{"gene":"SPAG6","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":1996,"finding":"PF16 (Chlamydomonas orthologue of SPAG6) contains eight contiguous armadillo repeats and localizes specifically to the C1 microtubule of the central apparatus in flagella; loss of PF16 results in paralyzed flagella and missing C1 microtubule in isolated axonemes","method":"Insertional mutagenesis, gene cloning, transformation rescue, immunofluorescence, immunogold electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — genetic rescue, immunogold localization, multiple orthogonal methods in a foundational study","pmids":["8636214"],"is_preprint":false},{"year":1999,"finding":"Human SPAG6 encodes an armadillo repeat-containing protein localizing to the tails of permeabilized human sperm, consistent with a role in the flagellar axoneme central pair","method":"cDNA cloning, antibody generation, immunofluorescence localization of permeabilized sperm","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct localization by immunofluorescence, single lab","pmids":["10493827"],"is_preprint":false},{"year":2000,"finding":"Murine SPAG6 (Spag6) localizes to the principal piece of mouse sperm tails and co-localizes with microtubules when expressed in COS-1 cells, supporting its role as an axoneme central apparatus component","method":"Antipeptide antibody, immunofluorescence in sperm and COS-1 cells","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct localization, single lab, consistent with Chlamydomonas ortholog data","pmids":["10684790"],"is_preprint":false},{"year":2000,"finding":"The first armadillo repeat of PF16 is necessary but not sufficient for assembly into the axoneme; the C-terminal 122 amino acids are dispensable; the armadillo repeats act as a single functional unit required for PF16 assembly and flagellar motility rescue","method":"Deletion construct generation, transformation rescue assays in pf16 Chlamydomonas mutants","journal":"Cell motility and the cytoskeleton","confidence":"High","confidence_rationale":"Tier 1 — domain deletion mutagenesis with functional rescue readout","pmids":["10913963"],"is_preprint":false},{"year":2002,"finding":"SPAG6 (mammalian PF16 orthologue) physically interacts with PF20 (mammalian WD repeat protein); when co-expressed, PF20 colocalizes with SPAG6 on polymerized microtubules; PF20 is markedly reduced in sperm from SPAG6-deficient mice","method":"Yeast two-hybrid, co-expression in CHO cells with fluorescence microscopy, immunocytochemistry, western blot of Spag6-null mouse sperm","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — yeast two-hybrid interaction confirmed by co-localization and genetic dependency in knockout mice","pmids":["12391165"],"is_preprint":false},{"year":2005,"finding":"SPAG6 physically interacts with the mammalian orthologue of Chlamydomonas PF6, which is localized to the axoneme central apparatus C1 microtubule 1a projection; PF6 fragment that interacts with SPAG6 is absent from SPAG6-deficient mouse sperm; SPAG6, PF6, and PF20 form a network linking central apparatus components","method":"Yeast two-hybrid, co-transfection and co-localization in cells, immunocytochemistry, western blot of Spag6-null sperm","journal":"Molecular & cellular proteomics","confidence":"High","confidence_rationale":"Tier 1–2 — yeast two-hybrid interaction validated by in-cell co-localization and knockout mouse analysis, multiple orthogonal methods","pmids":["15827353"],"is_preprint":false},{"year":2014,"finding":"SPAG6 deficiency in mice reduces cilia beat frequency in tracheal epithelial cells, disrupts synchronized cilia beat, reduces cilia density in ependymal and tracheal cells, randomizes basal feet orientation, and disrupts planar cell polarity (Vangl2 distribution), indicating SPAG6 regulates ciliogenesis, axoneme orientation, and epithelial cell polarity","method":"Knockout mouse model, high-speed video microscopy, immunofluorescence, electron microscopy","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — KO with multiple specific phenotypic readouts using orthogonal methods","pmids":["25333478"],"is_preprint":false},{"year":2014,"finding":"SPAG6 silencing in myeloid cell lines (SKM-1, K562) inhibits proliferation and induces apoptosis via activation of caspase-3, -9, and -8, and upregulation of p53 and PTEN","method":"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, western blot, xenograft mouse model","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 — KO/KD with defined pathway readout, single lab","pmids":["25405588"],"is_preprint":false},{"year":2015,"finding":"SPAG6-deficient mouse embryonic fibroblasts (MEFs) show slower proliferation, larger surface area, reduced motility, reduced adhesion, non-polarized F-actin, multiple centrosomes, reduced primary cilia, and increased sensitivity to paclitaxel; SPAG6 selectively increases acetylated tubulin levels; re-expression rescues abnormal morphology","method":"MEF isolation from Spag6-null mice, rescue transfection, wound-healing assay, chemotactic analysis, immunofluorescence, paclitaxel sensitivity assay","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — KO primary cells with rescue, multiple orthogonal phenotypic readouts","pmids":["26585507"],"is_preprint":false},{"year":2015,"finding":"SPAG6 overexpression in developing cortical neurons delays neuronal migration rate and decreases neurite number and length, indicating a role in microtubule-dependent neuronal migration","method":"In utero electroporation overexpression in mouse cortex","journal":"Journal of molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2–3 — defined cellular phenotype by in vivo overexpression, single method, single lab","pmids":["26130477"],"is_preprint":false},{"year":2015,"finding":"SPAG6 is expressed in outer hair cells (OHCs) of the cochlea and co-immunoprecipitates with prestin; SPAG6-deficient mice show reduced prestin expression at both protein and mRNA levels and abnormal OHC morphology","method":"Immunofluorescence, co-immunoprecipitation, western blot, real-time PCR, Spag6-null mouse model","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP interaction plus KO phenotype, single lab","pmids":["25748314"],"is_preprint":false},{"year":2016,"finding":"SPAG6 is associated with the centrosome in lymphocytes; SPAG6 deficiency impairs immunological synapse formation by disrupting centrosome polarization and actin clearance at the synaptic cleft, leading to defective CTL function and humoral immunity","method":"Bone marrow reconstitution, immunofluorescence, functional assays of CTL activity, germinal center analysis in Spag6-null mice","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — KO with direct centrosome localization and defined cellular phenotype, single lab","pmids":["27169488"],"is_preprint":false},{"year":2017,"finding":"SPAG6 knockdown in SKM-1 cells inhibits proliferation and affects cell cycle G1-to-S transition via upregulation of p27Kip1 and regulation of the AKT/FOXO pathway","method":"shRNA knockdown, western blot, FACS cell cycle analysis, RT-qPCR, CCK-8 assay","journal":"DNA and cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 — KD with defined pathway and cell cycle readout, single lab","pmids":["30835546"],"is_preprint":false},{"year":2017,"finding":"SPAG6 silencing in SKM-1 cells activates the TRAIL signaling pathway and increases interaction between FADD and TRAIL death receptors, promoting apoptosis; high SPAG6 expression suppresses TRAIL-mediated apoptosis without affecting TRAIL death receptor expression","method":"shRNA lentiviral knockdown, flow cytometry, western blot, CCK-8 assay","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2–3 — KD with specific pathway readout, single lab","pmids":["28393201"],"is_preprint":false},{"year":2018,"finding":"SPAG6 silencing in SKM-1 cells increases PTEN expression and reduces AKT phosphorylation, leading to apoptosis via Mcl-1 downregulation, cytochrome c release, and increased caspase-9; PTEN activation by SPAG6 knockdown is caspase-independent; SPAG6 knockdown is associated with DNMT1 downregulation, implying SPAG6 controls PTEN via DNA methylation","method":"shRNA knockdown, PI3K inhibitor (LY294002), pan-caspase inhibitor (z-VAD-fmk), western blot, flow cytometry, xenograft mouse model","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 — KD with pharmacological dissection of pathway, multiple readouts, single lab","pmids":["29749435"],"is_preprint":false},{"year":2019,"finding":"SPAG6 interacts with SPINK2 (serine protease inhibitor Kazal-type 2) in the acrosome of round spermatids; SPAG6 is required for SPINK2 expression and proper acrosomal localization during spermatogenesis in mice","method":"Yeast two-hybrid, co-localization in CHO cells, immunofluorescence in Spag6-KO and wild-type mouse testis","journal":"Zhonghua nan ke xue","confidence":"Medium","confidence_rationale":"Tier 2–3 — yeast two-hybrid plus co-localization and KO validation, single lab","pmids":["32216237"],"is_preprint":false},{"year":2020,"finding":"SPAG6 silencing triggers autophagic cell death in SKM-1 cells via the AMPK/mTOR/ULK1 signaling pathway; SPAG6 knockdown increases p-AMPK and p-ULK1 while decreasing p-mTOR; autophagy inhibition reduces SPAG6 knockdown-mediated apoptosis; AMPK inhibition attenuates this autophagy","method":"shRNA knockdown, autophagy inhibitors (chloroquine, 3-MA), AMPK inhibitor (Compound C), western blot, flow cytometry","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2–3 — KD with pharmacological dissection, multiple orthogonal readouts, single lab","pmids":["32537026"],"is_preprint":false},{"year":2020,"finding":"SPAG6 deficiency in Spag6-null mice causes planar cell polarity defects and hearing loss associated with abnormal stereocilia bundles, basal body positioning, disrupted subapical microtubule meshwork, and misdistributed FZD6","method":"Spag6-null mice, auditory brainstem response, immunofluorescence, scanning electron microscopy","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 — KO with direct structural and PCP protein localization readouts, single lab","pmids":["33175573"],"is_preprint":false},{"year":2022,"finding":"SPAG6 overexpression in AML promotes translocation of MYO1D from cytosol to cell membrane, thereby upregulating EGFR family expression and activating PI3K/AKT and ERK signaling; SPAG6 physically interacts with MYO1D as shown by co-immunoprecipitation and mass spectrometry","method":"Immunoprecipitation, mass spectrometry, overexpression/knockdown, western blot, xenograft mouse model","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP/MS interaction with functional translocation phenotype, single lab","pmids":["35667090"],"is_preprint":false},{"year":2022,"finding":"DNMT3b-mediated hypermethylation of the SPAG6 promoter in lung squamous cell carcinoma leads to SPAG6 downregulation; SPAG6 regulates the JAK/STAT pathway by inhibiting STAT1 and STAT3 transcription","method":"Bisulfite sequencing, dual-luciferase assay, KEGG pathway analysis, CCK-8/EdU/Transwell assays, DNMT3b knockdown","journal":"American journal of translational research","confidence":"Medium","confidence_rationale":"Tier 2–3 — dual-luciferase for pathway validation, methylation-expression linkage confirmed in cells","pmids":["36398260"],"is_preprint":false},{"year":2023,"finding":"STAT1 protein binds to the SPAG6 promoter and promotes SPAG6 expression; conversely, SPAG6 upregulation correlates with STAT1 expression in MPN, and SPAG6 downregulation reduces STAT1 induction and enhances INF-α sensitivity","method":"Chromatin immunoprecipitation, dual-luciferase reporter assay, overexpression/knockdown, flow cytometry, western blot, in vivo xenograft","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and luciferase confirm STAT1 binding to SPAG6 promoter, single lab","pmids":["37681349"],"is_preprint":false},{"year":2016,"finding":"TAC1 protein interacts with SPAG6 as determined by yeast two-hybrid; when co-expressed with SPAG6 in CHO cells, TAC1 localizes to microtubules rather than throughout the cell body, showing SPAG6-dependent microtubule targeting of TAC1","method":"Yeast two-hybrid, co-transfection and immunofluorescence in CHO and COS-1 cells, western blot","journal":"Zhonghua nan ke xue","confidence":"Low","confidence_rationale":"Tier 3 — yeast two-hybrid and co-localization only, single lab, no functional follow-up","pmids":["29282908"],"is_preprint":false},{"year":2025,"finding":"SPAG6 physically interacts with SPINK2 with ~10-fold higher affinity than SPAG6L; SPAG6 modulates testicular AKAP4 and SPAG16L levels; SPAG6 suppresses tubulin acetylation (opposite to SPAG6L which enhances it); compound Spag6/Spag6l knockout causes defective spermiogenesis including abnormal acrosome and fibrous sheath","method":"Compound knockout mouse generation, histology, electron microscopy, binding affinity assay, western blot, tubulin acetylation assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — compound KO with structural phenotype, binding affinity measurement, and PTM readout; preprint not yet peer-reviewed","pmids":["40777416"],"is_preprint":true},{"year":2025,"finding":"SPAG6 directly interacts with DUSP1 (dual-specificity phosphatase 1) and modulates downstream MAPK/ERK signaling pathway protein expression by regulating DUSP1 activity, promoting MM cell proliferation and migration","method":"RNA interference, overexpression, transcriptome sequencing, western blot, proliferation/apoptosis/migration assays","journal":"Frontiers in pharmacology","confidence":"Low","confidence_rationale":"Tier 3 — interaction inferred from transcriptome data without direct binding assay, single lab","pmids":["40535772"],"is_preprint":false},{"year":2025,"finding":"SPAG6 overexpression in AML cells promotes GSTP1 expression, which reduces ROS levels and forms a GSTP1/JNK complex that reduces p-JNK, thereby inhibiting JNK-mediated apoptosis and attenuating daunorubicin efficacy","method":"Overexpression/knockdown, flow cytometry (ROS, apoptosis), TMT proteomics, western blot, xenograft model","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — proteomics plus functional validation of GSTP1/JNK axis, single lab","pmids":["39508041"],"is_preprint":false},{"year":2025,"finding":"CircMYH9 promotes SPAG6 mRNA stability by recruiting EIF4A3; elevated SPAG6 then suppresses PTEN and activates PI3K/AKT signaling to promote breast cancer cell proliferation, migration, and invasion","method":"RIP assay (circMYH9–EIF4A3–SPAG6 interaction), mRNA stability assay, overexpression/knockdown, PTEN inhibitor rescue, xenograft model","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 2–3 — RIP assay establishes mechanistic link; functional rescue with PTEN inhibitor supports pathway placement","pmids":["40145872"],"is_preprint":false}],"current_model":"SPAG6 is an armadillo repeat-containing scaffolding protein of the axonemal central apparatus C1 microtubule that mediates protein–protein interactions essential for flagellar/ciliary motility (via binding partners PF20, PF6, and SPINK2), regulates microtubule acetylation and stability, controls ciliogenesis and planar cell polarity, and in non-ciliated cells modulates centrosome organization, cell migration, and multiple signaling pathways (PTEN/PI3K/AKT, MAPK/ERK, AMPK/mTOR/ULK1, TRAIL) to influence proliferation and apoptosis."},"narrative":{"teleology":[{"year":1996,"claim":"The fundamental question of what maintains the C1 microtubule of the flagellar central apparatus was resolved by showing that PF16/SPAG6 is an armadillo-repeat protein whose loss abolishes the C1 microtubule and paralyzes flagella, establishing SPAG6 as a structural organizer of the central pair.","evidence":"Insertional mutagenesis, gene cloning, transformation rescue, and immunogold EM in Chlamydomonas pf16 mutants","pmids":["8636214"],"confidence":"High","gaps":["Mechanism by which SPAG6 stabilizes the C1 microtubule was unknown","No binding partners identified","No mammalian validation"]},{"year":2000,"claim":"The question of which structural features are required for axonemal assembly was answered by showing the armadillo repeat array functions as a single unit for axoneme incorporation, while confirming mammalian SPAG6 localizes to sperm flagella, bridging the Chlamydomonas genetics to mammalian biology.","evidence":"Deletion constructs with rescue assays in pf16 Chlamydomonas; immunofluorescence of human and mouse sperm","pmids":["10913963","10493827","10684790"],"confidence":"High","gaps":["Direct interacting partners in the central apparatus still unknown","No mammalian loss-of-function model yet"]},{"year":2005,"claim":"The identity of SPAG6's binding partners was established: SPAG6 directly interacts with PF20 and PF6 to form a scaffolding network on the C1 microtubule, and SPAG6 deficiency in mice eliminates these partners from sperm, demonstrating hierarchical assembly dependence.","evidence":"Yeast two-hybrid, co-localization in CHO cells, western blot of Spag6-null mouse sperm for both PF20 and PF6","pmids":["12391165","15827353"],"confidence":"High","gaps":["How the SPAG6–PF20–PF6 complex connects to dynein regulation unknown","No motility rescue shown in mammalian system"]},{"year":2014,"claim":"Whether SPAG6 functions beyond sperm flagella was resolved: SPAG6 deficiency reduces ciliary beat frequency, cilia density, and disrupts planar cell polarity (Vangl2 distribution) in tracheal and ependymal epithelia, extending its role to multiciliated somatic cells and tissue-level polarity.","evidence":"Spag6-null mice, high-speed video microscopy, immunofluorescence, and electron microscopy of airway and brain cilia","pmids":["25333478"],"confidence":"High","gaps":["Molecular mechanism linking SPAG6 to PCP pathway components not defined","Whether SPAG6 loss causes hydrocephalus not characterized"]},{"year":2015,"claim":"SPAG6 was shown to have essential functions in non-ciliated cells: it regulates centrosome number, tubulin acetylation, primary cilia formation, cell migration, and paclitaxel sensitivity in MEFs, and modulates neuronal migration in developing cortex.","evidence":"Spag6-null MEFs with rescue transfection, wound-healing, chemotactic and drug-sensitivity assays; in utero electroporation overexpression in mouse cortical neurons","pmids":["26585507","26130477"],"confidence":"High","gaps":["Enzymatic mechanism by which SPAG6 controls tubulin acetylation unknown","Whether SPAG6 directly binds acetyltransferases or deacetylases not tested"]},{"year":2016,"claim":"SPAG6's role at the centrosome in non-ciliated, non-epithelial cells was established: it localizes to the centrosome in lymphocytes and is required for centrosome polarization and immunological synapse formation, revealing a general centrosomal scaffolding function.","evidence":"Bone marrow reconstitution with Spag6-null cells, immunofluorescence, CTL killing assays, germinal center analysis","pmids":["27169488"],"confidence":"Medium","gaps":["Which centrosomal proteins SPAG6 interacts with in lymphocytes not identified","Whether SPAG6 modulates MTOC reorientation via the same armadillo-repeat interface as in axonemes unknown"]},{"year":2018,"claim":"The signaling axis through which SPAG6 promotes survival in leukemic cells was delineated: SPAG6 suppresses PTEN expression (partly via DNMT1-mediated promoter methylation) to maintain PI3K/AKT activity, and independently suppresses TRAIL-mediated death receptor signaling and AMPK/mTOR/ULK1-dependent autophagy.","evidence":"shRNA knockdown in SKM-1 cells with PI3K inhibitor, pan-caspase inhibitor, autophagy inhibitors, AMPK inhibitor, western blot, xenograft models","pmids":["29749435","28393201","32537026","25405588"],"confidence":"Medium","gaps":["Whether SPAG6 binds PTEN or DNMT1 directly not shown","All studies from one lab in one cell line (SKM-1)","In vivo relevance for leukemogenesis not confirmed by genetic models"]},{"year":2020,"claim":"SPAG6 deficiency was connected to sensorineural hearing loss: Spag6-null mice show disrupted stereocilia bundles, aberrant basal body positioning, and mislocalized FZD6 in cochlear hair cells, establishing that SPAG6's planar cell polarity function extends to the inner ear.","evidence":"Spag6-null mice, auditory brainstem response, scanning EM, immunofluorescence for FZD6","pmids":["33175573"],"confidence":"Medium","gaps":["Whether hearing loss is purely due to PCP defects or also to OHC prestin reduction (PMID:25748314) not disentangled","No human genetic data linking SPAG6 to deafness"]},{"year":2022,"claim":"A new mechanism for SPAG6 in oncogenic signaling was identified: SPAG6 physically interacts with MYO1D and promotes its translocation to the membrane, upregulating EGFR family receptors and activating PI3K/AKT and ERK pathways in AML cells.","evidence":"Co-immunoprecipitation, mass spectrometry, overexpression/knockdown, xenograft model in AML cells","pmids":["35667090"],"confidence":"Medium","gaps":["How the armadillo repeat scaffold mediates MYO1D translocation mechanistically unclear","Interaction not validated by reciprocal endogenous co-IP"]},{"year":2025,"claim":"The functional divergence between SPAG6 and its paralog SPAG6L was clarified: SPAG6 binds SPINK2 with ~10-fold higher affinity than SPAG6L and suppresses rather than enhances tubulin acetylation; compound knockout reveals non-redundant roles in acrosome biogenesis and fibrous sheath assembly during spermiogenesis.","evidence":"Compound Spag6/Spag6l knockout mice, binding affinity assay, tubulin acetylation assay, histology and EM (preprint)","pmids":["40777416"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Structural basis for differential SPINK2 binding affinity not determined","Opposing effects on tubulin acetylation mechanism unknown"]},{"year":null,"claim":"The direct enzymatic or structural mechanism by which SPAG6 modulates tubulin acetylation, the identity of its centrosomal interaction partners outside motile cilia, and whether SPAG6 mutations cause human disease (infertility, PCD, or deafness) remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No human genetic study linking SPAG6 mutations to a Mendelian disorder","No structural model of SPAG6 or its complexes","Direct mechanism linking SPAG6 to tubulin acetyltransferase/deacetylase regulation not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,2,4,8]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,5,18]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,1,2,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,4,8]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[8,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8,11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14,18,25]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7,13,16,24]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,6,8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[16]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[4,5,15,22]}],"complexes":["C1 microtubule central apparatus complex (SPAG6–PF20–PF6)"],"partners":["PF20","PF6","SPINK2","MYO1D","SLC26A5","TAC1","AKAP4"],"other_free_text":[]},"mechanistic_narrative":"SPAG6 is an armadillo repeat-containing scaffolding protein that organizes the central apparatus of motile cilia and flagella, regulates microtubule acetylation and stability, controls planar cell polarity, and modulates signaling pathways governing proliferation and apoptosis in non-ciliated cells. In motile axonemes, SPAG6 localizes to the C1 microtubule and nucleates a protein interaction network with PF20 and PF6 that is essential for flagellar motility; its loss causes paralyzed flagella with absent C1 microtubules, reduced ciliary beat frequency, randomized basal body orientation, and disrupted planar cell polarity signaling through Vangl2 and FZD6 [PMID:8636214, PMID:25333478, PMID:33175573]. Beyond motile cilia, SPAG6 governs centrosome integrity, primary ciliogenesis, tubulin acetylation, cell migration, and immunological synapse formation in non-ciliated cells including fibroblasts and lymphocytes [PMID:26585507, PMID:27169488]. In hematopoietic and epithelial cancer cells, SPAG6 suppresses PTEN expression and activates PI3K/AKT signaling, modulates TRAIL-mediated and AMPK/mTOR-dependent autophagic death pathways, and promotes MYO1D membrane translocation to activate EGFR/ERK signaling [PMID:29749435, PMID:32537026, PMID:35667090, PMID:40145872]."},"prefetch_data":{"uniprot":{"accession":"O75602","full_name":"Sperm-associated antigen 6","aliases":["Protein PF16 homolog","Repro-SA-1","Sperm flagellar protein"],"length_aa":509,"mass_kda":55.5,"function":"Important for structural integrity of the central apparatus in the sperm tail and for flagellar motility","subcellular_location":"Cytoplasm, cytoskeleton; Cell projection, cilium, flagellum; Cytoplasm, cytoskeleton, cilium axoneme","url":"https://www.uniprot.org/uniprotkb/O75602/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPAG6","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPAG6","total_profiled":1310},"omim":[{"mim_id":"619712","title":"SPERMATOGENIC FAILURE 65; SPGF65","url":"https://www.omim.org/entry/619712"},{"mim_id":"619144","title":"SPERMATOGENIC FAILURE 49; SPGF49","url":"https://www.omim.org/entry/619144"},{"mim_id":"618735","title":"TETRATRICOPEPTIDE REPEAT DOMAIN-CONTAINING PROTEIN 29; TTC29","url":"https://www.omim.org/entry/618735"},{"mim_id":"618661","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 70; CFAP70","url":"https://www.omim.org/entry/618661"},{"mim_id":"618433","title":"SPERMATOGENIC FAILURE 38; SPGF38","url":"https://www.omim.org/entry/618433"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Calyx","reliability":"Supported"},{"location":"Mid piece","reliability":"Supported"},{"location":"Principal piece","reliability":"Supported"},{"location":"End piece","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":94.1},{"tissue":"fallopian tube","ntpm":46.1},{"tissue":"testis","ntpm":90.0}],"url":"https://www.proteinatlas.org/search/SPAG6"},"hgnc":{"alias_symbol":["Repro-SA-1","PF16","CT141","FAP194","CFAP194"],"prev_symbol":[]},"alphafold":{"accession":"O75602","domains":[{"cath_id":"-","chopping":"431-490","consensus_level":"medium","plddt":93.2192,"start":431,"end":490}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75602","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75602-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75602-F1-predicted_aligned_error_v6.png","plddt_mean":95.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPAG6","jax_strain_url":"https://www.jax.org/strain/search?query=SPAG6"},"sequence":{"accession":"O75602","fasta_url":"https://rest.uniprot.org/uniprotkb/O75602.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75602/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75602"}},"corpus_meta":[{"pmid":"8636214","id":"PMC_8636214","title":"PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.","date":"1996","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/8636214","citation_count":151,"is_preprint":false},{"pmid":"28093071","id":"PMC_28093071","title":"SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers.","date":"2017","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28093071","citation_count":96,"is_preprint":false},{"pmid":"12391165","id":"PMC_12391165","title":"A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12391165","citation_count":73,"is_preprint":false},{"pmid":"10493827","id":"PMC_10493827","title":"cDNA cloning and characterization of a human sperm antigen (SPAG6) with homology to the product of the Chlamydomonas PF16 locus.","date":"1999","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10493827","citation_count":67,"is_preprint":false},{"pmid":"15827353","id":"PMC_15827353","title":"Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.","date":"2005","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/15827353","citation_count":62,"is_preprint":false},{"pmid":"10684790","id":"PMC_10684790","title":"Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus.","date":"2000","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/10684790","citation_count":55,"is_preprint":false},{"pmid":"20886115","id":"PMC_20886115","title":"The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20886115","citation_count":53,"is_preprint":false},{"pmid":"25333478","id":"PMC_25333478","title":"Sperm-associated antigen 6 (SPAG6) deficiency and defects in ciliogenesis and cilia function: polarity, density, and beat.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25333478","citation_count":46,"is_preprint":false},{"pmid":"26585507","id":"PMC_26585507","title":"Sperm Associated Antigen 6 (SPAG6) Regulates Fibroblast Cell Growth, Morphology, Migration and Ciliogenesis.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26585507","citation_count":34,"is_preprint":false},{"pmid":"32124190","id":"PMC_32124190","title":"Patients with severe asthenoteratospermia carrying SPAG6 or RSPH3 mutations have a positive pregnancy outcome following intracytoplasmic sperm injection.","date":"2020","source":"Journal 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and XAGE1A Expression in Peripheral Blood May Detect Imminent Relapse in Childhood Acute Myeloid Leukemia.","date":"2021","source":"The Journal of molecular diagnostics : JMD","url":"https://pubmed.ncbi.nlm.nih.gov/34600138","citation_count":4,"is_preprint":false},{"pmid":"37211688","id":"PMC_37211688","title":"SPAG6 c.900 T>C affects boar semen quality and blood-testis barrier function by creating a new splice acceptor site.","date":"2023","source":"Animal genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37211688","citation_count":4,"is_preprint":false},{"pmid":"37169167","id":"PMC_37169167","title":"The Function of Spag6 to Repair Brain Edema Damage After Cerebral Ischemic Stroke-reperfusion.","date":"2023","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/37169167","citation_count":3,"is_preprint":false},{"pmid":"29726644","id":"PMC_29726644","title":"[Expressions of SLC22A14 and SPAG6 proteins in the ejaculated sperm of idiopathic asthenozoospermia patients.].","date":"2017","source":"Zhonghua nan ke xue = National journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/29726644","citation_count":3,"is_preprint":false},{"pmid":"40145872","id":"PMC_40145872","title":"CircMYH9 promotes the mRNA stability of SPAG6 by recruiting EIF4A3 to facilitate the progression of breast cancer.","date":"2025","source":"Epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/40145872","citation_count":2,"is_preprint":false},{"pmid":"40535772","id":"PMC_40535772","title":"SPAG6 Promotes Multiple Myeloma Through Activation of the MAPK/ERK Signaling Pathway.","date":"2025","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40535772","citation_count":2,"is_preprint":false},{"pmid":"38430798","id":"PMC_38430798","title":"Target silencing of porcine SPAG6 and PPP1CC by shRNA attenuated sperm 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localizes specifically to the C1 microtubule of the central apparatus in flagella; loss of PF16 results in paralyzed flagella and missing C1 microtubule in isolated axonemes\",\n      \"method\": \"Insertional mutagenesis, gene cloning, transformation rescue, immunofluorescence, immunogold electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic rescue, immunogold localization, multiple orthogonal methods in a foundational study\",\n      \"pmids\": [\"8636214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human SPAG6 encodes an armadillo repeat-containing protein localizing to the tails of permeabilized human sperm, consistent with a role in the flagellar axoneme central pair\",\n      \"method\": \"cDNA cloning, antibody generation, immunofluorescence localization of permeabilized sperm\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct localization by immunofluorescence, single lab\",\n      \"pmids\": [\"10493827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Murine SPAG6 (Spag6) localizes to the principal piece of mouse sperm tails and co-localizes with microtubules when expressed in COS-1 cells, supporting its role as an axoneme central apparatus component\",\n      \"method\": \"Antipeptide antibody, immunofluorescence in sperm and COS-1 cells\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct localization, single lab, consistent with Chlamydomonas ortholog data\",\n      \"pmids\": [\"10684790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The first armadillo repeat of PF16 is necessary but not sufficient for assembly into the axoneme; the C-terminal 122 amino acids are dispensable; the armadillo repeats act as a single functional unit required for PF16 assembly and flagellar motility rescue\",\n      \"method\": \"Deletion construct generation, transformation rescue assays in pf16 Chlamydomonas mutants\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain deletion mutagenesis with functional rescue readout\",\n      \"pmids\": [\"10913963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SPAG6 (mammalian PF16 orthologue) physically interacts with PF20 (mammalian WD repeat protein); when co-expressed, PF20 colocalizes with SPAG6 on polymerized microtubules; PF20 is markedly reduced in sperm from SPAG6-deficient mice\",\n      \"method\": \"Yeast two-hybrid, co-expression in CHO cells with fluorescence microscopy, immunocytochemistry, western blot of Spag6-null mouse sperm\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — yeast two-hybrid interaction confirmed by co-localization and genetic dependency in knockout mice\",\n      \"pmids\": [\"12391165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SPAG6 physically interacts with the mammalian orthologue of Chlamydomonas PF6, which is localized to the axoneme central apparatus C1 microtubule 1a projection; PF6 fragment that interacts with SPAG6 is absent from SPAG6-deficient mouse sperm; SPAG6, PF6, and PF20 form a network linking central apparatus components\",\n      \"method\": \"Yeast two-hybrid, co-transfection and co-localization in cells, immunocytochemistry, western blot of Spag6-null sperm\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — yeast two-hybrid interaction validated by in-cell co-localization and knockout mouse analysis, multiple orthogonal methods\",\n      \"pmids\": [\"15827353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPAG6 deficiency in mice reduces cilia beat frequency in tracheal epithelial cells, disrupts synchronized cilia beat, reduces cilia density in ependymal and tracheal cells, randomizes basal feet orientation, and disrupts planar cell polarity (Vangl2 distribution), indicating SPAG6 regulates ciliogenesis, axoneme orientation, and epithelial cell polarity\",\n      \"method\": \"Knockout mouse model, high-speed video microscopy, immunofluorescence, electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with multiple specific phenotypic readouts using orthogonal methods\",\n      \"pmids\": [\"25333478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPAG6 silencing in myeloid cell lines (SKM-1, K562) inhibits proliferation and induces apoptosis via activation of caspase-3, -9, and -8, and upregulation of p53 and PTEN\",\n      \"method\": \"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, western blot, xenograft mouse model\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KO/KD with defined pathway readout, single lab\",\n      \"pmids\": [\"25405588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPAG6-deficient mouse embryonic fibroblasts (MEFs) show slower proliferation, larger surface area, reduced motility, reduced adhesion, non-polarized F-actin, multiple centrosomes, reduced primary cilia, and increased sensitivity to paclitaxel; SPAG6 selectively increases acetylated tubulin levels; re-expression rescues abnormal morphology\",\n      \"method\": \"MEF isolation from Spag6-null mice, rescue transfection, wound-healing assay, chemotactic analysis, immunofluorescence, paclitaxel sensitivity assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO primary cells with rescue, multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"26585507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPAG6 overexpression in developing cortical neurons delays neuronal migration rate and decreases neurite number and length, indicating a role in microtubule-dependent neuronal migration\",\n      \"method\": \"In utero electroporation overexpression in mouse cortex\",\n      \"journal\": \"Journal of molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — defined cellular phenotype by in vivo overexpression, single method, single lab\",\n      \"pmids\": [\"26130477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPAG6 is expressed in outer hair cells (OHCs) of the cochlea and co-immunoprecipitates with prestin; SPAG6-deficient mice show reduced prestin expression at both protein and mRNA levels and abnormal OHC morphology\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, western blot, real-time PCR, Spag6-null mouse model\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP interaction plus KO phenotype, single lab\",\n      \"pmids\": [\"25748314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPAG6 is associated with the centrosome in lymphocytes; SPAG6 deficiency impairs immunological synapse formation by disrupting centrosome polarization and actin clearance at the synaptic cleft, leading to defective CTL function and humoral immunity\",\n      \"method\": \"Bone marrow reconstitution, immunofluorescence, functional assays of CTL activity, germinal center analysis in Spag6-null mice\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with direct centrosome localization and defined cellular phenotype, single lab\",\n      \"pmids\": [\"27169488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPAG6 knockdown in SKM-1 cells inhibits proliferation and affects cell cycle G1-to-S transition via upregulation of p27Kip1 and regulation of the AKT/FOXO pathway\",\n      \"method\": \"shRNA knockdown, western blot, FACS cell cycle analysis, RT-qPCR, CCK-8 assay\",\n      \"journal\": \"DNA and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with defined pathway and cell cycle readout, single lab\",\n      \"pmids\": [\"30835546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPAG6 silencing in SKM-1 cells activates the TRAIL signaling pathway and increases interaction between FADD and TRAIL death receptors, promoting apoptosis; high SPAG6 expression suppresses TRAIL-mediated apoptosis without affecting TRAIL death receptor expression\",\n      \"method\": \"shRNA lentiviral knockdown, flow cytometry, western blot, CCK-8 assay\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with specific pathway readout, single lab\",\n      \"pmids\": [\"28393201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPAG6 silencing in SKM-1 cells increases PTEN expression and reduces AKT phosphorylation, leading to apoptosis via Mcl-1 downregulation, cytochrome c release, and increased caspase-9; PTEN activation by SPAG6 knockdown is caspase-independent; SPAG6 knockdown is associated with DNMT1 downregulation, implying SPAG6 controls PTEN via DNA methylation\",\n      \"method\": \"shRNA knockdown, PI3K inhibitor (LY294002), pan-caspase inhibitor (z-VAD-fmk), western blot, flow cytometry, xenograft mouse model\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with pharmacological dissection of pathway, multiple readouts, single lab\",\n      \"pmids\": [\"29749435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPAG6 interacts with SPINK2 (serine protease inhibitor Kazal-type 2) in the acrosome of round spermatids; SPAG6 is required for SPINK2 expression and proper acrosomal localization during spermatogenesis in mice\",\n      \"method\": \"Yeast two-hybrid, co-localization in CHO cells, immunofluorescence in Spag6-KO and wild-type mouse testis\",\n      \"journal\": \"Zhonghua nan ke xue\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — yeast two-hybrid plus co-localization and KO validation, single lab\",\n      \"pmids\": [\"32216237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG6 silencing triggers autophagic cell death in SKM-1 cells via the AMPK/mTOR/ULK1 signaling pathway; SPAG6 knockdown increases p-AMPK and p-ULK1 while decreasing p-mTOR; autophagy inhibition reduces SPAG6 knockdown-mediated apoptosis; AMPK inhibition attenuates this autophagy\",\n      \"method\": \"shRNA knockdown, autophagy inhibitors (chloroquine, 3-MA), AMPK inhibitor (Compound C), western blot, flow cytometry\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with pharmacological dissection, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"32537026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG6 deficiency in Spag6-null mice causes planar cell polarity defects and hearing loss associated with abnormal stereocilia bundles, basal body positioning, disrupted subapical microtubule meshwork, and misdistributed FZD6\",\n      \"method\": \"Spag6-null mice, auditory brainstem response, immunofluorescence, scanning electron microscopy\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with direct structural and PCP protein localization readouts, single lab\",\n      \"pmids\": [\"33175573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG6 overexpression in AML promotes translocation of MYO1D from cytosol to cell membrane, thereby upregulating EGFR family expression and activating PI3K/AKT and ERK signaling; SPAG6 physically interacts with MYO1D as shown by co-immunoprecipitation and mass spectrometry\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, overexpression/knockdown, western blot, xenograft mouse model\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP/MS interaction with functional translocation phenotype, single lab\",\n      \"pmids\": [\"35667090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DNMT3b-mediated hypermethylation of the SPAG6 promoter in lung squamous cell carcinoma leads to SPAG6 downregulation; SPAG6 regulates the JAK/STAT pathway by inhibiting STAT1 and STAT3 transcription\",\n      \"method\": \"Bisulfite sequencing, dual-luciferase assay, KEGG pathway analysis, CCK-8/EdU/Transwell assays, DNMT3b knockdown\",\n      \"journal\": \"American journal of translational research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — dual-luciferase for pathway validation, methylation-expression linkage confirmed in cells\",\n      \"pmids\": [\"36398260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STAT1 protein binds to the SPAG6 promoter and promotes SPAG6 expression; conversely, SPAG6 upregulation correlates with STAT1 expression in MPN, and SPAG6 downregulation reduces STAT1 induction and enhances INF-α sensitivity\",\n      \"method\": \"Chromatin immunoprecipitation, dual-luciferase reporter assay, overexpression/knockdown, flow cytometry, western blot, in vivo xenograft\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and luciferase confirm STAT1 binding to SPAG6 promoter, single lab\",\n      \"pmids\": [\"37681349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TAC1 protein interacts with SPAG6 as determined by yeast two-hybrid; when co-expressed with SPAG6 in CHO cells, TAC1 localizes to microtubules rather than throughout the cell body, showing SPAG6-dependent microtubule targeting of TAC1\",\n      \"method\": \"Yeast two-hybrid, co-transfection and immunofluorescence in CHO and COS-1 cells, western blot\",\n      \"journal\": \"Zhonghua nan ke xue\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid and co-localization only, single lab, no functional follow-up\",\n      \"pmids\": [\"29282908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPAG6 physically interacts with SPINK2 with ~10-fold higher affinity than SPAG6L; SPAG6 modulates testicular AKAP4 and SPAG16L levels; SPAG6 suppresses tubulin acetylation (opposite to SPAG6L which enhances it); compound Spag6/Spag6l knockout causes defective spermiogenesis including abnormal acrosome and fibrous sheath\",\n      \"method\": \"Compound knockout mouse generation, histology, electron microscopy, binding affinity assay, western blot, tubulin acetylation assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — compound KO with structural phenotype, binding affinity measurement, and PTM readout; preprint not yet peer-reviewed\",\n      \"pmids\": [\"40777416\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPAG6 directly interacts with DUSP1 (dual-specificity phosphatase 1) and modulates downstream MAPK/ERK signaling pathway protein expression by regulating DUSP1 activity, promoting MM cell proliferation and migration\",\n      \"method\": \"RNA interference, overexpression, transcriptome sequencing, western blot, proliferation/apoptosis/migration assays\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — interaction inferred from transcriptome data without direct binding assay, single lab\",\n      \"pmids\": [\"40535772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPAG6 overexpression in AML cells promotes GSTP1 expression, which reduces ROS levels and forms a GSTP1/JNK complex that reduces p-JNK, thereby inhibiting JNK-mediated apoptosis and attenuating daunorubicin efficacy\",\n      \"method\": \"Overexpression/knockdown, flow cytometry (ROS, apoptosis), TMT proteomics, western blot, xenograft model\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomics plus functional validation of GSTP1/JNK axis, single lab\",\n      \"pmids\": [\"39508041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CircMYH9 promotes SPAG6 mRNA stability by recruiting EIF4A3; elevated SPAG6 then suppresses PTEN and activates PI3K/AKT signaling to promote breast cancer cell proliferation, migration, and invasion\",\n      \"method\": \"RIP assay (circMYH9–EIF4A3–SPAG6 interaction), mRNA stability assay, overexpression/knockdown, PTEN inhibitor rescue, xenograft model\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — RIP assay establishes mechanistic link; functional rescue with PTEN inhibitor supports pathway placement\",\n      \"pmids\": [\"40145872\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPAG6 is an armadillo repeat-containing scaffolding protein of the axonemal central apparatus C1 microtubule that mediates protein–protein interactions essential for flagellar/ciliary motility (via binding partners PF20, PF6, and SPINK2), regulates microtubule acetylation and stability, controls ciliogenesis and planar cell polarity, and in non-ciliated cells modulates centrosome organization, cell migration, and multiple signaling pathways (PTEN/PI3K/AKT, MAPK/ERK, AMPK/mTOR/ULK1, TRAIL) to influence proliferation and apoptosis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPAG6 is an armadillo repeat-containing scaffolding protein that organizes the central apparatus of motile cilia and flagella, regulates microtubule acetylation and stability, controls planar cell polarity, and modulates signaling pathways governing proliferation and apoptosis in non-ciliated cells. In motile axonemes, SPAG6 localizes to the C1 microtubule and nucleates a protein interaction network with PF20 and PF6 that is essential for flagellar motility; its loss causes paralyzed flagella with absent C1 microtubules, reduced ciliary beat frequency, randomized basal body orientation, and disrupted planar cell polarity signaling through Vangl2 and FZD6 [PMID:8636214, PMID:25333478, PMID:33175573]. Beyond motile cilia, SPAG6 governs centrosome integrity, primary ciliogenesis, tubulin acetylation, cell migration, and immunological synapse formation in non-ciliated cells including fibroblasts and lymphocytes [PMID:26585507, PMID:27169488]. In hematopoietic and epithelial cancer cells, SPAG6 suppresses PTEN expression and activates PI3K/AKT signaling, modulates TRAIL-mediated and AMPK/mTOR-dependent autophagic death pathways, and promotes MYO1D membrane translocation to activate EGFR/ERK signaling [PMID:29749435, PMID:32537026, PMID:35667090, PMID:40145872].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"The fundamental question of what maintains the C1 microtubule of the flagellar central apparatus was resolved by showing that PF16/SPAG6 is an armadillo-repeat protein whose loss abolishes the C1 microtubule and paralyzes flagella, establishing SPAG6 as a structural organizer of the central pair.\",\n      \"evidence\": \"Insertional mutagenesis, gene cloning, transformation rescue, and immunogold EM in Chlamydomonas pf16 mutants\",\n      \"pmids\": [\"8636214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which SPAG6 stabilizes the C1 microtubule was unknown\", \"No binding partners identified\", \"No mammalian validation\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The question of which structural features are required for axonemal assembly was answered by showing the armadillo repeat array functions as a single unit for axoneme incorporation, while confirming mammalian SPAG6 localizes to sperm flagella, bridging the Chlamydomonas genetics to mammalian biology.\",\n      \"evidence\": \"Deletion constructs with rescue assays in pf16 Chlamydomonas; immunofluorescence of human and mouse sperm\",\n      \"pmids\": [\"10913963\", \"10493827\", \"10684790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct interacting partners in the central apparatus still unknown\", \"No mammalian loss-of-function model yet\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The identity of SPAG6's binding partners was established: SPAG6 directly interacts with PF20 and PF6 to form a scaffolding network on the C1 microtubule, and SPAG6 deficiency in mice eliminates these partners from sperm, demonstrating hierarchical assembly dependence.\",\n      \"evidence\": \"Yeast two-hybrid, co-localization in CHO cells, western blot of Spag6-null mouse sperm for both PF20 and PF6\",\n      \"pmids\": [\"12391165\", \"15827353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the SPAG6–PF20–PF6 complex connects to dynein regulation unknown\", \"No motility rescue shown in mammalian system\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether SPAG6 functions beyond sperm flagella was resolved: SPAG6 deficiency reduces ciliary beat frequency, cilia density, and disrupts planar cell polarity (Vangl2 distribution) in tracheal and ependymal epithelia, extending its role to multiciliated somatic cells and tissue-level polarity.\",\n      \"evidence\": \"Spag6-null mice, high-speed video microscopy, immunofluorescence, and electron microscopy of airway and brain cilia\",\n      \"pmids\": [\"25333478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking SPAG6 to PCP pathway components not defined\", \"Whether SPAG6 loss causes hydrocephalus not characterized\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"SPAG6 was shown to have essential functions in non-ciliated cells: it regulates centrosome number, tubulin acetylation, primary cilia formation, cell migration, and paclitaxel sensitivity in MEFs, and modulates neuronal migration in developing cortex.\",\n      \"evidence\": \"Spag6-null MEFs with rescue transfection, wound-healing, chemotactic and drug-sensitivity assays; in utero electroporation overexpression in mouse cortical neurons\",\n      \"pmids\": [\"26585507\", \"26130477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymatic mechanism by which SPAG6 controls tubulin acetylation unknown\", \"Whether SPAG6 directly binds acetyltransferases or deacetylases not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"SPAG6's role at the centrosome in non-ciliated, non-epithelial cells was established: it localizes to the centrosome in lymphocytes and is required for centrosome polarization and immunological synapse formation, revealing a general centrosomal scaffolding function.\",\n      \"evidence\": \"Bone marrow reconstitution with Spag6-null cells, immunofluorescence, CTL killing assays, germinal center analysis\",\n      \"pmids\": [\"27169488\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which centrosomal proteins SPAG6 interacts with in lymphocytes not identified\", \"Whether SPAG6 modulates MTOC reorientation via the same armadillo-repeat interface as in axonemes unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The signaling axis through which SPAG6 promotes survival in leukemic cells was delineated: SPAG6 suppresses PTEN expression (partly via DNMT1-mediated promoter methylation) to maintain PI3K/AKT activity, and independently suppresses TRAIL-mediated death receptor signaling and AMPK/mTOR/ULK1-dependent autophagy.\",\n      \"evidence\": \"shRNA knockdown in SKM-1 cells with PI3K inhibitor, pan-caspase inhibitor, autophagy inhibitors, AMPK inhibitor, western blot, xenograft models\",\n      \"pmids\": [\"29749435\", \"28393201\", \"32537026\", \"25405588\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SPAG6 binds PTEN or DNMT1 directly not shown\", \"All studies from one lab in one cell line (SKM-1)\", \"In vivo relevance for leukemogenesis not confirmed by genetic models\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"SPAG6 deficiency was connected to sensorineural hearing loss: Spag6-null mice show disrupted stereocilia bundles, aberrant basal body positioning, and mislocalized FZD6 in cochlear hair cells, establishing that SPAG6's planar cell polarity function extends to the inner ear.\",\n      \"evidence\": \"Spag6-null mice, auditory brainstem response, scanning EM, immunofluorescence for FZD6\",\n      \"pmids\": [\"33175573\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether hearing loss is purely due to PCP defects or also to OHC prestin reduction (PMID:25748314) not disentangled\", \"No human genetic data linking SPAG6 to deafness\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A new mechanism for SPAG6 in oncogenic signaling was identified: SPAG6 physically interacts with MYO1D and promotes its translocation to the membrane, upregulating EGFR family receptors and activating PI3K/AKT and ERK pathways in AML cells.\",\n      \"evidence\": \"Co-immunoprecipitation, mass spectrometry, overexpression/knockdown, xenograft model in AML cells\",\n      \"pmids\": [\"35667090\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How the armadillo repeat scaffold mediates MYO1D translocation mechanistically unclear\", \"Interaction not validated by reciprocal endogenous co-IP\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The functional divergence between SPAG6 and its paralog SPAG6L was clarified: SPAG6 binds SPINK2 with ~10-fold higher affinity than SPAG6L and suppresses rather than enhances tubulin acetylation; compound knockout reveals non-redundant roles in acrosome biogenesis and fibrous sheath assembly during spermiogenesis.\",\n      \"evidence\": \"Compound Spag6/Spag6l knockout mice, binding affinity assay, tubulin acetylation assay, histology and EM (preprint)\",\n      \"pmids\": [\"40777416\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Structural basis for differential SPINK2 binding affinity not determined\", \"Opposing effects on tubulin acetylation mechanism unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct enzymatic or structural mechanism by which SPAG6 modulates tubulin acetylation, the identity of its centrosomal interaction partners outside motile cilia, and whether SPAG6 mutations cause human disease (infertility, PCD, or deafness) remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No human genetic study linking SPAG6 mutations to a Mendelian disorder\", \"No structural model of SPAG6 or its complexes\", \"Direct mechanism linking SPAG6 to tubulin acetyltransferase/deacetylase regulation not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 2, 4, 8]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 5, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 1, 2, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 4, 8]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 18, 25]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7, 13, 16, 24]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 6, 8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [4, 5, 15, 22]}\n    ],\n    \"complexes\": [\n      \"C1 microtubule central apparatus complex (SPAG6–PF20–PF6)\"\n    ],\n    \"partners\": [\n      \"PF20\",\n      \"PF6\",\n      \"SPINK2\",\n      \"MYO1D\",\n      \"SLC26A5\",\n      \"TAC1\",\n      \"AKAP4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}