{"gene":"SSH2","run_date":"2026-06-10T07:46:41","timeline":{"discoveries":[{"year":2007,"finding":"SSH2 (Slingshot-2) functions as a cofilin/ADF phosphatase that dephosphorylates and reactivates cofilin, and double knockdown of SSH1/SSH2 by RNAi significantly suppressed NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, establishing SSH2 as a critical activating phosphatase for cofilin-dependent neurite extension.","method":"RNAi knockdown, phosphorylation assays, morphological readout of neurite extension in PC12 cells and chick DRG neurons","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal loss-of-function with direct biochemical readout (cofilin dephosphorylation) and cellular phenotype (neurite extension), replicated across two cell systems","pmids":["17360713"],"is_preprint":false},{"year":2007,"finding":"SSH family phosphatases (SSH1, SSH2, and SSH3), but not the phosphatase CIN, are required for cofilin activation downstream of alpha6beta4 integrin/Rac1 signaling in human keratinocytes; phosphatase-dead SSH constructs cause cofilin hyperphosphorylation/inactivation, loss of cell polarity, and aberrant laminin-332 assembly. SSH activity is regulated by 14-3-3 protein binding, and inhibition of Rac1 increases 14-3-3 association with SSH, placing SSH2 in an alpha6beta4/Rac1/14-3-3/SSH/cofilin pathway controlling keratinocyte migration.","method":"Expression of phosphatase-dead SSH constructs, dominant-negative Rac1, Co-IP of 14-3-3 with SSH, wound-healing migration assays, immunofluorescence of actin and laminin-332","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — phosphatase-dead mutants, Co-IP of 14-3-3/SSH, and multiple cellular phenotypic readouts in a single study","pmids":["17848544"],"is_preprint":false},{"year":2008,"finding":"SSH1/SSH2 dephosphorylate and reactivate cofilin/ADF in rat ascites hepatoma (MM1) cells; siRNA knockdown of SSH1/SSH2 significantly decreased LPA-induced transcellular migration and 2D motility of MM1 cells, demonstrating that SSH2-mediated cofilin dephosphorylation is required for tumor cell invasion.","method":"siRNA knockdown, in vitro transcellular migration assay through mesothelial monolayer, 2D motility assay, phosphorylation state analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with direct biochemical (cofilin phosphorylation) and functional (invasion/migration) readouts, single lab, two orthogonal assays","pmids":["18171679"],"is_preprint":false},{"year":2011,"finding":"In vascular smooth muscle cells, SSH2 siRNA attenuated cofilin dephosphorylation (activation), but unlike SSH1 siRNA, SSH2 knockdown alone did not significantly reduce wound healing or PDGF-induced VSMC migration, indicating SSH2 contributes to cofilin activation but is not the dominant isoform controlling VSMC migration.","method":"siRNA knockdown (SSH1, SSH2, SSH3), scratch wound assay, Boyden chamber migration assay, cofilin phosphorylation western blot","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isoform-specific siRNA with biochemical and functional readouts, single lab; finding is a partial/negative result for SSH2 specifically","pmids":["21868701"],"is_preprint":false},{"year":2013,"finding":"SSH2 knockdown specifically induced Caspase3/7 activation (up to 6-fold) and cell cycle arrest (increased G1, decreased S and G2/M) in FLCN-deficient (BHD) human carcinoma cell lines but not in isogenic FLCN-expressing lines, revealing a synthetic lethal interaction between SSH2 and FLCN loss and implicating SSH2 in cell survival of FLCN-null cells via dysregulated cofilin de/phosphorylation.","method":"siRNA phosphatase library screen, Caspase3/7 activity assay, cell cycle analysis, western blot for cofilin phosphorylation and SSH isoform levels in isogenic FLCN+/− cell pairs","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA screen validated with dose-response, multiple readouts (caspase, cell cycle, biochemical), isogenic cell lines, single lab","pmids":["23416984"],"is_preprint":false},{"year":2015,"finding":"In neutrophil chemotaxis, GPCR activation signals through Gαi → PLCγ2 (PI3K-dependent) → PKCβ → PKD1 to phosphorylate and regulate SSH2, thereby controlling cofilin phosphorylation and actin cytoskeletal remodeling; PKD1 was identified as a direct upstream kinase that targets SSH2 in this pathway.","method":"Co-immunoprecipitation (PKCβ–PKD1 interaction), genetic epistasis/knockdown of pathway components, cofilin phosphorylation assays, chemotaxis assays in neutrophils; SSH2 identified as PKD1 substrate","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, pathway epistasis by sequential knockdowns, biochemical (cofilin phosphorylation) and functional (chemotaxis) readouts, multiple orthogonal methods in single study","pmids":["25568344"],"is_preprint":false},{"year":2019,"finding":"miR-194 directly targets the 3′UTR of SSH2 mRNA (confirmed by luciferase reporter assay), reducing SSH2 protein expression; this suppresses proliferation and promotes apoptosis of CRC stem cells, placing SSH2 as a downstream effector of miR-194 in colon cancer stem cell growth.","method":"Luciferase reporter assay, RT-PCR, western blot, colony formation, MTT, apoptosis/cell cycle assays, in vivo xenograft","journal":"Cancer management and research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — luciferase reporter validates direct miR-194/SSH2 3′UTR interaction; functional rescue not shown for SSH2 specifically; single lab","pmids":["31824193"],"is_preprint":false},{"year":2014,"finding":"ALK-SSH2 fusion transcript identified by next-generation sequencing in a lung adenocarcinoma patient co-harboring EML4-ALK; the ALK-SSH2 fusion conferred sensitivity to the ALK inhibitor crizotinib, indicating the fusion retains ALK kinase signaling.","method":"Next-generation sequencing (NGS) of tumor tissue, clinical response to crizotinib as functional readout","journal":"Diagnostic pathology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single clinical case, NGS identification only; no in vitro/biochemical validation of fusion kinase activity","pmids":["35144623"],"is_preprint":false}],"current_model":"SSH2 (Slingshot-2) is a serine phosphatase that directly dephosphorylates and reactivates cofilin/ADF, and is regulated upstream by a GPCR→Gαi→PLCγ2→PKCβ→PKD1 signaling axis (which targets SSH2) as well as by 14-3-3 protein binding downstream of Rac1; active SSH2 promotes actin cytoskeletal remodeling that is required for neurite extension, keratinocyte migration, tumor cell invasion, and neutrophil chemotaxis, and SSH2 loss triggers apoptosis specifically in FLCN-deficient cells via dysregulated cofilin phosphorylation."},"narrative":{"mechanistic_narrative":"SSH2 (Slingshot-2) is a cofilin/ADF phosphatase that directly dephosphorylates and reactivates cofilin to drive actin cytoskeletal remodeling across multiple migratory and morphogenic contexts [PMID:17360713, PMID:18171679]. Through this activity SSH2 contributes to NGF-induced neurite extension [PMID:17360713], alpha6beta4 integrin/Rac1-dependent keratinocyte migration and laminin-332 assembly [PMID:17848544], and LPA-induced tumor cell transcellular invasion [PMID:18171679], with isoform-specific contributions that vary by cell type—for example, SSH2 supports cofilin activation in vascular smooth muscle cells but is not the dominant isoform there [PMID:21868701]. SSH2 activity is controlled both downstream of Rac1 via 14-3-3 protein binding, which increases when Rac1 is inhibited [PMID:17848544], and through a GPCR→Gαi→PLCγ2→PKCβ→PKD1 kinase axis in which PKD1 directly targets SSH2 to regulate cofilin and neutrophil chemotaxis [PMID:25568344]. SSH2 expression is restrained post-transcriptionally by miR-194 acting on its 3′UTR [PMID:31824193], and SSH2 loss is synthetically lethal with FLCN deficiency, triggering caspase-3/7 activation and cell cycle arrest specifically in FLCN-null carcinoma cells [PMID:23416984].","teleology":[{"year":2007,"claim":"Established the core biochemical activity of SSH2 by showing it dephosphorylates and reactivates cofilin, linking this activity to a cellular phenotype (neurite extension).","evidence":"RNAi double knockdown of SSH1/SSH2 with cofilin phosphorylation and neurite extension readouts in PC12 cells and chick DRG neurons","pmids":["17360713"],"confidence":"High","gaps":["Knockdown was combined SSH1/SSH2, leaving SSH2-specific contribution unresolved","No structural or kinetic characterization of SSH2 catalysis"]},{"year":2007,"claim":"Placed SSH2 in an upstream signaling context, showing its activity is regulated by 14-3-3 binding downstream of alpha6beta4 integrin/Rac1 and required for keratinocyte migration.","evidence":"Phosphatase-dead SSH constructs, dominant-negative Rac1, 14-3-3 Co-IP, and wound-healing/immunofluorescence assays in human keratinocytes","pmids":["17848544"],"confidence":"High","gaps":["14-3-3 binding shown for SSH family broadly, not SSH2 isoform-specifically","Mechanism by which Rac1 modulates 14-3-3 association not resolved at the molecular level"]},{"year":2008,"claim":"Extended the cofilin-activation role of SSH2 to cancer cell invasion, demonstrating it is required for LPA-induced transcellular migration.","evidence":"siRNA knockdown with transcellular migration, 2D motility, and cofilin phosphorylation assays in MM1 hepatoma cells","pmids":["18171679"],"confidence":"High","gaps":["SSH1 and SSH2 contributions not fully separated","Upstream signal coupling LPA to SSH2 in these cells unidentified"]},{"year":2011,"claim":"Refined isoform specificity, showing SSH2 contributes to cofilin activation but is not the dominant migration-controlling isoform in vascular smooth muscle cells.","evidence":"Isoform-specific siRNA with scratch wound, Boyden chamber, and cofilin phosphorylation assays in VSMCs","pmids":["21868701"],"confidence":"Medium","gaps":["Negative migration result for SSH2 specific to VSMC context","Functional redundancy with SSH1 not directly tested by double knockdown"]},{"year":2013,"claim":"Revealed a context-dependent survival role, identifying a synthetic lethal interaction between SSH2 loss and FLCN deficiency.","evidence":"siRNA phosphatase screen, caspase-3/7 and cell cycle assays in isogenic FLCN+/− carcinoma cell pairs","pmids":["23416984"],"confidence":"Medium","gaps":["Mechanistic link between FLCN status and SSH2 dependency via cofilin not fully resolved","Single lab, in vitro only"]},{"year":2014,"claim":"Reported SSH2 as a fusion partner in lung adenocarcinoma, where an ALK-SSH2 fusion conferred crizotinib sensitivity.","evidence":"NGS of tumor tissue with clinical crizotinib response as readout in a single case","pmids":["35144623"],"confidence":"Low","gaps":["Single clinical case with no in vitro validation of fusion kinase activity","Functional contribution of the SSH2 portion of the fusion unknown"]},{"year":2015,"claim":"Defined the upstream kinase cascade regulating SSH2, identifying PKD1 as a direct kinase targeting SSH2 within a GPCR→Gαi→PLCγ2→PKCβ→PKD1 chemotaxis pathway.","evidence":"Co-IP, sequential knockdown epistasis, cofilin phosphorylation and chemotaxis assays in neutrophils with SSH2 as PKD1 substrate","pmids":["25568344"],"confidence":"High","gaps":["PKD1 phosphorylation site(s) on SSH2 and their effect on phosphatase activity not mapped","Whether this axis operates in non-neutrophil contexts untested"]},{"year":2019,"claim":"Established post-transcriptional control of SSH2, showing miR-194 directly targets its 3′UTR to suppress expression in colon cancer stem cells.","evidence":"Luciferase reporter, RT-PCR, western blot, proliferation/apoptosis assays and xenograft","pmids":["31824193"],"confidence":"Medium","gaps":["Functional rescue of the phenotype by SSH2 specifically not demonstrated","Whether SSH2 cofilin-phosphatase activity mediates the miR-194 phenotype unknown"]},{"year":null,"claim":"How SSH2 phosphorylation by upstream kinases and 14-3-3 binding are integrated to set cofilin-phosphatase output, and how this is wired into context-specific survival (FLCN) and disease phenotypes, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of SSH2 or its regulatory phosphosites","Substrate specificity beyond cofilin/ADF uncharacterized","Mechanistic basis of SSH2-FLCN synthetic lethality not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,5]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,2]}],"localization":[],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,5]}],"complexes":[],"partners":["CFL1","YWHAZ","PRKD1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q76I76","full_name":"Protein phosphatase Slingshot homolog 2","aliases":["SSH-like protein 2","SSH-2L","hSSH-2L"],"length_aa":1423,"mass_kda":158.2,"function":"Protein phosphatase which regulates actin filament dynamics. Dephosphorylates and activates the actin binding/depolymerizing factor cofilin, which subsequently binds to actin filaments and stimulates their disassembly. Inhibitory phosphorylation of cofilin is mediated by LIMK1, which may also be dephosphorylated and inactivated by this protein (PubMed:11832213). Required for spermatogenesis (By similarity). Involved in acrosome biogenesis, probably by regulating cofilin-mediated actin cytoskeleton remodeling during proacrosomal vesicle fusion and/or Golgi to perinuclear vesicle trafficking (By similarity)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton; Cell junction, focal adhesion; Cytoplasmic vesicle, secretory vesicle, acrosome","url":"https://www.uniprot.org/uniprotkb/Q76I76/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SSH2","classification":"Not Classified","n_dependent_lines":80,"n_total_lines":1208,"dependency_fraction":0.06622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SSH2","total_profiled":1310},"omim":[{"mim_id":"613675","title":"CHROMOSOME 17q11.2 DELETION SYNDROME, 1.4-MB","url":"https://www.omim.org/entry/613675"},{"mim_id":"606780","title":"SLINGSHOT PROTEIN PHOSPHATASE 3; SSH3","url":"https://www.omim.org/entry/606780"},{"mim_id":"606779","title":"SLINGSHOT PROTEIN PHOSPHATASE 2; SSH2","url":"https://www.omim.org/entry/606779"},{"mim_id":"606778","title":"SLINGSHOT PROTEIN PHOSPHATASE 1; SSH1","url":"https://www.omim.org/entry/606778"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":92.5}],"url":"https://www.proteinatlas.org/search/SSH2"},"hgnc":{"alias_symbol":["KIAA1725"],"prev_symbol":[]},"alphafold":{"accession":"Q76I76","domains":[{"cath_id":"2.30.29.110","chopping":"70-228","consensus_level":"medium","plddt":86.1089,"start":70,"end":228},{"cath_id":"3.90.190.10","chopping":"308-452","consensus_level":"medium","plddt":93.3238,"start":308,"end":452},{"cath_id":"1.10.10","chopping":"239-306","consensus_level":"medium","plddt":84.1174,"start":239,"end":306}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q76I76","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q76I76-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q76I76-F1-predicted_aligned_error_v6.png","plddt_mean":47.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SSH2","jax_strain_url":"https://www.jax.org/strain/search?query=SSH2"},"sequence":{"accession":"Q76I76","fasta_url":"https://rest.uniprot.org/uniprotkb/Q76I76.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q76I76/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q76I76"}},"corpus_meta":[{"pmid":"17360713","id":"PMC_17360713","title":"LIM kinase and slingshot are critical for neurite extension.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17360713","citation_count":107,"is_preprint":false},{"pmid":"17848544","id":"PMC_17848544","title":"The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17848544","citation_count":71,"is_preprint":false},{"pmid":"24497979","id":"PMC_24497979","title":"Identification of STAT5A and STAT5B target genes in human T cells.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24497979","citation_count":68,"is_preprint":false},{"pmid":"18171679","id":"PMC_18171679","title":"Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18171679","citation_count":48,"is_preprint":false},{"pmid":"25568344","id":"PMC_25568344","title":"GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis.","date":"2015","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/25568344","citation_count":42,"is_preprint":false},{"pmid":"21868701","id":"PMC_21868701","title":"Slingshot isoform-specific regulation of cofilin-mediated vascular smooth muscle cell migration and neointima formation.","date":"2011","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21868701","citation_count":33,"is_preprint":false},{"pmid":"27880036","id":"PMC_27880036","title":"Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.","date":"2017","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/27880036","citation_count":22,"is_preprint":false},{"pmid":"38092781","id":"PMC_38092781","title":"Identification of candidate DNA methylation biomarkers related to Alzheimer's disease risk by integrating genome and blood methylome data.","date":"2023","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/38092781","citation_count":22,"is_preprint":false},{"pmid":"20171024","id":"PMC_20171024","title":"Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.","date":"2010","source":"Veterinary microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/20171024","citation_count":21,"is_preprint":false},{"pmid":"30809968","id":"PMC_30809968","title":"Exome sequencing in 51 early onset non-familial CRC cases.","date":"2019","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30809968","citation_count":21,"is_preprint":false},{"pmid":"31824193","id":"PMC_31824193","title":"miR-194 Inhibits the Proliferation of SW620 Colon Cancer Stem Cells Through Downregulation of SSH2 Expression.","date":"2019","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/31824193","citation_count":17,"is_preprint":false},{"pmid":"20477815","id":"PMC_20477815","title":"Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability.","date":"2010","source":"APMIS : acta pathologica, microbiologica, et immunologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/20477815","citation_count":16,"is_preprint":false},{"pmid":"35793933","id":"PMC_35793933","title":"Genetic Odyssey to Ossification of the Posterior Longitudinal Ligament in the Cervical Spine: A Systematic Review.","date":"2022","source":"Neurospine","url":"https://pubmed.ncbi.nlm.nih.gov/35793933","citation_count":12,"is_preprint":false},{"pmid":"35144623","id":"PMC_35144623","title":"Concomitant novel ALK-SSH2, EML4-ALK and ARID2-ALK, EML4-ALK double-fusion variants and confer sensitivity to crizotinib in two lung adenocarcinoma patients, respectively.","date":"2022","source":"Diagnostic pathology","url":"https://pubmed.ncbi.nlm.nih.gov/35144623","citation_count":11,"is_preprint":false},{"pmid":"36597771","id":"PMC_36597771","title":"A PREVENTIVE TOOL FOR PREDICTING BLOODSTREAM INFECTIONS IN CHILDREN WITH BURNS.","date":"2023","source":"Shock (Augusta, Ga.)","url":"https://pubmed.ncbi.nlm.nih.gov/36597771","citation_count":11,"is_preprint":false},{"pmid":"26748257","id":"PMC_26748257","title":"Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.","date":"2015","source":"Behavioural brain research","url":"https://pubmed.ncbi.nlm.nih.gov/26748257","citation_count":9,"is_preprint":false},{"pmid":"34222341","id":"PMC_34222341","title":"LINC00174 Facilitates Cell Proliferation, Cell Migration and Tumor Growth of Osteosarcoma via Regulating the TGF-β/SMAD Signaling Pathway and Upregulating SSH2 Expression.","date":"2021","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/34222341","citation_count":8,"is_preprint":false},{"pmid":"30143211","id":"PMC_30143211","title":"Development of novel affinity reagents for detecting protein tyrosine phosphorylation based on superbinder SH2 domain in tumor cells.","date":"2018","source":"Analytica chimica acta","url":"https://pubmed.ncbi.nlm.nih.gov/30143211","citation_count":8,"is_preprint":false},{"pmid":"23416984","id":"PMC_23416984","title":"Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt-Hogg-Dubé tumour suppressor gene (FLCN).","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23416984","citation_count":7,"is_preprint":false},{"pmid":"39753851","id":"PMC_39753851","title":"Integrated Mendelian Randomization and Single-Cell Transcriptomics Analysis Identifies Critical Blood Biomarkers and Potential Mechanisms in Epilepsy.","date":"2025","source":"CNS neuroscience & therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/39753851","citation_count":4,"is_preprint":false},{"pmid":"37731406","id":"PMC_37731406","title":"Genome-Wide Identification of lncRNA and mRNA for Diagnosing Type 2 Diabetes in Saudi Arabia.","date":"2023","source":"Pharmacogenomics and personalized medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37731406","citation_count":2,"is_preprint":false},{"pmid":"40003656","id":"PMC_40003656","title":"Epigenetic Modulation Directs Recovery Post LASIK and SMILE Surgery: An Experimental Study.","date":"2025","source":"Life (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/40003656","citation_count":2,"is_preprint":false},{"pmid":"39560568","id":"PMC_39560568","title":"Integrative analysis of single-nucleus RNA sequencing and Mendelian randomization to explore novel risk genes for Alzheimer's disease.","date":"2024","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39560568","citation_count":1,"is_preprint":false},{"pmid":"40787599","id":"PMC_40787599","title":"Identification of novel candidate loci for Alzheimer's disease and related dementias by leveraging the shared genetic basis with hippocampal volume.","date":"2025","source":"Aging brain","url":"https://pubmed.ncbi.nlm.nih.gov/40787599","citation_count":1,"is_preprint":false},{"pmid":"41370394","id":"PMC_41370394","title":"Evaluating the effects of archaic protein-altering variants in living human adults.","date":"2025","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/41370394","citation_count":1,"is_preprint":false},{"pmid":"37214336","id":"PMC_37214336","title":"Retraction: LINC00174 facilitates cell proliferation, cell migration and tumor growth of osteosarcoma via regulating the TGF-β/SMAD signaling pathway and upregulating SSH2 expression.","date":"2023","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/37214336","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.01.24314738","title":"Identification of novel associations of candidate loci with Alzheimer’s disease by leveraging the shared genetic basis with hippocampal volume","date":"2024-10-02","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.01.24314738","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.07.25339770","title":"Genome-Wide Association Study of Risk for Eosinophilic Granulomatosis with Polyangiitis","date":"2025-11-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.07.25339770","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16163,"output_tokens":2314,"usd":0.041599,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9491,"output_tokens":2896,"usd":0.059928,"stage2_stop_reason":"end_turn"},"total_usd":0.101527,"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\": 2007,\n      \"finding\": \"SSH2 (Slingshot-2) functions as a cofilin/ADF phosphatase that dephosphorylates and reactivates cofilin, and double knockdown of SSH1/SSH2 by RNAi significantly suppressed NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, establishing SSH2 as a critical activating phosphatase for cofilin-dependent neurite extension.\",\n      \"method\": \"RNAi knockdown, phosphorylation assays, morphological readout of neurite extension in PC12 cells and chick DRG neurons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal loss-of-function with direct biochemical readout (cofilin dephosphorylation) and cellular phenotype (neurite extension), replicated across two cell systems\",\n      \"pmids\": [\"17360713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SSH family phosphatases (SSH1, SSH2, and SSH3), but not the phosphatase CIN, are required for cofilin activation downstream of alpha6beta4 integrin/Rac1 signaling in human keratinocytes; phosphatase-dead SSH constructs cause cofilin hyperphosphorylation/inactivation, loss of cell polarity, and aberrant laminin-332 assembly. SSH activity is regulated by 14-3-3 protein binding, and inhibition of Rac1 increases 14-3-3 association with SSH, placing SSH2 in an alpha6beta4/Rac1/14-3-3/SSH/cofilin pathway controlling keratinocyte migration.\",\n      \"method\": \"Expression of phosphatase-dead SSH constructs, dominant-negative Rac1, Co-IP of 14-3-3 with SSH, wound-healing migration assays, immunofluorescence of actin and laminin-332\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphatase-dead mutants, Co-IP of 14-3-3/SSH, and multiple cellular phenotypic readouts in a single study\",\n      \"pmids\": [\"17848544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SSH1/SSH2 dephosphorylate and reactivate cofilin/ADF in rat ascites hepatoma (MM1) cells; siRNA knockdown of SSH1/SSH2 significantly decreased LPA-induced transcellular migration and 2D motility of MM1 cells, demonstrating that SSH2-mediated cofilin dephosphorylation is required for tumor cell invasion.\",\n      \"method\": \"siRNA knockdown, in vitro transcellular migration assay through mesothelial monolayer, 2D motility assay, phosphorylation state analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with direct biochemical (cofilin phosphorylation) and functional (invasion/migration) readouts, single lab, two orthogonal assays\",\n      \"pmids\": [\"18171679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In vascular smooth muscle cells, SSH2 siRNA attenuated cofilin dephosphorylation (activation), but unlike SSH1 siRNA, SSH2 knockdown alone did not significantly reduce wound healing or PDGF-induced VSMC migration, indicating SSH2 contributes to cofilin activation but is not the dominant isoform controlling VSMC migration.\",\n      \"method\": \"siRNA knockdown (SSH1, SSH2, SSH3), scratch wound assay, Boyden chamber migration assay, cofilin phosphorylation western blot\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific siRNA with biochemical and functional readouts, single lab; finding is a partial/negative result for SSH2 specifically\",\n      \"pmids\": [\"21868701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SSH2 knockdown specifically induced Caspase3/7 activation (up to 6-fold) and cell cycle arrest (increased G1, decreased S and G2/M) in FLCN-deficient (BHD) human carcinoma cell lines but not in isogenic FLCN-expressing lines, revealing a synthetic lethal interaction between SSH2 and FLCN loss and implicating SSH2 in cell survival of FLCN-null cells via dysregulated cofilin de/phosphorylation.\",\n      \"method\": \"siRNA phosphatase library screen, Caspase3/7 activity assay, cell cycle analysis, western blot for cofilin phosphorylation and SSH isoform levels in isogenic FLCN+/− cell pairs\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA screen validated with dose-response, multiple readouts (caspase, cell cycle, biochemical), isogenic cell lines, single lab\",\n      \"pmids\": [\"23416984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In neutrophil chemotaxis, GPCR activation signals through Gαi → PLCγ2 (PI3K-dependent) → PKCβ → PKD1 to phosphorylate and regulate SSH2, thereby controlling cofilin phosphorylation and actin cytoskeletal remodeling; PKD1 was identified as a direct upstream kinase that targets SSH2 in this pathway.\",\n      \"method\": \"Co-immunoprecipitation (PKCβ–PKD1 interaction), genetic epistasis/knockdown of pathway components, cofilin phosphorylation assays, chemotaxis assays in neutrophils; SSH2 identified as PKD1 substrate\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, pathway epistasis by sequential knockdowns, biochemical (cofilin phosphorylation) and functional (chemotaxis) readouts, multiple orthogonal methods in single study\",\n      \"pmids\": [\"25568344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-194 directly targets the 3′UTR of SSH2 mRNA (confirmed by luciferase reporter assay), reducing SSH2 protein expression; this suppresses proliferation and promotes apoptosis of CRC stem cells, placing SSH2 as a downstream effector of miR-194 in colon cancer stem cell growth.\",\n      \"method\": \"Luciferase reporter assay, RT-PCR, western blot, colony formation, MTT, apoptosis/cell cycle assays, in vivo xenograft\",\n      \"journal\": \"Cancer management and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — luciferase reporter validates direct miR-194/SSH2 3′UTR interaction; functional rescue not shown for SSH2 specifically; single lab\",\n      \"pmids\": [\"31824193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ALK-SSH2 fusion transcript identified by next-generation sequencing in a lung adenocarcinoma patient co-harboring EML4-ALK; the ALK-SSH2 fusion conferred sensitivity to the ALK inhibitor crizotinib, indicating the fusion retains ALK kinase signaling.\",\n      \"method\": \"Next-generation sequencing (NGS) of tumor tissue, clinical response to crizotinib as functional readout\",\n      \"journal\": \"Diagnostic pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single clinical case, NGS identification only; no in vitro/biochemical validation of fusion kinase activity\",\n      \"pmids\": [\"35144623\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SSH2 (Slingshot-2) is a serine phosphatase that directly dephosphorylates and reactivates cofilin/ADF, and is regulated upstream by a GPCR→Gαi→PLCγ2→PKCβ→PKD1 signaling axis (which targets SSH2) as well as by 14-3-3 protein binding downstream of Rac1; active SSH2 promotes actin cytoskeletal remodeling that is required for neurite extension, keratinocyte migration, tumor cell invasion, and neutrophil chemotaxis, and SSH2 loss triggers apoptosis specifically in FLCN-deficient cells via dysregulated cofilin phosphorylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SSH2 (Slingshot-2) is a cofilin/ADF phosphatase that directly dephosphorylates and reactivates cofilin to drive actin cytoskeletal remodeling across multiple migratory and morphogenic contexts [#0, #2]. Through this activity SSH2 contributes to NGF-induced neurite extension [#0], alpha6beta4 integrin/Rac1-dependent keratinocyte migration and laminin-332 assembly [#1], and LPA-induced tumor cell transcellular invasion [#2], with isoform-specific contributions that vary by cell type—for example, SSH2 supports cofilin activation in vascular smooth muscle cells but is not the dominant isoform there [#3]. SSH2 activity is controlled both downstream of Rac1 via 14-3-3 protein binding, which increases when Rac1 is inhibited [#1], and through a GPCR\\u2192G\\u03b1i\\u2192PLC\\u03b32\\u2192PKC\\u03b2\\u2192PKD1 kinase axis in which PKD1 directly targets SSH2 to regulate cofilin and neutrophil chemotaxis [#5]. SSH2 expression is restrained post-transcriptionally by miR-194 acting on its 3\\u2032UTR [#6], and SSH2 loss is synthetically lethal with FLCN deficiency, triggering caspase-3/7 activation and cell cycle arrest specifically in FLCN-null carcinoma cells [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the core biochemical activity of SSH2 by showing it dephosphorylates and reactivates cofilin, linking this activity to a cellular phenotype (neurite extension).\",\n      \"evidence\": \"RNAi double knockdown of SSH1/SSH2 with cofilin phosphorylation and neurite extension readouts in PC12 cells and chick DRG neurons\",\n      \"pmids\": [\"17360713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Knockdown was combined SSH1/SSH2, leaving SSH2-specific contribution unresolved\",\n        \"No structural or kinetic characterization of SSH2 catalysis\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed SSH2 in an upstream signaling context, showing its activity is regulated by 14-3-3 binding downstream of alpha6beta4 integrin/Rac1 and required for keratinocyte migration.\",\n      \"evidence\": \"Phosphatase-dead SSH constructs, dominant-negative Rac1, 14-3-3 Co-IP, and wound-healing/immunofluorescence assays in human keratinocytes\",\n      \"pmids\": [\"17848544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"14-3-3 binding shown for SSH family broadly, not SSH2 isoform-specifically\",\n        \"Mechanism by which Rac1 modulates 14-3-3 association not resolved at the molecular level\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extended the cofilin-activation role of SSH2 to cancer cell invasion, demonstrating it is required for LPA-induced transcellular migration.\",\n      \"evidence\": \"siRNA knockdown with transcellular migration, 2D motility, and cofilin phosphorylation assays in MM1 hepatoma cells\",\n      \"pmids\": [\"18171679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"SSH1 and SSH2 contributions not fully separated\",\n        \"Upstream signal coupling LPA to SSH2 in these cells unidentified\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Refined isoform specificity, showing SSH2 contributes to cofilin activation but is not the dominant migration-controlling isoform in vascular smooth muscle cells.\",\n      \"evidence\": \"Isoform-specific siRNA with scratch wound, Boyden chamber, and cofilin phosphorylation assays in VSMCs\",\n      \"pmids\": [\"21868701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Negative migration result for SSH2 specific to VSMC context\",\n        \"Functional redundancy with SSH1 not directly tested by double knockdown\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a context-dependent survival role, identifying a synthetic lethal interaction between SSH2 loss and FLCN deficiency.\",\n      \"evidence\": \"siRNA phosphatase screen, caspase-3/7 and cell cycle assays in isogenic FLCN+/\\u2212 carcinoma cell pairs\",\n      \"pmids\": [\"23416984\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanistic link between FLCN status and SSH2 dependency via cofilin not fully resolved\",\n        \"Single lab, in vitro only\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Reported SSH2 as a fusion partner in lung adenocarcinoma, where an ALK-SSH2 fusion conferred crizotinib sensitivity.\",\n      \"evidence\": \"NGS of tumor tissue with clinical crizotinib response as readout in a single case\",\n      \"pmids\": [\"35144623\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single clinical case with no in vitro validation of fusion kinase activity\",\n        \"Functional contribution of the SSH2 portion of the fusion unknown\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the upstream kinase cascade regulating SSH2, identifying PKD1 as a direct kinase targeting SSH2 within a GPCR\\u2192G\\u03b1i\\u2192PLC\\u03b32\\u2192PKC\\u03b2\\u2192PKD1 chemotaxis pathway.\",\n      \"evidence\": \"Co-IP, sequential knockdown epistasis, cofilin phosphorylation and chemotaxis assays in neutrophils with SSH2 as PKD1 substrate\",\n      \"pmids\": [\"25568344\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"PKD1 phosphorylation site(s) on SSH2 and their effect on phosphatase activity not mapped\",\n        \"Whether this axis operates in non-neutrophil contexts untested\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established post-transcriptional control of SSH2, showing miR-194 directly targets its 3\\u2032UTR to suppress expression in colon cancer stem cells.\",\n      \"evidence\": \"Luciferase reporter, RT-PCR, western blot, proliferation/apoptosis assays and xenograft\",\n      \"pmids\": [\"31824193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional rescue of the phenotype by SSH2 specifically not demonstrated\",\n        \"Whether SSH2 cofilin-phosphatase activity mediates the miR-194 phenotype unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SSH2 phosphorylation by upstream kinases and 14-3-3 binding are integrated to set cofilin-phosphatase output, and how this is wired into context-specific survival (FLCN) and disease phenotypes, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural model of SSH2 or its regulatory phosphosites\",\n        \"Substrate specificity beyond cofilin/ADF uncharacterized\",\n        \"Mechanistic basis of SSH2-FLCN synthetic lethality not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 5]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CFL1\", \"YWHAZ\", \"PRKD1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":4,"faith_total":4,"faith_pct":100.0}}