{"gene":"SH2D2A","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":1998,"finding":"TSAd (F2771/SH2D2A) was identified as a novel adapter protein containing an SH2 domain and putative SH3/phosphotyrosine-binding motifs but no catalytic domains. The protein is expressed in activated T cells, localizes to the cytoplasm when expressed in COS cells, and is itself tyrosine-phosphorylated (~52 kDa) upon PHA stimulation of peripheral blood mononuclear cells.","method":"cDNA cloning, transfection in COS cells with localization by immunostaining, immunoprecipitation with anti-phosphotyrosine antibody","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — initial characterization with multiple orthogonal methods (cloning, cell expression, immunoprecipitation), single lab","pmids":["9468509"],"is_preprint":false},{"year":2001,"finding":"TSAd localizes to the T cell nucleus via an active import process that requires a functional SH2 domain recognizing a phosphotyrosine-containing ligand (~95-100 kDa). TSAd acts as a transcriptional activator in T cells and upregulates IL-2 promoter activity in a manner dependent on an intact SH2 domain, but does not modulate NF-κB, NFAT, or AP-1 activity.","method":"Subcellular fractionation, nuclear import assays, SH2 domain mutant transfection in T cells, IL-2 promoter-reporter cotransfection assays","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (fractionation, reporter assay, mutagenesis) in single lab","pmids":["11413197"],"is_preprint":false},{"year":2004,"finding":"The TSAd SH2 domain ligand involved in nuclear import was identified as p97 Valosin-containing protein (VCP), which is phosphorylated on Y805 by TCR-activated tyrosine kinases. Direct interaction between endogenous TSAd and VCP was demonstrated in T cells. VCP/CDC48 (yeast homolog) is required for TSAd nuclear transport, established using CDC48 mutant yeast.","method":"Microchemical identification (mass spectrometry), co-immunoprecipitation of endogenous proteins, direct interaction assay, CDC48 mutant yeast complementation","journal":"Immunology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous proteins, genetic epistasis in yeast, identification by MS; single lab","pmids":["15752563"],"is_preprint":false},{"year":2004,"finding":"Transcriptional activation of SH2D2A in T cells requires a cAMP response element (CRE) centered at position -117 bp from the first coding ATG. CD3 stimulation-induced SH2D2A expression requires cAMP-dependent protein kinase activity, and cAMP analogs potently induce SH2D2A mRNA.","method":"Reporter gene assays (luciferase), EMSA with nuclear extracts, real-time RT-PCR, PKA inhibitor treatment","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (reporter, EMSA, RT-PCR, pharmacological inhibition), single lab","pmids":["15128801"],"is_preprint":false},{"year":2005,"finding":"TSAd interacts with Lck through Lck's SH2 and SH3 domains, and TSAd is a substrate for Lck phosphorylation. The TSAd C-terminus (proline-rich region and five tyrosines) is both necessary and sufficient for interaction with and phosphorylation by Lck. TSAd expression causes hyperphosphorylation of Lck at Y394 and Y505, reducing the Y394/Y505 ratio, thereby inhibiting Lck activity. The C-terminus alone is sufficient to inhibit the hyperactive Lck Y505F mutant.","method":"Co-immunoprecipitation, Lck SH2/SH3 domain pull-down, Lck kinase activity assays, TSAd deletion mutant transfection in Jurkat T cells, phosphospecific immunoblotting","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro binding assays, kinase assays with mutagenesis, and functional read-out in cells; multiple orthogonal methods in single lab with rigorous domain mapping","pmids":["15827961"],"is_preprint":false},{"year":2005,"finding":"VEGFR-2 phosphorylation at Y951 (within the kinase insert domain) creates a binding site for TSAd. TSAd is expressed in tumor vessels, and Y951-TSAd signaling is required for VEGF-A-induced actin stress fiber formation and endothelial cell migration (but not mitogenesis). TSAd-deficient mice show reduced tumor vascularization and growth.","method":"Pan-phosphorylation site mapping of VEGFR-2, phosphopeptide binding assays, TSAd siRNA in endothelial cells, Y951F VEGFR-2 mutation, TSAd knockout mice with tumor implantation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (phosphosite mapping, peptide competition, siRNA, KO mice with phenotype), replicated in subsequent studies","pmids":["15962004"],"is_preprint":false},{"year":2006,"finding":"TSAd interacts with Lck through three modes: (1) TSAd aa239-256 binds the Lck-SH3 domain; (2) one or more tyrosines within TSAd aa239-334 (exon 7) bind the Lck-SH2 domain; (3) the TSAd SH2 domain also interacts with Lck. The exon 7-encoded region (aa239-334) is essential for Lck modulation and for TSAd nuclear translocation. TSAd-Lck interaction was confirmed in human CD4+ T cells ex vivo.","method":"Co-immunoprecipitation with TSAd isoform/deletion mutants in Jurkat cells and primary CD4+ T cells, domain-binding assays","journal":"BMC immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic domain mapping with multiple isoforms, confirmed in primary T cells; single lab","pmids":["16839418"],"is_preprint":false},{"year":2006,"finding":"TSAd interacts with Smad2 and Smad3, primarily through the TSAd type I SH2 domain. Both Smad2 and Smad3 also interact with the Lck type I SH2 domain but not the PI3K type III SH2 domain.","method":"Yeast two-hybrid, co-immunoprecipitation, GST pull-down assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — interaction shown by yeast two-hybrid, co-IP and pull-down in single lab; no functional validation of the TSAd-Smad interaction consequence","pmids":["16806069"],"is_preprint":false},{"year":2007,"finding":"SH2D2A expression is regulated at both transcriptional and translational levels. cAMP signaling alone induces TSAd mRNA but not protein. TCR engagement is required for both transcription and translation of TSAd. cAMP signaling primes T cells for more rapid TSAd protein expression upon TCR stimulation.","method":"RT-PCR for mRNA quantification, immunoblotting for protein levels, pharmacological modulation of cAMP and TCR signaling in primary T cells","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — orthogonal mRNA/protein measurements with multiple signaling conditions, single lab","pmids":["18160104"],"is_preprint":false},{"year":2009,"finding":"TSAd interacts with the 67 kDa laminin-binding protein (LBP) as identified by yeast two-hybrid and confirmed by co-immunoprecipitation in D1.1 T cells. TSAd-LBP interaction occurs upon TCR plus laminin or TCR plus integrin α6 costimulation. Overexpression of TSAd enhances TCR+laminin-induced T cell migration, while dominant-negative TSAd or TSAd shRNA disrupts this migration.","method":"Yeast two-hybrid screening, co-immunoprecipitation, overexpression and shRNA knockdown in D1.1 T cells with migration assays","journal":"Experimental & molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-IP and functional assay in single lab; no domain-level mapping of the TSAd-LBP interaction","pmids":["19561400"],"is_preprint":false},{"year":2010,"finding":"TSAd is required for tyrosine phosphorylation of the Lck substrate Itk (at Y511). TSAd promotes Itk phosphorylation by Lck by bridging the two kinases. TSAd interaction with Itk through its proline-rich region is necessary for TSAd to enhance CXCL12-induced actin polymerization and migration of Jurkat T cells. TSAd-deficient murine thymocytes show reduced Itk phosphorylation, actin polymerization, and migration in response to CXCL12.","method":"Co-immunoprecipitation, Itk phospho-specific immunoblotting, TSAd proline-rich region mutants, Jurkat migration assays, TSAd-deficient mouse thymocyte assays, actin polymerization (flow cytometry)","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, phospho-blot, domain mutants, KO mouse cells, functional assays), replicated in cell line and primary cells","pmids":["20305788"],"is_preprint":false},{"year":2012,"finding":"VEGFR2 Y951 phosphorylation facilitates binding of VEGFR2 to the SH2 domain of TSAd. TSAd in turn regulates VEGF-induced c-Src activation (increased pY418, reduced pY527). TSAd silencing blocked VEGF-induced c-Src activation but not PLCγ, ERK, or eNOS pathways. TSAd forms a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. TSAd-deficient mice show impaired VEGF-induced vascular permeability (Evans blue, dextran, microsphere extravasation) but normal histamine-induced permeability.","method":"Co-immunoprecipitation of endogenous proteins, phospho-specific immunoblotting, Tsad siRNA in endothelial cells, tsad-/- mice with permeability assays (Evans blue, dextran, microspheres), confocal microscopy of VE-cadherin junctions","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, multiple KO mouse permeability assays, siRNA with pathway-specific read-outs; multiple orthogonal methods, independent replication of VEGFR2-TSAd interaction","pmids":["22689825"],"is_preprint":false},{"year":2014,"finding":"Itk and TSAd promote TCR-dependent phosphorylation of Lck at Tyr192 within the Lck SH2 domain. Phospho-Tyr192 of Lck preferentially recruits actin cytoskeleton regulators (including Itk and TSAd) compared to unphosphorylated Lck. Lck Y192 phosphorylation is required for proper T cell activation and T cell–APC conjugate formation.","method":"Phosphopeptide arrays, biochemical binding assays, phospho-specific immunoblotting, T cell–APC conjugation assay, Lck point mutant transfection","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphopeptide arrays plus biochemical assays plus functional T cell assays, single lab","pmids":["25492967"],"is_preprint":false},{"year":2015,"finding":"TSAd binds Nck via Nck SH2 domain interaction with TSAd phospho-Tyr280 and phospho-Tyr305, and via Nck SH3 domains interacting with TSAd's proline-rich region. TSAd promotes Nck interaction with Lck and SLP-76 (but not Vav1) in T cells. TSAd expression increases polymerized actin in a manner dependent on TSAd exon 7 (which encodes Nck and Lck interaction sites). TSAd and Nck co-localize in Jurkat cells.","method":"SMALI algorithm prediction, peptide array, pull-down, co-immunoprecipitation, confocal microscopy, imaging flow cytometry, actin polymerization assay in TSAd-deficient Jurkat cells","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal binding methods (peptide array, pull-down, co-IP), functional actin assay with genetic deletion, single lab","pmids":["26163016"],"is_preprint":false},{"year":2016,"finding":"The SH2 and proline-rich domains of TSAd bridge VEGFR2 and c-Src, and this bridging is required for localization of activated c-Src to endothelial junctions and elongation of growing angiogenic sprouts. TSAd-deficient embryoid bodies fail to sprout in response to VEGF; TSAd-deficient mice show impaired tracheal vessel development and reduced sprouting in VEGF-Matrigel plugs (but not FGF-Matrigel plugs). TSAd-deficient embryoid bodies fail to phosphorylate/turn over VE-cadherin at junctions.","method":"Domain deletion mutants in endothelial cells, tsad-/- embryoid body sprouting assay, tsad-/- mouse tracheal and retinal vasculature analysis, VEGF/FGF Matrigel plugs, c-Src localization by immunofluorescence","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple tissue/assay systems, domain-mapping in cells, multiple orthogonal phenotypic read-outs; single lab but rigorous","pmids":["27436360"],"is_preprint":false},{"year":2018,"finding":"TSAd modulates polarization of CD4+ T cells towards the APC. Sh2d2a-/- CD4+ T cells show impaired polarization of F-actin and TCR to the immunological synapse, and defective polarization of PKCξ, PAR3, and the MTOC. TSAd-deficient T cells preferentially differentiate toward effector memory (Tem) rather than central memory (Tcm) phenotype.","method":"Imaging flow cytometry of Sh2d2a-/- T cells with APC, F-actin/TCR/PKCξ/PAR3/MTOC localization, in vitro T cell differentiation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with direct localization readouts and differentiation assay; single lab","pmids":["30190583"],"is_preprint":false},{"year":2020,"finding":"TSAd is regulated epigenetically downstream of Myo9b: Myo9b deficiency causes H3K27me3 enrichment at the TSAd promoter, reducing TSAd expression. TSAd knockdown in naive T cells recapitulates the Myo9b-/- phenotype of reduced TH1 and increased TH17 differentiation. TSAd siRNA in vivo suppresses malignant pleural effusion development and shifts TH1/TH17 balance.","method":"Chromatin immunoprecipitation (ChIP) for H3K27me3 at TSAd promoter, TSAd siRNA in primary T cells with TH1/TH17 differentiation assay, in vivo siRNA delivery with pleural effusion model","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP demonstrating epigenetic regulation plus functional siRNA rescue experiments in vitro and in vivo; single lab","pmids":["33046503"],"is_preprint":false},{"year":2025,"finding":"Affinity-purification mass spectrometry identified DOK2 and PTPN11 as novel ligands of the TSAd SH2 domain. The specific phosphotyrosines on DOK2 and PTPN11 responsible for TSAd SH2 interaction were determined. CRISPR/Cas9 ablation of TSAd and DOK2 in Jurkat T cells resulted in altered tyrosine phosphorylation, suggesting TSAd functions as a negative signaling node via DOK2 and PTPN11.","method":"Affinity-purification mass spectrometry (AP-MS), phosphopeptide mapping, CRISPR/Cas9 knockouts in Jurkat T cells, phosphotyrosine immunoblotting","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased AP-MS with functional CRISPR validation; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.02.11.636929"],"is_preprint":true},{"year":2025,"finding":"Multi-omics data (single-cell RNA-seq, bulk RNA-seq, epigenomic/ATAC, mass spectrometry-based proteomics) from multiple organs indicate that TSAd transcripts are largely absent from endothelial cells, the TSAd promoter resides in closed chromatin in ECs, and TSAd protein is typically undetected in EC proteomes. This challenges the proposed universal role of TSAd in VEGFR2-mediated vascular permeability signaling in ECs.","method":"Mining of public scRNA-seq, bulk RNA-seq, epigenomic (chromatin accessibility), and MS proteomics datasets from endothelial cells across multiple organs","journal":"Angiogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal omics data types across organs; negative/contradictory finding that challenges prior mechanistic claims; single published study","pmids":["40080216"],"is_preprint":false}],"current_model":"TSAd (SH2D2A) is a T cell- and endothelial cell-expressed adapter protein that lacks catalytic activity but contains an SH2 domain, proline-rich region, and multiple tyrosines; it is tyrosine-phosphorylated upon activation and (1) in T cells, binds Lck (via SH3/SH2 interactions) to modulate Lck activity and promote phosphorylation of Itk at Y511, scaffolds Nck-Lck-SLP-76 complexes to drive actin polymerization, undergoes SH2-domain-dependent nuclear import via VCP/p97, and acts as a transcriptional activator for IL-2; (2) in endothelial cells, the SH2 domain binds phospho-Y951 of VEGFR2 while the proline-rich domain engages c-Src, thereby localizing active c-Src to endothelial junctions to regulate VE-cadherin turnover, vascular permeability, and angiogenic sprouting in a tissue-specific manner, though recent omics evidence questions whether TSAd is broadly expressed in endothelial cells across all organs."},"narrative":{"mechanistic_narrative":"SH2D2A (TSAd) is a catalytically inactive adapter protein that couples antigen- and growth-factor receptor signaling to cytoskeletal remodeling, migration, and transcription through an SH2 domain, a proline-rich region, and multiple phosphorylatable tyrosines [PMID:9468509]. In T cells, TSAd expression is induced upon TCR engagement and is regulated both transcriptionally—via a CRE element requiring PKA activity, with cAMP priming cells for rapid protein production—and translationally [PMID:15128801, PMID:18160104]. Once expressed and tyrosine-phosphorylated, TSAd engages Lck through three modes (Lck-SH3, Lck-SH2, and the TSAd SH2 domain) and acts as a substrate that drives Lck hyperphosphorylation at Y394 and Y505 to modulate its activity, and promotes Lck phosphorylation of Lck Y192 within the Lck SH2 domain [PMID:15827961, PMID:16839418, PMID:25492967]. By bridging Lck to its substrate Itk, TSAd is required for Itk Y511 phosphorylation, and it scaffolds Nck with Lck and SLP-76 via phospho-Y280/Y305 and its proline-rich region to drive actin polymerization and chemokine-induced migration [PMID:20305788, PMID:26163016]. These activities underlie TSAd control of CD4+ T cell polarization toward the APC, immunological synapse organization, and effector-versus-central memory differentiation [PMID:30190583]. TSAd also undergoes SH2-domain-dependent nuclear import—using VCP/p97 (phosphorylated on Y805) as its phosphotyrosine ligand—where it activates the IL-2 promoter [PMID:11413197, PMID:15752563]. In endothelial cells, the TSAd SH2 domain binds VEGFR2 phospho-Y951 while its proline-rich region engages c-Src, bridging the two within a VE-cadherin/VEGFR2/c-Src complex to localize active c-Src to junctions and thereby regulate VE-cadherin turnover, VEGF-induced vascular permeability, and angiogenic sprouting [PMID:15962004, PMID:22689825, PMID:27436360]. The breadth of this endothelial role across organs is contested by multi-omics data indicating TSAd is largely absent from endothelial cells [PMID:40080216].","teleology":[{"year":1998,"claim":"Established TSAd as a non-catalytic adapter, defining the structural toolkit (SH2 domain, phosphotyrosine-binding motifs) through which it would later be shown to act.","evidence":"cDNA cloning, COS-cell expression with immunostaining, and anti-phosphotyrosine immunoprecipitation in PHA-stimulated PBMCs","pmids":["9468509"],"confidence":"Medium","gaps":["No binding partners identified","No functional readout beyond inducible phosphorylation"]},{"year":2001,"claim":"Showed TSAd has a nuclear, transcriptional function, answering whether this cytoplasmic adapter influences gene expression and linking its SH2 domain to both nuclear import and IL-2 promoter activation.","evidence":"Subcellular fractionation, SH2-mutant transfection, and IL-2 promoter-reporter assays in T cells","pmids":["11413197"],"confidence":"Medium","gaps":["Identity of the ~95-100 kDa SH2 ligand unknown at this stage","Mechanism of transcriptional activation not defined"]},{"year":2004,"claim":"Identified VCP/p97 as the phosphotyrosine ligand driving TSAd nuclear import and defined how SH2D2A transcription is controlled, connecting receptor signaling to TSAd induction and trafficking.","evidence":"MS identification and endogenous co-IP of VCP, CDC48 mutant yeast complementation (#2); luciferase reporters, EMSA, RT-PCR and PKA inhibition (#3)","pmids":["15752563","15128801"],"confidence":"Medium","gaps":["Functional consequence of nuclear TSAd-VCP interaction on specific target genes unresolved","Whether VCP-dependent import is required for IL-2 activation not directly tested"]},{"year":2005,"claim":"Defined two distinct receptor-proximal roles: TSAd as a multivalent Lck binder and negative regulator of Lck activity in T cells, and as a VEGFR2 pY951 effector required for endothelial migration and tumor vascularization.","evidence":"Lck SH2/SH3 pull-downs, kinase assays and deletion mutants in Jurkat cells (#4); VEGFR2 phosphosite mapping, siRNA, Y951F mutation and TSAd-KO mouse tumor implantation (#5)","pmids":["15827961","15962004"],"confidence":"High","gaps":["How TSAd reconciles opposing roles in two cell types unclear","Downstream effectors of VEGFR2-TSAd not yet identified"]},{"year":2006,"claim":"Mapped the TSAd-Lck interaction to three binding modes and localized the exon 7 region essential for both Lck modulation and nuclear translocation, and reported TSAd binding to Smad2/3.","evidence":"Domain/isoform co-IP in Jurkat and primary CD4+ T cells (#6); yeast two-hybrid, co-IP and GST pull-down for Smad2/3 (#7)","pmids":["16839418","16806069"],"confidence":"Medium","gaps":["Functional consequence of TSAd-Smad2/3 interaction never validated","Specific tyrosines binding Lck-SH2 not pinpointed"]},{"year":2007,"claim":"Resolved that SH2D2A is dual-controlled at transcription and translation, explaining how cAMP priming and TCR engagement jointly gate TSAd protein output.","evidence":"RT-PCR, immunoblotting and pharmacological cAMP/TCR modulation in primary T cells","pmids":["18160104"],"confidence":"Medium","gaps":["Molecular basis of translational control not defined","Identity of translation-regulating signals downstream of TCR unknown"]},{"year":2009,"claim":"Implicated TSAd in laminin/integrin-costimulated T cell migration via interaction with the 67 kDa laminin-binding protein.","evidence":"Yeast two-hybrid, co-IP, overexpression and shRNA migration assays in D1.1 T cells","pmids":["19561400"],"confidence":"Low","gaps":["No domain-level mapping of the TSAd-LBP interaction","Single lab, no reciprocal validation","Connection to canonical TSAd-Lck/Nck pathway unestablished"]},{"year":2010,"claim":"Demonstrated that TSAd bridges Lck and Itk to enable Itk Y511 phosphorylation, providing a mechanistic basis for TSAd-driven chemokine-induced actin polymerization and migration.","evidence":"Co-IP, phospho-Itk blotting, proline-rich mutants, Jurkat migration assays, and TSAd-deficient mouse thymocyte assays with actin polymerization readouts","pmids":["20305788"],"confidence":"High","gaps":["Stoichiometry of the Lck-TSAd-Itk ternary assembly not defined","Whether nuclear and cytoskeletal pools of TSAd are functionally separable unaddressed"]},{"year":2012,"claim":"Defined the endothelial TSAd permeability module, showing TSAd assembles a VE-cadherin/VEGFR2/c-Src complex at junctions and is required for VEGF-induced c-Src activation and vascular permeability.","evidence":"Reciprocal endogenous co-IP, phospho-blotting, siRNA with pathway-specific readouts, and tsad-/- mouse permeability assays with confocal junction imaging","pmids":["22689825"],"confidence":"High","gaps":["Selectivity for c-Src over PLCγ/ERK/eNOS mechanistically unexplained","Direct contribution of nuclear TSAd functions to EC phenotype untested"]},{"year":2014,"claim":"Showed TSAd and Itk drive Lck Y192 phosphorylation within the Lck SH2 domain, creating a docking surface that recruits cytoskeletal regulators and supports T cell-APC conjugate formation.","evidence":"Phosphopeptide arrays, biochemical binding, phospho-specific blotting, Lck point mutants and T cell-APC conjugation assays","pmids":["25492967"],"confidence":"Medium","gaps":["Whether Lck Y192 phosphorylation is direct or indirect not fully resolved","Quantitative contribution of TSAd versus Itk to Y192 phosphorylation unclear"]},{"year":2015,"claim":"Established TSAd as a Nck-recruiting scaffold, linking phospho-Y280/Y305 and the proline-rich region to assembly of Nck-Lck-SLP-76 complexes that drive actin polymerization.","evidence":"SMALI prediction, peptide arrays, pull-down, co-IP, confocal imaging and actin assays in TSAd-deficient Jurkat cells","pmids":["26163016"],"confidence":"Medium","gaps":["Why Vav1 is excluded from the complex not explained","In vivo relevance of the Nck-Lck-SLP-76 assembly not tested in primary cells"]},{"year":2018,"claim":"Connected TSAd's cytoskeletal scaffolding role to T cell biology, showing it controls synapse polarization, MTOC orientation and effector/central memory differentiation.","evidence":"Imaging flow cytometry of Sh2d2a-/- T cells with APC, polarity-marker localization and in vitro differentiation assays","pmids":["30190583"],"confidence":"Medium","gaps":["Molecular link between polarity defects and memory skewing not defined","Single-cell heterogeneity of the phenotype unaddressed"]},{"year":2020,"claim":"Placed TSAd downstream of an epigenetic regulatory axis, showing Myo9b loss silences TSAd via H3K27me3 and that TSAd controls TH1/TH17 balance with disease relevance in pleural effusion.","evidence":"ChIP for H3K27me3 at the TSAd promoter, TSAd siRNA differentiation assays, and in vivo siRNA in a malignant pleural effusion model","pmids":["33046503"],"confidence":"Medium","gaps":["Direct transcriptional targets of TSAd in TH differentiation unidentified","Mechanism coupling TSAd adapter activity to lineage commitment unclear"]},{"year":2025,"claim":"Expanded the TSAd SH2 interactome to negative-signaling phosphatases/adapters DOK2 and PTPN11, recasting TSAd as a potential negative signaling node in T cells.","evidence":"Affinity-purification mass spectrometry, phosphopeptide mapping and CRISPR/Cas9 TSAd/DOK2 knockouts in Jurkat cells (preprint)","pmids":["bio_10.1101_2025.02.11.636929"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Functional consequence of DOK2/PTPN11 recruitment on specific pathways not resolved","Reconciliation with TSAd's positive scaffolding roles unaddressed"]},{"year":2025,"claim":"Challenged the universality of the endothelial TSAd model by showing TSAd transcripts and protein are largely absent from endothelial cells across organs.","evidence":"Mining of public scRNA-seq, bulk RNA-seq, chromatin accessibility and MS proteomics datasets from endothelial cells across multiple organs","pmids":["40080216"],"confidence":"Medium","gaps":["Does not exclude regulated or context-specific EC expression","Discordance with functional KO permeability phenotypes unresolved","Single study, detection-sensitivity limits not fully addressed"]},{"year":null,"claim":"How TSAd integrates its opposing positive (Lck/Itk/Nck scaffolding, IL-2 activation) and negative (Lck inhibition, DOK2/PTPN11 recruitment) functions, and in which cell types the VEGFR2 module is physiologically operative, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling positive and negative signaling roles","Definitive cell-type-resolved expression map of TSAd lacking","No structural model of TSAd in any signaling complex"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4,10,13,14]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,11]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11,14]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,10,15,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,11,14]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[11,14]}],"complexes":["VE-cadherin/VEGFR2/c-Src junctional complex","Nck-Lck-SLP-76 complex"],"partners":["LCK","ITK","NCK1","VEGFR2","SRC","VCP","DOK2","PTPN11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NP31","full_name":"SH2 domain-containing protein 2A","aliases":["SH2 domain-containing adapter protein","T cell-specific adapter protein","TSAd","VEGF receptor-associated protein"],"length_aa":389,"mass_kda":42.9,"function":"Could be a T-cell-specific adapter protein involved in the control of T-cell activation. May play a role in the CD4-p56-LCK-dependent signal transduction pathway. Could also play an important role in normal and pathological angiogenesis. Could be an adapter protein that facilitates and regulates interaction of KDR with effector proteins important to endothelial cell survival and proliferation","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9NP31/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SH2D2A","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SH2D2A","total_profiled":1310},"omim":[{"mim_id":"614968","title":"SH2 DOMAIN-CONTAINING PROTEIN 4A; SH2D4A","url":"https://www.omim.org/entry/614968"},{"mim_id":"604514","title":"SH2 DOMAIN PROTEIN 2A; SH2D2A","url":"https://www.omim.org/entry/604514"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":13.8}],"url":"https://www.proteinatlas.org/search/SH2D2A"},"hgnc":{"alias_symbol":["TSAd","F2771"],"prev_symbol":[]},"alphafold":{"accession":"Q9NP31","domains":[{"cath_id":"3.30.505.10","chopping":"67-186","consensus_level":"high","plddt":93.9623,"start":67,"end":186}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NP31","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NP31-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NP31-F1-predicted_aligned_error_v6.png","plddt_mean":61.16},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SH2D2A","jax_strain_url":"https://www.jax.org/strain/search?query=SH2D2A"},"sequence":{"accession":"Q9NP31","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NP31.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NP31/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NP31"}},"corpus_meta":[{"pmid":"15962004","id":"PMC_15962004","title":"VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis.","date":"2005","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/15962004","citation_count":221,"is_preprint":false},{"pmid":"22689825","id":"PMC_22689825","title":"VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd.","date":"2012","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/22689825","citation_count":195,"is_preprint":false},{"pmid":"9468509","id":"PMC_9468509","title":"Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9468509","citation_count":59,"is_preprint":false},{"pmid":"11528519","id":"PMC_11528519","title":"The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple 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The protein is expressed in activated T cells, localizes to the cytoplasm when expressed in COS cells, and is itself tyrosine-phosphorylated (~52 kDa) upon PHA stimulation of peripheral blood mononuclear cells.\",\n      \"method\": \"cDNA cloning, transfection in COS cells with localization by immunostaining, immunoprecipitation with anti-phosphotyrosine antibody\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — initial characterization with multiple orthogonal methods (cloning, cell expression, immunoprecipitation), single lab\",\n      \"pmids\": [\"9468509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TSAd localizes to the T cell nucleus via an active import process that requires a functional SH2 domain recognizing a phosphotyrosine-containing ligand (~95-100 kDa). TSAd acts as a transcriptional activator in T cells and upregulates IL-2 promoter activity in a manner dependent on an intact SH2 domain, but does not modulate NF-κB, NFAT, or AP-1 activity.\",\n      \"method\": \"Subcellular fractionation, nuclear import assays, SH2 domain mutant transfection in T cells, IL-2 promoter-reporter cotransfection assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (fractionation, reporter assay, mutagenesis) in single lab\",\n      \"pmids\": [\"11413197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The TSAd SH2 domain ligand involved in nuclear import was identified as p97 Valosin-containing protein (VCP), which is phosphorylated on Y805 by TCR-activated tyrosine kinases. Direct interaction between endogenous TSAd and VCP was demonstrated in T cells. VCP/CDC48 (yeast homolog) is required for TSAd nuclear transport, established using CDC48 mutant yeast.\",\n      \"method\": \"Microchemical identification (mass spectrometry), co-immunoprecipitation of endogenous proteins, direct interaction assay, CDC48 mutant yeast complementation\",\n      \"journal\": \"Immunology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous proteins, genetic epistasis in yeast, identification by MS; single lab\",\n      \"pmids\": [\"15752563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Transcriptional activation of SH2D2A in T cells requires a cAMP response element (CRE) centered at position -117 bp from the first coding ATG. CD3 stimulation-induced SH2D2A expression requires cAMP-dependent protein kinase activity, and cAMP analogs potently induce SH2D2A mRNA.\",\n      \"method\": \"Reporter gene assays (luciferase), EMSA with nuclear extracts, real-time RT-PCR, PKA inhibitor treatment\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (reporter, EMSA, RT-PCR, pharmacological inhibition), single lab\",\n      \"pmids\": [\"15128801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TSAd interacts with Lck through Lck's SH2 and SH3 domains, and TSAd is a substrate for Lck phosphorylation. The TSAd C-terminus (proline-rich region and five tyrosines) is both necessary and sufficient for interaction with and phosphorylation by Lck. TSAd expression causes hyperphosphorylation of Lck at Y394 and Y505, reducing the Y394/Y505 ratio, thereby inhibiting Lck activity. The C-terminus alone is sufficient to inhibit the hyperactive Lck Y505F mutant.\",\n      \"method\": \"Co-immunoprecipitation, Lck SH2/SH3 domain pull-down, Lck kinase activity assays, TSAd deletion mutant transfection in Jurkat T cells, phosphospecific immunoblotting\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro binding assays, kinase assays with mutagenesis, and functional read-out in cells; multiple orthogonal methods in single lab with rigorous domain mapping\",\n      \"pmids\": [\"15827961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VEGFR-2 phosphorylation at Y951 (within the kinase insert domain) creates a binding site for TSAd. TSAd is expressed in tumor vessels, and Y951-TSAd signaling is required for VEGF-A-induced actin stress fiber formation and endothelial cell migration (but not mitogenesis). TSAd-deficient mice show reduced tumor vascularization and growth.\",\n      \"method\": \"Pan-phosphorylation site mapping of VEGFR-2, phosphopeptide binding assays, TSAd siRNA in endothelial cells, Y951F VEGFR-2 mutation, TSAd knockout mice with tumor implantation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (phosphosite mapping, peptide competition, siRNA, KO mice with phenotype), replicated in subsequent studies\",\n      \"pmids\": [\"15962004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TSAd interacts with Lck through three modes: (1) TSAd aa239-256 binds the Lck-SH3 domain; (2) one or more tyrosines within TSAd aa239-334 (exon 7) bind the Lck-SH2 domain; (3) the TSAd SH2 domain also interacts with Lck. The exon 7-encoded region (aa239-334) is essential for Lck modulation and for TSAd nuclear translocation. TSAd-Lck interaction was confirmed in human CD4+ T cells ex vivo.\",\n      \"method\": \"Co-immunoprecipitation with TSAd isoform/deletion mutants in Jurkat cells and primary CD4+ T cells, domain-binding assays\",\n      \"journal\": \"BMC immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic domain mapping with multiple isoforms, confirmed in primary T cells; single lab\",\n      \"pmids\": [\"16839418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TSAd interacts with Smad2 and Smad3, primarily through the TSAd type I SH2 domain. Both Smad2 and Smad3 also interact with the Lck type I SH2 domain but not the PI3K type III SH2 domain.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, GST pull-down assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — interaction shown by yeast two-hybrid, co-IP and pull-down in single lab; no functional validation of the TSAd-Smad interaction consequence\",\n      \"pmids\": [\"16806069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SH2D2A expression is regulated at both transcriptional and translational levels. cAMP signaling alone induces TSAd mRNA but not protein. TCR engagement is required for both transcription and translation of TSAd. cAMP signaling primes T cells for more rapid TSAd protein expression upon TCR stimulation.\",\n      \"method\": \"RT-PCR for mRNA quantification, immunoblotting for protein levels, pharmacological modulation of cAMP and TCR signaling in primary T cells\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — orthogonal mRNA/protein measurements with multiple signaling conditions, single lab\",\n      \"pmids\": [\"18160104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TSAd interacts with the 67 kDa laminin-binding protein (LBP) as identified by yeast two-hybrid and confirmed by co-immunoprecipitation in D1.1 T cells. TSAd-LBP interaction occurs upon TCR plus laminin or TCR plus integrin α6 costimulation. Overexpression of TSAd enhances TCR+laminin-induced T cell migration, while dominant-negative TSAd or TSAd shRNA disrupts this migration.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, overexpression and shRNA knockdown in D1.1 T cells with migration assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP and functional assay in single lab; no domain-level mapping of the TSAd-LBP interaction\",\n      \"pmids\": [\"19561400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TSAd is required for tyrosine phosphorylation of the Lck substrate Itk (at Y511). TSAd promotes Itk phosphorylation by Lck by bridging the two kinases. TSAd interaction with Itk through its proline-rich region is necessary for TSAd to enhance CXCL12-induced actin polymerization and migration of Jurkat T cells. TSAd-deficient murine thymocytes show reduced Itk phosphorylation, actin polymerization, and migration in response to CXCL12.\",\n      \"method\": \"Co-immunoprecipitation, Itk phospho-specific immunoblotting, TSAd proline-rich region mutants, Jurkat migration assays, TSAd-deficient mouse thymocyte assays, actin polymerization (flow cytometry)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, phospho-blot, domain mutants, KO mouse cells, functional assays), replicated in cell line and primary cells\",\n      \"pmids\": [\"20305788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VEGFR2 Y951 phosphorylation facilitates binding of VEGFR2 to the SH2 domain of TSAd. TSAd in turn regulates VEGF-induced c-Src activation (increased pY418, reduced pY527). TSAd silencing blocked VEGF-induced c-Src activation but not PLCγ, ERK, or eNOS pathways. TSAd forms a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. TSAd-deficient mice show impaired VEGF-induced vascular permeability (Evans blue, dextran, microsphere extravasation) but normal histamine-induced permeability.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, phospho-specific immunoblotting, Tsad siRNA in endothelial cells, tsad-/- mice with permeability assays (Evans blue, dextran, microspheres), confocal microscopy of VE-cadherin junctions\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, multiple KO mouse permeability assays, siRNA with pathway-specific read-outs; multiple orthogonal methods, independent replication of VEGFR2-TSAd interaction\",\n      \"pmids\": [\"22689825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Itk and TSAd promote TCR-dependent phosphorylation of Lck at Tyr192 within the Lck SH2 domain. Phospho-Tyr192 of Lck preferentially recruits actin cytoskeleton regulators (including Itk and TSAd) compared to unphosphorylated Lck. Lck Y192 phosphorylation is required for proper T cell activation and T cell–APC conjugate formation.\",\n      \"method\": \"Phosphopeptide arrays, biochemical binding assays, phospho-specific immunoblotting, T cell–APC conjugation assay, Lck point mutant transfection\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphopeptide arrays plus biochemical assays plus functional T cell assays, single lab\",\n      \"pmids\": [\"25492967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TSAd binds Nck via Nck SH2 domain interaction with TSAd phospho-Tyr280 and phospho-Tyr305, and via Nck SH3 domains interacting with TSAd's proline-rich region. TSAd promotes Nck interaction with Lck and SLP-76 (but not Vav1) in T cells. TSAd expression increases polymerized actin in a manner dependent on TSAd exon 7 (which encodes Nck and Lck interaction sites). TSAd and Nck co-localize in Jurkat cells.\",\n      \"method\": \"SMALI algorithm prediction, peptide array, pull-down, co-immunoprecipitation, confocal microscopy, imaging flow cytometry, actin polymerization assay in TSAd-deficient Jurkat cells\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal binding methods (peptide array, pull-down, co-IP), functional actin assay with genetic deletion, single lab\",\n      \"pmids\": [\"26163016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The SH2 and proline-rich domains of TSAd bridge VEGFR2 and c-Src, and this bridging is required for localization of activated c-Src to endothelial junctions and elongation of growing angiogenic sprouts. TSAd-deficient embryoid bodies fail to sprout in response to VEGF; TSAd-deficient mice show impaired tracheal vessel development and reduced sprouting in VEGF-Matrigel plugs (but not FGF-Matrigel plugs). TSAd-deficient embryoid bodies fail to phosphorylate/turn over VE-cadherin at junctions.\",\n      \"method\": \"Domain deletion mutants in endothelial cells, tsad-/- embryoid body sprouting assay, tsad-/- mouse tracheal and retinal vasculature analysis, VEGF/FGF Matrigel plugs, c-Src localization by immunofluorescence\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple tissue/assay systems, domain-mapping in cells, multiple orthogonal phenotypic read-outs; single lab but rigorous\",\n      \"pmids\": [\"27436360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TSAd modulates polarization of CD4+ T cells towards the APC. Sh2d2a-/- CD4+ T cells show impaired polarization of F-actin and TCR to the immunological synapse, and defective polarization of PKCξ, PAR3, and the MTOC. TSAd-deficient T cells preferentially differentiate toward effector memory (Tem) rather than central memory (Tcm) phenotype.\",\n      \"method\": \"Imaging flow cytometry of Sh2d2a-/- T cells with APC, F-actin/TCR/PKCξ/PAR3/MTOC localization, in vitro T cell differentiation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with direct localization readouts and differentiation assay; single lab\",\n      \"pmids\": [\"30190583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TSAd is regulated epigenetically downstream of Myo9b: Myo9b deficiency causes H3K27me3 enrichment at the TSAd promoter, reducing TSAd expression. TSAd knockdown in naive T cells recapitulates the Myo9b-/- phenotype of reduced TH1 and increased TH17 differentiation. TSAd siRNA in vivo suppresses malignant pleural effusion development and shifts TH1/TH17 balance.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for H3K27me3 at TSAd promoter, TSAd siRNA in primary T cells with TH1/TH17 differentiation assay, in vivo siRNA delivery with pleural effusion model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP demonstrating epigenetic regulation plus functional siRNA rescue experiments in vitro and in vivo; single lab\",\n      \"pmids\": [\"33046503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Affinity-purification mass spectrometry identified DOK2 and PTPN11 as novel ligands of the TSAd SH2 domain. The specific phosphotyrosines on DOK2 and PTPN11 responsible for TSAd SH2 interaction were determined. CRISPR/Cas9 ablation of TSAd and DOK2 in Jurkat T cells resulted in altered tyrosine phosphorylation, suggesting TSAd functions as a negative signaling node via DOK2 and PTPN11.\",\n      \"method\": \"Affinity-purification mass spectrometry (AP-MS), phosphopeptide mapping, CRISPR/Cas9 knockouts in Jurkat T cells, phosphotyrosine immunoblotting\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased AP-MS with functional CRISPR validation; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.02.11.636929\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Multi-omics data (single-cell RNA-seq, bulk RNA-seq, epigenomic/ATAC, mass spectrometry-based proteomics) from multiple organs indicate that TSAd transcripts are largely absent from endothelial cells, the TSAd promoter resides in closed chromatin in ECs, and TSAd protein is typically undetected in EC proteomes. This challenges the proposed universal role of TSAd in VEGFR2-mediated vascular permeability signaling in ECs.\",\n      \"method\": \"Mining of public scRNA-seq, bulk RNA-seq, epigenomic (chromatin accessibility), and MS proteomics datasets from endothelial cells across multiple organs\",\n      \"journal\": \"Angiogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal omics data types across organs; negative/contradictory finding that challenges prior mechanistic claims; single published study\",\n      \"pmids\": [\"40080216\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSAd (SH2D2A) is a T cell- and endothelial cell-expressed adapter protein that lacks catalytic activity but contains an SH2 domain, proline-rich region, and multiple tyrosines; it is tyrosine-phosphorylated upon activation and (1) in T cells, binds Lck (via SH3/SH2 interactions) to modulate Lck activity and promote phosphorylation of Itk at Y511, scaffolds Nck-Lck-SLP-76 complexes to drive actin polymerization, undergoes SH2-domain-dependent nuclear import via VCP/p97, and acts as a transcriptional activator for IL-2; (2) in endothelial cells, the SH2 domain binds phospho-Y951 of VEGFR2 while the proline-rich domain engages c-Src, thereby localizing active c-Src to endothelial junctions to regulate VE-cadherin turnover, vascular permeability, and angiogenic sprouting in a tissue-specific manner, though recent omics evidence questions whether TSAd is broadly expressed in endothelial cells across all organs.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SH2D2A (TSAd) is a catalytically inactive adapter protein that couples antigen- and growth-factor receptor signaling to cytoskeletal remodeling, migration, and transcription through an SH2 domain, a proline-rich region, and multiple phosphorylatable tyrosines [#0]. In T cells, TSAd expression is induced upon TCR engagement and is regulated both transcriptionally—via a CRE element requiring PKA activity, with cAMP priming cells for rapid protein production—and translationally [#3, #8]. Once expressed and tyrosine-phosphorylated, TSAd engages Lck through three modes (Lck-SH3, Lck-SH2, and the TSAd SH2 domain) and acts as a substrate that drives Lck hyperphosphorylation at Y394 and Y505 to modulate its activity, and promotes Lck phosphorylation of Lck Y192 within the Lck SH2 domain [#4, #6, #12]. By bridging Lck to its substrate Itk, TSAd is required for Itk Y511 phosphorylation, and it scaffolds Nck with Lck and SLP-76 via phospho-Y280/Y305 and its proline-rich region to drive actin polymerization and chemokine-induced migration [#10, #13]. These activities underlie TSAd control of CD4+ T cell polarization toward the APC, immunological synapse organization, and effector-versus-central memory differentiation [#15]. TSAd also undergoes SH2-domain-dependent nuclear import—using VCP/p97 (phosphorylated on Y805) as its phosphotyrosine ligand—where it activates the IL-2 promoter [#1, #2]. In endothelial cells, the TSAd SH2 domain binds VEGFR2 phospho-Y951 while its proline-rich region engages c-Src, bridging the two within a VE-cadherin/VEGFR2/c-Src complex to localize active c-Src to junctions and thereby regulate VE-cadherin turnover, VEGF-induced vascular permeability, and angiogenic sprouting [#5, #11, #14]. The breadth of this endothelial role across organs is contested by multi-omics data indicating TSAd is largely absent from endothelial cells [#18].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established TSAd as a non-catalytic adapter, defining the structural toolkit (SH2 domain, phosphotyrosine-binding motifs) through which it would later be shown to act.\",\n      \"evidence\": \"cDNA cloning, COS-cell expression with immunostaining, and anti-phosphotyrosine immunoprecipitation in PHA-stimulated PBMCs\",\n      \"pmids\": [\"9468509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No binding partners identified\", \"No functional readout beyond inducible phosphorylation\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed TSAd has a nuclear, transcriptional function, answering whether this cytoplasmic adapter influences gene expression and linking its SH2 domain to both nuclear import and IL-2 promoter activation.\",\n      \"evidence\": \"Subcellular fractionation, SH2-mutant transfection, and IL-2 promoter-reporter assays in T cells\",\n      \"pmids\": [\"11413197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the ~95-100 kDa SH2 ligand unknown at this stage\", \"Mechanism of transcriptional activation not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified VCP/p97 as the phosphotyrosine ligand driving TSAd nuclear import and defined how SH2D2A transcription is controlled, connecting receptor signaling to TSAd induction and trafficking.\",\n      \"evidence\": \"MS identification and endogenous co-IP of VCP, CDC48 mutant yeast complementation (#2); luciferase reporters, EMSA, RT-PCR and PKA inhibition (#3)\",\n      \"pmids\": [\"15752563\", \"15128801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of nuclear TSAd-VCP interaction on specific target genes unresolved\", \"Whether VCP-dependent import is required for IL-2 activation not directly tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined two distinct receptor-proximal roles: TSAd as a multivalent Lck binder and negative regulator of Lck activity in T cells, and as a VEGFR2 pY951 effector required for endothelial migration and tumor vascularization.\",\n      \"evidence\": \"Lck SH2/SH3 pull-downs, kinase assays and deletion mutants in Jurkat cells (#4); VEGFR2 phosphosite mapping, siRNA, Y951F mutation and TSAd-KO mouse tumor implantation (#5)\",\n      \"pmids\": [\"15827961\", \"15962004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TSAd reconciles opposing roles in two cell types unclear\", \"Downstream effectors of VEGFR2-TSAd not yet identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapped the TSAd-Lck interaction to three binding modes and localized the exon 7 region essential for both Lck modulation and nuclear translocation, and reported TSAd binding to Smad2/3.\",\n      \"evidence\": \"Domain/isoform co-IP in Jurkat and primary CD4+ T cells (#6); yeast two-hybrid, co-IP and GST pull-down for Smad2/3 (#7)\",\n      \"pmids\": [\"16839418\", \"16806069\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of TSAd-Smad2/3 interaction never validated\", \"Specific tyrosines binding Lck-SH2 not pinpointed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved that SH2D2A is dual-controlled at transcription and translation, explaining how cAMP priming and TCR engagement jointly gate TSAd protein output.\",\n      \"evidence\": \"RT-PCR, immunoblotting and pharmacological cAMP/TCR modulation in primary T cells\",\n      \"pmids\": [\"18160104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of translational control not defined\", \"Identity of translation-regulating signals downstream of TCR unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Implicated TSAd in laminin/integrin-costimulated T cell migration via interaction with the 67 kDa laminin-binding protein.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, overexpression and shRNA migration assays in D1.1 T cells\",\n      \"pmids\": [\"19561400\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No domain-level mapping of the TSAd-LBP interaction\", \"Single lab, no reciprocal validation\", \"Connection to canonical TSAd-Lck/Nck pathway unestablished\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated that TSAd bridges Lck and Itk to enable Itk Y511 phosphorylation, providing a mechanistic basis for TSAd-driven chemokine-induced actin polymerization and migration.\",\n      \"evidence\": \"Co-IP, phospho-Itk blotting, proline-rich mutants, Jurkat migration assays, and TSAd-deficient mouse thymocyte assays with actin polymerization readouts\",\n      \"pmids\": [\"20305788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the Lck-TSAd-Itk ternary assembly not defined\", \"Whether nuclear and cytoskeletal pools of TSAd are functionally separable unaddressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the endothelial TSAd permeability module, showing TSAd assembles a VE-cadherin/VEGFR2/c-Src complex at junctions and is required for VEGF-induced c-Src activation and vascular permeability.\",\n      \"evidence\": \"Reciprocal endogenous co-IP, phospho-blotting, siRNA with pathway-specific readouts, and tsad-/- mouse permeability assays with confocal junction imaging\",\n      \"pmids\": [\"22689825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity for c-Src over PLCγ/ERK/eNOS mechanistically unexplained\", \"Direct contribution of nuclear TSAd functions to EC phenotype untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed TSAd and Itk drive Lck Y192 phosphorylation within the Lck SH2 domain, creating a docking surface that recruits cytoskeletal regulators and supports T cell-APC conjugate formation.\",\n      \"evidence\": \"Phosphopeptide arrays, biochemical binding, phospho-specific blotting, Lck point mutants and T cell-APC conjugation assays\",\n      \"pmids\": [\"25492967\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Lck Y192 phosphorylation is direct or indirect not fully resolved\", \"Quantitative contribution of TSAd versus Itk to Y192 phosphorylation unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established TSAd as a Nck-recruiting scaffold, linking phospho-Y280/Y305 and the proline-rich region to assembly of Nck-Lck-SLP-76 complexes that drive actin polymerization.\",\n      \"evidence\": \"SMALI prediction, peptide arrays, pull-down, co-IP, confocal imaging and actin assays in TSAd-deficient Jurkat cells\",\n      \"pmids\": [\"26163016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why Vav1 is excluded from the complex not explained\", \"In vivo relevance of the Nck-Lck-SLP-76 assembly not tested in primary cells\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected TSAd's cytoskeletal scaffolding role to T cell biology, showing it controls synapse polarization, MTOC orientation and effector/central memory differentiation.\",\n      \"evidence\": \"Imaging flow cytometry of Sh2d2a-/- T cells with APC, polarity-marker localization and in vitro differentiation assays\",\n      \"pmids\": [\"30190583\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between polarity defects and memory skewing not defined\", \"Single-cell heterogeneity of the phenotype unaddressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed TSAd downstream of an epigenetic regulatory axis, showing Myo9b loss silences TSAd via H3K27me3 and that TSAd controls TH1/TH17 balance with disease relevance in pleural effusion.\",\n      \"evidence\": \"ChIP for H3K27me3 at the TSAd promoter, TSAd siRNA differentiation assays, and in vivo siRNA in a malignant pleural effusion model\",\n      \"pmids\": [\"33046503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets of TSAd in TH differentiation unidentified\", \"Mechanism coupling TSAd adapter activity to lineage commitment unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the TSAd SH2 interactome to negative-signaling phosphatases/adapters DOK2 and PTPN11, recasting TSAd as a potential negative signaling node in T cells.\",\n      \"evidence\": \"Affinity-purification mass spectrometry, phosphopeptide mapping and CRISPR/Cas9 TSAd/DOK2 knockouts in Jurkat cells (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.02.11.636929\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Functional consequence of DOK2/PTPN11 recruitment on specific pathways not resolved\", \"Reconciliation with TSAd's positive scaffolding roles unaddressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Challenged the universality of the endothelial TSAd model by showing TSAd transcripts and protein are largely absent from endothelial cells across organs.\",\n      \"evidence\": \"Mining of public scRNA-seq, bulk RNA-seq, chromatin accessibility and MS proteomics datasets from endothelial cells across multiple organs\",\n      \"pmids\": [\"40080216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not exclude regulated or context-specific EC expression\", \"Discordance with functional KO permeability phenotypes unresolved\", \"Single study, detection-sensitivity limits not fully addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TSAd integrates its opposing positive (Lck/Itk/Nck scaffolding, IL-2 activation) and negative (Lck inhibition, DOK2/PTPN11 recruitment) functions, and in which cell types the VEGFR2 module is physiologically operative, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling positive and negative signaling roles\", \"Definitive cell-type-resolved expression map of TSAd lacking\", \"No structural model of TSAd in any signaling complex\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4, 10, 13, 14]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 10, 15, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 11, 14]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [11, 14]}\n    ],\n    \"complexes\": [\n      \"VE-cadherin/VEGFR2/c-Src junctional complex\",\n      \"Nck-Lck-SLP-76 complex\"\n    ],\n    \"partners\": [\n      \"LCK\",\n      \"ITK\",\n      \"NCK1\",\n      \"VEGFR2\",\n      \"SRC\",\n      \"VCP\",\n      \"DOK2\",\n      \"PTPN11\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}