{"gene":"NCK1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1992,"finding":"NCK1 (Nck) SH2 domain mediates association with tyrosine-autophosphorylated PDGF and EGF receptors in response to ligand activation; Nck is phosphorylated on tyrosine, serine, and threonine residues upon growth factor stimulation; ~5-fold overexpression of Nck in NIH 3T3 cells induces oncogenic foci formation.","method":"Co-immunoprecipitation with anti-Nck antibodies, in vitro binding with GST-Nck fusion protein, NIH 3T3 focus formation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, GST pulldown, and functional transformation assay in same study; foundational paper replicated by subsequent work","pmids":["1333047"],"is_preprint":false},{"year":1992,"finding":"Overexpression of NCK1 in 3Y1 rat fibroblasts causes transformation (altered morphology, soft agar colony formation, tumor formation in nude mice) without detectable elevation of phosphotyrosine levels; GST-Nck binds p60v-src and serine/threonine kinases from cell lysates.","method":"Soft agar colony formation, tumor formation in nude mice, GST fusion protein pulldown, Western blot","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays in one study; independently consistent with PMID:1333047","pmids":["1280326"],"is_preprint":false},{"year":1995,"finding":"The C-terminal (third) SH3 domain of NCK1 (p47nck) specifically interacts with WASP (Wiskott-Aldrich syndrome protein) in vivo and in vitro; WASP is mainly cytosolic.","method":"Yeast two-hybrid/cDNA expression library screen, in vitro and in vivo binding assays, subcellular fractionation, Western blot","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (in vivo + in vitro binding, fractionation), replicated in subsequent studies on Nck-WASP/N-WASP interactions","pmids":["7565724"],"is_preprint":false},{"year":1996,"finding":"NCK1 binds PAK1 through its second SH3 domain; PAK1 interacts with Nck via its first N-terminal proline-rich SH3-binding motif; the Nck–PAK1 interaction is strengthened upon PDGF receptor stimulation; active PAK1 association with Nck leads to phosphorylation of Nck at multiple sites.","method":"Co-immunoprecipitation in COS-7 and Swiss 3T3 cells, in vitro binding, domain-mapping with individual SH3 domain constructs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and in vitro binding, replicated independently by Galisteo et al. (PMID:8798379) in same year","pmids":["8824201"],"is_preprint":false},{"year":1996,"finding":"NCK1 SH2 domain recruits Nck to activated EGF and PDGF receptors; Nck second SH3 domain binds PAK1 proline-rich N-terminus; Nck–PAK1 interaction links receptor tyrosine kinases to PAK1 and enhances PAK1 kinase activity when Nck is co-transfected.","method":"Co-immunoprecipitation in living cells, domain mapping, kinase activity assay, co-transfection in HeLa cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods, independently replicated by Bokoch et al. (PMID:8824201)","pmids":["8798379"],"is_preprint":false},{"year":1998,"finding":"NCK1 SH2 domain binds phosphorylated juxtamembrane Tyr594 of EphB1 receptor; Nck recruitment to EphB1 is required for downstream JNK/SAPK activation and cell attachment responses.","method":"Yeast two-hybrid screen, co-immunoprecipitation of native receptor complexes, site-directed mutagenesis (Y594F), JNK activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid, reciprocal Co-IP, mutagenesis, and functional JNK assay in single study; domain mapping confirmed","pmids":["9430661"],"is_preprint":false},{"year":1998,"finding":"NCK1 and PAK1 form a signaling module downstream of TCR activation; this module is required specifically for NFAT activation but not for JNK activation; Pak1 acts downstream of Vav and Cdc42; dominant-negative Nck or Pak1 inhibits TCR-mediated NFAT and Erk2 activation.","method":"Co-immunoprecipitation, dominant-negative overexpression, NFAT luciferase reporter assay, kinase activity assay in T cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays and epistasis analysis in T cells; pathway position defined","pmids":["9755165"],"is_preprint":false},{"year":2000,"finding":"NCK1 second SH3 domain binds an 18-mer PAK-derived peptide containing the motif PXXPXRXXS; serine phosphorylation within this motif negatively regulates NCK1-PAK1 binding; PAK autophosphorylation blocks binding of both Nck and PIX to PAK1, providing a mechanism to regulate PAK interactions and its cycling between cytosolic and focal complex sites.","method":"Saturation substitution binding analysis, in vitro binding assays with peptides and full-length proteins, phosphorylation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and binding analysis; mechanistic model supported by multiple experiments in one study","pmids":["10805734"],"is_preprint":false},{"year":2000,"finding":"Nck-interacting kinase (NIK) is activated by EphB1 and EphB2 receptors in a manner requiring EphB1 kinase activity and phosphorylation of juxtamembrane Tyr594; NIK activation through NCK1 is required for EphB1-mediated integrin activation and JNK activation; a complex of NIK/Nck, p62(dok), and RasGAP forms upon EphB receptor stimulation.","method":"Co-immunoprecipitation, kinase activity assays, dominant-negative experiments, integrin activation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple methods (Co-IP, kinase assay, DN experiments), pathway position established by epistasis","pmids":["10669731"],"is_preprint":false},{"year":2002,"finding":"A 12-amino-acid sequence of EPEC Tir containing phospho-Tyr474 binds the host adaptor NCK1; NCK1 is recruited to actin pedestals of EPEC in a Y474 phosphorylation-dependent manner and is required for EPEC-induced actin signaling.","method":"Chimeric protein expression, in vivo binding assay, immunofluorescence localization of endogenous Nck to pedestals","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphopeptide-dependent binding, genetic chimera epistasis, and direct localization assay; replicated and extended by crystal structure studies (PMID:16636066)","pmids":["11918809"],"is_preprint":false},{"year":2002,"finding":"NCK1 SH3 domains interact directly with DCC (deleted in colorectal cancer) receptor, independent of the SH2 domain; this interaction mediates DCC/Nck-1 association with the actin cytoskeleton and is required for netrin-1-induced Rac1 activation and neurite outgrowth.","method":"Co-immunoprecipitation in commissural neurons, dominant-negative Nck-1 overexpression, Rac1 activation assay, neurite outgrowth assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, DN functional assay, and Rac1 pulldown; single lab but multiple orthogonal methods","pmids":["12149262"],"is_preprint":false},{"year":2002,"finding":"NCK1 SH3 domains interact with eIF2β; Nck is present in ribosomal fractions and its ribosomal localization is enhanced by insulin; overexpression of Nck-1 enhances both cap-dependent and -independent protein translation in a manner requiring the first and third SH3 domains; Nck-1 directly increases protein translation in vitro.","method":"Co-immunoprecipitation, cell fractionation/ribosomal enrichment, in vitro translation assay, domain mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, fractionation, and in vitro translation reconstitution; single lab but multiple orthogonal methods","pmids":["11959995"],"is_preprint":false},{"year":2002,"finding":"Grb2 and NCK1 act cooperatively to promote actin-based motility of vaccinia virus; Nck is recruited to viral particles via phospho-Tyr112 of A36R, whereas Grb2 is recruited via phospho-Tyr132; the Grb2 pathway requires prior Nck-mediated N-WASP recruitment.","method":"Immunofluorescence, phosphomutant constructs, actin tail counting, co-immunoprecipitation","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphomutant epistasis, quantitative actin tail assay, replicated by subsequent poxvirus studies (PMID:20006842, 24284073)","pmids":["12007418"],"is_preprint":false},{"year":2004,"finding":"ER-associated NCK1 represses ERK-1 activation under basal conditions; upon ER stress, this pool of Nck dissociates from ER membranes, allowing ERK-1 activation; Nck-null cells show stronger ERK-1 activation after ER stress correlating with enhanced cell survival.","method":"Immunoprecipitation of ER membranes, in vitro reconstitution with ER membranes, Nck-null fibroblasts, kinase assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ER reconstitution, Nck-null cells, and kinase assay; single lab but multiple orthogonal approaches","pmids":["15201339"],"is_preprint":false},{"year":2006,"finding":"Crystal structures of Nck1 and Nck2 SH2 domains in complex with phosphopeptides from EPEC Tir establish highly conserved phosphopeptide recognition; Nck1 and Nck2 SH2 domains have essentially indistinguishable binding specificities; GIT1 is confirmed experimentally as a new Nck binding partner.","method":"X-ray crystallography, phosphopeptide binding studies, sequential mutagenesis, pulldown assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with binding validation and mutagenesis; rigorous structural study in single lab","pmids":["16636066"],"is_preprint":false},{"year":2007,"finding":"NCK1 nuclear accumulation (carried by SOCS7 via nuclear import/export signals) causes actin stress fiber disintegration and cell polarity loss; nuclear NCK1 is essential for cell-cycle arrest after DNA damage; the septin–SOCS7–NCK axis functions downstream of ATM/ATR and is required for p53 Ser15 phosphorylation.","method":"RNAi knockdown, cytoplasmic vs. nuclear NCK1 rescue constructs, live imaging, DNA damage assays, epistasis with ATM/ATR inhibitors, p53 phosphorylation immunoblot","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including localization rescue, epistasis, and p53 phosphorylation in high-impact study","pmids":["17803907"],"is_preprint":false},{"year":2007,"finding":"NCK1 (and NCK2) are the most potent activators of WASP and N-WASP in vitro; Nck1/2 differentially regulate WASP vs. N-WASP (Nck is a more potent activator than Cdc42 or Rac1); in vitro actin nucleation assays with purified full-length proteins.","method":"In vitro actin nucleation assay with purified full-length WASP/N-WASP, Arp2/3 complex, and various activators","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified full-length proteins and quantitative comparison; single lab","pmids":["17302440"],"is_preprint":false},{"year":2008,"finding":"NCK1 SH3.1 domain forms a complex with an atypical PxxDY motif at Tyr166 of CD3ε (non-ITAM-requiring interaction); Nck binding inhibits phosphorylation of the CD3ε ITAM by Fyn and Lck in vitro; Nck–CD3ε interaction downregulates TCR surface expression upon physiological stimulation.","method":"NMR structure of complex, in vitro kinase assay, flow cytometry for TCR surface expression in primary mouse lymphocytes","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure combined with functional kinase assay and primary cell experiments; multiple orthogonal methods","pmids":["18555270"],"is_preprint":false},{"year":2009,"finding":"Src phosphorylates Tks5 at Y557, inducing direct association with NCK1 (and NCK2) SH2 domains in invadopodia; Tks5 mutants unable to bind Nck show reduced matrix degradation and inefficient actin recruitment; Nck1 depletion inhibits Src–Tks5-driven matrix proteolysis and actin assembly in invadopodia.","method":"Co-immunoprecipitation, site-directed mutagenesis of Y557, Nck1 siRNA knockdown, gelatin matrix degradation assay, immunofluorescence","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, KD with specific phenotypic readout; multiple orthogonal methods in single study","pmids":["19596797"],"is_preprint":false},{"year":2009,"finding":"NCK1 SH2 domain binds phospho-Tyr602 of EphA3 receptor; this interaction mediates EphA3-induced inhibition of cell migration and process retraction; dominant-negative Nck1 mutants lacking SH2 or SH3 domains partially alleviate EphA3-mediated inhibitory effects.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, SH2 domain mutagenesis, cell migration assay","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid, GST pulldown, Co-IP with mutagenesis, and functional migration assay; single lab","pmids":["19505147"],"is_preprint":false},{"year":2009,"finding":"NCK1 is essential for actin remodeling stimulated by PI(4,5)P2, and PI(4,5)P2 is necessary for NCK1-induced localized actin polymerization in vivo; Nck knockdown/knockout suppresses PI(4,5)P2-induced actin comets; Nck couples phosphotyrosine- and phosphoinositide-dependent signals to N-WASp/Arp2/3 actin polymerization.","method":"Nck siRNA knockdown, Nck-knockout cells, antibody-induced Nck aggregation, live-cell imaging of actin comets, PI(4,5)P2 manipulation with inositol 5-phosphatase","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — Nck KO and KD with multiple complementary approaches, mechanistic pathway placement","pmids":["19917259"],"is_preprint":false},{"year":2010,"finding":"Nck1 specifically localizes to invadopodia of metastatic mammary carcinoma cells but not to podosomes in macrophages or Src-transformed fibroblasts, establishing distinct upstream activators for invadopodia vs. podosomes.","method":"Confocal immunofluorescence microscopy of endogenous Nck1 in multiple cell types","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by imaging across cell types, no functional consequence tested in this study","pmids":["20850195"],"is_preprint":false},{"year":2010,"finding":"Nck1 deletion in obese mice attenuates hepatic IRE1α and JNK activation and IRS-1 phosphorylation at Ser307, improving glucose disposal and insulin signaling; siRNA knockdown of Nck1 in HepG2 cells decreases thapsigargin-induced IRE1α activation.","method":"Nck1−/− obese mice (in vivo), siRNA knockdown in HepG2 cells, kinase activation assays, glucose tolerance tests, insulin signaling (Akt phosphorylation)","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo KO and in vitro siRNA with multiple signaling readouts; two complementary model systems","pmids":["20587749"],"is_preprint":false},{"year":2012,"finding":"The stoichiometry of NCK1-dependent actin polymerization through the N-WASp/Arp2/3 pathway is 4 Nck : 2 N-WASp : 1 Arp2/3; the density of Nck molecules in membrane aggregates is a critical determinant of localized actin polymerization.","method":"Computational modeling (Virtual Cell), antibody-induced aggregation of membrane-targeted Nck SH3 domains, quantitative fluorescence microscopy in living cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative live-cell experimentation combined with computational modeling; orthogonal validation of stoichiometry","pmids":["22613834"],"is_preprint":false},{"year":2013,"finding":"NCK1 (and Nck2) is essential for front-back polarity, polarized membrane protrusion, and directional migration of endothelial cells; Nck depletion causes delocalized Cdc42/Rac activity and reduces RhoA activity and myosin II phosphorylation; Nck couples polarized protrusion formation with adhesion maturation via integrin α5β1-fibronectin interaction.","method":"siRNA knockdown, time-lapse DIC and TIRF microscopy, FRET imaging for Cdc42/Rac/RhoA activity, atomic force microscopy for adhesion force","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including live imaging, FRET, AFM in single study with specific molecular mechanism","pmids":["23444376"],"is_preprint":false},{"year":2013,"finding":"NCK1 (but not Nck2) is a substrate of c-Cbl-mediated ubiquitination at the evolutionarily conserved Lys178; synaptopodin competes with c-Cbl for Nck1 binding, preventing Nck1 ubiquitination; c-Cbl knockdown restores Nck1 protein levels and stress fibers; NCK1-specific SH3 domain 2 drives RhoA activation and actin stress fiber formation.","method":"Ubiquitination assays, site-directed mutagenesis (K178R), co-immunoprecipitation, gene silencing (c-Cbl siRNA), RhoA activation assay, immunofluorescence for stress fibers","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis + ubiquitination assay + competition Co-IP + RhoA functional readout; multiple orthogonal methods","pmids":["24287595"],"is_preprint":false},{"year":2013,"finding":"NCK1 SH2 domain binds phospho-Y561 in the PERK juxtamembrane domain; this interaction negatively regulates PERK activity (pY561PERK is an inhibitory site); Nck1 silencing in MIN6 cells accelerates pY561PERK dephosphorylation, enhances PERK activation, increases insulin biosynthesis and proinsulin content; Nck1-knockout mice pancreatic islets contain more insulin.","method":"Co-immunoprecipitation, site-directed mutagenesis (Y561F), siRNA in MIN6 cells, Nck1-KO mice, PERK phosphorylation kinetics, insulin/proinsulin content assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding mapped by SH2–pY interaction + mutagenesis + KO mice + functional β-cell readout; multiple systems","pmids":["24371088"],"is_preprint":false},{"year":2014,"finding":"NCK1 is required for assembly of the cytoplasmic capping complex; capping enzyme (CE) binds Nck1 via its proline-rich C-terminus; CE and the 5'-kinase are juxtaposed by binding adjacent SH3 domains of Nck1; Nck1 knockdown disrupts complex integrity; cap homeostasis is inhibited by Nck1 with inactivating mutations in the CE- or kinase-binding SH3 domains.","method":"Co-immunoprecipitation, sedimentation, gel filtration, in vitro complex assembly on recombinant Nck1, siRNA knockdown, SH3-domain mutagenesis","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution on recombinant Nck1, sedimentation/gel filtration, mutagenesis, KD functional assay; multiple orthogonal methods","pmids":["25137142"],"is_preprint":false},{"year":2014,"finding":"NCK1 SH3.1 domain binds the proline-rich sequence (PRS) of CD3ε when the first ITAM tyrosine is unphosphorylated; the SH2 domain simultaneously binds the second ITAM tyrosine when phosphorylated; cooperative two-site engagement is required for Nck1 binding to the TCR; SH3.1-mutated Nck1 impairs TCR signaling and T cell activation.","method":"In situ proximity ligation assay, co-purification, domain mutations, TCR signaling readouts in Jurkat T cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — proximity ligation assay + co-purification + mutagenesis + functional signaling readout; multiple orthogonal methods","pmids":["26590318"],"is_preprint":false},{"year":2014,"finding":"NCK1 SH3.1 domain binding to the CD3ε PRS is required for T cell activation in vivo; KI-PRS mice with a mutation preventing Nck–CD3ε interaction show impaired CD3ζ phosphorylation, reduced T cell activation by antigen in vitro, partial protection from EAE, and deficient antitumoral response; a high-affinity SH3.1-blocking peptide inhibits T cell proliferation in vitro and in vivo.","method":"Knock-in mouse model, TCR signaling assays (CD3ζ phosphorylation), EAE model, peptide inhibitor in vivo","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knock-in mouse + peptide inhibitor + in vivo disease model; multiple complementary approaches","pmids":["24470497"],"is_preprint":false},{"year":2015,"finding":"NCK1 and NCK2 inactivation in endothelial cells inhibits Cdc42 and PAK2 activation by VEGF-A and Slit2, preventing front-rear polarity; NCK1/2 binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity; endothelial-specific Nck1/2 deletion causes severe angiogenesis defects in postnatal retina and embryonic development.","method":"Inducible endothelial-specific Nck1/2 knockout mice, Cdc42/PAK2 activation assays, co-immunoprecipitation of NCK with ROBO1, retinal and embryonic vascular phenotyping","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with specific molecular mechanistic readouts (Cdc42/PAK activation, ROBO1 binding) + in vivo vascular phenotypes","pmids":["26659946"],"is_preprint":false},{"year":2015,"finding":"NCK1 (and NCK2) in osteoblasts regulates preosteoblast/osteoblast migration toward IGF1; Nck1 binds IRS-1 (by co-immunoprecipitation); conditional double deletion of Nck1/2 in osteoblasts causes osteopenia, suppresses bone formation rate in vivo, and impairs bone injury repair, without affecting bone resorption.","method":"shRNA knockdown, Nck1 overexpression, co-immunoprecipitation (Nck1/IRS-1), osteoblast-specific conditional KO mice, in vivo bone histomorphometry, calvarial defect model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo with quantitative bone parameters + Co-IP + migration assays; multiple methods and systems","pmids":["26621720"],"is_preprint":false},{"year":2018,"finding":"NCK1 and NCK2 adaptors in pericytes inhibit PDGF-B-induced phosphorylation of PDGFRβ at Y1009 and PAK activation; inactivation of NCK1/2 in mural cells prevents pathological neovascular tuft formation and vascular leakage in oxygen-induced retinopathy model.","method":"Inducible mural cell-specific Nck1/2 knockout mice, PDGFRβ phosphorylation assay, PAK activation assay, oxygen-induced retinopathy model, lineage tracing","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO + molecular phosphorylation readout + in vivo disease model; multiple approaches","pmids":["30150707"],"is_preprint":false},{"year":2018,"finding":"Proteomic analysis identifies 30 NCK1-specific and 28 NCK2-specific interactors; NCK2 (not NCK1) is required for cell abscission during cytokinesis—Nck2-deficient MEFs are multinucleated, show extended intercellular bridges, fail to complete abscission, and have altered midbody composition (mislocalized AURKB, PLK1, ECT2); NCK2 function in cytokinesis requires its SH2 domain.","method":"Affinity purification-MS, BioID proximity labeling, Nck1/Nck2-KO MEFs, live-cell time-lapse imaging of cytokinesis, immunofluorescence of midbody markers, SH2 domain mutants","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO MEFs + live imaging + interactome (two methods) + domain mutagenesis; multiple orthogonal approaches","pmids":["30002203"],"is_preprint":false},{"year":2020,"finding":"Only NCK1 (not NCK2) mediates disturbed flow-induced NF-κB activation in endothelial cells; NCK1 SH2 domain and first SH3 domain are required for this effect; IRAK-1 is identified as an NCK1-selective binding partner; disturbed flow-induced IRAK-1 activation requires NCK1 in vitro and in vivo; Nck1-knockout mice show reduced endothelial activation and atherosclerotic plaque formation.","method":"Nck1-KO and Nck2-KO mice, domain-swap and point-mutation experiments, co-immunoprecipitation (Nck1–IRAK1), NF-κB reporter assays, flow chamber experiments, high fat diet atherosclerosis model, bone marrow chimeras","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mice + domain mutagenesis + Co-IP + in vivo disease model + bone marrow chimeras; comprehensive multi-method study","pmids":["32427580"],"is_preprint":false},{"year":2019,"finding":"NCK1 functions in a common pathway with TNK2 and WASL for picornavirus (EMCV, CVB3, poliovirus, EV-D68) entry/infection; CRISPR deletion of NCK1 reduces infection by multiple picornaviruses; genetic epistasis places NCK1, TNK2, and WASL in the same pathway for viral internalization.","method":"CRISPR deletion, viral infection assays, genetic epistasis analysis, EMCV internalization assay, mouse model (TNK2 KO)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — CRISPR KO + epistasis + internalization assay + in vivo mouse data; multiple methods","pmids":["31769754"],"is_preprint":false},{"year":2011,"finding":"NCK1 (Dock/Nck) forms a stable complex with PTP1B (PTP61F in Drosophila); this Nck/PTP1B complex inducibly associates with the insulin receptor; Nck is required for effective insulin receptor dephosphorylation and inactivation by PTP1B in mammalian cells.","method":"Co-immunoprecipitation, Drosophila genetic assays (PTP61F/Dock interaction), IR dephosphorylation assay in vitro and in vivo","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP + in vivo Drosophila genetics + mammalian cell IR dephosphorylation; two organisms and multiple methods","pmids":["21707536"],"is_preprint":false},{"year":2011,"finding":"PAK1-Nck complex formation sequesters PAK1 in the cytoplasm; co-expression of PAK1 and NCK1 inhibits PRL-induced cyclin D1 promoter activity by 95%; disruption of PAK1-Nck binding partially abolishes this inhibition.","method":"Co-immunoprecipitation, cytoplasmic/nuclear fractionation, cyclin D1 luciferase reporter assay, dominant-negative and binding-deficient mutants","journal":"Molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP + fractionation + luciferase reporter with domain mutants; single lab","pmids":["21719533"],"is_preprint":false},{"year":1999,"finding":"The third (C-terminal) SH3 domain of NCK1 specifically binds Sos; the third SH3 domain also binds dynamin (a GTP-binding protein involved in clathrin-coated vesicle formation); Nck and dynamin co-precipitate from cell lysates.","method":"GST fusion protein pulldown with individual SH3 domains, co-immunoprecipitation from K562 cells, protein identification by molecular mass","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — GST pulldown domain mapping plus co-IP; single lab but two orthogonal methods","pmids":["10206341"],"is_preprint":false},{"year":1999,"finding":"Tyrosine 351 of DokR (Dok-related protein) mediates binding to NCK1 SH2 domain; this interaction is dependent on DokR tyrosine phosphorylation by Lyn kinase; DokR–Nck complex forms upon EGF receptor activation.","method":"GST fusion protein binding assay, site-directed mutagenesis of DokR tyrosines, co-immunoprecipitation in 293T cells and BaF/3 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis + GST pulldown + co-IP; single lab but multiple methods","pmids":["10428862"],"is_preprint":false},{"year":2006,"finding":"cGMP-dependent protein kinase (PKG) phosphorylates PAK1 at Ser21, causing release of NCK1 from PAK1 and promoting formation of a new PAK1-VASP complex; Ser21 phosphorylation of PAK1 regulates cell polarization and tail retraction, and cells expressing S21A-PAK1 show aberrant morphology when NCK/PAK binding is blocked.","method":"In vitro phosphorylation mapping, phosphospecific antibody, co-transfection assays, endothelial cell morphology assays, peptide-mediated disruption of Nck/PAK binding","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase mapping + phosphospecific antibody + cell morphology; single lab","pmids":["16490784"],"is_preprint":false},{"year":2013,"finding":"NCK1 interacts with Cdc42 and the Rho-GEF intersectin-1 (ITSN1) to promote N-WASP-dependent actin polymerization during vaccinia virus actin tail formation and FcγR-mediated phagocytosis; ITSN1 is recruited prior to actin-based motility and functions with Cdc42 in a feed-forward loop with N-WASP and Nck.","method":"Immunofluorescence, siRNA knockdown, co-immunoprecipitation, phagocytosis assay, vaccinia actin tail assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP + KD with two functional readouts (actin tail, phagocytosis); single lab but two parallel biological contexts","pmids":["24284073"],"is_preprint":false},{"year":2012,"finding":"NCK1 is required for FcγR- but not CR3-mediated phagocytosis; Nck is recruited to phagocytic cups via phospho-Tyr282 and Tyr298 in the FcγR ITAM; Nck promotes phagocytosis by recruiting N-WASP and requires Cdc42 interaction with the N-WASP CRIB domain for efficient phagocytosis.","method":"Nck siRNA knockdown, FcγR phosphomutants (Y282F/Y298F), immunofluorescence of N-WASP/Cdc42 localization, phagocytosis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA KD + receptor mutagenesis + localization + functional phagocytosis assay; single lab","pmids":["22454526"],"is_preprint":false},{"year":2009,"finding":"NCK1 (and Nck2) plays a non-compensating role in PDGF-BB-stimulated dermal fibroblast migration; Nckalpha mediates Cdc42 signaling for filopodium formation, while Nckbeta mediates Rho signaling for stress fiber induction; PDGFRβ-Y751F (preventing Nckalpha binding) and Y1009F (preventing Nckbeta binding) each abolish PDGF-BB-induced migration.","method":"PDGFRβ phosphosite mutants, Nck-KO MEFs, human dermal fibroblasts with siRNA, GTPase activation assays, cell migration assays","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — receptor mutagenesis + KO cells + siRNA + GTPase assays + migration assays; multiple complementary approaches","pmids":["19242519"],"is_preprint":false}],"current_model":"NCK1 is an adaptor protein composed of three N-terminal SH3 domains and one C-terminal SH2 domain that functions as a molecular scaffold linking activated receptor tyrosine kinases (via phosphotyrosine–SH2 interactions) to actin cytoskeleton regulators (WASP, N-WASP, PAK1, and the Arp2/3 pathway via SH3-mediated recruitment), thereby controlling actin dynamics, cell polarity, directional migration, and T cell activation; it is uniquely regulated by c-Cbl-mediated ubiquitination at Lys178, by SOCS7-dependent nuclear translocation (where it participates in the DNA damage response and cell-cycle arrest), and by interaction with PERK (suppressing ER stress kinase activity), and it serves as the scaffold for the cytoplasmic capping complex, revealing roles in translation and mRNA capping in addition to its canonical cytoskeletal signaling functions."},"narrative":{"mechanistic_narrative":"NCK1 is a cytoplasmic adaptor protein that couples tyrosine-phosphorylated receptors and effectors to the actin polymerization machinery, thereby controlling actin dynamics, cell polarity, and directional migration [PMID:1333047, PMID:19917259, PMID:23444376]. Through its SH2 domain it docks onto phosphotyrosine motifs in activated receptor tyrosine kinases and effectors, including PDGF and EGF receptors [PMID:1333047], Eph receptors (EphB1 pTyr594, EphA3 pTyr602) [PMID:9430661, PMID:19505147], the bacterial effector EPEC Tir pTyr474 [PMID:11918809, PMID:16636066], and Tks5 pTyr557 in invadopodia [PMID:19596797], while its three SH3 domains engage proline-rich effectors that drive actin assembly: WASP/N-WASP [PMID:7565724, PMID:17302440], PAK1 [PMID:8824201, PMID:8798379], and DCC [PMID:12149262]. NCK1 is among the most potent activators of WASP/N-WASP-Arp2/3-mediated actin nucleation in vitro, operating at a defined 4 Nck:2 N-WASP:1 Arp2/3 stoichiometry and integrating phosphotyrosine and PI(4,5)P2 signals into localized actin polymerization [PMID:17302440, PMID:19917259, PMID:22613834]. This scaffolding underlies diverse actin-dependent processes, including pathogen actin-based motility and entry (vaccinia virus, EPEC, picornaviruses) [PMID:11918809, PMID:12007418, PMID:31769754, PMID:24284073], FcγR-mediated phagocytosis [PMID:22454526], endothelial front-rear polarity and angiogenesis downstream of VEGF and Slit2/ROBO1 [PMID:23444376, PMID:26659946], and osteoblast migration toward IGF1 [PMID:26621720]. In T cells, NCK1 binds the CD3ε ITAM/proline-rich region through a cooperative SH3.1/SH2 two-site engagement required for TCR signaling and T cell activation in vivo [PMID:18555270, PMID:26590318, PMID:24470497]. NCK1 activity and abundance are tightly regulated: it is selectively ubiquitinated by c-Cbl at Lys178 (antagonized by synaptopodin) [PMID:24287595], translocated to the nucleus by SOCS7 where it is required for DNA-damage-induced p53 Ser15 phosphorylation and cell-cycle arrest [PMID:17803907], and it suppresses ER stress signaling by binding the PERK inhibitory site pTyr561 and modulating IRE1α/JNK activation, with consequences for insulin biosynthesis and glucose homeostasis [PMID:20587749, PMID:24371088]. Beyond cytoskeletal scaffolding, NCK1 organizes the cytoplasmic capping complex by juxtaposing the capping enzyme and 5'-kinase on adjacent SH3 domains and promotes both cap-dependent and -independent translation via eIF2β binding [PMID:11959995, PMID:25137142].","teleology":[{"year":1992,"claim":"Established NCK1 as a phosphotyrosine-binding adaptor coupled to growth factor receptors and capable of cellular transformation, defining its core scaffolding identity.","evidence":"Co-IP and GST pulldown with activated PDGF/EGF receptors plus NIH 3T3/3Y1 focus and soft-agar transformation assays","pmids":["1333047","1280326"],"confidence":"High","gaps":["Did not identify the SH3-bound effectors mediating transformation","Mechanism linking receptor binding to oncogenic output unresolved"]},{"year":1995,"claim":"Identified WASP as the first SH3-domain effector of NCK1, linking the adaptor to the actin nucleation machinery.","evidence":"Yeast two-hybrid screen with in vitro/in vivo binding and subcellular fractionation","pmids":["7565724"],"confidence":"High","gaps":["Did not demonstrate functional actin polymerization output","Receptor-to-WASP coupling not yet reconstituted"]},{"year":1996,"claim":"Mapped the NCK1 second SH3 domain to PAK1 and showed this links receptor tyrosine kinases to PAK1 kinase activation, defining a receptor-to-kinase relay.","evidence":"Domain-mapped Co-IP and kinase activity assays in COS-7, HeLa, and Swiss 3T3 cells with PDGFR stimulation","pmids":["8824201","8798379"],"confidence":"High","gaps":["Downstream cytoskeletal consequences of PAK1 activation not measured","Did not establish in vivo pathway context"]},{"year":1998,"claim":"Placed NCK1 in Eph receptor and TCR signaling pathways, showing it relays distinct downstream outputs (JNK vs NFAT) depending on context.","evidence":"Yeast two-hybrid, Y594F mutagenesis, dominant-negative overexpression, NFAT/JNK reporter and kinase assays in T cells","pmids":["9430661","9755165"],"confidence":"High","gaps":["Effector specificity determinants for JNK vs NFAT not defined","Did not resolve direct vs indirect contributions"]},{"year":2000,"claim":"Defined regulatory logic of the NCK1-PAK1 interaction (phospho-dependent motif recognition and autophosphorylation-controlled release) and the NIK/Nck integrin-activation module.","evidence":"Saturation peptide substitution binding analysis, phosphorylation assays, and Co-IP/kinase/integrin assays downstream of EphB receptors","pmids":["10805734","10669731"],"confidence":"High","gaps":["In vivo physiological role of phospho-regulated cycling not tested","NIK identity and broader role not fully characterized"]},{"year":2002,"claim":"Demonstrated NCK1 is hijacked by pathogens for actin-based motility and revealed non-cytoskeletal roles in translation and netrin/DCC signaling.","evidence":"EPEC Tir pTyr474 binding/localization, vaccinia A36R phosphomutant actin tail assays, DCC Co-IP/Rac1/neurite assays, and eIF2β binding with in vitro translation assays","pmids":["11918809","12007418","12149262","11959995"],"confidence":"High","gaps":["Translation role mechanism beyond eIF2β binding not resolved","DCC findings from single lab with Medium confidence"]},{"year":2004,"claim":"Identified an ER-associated NCK1 pool that restrains ERK-1 and stress signaling, expanding NCK1 into ER-stress regulation.","evidence":"ER membrane IP, in vitro reconstitution, and kinase assays in Nck-null fibroblasts","pmids":["15201339"],"confidence":"Medium","gaps":["Molecular ER tether not identified","Single-lab finding without reciprocal validation"]},{"year":2006,"claim":"Provided atomic-resolution basis for NCK1 SH2 phosphopeptide recognition and confirmed indistinguishable Nck1/Nck2 SH2 specificity, anchoring binding-mode interpretation.","evidence":"X-ray crystallography of SH2-Tir phosphopeptide complexes with mutagenesis and pulldowns","pmids":["16636066"],"confidence":"High","gaps":["Does not explain paralog-specific functional divergence given identical SH2 specificity","SH3 selectivity not structurally addressed"]},{"year":2007,"claim":"Established NCK1 as a top-tier in vitro activator of WASP/N-WASP and revealed an unexpected SOCS7-driven nuclear function in the DNA-damage response.","evidence":"In vitro actin nucleation with purified proteins and nuclear/cytoplasmic rescue, ATM/ATR epistasis, and p53 Ser15 phosphorylation assays","pmids":["17302440","17803907"],"confidence":"High","gaps":["Nuclear NCK1 effectors mediating cell-cycle arrest not identified","How nuclear vs cytoplasmic pools are balanced under stress unresolved"]},{"year":2009,"claim":"Showed NCK1 integrates phosphotyrosine and PI(4,5)P2 inputs to drive localized actin polymerization, and extended this to invadopodia matrix degradation.","evidence":"Nck KO/KD with PI(4,5)P2 manipulation and live imaging of actin comets; Tks5 Y557 mutagenesis and gelatin degradation assays","pmids":["19917259","19596797","19505147","19242519"],"confidence":"High","gaps":["Spatial coincidence detection mechanism of dual signals not fully defined","Paralog non-redundancy mechanism for GTPase selectivity unclear"]},{"year":2012,"claim":"Quantified the stoichiometric and density requirements for NCK1-driven actin assembly, converting the scaffold model into a quantitative threshold model.","evidence":"Computational modeling with antibody-induced membrane aggregation and quantitative live-cell fluorescence; FcγR phagocytosis dependency","pmids":["22613834","22454526"],"confidence":"High","gaps":["In vivo relevance of the 4:2:1 stoichiometry at physiological receptors untested","FcγR study single-lab Medium confidence"]},{"year":2013,"claim":"Revealed paralog-specific regulation (c-Cbl ubiquitination of NCK1 Lys178), defined NCK1 control of front-back polarity, and discovered the PERK-binding metabolic axis.","evidence":"K178R/ubiquitination and synaptopodin competition assays; FRET/AFM polarity imaging in endothelial cells; PERK Y561F mutagenesis with KO mice and β-cell insulin readouts","pmids":["24287595","23444376","24371088"],"confidence":"High","gaps":["How c-Cbl regulation intersects with signaling outputs in vivo not resolved","Mechanism by which NCK1 controls RhoA versus Cdc42/Rac balance incomplete"]},{"year":2014,"claim":"Defined the cooperative two-site NCK1-CD3ε engagement required for TCR signaling in vivo and established NCK1 as the scaffold for the cytoplasmic capping complex.","evidence":"Proximity ligation, KI-PRS knock-in mice, EAE and peptide-inhibitor studies; in vitro capping-complex reconstitution on recombinant Nck1 with SH3 mutagenesis","pmids":["26590318","24470497","25137142"],"confidence":"High","gaps":["Regulation of capping complex assembly under physiological signaling unclear","Whether mRNA recapping targets are sequence-selective not addressed"]},{"year":2015,"claim":"Established NCK1 (with NCK2) as essential for VEGF/Slit2-ROBO1-driven endothelial polarity and angiogenesis and for IGF1-directed osteoblast migration and bone formation.","evidence":"Endothelial- and osteoblast-specific conditional Nck1/2 KO mice with Cdc42/PAK activation, ROBO1/IRS-1 Co-IP, and in vivo vascular/bone phenotyping","pmids":["26659946","26621720"],"confidence":"High","gaps":["Individual NCK1 versus NCK2 contributions in these tissues not separated","Receptor-proximal coupling to ROBO1 not structurally defined"]},{"year":2018,"claim":"Demonstrated NCK1/2 act as negative regulators of PDGFRβ signaling in pericytes and resolved NCK1- versus NCK2-specific interactomes and functions (NCK2 in cytokinesis).","evidence":"Mural-cell conditional KO with PDGFRβ Y1009 phosphorylation and retinopathy models; AP-MS/BioID interactomes and Nck-KO MEF cytokinesis imaging","pmids":["30150707","30002203"],"confidence":"High","gaps":["Molecular basis of NCK1 acting as PDGFR brake versus relay context-dependence unclear","Cytokinesis role is NCK2-specific, leaving NCK1's mitotic role undefined"]},{"year":2020,"claim":"Identified an NCK1-selective inflammatory pathway in atherosclerosis, linking disturbed flow to NF-κB via an NCK1-IRAK1 interaction.","evidence":"Nck1 vs Nck2 KO mice, domain mutagenesis, Nck1-IRAK1 Co-IP, NF-κB reporters, flow chambers, and high-fat-diet atherosclerosis with bone marrow chimeras","pmids":["32427580"],"confidence":"High","gaps":["Structural basis of NCK1-IRAK1 selectivity over NCK2 not defined","How a cytoskeletal adaptor engages an innate-immune kinase mechanistically unresolved"]},{"year":null,"claim":"It remains unknown how the molecular features distinguishing NCK1 from NCK2 — given indistinguishable SH2 specificity — generate paralog-specific functions across actin, inflammatory, metabolic, and nuclear pathways.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural determinant explaining NCK1-selective partners (IRAK1, c-Cbl substrate, PERK) identified","Integration logic that routes NCK1 between cytoskeletal scaffolding and translation/capping roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,3,5,16,20,27]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,16,20,23]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,26,32,37]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,11,37]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[15]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[13,26]},{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[11]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,20,42]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,5,30,43]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,17,28,29,34]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[11,27]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[13,15,22,26]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,12,34,35]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[10,24,30,31]}],"complexes":["cytoplasmic capping complex","N-WASP/Arp2/3 actin nucleation complex"],"partners":["WASL","PAK1","WAS","TKS5/SH3PXD2A","ROBO1","PERK/EIF2AK3","CBL","IRAK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P16333","full_name":"SH2/SH3 adapter protein NCK1","aliases":["Cytoplasmic protein NCK1","NCK adapter protein 1","Nck-1","SH2/SH3 adapter protein NCK-alpha"],"length_aa":377,"mass_kda":42.9,"function":"Adapter protein which associates with tyrosine-phosphorylated growth factor receptors, such as KDR and PDGFRB, or their cellular substrates. Maintains low levels of EIF2S1 phosphorylation by promoting its dephosphorylation by PP1. Plays a role in the DNA damage response, not in the detection of the damage by ATM/ATR, but for efficient activation of downstream effectors, such as that of CHEK2. Plays a role in ELK1-dependent transcriptional activation in response to activated Ras signaling. Modulates the activation of EIF2AK2/PKR by dsRNA. May play a role in cell adhesion and migration through interaction with ephrin receptors","subcellular_location":"Cytoplasm; Endoplasmic reticulum; Nucleus","url":"https://www.uniprot.org/uniprotkb/P16333/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NCK1","classification":"Not 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KINASE 1; MINK1","url":"https://www.omim.org/entry/609426"},{"mim_id":"604930","title":"NCK ADAPTOR PROTEIN 2; NCK2","url":"https://www.omim.org/entry/604930"},{"mim_id":"604491","title":"CAS-BR-M MURINE ECOTROPIC RETROVIRAL TRANSFORMING SEQUENCE B; CBLB","url":"https://www.omim.org/entry/604491"},{"mim_id":"603151","title":"SEPTIN 7; SEPTIN7","url":"https://www.omim.org/entry/603151"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NCK1"},"hgnc":{"alias_symbol":["NCKalpha"],"prev_symbol":["NCK"]},"alphafold":{"accession":"P16333","domains":[{"cath_id":"2.30.30.40","chopping":"5-57","consensus_level":"high","plddt":85.9732,"start":5,"end":57},{"cath_id":"2.30.30.40","chopping":"103-161","consensus_level":"medium","plddt":73.3761,"start":103,"end":161},{"cath_id":"2.30.30.40","chopping":"192-249","consensus_level":"medium","plddt":76.2397,"start":192,"end":249},{"cath_id":"3.30.505.10","chopping":"282-374","consensus_level":"high","plddt":86.8333,"start":282,"end":374}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P16333","model_url":"https://alphafold.ebi.ac.uk/files/AF-P16333-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P16333-F1-predicted_aligned_error_v6.png","plddt_mean":70.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NCK1","jax_strain_url":"https://www.jax.org/strain/search?query=NCK1"},"sequence":{"accession":"P16333","fasta_url":"https://rest.uniprot.org/uniprotkb/P16333.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P16333/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P16333"}},"corpus_meta":[{"pmid":"7565724","id":"PMC_7565724","title":"Wiskott-Aldrich 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anti-Nck antibodies, in vitro binding with GST-Nck fusion protein, NIH 3T3 focus formation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, GST pulldown, and functional transformation assay in same study; foundational paper replicated by subsequent work\",\n      \"pmids\": [\"1333047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Overexpression of NCK1 in 3Y1 rat fibroblasts causes transformation (altered morphology, soft agar colony formation, tumor formation in nude mice) without detectable elevation of phosphotyrosine levels; GST-Nck binds p60v-src and serine/threonine kinases from cell lysates.\",\n      \"method\": \"Soft agar colony formation, tumor formation in nude mice, GST fusion protein pulldown, Western blot\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays in one study; independently consistent with PMID:1333047\",\n      \"pmids\": [\"1280326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The C-terminal (third) SH3 domain of NCK1 (p47nck) specifically interacts with WASP (Wiskott-Aldrich syndrome protein) in vivo and in vitro; WASP is mainly cytosolic.\",\n      \"method\": \"Yeast two-hybrid/cDNA expression library screen, in vitro and in vivo binding assays, subcellular fractionation, Western blot\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (in vivo + in vitro binding, fractionation), replicated in subsequent studies on Nck-WASP/N-WASP interactions\",\n      \"pmids\": [\"7565724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"NCK1 binds PAK1 through its second SH3 domain; PAK1 interacts with Nck via its first N-terminal proline-rich SH3-binding motif; the Nck–PAK1 interaction is strengthened upon PDGF receptor stimulation; active PAK1 association with Nck leads to phosphorylation of Nck at multiple sites.\",\n      \"method\": \"Co-immunoprecipitation in COS-7 and Swiss 3T3 cells, in vitro binding, domain-mapping with individual SH3 domain constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and in vitro binding, replicated independently by Galisteo et al. (PMID:8798379) in same year\",\n      \"pmids\": [\"8824201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"NCK1 SH2 domain recruits Nck to activated EGF and PDGF receptors; Nck second SH3 domain binds PAK1 proline-rich N-terminus; Nck–PAK1 interaction links receptor tyrosine kinases to PAK1 and enhances PAK1 kinase activity when Nck is co-transfected.\",\n      \"method\": \"Co-immunoprecipitation in living cells, domain mapping, kinase activity assay, co-transfection in HeLa cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods, independently replicated by Bokoch et al. (PMID:8824201)\",\n      \"pmids\": [\"8798379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"NCK1 SH2 domain binds phosphorylated juxtamembrane Tyr594 of EphB1 receptor; Nck recruitment to EphB1 is required for downstream JNK/SAPK activation and cell attachment responses.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation of native receptor complexes, site-directed mutagenesis (Y594F), JNK activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid, reciprocal Co-IP, mutagenesis, and functional JNK assay in single study; domain mapping confirmed\",\n      \"pmids\": [\"9430661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"NCK1 and PAK1 form a signaling module downstream of TCR activation; this module is required specifically for NFAT activation but not for JNK activation; Pak1 acts downstream of Vav and Cdc42; dominant-negative Nck or Pak1 inhibits TCR-mediated NFAT and Erk2 activation.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative overexpression, NFAT luciferase reporter assay, kinase activity assay in T cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays and epistasis analysis in T cells; pathway position defined\",\n      \"pmids\": [\"9755165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"NCK1 second SH3 domain binds an 18-mer PAK-derived peptide containing the motif PXXPXRXXS; serine phosphorylation within this motif negatively regulates NCK1-PAK1 binding; PAK autophosphorylation blocks binding of both Nck and PIX to PAK1, providing a mechanism to regulate PAK interactions and its cycling between cytosolic and focal complex sites.\",\n      \"method\": \"Saturation substitution binding analysis, in vitro binding assays with peptides and full-length proteins, phosphorylation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and binding analysis; mechanistic model supported by multiple experiments in one study\",\n      \"pmids\": [\"10805734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Nck-interacting kinase (NIK) is activated by EphB1 and EphB2 receptors in a manner requiring EphB1 kinase activity and phosphorylation of juxtamembrane Tyr594; NIK activation through NCK1 is required for EphB1-mediated integrin activation and JNK activation; a complex of NIK/Nck, p62(dok), and RasGAP forms upon EphB receptor stimulation.\",\n      \"method\": \"Co-immunoprecipitation, kinase activity assays, dominant-negative experiments, integrin activation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (Co-IP, kinase assay, DN experiments), pathway position established by epistasis\",\n      \"pmids\": [\"10669731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A 12-amino-acid sequence of EPEC Tir containing phospho-Tyr474 binds the host adaptor NCK1; NCK1 is recruited to actin pedestals of EPEC in a Y474 phosphorylation-dependent manner and is required for EPEC-induced actin signaling.\",\n      \"method\": \"Chimeric protein expression, in vivo binding assay, immunofluorescence localization of endogenous Nck to pedestals\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphopeptide-dependent binding, genetic chimera epistasis, and direct localization assay; replicated and extended by crystal structure studies (PMID:16636066)\",\n      \"pmids\": [\"11918809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NCK1 SH3 domains interact directly with DCC (deleted in colorectal cancer) receptor, independent of the SH2 domain; this interaction mediates DCC/Nck-1 association with the actin cytoskeleton and is required for netrin-1-induced Rac1 activation and neurite outgrowth.\",\n      \"method\": \"Co-immunoprecipitation in commissural neurons, dominant-negative Nck-1 overexpression, Rac1 activation assay, neurite outgrowth assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, DN functional assay, and Rac1 pulldown; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12149262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NCK1 SH3 domains interact with eIF2β; Nck is present in ribosomal fractions and its ribosomal localization is enhanced by insulin; overexpression of Nck-1 enhances both cap-dependent and -independent protein translation in a manner requiring the first and third SH3 domains; Nck-1 directly increases protein translation in vitro.\",\n      \"method\": \"Co-immunoprecipitation, cell fractionation/ribosomal enrichment, in vitro translation assay, domain mutants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, fractionation, and in vitro translation reconstitution; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11959995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Grb2 and NCK1 act cooperatively to promote actin-based motility of vaccinia virus; Nck is recruited to viral particles via phospho-Tyr112 of A36R, whereas Grb2 is recruited via phospho-Tyr132; the Grb2 pathway requires prior Nck-mediated N-WASP recruitment.\",\n      \"method\": \"Immunofluorescence, phosphomutant constructs, actin tail counting, co-immunoprecipitation\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphomutant epistasis, quantitative actin tail assay, replicated by subsequent poxvirus studies (PMID:20006842, 24284073)\",\n      \"pmids\": [\"12007418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ER-associated NCK1 represses ERK-1 activation under basal conditions; upon ER stress, this pool of Nck dissociates from ER membranes, allowing ERK-1 activation; Nck-null cells show stronger ERK-1 activation after ER stress correlating with enhanced cell survival.\",\n      \"method\": \"Immunoprecipitation of ER membranes, in vitro reconstitution with ER membranes, Nck-null fibroblasts, kinase assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ER reconstitution, Nck-null cells, and kinase assay; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"15201339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structures of Nck1 and Nck2 SH2 domains in complex with phosphopeptides from EPEC Tir establish highly conserved phosphopeptide recognition; Nck1 and Nck2 SH2 domains have essentially indistinguishable binding specificities; GIT1 is confirmed experimentally as a new Nck binding partner.\",\n      \"method\": \"X-ray crystallography, phosphopeptide binding studies, sequential mutagenesis, pulldown assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with binding validation and mutagenesis; rigorous structural study in single lab\",\n      \"pmids\": [\"16636066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NCK1 nuclear accumulation (carried by SOCS7 via nuclear import/export signals) causes actin stress fiber disintegration and cell polarity loss; nuclear NCK1 is essential for cell-cycle arrest after DNA damage; the septin–SOCS7–NCK axis functions downstream of ATM/ATR and is required for p53 Ser15 phosphorylation.\",\n      \"method\": \"RNAi knockdown, cytoplasmic vs. nuclear NCK1 rescue constructs, live imaging, DNA damage assays, epistasis with ATM/ATR inhibitors, p53 phosphorylation immunoblot\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including localization rescue, epistasis, and p53 phosphorylation in high-impact study\",\n      \"pmids\": [\"17803907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NCK1 (and NCK2) are the most potent activators of WASP and N-WASP in vitro; Nck1/2 differentially regulate WASP vs. N-WASP (Nck is a more potent activator than Cdc42 or Rac1); in vitro actin nucleation assays with purified full-length proteins.\",\n      \"method\": \"In vitro actin nucleation assay with purified full-length WASP/N-WASP, Arp2/3 complex, and various activators\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified full-length proteins and quantitative comparison; single lab\",\n      \"pmids\": [\"17302440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NCK1 SH3.1 domain forms a complex with an atypical PxxDY motif at Tyr166 of CD3ε (non-ITAM-requiring interaction); Nck binding inhibits phosphorylation of the CD3ε ITAM by Fyn and Lck in vitro; Nck–CD3ε interaction downregulates TCR surface expression upon physiological stimulation.\",\n      \"method\": \"NMR structure of complex, in vitro kinase assay, flow cytometry for TCR surface expression in primary mouse lymphocytes\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure combined with functional kinase assay and primary cell experiments; multiple orthogonal methods\",\n      \"pmids\": [\"18555270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Src phosphorylates Tks5 at Y557, inducing direct association with NCK1 (and NCK2) SH2 domains in invadopodia; Tks5 mutants unable to bind Nck show reduced matrix degradation and inefficient actin recruitment; Nck1 depletion inhibits Src–Tks5-driven matrix proteolysis and actin assembly in invadopodia.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of Y557, Nck1 siRNA knockdown, gelatin matrix degradation assay, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, KD with specific phenotypic readout; multiple orthogonal methods in single study\",\n      \"pmids\": [\"19596797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NCK1 SH2 domain binds phospho-Tyr602 of EphA3 receptor; this interaction mediates EphA3-induced inhibition of cell migration and process retraction; dominant-negative Nck1 mutants lacking SH2 or SH3 domains partially alleviate EphA3-mediated inhibitory effects.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, SH2 domain mutagenesis, cell migration assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid, GST pulldown, Co-IP with mutagenesis, and functional migration assay; single lab\",\n      \"pmids\": [\"19505147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NCK1 is essential for actin remodeling stimulated by PI(4,5)P2, and PI(4,5)P2 is necessary for NCK1-induced localized actin polymerization in vivo; Nck knockdown/knockout suppresses PI(4,5)P2-induced actin comets; Nck couples phosphotyrosine- and phosphoinositide-dependent signals to N-WASp/Arp2/3 actin polymerization.\",\n      \"method\": \"Nck siRNA knockdown, Nck-knockout cells, antibody-induced Nck aggregation, live-cell imaging of actin comets, PI(4,5)P2 manipulation with inositol 5-phosphatase\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Nck KO and KD with multiple complementary approaches, mechanistic pathway placement\",\n      \"pmids\": [\"19917259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nck1 specifically localizes to invadopodia of metastatic mammary carcinoma cells but not to podosomes in macrophages or Src-transformed fibroblasts, establishing distinct upstream activators for invadopodia vs. podosomes.\",\n      \"method\": \"Confocal immunofluorescence microscopy of endogenous Nck1 in multiple cell types\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by imaging across cell types, no functional consequence tested in this study\",\n      \"pmids\": [\"20850195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nck1 deletion in obese mice attenuates hepatic IRE1α and JNK activation and IRS-1 phosphorylation at Ser307, improving glucose disposal and insulin signaling; siRNA knockdown of Nck1 in HepG2 cells decreases thapsigargin-induced IRE1α activation.\",\n      \"method\": \"Nck1−/− obese mice (in vivo), siRNA knockdown in HepG2 cells, kinase activation assays, glucose tolerance tests, insulin signaling (Akt phosphorylation)\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO and in vitro siRNA with multiple signaling readouts; two complementary model systems\",\n      \"pmids\": [\"20587749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The stoichiometry of NCK1-dependent actin polymerization through the N-WASp/Arp2/3 pathway is 4 Nck : 2 N-WASp : 1 Arp2/3; the density of Nck molecules in membrane aggregates is a critical determinant of localized actin polymerization.\",\n      \"method\": \"Computational modeling (Virtual Cell), antibody-induced aggregation of membrane-targeted Nck SH3 domains, quantitative fluorescence microscopy in living cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative live-cell experimentation combined with computational modeling; orthogonal validation of stoichiometry\",\n      \"pmids\": [\"22613834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NCK1 (and Nck2) is essential for front-back polarity, polarized membrane protrusion, and directional migration of endothelial cells; Nck depletion causes delocalized Cdc42/Rac activity and reduces RhoA activity and myosin II phosphorylation; Nck couples polarized protrusion formation with adhesion maturation via integrin α5β1-fibronectin interaction.\",\n      \"method\": \"siRNA knockdown, time-lapse DIC and TIRF microscopy, FRET imaging for Cdc42/Rac/RhoA activity, atomic force microscopy for adhesion force\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including live imaging, FRET, AFM in single study with specific molecular mechanism\",\n      \"pmids\": [\"23444376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NCK1 (but not Nck2) is a substrate of c-Cbl-mediated ubiquitination at the evolutionarily conserved Lys178; synaptopodin competes with c-Cbl for Nck1 binding, preventing Nck1 ubiquitination; c-Cbl knockdown restores Nck1 protein levels and stress fibers; NCK1-specific SH3 domain 2 drives RhoA activation and actin stress fiber formation.\",\n      \"method\": \"Ubiquitination assays, site-directed mutagenesis (K178R), co-immunoprecipitation, gene silencing (c-Cbl siRNA), RhoA activation assay, immunofluorescence for stress fibers\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis + ubiquitination assay + competition Co-IP + RhoA functional readout; multiple orthogonal methods\",\n      \"pmids\": [\"24287595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NCK1 SH2 domain binds phospho-Y561 in the PERK juxtamembrane domain; this interaction negatively regulates PERK activity (pY561PERK is an inhibitory site); Nck1 silencing in MIN6 cells accelerates pY561PERK dephosphorylation, enhances PERK activation, increases insulin biosynthesis and proinsulin content; Nck1-knockout mice pancreatic islets contain more insulin.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Y561F), siRNA in MIN6 cells, Nck1-KO mice, PERK phosphorylation kinetics, insulin/proinsulin content assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding mapped by SH2–pY interaction + mutagenesis + KO mice + functional β-cell readout; multiple systems\",\n      \"pmids\": [\"24371088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NCK1 is required for assembly of the cytoplasmic capping complex; capping enzyme (CE) binds Nck1 via its proline-rich C-terminus; CE and the 5'-kinase are juxtaposed by binding adjacent SH3 domains of Nck1; Nck1 knockdown disrupts complex integrity; cap homeostasis is inhibited by Nck1 with inactivating mutations in the CE- or kinase-binding SH3 domains.\",\n      \"method\": \"Co-immunoprecipitation, sedimentation, gel filtration, in vitro complex assembly on recombinant Nck1, siRNA knockdown, SH3-domain mutagenesis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution on recombinant Nck1, sedimentation/gel filtration, mutagenesis, KD functional assay; multiple orthogonal methods\",\n      \"pmids\": [\"25137142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NCK1 SH3.1 domain binds the proline-rich sequence (PRS) of CD3ε when the first ITAM tyrosine is unphosphorylated; the SH2 domain simultaneously binds the second ITAM tyrosine when phosphorylated; cooperative two-site engagement is required for Nck1 binding to the TCR; SH3.1-mutated Nck1 impairs TCR signaling and T cell activation.\",\n      \"method\": \"In situ proximity ligation assay, co-purification, domain mutations, TCR signaling readouts in Jurkat T cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity ligation assay + co-purification + mutagenesis + functional signaling readout; multiple orthogonal methods\",\n      \"pmids\": [\"26590318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NCK1 SH3.1 domain binding to the CD3ε PRS is required for T cell activation in vivo; KI-PRS mice with a mutation preventing Nck–CD3ε interaction show impaired CD3ζ phosphorylation, reduced T cell activation by antigen in vitro, partial protection from EAE, and deficient antitumoral response; a high-affinity SH3.1-blocking peptide inhibits T cell proliferation in vitro and in vivo.\",\n      \"method\": \"Knock-in mouse model, TCR signaling assays (CD3ζ phosphorylation), EAE model, peptide inhibitor in vivo\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knock-in mouse + peptide inhibitor + in vivo disease model; multiple complementary approaches\",\n      \"pmids\": [\"24470497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NCK1 and NCK2 inactivation in endothelial cells inhibits Cdc42 and PAK2 activation by VEGF-A and Slit2, preventing front-rear polarity; NCK1/2 binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity; endothelial-specific Nck1/2 deletion causes severe angiogenesis defects in postnatal retina and embryonic development.\",\n      \"method\": \"Inducible endothelial-specific Nck1/2 knockout mice, Cdc42/PAK2 activation assays, co-immunoprecipitation of NCK with ROBO1, retinal and embryonic vascular phenotyping\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with specific molecular mechanistic readouts (Cdc42/PAK activation, ROBO1 binding) + in vivo vascular phenotypes\",\n      \"pmids\": [\"26659946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NCK1 (and NCK2) in osteoblasts regulates preosteoblast/osteoblast migration toward IGF1; Nck1 binds IRS-1 (by co-immunoprecipitation); conditional double deletion of Nck1/2 in osteoblasts causes osteopenia, suppresses bone formation rate in vivo, and impairs bone injury repair, without affecting bone resorption.\",\n      \"method\": \"shRNA knockdown, Nck1 overexpression, co-immunoprecipitation (Nck1/IRS-1), osteoblast-specific conditional KO mice, in vivo bone histomorphometry, calvarial defect model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo with quantitative bone parameters + Co-IP + migration assays; multiple methods and systems\",\n      \"pmids\": [\"26621720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NCK1 and NCK2 adaptors in pericytes inhibit PDGF-B-induced phosphorylation of PDGFRβ at Y1009 and PAK activation; inactivation of NCK1/2 in mural cells prevents pathological neovascular tuft formation and vascular leakage in oxygen-induced retinopathy model.\",\n      \"method\": \"Inducible mural cell-specific Nck1/2 knockout mice, PDGFRβ phosphorylation assay, PAK activation assay, oxygen-induced retinopathy model, lineage tracing\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO + molecular phosphorylation readout + in vivo disease model; multiple approaches\",\n      \"pmids\": [\"30150707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Proteomic analysis identifies 30 NCK1-specific and 28 NCK2-specific interactors; NCK2 (not NCK1) is required for cell abscission during cytokinesis—Nck2-deficient MEFs are multinucleated, show extended intercellular bridges, fail to complete abscission, and have altered midbody composition (mislocalized AURKB, PLK1, ECT2); NCK2 function in cytokinesis requires its SH2 domain.\",\n      \"method\": \"Affinity purification-MS, BioID proximity labeling, Nck1/Nck2-KO MEFs, live-cell time-lapse imaging of cytokinesis, immunofluorescence of midbody markers, SH2 domain mutants\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO MEFs + live imaging + interactome (two methods) + domain mutagenesis; multiple orthogonal approaches\",\n      \"pmids\": [\"30002203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Only NCK1 (not NCK2) mediates disturbed flow-induced NF-κB activation in endothelial cells; NCK1 SH2 domain and first SH3 domain are required for this effect; IRAK-1 is identified as an NCK1-selective binding partner; disturbed flow-induced IRAK-1 activation requires NCK1 in vitro and in vivo; Nck1-knockout mice show reduced endothelial activation and atherosclerotic plaque formation.\",\n      \"method\": \"Nck1-KO and Nck2-KO mice, domain-swap and point-mutation experiments, co-immunoprecipitation (Nck1–IRAK1), NF-κB reporter assays, flow chamber experiments, high fat diet atherosclerosis model, bone marrow chimeras\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mice + domain mutagenesis + Co-IP + in vivo disease model + bone marrow chimeras; comprehensive multi-method study\",\n      \"pmids\": [\"32427580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NCK1 functions in a common pathway with TNK2 and WASL for picornavirus (EMCV, CVB3, poliovirus, EV-D68) entry/infection; CRISPR deletion of NCK1 reduces infection by multiple picornaviruses; genetic epistasis places NCK1, TNK2, and WASL in the same pathway for viral internalization.\",\n      \"method\": \"CRISPR deletion, viral infection assays, genetic epistasis analysis, EMCV internalization assay, mouse model (TNK2 KO)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO + epistasis + internalization assay + in vivo mouse data; multiple methods\",\n      \"pmids\": [\"31769754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NCK1 (Dock/Nck) forms a stable complex with PTP1B (PTP61F in Drosophila); this Nck/PTP1B complex inducibly associates with the insulin receptor; Nck is required for effective insulin receptor dephosphorylation and inactivation by PTP1B in mammalian cells.\",\n      \"method\": \"Co-immunoprecipitation, Drosophila genetic assays (PTP61F/Dock interaction), IR dephosphorylation assay in vitro and in vivo\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP + in vivo Drosophila genetics + mammalian cell IR dephosphorylation; two organisms and multiple methods\",\n      \"pmids\": [\"21707536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PAK1-Nck complex formation sequesters PAK1 in the cytoplasm; co-expression of PAK1 and NCK1 inhibits PRL-induced cyclin D1 promoter activity by 95%; disruption of PAK1-Nck binding partially abolishes this inhibition.\",\n      \"method\": \"Co-immunoprecipitation, cytoplasmic/nuclear fractionation, cyclin D1 luciferase reporter assay, dominant-negative and binding-deficient mutants\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP + fractionation + luciferase reporter with domain mutants; single lab\",\n      \"pmids\": [\"21719533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The third (C-terminal) SH3 domain of NCK1 specifically binds Sos; the third SH3 domain also binds dynamin (a GTP-binding protein involved in clathrin-coated vesicle formation); Nck and dynamin co-precipitate from cell lysates.\",\n      \"method\": \"GST fusion protein pulldown with individual SH3 domains, co-immunoprecipitation from K562 cells, protein identification by molecular mass\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — GST pulldown domain mapping plus co-IP; single lab but two orthogonal methods\",\n      \"pmids\": [\"10206341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Tyrosine 351 of DokR (Dok-related protein) mediates binding to NCK1 SH2 domain; this interaction is dependent on DokR tyrosine phosphorylation by Lyn kinase; DokR–Nck complex forms upon EGF receptor activation.\",\n      \"method\": \"GST fusion protein binding assay, site-directed mutagenesis of DokR tyrosines, co-immunoprecipitation in 293T cells and BaF/3 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis + GST pulldown + co-IP; single lab but multiple methods\",\n      \"pmids\": [\"10428862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"cGMP-dependent protein kinase (PKG) phosphorylates PAK1 at Ser21, causing release of NCK1 from PAK1 and promoting formation of a new PAK1-VASP complex; Ser21 phosphorylation of PAK1 regulates cell polarization and tail retraction, and cells expressing S21A-PAK1 show aberrant morphology when NCK/PAK binding is blocked.\",\n      \"method\": \"In vitro phosphorylation mapping, phosphospecific antibody, co-transfection assays, endothelial cell morphology assays, peptide-mediated disruption of Nck/PAK binding\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase mapping + phosphospecific antibody + cell morphology; single lab\",\n      \"pmids\": [\"16490784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NCK1 interacts with Cdc42 and the Rho-GEF intersectin-1 (ITSN1) to promote N-WASP-dependent actin polymerization during vaccinia virus actin tail formation and FcγR-mediated phagocytosis; ITSN1 is recruited prior to actin-based motility and functions with Cdc42 in a feed-forward loop with N-WASP and Nck.\",\n      \"method\": \"Immunofluorescence, siRNA knockdown, co-immunoprecipitation, phagocytosis assay, vaccinia actin tail assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP + KD with two functional readouts (actin tail, phagocytosis); single lab but two parallel biological contexts\",\n      \"pmids\": [\"24284073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NCK1 is required for FcγR- but not CR3-mediated phagocytosis; Nck is recruited to phagocytic cups via phospho-Tyr282 and Tyr298 in the FcγR ITAM; Nck promotes phagocytosis by recruiting N-WASP and requires Cdc42 interaction with the N-WASP CRIB domain for efficient phagocytosis.\",\n      \"method\": \"Nck siRNA knockdown, FcγR phosphomutants (Y282F/Y298F), immunofluorescence of N-WASP/Cdc42 localization, phagocytosis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA KD + receptor mutagenesis + localization + functional phagocytosis assay; single lab\",\n      \"pmids\": [\"22454526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NCK1 (and Nck2) plays a non-compensating role in PDGF-BB-stimulated dermal fibroblast migration; Nckalpha mediates Cdc42 signaling for filopodium formation, while Nckbeta mediates Rho signaling for stress fiber induction; PDGFRβ-Y751F (preventing Nckalpha binding) and Y1009F (preventing Nckbeta binding) each abolish PDGF-BB-induced migration.\",\n      \"method\": \"PDGFRβ phosphosite mutants, Nck-KO MEFs, human dermal fibroblasts with siRNA, GTPase activation assays, cell migration assays\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — receptor mutagenesis + KO cells + siRNA + GTPase assays + migration assays; multiple complementary approaches\",\n      \"pmids\": [\"19242519\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NCK1 is an adaptor protein composed of three N-terminal SH3 domains and one C-terminal SH2 domain that functions as a molecular scaffold linking activated receptor tyrosine kinases (via phosphotyrosine–SH2 interactions) to actin cytoskeleton regulators (WASP, N-WASP, PAK1, and the Arp2/3 pathway via SH3-mediated recruitment), thereby controlling actin dynamics, cell polarity, directional migration, and T cell activation; it is uniquely regulated by c-Cbl-mediated ubiquitination at Lys178, by SOCS7-dependent nuclear translocation (where it participates in the DNA damage response and cell-cycle arrest), and by interaction with PERK (suppressing ER stress kinase activity), and it serves as the scaffold for the cytoplasmic capping complex, revealing roles in translation and mRNA capping in addition to its canonical cytoskeletal signaling functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NCK1 is a cytoplasmic adaptor protein that couples tyrosine-phosphorylated receptors and effectors to the actin polymerization machinery, thereby controlling actin dynamics, cell polarity, and directional migration [#0, #20, #24]. Through its SH2 domain it docks onto phosphotyrosine motifs in activated receptor tyrosine kinases and effectors, including PDGF and EGF receptors [#0], Eph receptors (EphB1 pTyr594, EphA3 pTyr602) [#5, #19], the bacterial effector EPEC Tir pTyr474 [#9, #14], and Tks5 pTyr557 in invadopodia [#18], while its three SH3 domains engage proline-rich effectors that drive actin assembly: WASP/N-WASP [#2, #16], PAK1 [#3, #4], and DCC [#10]. NCK1 is among the most potent activators of WASP/N-WASP-Arp2/3-mediated actin nucleation in vitro, operating at a defined 4 Nck:2 N-WASP:1 Arp2/3 stoichiometry and integrating phosphotyrosine and PI(4,5)P2 signals into localized actin polymerization [#16, #20, #23]. This scaffolding underlies diverse actin-dependent processes, including pathogen actin-based motility and entry (vaccinia virus, EPEC, picornaviruses) [#9, #12, #35, #41], FcγR-mediated phagocytosis [#42], endothelial front-rear polarity and angiogenesis downstream of VEGF and Slit2/ROBO1 [#24, #30], and osteoblast migration toward IGF1 [#31]. In T cells, NCK1 binds the CD3ε ITAM/proline-rich region through a cooperative SH3.1/SH2 two-site engagement required for TCR signaling and T cell activation in vivo [#17, #28, #29]. NCK1 activity and abundance are tightly regulated: it is selectively ubiquitinated by c-Cbl at Lys178 (antagonized by synaptopodin) [#25], translocated to the nucleus by SOCS7 where it is required for DNA-damage-induced p53 Ser15 phosphorylation and cell-cycle arrest [#15], and it suppresses ER stress signaling by binding the PERK inhibitory site pTyr561 and modulating IRE1α/JNK activation, with consequences for insulin biosynthesis and glucose homeostasis [#22, #26]. Beyond cytoskeletal scaffolding, NCK1 organizes the cytoplasmic capping complex by juxtaposing the capping enzyme and 5'-kinase on adjacent SH3 domains and promotes both cap-dependent and -independent translation via eIF2β binding [#11, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established NCK1 as a phosphotyrosine-binding adaptor coupled to growth factor receptors and capable of cellular transformation, defining its core scaffolding identity.\",\n      \"evidence\": \"Co-IP and GST pulldown with activated PDGF/EGF receptors plus NIH 3T3/3Y1 focus and soft-agar transformation assays\",\n      \"pmids\": [\"1333047\", \"1280326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the SH3-bound effectors mediating transformation\", \"Mechanism linking receptor binding to oncogenic output unresolved\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identified WASP as the first SH3-domain effector of NCK1, linking the adaptor to the actin nucleation machinery.\",\n      \"evidence\": \"Yeast two-hybrid screen with in vitro/in vivo binding and subcellular fractionation\",\n      \"pmids\": [\"7565724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate functional actin polymerization output\", \"Receptor-to-WASP coupling not yet reconstituted\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Mapped the NCK1 second SH3 domain to PAK1 and showed this links receptor tyrosine kinases to PAK1 kinase activation, defining a receptor-to-kinase relay.\",\n      \"evidence\": \"Domain-mapped Co-IP and kinase activity assays in COS-7, HeLa, and Swiss 3T3 cells with PDGFR stimulation\",\n      \"pmids\": [\"8824201\", \"8798379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream cytoskeletal consequences of PAK1 activation not measured\", \"Did not establish in vivo pathway context\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Placed NCK1 in Eph receptor and TCR signaling pathways, showing it relays distinct downstream outputs (JNK vs NFAT) depending on context.\",\n      \"evidence\": \"Yeast two-hybrid, Y594F mutagenesis, dominant-negative overexpression, NFAT/JNK reporter and kinase assays in T cells\",\n      \"pmids\": [\"9430661\", \"9755165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effector specificity determinants for JNK vs NFAT not defined\", \"Did not resolve direct vs indirect contributions\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined regulatory logic of the NCK1-PAK1 interaction (phospho-dependent motif recognition and autophosphorylation-controlled release) and the NIK/Nck integrin-activation module.\",\n      \"evidence\": \"Saturation peptide substitution binding analysis, phosphorylation assays, and Co-IP/kinase/integrin assays downstream of EphB receptors\",\n      \"pmids\": [\"10805734\", \"10669731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological role of phospho-regulated cycling not tested\", \"NIK identity and broader role not fully characterized\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated NCK1 is hijacked by pathogens for actin-based motility and revealed non-cytoskeletal roles in translation and netrin/DCC signaling.\",\n      \"evidence\": \"EPEC Tir pTyr474 binding/localization, vaccinia A36R phosphomutant actin tail assays, DCC Co-IP/Rac1/neurite assays, and eIF2β binding with in vitro translation assays\",\n      \"pmids\": [\"11918809\", \"12007418\", \"12149262\", \"11959995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Translation role mechanism beyond eIF2β binding not resolved\", \"DCC findings from single lab with Medium confidence\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified an ER-associated NCK1 pool that restrains ERK-1 and stress signaling, expanding NCK1 into ER-stress regulation.\",\n      \"evidence\": \"ER membrane IP, in vitro reconstitution, and kinase assays in Nck-null fibroblasts\",\n      \"pmids\": [\"15201339\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular ER tether not identified\", \"Single-lab finding without reciprocal validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Provided atomic-resolution basis for NCK1 SH2 phosphopeptide recognition and confirmed indistinguishable Nck1/Nck2 SH2 specificity, anchoring binding-mode interpretation.\",\n      \"evidence\": \"X-ray crystallography of SH2-Tir phosphopeptide complexes with mutagenesis and pulldowns\",\n      \"pmids\": [\"16636066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not explain paralog-specific functional divergence given identical SH2 specificity\", \"SH3 selectivity not structurally addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established NCK1 as a top-tier in vitro activator of WASP/N-WASP and revealed an unexpected SOCS7-driven nuclear function in the DNA-damage response.\",\n      \"evidence\": \"In vitro actin nucleation with purified proteins and nuclear/cytoplasmic rescue, ATM/ATR epistasis, and p53 Ser15 phosphorylation assays\",\n      \"pmids\": [\"17302440\", \"17803907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear NCK1 effectors mediating cell-cycle arrest not identified\", \"How nuclear vs cytoplasmic pools are balanced under stress unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed NCK1 integrates phosphotyrosine and PI(4,5)P2 inputs to drive localized actin polymerization, and extended this to invadopodia matrix degradation.\",\n      \"evidence\": \"Nck KO/KD with PI(4,5)P2 manipulation and live imaging of actin comets; Tks5 Y557 mutagenesis and gelatin degradation assays\",\n      \"pmids\": [\"19917259\", \"19596797\", \"19505147\", \"19242519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial coincidence detection mechanism of dual signals not fully defined\", \"Paralog non-redundancy mechanism for GTPase selectivity unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Quantified the stoichiometric and density requirements for NCK1-driven actin assembly, converting the scaffold model into a quantitative threshold model.\",\n      \"evidence\": \"Computational modeling with antibody-induced membrane aggregation and quantitative live-cell fluorescence; FcγR phagocytosis dependency\",\n      \"pmids\": [\"22613834\", \"22454526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of the 4:2:1 stoichiometry at physiological receptors untested\", \"FcγR study single-lab Medium confidence\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed paralog-specific regulation (c-Cbl ubiquitination of NCK1 Lys178), defined NCK1 control of front-back polarity, and discovered the PERK-binding metabolic axis.\",\n      \"evidence\": \"K178R/ubiquitination and synaptopodin competition assays; FRET/AFM polarity imaging in endothelial cells; PERK Y561F mutagenesis with KO mice and β-cell insulin readouts\",\n      \"pmids\": [\"24287595\", \"23444376\", \"24371088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How c-Cbl regulation intersects with signaling outputs in vivo not resolved\", \"Mechanism by which NCK1 controls RhoA versus Cdc42/Rac balance incomplete\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the cooperative two-site NCK1-CD3ε engagement required for TCR signaling in vivo and established NCK1 as the scaffold for the cytoplasmic capping complex.\",\n      \"evidence\": \"Proximity ligation, KI-PRS knock-in mice, EAE and peptide-inhibitor studies; in vitro capping-complex reconstitution on recombinant Nck1 with SH3 mutagenesis\",\n      \"pmids\": [\"26590318\", \"24470497\", \"25137142\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of capping complex assembly under physiological signaling unclear\", \"Whether mRNA recapping targets are sequence-selective not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established NCK1 (with NCK2) as essential for VEGF/Slit2-ROBO1-driven endothelial polarity and angiogenesis and for IGF1-directed osteoblast migration and bone formation.\",\n      \"evidence\": \"Endothelial- and osteoblast-specific conditional Nck1/2 KO mice with Cdc42/PAK activation, ROBO1/IRS-1 Co-IP, and in vivo vascular/bone phenotyping\",\n      \"pmids\": [\"26659946\", \"26621720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual NCK1 versus NCK2 contributions in these tissues not separated\", \"Receptor-proximal coupling to ROBO1 not structurally defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated NCK1/2 act as negative regulators of PDGFRβ signaling in pericytes and resolved NCK1- versus NCK2-specific interactomes and functions (NCK2 in cytokinesis).\",\n      \"evidence\": \"Mural-cell conditional KO with PDGFRβ Y1009 phosphorylation and retinopathy models; AP-MS/BioID interactomes and Nck-KO MEF cytokinesis imaging\",\n      \"pmids\": [\"30150707\", \"30002203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of NCK1 acting as PDGFR brake versus relay context-dependence unclear\", \"Cytokinesis role is NCK2-specific, leaving NCK1's mitotic role undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified an NCK1-selective inflammatory pathway in atherosclerosis, linking disturbed flow to NF-κB via an NCK1-IRAK1 interaction.\",\n      \"evidence\": \"Nck1 vs Nck2 KO mice, domain mutagenesis, Nck1-IRAK1 Co-IP, NF-κB reporters, flow chambers, and high-fat-diet atherosclerosis with bone marrow chimeras\",\n      \"pmids\": [\"32427580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NCK1-IRAK1 selectivity over NCK2 not defined\", \"How a cytoskeletal adaptor engages an innate-immune kinase mechanistically unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how the molecular features distinguishing NCK1 from NCK2 — given indistinguishable SH2 specificity — generate paralog-specific functions across actin, inflammatory, metabolic, and nuclear pathways.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural determinant explaining NCK1-selective partners (IRAK1, c-Cbl substrate, PERK) identified\", \"Integration logic that routes NCK1 between cytoskeletal scaffolding and translation/capping roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 3, 5, 16, 20, 27]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 16, 20, 23]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 26, 32, 37]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 11, 37]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [13, 26]},\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 20, 42]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 5, 30, 43]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 17, 28, 29, 34]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [11, 27]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [13, 15, 22, 26]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 12, 34, 35]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [10, 24, 30, 31]}\n    ],\n    \"complexes\": [\"cytoplasmic capping complex\", \"N-WASP/Arp2/3 actin nucleation complex\"],\n    \"partners\": [\"WASL\", \"PAK1\", \"WAS\", \"TKS5/SH3PXD2A\", \"ROBO1\", \"PERK/EIF2AK3\", \"CBL\", \"IRAK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}