{"gene":"SUFU","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2002,"finding":"SUFU mutations (including truncations) abolish its ability to export GLI transcription factors from nucleus to cytoplasm, resulting in activation of SHH signaling; SUFU functions as a tumor suppressor by modulating SHH signaling through nuclear-cytoplasmic regulation of GLI.","method":"Mutational analysis of patient-derived SUFU truncations with functional readout of GLI nuclear localization","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived loss-of-function mutations with defined cellular phenotype (GLI nuclear retention), single study but mechanistically specific","pmids":["12068298"],"is_preprint":false},{"year":2002,"finding":"SUFU directly interacts with GLI proteins via a conserved SYGH motif in GLI/Ci; residues Gly122 and His123 in this motif are critical for binding. A C-terminal 19-amino acid deletion in SUFU (Δ465) abolishes interaction with GLI1. GLI mutants lacking the SYGH motif are no longer suppressed by SUFU co-expression.","method":"Co-immunoprecipitation, Far Western assays, truncation and site-directed mutagenesis, luciferase reporter functional assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reciprocal binding assays combined with mutagenesis and functional reporter assay in a single rigorous study","pmids":["12426310"],"is_preprint":false},{"year":2004,"finding":"SUFU interacts with SAP18 (Sin3-associated polypeptide 18) and Galectin3 as nuclear binding partners. Co-expression with SAP18 translocates SUFU to the nucleus, while co-expression with SUFU translocates Galectin3 to the cytoplasm. SUFU is predominantly cytoplasmic but becomes mostly nuclear when its putative nuclear export signal is mutated or after leptomycin B treatment, establishing SUFU as a nucleocytoplasmic shuttle protein.","method":"Yeast two-hybrid screen, co-immunoprecipitation, cell transfection with NES mutant and leptomycin B treatment, subcellular fractionation/imaging","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by independent biochemical assays, localization determined by direct experiment with functional NES mutation, single lab","pmids":["14611647"],"is_preprint":false},{"year":2006,"finding":"GSK3β binds to SUFU both in vitro and in vivo and phosphorylates SUFU; a phosphorylation-mimicking SUFU mutant shows significantly decreased ability to bind GLI1 and cannot suppress GLI-mediated reporter gene expression. GSK3β knockdown attenuates Hedgehog signaling, suggesting GSK3β functions as a positive regulator of Hedgehog signaling through phosphorylation of SUFU.","method":"Mass spectrometric identification of binding partner, GST pull-down and co-immunoprecipitation (in vitro and in vivo), in vitro kinase assay, RNAi knockdown, luciferase reporter assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay combined with co-IP, phosphomimetic mutant functional analysis, and RNAi phenotype, single lab with multiple orthogonal methods","pmids":["17182001"],"is_preprint":false},{"year":2008,"finding":"Hedgehog signaling promotes ubiquitination of SUFU, leading to its proteasomal degradation. Lysine 257 (K257) is the ubiquitin attachment site; the K257R mutant of SUFU is more stable, more potent as a transcription repressor, and a stronger cell growth inhibitor.","method":"In vivo ubiquitination assay, site-directed mutagenesis (K257R), proteasome inhibitor experiments, transcription repressor and cell growth assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of ubiquitin acceptor site combined with functional readouts (stability, transcriptional repression, growth inhibition), single lab multiple orthogonal methods","pmids":["18997815"],"is_preprint":false},{"year":2009,"finding":"SUFU promotes Gli3 processing into its repressor form by recruiting GSK3β; SUFU can bind both GSK3β and Gli3 simultaneously, forming a trimolecular complex Gli3/SUFU/GSK3β that facilitates phosphorylation of Gli3 by GSK3β. Sonic Hh stimulation dissociates the SUFU/GSK3β complex from Gli3, blocking Gli3 processing. In SUFU-deficient MEFs, Gli3 processing is severely impaired.","method":"Co-immunoprecipitation, siRNA knockdown, Sufu−/− mouse embryonic fibroblasts with functional Gli3 processing readout","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP establishing trimolecular complex, KO MEF model with defined processing phenotype, single lab multiple methods","pmids":["19622347"],"is_preprint":false},{"year":2009,"finding":"SUFU controls protein levels of full-length Gli transcription factors (Gli activators and repressors) in a cilium-independent manner, partly by antagonizing the activity of Spop, a Gli-degrading factor. Zebrafish and fly Sufu can restore Gli protein function in Sufu-deficient mammalian cells, indicating evolutionary conservation of this mechanism.","method":"Sufu conditional knockout mice, cell-based reconstitution with heterologous Sufu orthologs, Spop epistasis analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in KO cells combined with cross-species functional rescue, multiple orthogonal methods, replicated across organisms","pmids":["19684112"],"is_preprint":false},{"year":2010,"finding":"In vertebrates, Hedgehog stimulation rapidly recruits endogenous SUFU-Gli complexes to primary cilia, then causes rapid dissociation of SUFU-Gli complexes, allowing Gli to enter the nucleus and activate transcription. PKA activation blocks ciliary localization of SUFU-Gli complexes, preventing their Hh-induced dissociation.","method":"Live-cell imaging of endogenous SUFU-Gli complexes at cilia, pharmacological PKA activation/inhibition, colocalization and biochemical dissociation assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct imaging of endogenous complexes combined with biochemical dissociation assay and PKA perturbation, multiple orthogonal methods, rigorous controls","pmids":["20956384"],"is_preprint":false},{"year":2011,"finding":"SUFU is phosphorylated at Ser-342 by GSK3β and at Ser-346 by PKA; dual phosphorylation at these sites stabilizes SUFU against Shh-induced degradation. Shh signaling induces trafficking of phospho-SUFU to the primary cilium in a complex with Gli2/3, and dephosphorylation triggers retrograde export and ubiquitin-proteasome degradation of SUFU.","method":"Site-directed mutagenesis (S342A, S346A, phosphomimetic S342D/S346D), PKA inhibitors, ciliary localization assays, ubiquitin-proteasome pathway analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with ciliary localization assay and degradation assay, multiple orthogonal methods, single lab","pmids":["21317289"],"is_preprint":false},{"year":2012,"finding":"Kif7 promotes turnover of SUFU and exclusion of SUFU-Gli complexes from the primary cilium, thereby restricting SUFU's inhibitory function. Halving the dose of Sufu restores normal Hedgehog pathway activity and chondrocyte development in Kif7-null mice, establishing that Kif7's positive role in Hh signaling is to restrict SUFU activity.","method":"Kif7-null and compound mutant mouse genetics (epistasis), ciliary localization of SUFU-Gli complexes, SUFU protein level analysis","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis in mouse models with defined quantitative rescue of pathway activity and cell fate, replicated across labs","pmids":["21795282"],"is_preprint":false},{"year":2012,"finding":"Sufu restricts Gli2 activity through cytoplasmic sequestration in keratinocytes; Kif7 promotes Hh pathway activity by dissociating the SUFU-Gli2 complex. Simultaneous deletion of SUFU and Kif7 in adult epidermis induces basal cell carcinoma, whereas deletion of either alone cannot.","method":"Conditional knockout mice (skin-specific), genetic epistasis (single and double mutants), subcellular localization analysis","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with compound conditional KO mouse models, defined BCC phenotypic readout, mechanistic localization analysis","pmids":["23034632"],"is_preprint":false},{"year":2013,"finding":"Crystal structure and SAXS structure of full-length human SUFU and its complex with the conserved SYGHL motif of GLI reveal that GLI binding induces major conformational changes in SUFU, including rearrangement of an intrinsically disordered loop crucial for pathway activation.","method":"X-ray crystallography, small-angle X-ray scattering (SAXS), structural analysis of SUFU-GLI peptide complex","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure and SAXS of full-length human protein with ligand, rigorous structural validation, single study","pmids":["24311597"],"is_preprint":false},{"year":2014,"finding":"SUFU can bind Ci/Gli through two distinct sites: the N-terminal Sufu-interacting site (SIN) and a newly identified C-terminal Sufu-interacting site (SIC). Binding via SIN mediates cytoplasmic sequestration of Ci/Gli, while binding via SIC inhibits Ci/Gli activity in the nucleus by masking the CBP coactivator binding site in the C-terminal region of Ci.","method":"Binding site mutagenesis, cytoplasmic/nuclear localization assays, Drosophila genetics, CBP binding competition assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — site mutagenesis combined with localization assays and transcriptional coactivator competition analysis, mechanistic dissection with multiple orthogonal methods","pmids":["25941387"],"is_preprint":false},{"year":2014,"finding":"Importin β1 and SUFU bind to overlapping sites on the GLI1 N-terminus with high nanomolar affinity; they compete for binding to GLI1, such that relative intracellular concentrations of SUFU and importin β1 determine GLI1 nuclear localization. SUFU co-expression specifically inhibits GFP-Gli1 nuclear accumulation.","method":"Quantitative live mammalian cell imaging, direct binding affinity measurement, siRNA knockdown of importin β1, competition binding assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct affinity measurement combined with live-cell imaging, specific knockdown, and competition assay, multiple orthogonal methods, single lab","pmids":["24854174"],"is_preprint":false},{"year":2014,"finding":"In the nucleus, SUFU recruits p66β (a NuRD repressor complex member) to block Gli-mediated Hh target gene expression; Mycbp forms a complex with Gli and SUFU without Hh stimulation but is kept inactive. Hh activation leads to dissociation of SUFU/p66β from Gli, enabling Mycbp to promote Gli activity. SUFU, p66β, Mycbp, and Gli are detected on promoters of Hh targets in a dynamic manner.","method":"Proteomic identification of Sufu-interacting proteins, cell-based signaling assays, zebrafish epistasis, chromatin immunoprecipitation (ChIP)","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomic ID confirmed by ChIP and cell-based assays and in vivo zebrafish epistasis, multiple orthogonal methods across species","pmids":["25403183"],"is_preprint":false},{"year":2016,"finding":"The F-box protein Fbxl17 targets SUFU for proteolysis specifically in the nucleus, mediating its polyubiquitylation and allowing Gli1 release for Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling and impaired medulloblastoma tumor growth. A Gorlin syndrome-derived SUFU mutation increases SUFU turnover through enhanced Fbxl17-mediated polyubiquitylation.","method":"Co-immunoprecipitation, ubiquitylation assay, nuclear-targeted constructs, Fbxl17 depletion in cells and tumor models, patient-derived mutation analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitylation, nuclear targeting experiments, and in vivo tumor model, multiple orthogonal methods, single lab","pmids":["27234298"],"is_preprint":false},{"year":2016,"finding":"Nek2A binds SUFU (identified by yeast two-hybrid, GST pull-down and co-immunoprecipitation), phosphorylates SUFU, and stabilizes it against ubiquitin/proteasome degradation. Phosphorylated SUFU inhibits nuclear localization and transcriptional activity of Gli2. In turn, Gli1 and Gli2 directly bind the NEK2A gene promoter to induce its transcription, forming a negative-feedback loop.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, in vitro kinase assay, chromatin immunoprecipitation (ChIP), reporter assay","journal":"Cellular signalling / International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding and kinase assays, ChIP, functional reporter, single lab","pmids":["27297360","28035348"],"is_preprint":false},{"year":2017,"finding":"BCL-2 family anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL) found outside the outer mitochondrial membrane engage a BH domain sequence in SUFU, directly promote SUFU turnover, inhibit SUFU-GLI interaction, and thereby induce expression of GLI target genes including BCL-2, MCL-1, and BCL-XL (feedforward loop). BH3 mimetics can disable this interaction.","method":"Co-immunoprecipitation, SUFU-GLI interaction assays, SUFU stability assays, BH3 mimetic pharmacological disruption, GLI target gene expression","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interaction and functional mechanistic dissection with multiple methods including pharmacological disruption, cancer cell and in vivo models, single lab but rigorous","pmids":["28945232"],"is_preprint":false},{"year":2018,"finding":"The Itch/β-arrestin2 complex binds SUFU and induces its Lys63-linked (non-proteolytic) polyubiquitylation, which increases SUFU association with Gli3 and promotes conversion of Gli3 into its repressor form, keeping Hh signaling off. Hh activation antagonizes Itch-dependent polyubiquitylation of SUFU. SUFU mutations found in medulloblastoma patients are insensitive to Itch activity.","method":"Co-immunoprecipitation, ubiquitylation assays (K63-specific), Gli3 processing assays, patient-derived mutation analysis, Hh pathway activation/inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mechanistically precise ubiquitylation (K63-specific) combined with Gli3 processing readout, patient mutations tested, multiple orthogonal methods","pmids":["29515120"],"is_preprint":false},{"year":2019,"finding":"PRMT7 methylates GLI2 at arginine residues R225 and R227, which are located near the SUFU-binding region. This methylation interferes with GLI2-SUFU binding, leading to increased GLI2 nuclear accumulation and enhanced Shh signaling. PRMT7-deficient MEFs show increased GLI2-SUFU interaction and reduced Shh signaling.","method":"Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis (R225/R227), GLI2 nuclear localization assay, PRMT7 KO MEFs","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro methylation with mutagenesis of target residues, binding competition assay, and KO MEF validation, multiple orthogonal methods, single lab","pmids":["31000813"],"is_preprint":false},{"year":2021,"finding":"SNEP1, a Hh transcriptional target, enhances SUFU ubiquitination and proteasomal degradation by recruiting the E3 ubiquitin ligase LNX1, thereby promoting Hh signaling in a positive feedback manner.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor experiments, LNX1 knockdown, Hh reporter assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with LNX1, single lab with defined mechanistic readout","pmids":["33608498"],"is_preprint":false},{"year":2022,"finding":"SUFU suppresses ferroptosis sensitivity in breast cancer cells by activating LATS1 to suppress YAP, thereby reducing ACSL4 expression. Deletion of SUFU residues 174-385 disrupts SUFU binding to LATS1, abolishing its regulation of the YAP-ACSL4 axis and sensitivity to ferroptosis.","method":"Co-immunoprecipitation, SUFU deletion mutant mapping (residues 174-385), ACSL4 expression analysis, RSL3-triggered ferroptosis assay, ectopic expression and depletion","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mapping with functional ferroptosis readout and LATS1 Co-IP, single lab","pmids":["35800779"],"is_preprint":false},{"year":2022,"finding":"ERK2 phosphorylates GLI1 at three evolutionarily conserved sites (S102, S116, S130) near the high-affinity SUFU-binding site; multisite phosphorylation cooperates to weaken GLI1-SUFU binding by over 25-fold. Phosphomimetic substitutions at all three sites enhance GLI1 transcriptional activity in cells. Single or pairwise phosphorylations do not achieve the same level of SUFU release.","method":"In vitro ERK2 kinase assay, site-directed mutagenesis (phosphomimetic and phospho-dead substitutions), quantitative binding affinity measurement, transcriptional reporter assay","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay combined with mutagenesis, quantitative affinity measurement, and functional reporter, mechanistically rigorous single study","pmids":["35831023"],"is_preprint":false},{"year":2022,"finding":"SUFU binds the transcription factor ZNF281 (a mammalian-specific protein with no Drosophila homolog) and retains it in the cytoplasm by masking ZNF281's nuclear localization signal; SUFU also hampers ZNF281 interaction with target gene promoters, suppressing tumor cell migration independent of the canonical Hh pathway.","method":"Co-immunoprecipitation, subcellular localization assays, ChIP/promoter-binding assays, in vivo tumor migration model, NLS masking mutant analysis","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, localization, promoter-binding, and in vivo model, single lab, novel pathway but well-controlled","pmids":["36220888"],"is_preprint":false},{"year":2023,"finding":"SUFU directly interacts with TRAF6 and prevents TRAF6 oligomerization, phase separation (liquid-liquid phase separation into droplets), and autoubiquitination. SUFU inhibits NF-κB activation upon LPS stimulation by suppressing TRAF6 phase-separated droplet formation. In myeloid cell-specific Sufu-KO mice, LPS-induced inflammation and lethality are aggravated and rescued by TRAF6 depletion.","method":"Co-immunoprecipitation, in vitro phase separation assay with purified proteins, FRAP, Native-PAGE, myeloid cell-specific KO mice with LPS-induced septic shock and CLP models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reconstitution with purified proteins for phase separation, FRAP, reciprocal Co-IP, and in vivo genetic rescue, multiple orthogonal methods","pmids":["37441604"],"is_preprint":false},{"year":2015,"finding":"SUFU depletion in recipient dendritic cells leads to increased surface HLA-DR expression and augmented allogeneic mixed leukocyte response, while ectopic SUFU expression reduces HLA-DR and suppresses MLR, demonstrating a role for SUFU in regulating dendritic cell antigen presentation.","method":"Ectopic expression and siRNA knockdown of SUFU in THP-1-derived DCs, HLA-DR surface expression measurement, allogeneic mixed leukocyte response assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — paired gain- and loss-of-function in DCs with defined molecular (HLA-DR) and functional (MLR) readouts, single lab","pmids":["26067905"],"is_preprint":false},{"year":2004,"finding":"A medulloblastoma-derived mutant of SUFU loses the ability to decrease nuclear levels of β-catenin and cannot inhibit β-catenin/TCF-mediated transcription, indicating that wild-type SUFU can bind β-catenin, export it from the nucleus, and repress WNT signaling.","method":"Nuclear β-catenin localization assay, β-catenin/TCF luciferase reporter assay with wild-type vs. mutant SUFU","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived mutant used to dissect function, dual localization and reporter assays, single lab","pmids":["15077159"],"is_preprint":false},{"year":2006,"finding":"Drosophila Su(fu) enters the nucleus in concert with Ci in response to Hh signaling; Ci can bring Su(fu) with it to a multimerized Ci DNA-binding site, and Su(fu) nuclear import depends on the Ci nuclear localization signal.","method":"Cell biological localization assay, NLS mutant analysis, multimerized DNA binding site tethering assay","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging and DNA-binding site tethering demonstrate nuclear co-import mechanism, single lab","pmids":["16595130"],"is_preprint":false},{"year":2017,"finding":"Recessive hypomorphic missense variants in SUFU reduce SUFU protein stability and its capacity to bind GLI3 and promote GLI3 cleavage into the repressor form GLI3R, impairing SUFU-mediated repression of the SHH pathway and causing Joubert syndrome.","method":"Molecular dynamics simulation, functional studies in cellular models and patient fibroblasts (SUFU stability, GLI3 binding, GLI3 cleavage, Hh target gene expression)","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient variant functional validation in fibroblasts with binding and processing assays, supported by molecular dynamics, single study","pmids":["28965847"],"is_preprint":false},{"year":2013,"finding":"RIO kinase 3 (RIOK3) modulates SUFU subcellular distribution; a SUFU construct experimentally tethered exclusively to the nucleus is unable to antagonize endogenous Hh signaling (negative finding: nuclear-only SUFU loses inhibitory function), indicating that normal nucleo-cytoplasmic distribution of SUFU is required for its negative regulatory role.","method":"Kinase screen in human cells, nuclear-tethered SUFU construct functional assay, RIOK3 overexpression and SUFU localization analysis","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear tethering experiment with defined functional consequence establishes localization requirement, kinase screen identifies regulator, single lab","pmids":["24018050"],"is_preprint":false}],"current_model":"SUFU is an essential, multifunctional negative regulator of the Hedgehog (and, to a lesser extent, WNT) signaling pathway that operates through at least four distinct mechanisms: (1) it forms a high-affinity complex with GLI transcription factors via conserved N-terminal (SIN) and C-terminal (SIC) binding motifs—structurally defined by crystallography—sequestering GLI in the cytoplasm through SIN and blocking nuclear GLI activity through SIC by masking the CBP coactivator site; (2) in response to Hh stimulation, SUFU-Gli complexes are recruited to primary cilia where SUFU undergoes PKA/GSK3β dual phosphorylation and subsequent Shh-triggered dephosphorylation that drives retrograde export and ubiquitin-proteasome degradation; (3) SUFU stability and activity are dynamically regulated by multiple post-translational modifications—proteolytic K48/K63 polyubiquitylation by Fbxl17/LNX1 (degradative) and K63-linked non-proteolytic ubiquitylation by the Itch/β-arrestin2 complex (enhances Gli3 repressor formation), phosphorylation by GSK3β and PKA (stabilizing), Nek2A (stabilizing), and weakening by ERK2-mediated multisite phosphorylation of GLI1 (S102/S116/S130); (4) beyond Hh, SUFU acts as a tumor suppressor through Hh-independent mechanisms including retention of ZNF281 in the cytoplasm to suppress tumor cell migration, activation of LATS1 to suppress YAP/ACSL4 and thereby modulate ferroptosis sensitivity, and direct binding to TRAF6 to inhibit its phase separation, autoubiquitination, and NF-κB activation in innate immune signaling."},"narrative":{"mechanistic_narrative":"SUFU is an essential negative regulator of Hedgehog (Hh) signaling and a tumor suppressor that controls the activity, stability, and subcellular distribution of GLI/Ci transcription factors [PMID:12068298, PMID:12426310]. It binds GLI directly through a conserved SYGH motif (Gly122/His123 critical), and structural analysis of full-length human SUFU bound to the GLI SYGHL peptide shows that GLI engagement drives major conformational rearrangement including an intrinsically disordered loop required for pathway activation [PMID:12426310, PMID:24311597]. SUFU engages Ci/GLI at two sites: an N-terminal SIN site that mediates cytoplasmic sequestration and a C-terminal SIC site that represses nuclear GLI by masking the CBP coactivator surface [PMID:25941387]. As a nucleocytoplasmic shuttle, SUFU competes with importin β1 for the GLI1 N-terminus and recruits nuclear corepressors such as p66β (NuRD) onto Hh target promoters, and its balanced nucleo-cytoplasmic distribution is itself required for repressive function [PMID:24854174, PMID:25403183, PMID:24018050]. SUFU also promotes formation of the GLI3 repressor by assembling a trimolecular GLI3/SUFU/GSK3β complex that licenses GLI3 phosphorylation and processing [PMID:19622347], and stabilizes full-length GLI by antagonizing the GLI-degrading factor SPOP [PMID:19684112]. Hh stimulation recruits SUFU-GLI complexes to the primary cilium and triggers their dissociation, releasing GLI to the nucleus; PKA activity restrains ciliary localization, and dual phosphorylation of SUFU at Ser342 (GSK3β) and Ser346 (PKA) stabilizes SUFU until Hh-induced dephosphorylation drives retrograde export and proteasomal degradation [PMID:20956384, PMID:21317289]. SUFU abundance is set by a network of ubiquitin ligases and kinases—degradative polyubiquitylation by Fbxl17 (nuclear) and LNX1, K63-linked non-proteolytic ubiquitylation by Itch/β-arrestin2 that enhances GLI3 repressor formation, and stabilizing phosphorylation by Nek2A—while GLI-SUFU affinity is weakened by ERK2 multisite phosphorylation of GLI1 (S102/S116/S130) and PRMT7 methylation of GLI2 [PMID:27234298, PMID:27297360, PMID:28035348, PMID:29515120, PMID:33608498, PMID:35831023, PMID:31000813]. Beyond Hh, SUFU acts through additional mechanisms: cytoplasmic retention of ZNF281 to suppress tumor cell migration, activation of LATS1 to suppress the YAP/ACSL4 axis and modulate ferroptosis sensitivity, repression of WNT/β-catenin signaling, and direct binding to TRAF6 to block its phase separation, autoubiquitination, and NF-κB activation in innate immunity [PMID:36220888, PMID:35800779, PMID:15077159, PMID:37441604]. Loss-of-function and hypomorphic SUFU variants cause disease: recessive missense variants that destabilize SUFU and impair GLI3 processing cause Joubert syndrome [PMID:28965847].","teleology":[{"year":2002,"claim":"Established SUFU as a tumor suppressor acting by controlling GLI nuclear-cytoplasmic localization, answering how SUFU restrains SHH signaling at the level of transcription factor compartmentalization.","evidence":"Patient-derived SUFU truncation mutants assayed for GLI nuclear export","pmids":["12068298"],"confidence":"Medium","gaps":["Does not define the direct SUFU-GLI binding interface","Mechanism of export (shuttle vs. retention) not resolved"]},{"year":2002,"claim":"Mapped the direct physical SUFU-GLI interaction to a conserved SYGH motif, establishing the molecular basis of GLI recognition.","evidence":"Co-IP, Far Western, site-directed mutagenesis (Gly122/His123), luciferase reporter","pmids":["12426310"],"confidence":"High","gaps":["Structural mechanism of binding not yet visualized","Whether a single binding mode accounts for both cytoplasmic and nuclear repression unknown"]},{"year":2004,"claim":"Defined SUFU as a nucleocytoplasmic shuttle protein with nuclear and cytoplasmic partners, and extended its repressive role to WNT/β-catenin signaling.","evidence":"Yeast two-hybrid (SAP18, Galectin3), NES mutant and leptomycin B; β-catenin/TCF reporter with medulloblastoma mutant","pmids":["14611647","15077159"],"confidence":"Medium","gaps":["Physiological significance of SAP18/Galectin3 interactions in Hh unclear","Directness of SUFU-β-catenin binding not biochemically resolved"]},{"year":2009,"claim":"Showed SUFU promotes GLI3 repressor formation and controls full-length GLI levels, distinguishing its scaffolding (GSK3β recruitment) and stabilizing (SPOP antagonism) roles.","evidence":"Co-IP of GLI3/SUFU/GSK3β trimolecular complex, Sufu-/- MEFs, SPOP epistasis, cross-species rescue","pmids":["19622347","19684112"],"confidence":"High","gaps":["Quantitative contribution of processing vs. stabilization to repression not separated","Cilium-dependence of these steps not fully delineated"]},{"year":2011,"claim":"Defined ciliary recruitment and phospho-regulated stability as the dynamic switch controlling SUFU release of GLI, answering how Hh stimulation reverses SUFU repression.","evidence":"Live imaging of endogenous SUFU-Gli at cilia, PKA perturbation; S342/S346 phosphosite mutagenesis and degradation assays","pmids":["20956384","21317289"],"confidence":"High","gaps":["Identity of the Shh-triggered phosphatase not defined","Coupling between dephosphorylation and the degradation machinery unresolved"]},{"year":2012,"claim":"Genetically established Kif7 as the positive regulator that restricts SUFU by promoting its turnover and ciliary exclusion, with combined loss driving basal cell carcinoma.","evidence":"Kif7-null and compound mutant mouse genetics, ciliary localization, skin-specific double KO","pmids":["21795282","23034632"],"confidence":"High","gaps":["Molecular mechanism by which Kif7 promotes SUFU turnover not defined","Tissue specificity of the SUFU-Kif7 balance not generalized"]},{"year":2014,"claim":"Resolved the structural and dual-site basis of SUFU-GLI binding and added nuclear corepressor recruitment and importin competition as parallel repressive mechanisms.","evidence":"X-ray crystallography and SAXS of SUFU-GLI; SIN/SIC site mutagenesis and CBP competition; importin β1 affinity and competition; p66β/Mycbp proteomics and ChIP","pmids":["24311597","25941387","24854174","25403183"],"confidence":"High","gaps":["How the SIN/SIC sites are coordinated in vivo unclear","Dynamics of corepressor exchange at promoters not time-resolved"]},{"year":2018,"claim":"Defined an extensive ubiquitin/kinase network setting SUFU abundance and GLI-binding affinity, linking post-translational control to disease mutations.","evidence":"Fbxl17 (nuclear) and Itch/β-arrestin2 (K63) ubiquitylation assays, Nek2A kinase/ChIP feedback, K257 ubiquitin site, ERK2 and PRMT7 modification of GLI, LNX1/SNEP1 feedback","pmids":["27234298","29515120","27297360","28035348","18997815","35831023","31000813","33608498"],"confidence":"High","gaps":["Hierarchy and crosstalk among the competing modifications unresolved","Spatial (nuclear vs. ciliary) compartmentalization of each ligase not fully mapped"]},{"year":2017,"claim":"Identified Hh-independent and tumor-relevant SUFU functions, broadening its role beyond GLI regulation.","evidence":"BCL-2 family BH-domain engagement of SUFU; ZNF281 cytoplasmic retention; LATS1/YAP/ACSL4 ferroptosis axis; dendritic cell HLA-DR/MLR regulation","pmids":["28945232","36220888","35800779","26067905"],"confidence":"Medium","gaps":["Degree of GLI-independence varies and is not always cleanly separated","Physiological contexts for each non-canonical function not fully established"]},{"year":2017,"claim":"Connected SUFU loss-of-function to a Mendelian ciliopathy, showing hypomorphic variants impair GLI3 processing.","evidence":"Patient fibroblast functional studies of stability, GLI3 binding and cleavage; molecular dynamics","pmids":["28965847"],"confidence":"Medium","gaps":["Genotype-phenotype correlation across variants not established","Tissue-specific consequences during development not defined"]},{"year":2023,"claim":"Revealed a direct innate immune function of SUFU through inhibition of TRAF6 phase separation and NF-κB activation, defining a Hh-independent role in inflammation.","evidence":"Reconstituted phase separation with purified proteins, FRAP, Native-PAGE, reciprocal Co-IP, myeloid-specific Sufu KO with LPS/CLP and TRAF6 rescue","pmids":["37441604"],"confidence":"High","gaps":["Whether SUFU regulates other phase-separating signaling hubs unknown","Relationship between SUFU's Hh and TRAF6 pools not addressed"]},{"year":null,"claim":"How the competing post-translational modifications, ciliary trafficking, and Hh-independent partners are integrated to set SUFU dosage in a given cell type remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of SUFU stability control across compartments","The Shh-induced phosphatase acting on SUFU is unidentified","Mechanistic basis for selecting Hh vs. non-canonical SUFU functions is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,12,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,5,24]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[12,13,23]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,14]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,2,12]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,2,15,29]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[7,8,9]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,7,12]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[12,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15,18,28]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[24,25]}],"complexes":["GLI3/SUFU/GSK3β complex","SUFU-GLI complex","Itch/β-arrestin2 complex"],"partners":["GLI1","GLI2","GLI3","GSK3B","FBXL17","ITCH","TRAF6","ZNF281"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UMX1","full_name":"Suppressor of fused homolog","aliases":[],"length_aa":484,"mass_kda":53.9,"function":"Negative regulator in the hedgehog/smoothened signaling pathway (PubMed:10559945, PubMed:10564661, PubMed:10806483, PubMed:12068298, PubMed:12975309, PubMed:15367681, PubMed:22365972, PubMed:24217340, PubMed:24311597, PubMed:27234298, PubMed:28965847). Down-regulates GLI1-mediated transactivation of target genes (PubMed:15367681, PubMed:24217340, PubMed:24311597). Down-regulates GLI2-mediated transactivation of target genes (PubMed:24217340, PubMed:24311597). Part of a corepressor complex that acts on DNA-bound GLI1. May also act by linking GLI1 to BTRC and thereby targeting GLI1 to degradation by the proteasome (PubMed:10559945, PubMed:10564661, PubMed:10806483, PubMed:24217340). Sequesters GLI1, GLI2 and GLI3 in the cytoplasm, this effect is overcome by binding of STK36 to both SUFU and a GLI protein (PubMed:10559945, PubMed:10564661, PubMed:10806483, PubMed:24217340). Negative regulator of beta-catenin signaling (By similarity). Regulates the formation of either the repressor form (GLI3R) or the activator form (GLI3A) of the full-length form of GLI3 (GLI3FL) (PubMed:24311597, PubMed:28965847). GLI3FL is complexed with SUFU in the cytoplasm and is maintained in a neutral state (PubMed:24311597, PubMed:28965847). Without the Hh signal, the SUFU-GLI3 complex is recruited to cilia, leading to the efficient processing of GLI3FL into GLI3R (PubMed:24311597, PubMed:28965847). When Hh signaling is initiated, SUFU dissociates from GLI3FL and the latter translocates to the nucleus, where it is phosphorylated, destabilized, and converted to a transcriptional activator (GLI3A) (PubMed:24311597, PubMed:28965847). Required for normal embryonic development (By similarity). Required for the proper formation of hair follicles and the control of epidermal differentiation (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UMX1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SUFU","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SUFU","total_profiled":1310},"omim":[{"mim_id":"621143","title":"HOLOPROSENCEPHALY 10; HPE10","url":"https://www.omim.org/entry/621143"},{"mim_id":"620343","title":"BASAL CELL NEVUS SYNDROME 2; BCNS2","url":"https://www.omim.org/entry/620343"},{"mim_id":"617757","title":"JOUBERT SYNDROME 32; JBTS32","url":"https://www.omim.org/entry/617757"},{"mim_id":"612250","title":"G PROTEIN-COUPLED RECEPTOR 161: GPR161","url":"https://www.omim.org/entry/612250"},{"mim_id":"611957","title":"MICRO RNA 378; MIRN378","url":"https://www.omim.org/entry/611957"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SUFU"},"hgnc":{"alias_symbol":["SUFUH","SUFUXL","PRO1280"],"prev_symbol":[]},"alphafold":{"accession":"Q9UMX1","domains":[{"cath_id":"-","chopping":"31-261","consensus_level":"high","plddt":96.4678,"start":31,"end":261},{"cath_id":"3.30.1360.230","chopping":"272-280_303-308_362-477","consensus_level":"high","plddt":91.3272,"start":272,"end":477}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UMX1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UMX1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UMX1-F1-predicted_aligned_error_v6.png","plddt_mean":82.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SUFU","jax_strain_url":"https://www.jax.org/strain/search?query=SUFU"},"sequence":{"accession":"Q9UMX1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UMX1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UMX1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UMX1"}},"corpus_meta":[{"pmid":"12068298","id":"PMC_12068298","title":"Mutations in SUFU predispose to medulloblastoma.","date":"2002","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12068298","citation_count":608,"is_preprint":false},{"pmid":"18077375","id":"PMC_18077375","title":"MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/18077375","citation_count":450,"is_preprint":false},{"pmid":"20956384","id":"PMC_20956384","title":"A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20956384","citation_count":319,"is_preprint":false},{"pmid":"15656799","id":"PMC_15656799","title":"Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas.","date":"2005","source":"The British journal of dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/15656799","citation_count":313,"is_preprint":false},{"pmid":"19684112","id":"PMC_19684112","title":"Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved.","date":"2009","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/19684112","citation_count":300,"is_preprint":false},{"pmid":"25403219","id":"PMC_25403219","title":"Germline mutations in SUFU cause Gorlin syndrome-associated childhood medulloblastoma and redefine the risk associated with PTCH1 mutations.","date":"2014","source":"Journal of clinical oncology : official journal of the American Society of Clinical Oncology","url":"https://pubmed.ncbi.nlm.nih.gov/25403219","citation_count":214,"is_preprint":false},{"pmid":"16294371","id":"PMC_16294371","title":"Deregulation of the hedgehog signalling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development.","date":"2006","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/16294371","citation_count":188,"is_preprint":false},{"pmid":"12426310","id":"PMC_12426310","title":"Characterization of the physical interaction of Gli proteins with SUFU proteins.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12426310","citation_count":147,"is_preprint":false},{"pmid":"19533801","id":"PMC_19533801","title":"Identification of a SUFU germline mutation in a family with Gorlin syndrome.","date":"2009","source":"American journal of medical genetics. 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therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33143723","citation_count":16,"is_preprint":false},{"pmid":"28640224","id":"PMC_28640224","title":"Nomegestrol Acetate Suppresses Human Endometrial Cancer RL95-2 Cells Proliferation In Vitro and In Vivo Possibly Related to Upregulating Expression of SUFU and Wnt7a.","date":"2017","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/28640224","citation_count":16,"is_preprint":false},{"pmid":"25287320","id":"PMC_25287320","title":"Multiple skin hamartomata: a possible novel clinical presentation of SUFU neoplasia syndrome.","date":"2015","source":"Familial cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25287320","citation_count":16,"is_preprint":false},{"pmid":"15028872","id":"PMC_15028872","title":"Mucoraceous moulds involved in the commercial fermentation of Sufu Pehtze.","date":"2004","source":"Antonie van 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functional readout of GLI nuclear localization\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived loss-of-function mutations with defined cellular phenotype (GLI nuclear retention), single study but mechanistically specific\",\n      \"pmids\": [\"12068298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SUFU directly interacts with GLI proteins via a conserved SYGH motif in GLI/Ci; residues Gly122 and His123 in this motif are critical for binding. A C-terminal 19-amino acid deletion in SUFU (Δ465) abolishes interaction with GLI1. GLI mutants lacking the SYGH motif are no longer suppressed by SUFU co-expression.\",\n      \"method\": \"Co-immunoprecipitation, Far Western assays, truncation and site-directed mutagenesis, luciferase reporter functional assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reciprocal binding assays combined with mutagenesis and functional reporter assay in a single rigorous study\",\n      \"pmids\": [\"12426310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SUFU interacts with SAP18 (Sin3-associated polypeptide 18) and Galectin3 as nuclear binding partners. Co-expression with SAP18 translocates SUFU to the nucleus, while co-expression with SUFU translocates Galectin3 to the cytoplasm. SUFU is predominantly cytoplasmic but becomes mostly nuclear when its putative nuclear export signal is mutated or after leptomycin B treatment, establishing SUFU as a nucleocytoplasmic shuttle protein.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, cell transfection with NES mutant and leptomycin B treatment, subcellular fractionation/imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by independent biochemical assays, localization determined by direct experiment with functional NES mutation, single lab\",\n      \"pmids\": [\"14611647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GSK3β binds to SUFU both in vitro and in vivo and phosphorylates SUFU; a phosphorylation-mimicking SUFU mutant shows significantly decreased ability to bind GLI1 and cannot suppress GLI-mediated reporter gene expression. GSK3β knockdown attenuates Hedgehog signaling, suggesting GSK3β functions as a positive regulator of Hedgehog signaling through phosphorylation of SUFU.\",\n      \"method\": \"Mass spectrometric identification of binding partner, GST pull-down and co-immunoprecipitation (in vitro and in vivo), in vitro kinase assay, RNAi knockdown, luciferase reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay combined with co-IP, phosphomimetic mutant functional analysis, and RNAi phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17182001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Hedgehog signaling promotes ubiquitination of SUFU, leading to its proteasomal degradation. Lysine 257 (K257) is the ubiquitin attachment site; the K257R mutant of SUFU is more stable, more potent as a transcription repressor, and a stronger cell growth inhibitor.\",\n      \"method\": \"In vivo ubiquitination assay, site-directed mutagenesis (K257R), proteasome inhibitor experiments, transcription repressor and cell growth assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of ubiquitin acceptor site combined with functional readouts (stability, transcriptional repression, growth inhibition), single lab multiple orthogonal methods\",\n      \"pmids\": [\"18997815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SUFU promotes Gli3 processing into its repressor form by recruiting GSK3β; SUFU can bind both GSK3β and Gli3 simultaneously, forming a trimolecular complex Gli3/SUFU/GSK3β that facilitates phosphorylation of Gli3 by GSK3β. Sonic Hh stimulation dissociates the SUFU/GSK3β complex from Gli3, blocking Gli3 processing. In SUFU-deficient MEFs, Gli3 processing is severely impaired.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, Sufu−/− mouse embryonic fibroblasts with functional Gli3 processing readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP establishing trimolecular complex, KO MEF model with defined processing phenotype, single lab multiple methods\",\n      \"pmids\": [\"19622347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SUFU controls protein levels of full-length Gli transcription factors (Gli activators and repressors) in a cilium-independent manner, partly by antagonizing the activity of Spop, a Gli-degrading factor. Zebrafish and fly Sufu can restore Gli protein function in Sufu-deficient mammalian cells, indicating evolutionary conservation of this mechanism.\",\n      \"method\": \"Sufu conditional knockout mice, cell-based reconstitution with heterologous Sufu orthologs, Spop epistasis analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in KO cells combined with cross-species functional rescue, multiple orthogonal methods, replicated across organisms\",\n      \"pmids\": [\"19684112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In vertebrates, Hedgehog stimulation rapidly recruits endogenous SUFU-Gli complexes to primary cilia, then causes rapid dissociation of SUFU-Gli complexes, allowing Gli to enter the nucleus and activate transcription. PKA activation blocks ciliary localization of SUFU-Gli complexes, preventing their Hh-induced dissociation.\",\n      \"method\": \"Live-cell imaging of endogenous SUFU-Gli complexes at cilia, pharmacological PKA activation/inhibition, colocalization and biochemical dissociation assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct imaging of endogenous complexes combined with biochemical dissociation assay and PKA perturbation, multiple orthogonal methods, rigorous controls\",\n      \"pmids\": [\"20956384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SUFU is phosphorylated at Ser-342 by GSK3β and at Ser-346 by PKA; dual phosphorylation at these sites stabilizes SUFU against Shh-induced degradation. Shh signaling induces trafficking of phospho-SUFU to the primary cilium in a complex with Gli2/3, and dephosphorylation triggers retrograde export and ubiquitin-proteasome degradation of SUFU.\",\n      \"method\": \"Site-directed mutagenesis (S342A, S346A, phosphomimetic S342D/S346D), PKA inhibitors, ciliary localization assays, ubiquitin-proteasome pathway analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with ciliary localization assay and degradation assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"21317289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Kif7 promotes turnover of SUFU and exclusion of SUFU-Gli complexes from the primary cilium, thereby restricting SUFU's inhibitory function. Halving the dose of Sufu restores normal Hedgehog pathway activity and chondrocyte development in Kif7-null mice, establishing that Kif7's positive role in Hh signaling is to restrict SUFU activity.\",\n      \"method\": \"Kif7-null and compound mutant mouse genetics (epistasis), ciliary localization of SUFU-Gli complexes, SUFU protein level analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis in mouse models with defined quantitative rescue of pathway activity and cell fate, replicated across labs\",\n      \"pmids\": [\"21795282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sufu restricts Gli2 activity through cytoplasmic sequestration in keratinocytes; Kif7 promotes Hh pathway activity by dissociating the SUFU-Gli2 complex. Simultaneous deletion of SUFU and Kif7 in adult epidermis induces basal cell carcinoma, whereas deletion of either alone cannot.\",\n      \"method\": \"Conditional knockout mice (skin-specific), genetic epistasis (single and double mutants), subcellular localization analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with compound conditional KO mouse models, defined BCC phenotypic readout, mechanistic localization analysis\",\n      \"pmids\": [\"23034632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure and SAXS structure of full-length human SUFU and its complex with the conserved SYGHL motif of GLI reveal that GLI binding induces major conformational changes in SUFU, including rearrangement of an intrinsically disordered loop crucial for pathway activation.\",\n      \"method\": \"X-ray crystallography, small-angle X-ray scattering (SAXS), structural analysis of SUFU-GLI peptide complex\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure and SAXS of full-length human protein with ligand, rigorous structural validation, single study\",\n      \"pmids\": [\"24311597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SUFU can bind Ci/Gli through two distinct sites: the N-terminal Sufu-interacting site (SIN) and a newly identified C-terminal Sufu-interacting site (SIC). Binding via SIN mediates cytoplasmic sequestration of Ci/Gli, while binding via SIC inhibits Ci/Gli activity in the nucleus by masking the CBP coactivator binding site in the C-terminal region of Ci.\",\n      \"method\": \"Binding site mutagenesis, cytoplasmic/nuclear localization assays, Drosophila genetics, CBP binding competition assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site mutagenesis combined with localization assays and transcriptional coactivator competition analysis, mechanistic dissection with multiple orthogonal methods\",\n      \"pmids\": [\"25941387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Importin β1 and SUFU bind to overlapping sites on the GLI1 N-terminus with high nanomolar affinity; they compete for binding to GLI1, such that relative intracellular concentrations of SUFU and importin β1 determine GLI1 nuclear localization. SUFU co-expression specifically inhibits GFP-Gli1 nuclear accumulation.\",\n      \"method\": \"Quantitative live mammalian cell imaging, direct binding affinity measurement, siRNA knockdown of importin β1, competition binding assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct affinity measurement combined with live-cell imaging, specific knockdown, and competition assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"24854174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In the nucleus, SUFU recruits p66β (a NuRD repressor complex member) to block Gli-mediated Hh target gene expression; Mycbp forms a complex with Gli and SUFU without Hh stimulation but is kept inactive. Hh activation leads to dissociation of SUFU/p66β from Gli, enabling Mycbp to promote Gli activity. SUFU, p66β, Mycbp, and Gli are detected on promoters of Hh targets in a dynamic manner.\",\n      \"method\": \"Proteomic identification of Sufu-interacting proteins, cell-based signaling assays, zebrafish epistasis, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomic ID confirmed by ChIP and cell-based assays and in vivo zebrafish epistasis, multiple orthogonal methods across species\",\n      \"pmids\": [\"25403183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The F-box protein Fbxl17 targets SUFU for proteolysis specifically in the nucleus, mediating its polyubiquitylation and allowing Gli1 release for Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling and impaired medulloblastoma tumor growth. A Gorlin syndrome-derived SUFU mutation increases SUFU turnover through enhanced Fbxl17-mediated polyubiquitylation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, nuclear-targeted constructs, Fbxl17 depletion in cells and tumor models, patient-derived mutation analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitylation, nuclear targeting experiments, and in vivo tumor model, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"27234298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Nek2A binds SUFU (identified by yeast two-hybrid, GST pull-down and co-immunoprecipitation), phosphorylates SUFU, and stabilizes it against ubiquitin/proteasome degradation. Phosphorylated SUFU inhibits nuclear localization and transcriptional activity of Gli2. In turn, Gli1 and Gli2 directly bind the NEK2A gene promoter to induce its transcription, forming a negative-feedback loop.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, in vitro kinase assay, chromatin immunoprecipitation (ChIP), reporter assay\",\n      \"journal\": \"Cellular signalling / International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding and kinase assays, ChIP, functional reporter, single lab\",\n      \"pmids\": [\"27297360\", \"28035348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BCL-2 family anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL) found outside the outer mitochondrial membrane engage a BH domain sequence in SUFU, directly promote SUFU turnover, inhibit SUFU-GLI interaction, and thereby induce expression of GLI target genes including BCL-2, MCL-1, and BCL-XL (feedforward loop). BH3 mimetics can disable this interaction.\",\n      \"method\": \"Co-immunoprecipitation, SUFU-GLI interaction assays, SUFU stability assays, BH3 mimetic pharmacological disruption, GLI target gene expression\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interaction and functional mechanistic dissection with multiple methods including pharmacological disruption, cancer cell and in vivo models, single lab but rigorous\",\n      \"pmids\": [\"28945232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The Itch/β-arrestin2 complex binds SUFU and induces its Lys63-linked (non-proteolytic) polyubiquitylation, which increases SUFU association with Gli3 and promotes conversion of Gli3 into its repressor form, keeping Hh signaling off. Hh activation antagonizes Itch-dependent polyubiquitylation of SUFU. SUFU mutations found in medulloblastoma patients are insensitive to Itch activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays (K63-specific), Gli3 processing assays, patient-derived mutation analysis, Hh pathway activation/inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mechanistically precise ubiquitylation (K63-specific) combined with Gli3 processing readout, patient mutations tested, multiple orthogonal methods\",\n      \"pmids\": [\"29515120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PRMT7 methylates GLI2 at arginine residues R225 and R227, which are located near the SUFU-binding region. This methylation interferes with GLI2-SUFU binding, leading to increased GLI2 nuclear accumulation and enhanced Shh signaling. PRMT7-deficient MEFs show increased GLI2-SUFU interaction and reduced Shh signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis (R225/R227), GLI2 nuclear localization assay, PRMT7 KO MEFs\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro methylation with mutagenesis of target residues, binding competition assay, and KO MEF validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"31000813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SNEP1, a Hh transcriptional target, enhances SUFU ubiquitination and proteasomal degradation by recruiting the E3 ubiquitin ligase LNX1, thereby promoting Hh signaling in a positive feedback manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor experiments, LNX1 knockdown, Hh reporter assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with LNX1, single lab with defined mechanistic readout\",\n      \"pmids\": [\"33608498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SUFU suppresses ferroptosis sensitivity in breast cancer cells by activating LATS1 to suppress YAP, thereby reducing ACSL4 expression. Deletion of SUFU residues 174-385 disrupts SUFU binding to LATS1, abolishing its regulation of the YAP-ACSL4 axis and sensitivity to ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, SUFU deletion mutant mapping (residues 174-385), ACSL4 expression analysis, RSL3-triggered ferroptosis assay, ectopic expression and depletion\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mapping with functional ferroptosis readout and LATS1 Co-IP, single lab\",\n      \"pmids\": [\"35800779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERK2 phosphorylates GLI1 at three evolutionarily conserved sites (S102, S116, S130) near the high-affinity SUFU-binding site; multisite phosphorylation cooperates to weaken GLI1-SUFU binding by over 25-fold. Phosphomimetic substitutions at all three sites enhance GLI1 transcriptional activity in cells. Single or pairwise phosphorylations do not achieve the same level of SUFU release.\",\n      \"method\": \"In vitro ERK2 kinase assay, site-directed mutagenesis (phosphomimetic and phospho-dead substitutions), quantitative binding affinity measurement, transcriptional reporter assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay combined with mutagenesis, quantitative affinity measurement, and functional reporter, mechanistically rigorous single study\",\n      \"pmids\": [\"35831023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SUFU binds the transcription factor ZNF281 (a mammalian-specific protein with no Drosophila homolog) and retains it in the cytoplasm by masking ZNF281's nuclear localization signal; SUFU also hampers ZNF281 interaction with target gene promoters, suppressing tumor cell migration independent of the canonical Hh pathway.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization assays, ChIP/promoter-binding assays, in vivo tumor migration model, NLS masking mutant analysis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, localization, promoter-binding, and in vivo model, single lab, novel pathway but well-controlled\",\n      \"pmids\": [\"36220888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SUFU directly interacts with TRAF6 and prevents TRAF6 oligomerization, phase separation (liquid-liquid phase separation into droplets), and autoubiquitination. SUFU inhibits NF-κB activation upon LPS stimulation by suppressing TRAF6 phase-separated droplet formation. In myeloid cell-specific Sufu-KO mice, LPS-induced inflammation and lethality are aggravated and rescued by TRAF6 depletion.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phase separation assay with purified proteins, FRAP, Native-PAGE, myeloid cell-specific KO mice with LPS-induced septic shock and CLP models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reconstitution with purified proteins for phase separation, FRAP, reciprocal Co-IP, and in vivo genetic rescue, multiple orthogonal methods\",\n      \"pmids\": [\"37441604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SUFU depletion in recipient dendritic cells leads to increased surface HLA-DR expression and augmented allogeneic mixed leukocyte response, while ectopic SUFU expression reduces HLA-DR and suppresses MLR, demonstrating a role for SUFU in regulating dendritic cell antigen presentation.\",\n      \"method\": \"Ectopic expression and siRNA knockdown of SUFU in THP-1-derived DCs, HLA-DR surface expression measurement, allogeneic mixed leukocyte response assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — paired gain- and loss-of-function in DCs with defined molecular (HLA-DR) and functional (MLR) readouts, single lab\",\n      \"pmids\": [\"26067905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"A medulloblastoma-derived mutant of SUFU loses the ability to decrease nuclear levels of β-catenin and cannot inhibit β-catenin/TCF-mediated transcription, indicating that wild-type SUFU can bind β-catenin, export it from the nucleus, and repress WNT signaling.\",\n      \"method\": \"Nuclear β-catenin localization assay, β-catenin/TCF luciferase reporter assay with wild-type vs. mutant SUFU\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived mutant used to dissect function, dual localization and reporter assays, single lab\",\n      \"pmids\": [\"15077159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Drosophila Su(fu) enters the nucleus in concert with Ci in response to Hh signaling; Ci can bring Su(fu) with it to a multimerized Ci DNA-binding site, and Su(fu) nuclear import depends on the Ci nuclear localization signal.\",\n      \"method\": \"Cell biological localization assay, NLS mutant analysis, multimerized DNA binding site tethering assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging and DNA-binding site tethering demonstrate nuclear co-import mechanism, single lab\",\n      \"pmids\": [\"16595130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Recessive hypomorphic missense variants in SUFU reduce SUFU protein stability and its capacity to bind GLI3 and promote GLI3 cleavage into the repressor form GLI3R, impairing SUFU-mediated repression of the SHH pathway and causing Joubert syndrome.\",\n      \"method\": \"Molecular dynamics simulation, functional studies in cellular models and patient fibroblasts (SUFU stability, GLI3 binding, GLI3 cleavage, Hh target gene expression)\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient variant functional validation in fibroblasts with binding and processing assays, supported by molecular dynamics, single study\",\n      \"pmids\": [\"28965847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RIO kinase 3 (RIOK3) modulates SUFU subcellular distribution; a SUFU construct experimentally tethered exclusively to the nucleus is unable to antagonize endogenous Hh signaling (negative finding: nuclear-only SUFU loses inhibitory function), indicating that normal nucleo-cytoplasmic distribution of SUFU is required for its negative regulatory role.\",\n      \"method\": \"Kinase screen in human cells, nuclear-tethered SUFU construct functional assay, RIOK3 overexpression and SUFU localization analysis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear tethering experiment with defined functional consequence establishes localization requirement, kinase screen identifies regulator, single lab\",\n      \"pmids\": [\"24018050\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SUFU is an essential, multifunctional negative regulator of the Hedgehog (and, to a lesser extent, WNT) signaling pathway that operates through at least four distinct mechanisms: (1) it forms a high-affinity complex with GLI transcription factors via conserved N-terminal (SIN) and C-terminal (SIC) binding motifs—structurally defined by crystallography—sequestering GLI in the cytoplasm through SIN and blocking nuclear GLI activity through SIC by masking the CBP coactivator site; (2) in response to Hh stimulation, SUFU-Gli complexes are recruited to primary cilia where SUFU undergoes PKA/GSK3β dual phosphorylation and subsequent Shh-triggered dephosphorylation that drives retrograde export and ubiquitin-proteasome degradation; (3) SUFU stability and activity are dynamically regulated by multiple post-translational modifications—proteolytic K48/K63 polyubiquitylation by Fbxl17/LNX1 (degradative) and K63-linked non-proteolytic ubiquitylation by the Itch/β-arrestin2 complex (enhances Gli3 repressor formation), phosphorylation by GSK3β and PKA (stabilizing), Nek2A (stabilizing), and weakening by ERK2-mediated multisite phosphorylation of GLI1 (S102/S116/S130); (4) beyond Hh, SUFU acts as a tumor suppressor through Hh-independent mechanisms including retention of ZNF281 in the cytoplasm to suppress tumor cell migration, activation of LATS1 to suppress YAP/ACSL4 and thereby modulate ferroptosis sensitivity, and direct binding to TRAF6 to inhibit its phase separation, autoubiquitination, and NF-κB activation in innate immune signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SUFU is an essential negative regulator of Hedgehog (Hh) signaling and a tumor suppressor that controls the activity, stability, and subcellular distribution of GLI/Ci transcription factors [#0, #1]. It binds GLI directly through a conserved SYGH motif (Gly122/His123 critical), and structural analysis of full-length human SUFU bound to the GLI SYGHL peptide shows that GLI engagement drives major conformational rearrangement including an intrinsically disordered loop required for pathway activation [#1, #11]. SUFU engages Ci/GLI at two sites: an N-terminal SIN site that mediates cytoplasmic sequestration and a C-terminal SIC site that represses nuclear GLI by masking the CBP coactivator surface [#12]. As a nucleocytoplasmic shuttle, SUFU competes with importin \\u03b21 for the GLI1 N-terminus and recruits nuclear corepressors such as p66\\u03b2 (NuRD) onto Hh target promoters, and its balanced nucleo-cytoplasmic distribution is itself required for repressive function [#13, #14, #29]. SUFU also promotes formation of the GLI3 repressor by assembling a trimolecular GLI3/SUFU/GSK3\\u03b2 complex that licenses GLI3 phosphorylation and processing [#5], and stabilizes full-length GLI by antagonizing the GLI-degrading factor SPOP [#6]. Hh stimulation recruits SUFU-GLI complexes to the primary cilium and triggers their dissociation, releasing GLI to the nucleus; PKA activity restrains ciliary localization, and dual phosphorylation of SUFU at Ser342 (GSK3\\u03b2) and Ser346 (PKA) stabilizes SUFU until Hh-induced dephosphorylation drives retrograde export and proteasomal degradation [#7, #8]. SUFU abundance is set by a network of ubiquitin ligases and kinases\\u2014degradative polyubiquitylation by Fbxl17 (nuclear) and LNX1, K63-linked non-proteolytic ubiquitylation by Itch/\\u03b2-arrestin2 that enhances GLI3 repressor formation, and stabilizing phosphorylation by Nek2A\\u2014while GLI-SUFU affinity is weakened by ERK2 multisite phosphorylation of GLI1 (S102/S116/S130) and PRMT7 methylation of GLI2 [#15, #16, #18, #20, #22, #19]. Beyond Hh, SUFU acts through additional mechanisms: cytoplasmic retention of ZNF281 to suppress tumor cell migration, activation of LATS1 to suppress the YAP/ACSL4 axis and modulate ferroptosis sensitivity, repression of WNT/\\u03b2-catenin signaling, and direct binding to TRAF6 to block its phase separation, autoubiquitination, and NF-\\u03baB activation in innate immunity [#23, #21, #26, #24]. Loss-of-function and hypomorphic SUFU variants cause disease: recessive missense variants that destabilize SUFU and impair GLI3 processing cause Joubert syndrome [#28].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established SUFU as a tumor suppressor acting by controlling GLI nuclear-cytoplasmic localization, answering how SUFU restrains SHH signaling at the level of transcription factor compartmentalization.\",\n      \"evidence\": \"Patient-derived SUFU truncation mutants assayed for GLI nuclear export\",\n      \"pmids\": [\"12068298\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define the direct SUFU-GLI binding interface\", \"Mechanism of export (shuttle vs. retention) not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the direct physical SUFU-GLI interaction to a conserved SYGH motif, establishing the molecular basis of GLI recognition.\",\n      \"evidence\": \"Co-IP, Far Western, site-directed mutagenesis (Gly122/His123), luciferase reporter\",\n      \"pmids\": [\"12426310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of binding not yet visualized\", \"Whether a single binding mode accounts for both cytoplasmic and nuclear repression unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined SUFU as a nucleocytoplasmic shuttle protein with nuclear and cytoplasmic partners, and extended its repressive role to WNT/\\u03b2-catenin signaling.\",\n      \"evidence\": \"Yeast two-hybrid (SAP18, Galectin3), NES mutant and leptomycin B; \\u03b2-catenin/TCF reporter with medulloblastoma mutant\",\n      \"pmids\": [\"14611647\", \"15077159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological significance of SAP18/Galectin3 interactions in Hh unclear\", \"Directness of SUFU-\\u03b2-catenin binding not biochemically resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed SUFU promotes GLI3 repressor formation and controls full-length GLI levels, distinguishing its scaffolding (GSK3\\u03b2 recruitment) and stabilizing (SPOP antagonism) roles.\",\n      \"evidence\": \"Co-IP of GLI3/SUFU/GSK3\\u03b2 trimolecular complex, Sufu-/- MEFs, SPOP epistasis, cross-species rescue\",\n      \"pmids\": [\"19622347\", \"19684112\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of processing vs. stabilization to repression not separated\", \"Cilium-dependence of these steps not fully delineated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined ciliary recruitment and phospho-regulated stability as the dynamic switch controlling SUFU release of GLI, answering how Hh stimulation reverses SUFU repression.\",\n      \"evidence\": \"Live imaging of endogenous SUFU-Gli at cilia, PKA perturbation; S342/S346 phosphosite mutagenesis and degradation assays\",\n      \"pmids\": [\"20956384\", \"21317289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Shh-triggered phosphatase not defined\", \"Coupling between dephosphorylation and the degradation machinery unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetically established Kif7 as the positive regulator that restricts SUFU by promoting its turnover and ciliary exclusion, with combined loss driving basal cell carcinoma.\",\n      \"evidence\": \"Kif7-null and compound mutant mouse genetics, ciliary localization, skin-specific double KO\",\n      \"pmids\": [\"21795282\", \"23034632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which Kif7 promotes SUFU turnover not defined\", \"Tissue specificity of the SUFU-Kif7 balance not generalized\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the structural and dual-site basis of SUFU-GLI binding and added nuclear corepressor recruitment and importin competition as parallel repressive mechanisms.\",\n      \"evidence\": \"X-ray crystallography and SAXS of SUFU-GLI; SIN/SIC site mutagenesis and CBP competition; importin \\u03b21 affinity and competition; p66\\u03b2/Mycbp proteomics and ChIP\",\n      \"pmids\": [\"24311597\", \"25941387\", \"24854174\", \"25403183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the SIN/SIC sites are coordinated in vivo unclear\", \"Dynamics of corepressor exchange at promoters not time-resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined an extensive ubiquitin/kinase network setting SUFU abundance and GLI-binding affinity, linking post-translational control to disease mutations.\",\n      \"evidence\": \"Fbxl17 (nuclear) and Itch/\\u03b2-arrestin2 (K63) ubiquitylation assays, Nek2A kinase/ChIP feedback, K257 ubiquitin site, ERK2 and PRMT7 modification of GLI, LNX1/SNEP1 feedback\",\n      \"pmids\": [\"27234298\", \"29515120\", \"27297360\", \"28035348\", \"18997815\", \"35831023\", \"31000813\", \"33608498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy and crosstalk among the competing modifications unresolved\", \"Spatial (nuclear vs. ciliary) compartmentalization of each ligase not fully mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified Hh-independent and tumor-relevant SUFU functions, broadening its role beyond GLI regulation.\",\n      \"evidence\": \"BCL-2 family BH-domain engagement of SUFU; ZNF281 cytoplasmic retention; LATS1/YAP/ACSL4 ferroptosis axis; dendritic cell HLA-DR/MLR regulation\",\n      \"pmids\": [\"28945232\", \"36220888\", \"35800779\", \"26067905\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degree of GLI-independence varies and is not always cleanly separated\", \"Physiological contexts for each non-canonical function not fully established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected SUFU loss-of-function to a Mendelian ciliopathy, showing hypomorphic variants impair GLI3 processing.\",\n      \"evidence\": \"Patient fibroblast functional studies of stability, GLI3 binding and cleavage; molecular dynamics\",\n      \"pmids\": [\"28965847\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype correlation across variants not established\", \"Tissue-specific consequences during development not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a direct innate immune function of SUFU through inhibition of TRAF6 phase separation and NF-\\u03baB activation, defining a Hh-independent role in inflammation.\",\n      \"evidence\": \"Reconstituted phase separation with purified proteins, FRAP, Native-PAGE, reciprocal Co-IP, myeloid-specific Sufu KO with LPS/CLP and TRAF6 rescue\",\n      \"pmids\": [\"37441604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SUFU regulates other phase-separating signaling hubs unknown\", \"Relationship between SUFU's Hh and TRAF6 pools not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the competing post-translational modifications, ciliary trafficking, and Hh-independent partners are integrated to set SUFU dosage in a given cell type remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of SUFU stability control across compartments\", \"The Shh-induced phosphatase acting on SUFU is unidentified\", \"Mechanistic basis for selecting Hh vs. non-canonical SUFU functions is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 12, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 5, 24]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [12, 13, 23]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 2, 12]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 2, 15, 29]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7, 8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 7, 12]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [12, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 18, 28]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [24, 25]}\n    ],\n    \"complexes\": [\n      \"GLI3/SUFU/GSK3\\u03b2 complex\",\n      \"SUFU-GLI complex\",\n      \"Itch/\\u03b2-arrestin2 complex\"\n    ],\n    \"partners\": [\n      \"GLI1\",\n      \"GLI2\",\n      \"GLI3\",\n      \"GSK3B\",\n      \"FBXL17\",\n      \"ITCH\",\n      \"TRAF6\",\n      \"ZNF281\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}