the mother centriole and is present at mitotic spindle poles, the basal body of primary cilia, and spermatozoa tails. ZO-2 depletion alters centriolar protein levels (CEP164, centriolin, CEP135), inhibits astral and mitotic spindle microtubule growth, increases NuMA and decreases KIF14/TPX2/p-Aurora at spindle poles, reduces mitotic spindle length, and blocks primary cilia development. KIF14, NuMA, and p-Aurora co-immunoprecipitate with ZO-2; NuMA and Aurora-A bind distinct ZO-2 segments.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, siRNA knockdown, domain binding assays\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional KD phenotypes, single lab with multiple readouts\",\n      \"pmids\": [\"40728639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZO-2 is identified as a novel NTCP-binding protein by immunopurification/LC-MS/MS; ZO-2 knockdown or knockout reduces NTCP at the cell surface, decreasing HBV attachment and infection. HBV surface element preS1 dissociates NTCP from ZO-2 and promotes formation of NTCP-preS1-actin complexes that are internalized; actin polymerization is required for preS1 internalization and HBV infection.\",\n      \"method\": \"Immunopurification + LC-MS/MS, siRNA knockdown/knockout, HBV infection assay, co-immunoprecipitation, actin inhibitor (latrunculin A)\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomic identification confirmed by KO functional assay with mechanistic dissection of receptor trafficking\",\n      \"pmids\": [\"41870046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"c-Abl directly binds to and phosphorylates the C-terminus of ZO-2; c-Abl also stimulates JAK1 activity, which subsequently phosphorylates the N-terminus of ZO-2. By RNAi knockdown/rescue, c-Abl regulates cellular morphology and migration through ZO-2 phosphorylation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, RNAi knockdown/rescue, traction force microscopy\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with defined substrate plus functional rescue experiments\",\n      \"pmids\": [\"41259016\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TJP2/ZO-2 is a MAGUK scaffold protein that localizes to epithelial tight junctions and, in sparse/subconfluent cells, to the nucleus; at tight junctions it directly binds claudins (via PDZ1), occludin, ZO-1, cingulin, JAM-A, and the actin cytoskeleton to organize the paracellular barrier and drive claudin polymerization together with ZO-1; in the nucleus it associates with transcription factors (AP-1/Jun/Fos/C/EBP, c-Myc, TEAD) and the SAF-B chromatin protein to suppress proliferative gene expression (cyclin D1, Wnt/β-catenin targets); it acts as a scaffold for the Hippo pathway by recruiting LATS1 and YAP via its SH3 and PDZ domains to promote YAP phosphorylation and cytoplasmic retention, thereby enforcing contact inhibition; its intracellular traffic between TJ, cytoplasm, and nucleus is controlled by multiple NLS/NES sequences, SUMOylation at K730, K48-polyubiquitination at K759/K992 for proteasomal turnover, interaction with 14-3-3 proteins in low-calcium conditions, and phosphorylation cascades including PKC/WNK4 and c-Abl/JAK1; loss-of-function mutations cause progressive cholestatic liver disease (PFIC4) by disrupting TJ localization of ZO-2, reducing claudin-1 and bile acid transporter (BSEP/ABCB11) expression, and deregulating YAP/Taz-mediated hepatocyte transdifferentiation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TJP2/ZO-2 is a MAGUK family scaffold protein that organizes epithelial tight junctions, suppresses proliferative gene expression in the nucleus, and enforces contact inhibition through the Hippo pathway. At tight junctions, ZO-2 binds claudins via its PDZ1 domain, occludin, ZO-1, cingulin, JAM-A, and F-actin, and together with ZO-1 is essential for claudin polymerization into TJ strands and paracellular barrier formation; its absence causes loss of barrier integrity, reduced apical membrane rigidity, and in the liver leads to progressive cholestatic disease (PFIC4) with diminished claudin-1 and BSEP expression [PMID:10601346, PMID:16923393, PMID:24614073, PMID:33465371]. In the nucleus, ZO-2 represses AP-1- and β-catenin/TCF-dependent transcription by associating with Jun/Fos, c-Myc, and SAF-B, downregulates cyclin D1 via c-Myc/HDAC1 recruitment, and acts as a Hippo pathway scaffold that bridges LATS1 (via its SH3 domain) and YAP (via its PDZ domain) to promote YAP phosphorylation and cytoplasmic retention [PMID:14720506, PMID:17881732, PMID:39462647, PMID:12403786]. ZO-2 subcellular distribution is controlled by multiple NLS/NES sequences regulated by PKC, c-Abl/JAK1 phosphorylation, SUMOylation at K730, K48-linked polyubiquitination at K759/K992 for proteasomal turnover, and calcium-dependent 14-3-3 association [PMID:16920099, PMID:31318316, PMID:36291162, PMID:27604867, PMID:41259016].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Identifying ZO-2 as a ZO-1-interacting MAGUK protein at tight junctions established the existence of a multi-protein scaffold at the cytoplasmic face of TJs beyond ZO-1 alone.\",\n      \"evidence\": \"Co-immunoprecipitation from MDCK cells with immunofluorescence localization\",\n      \"pmids\": [\"8132716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding domains not yet mapped\", \"Functional role at TJ unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Determination of the full domain architecture (three PDZ, SH3, GuK domains) revealed the multi-interaction capacity of ZO-2 and explained how it could serve as a hub scaffold.\",\n      \"evidence\": \"cDNA cloning and sequence analysis with alternative splice isoform characterization\",\n      \"pmids\": [\"8824195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligands for each domain not identified\", \"No functional assays for individual domains\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstration that ZO-2 directly binds claudins (via PDZ1), occludin, ZO-1, cingulin, and F-actin defined the molecular connections through which ZO-2 links transmembrane TJ proteins to the cytoskeleton.\",\n      \"evidence\": \"In vitro binding/cosedimentation assays, co-immunoprecipitation, and transfection of claudins into fibroblasts\",\n      \"pmids\": [\"10601346\", \"10575001\", \"10613913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZO-2 is sufficient for TJ strand formation unknown\", \"Regulation of these interactions uncharacterized\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery that ZO-2 shuttles to the nucleus in subconfluent cells and interacts with SAF-B via PDZ1 revealed a non-redundant nuclear role distinct from ZO-1, linking TJ disassembly to changes in nuclear function.\",\n      \"evidence\": \"Immunofluorescence with leptomycin B and calcium switch; yeast two-hybrid and co-IP for SAF-B interaction\",\n      \"pmids\": [\"11855865\", \"12403786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcriptional targets of nuclear ZO-2 unknown\", \"NLS/NES sequences not yet mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"A PDZ1 mutation causing familial hypercholanemia linked ZO-2 to human liver disease and showed that PDZ1 integrity is essential for hepatic TJ function.\",\n      \"evidence\": \"Patient genetic analysis with in vitro PDZ domain stability/binding assays and liver histology\",\n      \"pmids\": [\"12704386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of cholestasis not resolved\", \"Whether complete loss of function causes more severe disease unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of ZO-2 as a transcriptional repressor of AP-1 targets through association with Jun/Fos/C/EBP, and mapping of multiple NLS/NES sequences, established how ZO-2 nuclear shuttling translates into gene regulation.\",\n      \"evidence\": \"GST pull-down, EMSA, reporter assays, NLS deletion analysis, nuclear export peptide assays\",\n      \"pmids\": [\"14720506\", \"15194440\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide transcriptional targets not defined\", \"Phosphorylation-dependent regulation of NES not yet shown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Double knockout/knockdown of ZO-1 and ZO-2 abolished TJ strand formation, proving these two proteins are the essential and sufficient scaffolds for claudin polymerization; four NES sequences with phosphorylation-dependent activation were mapped.\",\n      \"evidence\": \"Genetic epistasis (KO/KD/rescue in EpH4 cells); NES-ovalbumin injection assays with NES mutants\",\n      \"pmids\": [\"16923393\", \"16920099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of ZO-1 vs ZO-2 not fully separated\", \"In vivo confirmation of NES phosphoregulation lacking\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"ZO-2 was shown to repress cyclin D1 transcription through c-Myc/HDAC1 recruitment to the E-box, and ZO-2 knockdown increased paracellular permeability and disrupted fence function, connecting nuclear and junctional roles to cell cycle control and polarity.\",\n      \"evidence\": \"ChIP, reporter assays, co-IP for c-Myc; siRNA KD in MDCK with TEER, dextran permeability, and calcium switch\",\n      \"pmids\": [\"17881732\", \"17374535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cyclin D1 repression is the main growth-inhibitory mechanism unknown\", \"Redundancy with ZO-1 at TJs not fully resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"ZO-2 knockout embryos died at gastrulation with proliferation defects, apoptosis, and barrier failure, proving ZO-2 is essential for early mammalian development and non-redundant with ZO-1 in vivo.\",\n      \"evidence\": \"Gene knockout mice with embryo histology, permeability tracer, proliferation/apoptosis assays\",\n      \"pmids\": [\"18172007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type-specific requirements in later development unknown\", \"Molecular basis of embryonic lethality not dissected\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"ZO-2 was found to interact with YAP2 via PDZ1 and facilitate its nuclear localization and pro-apoptotic activity, establishing ZO-2 as a Hippo pathway component; separately, TJP2 duplication causing overexpression was linked to progressive hearing loss (DFNA51).\",\n      \"evidence\": \"Co-IP, domain-deletion analysis, apoptosis assays (YAP2); family genomic analysis with phosphorylation readouts (DFNA51)\",\n      \"pmids\": [\"20868367\", \"20602916\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZO-2 scaffolds LATS-YAP phosphorylation not yet tested\", \"DFNA51 mechanism remains correlative\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Protein-truncating TJP2 mutations were shown to cause severe progressive familial intrahepatic cholestasis (PFIC4) with TJ ultrastructural disruption, establishing complete ZO-2 loss as a cause of Mendelian liver disease.\",\n      \"evidence\": \"Patient genetic analysis with immunolocalization and electron microscopy of hepatic TJs\",\n      \"pmids\": [\"24614073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream molecular pathology (claudin, transporter levels) not fully characterized at this point\", \"No animal model recapitulating PFIC4\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"SUMOylation at K730 was identified as a switch controlling ZO-2 subcellular localization and its ability to inhibit β-catenin/Wnt signaling; ZO-2 silencing was shown to cause cell hypertrophy via nuclear YAP/Akt/mTOR activation, integrating post-translational control with growth regulation.\",\n      \"evidence\": \"Ubc9-directed SUMOylation, site mutagenesis, reporter assays; siRNA KD with cell cycle/mTOR pathway analysis and in vivo uninephrectomy model\",\n      \"pmids\": [\"27604867\", \"27009203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO ligase identity unknown\", \"Whether SUMOylation and ubiquitination are coordinated not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Ca2+-sensing receptor/PKC/WNK4 signaling was found to drive ZO-2 phosphorylation and TJ recruitment, while 14-3-3 proteins protect ZO-2 from degradation in low calcium, revealing how extracellular calcium signals control ZO-2 trafficking between cytoplasm and TJs.\",\n      \"evidence\": \"Co-IP, kinase inhibitors, calcium switch, domain mutagenesis including S261 in NLS\",\n      \"pmids\": [\"31318316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct WNK4 phosphorylation sites on ZO-2 not mapped\", \"14-3-3 binding stoichiometry and specificity not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Liver-specific Tjp2 deletion in mice recapitulated key PFIC4 features including reduced claudin-1, BSEP/Abcb11, and canalicular defects, while nuclear ZO-2 was shown to co-import TEAD via PKCδ/ε-regulated shuttling, expanding ZO-2's nuclear transcriptional partnerships.\",\n      \"evidence\": \"Hepatocyte-specific conditional KO with EM, biochemistry, and dietary challenge; proximity ligation and kinase inhibitors for TEAD interaction\",\n      \"pmids\": [\"33465371\", \"34010016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TEAD nuclear import is the key driver of cholestasis phenotype untested\", \"ZO-2-LATS scaffolding mechanism not yet reconstituted\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"K48-linked polyubiquitination at K759 and K992 was shown to target ZO-2 for proteasomal degradation, and interplay with K730 SUMOylation was demonstrated, establishing a post-translational code controlling ZO-2 turnover and TJ sealing; hepatocyte-specific Tjp2 loss was shown to derepress Yap/Taz and enable hepatocyte-to-cholangiocyte transdifferentiation.\",\n      \"evidence\": \"TUBES ubiquitin capture, site-directed mutagenesis, CHX chase, TEER; hepatocyte- vs cholangiocyte-specific KO with dietary DDC challenge\",\n      \"pmids\": [\"36291162\", \"36151109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ubiquitin ligase identity unknown\", \"Full ubiquitin/SUMO crosstalk mechanism not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"ZO-2 was identified as essential for p190A RhoGAP-mediated LATS activation and as an ERK2-binding protein suppressing ERK phosphorylation, broadening ZO-2's scaffolding role to RhoGAP-Hippo and MAPK signaling.\",\n      \"evidence\": \"Co-IP, siRNA KD, LATS kinase activity assays, tumor growth assays (p190A); TMT proteomics, co-IP, orthotopic tumor models (ERK2)\",\n      \"pmids\": [\"37995182\", \"36966163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface of p190A-ZO-2 not mapped\", \"ERK2 interaction confirmed in single lab only\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The scaffolding mechanism for Hippo signaling was reconstituted: ZO-2 bridges LATS1 via SH3 and YAP via PDZ domains to enable LATS1-dependent YAP phosphorylation; biophysically, ZO-2 loss reduces apical rigidity and increases TJ mechanical tension.\",\n      \"evidence\": \"Domain-deletion co-IP with LATS1 kinase/YAP phosphorylation assays; AFM and FRET tension probes in KD cells\",\n      \"pmids\": [\"39462647\", \"38473701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of SH3-LATS1 and PDZ-YAP interfaces not determined\", \"How mechanical tension feedback integrates with Hippo signaling unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ZO-2 was localized to centrosomes and the basal body of primary cilia and shown to regulate mitotic spindle organization, and was independently identified as a stabilizer of the bile acid transporter NTCP at the cell surface required for HBV entry; c-Abl/JAK1 were identified as kinases that directly phosphorylate ZO-2 to control cell morphology and migration.\",\n      \"evidence\": \"Co-IP with spindle/centrosomal proteins and siRNA KD (cilia/spindle); LC-MS/MS immunopurification with KO functional HBV assay (NTCP); in vitro kinase assays with RNAi rescue (c-Abl/JAK1)\",\n      \"pmids\": [\"40728639\", \"41870046\", \"41259016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Centrosomal localization confirmed in single lab only\", \"Whether NTCP stabilization contributes to PFIC4 pathology untested\", \"JAK1 phosphorylation sites on ZO-2 not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the E3 ubiquitin ligase(s) targeting ZO-2, the structural basis for ZO-2's scaffolding of LATS1-YAP, the genome-wide transcriptional program controlled by nuclear ZO-2, and how ZO-2's centrosomal and ciliary functions relate to its established junctional and nuclear roles.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"E3 ligase unknown\", \"No high-resolution structure of ZO-2 scaffold complexes\", \"No genome-wide ChIP-seq or nuclear transcriptomics for ZO-2\", \"Centrosome/cilia role not independently replicated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 15, 22, 33, 37, 38]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [11, 18, 27, 32]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 5, 39]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [15, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 8, 11, 12, 18, 32]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 15, 19, 30]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 5, 39, 40]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [40]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [40]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [27, 30, 32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 2, 15, 19, 26]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [22, 28, 33, 37, 38]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [11, 18, 27, 32]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [18, 28, 40]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 26, 31, 35]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [27, 34]}\n    ],\n    \"complexes\": [\n      \"ZO-1/ZO-2 heterodimer\",\n      \"Tight junction plaque complex\"\n    ],\n    \"partners\": [\n      \"TJP1\",\n      \"OCLN\",\n      \"CLDN1\",\n      \"CGN\",\n      \"F11R\",\n      \"LATS1\",\n      \"YAP1\",\n      \"ABL1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}