{"gene":"SH3RF1","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":1998,"finding":"POSH (SH3RF1) was identified as a direct target of activated Rac GTPase via yeast two-hybrid selection. POSH contains four SH3 domains and its ectopic expression activates the JNK pathway and induces nuclear translocation of NF-κB. Overexpression in fibroblasts induces apoptosis, establishing POSH as a Rac effector scaffold linking Rac to JNK/NF-κB signaling.","method":"Yeast two-hybrid selection, ectopic overexpression with JNK/NF-κB activity assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — foundational discovery paper with yeast two-hybrid identification, replicated by multiple subsequent labs","pmids":["9482736"],"is_preprint":false},{"year":2003,"finding":"POSH acts as a scaffold for a multiprotein JNK apoptotic complex. POSH binds GTP-Rac1 and also binds mixed-lineage kinases (MLKs) both in vivo and in vitro, and complexes with MKK4, MKK7, and JNKs. POSH overexpression promotes apoptotic neuronal death suppressed by dominant-negative MLKs, MKK4/7, and c-Jun; POSH antisense oligonucleotides and siRNA suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal.","method":"Co-immunoprecipitation (in vivo and in vitro binding), dominant-negative epistasis, antisense oligonucleotides, siRNA knockdown in primary neurons","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple dominant-negative epistasis experiments, siRNA knockdown, replicated in subsequent studies","pmids":["12514131"],"is_preprint":false},{"year":2003,"finding":"Akt2 negatively regulates the POSH-MLK-JNK signaling complex. POSH binds Akt2; a POSH W489A mutant unable to bind Akt2 shows enhanced MLK3 binding and increased JNK activation. Inhibition of PI3K/Akt signaling increases MLK3 association with POSH. Akt2 phosphorylates MLK3 within the POSH complex, promoting complex disassembly and JNK pathway downregulation.","method":"Co-immunoprecipitation, mutagenesis (POSH W489A), PI3K/Akt inhibitor treatment, kinase assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis combined with Co-IP and kinase assay, single lab with multiple orthogonal methods","pmids":["14504284"],"is_preprint":false},{"year":2005,"finding":"Human POSH (hPOSH) is localized to the trans-Golgi network and is essential for HIV-1 Gag targeting to the plasma membrane. siRNA silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native but not a RING finger mutant hPOSH restores virus release, indicating that POSH's E3 ubiquitin ligase (RING finger) activity is required for protein sorting at the trans-Golgi network.","method":"siRNA knockdown, RING finger mutant complementation, confocal microscopy, virus release assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with RING mutant rescue, multiple functional readouts in single lab","pmids":["15659549"],"is_preprint":false},{"year":2005,"finding":"Siah1 is a binding partner of POSH and promotes apoptosis by activating the JNK pathway through POSH. Siah1's E3 ligase activity is required for its proapoptotic function. Apoptotic stimuli stabilize Siah1 protein in a JNK pathway-dependent manner requiring interaction with POSH; phosphorylation of Siah1 at tyrosines 100 and 126 enhances this stabilization, forming a positive feedback loop.","method":"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, mutagenesis of Siah1 phosphorylation sites","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, siRNA knockdown with apoptosis readout, multiple methods in single lab","pmids":["16230351"],"is_preprint":false},{"year":2005,"finding":"POSH is an E3 ubiquitin ligase for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) on early endosomes. POSH colocalizes with Hrs on early endosomes via interaction with POSH's two C-terminal SH3 domains. The RING domain of POSH regulates Hrs stability through ubiquitin-proteasomal degradation. JNK1 binding to POSH reduces POSH-catalyzed ubiquitination of Hrs, indicating reciprocal regulation between POSH's scaffold and E3 ligase activities.","method":"Co-immunoprecipitation, confocal colocalization, ubiquitination assay, domain mapping","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, single lab with multiple methods","pmids":["16084064"],"is_preprint":false},{"year":2005,"finding":"POSH knockdown in rat hippocampus is neuroprotective after cerebral ischemia by reducing activation of the MLK3-MKK4-JNK pathway. Co-immunoprecipitation showed enhanced interactions of MLK3, MKK4, and phospho-JNKs with POSH during reperfusion. POSH antisense oligodeoxynucleotides reduced POSH protein, decreased these interactions, attenuated JNK pathway activation, and increased neuronal survival in CA1.","method":"Co-immunoprecipitation, antisense oligodeoxynucleotides (intracerebroventricular infusion), immunohistochemistry, western blot","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus antisense knockdown with functional neuroprotection readout in vivo, single lab","pmids":["16248889"],"is_preprint":false},{"year":2006,"finding":"POSH and JIP scaffold proteins directly associate to form a POSH-JIP apoptotic complex (PJAC) containing all kinase components of the apoptotic JNK pathway (MLKs, MKK4/7, JNKs). This POSH-JIP interaction is required for JNK activation and apoptotic cell death in response to apoptotic stimuli.","method":"Co-immunoprecipitation, direct binding assays, dominant-negative interference, epistasis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding assays combined with functional epistasis, builds on prior replicated scaffold findings","pmids":["16571722"],"is_preprint":false},{"year":2006,"finding":"Nix (a pro-apoptotic BH3-only protein) directly interacts with POSH via yeast two-hybrid and co-immunoprecipitation. Nix promotes apoptosis and JNK/c-Jun activation in a POSH-dependent manner; in cells lacking POSH, Nix does not promote JNK/c-Jun phosphorylation or apoptosis. Nix and POSH mutually stabilize each other.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, loss-of-function (POSH-negative cells), overexpression with JNK/apoptosis readout","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus POSH-null cell epistasis, single lab","pmids":["17095503"],"is_preprint":false},{"year":2006,"finding":"Drosophila POSH forms a complex with ALG-2 and ALIX in a calcium-dependent manner. Overexpression of ALG-2 or ALIX induces ectopic JNK activation, and POSH co-overexpression enhances these phenotypes, suggesting POSH/ALG-2/ALIX function together in regulation of the JNK pathway.","method":"Co-immunoprecipitation, Drosophila genetic overexpression, JNK activation assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP in calcium-dependent manner plus Drosophila genetic epistasis, single lab","pmids":["16698022"],"is_preprint":false},{"year":2007,"finding":"POSH is an E3 ubiquitin ligase that regulates calcium homeostasis through spatial control of Herp. Herp is both a substrate and activator of POSH; Herp-mediated POSH activation requires the Ubl domain and promotes K63-linked polyubiquitination exclusively. Calcium perturbation (thapsigargin) induces POSH-dependent K63 ubiquitination of Herp which is required for Herp relocalization from trans-Golgi network to ER. POSH overexpression attenuates while dominant-negative POSH or siRNA enhances thapsigargin-induced calcium burst.","method":"In vitro ubiquitination assay, confocal microscopy, dominant-negative overexpression, siRNA, K63-linkage-specific ubiquitination assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitination assay, dominant-negative, siRNA, and mutagenesis of lysine residues, multiple orthogonal methods","pmids":["17420289"],"is_preprint":false},{"year":2007,"finding":"POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro. Serine 304 within the Rac-binding domain is a major Akt phosphorylation site. Phosphorylation at S304 reduces POSH binding to activated Rac, and phosphomimetic mutations S304D and S304E reduce both Rac binding and POSH-induced apoptosis.","method":"In vitro kinase assay, site-directed mutagenesis (S304D, S304E), co-immunoprecipitation, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis with functional apoptosis readout, single lab with multiple orthogonal methods","pmids":["17535800"],"is_preprint":false},{"year":2008,"finding":"POSH negatively regulates axon outgrowth via its third SH3 domain interaction with the actomyosin regulatory protein Shroom3. POSH RNAi enhances axon outgrowth; complementation requires the third SH3 domain. Shroom3 recruits Rho kinase to inhibit process outgrowth, and myosin II inhibition reverses the POSH/Shroom3 RNAi phenotype, indicating POSH-Shroom3 inhibit outgrowth through the actin-myosin network.","method":"RNAi knockdown in primary neurons, SH3 domain complementation analysis, dominant-negative approaches, myosin II inhibition","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with domain complementation and pharmacological epistasis, single lab with multiple methods","pmids":["18829867"],"is_preprint":false},{"year":2009,"finding":"POSH acts as an E3 ubiquitin ligase for ROMK1 (Kir1.1) potassium channels, stimulating their ubiquitination and clathrin-independent, dynamin-dependent endocytosis. POSH binds ROMK1 at its N-terminus. POSH overexpression decreases surface expression and potassium currents in a RING-domain-dependent manner; POSH deltaRING mutant abolishes both ubiquitination and current inhibition. In vitro ubiquitination assays confirm POSH E3 activity toward ROMK.","method":"Co-immunoprecipitation, GST pulldown, in vitro ubiquitination assay, biotinylation surface expression assay, electrophysiology, RING domain deletion mutant","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination assay plus electrophysiology plus surface biotinylation plus RING mutant, multiple orthogonal methods","pmids":["19710010"],"is_preprint":false},{"year":2009,"finding":"POSH interacts with ALIX and functions as its E3 ubiquitin ligase in human cells, inducing K63-linked ubiquitination of ALIX on multiple lysine residues in vivo and in vitro. Wild-type POSH but not the RING finger mutant (POSHV14A) enhances ALIX-mediated HIV-1 YPXnL-dependent virus release. This ubiquitination does not destabilize ALIX, suggesting a regulatory function.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, RING finger mutant, virus release assay","journal":"BMC biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay plus RING mutant complementation, single lab","pmids":["19393081"],"is_preprint":false},{"year":2010,"finding":"POSH is required for normal Drosophila embryogenesis and epidermal dorsal closure. In posh null mutants, F-actin accumulation and adherens junction formation are defective during dorsal closure. POSH acts downstream of dTAB2 and upstream of dTAK1 in the TNF (Eiger)-JNK signaling pathway.","method":"Drosophila null mutant analysis, genetic epistasis, F-actin staining, adherens junction imaging","journal":"Journal of genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — null mutant phenotyping with genetic epistasis placement, single lab","pmids":["20933214"],"is_preprint":false},{"year":2011,"finding":"Sh3rf2 (POSHER), a homologue of POSH, promotes proteasomal degradation of POSH through a mechanism requiring the RING domains of both proteins. Sh3rf2 knockdown stabilizes POSH protein, activates JNK signaling, and causes apoptosis of neuronal cells. Apoptotic stimuli rapidly decrease Sh3rf2 levels, thereby stabilizing POSH and activating JNK-driven death.","method":"siRNA knockdown of Sh3rf2 and POSH, proteasome inhibitor assays, dominant-negative RING mutants, western blot for protein stability","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA epistasis between Sh3rf2 and POSH with RING domain requirement, single lab with multiple cell types","pmids":["22128169"],"is_preprint":false},{"year":2011,"finding":"POSH and Siah2 physically interact (confirmed by yeast two-hybrid and co-immunoprecipitation); the RPxAxVxP motif in POSH's spacer region (aa 601-607) is the Siah2 binding site. Both POSH and Siah2 mediate resistance to death-receptor (TRAIL/Fas ligand)-induced caspase-8 activation in prostate cancer cells; POSH protein levels (not solely its E3 activity) are critical for maintaining viability.","method":"siRNA screen, co-immunoprecipitation, yeast two-hybrid domain mapping, caspase-8 activity assay","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with yeast two-hybrid domain mapping and functional siRNA data, single lab","pmids":["21586138"],"is_preprint":false},{"year":2012,"finding":"POSH is concentrated in the proximal cytoplasmic dilation of the leading process (PCDLP) of migratory neocortical neurons and is essential for neuronal migration in vivo. POSH knockdown impairs PCDLP formation, centrosome translocation, and nucleokinesis. POSH colocalizes with F-actin and activated Rac1; its knockdown impairs F-actin assembly and delocalizes activated Rac1. Disruption of Rac1 activity also disrupts F-actin assembly and PCDLP formation.","method":"In utero electroporation knockdown, confocal imaging, F-actin staining, activated Rac1 localization","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in utero knockdown with in vivo neuronal migration phenotype, colocalization and F-actin assays, multiple readouts","pmids":["22959435"],"is_preprint":false},{"year":2013,"finding":"POSH and JIP-1 form a multiprotein scaffold network (POSH/JIP-1) in CD8+ T cells that specifically regulates TCR-mediated JNK1 (not JNK2) activation. Disruption of the POSH/JIP-1 complex impairs JNK1 activation, reduces c-Jun, T-bet, and Eomesodermin induction, and results in defective proliferation, cytokine expression, and tumor control.","method":"Dominant-negative disruption of POSH/JIP-1 complex, immunoblotting for JNK1/2 activation, adoptive transfer tumor models","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative complex disruption with multiple functional T cell readouts, single lab","pmids":["23963642"],"is_preprint":false},{"year":2015,"finding":"Drosophila POSH and TAK1 regulate synaptic growth downstream of Rab8 at the neuromuscular junction. In Rab8 mutant synapses, POSH (as an endosomal JNK scaffold) and TAK1 (a JNK kinase kinase) are required for JNK/AP-1 overactivation that drives synaptic overgrowth. Recycling endosomes serve as a key compartment for POSH-mediated synaptic growth regulation.","method":"Drosophila genetic screen, mutant analysis, genetic epistasis, JNK/AP-1 signaling assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen plus epistasis in Drosophila, single lab","pmids":["25800055"],"is_preprint":false},{"year":2015,"finding":"POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66, acting downstream of the PirB receptor. POSH RNAi in cortical and cerebellar granule neurons releases cells from myelin/Nogo66 inhibition. Leucine zipper kinase (LZK) operates downstream of NogoA/PirB in a POSH-dependent manner; Shroom3 is also a POSH-associated effector in this pathway.","method":"RNAi knockdown in cortical/cerebellar neurons, suppression analysis (LZK epistasis), process outgrowth assays with Nogo66/myelin","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with genetic epistasis in multiple neuron types, single lab","pmids":["20926658"],"is_preprint":false},{"year":2016,"finding":"POSH regulates CD4+ T cell differentiation and survival through a Tak1-dependent mechanism. Disruption of POSH in CD4+ T cells leads to loss of Tak1-dependent JNK1/2 activation, decreased survival, and skewing toward Th2 differentiation. The POSH scaffold complex composition and POSH post-translational modifications differ between CD4+ and CD8+ T cells, indicating cell-type-specific mechanisms.","method":"POSH complex disruption, immunoblotting for JNK1/2 and Tak1, flow cytometry for T cell fate, apoptosis assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional T cell phenotyping with signaling pathway analysis, single lab","pmids":["27084103"],"is_preprint":false},{"year":2017,"finding":"SH3RF1 (POSH) is an E3 ubiquitin ligase that acts as a negative post-translational regulator of FAT1 cadherin protein levels. Identified by yeast two-hybrid screen against FAT1 cytoplasmic tail juxtamembrane region. siRNA-mediated ablation of SH3RF1 increases cellular FAT1 protein levels and stabilizes FAT1 at the cell surface, while SH3RF1 overexpression reduces FAT1 levels.","method":"Yeast two-hybrid, siRNA knockdown, overexpression, cell surface expression assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus siRNA knockdown and overexpression with functional readout, single lab","pmids":["28129444"],"is_preprint":false},{"year":2017,"finding":"POSH participates in epilepsy by increasing surface expression of NMDA receptors. Co-immunoprecipitation demonstrated POSH-NMDAR1 interaction. POSH overexpression increased while knockdown decreased mEPSCs and NMDAR-mediated currents, and altered surface NMDAR1 expression in hippocampus of epileptic mice.","method":"Co-immunoprecipitation, lentiviral knockdown/overexpression, whole-cell patch-clamp electrophysiology, western blot for surface NMDAR1","journal":"Expert opinion on therapeutic targets","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus electrophysiology plus surface expression in vivo mouse model, single lab","pmids":["29057721"],"is_preprint":false},{"year":2018,"finding":"POSH is a key regulator of Hippo signaling in Drosophila through ubiquitin-mediated degradation of Expanded (Ex). POSH binds the C-terminal of Ex and is essential for Crumbs-induced ubiquitination and degradation of Ex. POSH overexpression synergizes with loss of Kibra to induce tissue overgrowth and upregulation of Hippo target genes; POSH knockdown impedes Yorkie-dependent intestinal stem cell renewal.","method":"Genetic epistasis in Drosophila, co-immunoprecipitation, ubiquitination assay, tissue overgrowth/reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Drosophila genetic epistasis plus Co-IP plus ubiquitination assay with multiple genetic contexts, single lab with multiple orthogonal methods","pmids":["29440430"],"is_preprint":false},{"year":2018,"finding":"POSH/SH3RF1 mediates neuropathology in Drosophila and mammalian models of CHMP2BIntron5-associated frontotemporal dementia. CHMP2BIntron5 expression causes aberrant, AKT-dependent accumulation of POSH throughout the nervous system. POSH knockdown is neuroprotective, alleviating neuronal morphology defects, behavioral deficits, premature lethality, dendritic collapse, and reducing elevated JNK/apoptotic markers.","method":"Drosophila and rat primary neuron models, POSH knockdown, AKT inhibition, JNK/apoptotic marker quantification, behavioral assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-species knockdown experiments with multiple readouts, single lab","pmids":["29432529"],"is_preprint":false},{"year":2022,"finding":"POSH directly interacts with PSD-95 and SHANK2/3 at excitatory synapses, assembling the NMDAR/PSD-95/POSH/SHANK complex. In POSH conditional knockout mice, synaptic clustering of the NMDAR/PSD-95/SHANK complex is disrupted, accompanied by abnormal dendritic spine development, impaired glutamatergic transmission, and autism-like behaviors (social deficits, repetitive behaviors, learning/memory impairment).","method":"Co-immunoprecipitation, POSH conditional knockout mice, electrophysiology (glutamatergic transmission), dendritic spine imaging, behavioral assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout mouse with multiple orthogonal readouts (Co-IP, electrophysiology, imaging, behavior), single lab with rigorous controls","pmids":["35385725"],"is_preprint":false},{"year":2023,"finding":"SH3RF1 mediates K63-linked ubiquitination of snakehead vesiculovirus phosphoprotein (P) specifically at lysine 264, facilitating viral replication and maintaining P protein stability. Identified through RNAi screening of cellular E3 ligases; SH3RF1 interacted with and ubiquitinated SHVV-P, and K264R mutation abolished K264 ubiquitination and reduced SHVV replication.","method":"4D label-free ubiquitome analysis, RNAi screening, co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K264R), virus replication assay","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mutagenesis in viral model, single lab; note this is a fish virus model but demonstrates SH3RF1 E3 ligase mechanism","pmids":["37979762"],"is_preprint":false},{"year":2023,"finding":"Bruce (E2 ubiquitin-conjugating enzyme) synergizes with POSH (E3 ligase) to regulate ubiquitination-mediated degradation of Expanded (Ex) in Drosophila Hippo signaling. Bruce acts upstream of Ex but in parallel with canonical Hippo-Warts cascade. Loss of Bruce suppresses POSH-mediated wing overgrowth and Hippo target gene expression.","method":"Drosophila genetic screen, genetic epistasis, ubiquitination assay, tissue overgrowth quantification","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus ubiquitination assay in Drosophila, single lab","pmids":["37699871"],"is_preprint":false},{"year":2026,"finding":"POSH loss reduces SRC postsynaptic enrichment and weakens SRC-NMDAR-PSD-95 complex interactions. NMDAR activation triggers RAC1-GTP-dependent recruitment of POSH to the membrane, leading to NMDAR-induced SRC activation. Prolonged NMDAR activation depletes both POSH and SRC, establishing a negative feedback loop that prevents NMDAR hyperexcitation. POSH acts as a molecular rheostat integrating RAC1-driven membrane targeting with SRC activation.","method":"POSH conditional knockout mice, co-immunoprecipitation, synaptic fractionation, RAC1-GTP pulldown, SRC activity assays, live imaging","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with multiple biochemical assays, single lab, published 2026","pmids":["42058917"],"is_preprint":false},{"year":2025,"finding":"POSH is essential for coordinating JNK, NF-κB, and Akt signaling in CD8+ T cells. POSH conditional knockout (POSHfl/fl CD4-Cre and GzmB-Cre) mice show impaired induction of all three pathways, resulting in reduced differentiation into short-lived effector cells, delayed proliferation, and decreased survival of memory precursor cells during contraction phase.","method":"Conditional T cell POSH knockout reporter mouse models, flow cytometry, immunoblotting, adoptive transfer with VSV-OVA infection","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout mouse with in vivo infection model and multiple signaling pathway readouts, single lab","pmids":["40672960"],"is_preprint":false}],"current_model":"SH3RF1/POSH is a multidomain scaffold protein and RING-finger E3 ubiquitin ligase that directly binds GTP-Rac1 and assembles a pro-apoptotic JNK signaling complex (comprising MLKs, MKK4/7, and JNKs) in cooperation with JIP scaffold proteins; its activity is negatively regulated by Akt-mediated phosphorylation at S304 (reducing Rac1 binding) and by the paralog Sh3rf2/POSHER (which promotes POSH proteasomal degradation), while being positively amplified by Siah1 and Nix interactions; beyond JNK scaffolding, POSH functions as an E3 ligase for multiple substrates (Herp via K63 ubiquitination to regulate ER calcium homeostasis, Hrs on early endosomes, ROMK1 channels for clathrin-independent endocytosis, ALIX, FAT1, and Expanded in the Hippo pathway), localizes to the trans-Golgi network to direct HIV-1 Gag to the plasma membrane, concentrates in the leading process cytoplasmic dilation to localize activated Rac1 and control F-actin assembly for neuronal migration, assembles postsynaptic NMDAR/PSD-95/SHANK complexes at excitatory synapses (with RAC1-dependent membrane recruitment enabling SRC activation and a self-limiting degradation feedback), and acts as a signal integrator in T cells coordinating JNK, NF-κB, and Akt pathways to regulate effector differentiation."},"narrative":{"mechanistic_narrative":"SH3RF1 (POSH) is a multidomain Rac1-effector scaffold and RING-finger E3 ubiquitin ligase that couples Rho-GTPase signaling to JNK-dependent apoptosis, actin remodeling, and substrate ubiquitination across neuronal, immune, and developmental contexts [PMID:9482736, PMID:12514131]. Identified as a direct effector of activated Rac, POSH nucleates a multiprotein JNK apoptotic complex by binding GTP-Rac1 together with mixed-lineage kinases, MKK4/7, and JNKs, an assembly that drives c-Jun phosphorylation and neuronal death [PMID:12514131] and is reinforced through direct association with JIP scaffolds to form the POSH-JIP apoptotic complex [PMID:16571722]. This pro-apoptotic output is gated by the PI3K/Akt axis: Akt phosphorylates POSH at Ser304 within the Rac-binding domain to weaken Rac binding and apoptosis, and phosphorylates MLK3 within the complex to promote its disassembly [PMID:14504284, PMID:17535800]. Complex activity is further tuned by stabilizing partners Siah1 and Nix, which amplify JNK-driven apoptosis in a POSH-dependent manner [PMID:16230351, PMID:17095503], and by the homolog Sh3rf2/POSHER, which directs POSH for proteasomal degradation via a RING-dependent mechanism [PMID:22128169]. Independently of its scaffold role, POSH functions as a RING-dependent E3 ligase whose ubiquitination output regulates membrane and channel trafficking and protein stability: it controls trans-Golgi sorting required for HIV-1 Gag plasma-membrane targeting [PMID:15659549], ubiquitinates Hrs on early endosomes [PMID:16084064], drives endocytosis of ROMK1 potassium channels [PMID:19710010], catalyzes K63-linked ubiquitination of Herp to govern ER calcium homeostasis [PMID:17420289] and of ALIX [PMID:19393081], and degrades the Hippo-pathway component Expanded in Drosophila [PMID:29440430, PMID:37699871]. In the nervous system POSH localizes to the leading-process cytoplasmic dilation to position activated Rac1 and assemble F-actin for neuronal migration [PMID:22959435], and at excitatory synapses it assembles the NMDAR/PSD-95/SHANK complex, with RAC1-GTP-dependent membrane recruitment enabling SRC activation in a self-limiting feedback loop; its loss produces autism-like phenotypes [PMID:35385725, PMID:42058917]. In T cells POSH integrates JNK, NF-κB, and Akt signaling to control effector differentiation and survival [PMID:27084103, PMID:40672960].","teleology":[{"year":1998,"claim":"Established POSH as a direct Rac effector, answering how Rac is physically linked to downstream JNK/NF-κB activation and apoptosis.","evidence":"Yeast two-hybrid against activated Rac plus ectopic overexpression with JNK/NF-κB and apoptosis assays in fibroblasts","pmids":["9482736"],"confidence":"High","gaps":["Did not define the kinase intermediates between POSH and JNK","Overexpression-based apoptosis readout, no endogenous loss-of-function"]},{"year":2003,"claim":"Defined POSH as the physical scaffold assembling the full JNK kinase cascade, explaining how it transduces Rac signals into neuronal apoptosis.","evidence":"Reciprocal Co-IP (in vivo/in vitro), dominant-negative epistasis, antisense and siRNA in primary neurons","pmids":["12514131"],"confidence":"High","gaps":["Did not resolve stoichiometry or order of complex assembly","Mechanism of complex activation by upstream stimuli unclear"]},{"year":2003,"claim":"Showed the POSH-JNK module is negatively controlled by Akt, defining a survival brake on pro-apoptotic signaling.","evidence":"Co-IP, POSH W489A mutant, PI3K/Akt inhibitors, and kinase assays showing Akt2 phosphorylation of MLK3 within the complex","pmids":["14504284"],"confidence":"High","gaps":["Did not identify all Akt phosphosites on the complex","In vivo physiological relevance of disassembly not tested"]},{"year":2005,"claim":"Identified POSH's E3 ligase (RING) activity as essential for trans-Golgi protein sorting, extending its function beyond JNK scaffolding.","evidence":"siRNA knockdown with RING-mutant complementation, confocal microscopy, and HIV-1 Gag/virus release assays","pmids":["15659549"],"confidence":"High","gaps":["Endogenous cellular substrate at the TGN not identified","Link between sorting role and JNK scaffolding unresolved"]},{"year":2005,"claim":"Revealed positive-feedback amplification of POSH-driven apoptosis through Siah1 and through Hrs ubiquitination, refining the regulatory logic of the JNK complex.","evidence":"Yeast two-hybrid, Co-IP, siRNA, mutagenesis, and ubiquitination/domain-mapping assays (Siah1; Hrs on endosomes)","pmids":["16230351","16084064"],"confidence":"Medium","gaps":["Hrs ubiquitination linkage type not defined here","Single-lab findings without reciprocal cross-validation"]},{"year":2006,"claim":"Showed POSH directly partners with JIP scaffolds (PJAC) and with Nix/ALG-2/ALIX, defining the partner network that assembles and tunes the apoptotic JNK complex.","evidence":"Co-IP, direct binding, dominant-negative interference, yeast two-hybrid, and Drosophila genetic epistasis","pmids":["16571722","17095503","16698022"],"confidence":"High","gaps":["Spatial coordination of POSH-JIP assembly not resolved","Calcium-dependence of ALG-2/ALIX interaction mechanistically undefined"]},{"year":2007,"claim":"Established POSH as a K63-linkage E3 ligase coupling its activity to ER calcium homeostasis via Herp, and pinpointed Ser304 as the Akt site that disengages Rac binding.","evidence":"In vitro K63-specific ubiquitination assays, confocal microscopy, dominant-negative/siRNA, and in vitro kinase assay with S304D/S304E phosphomimetics","pmids":["17420289","17535800"],"confidence":"High","gaps":["How Herp both activates and is targeted by POSH not fully mechanized","Cellular triggers determining linkage specificity unknown"]},{"year":2009,"claim":"Extended POSH ubiquitin-ligase function to membrane channel trafficking and to non-degradative regulation, broadening its substrate repertoire.","evidence":"Co-IP, GST pulldown, in vitro ubiquitination, surface biotinylation, electrophysiology, and RING-mutant complementation (ROMK1; ALIX)","pmids":["19710010","19393081"],"confidence":"High","gaps":["Determinants of substrate selection across diverse targets unknown","ALIX ubiquitination consequence beyond virus release unclear"]},{"year":2011,"claim":"Defined POSH protein abundance as a controlled node via Sh3rf2/POSHER-mediated degradation and Siah2 binding, linking POSH levels to apoptotic threshold and cancer cell survival.","evidence":"siRNA epistasis, proteasome inhibition, RING-mutant analysis, yeast two-hybrid domain mapping, caspase-8 assays","pmids":["22128169","21586138"],"confidence":"Medium","gaps":["Whether Sh3rf2 directly ubiquitinates POSH not definitively shown","Scaffold vs. ligase contribution to survival not separated"]},{"year":2012,"claim":"Demonstrated POSH spatially concentrates activated Rac1 and F-actin in the leading process to drive neuronal migration in vivo, linking its Rac-effector role to cytoskeletal control.","evidence":"In utero electroporation knockdown, confocal imaging, F-actin staining, activated-Rac1 localization in migrating neurons","pmids":["22959435"],"confidence":"High","gaps":["Molecular mechanism of F-actin assembly downstream of POSH unresolved","Relationship to JNK scaffolding in migration not dissected"]},{"year":2008,"claim":"Linked POSH to actomyosin-based outgrowth inhibition and to the Nogo66/PirB axis through Shroom3, broadening its neuronal cytoskeletal roles.","evidence":"RNAi with SH3-domain complementation, myosin II inhibition, and LZK/Shroom3 epistasis in cortical/cerebellar neurons","pmids":["18829867","20926658"],"confidence":"Medium","gaps":["Direct biochemical mechanism connecting POSH to Rho-kinase/Shroom3 not defined","Single-lab epistasis without structural detail"]},{"year":2016,"claim":"Established POSH as a cell-type-specific signal integrator in T cells coordinating JNK and Tak1 signaling to control differentiation and survival.","evidence":"POSH/JIP-1 complex disruption, immunoblotting for JNK1/JNK2 and Tak1, flow cytometry, and adoptive transfer tumor models in CD8+ and CD4+ T cells","pmids":["23963642","27084103"],"confidence":"Medium","gaps":["Basis of CD4 vs CD8 complex-composition differences not molecularly resolved","Dominant-negative disruption not equivalent to clean genetic loss"]},{"year":2018,"claim":"Defined POSH as an E3 ligase regulating Hippo signaling through degradation of Expanded, identifying a conserved developmental growth-control role.","evidence":"Drosophila genetic epistasis, Co-IP, ubiquitination assays, and tissue overgrowth/reporter assays (with E2 partner Bruce in a later study)","pmids":["29440430","37699871"],"confidence":"High","gaps":["Conservation of Expanded regulation in mammals not shown","How Crumbs signals to POSH activation undefined"]},{"year":2022,"claim":"Identified POSH as a structural organizer of the postsynaptic NMDAR/PSD-95/SHANK complex required for normal synaptic transmission and behavior.","evidence":"Co-IP, POSH conditional knockout mice, electrophysiology, dendritic spine imaging, and behavioral assays","pmids":["35385725"],"confidence":"High","gaps":["Whether scaffold or ligase activity drives synaptic clustering not separated","Direct binding interfaces to PSD-95/SHANK not mapped"]},{"year":2026,"claim":"Defined POSH as a RAC1-GTP-gated rheostat that recruits to the membrane to activate SRC at NMDAR complexes within a self-limiting feedback loop.","evidence":"POSH conditional knockout mice, Co-IP, synaptic fractionation, RAC1-GTP pulldown, SRC activity assays, and live imaging","pmids":["42058917"],"confidence":"Medium","gaps":["Direct mechanism of SRC activation by POSH not biochemically resolved","How prolonged activation depletes POSH/SRC mechanistically unclear"]},{"year":2025,"claim":"Showed POSH coordinates JNK, NF-κB, and Akt simultaneously in CD8+ T cells to control effector and memory-precursor fate in vivo.","evidence":"POSH conditional knockout (CD4-Cre, GzmB-Cre) mice, flow cytometry, immunoblotting, and VSV-OVA adoptive-transfer infection","pmids":["40672960"],"confidence":"Medium","gaps":["Molecular basis for simultaneous three-pathway integration undefined","Whether E3 activity is required in T cells not tested"]},{"year":null,"claim":"It remains unresolved how POSH's substrate specificity is determined across its many ubiquitination targets and how its scaffold versus E3-ligase activities are partitioned within a given cell.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of POSH multidomain organization or substrate engagement","No unifying mechanism linking spatial localization to choice of substrate or signaling output"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3,10,13,14,25,28]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,10,13,23,28]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,7,27]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[18,12]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[3,10]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5,20]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[18,30]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[18,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,4,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,22,31]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[18,27,30]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,13,23,25]}],"complexes":["POSH-JIP apoptotic complex (PJAC)","NMDAR/PSD-95/POSH/SHANK postsynaptic complex"],"partners":["RAC1","MLK3","MKK4","MKK7","JIP1","AKT2","SIAH1","PSD-95"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z6J0","full_name":"E3 ubiquitin-protein ligase SH3RF1","aliases":["Plenty of SH3s","Protein POSH","RING finger protein 142","RING-type E3 ubiquitin transferase SH3RF1","SH3 domain-containing RING finger protein 1","SH3 multiple domains protein 2"],"length_aa":888,"mass_kda":93.1,"function":"Has E3 ubiquitin-protein ligase activity. In the absence of an external substrate, it can catalyze self-ubiquitination (PubMed:15659549, PubMed:20696164). Stimulates ubiquitination of potassium channel KCNJ1, enhancing its dynamin-dependent and clathrin-independent endocytosis (PubMed:19710010). Acts as a scaffold protein that coordinates with MAPK8IP1/JIP1 in organizing different components of the JNK pathway, including RAC1 or RAC2, MAP3K11/MLK3 or MAP3K7/TAK1, MAP2K7/MKK7, MAPK8/JNK1 and/or MAPK9/JNK2 into a functional multiprotein complex to ensure the effective activation of the JNK signaling pathway. Regulates the differentiation of CD4(+) and CD8(+) T-cells and promotes T-helper 1 (Th1) cell differentiation. Regulates the activation of MAPK8/JNK1 and MAPK9/JNK2 in CD4(+) T-cells and the activation of MAPK8/JNK1 in CD8(+) T-cells. Plays a crucial role in the migration of neocortical neurons in the developing brain. Controls proper cortical neuronal migration and the formation of proximal cytoplasmic dilation in the leading process (PCDLP) in migratory neocortical neurons by regulating the proper localization of activated RAC1 and F-actin assembly (By similarity) (Microbial infection) Plays an essential role in the targeting of HIV-1 Gag to the plasma membrane, this function is dependent on its RING domain, and hence its E3 ligase activity","subcellular_location":"Cytoplasm, perinuclear region; Cell projection, lamellipodium; Golgi apparatus, trans-Golgi network","url":"https://www.uniprot.org/uniprotkb/Q7Z6J0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SH3RF1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SH3RF1","total_profiled":1310},"omim":[{"mim_id":"618933","title":"SH3 DOMAIN-CONTAINING RING FINGER PROTEIN 3; SH3RF3","url":"https://www.omim.org/entry/618933"},{"mim_id":"618642","title":"SH3 DOMAIN-CONTAINING RING FINGER PROTEIN 1; SH3RF1","url":"https://www.omim.org/entry/618642"},{"mim_id":"608070","title":"HOMOCYSTEINE- AND ENDOPLASMIC RETICULUM STRESS-INDUCIBLE PROTEIN, UBIQUITIN-LIKE DOMAIN-CONTAINING, 1; HERPUD1","url":"https://www.omim.org/entry/608070"},{"mim_id":"604641","title":"MITOGEN-ACTIVATED PROTEIN KINASE 8-INTERACTING PROTEIN 1; MAPK8IP1","url":"https://www.omim.org/entry/604641"},{"mim_id":"600976","title":"FAT ATYPICAL CADHERIN 1; FAT1","url":"https://www.omim.org/entry/600976"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Intermediate filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SH3RF1"},"hgnc":{"alias_symbol":["POSH","RNF142","KIAA1494"],"prev_symbol":["SH3MD2"]},"alphafold":{"accession":"Q7Z6J0","domains":[{"cath_id":"3.30.40.10","chopping":"2-79","consensus_level":"medium","plddt":88.3453,"start":2,"end":79},{"cath_id":"2.30.30.40","chopping":"139-193","consensus_level":"high","plddt":92.4018,"start":139,"end":193},{"cath_id":"2.30.30.40","chopping":"201-267","consensus_level":"high","plddt":90.0419,"start":201,"end":267},{"cath_id":"2.30.30.40","chopping":"449-504","consensus_level":"high","plddt":93.0575,"start":449,"end":504},{"cath_id":"2.30.30.40","chopping":"831-888","consensus_level":"medium","plddt":92.4834,"start":831,"end":888}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6J0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z6J0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z6J0-F1-predicted_aligned_error_v6.png","plddt_mean":57.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SH3RF1","jax_strain_url":"https://www.jax.org/strain/search?query=SH3RF1"},"sequence":{"accession":"Q7Z6J0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7Z6J0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7Z6J0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6J0"}},"corpus_meta":[{"pmid":"29337092","id":"PMC_29337092","title":"Germline BRCA mutation and outcome in young-onset breast cancer (POSH): a prospective cohort study.","date":"2018","source":"The Lancet. 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selection","date":"2025-09-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.19.25336150","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25893,"output_tokens":8700,"usd":0.10409,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18364,"output_tokens":5506,"usd":0.114735,"stage2_stop_reason":"end_turn"},"total_usd":0.218825,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"POSH (SH3RF1) was identified as a direct target of activated Rac GTPase via yeast two-hybrid selection. POSH contains four SH3 domains and its ectopic expression activates the JNK pathway and induces nuclear translocation of NF-κB. Overexpression in fibroblasts induces apoptosis, establishing POSH as a Rac effector scaffold linking Rac to JNK/NF-κB signaling.\",\n      \"method\": \"Yeast two-hybrid selection, ectopic overexpression with JNK/NF-κB activity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — foundational discovery paper with yeast two-hybrid identification, replicated by multiple subsequent labs\",\n      \"pmids\": [\"9482736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"POSH acts as a scaffold for a multiprotein JNK apoptotic complex. POSH binds GTP-Rac1 and also binds mixed-lineage kinases (MLKs) both in vivo and in vitro, and complexes with MKK4, MKK7, and JNKs. POSH overexpression promotes apoptotic neuronal death suppressed by dominant-negative MLKs, MKK4/7, and c-Jun; POSH antisense oligonucleotides and siRNA suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal.\",\n      \"method\": \"Co-immunoprecipitation (in vivo and in vitro binding), dominant-negative epistasis, antisense oligonucleotides, siRNA knockdown in primary neurons\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple dominant-negative epistasis experiments, siRNA knockdown, replicated in subsequent studies\",\n      \"pmids\": [\"12514131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Akt2 negatively regulates the POSH-MLK-JNK signaling complex. POSH binds Akt2; a POSH W489A mutant unable to bind Akt2 shows enhanced MLK3 binding and increased JNK activation. Inhibition of PI3K/Akt signaling increases MLK3 association with POSH. Akt2 phosphorylates MLK3 within the POSH complex, promoting complex disassembly and JNK pathway downregulation.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis (POSH W489A), PI3K/Akt inhibitor treatment, kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis combined with Co-IP and kinase assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"14504284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human POSH (hPOSH) is localized to the trans-Golgi network and is essential for HIV-1 Gag targeting to the plasma membrane. siRNA silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native but not a RING finger mutant hPOSH restores virus release, indicating that POSH's E3 ubiquitin ligase (RING finger) activity is required for protein sorting at the trans-Golgi network.\",\n      \"method\": \"siRNA knockdown, RING finger mutant complementation, confocal microscopy, virus release assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with RING mutant rescue, multiple functional readouts in single lab\",\n      \"pmids\": [\"15659549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Siah1 is a binding partner of POSH and promotes apoptosis by activating the JNK pathway through POSH. Siah1's E3 ligase activity is required for its proapoptotic function. Apoptotic stimuli stabilize Siah1 protein in a JNK pathway-dependent manner requiring interaction with POSH; phosphorylation of Siah1 at tyrosines 100 and 126 enhances this stabilization, forming a positive feedback loop.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, mutagenesis of Siah1 phosphorylation sites\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, siRNA knockdown with apoptosis readout, multiple methods in single lab\",\n      \"pmids\": [\"16230351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"POSH is an E3 ubiquitin ligase for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) on early endosomes. POSH colocalizes with Hrs on early endosomes via interaction with POSH's two C-terminal SH3 domains. The RING domain of POSH regulates Hrs stability through ubiquitin-proteasomal degradation. JNK1 binding to POSH reduces POSH-catalyzed ubiquitination of Hrs, indicating reciprocal regulation between POSH's scaffold and E3 ligase activities.\",\n      \"method\": \"Co-immunoprecipitation, confocal colocalization, ubiquitination assay, domain mapping\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, single lab with multiple methods\",\n      \"pmids\": [\"16084064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"POSH knockdown in rat hippocampus is neuroprotective after cerebral ischemia by reducing activation of the MLK3-MKK4-JNK pathway. Co-immunoprecipitation showed enhanced interactions of MLK3, MKK4, and phospho-JNKs with POSH during reperfusion. POSH antisense oligodeoxynucleotides reduced POSH protein, decreased these interactions, attenuated JNK pathway activation, and increased neuronal survival in CA1.\",\n      \"method\": \"Co-immunoprecipitation, antisense oligodeoxynucleotides (intracerebroventricular infusion), immunohistochemistry, western blot\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus antisense knockdown with functional neuroprotection readout in vivo, single lab\",\n      \"pmids\": [\"16248889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"POSH and JIP scaffold proteins directly associate to form a POSH-JIP apoptotic complex (PJAC) containing all kinase components of the apoptotic JNK pathway (MLKs, MKK4/7, JNKs). This POSH-JIP interaction is required for JNK activation and apoptotic cell death in response to apoptotic stimuli.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assays, dominant-negative interference, epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding assays combined with functional epistasis, builds on prior replicated scaffold findings\",\n      \"pmids\": [\"16571722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Nix (a pro-apoptotic BH3-only protein) directly interacts with POSH via yeast two-hybrid and co-immunoprecipitation. Nix promotes apoptosis and JNK/c-Jun activation in a POSH-dependent manner; in cells lacking POSH, Nix does not promote JNK/c-Jun phosphorylation or apoptosis. Nix and POSH mutually stabilize each other.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, loss-of-function (POSH-negative cells), overexpression with JNK/apoptosis readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus POSH-null cell epistasis, single lab\",\n      \"pmids\": [\"17095503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Drosophila POSH forms a complex with ALG-2 and ALIX in a calcium-dependent manner. Overexpression of ALG-2 or ALIX induces ectopic JNK activation, and POSH co-overexpression enhances these phenotypes, suggesting POSH/ALG-2/ALIX function together in regulation of the JNK pathway.\",\n      \"method\": \"Co-immunoprecipitation, Drosophila genetic overexpression, JNK activation assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP in calcium-dependent manner plus Drosophila genetic epistasis, single lab\",\n      \"pmids\": [\"16698022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"POSH is an E3 ubiquitin ligase that regulates calcium homeostasis through spatial control of Herp. Herp is both a substrate and activator of POSH; Herp-mediated POSH activation requires the Ubl domain and promotes K63-linked polyubiquitination exclusively. Calcium perturbation (thapsigargin) induces POSH-dependent K63 ubiquitination of Herp which is required for Herp relocalization from trans-Golgi network to ER. POSH overexpression attenuates while dominant-negative POSH or siRNA enhances thapsigargin-induced calcium burst.\",\n      \"method\": \"In vitro ubiquitination assay, confocal microscopy, dominant-negative overexpression, siRNA, K63-linkage-specific ubiquitination assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitination assay, dominant-negative, siRNA, and mutagenesis of lysine residues, multiple orthogonal methods\",\n      \"pmids\": [\"17420289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro. Serine 304 within the Rac-binding domain is a major Akt phosphorylation site. Phosphorylation at S304 reduces POSH binding to activated Rac, and phosphomimetic mutations S304D and S304E reduce both Rac binding and POSH-induced apoptosis.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (S304D, S304E), co-immunoprecipitation, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis with functional apoptosis readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17535800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"POSH negatively regulates axon outgrowth via its third SH3 domain interaction with the actomyosin regulatory protein Shroom3. POSH RNAi enhances axon outgrowth; complementation requires the third SH3 domain. Shroom3 recruits Rho kinase to inhibit process outgrowth, and myosin II inhibition reverses the POSH/Shroom3 RNAi phenotype, indicating POSH-Shroom3 inhibit outgrowth through the actin-myosin network.\",\n      \"method\": \"RNAi knockdown in primary neurons, SH3 domain complementation analysis, dominant-negative approaches, myosin II inhibition\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with domain complementation and pharmacological epistasis, single lab with multiple methods\",\n      \"pmids\": [\"18829867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"POSH acts as an E3 ubiquitin ligase for ROMK1 (Kir1.1) potassium channels, stimulating their ubiquitination and clathrin-independent, dynamin-dependent endocytosis. POSH binds ROMK1 at its N-terminus. POSH overexpression decreases surface expression and potassium currents in a RING-domain-dependent manner; POSH deltaRING mutant abolishes both ubiquitination and current inhibition. In vitro ubiquitination assays confirm POSH E3 activity toward ROMK.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, in vitro ubiquitination assay, biotinylation surface expression assay, electrophysiology, RING domain deletion mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination assay plus electrophysiology plus surface biotinylation plus RING mutant, multiple orthogonal methods\",\n      \"pmids\": [\"19710010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"POSH interacts with ALIX and functions as its E3 ubiquitin ligase in human cells, inducing K63-linked ubiquitination of ALIX on multiple lysine residues in vivo and in vitro. Wild-type POSH but not the RING finger mutant (POSHV14A) enhances ALIX-mediated HIV-1 YPXnL-dependent virus release. This ubiquitination does not destabilize ALIX, suggesting a regulatory function.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, RING finger mutant, virus release assay\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay plus RING mutant complementation, single lab\",\n      \"pmids\": [\"19393081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"POSH is required for normal Drosophila embryogenesis and epidermal dorsal closure. In posh null mutants, F-actin accumulation and adherens junction formation are defective during dorsal closure. POSH acts downstream of dTAB2 and upstream of dTAK1 in the TNF (Eiger)-JNK signaling pathway.\",\n      \"method\": \"Drosophila null mutant analysis, genetic epistasis, F-actin staining, adherens junction imaging\",\n      \"journal\": \"Journal of genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — null mutant phenotyping with genetic epistasis placement, single lab\",\n      \"pmids\": [\"20933214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sh3rf2 (POSHER), a homologue of POSH, promotes proteasomal degradation of POSH through a mechanism requiring the RING domains of both proteins. Sh3rf2 knockdown stabilizes POSH protein, activates JNK signaling, and causes apoptosis of neuronal cells. Apoptotic stimuli rapidly decrease Sh3rf2 levels, thereby stabilizing POSH and activating JNK-driven death.\",\n      \"method\": \"siRNA knockdown of Sh3rf2 and POSH, proteasome inhibitor assays, dominant-negative RING mutants, western blot for protein stability\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA epistasis between Sh3rf2 and POSH with RING domain requirement, single lab with multiple cell types\",\n      \"pmids\": [\"22128169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"POSH and Siah2 physically interact (confirmed by yeast two-hybrid and co-immunoprecipitation); the RPxAxVxP motif in POSH's spacer region (aa 601-607) is the Siah2 binding site. Both POSH and Siah2 mediate resistance to death-receptor (TRAIL/Fas ligand)-induced caspase-8 activation in prostate cancer cells; POSH protein levels (not solely its E3 activity) are critical for maintaining viability.\",\n      \"method\": \"siRNA screen, co-immunoprecipitation, yeast two-hybrid domain mapping, caspase-8 activity assay\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with yeast two-hybrid domain mapping and functional siRNA data, single lab\",\n      \"pmids\": [\"21586138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"POSH is concentrated in the proximal cytoplasmic dilation of the leading process (PCDLP) of migratory neocortical neurons and is essential for neuronal migration in vivo. POSH knockdown impairs PCDLP formation, centrosome translocation, and nucleokinesis. POSH colocalizes with F-actin and activated Rac1; its knockdown impairs F-actin assembly and delocalizes activated Rac1. Disruption of Rac1 activity also disrupts F-actin assembly and PCDLP formation.\",\n      \"method\": \"In utero electroporation knockdown, confocal imaging, F-actin staining, activated Rac1 localization\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in utero knockdown with in vivo neuronal migration phenotype, colocalization and F-actin assays, multiple readouts\",\n      \"pmids\": [\"22959435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"POSH and JIP-1 form a multiprotein scaffold network (POSH/JIP-1) in CD8+ T cells that specifically regulates TCR-mediated JNK1 (not JNK2) activation. Disruption of the POSH/JIP-1 complex impairs JNK1 activation, reduces c-Jun, T-bet, and Eomesodermin induction, and results in defective proliferation, cytokine expression, and tumor control.\",\n      \"method\": \"Dominant-negative disruption of POSH/JIP-1 complex, immunoblotting for JNK1/2 activation, adoptive transfer tumor models\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative complex disruption with multiple functional T cell readouts, single lab\",\n      \"pmids\": [\"23963642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila POSH and TAK1 regulate synaptic growth downstream of Rab8 at the neuromuscular junction. In Rab8 mutant synapses, POSH (as an endosomal JNK scaffold) and TAK1 (a JNK kinase kinase) are required for JNK/AP-1 overactivation that drives synaptic overgrowth. Recycling endosomes serve as a key compartment for POSH-mediated synaptic growth regulation.\",\n      \"method\": \"Drosophila genetic screen, mutant analysis, genetic epistasis, JNK/AP-1 signaling assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen plus epistasis in Drosophila, single lab\",\n      \"pmids\": [\"25800055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66, acting downstream of the PirB receptor. POSH RNAi in cortical and cerebellar granule neurons releases cells from myelin/Nogo66 inhibition. Leucine zipper kinase (LZK) operates downstream of NogoA/PirB in a POSH-dependent manner; Shroom3 is also a POSH-associated effector in this pathway.\",\n      \"method\": \"RNAi knockdown in cortical/cerebellar neurons, suppression analysis (LZK epistasis), process outgrowth assays with Nogo66/myelin\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with genetic epistasis in multiple neuron types, single lab\",\n      \"pmids\": [\"20926658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"POSH regulates CD4+ T cell differentiation and survival through a Tak1-dependent mechanism. Disruption of POSH in CD4+ T cells leads to loss of Tak1-dependent JNK1/2 activation, decreased survival, and skewing toward Th2 differentiation. The POSH scaffold complex composition and POSH post-translational modifications differ between CD4+ and CD8+ T cells, indicating cell-type-specific mechanisms.\",\n      \"method\": \"POSH complex disruption, immunoblotting for JNK1/2 and Tak1, flow cytometry for T cell fate, apoptosis assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional T cell phenotyping with signaling pathway analysis, single lab\",\n      \"pmids\": [\"27084103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SH3RF1 (POSH) is an E3 ubiquitin ligase that acts as a negative post-translational regulator of FAT1 cadherin protein levels. Identified by yeast two-hybrid screen against FAT1 cytoplasmic tail juxtamembrane region. siRNA-mediated ablation of SH3RF1 increases cellular FAT1 protein levels and stabilizes FAT1 at the cell surface, while SH3RF1 overexpression reduces FAT1 levels.\",\n      \"method\": \"Yeast two-hybrid, siRNA knockdown, overexpression, cell surface expression assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus siRNA knockdown and overexpression with functional readout, single lab\",\n      \"pmids\": [\"28129444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"POSH participates in epilepsy by increasing surface expression of NMDA receptors. Co-immunoprecipitation demonstrated POSH-NMDAR1 interaction. POSH overexpression increased while knockdown decreased mEPSCs and NMDAR-mediated currents, and altered surface NMDAR1 expression in hippocampus of epileptic mice.\",\n      \"method\": \"Co-immunoprecipitation, lentiviral knockdown/overexpression, whole-cell patch-clamp electrophysiology, western blot for surface NMDAR1\",\n      \"journal\": \"Expert opinion on therapeutic targets\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus electrophysiology plus surface expression in vivo mouse model, single lab\",\n      \"pmids\": [\"29057721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"POSH is a key regulator of Hippo signaling in Drosophila through ubiquitin-mediated degradation of Expanded (Ex). POSH binds the C-terminal of Ex and is essential for Crumbs-induced ubiquitination and degradation of Ex. POSH overexpression synergizes with loss of Kibra to induce tissue overgrowth and upregulation of Hippo target genes; POSH knockdown impedes Yorkie-dependent intestinal stem cell renewal.\",\n      \"method\": \"Genetic epistasis in Drosophila, co-immunoprecipitation, ubiquitination assay, tissue overgrowth/reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Drosophila genetic epistasis plus Co-IP plus ubiquitination assay with multiple genetic contexts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29440430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"POSH/SH3RF1 mediates neuropathology in Drosophila and mammalian models of CHMP2BIntron5-associated frontotemporal dementia. CHMP2BIntron5 expression causes aberrant, AKT-dependent accumulation of POSH throughout the nervous system. POSH knockdown is neuroprotective, alleviating neuronal morphology defects, behavioral deficits, premature lethality, dendritic collapse, and reducing elevated JNK/apoptotic markers.\",\n      \"method\": \"Drosophila and rat primary neuron models, POSH knockdown, AKT inhibition, JNK/apoptotic marker quantification, behavioral assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-species knockdown experiments with multiple readouts, single lab\",\n      \"pmids\": [\"29432529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"POSH directly interacts with PSD-95 and SHANK2/3 at excitatory synapses, assembling the NMDAR/PSD-95/POSH/SHANK complex. In POSH conditional knockout mice, synaptic clustering of the NMDAR/PSD-95/SHANK complex is disrupted, accompanied by abnormal dendritic spine development, impaired glutamatergic transmission, and autism-like behaviors (social deficits, repetitive behaviors, learning/memory impairment).\",\n      \"method\": \"Co-immunoprecipitation, POSH conditional knockout mice, electrophysiology (glutamatergic transmission), dendritic spine imaging, behavioral assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout mouse with multiple orthogonal readouts (Co-IP, electrophysiology, imaging, behavior), single lab with rigorous controls\",\n      \"pmids\": [\"35385725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SH3RF1 mediates K63-linked ubiquitination of snakehead vesiculovirus phosphoprotein (P) specifically at lysine 264, facilitating viral replication and maintaining P protein stability. Identified through RNAi screening of cellular E3 ligases; SH3RF1 interacted with and ubiquitinated SHVV-P, and K264R mutation abolished K264 ubiquitination and reduced SHVV replication.\",\n      \"method\": \"4D label-free ubiquitome analysis, RNAi screening, co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K264R), virus replication assay\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mutagenesis in viral model, single lab; note this is a fish virus model but demonstrates SH3RF1 E3 ligase mechanism\",\n      \"pmids\": [\"37979762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bruce (E2 ubiquitin-conjugating enzyme) synergizes with POSH (E3 ligase) to regulate ubiquitination-mediated degradation of Expanded (Ex) in Drosophila Hippo signaling. Bruce acts upstream of Ex but in parallel with canonical Hippo-Warts cascade. Loss of Bruce suppresses POSH-mediated wing overgrowth and Hippo target gene expression.\",\n      \"method\": \"Drosophila genetic screen, genetic epistasis, ubiquitination assay, tissue overgrowth quantification\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus ubiquitination assay in Drosophila, single lab\",\n      \"pmids\": [\"37699871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"POSH loss reduces SRC postsynaptic enrichment and weakens SRC-NMDAR-PSD-95 complex interactions. NMDAR activation triggers RAC1-GTP-dependent recruitment of POSH to the membrane, leading to NMDAR-induced SRC activation. Prolonged NMDAR activation depletes both POSH and SRC, establishing a negative feedback loop that prevents NMDAR hyperexcitation. POSH acts as a molecular rheostat integrating RAC1-driven membrane targeting with SRC activation.\",\n      \"method\": \"POSH conditional knockout mice, co-immunoprecipitation, synaptic fractionation, RAC1-GTP pulldown, SRC activity assays, live imaging\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with multiple biochemical assays, single lab, published 2026\",\n      \"pmids\": [\"42058917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"POSH is essential for coordinating JNK, NF-κB, and Akt signaling in CD8+ T cells. POSH conditional knockout (POSHfl/fl CD4-Cre and GzmB-Cre) mice show impaired induction of all three pathways, resulting in reduced differentiation into short-lived effector cells, delayed proliferation, and decreased survival of memory precursor cells during contraction phase.\",\n      \"method\": \"Conditional T cell POSH knockout reporter mouse models, flow cytometry, immunoblotting, adoptive transfer with VSV-OVA infection\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout mouse with in vivo infection model and multiple signaling pathway readouts, single lab\",\n      \"pmids\": [\"40672960\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SH3RF1/POSH is a multidomain scaffold protein and RING-finger E3 ubiquitin ligase that directly binds GTP-Rac1 and assembles a pro-apoptotic JNK signaling complex (comprising MLKs, MKK4/7, and JNKs) in cooperation with JIP scaffold proteins; its activity is negatively regulated by Akt-mediated phosphorylation at S304 (reducing Rac1 binding) and by the paralog Sh3rf2/POSHER (which promotes POSH proteasomal degradation), while being positively amplified by Siah1 and Nix interactions; beyond JNK scaffolding, POSH functions as an E3 ligase for multiple substrates (Herp via K63 ubiquitination to regulate ER calcium homeostasis, Hrs on early endosomes, ROMK1 channels for clathrin-independent endocytosis, ALIX, FAT1, and Expanded in the Hippo pathway), localizes to the trans-Golgi network to direct HIV-1 Gag to the plasma membrane, concentrates in the leading process cytoplasmic dilation to localize activated Rac1 and control F-actin assembly for neuronal migration, assembles postsynaptic NMDAR/PSD-95/SHANK complexes at excitatory synapses (with RAC1-dependent membrane recruitment enabling SRC activation and a self-limiting degradation feedback), and acts as a signal integrator in T cells coordinating JNK, NF-κB, and Akt pathways to regulate effector differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SH3RF1 (POSH) is a multidomain Rac1-effector scaffold and RING-finger E3 ubiquitin ligase that couples Rho-GTPase signaling to JNK-dependent apoptosis, actin remodeling, and substrate ubiquitination across neuronal, immune, and developmental contexts [#0, #1]. Identified as a direct effector of activated Rac, POSH nucleates a multiprotein JNK apoptotic complex by binding GTP-Rac1 together with mixed-lineage kinases, MKK4/7, and JNKs, an assembly that drives c-Jun phosphorylation and neuronal death [#1] and is reinforced through direct association with JIP scaffolds to form the POSH-JIP apoptotic complex [#7]. This pro-apoptotic output is gated by the PI3K/Akt axis: Akt phosphorylates POSH at Ser304 within the Rac-binding domain to weaken Rac binding and apoptosis, and phosphorylates MLK3 within the complex to promote its disassembly [#2, #11]. Complex activity is further tuned by stabilizing partners Siah1 and Nix, which amplify JNK-driven apoptosis in a POSH-dependent manner [#4, #8], and by the homolog Sh3rf2/POSHER, which directs POSH for proteasomal degradation via a RING-dependent mechanism [#16]. Independently of its scaffold role, POSH functions as a RING-dependent E3 ligase whose ubiquitination output regulates membrane and channel trafficking and protein stability: it controls trans-Golgi sorting required for HIV-1 Gag plasma-membrane targeting [#3], ubiquitinates Hrs on early endosomes [#5], drives endocytosis of ROMK1 potassium channels [#13], catalyzes K63-linked ubiquitination of Herp to govern ER calcium homeostasis [#10] and of ALIX [#14], and degrades the Hippo-pathway component Expanded in Drosophila [#25, #29]. In the nervous system POSH localizes to the leading-process cytoplasmic dilation to position activated Rac1 and assemble F-actin for neuronal migration [#18], and at excitatory synapses it assembles the NMDAR/PSD-95/SHANK complex, with RAC1-GTP-dependent membrane recruitment enabling SRC activation in a self-limiting feedback loop; its loss produces autism-like phenotypes [#27, #30]. In T cells POSH integrates JNK, NF-\\u03baB, and Akt signaling to control effector differentiation and survival [#22, #31].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established POSH as a direct Rac effector, answering how Rac is physically linked to downstream JNK/NF-\\u03baB activation and apoptosis.\",\n      \"evidence\": \"Yeast two-hybrid against activated Rac plus ectopic overexpression with JNK/NF-\\u03baB and apoptosis assays in fibroblasts\",\n      \"pmids\": [\"9482736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the kinase intermediates between POSH and JNK\", \"Overexpression-based apoptosis readout, no endogenous loss-of-function\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined POSH as the physical scaffold assembling the full JNK kinase cascade, explaining how it transduces Rac signals into neuronal apoptosis.\",\n      \"evidence\": \"Reciprocal Co-IP (in vivo/in vitro), dominant-negative epistasis, antisense and siRNA in primary neurons\",\n      \"pmids\": [\"12514131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve stoichiometry or order of complex assembly\", \"Mechanism of complex activation by upstream stimuli unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed the POSH-JNK module is negatively controlled by Akt, defining a survival brake on pro-apoptotic signaling.\",\n      \"evidence\": \"Co-IP, POSH W489A mutant, PI3K/Akt inhibitors, and kinase assays showing Akt2 phosphorylation of MLK3 within the complex\",\n      \"pmids\": [\"14504284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify all Akt phosphosites on the complex\", \"In vivo physiological relevance of disassembly not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified POSH's E3 ligase (RING) activity as essential for trans-Golgi protein sorting, extending its function beyond JNK scaffolding.\",\n      \"evidence\": \"siRNA knockdown with RING-mutant complementation, confocal microscopy, and HIV-1 Gag/virus release assays\",\n      \"pmids\": [\"15659549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous cellular substrate at the TGN not identified\", \"Link between sorting role and JNK scaffolding unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed positive-feedback amplification of POSH-driven apoptosis through Siah1 and through Hrs ubiquitination, refining the regulatory logic of the JNK complex.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, siRNA, mutagenesis, and ubiquitination/domain-mapping assays (Siah1; Hrs on endosomes)\",\n      \"pmids\": [\"16230351\", \"16084064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hrs ubiquitination linkage type not defined here\", \"Single-lab findings without reciprocal cross-validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed POSH directly partners with JIP scaffolds (PJAC) and with Nix/ALG-2/ALIX, defining the partner network that assembles and tunes the apoptotic JNK complex.\",\n      \"evidence\": \"Co-IP, direct binding, dominant-negative interference, yeast two-hybrid, and Drosophila genetic epistasis\",\n      \"pmids\": [\"16571722\", \"17095503\", \"16698022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial coordination of POSH-JIP assembly not resolved\", \"Calcium-dependence of ALG-2/ALIX interaction mechanistically undefined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established POSH as a K63-linkage E3 ligase coupling its activity to ER calcium homeostasis via Herp, and pinpointed Ser304 as the Akt site that disengages Rac binding.\",\n      \"evidence\": \"In vitro K63-specific ubiquitination assays, confocal microscopy, dominant-negative/siRNA, and in vitro kinase assay with S304D/S304E phosphomimetics\",\n      \"pmids\": [\"17420289\", \"17535800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Herp both activates and is targeted by POSH not fully mechanized\", \"Cellular triggers determining linkage specificity unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended POSH ubiquitin-ligase function to membrane channel trafficking and to non-degradative regulation, broadening its substrate repertoire.\",\n      \"evidence\": \"Co-IP, GST pulldown, in vitro ubiquitination, surface biotinylation, electrophysiology, and RING-mutant complementation (ROMK1; ALIX)\",\n      \"pmids\": [\"19710010\", \"19393081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of substrate selection across diverse targets unknown\", \"ALIX ubiquitination consequence beyond virus release unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined POSH protein abundance as a controlled node via Sh3rf2/POSHER-mediated degradation and Siah2 binding, linking POSH levels to apoptotic threshold and cancer cell survival.\",\n      \"evidence\": \"siRNA epistasis, proteasome inhibition, RING-mutant analysis, yeast two-hybrid domain mapping, caspase-8 assays\",\n      \"pmids\": [\"22128169\", \"21586138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Sh3rf2 directly ubiquitinates POSH not definitively shown\", \"Scaffold vs. ligase contribution to survival not separated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated POSH spatially concentrates activated Rac1 and F-actin in the leading process to drive neuronal migration in vivo, linking its Rac-effector role to cytoskeletal control.\",\n      \"evidence\": \"In utero electroporation knockdown, confocal imaging, F-actin staining, activated-Rac1 localization in migrating neurons\",\n      \"pmids\": [\"22959435\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of F-actin assembly downstream of POSH unresolved\", \"Relationship to JNK scaffolding in migration not dissected\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked POSH to actomyosin-based outgrowth inhibition and to the Nogo66/PirB axis through Shroom3, broadening its neuronal cytoskeletal roles.\",\n      \"evidence\": \"RNAi with SH3-domain complementation, myosin II inhibition, and LZK/Shroom3 epistasis in cortical/cerebellar neurons\",\n      \"pmids\": [\"18829867\", \"20926658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical mechanism connecting POSH to Rho-kinase/Shroom3 not defined\", \"Single-lab epistasis without structural detail\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established POSH as a cell-type-specific signal integrator in T cells coordinating JNK and Tak1 signaling to control differentiation and survival.\",\n      \"evidence\": \"POSH/JIP-1 complex disruption, immunoblotting for JNK1/JNK2 and Tak1, flow cytometry, and adoptive transfer tumor models in CD8+ and CD4+ T cells\",\n      \"pmids\": [\"23963642\", \"27084103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basis of CD4 vs CD8 complex-composition differences not molecularly resolved\", \"Dominant-negative disruption not equivalent to clean genetic loss\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined POSH as an E3 ligase regulating Hippo signaling through degradation of Expanded, identifying a conserved developmental growth-control role.\",\n      \"evidence\": \"Drosophila genetic epistasis, Co-IP, ubiquitination assays, and tissue overgrowth/reporter assays (with E2 partner Bruce in a later study)\",\n      \"pmids\": [\"29440430\", \"37699871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conservation of Expanded regulation in mammals not shown\", \"How Crumbs signals to POSH activation undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified POSH as a structural organizer of the postsynaptic NMDAR/PSD-95/SHANK complex required for normal synaptic transmission and behavior.\",\n      \"evidence\": \"Co-IP, POSH conditional knockout mice, electrophysiology, dendritic spine imaging, and behavioral assays\",\n      \"pmids\": [\"35385725\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether scaffold or ligase activity drives synaptic clustering not separated\", \"Direct binding interfaces to PSD-95/SHANK not mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined POSH as a RAC1-GTP-gated rheostat that recruits to the membrane to activate SRC at NMDAR complexes within a self-limiting feedback loop.\",\n      \"evidence\": \"POSH conditional knockout mice, Co-IP, synaptic fractionation, RAC1-GTP pulldown, SRC activity assays, and live imaging\",\n      \"pmids\": [\"42058917\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism of SRC activation by POSH not biochemically resolved\", \"How prolonged activation depletes POSH/SRC mechanistically unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed POSH coordinates JNK, NF-\\u03baB, and Akt simultaneously in CD8+ T cells to control effector and memory-precursor fate in vivo.\",\n      \"evidence\": \"POSH conditional knockout (CD4-Cre, GzmB-Cre) mice, flow cytometry, immunoblotting, and VSV-OVA adoptive-transfer infection\",\n      \"pmids\": [\"40672960\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis for simultaneous three-pathway integration undefined\", \"Whether E3 activity is required in T cells not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how POSH's substrate specificity is determined across its many ubiquitination targets and how its scaffold versus E3-ligase activities are partitioned within a given cell.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of POSH multidomain organization or substrate engagement\", \"No unifying mechanism linking spatial localization to choice of substrate or signaling output\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3, 10, 13, 14, 25, 28]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 10, 13, 23, 28]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 7, 27]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [18, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [3, 10]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 20]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [18, 30]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [18, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 4, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 22, 31]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [18, 27, 30]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 13, 23, 25]}\n    ],\n    \"complexes\": [\n      \"POSH-JIP apoptotic complex (PJAC)\",\n      \"NMDAR/PSD-95/POSH/SHANK postsynaptic complex\"\n    ],\n    \"partners\": [\n      \"RAC1\",\n      \"MLK3\",\n      \"MKK4\",\n      \"MKK7\",\n      \"JIP1\",\n      \"AKT2\",\n      \"SIAH1\",\n      \"PSD-95\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}