Affinage

SPAG4

Sperm-associated antigen 4 protein · UniProt Q9NPE6

Length
437 aa
Mass
48.2 kDa
Annotated
2026-06-10
18 papers in source corpus 14 papers cited in narrative 13 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SPAG4 (SUN4) is a testis-enriched SUN-domain protein of the inner nuclear membrane that builds LINC complexes coupling the spermatid nuclear periphery to the cytoskeletal machinery of sperm head morphogenesis (PMID:26417726, PMID:36825599). It adopts classical SUN-protein topology, projecting its C-terminal SUN domain into the perinuclear space while its N-terminus faces the nucleoplasm, where it engages the spermiogenesis-specific lamin B3, and it forms heteromeric assemblies with SUN3 whose expression it also regulates (PMID:36825599). Within germ cells SPAG4 anchors a nuclear-envelope complex with SEPT12 and lamin, partners with SUN3/Nesprin1 LINC components at the posterior nuclear envelope, and binds the outer dense fiber protein Odf1 through a leucine zipper, while also self-associating (PMID:10373309, PMID:9691178, PMID:26621829, PMID:25775403). Loss of SPAG4 disorganizes the manchette, prevents nuclear elongation, and produces globozoospermia and infertility, and it is required for tight lateral anchorage of the sperm head-to-tail coupling apparatus rather than its initial assembly (PMID:26417726, PMID:26621829, PMID:29663073); the Drosophila ortholog performs an analogous role attaching the centriole/basal body to the spermatid nucleus via a Yuri Gagarin/dynein-dynactin pathway independent of KASH proteins (PMID:20647369). Beyond the germline, SPAG4 is reactivated in cancers, where hypoxia drives its expression through a HIF-1/VHL-dependent mechanism and it promotes migration and invasion (PMID:23818324); mechanistically it activates PI3K/Akt/mTOR signaling with elevated HIF-1α to drive aerobic glycolysis, engages a c-MYC/SULT2B1 axis to sustain glycolytic metabolism, and activates MAPK/ERK signaling to drive EMT and drug resistance (PMID:39410830, PMID:40287810, PMID:42126986). An oncogenic SPAG4-L splice isoform, generated when the tRF-3005a/RALY axis suppresses exon 8 skipping, activates GRB14/PI3K/AKT signaling to promote tumor progression (PMID:41872130).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 1999 High

    Established the first molecular partner of SPAG4, linking it physically to the sperm cytoskeleton and outer dense fibers and defining the leucine zipper as the binding interface.

    Evidence Yeast two-hybrid, co-IP, leucine zipper mutagenesis and immunofluorescence in spermatids

    PMID:10373309 PMID:9691178

    Open questions at the time
    • Did not define nuclear membrane topology
    • Functional consequence of the Odf1 interaction in vivo not tested
  2. 2010 Medium

    Showed the SUN ortholog mediates centriole/basal body attachment to the spermatid nucleus, placing it upstream of dynein-dynactin and Yuri Gagarin and outside the KASH-protein axis.

    Evidence Drosophila genetic mutant, epistasis/double-mutant analysis and immunolocalization

    PMID:20647369

    Open questions at the time
    • Ortholog-based; mammalian centriole-attachment role not directly tested
    • Direct binding partners at the nucleus not identified
  3. 2013 Medium

    Revealed cancer reactivation of SPAG4 and placed its expression under hypoxic HIF-1/VHL control while demonstrating a pro-invasive role.

    Evidence siRNA knockdown, overexpression, invasion/scratch assays in renal clear cell carcinoma

    PMID:23818324

    Open questions at the time
    • Downstream effector pathway not defined
    • Direct HIF-1 binding to SPAG4 regulatory region not mapped
  4. 2015 High

    Defined SPAG4 as an inner nuclear membrane LINC component essential for manchette-to-nucleus coupling and nuclear elongation, with a knockout phenotype of globozoospermia.

    Evidence Two independent knockout mouse models with immunofluorescence and LINC-component mislocalization analysis

    PMID:26417726 PMID:26621829

    Open questions at the time
    • Did not resolve membrane topology of the protein
    • Order of LINC assembly relative to SPAG4 not established
  5. 2015 Medium

    Identified a germ-cell nuclear-envelope complex of SPAG4 with SEPT12 and lamin, linking it to a human infertility mutation.

    Evidence Yeast two-hybrid, reciprocal co-IP and co-localization in male germ cells

    PMID:25775403

    Open questions at the time
    • Single lab; reciprocal validation across systems limited
    • Stoichiometry and direct vs indirect contacts within the complex unresolved
  6. 2018 Medium

    Distinguished SPAG4's role as anchoring rather than building the head-to-tail coupling apparatus, and extended its interactome to Nesprin3 in a cancer migration context.

    Evidence KO mouse EM ultrastructure; co-IP and BiFC with migration assays in lung cancer cells

    PMID:29663073 PMID:29901114

    Open questions at the time
    • Molecular basis of basal-plate anchorage not defined
    • Whether Nesprin3 interaction reflects germline LINC biology untested
  7. 2023 High

    Determined SPAG4's classical SUN topology and its nucleoplasmic engagement of lamin B3 plus SUN3 heteromer formation, providing the structural logic for LINC bridging.

    Evidence Protease protection/membrane fractionation topology, co-IP, immunofluorescence and Western blot

    PMID:36825599

    Open questions at the time
    • High-resolution structure of the SUN domain assembly not solved
    • Direct KASH-domain partner spanning the perinuclear space not identified here
  8. 2024 Medium

    Connected SPAG4 to oncogenic metabolic reprogramming via PI3K/Akt/mTOR activation and HIF-1α elevation driving aerobic glycolysis.

    Evidence Gain/loss-of-function, extracellular flux analysis and PI3K pharmacological rescue in colorectal cancer cells

    PMID:39410830

    Open questions at the time
    • How a SUN-domain INM protein activates cytoplasmic PI3K is unexplained
    • Direct molecular link between SPAG4 and pathway components not shown
  9. 2025 Medium

    Defined a c-MYC/SULT2B1 transcriptional-metabolic axis through which SPAG4 sustains glycolysis and proliferation.

    Evidence Knockdown/overexpression with SULT2B1 rescue and multiple metabolic readouts in HT29 cells

    PMID:40287810

    Open questions at the time
    • Mechanism by which SPAG4 regulates c-MYC not established
    • Single cell-line context
  10. 2026 Medium

    Showed SPAG4 drives EMT and lenvatinib resistance via MAPK/ERK signaling under DEHP exposure, and that an alternatively spliced SPAG4-L isoform generated by the tRF-3005a/RALY axis activates GRB14/PI3K/AKT to promote tumor progression.

    Evidence Genetic/pharmacological rescue with EMT and invasion readouts; RNA-seq, RNA-protein interaction and splicing analysis with co-IP

    PMID:41872130 PMID:42126986

    Open questions at the time
    • Isoform-specific functions of SPAG4-L vs canonical SPAG4 incompletely separated
    • Direct RALY-SPAG4 mRNA binding site not mapped

Open questions

Synthesis pass · forward-looking unresolved questions
  • How a single inner-nuclear-membrane LINC protein mechanistically activates cytoplasmic PI3K/Akt and MAPK/ERK signaling cascades in cancer remains unresolved.
  • No direct molecular bridge between SPAG4 and PI3K/MAPK components identified
  • Relationship between germline LINC function and oncogenic signaling unknown
  • No structure of SPAG4-containing complexes

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0005198 structural molecule activity 3 GO:0060089 molecular transducer activity 3 GO:0008092 cytoskeletal protein binding 2
Localization
GO:0005635 nuclear envelope 3 GO:0005856 cytoskeleton 1
Pathway
R-HSA-1474165 Reproduction 3 R-HSA-162582 Signal Transduction 3 R-HSA-1430728 Metabolism 2 R-HSA-1643685 Disease 2
Partners
LMNB3NESP1NESP3ODF1RALYSEPT12SUN3
Complex memberships
LINC complex

Evidence

Reading pass · 13 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 SPAG4 (Spag4) protein specifically interacts with the outer dense fiber protein Odf1 (ODF27), but not Odf2, via a leucine zipper motif. SPAG4 also self-associates. The protein localizes to microtubules of the manchette and axoneme in elongating spermatids and epididymal sperm. Yeast two-hybrid interaction screen, co-immunoprecipitation, leucine zipper mutagenesis, immunofluorescence localization Developmental biology High 10373309 9691178
2015 SUN4/SPAG4 is an inner nuclear membrane protein essential for coupling the manchette to the nuclear periphery during spermiogenesis. In SUN4-null mice, manchette microtubules are highly disorganized, the nucleus fails to elongate, and mice display globozoospermia and infertility. Knockout mouse model, immunofluorescence, nuclear morphology analysis Developmental biology High 26417726
2015 Sun4/SPAG4 localizes to the posterior nuclear envelope of spermatids, likely interacting with Sun3/Nesprin1 LINC components. Sun4 deficiency causes mislocalization of other LINC components and failure of manchette formation, resulting in a globozoospermia-like phenotype. Knockout mouse model, immunofluorescence, co-localization studies Biology open High 26621829
2015 SPAG4 (SUN4) interacts with SEPT12 in male germ cells, and SEPT12/SPAG4/LAMINB1 form a complex at the nuclear periphery of round spermatids. A SEPT12 mutation from an infertile man disrupts the integration of this nuclear envelope complex. Yeast two-hybrid, co-immunoprecipitation, co-localization by immunofluorescence PloS one Medium 25775403
2018 SPAG4-deficient sperm develop the head-to-tail coupling apparatus (HTCA) structurally, but the lateral attachment of the basal plate to the implantation fossa is diminished, indicating SPAG4 is required for tight anchorage of the sperm head-to-tail linkage rather than HTCA formation per se. Knockout mouse model, electron microscopy ultrastructural analysis Histochemistry and cell biology Medium 29663073
2010 Drosophila Spag4 (SUN protein ortholog) is required for centriole/basal body attachment to the spermatid nucleus. In spag4 mutants, nuclei and centrioles dissociate after meiosis. Epistasis studies placed Yuri Gagarin and dynein-dynactin activities downstream of spag4 in this centriole attachment pathway. This function does not involve either Drosophila KASH protein (Klarsicht or MSP-300). Genetic mutant analysis, immunofluorescence localization, epistasis/double-mutant analysis Journal of cell science Medium 20647369
2023 SUN4/SPAG4 is an inner nuclear membrane protein with a classical SUN domain protein topology: the C-terminal SUN domain localizes to the perinuclear space and the N-terminus is directed to the nucleoplasm where it interacts with the spermiogenesis-specific lamin B3. SUN4 also forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Topology analysis by protease protection/membrane fractionation, co-immunoprecipitation, immunofluorescence, Western blot Journal of cell science High 36825599
2018 SPAG4 interacts with Nesprin3 in lung cancer cells, as determined by co-immunoprecipitation and bimolecular fluorescence complementation. SPAG4 knockdown reduces migration of A549 lung cancer cells, and SPAG4 acts as a positive regulator of Nesprin3 localization and expression. Silencing Nesprin3 also reduces cell migration. Co-immunoprecipitation, bimolecular fluorescence complementation (BiFC), RNAi knockdown, scratch migration assay, immunofluorescence Oncology reports Medium 29901114
2013 SPAG4 expression is regulated by hypoxia via a HIF-1 and VHL-dependent mechanism. SPAG4 knockdown reduces invasion capability of renal clear cell carcinoma (RCC) cells in vitro, while overexpression enhances tumor cell migration. siRNA knockdown, transient overexpression, scratch assay, invasion assay, immunofluorescence, RNase protection assay Molecular carcinogenesis Medium 23818324
2024 SPAG4 overexpression activates the PI3K/Akt/mTOR signaling pathway and elevates HIF-1α in colorectal cancer cells, promoting mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PI3K (LY294002) reverses SPAG4 overexpression-induced effects on proliferation, migration, invasion, and glycolysis. siRNA knockdown, overexpression, XF24 extracellular flux analysis, CCK-8, Transwell assay, Western blot, pharmacological inhibition Journal of biochemical and molecular toxicology Medium 39410830
2025 SPAG4 promotes glycolytic metabolism in HT29 colorectal cancer cells through a c-MYC/SULT2B1 axis: SPAG4 knockdown reduces c-MYC expression, which decreases SULT2B1 expression, suppressing glycolysis and cell proliferation. Elevated SULT2B1 counteracts the glycolytic reduction induced by SPAG4 or c-MYC silencing. siRNA knockdown, overexpression, CCK-8, colony formation, glucose uptake, lactate production, ATP/ADP ratio, Western blot, qRT-PCR, 2-DG rescue Discovery medicine Medium 40287810
2026 DEHP-induced upregulation of SPAG4 activates the MAPK/ERK signaling pathway, driving epithelial-mesenchymal transition (EMT) and reduced lenvatinib responsiveness in hepatocellular carcinoma cells. Genetic silencing of SPAG4 or pharmacological MAPK/ERK inhibition reverses DEHP-induced EMT and drug resistance. siRNA knockdown, overexpression, pharmacological inhibition, EMT marker analysis, migration/invasion assays, Western blot Chemical research in toxicology Medium 42126986
2026 tRF-3005a binds RALY, enhancing its interaction with SPAG4 mRNA and suppressing exon 8 skipping of SPAG4, leading to increased production of the oncogenic SPAG4-L isoform that activates GRB14/PI3K/AKT signaling to promote gastric cancer progression. RNA sequencing, RNA-protein interaction assays, alternative splicing analysis, co-immunoprecipitation, Western blot, functional cell assays Cell death discovery Medium 41872130

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1999 Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. Developmental biology 134 10373309
2015 SUN4 is essential for nuclear remodeling during mammalian spermiogenesis. Developmental biology 63 26417726
2015 The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility. Biology open 60 26621829
2010 The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus. Journal of cell science 57 20647369
2000 The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation. Yeast (Chichester, England) 50 10870102
2015 SEPT12/SPAG4/LAMINB1 complexes are required for maintaining the integrity of the nuclear envelope in postmeiotic male germ cells. PloS one 48 25775403
2018 Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4. Histochemistry and cell biology 32 29663073
2018 Sperm‑associated antigen 4 (SPAG4) as a new cancer marker interacts with Nesprin3 to regulate cell migration in lung carcinoma. Oncology reports 29 29901114
2003 Human sperm associated antigen 4 (SPAG4) is a potential cancer marker. Cell and tissue research 29 14614621
2013 Hypoxia regulates the sperm associated antigen 4 (SPAG4) via HIF, which is expressed in renal clear cell carcinoma and promotes migration and invasion in vitro. Molecular carcinogenesis 26 23818324
1998 A novel testis-specific gene, SPAG4, whose product interacts specifically with outer dense fiber protein ODF27, maps to human chromosome 20q11.2. Cytogenetics and cell genetics 22 9691178
2023 SUN4 is a spermatid type II inner nuclear membrane protein that forms heteromeric assemblies with SUN3 and interacts with lamin B3. Journal of cell science 9 36825599
2024 SPAG4 enhances mitochondrial respiration and aerobic glycolysis in colorectal cancer cells by activating the PI3K/Akt signaling pathway. Journal of biochemical and molecular toxicology 4 39410830
2022 The impact of glutamine deprivation on the expression of MEIS3, SPAG4, LHX1, LHX2, and LHX6 genes in ERN1 knockdown U87 glioma cells. Endocrine regulations 4 35180817
2024 Integrated multi-level omics profiling of disulfidptosis identifis SPAG4 as an innovative immunotherapeutic target in glioblastoma. Frontiers in immunology 1 39539547
2026 tRF-3005a regulates exon skipping of SPAG4 by interacting with RALY to drive gastric cancer progression. Cell death discovery 0 41872130
2026 Di-2-ethylhexyl Phthalate Reprograms Epithelial-Mesenchymal Transition and Lenvatinib Responsiveness in Hepatoma Cells via SPAG4-Dependent MAPK/ERK Signaling. Chemical research in toxicology 0 42126986
2025 SPAG4 Regulates Glycolytic Metabolism in HT29 Cells as a Target via the c-MYC/SULT2B1 Pathway. Discovery medicine 0 40287810

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