{"gene":"DOK1","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1997,"finding":"p62(dok)/DOK1 is a constitutively tyrosine-phosphorylated protein in CML progenitor cells that associates with the p120 RasGTPase-activating protein (RasGAP); association with RasGAP correlates with its tyrosine phosphorylation. DOK1 is also rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor tyrosine kinase.","method":"Biochemical purification, co-immunoprecipitation, SDS-PAGE/Western blot from CML progenitor cells and hematopoietic cell lines","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — original identification by biochemical purification and co-IP, foundational paper, replicated extensively in subsequent work","pmids":["9008160"],"is_preprint":false},{"year":2000,"finding":"Tyrosine-phosphorylated p62(dok) directly inhibits RasGAP activity in vitro. p210(bcr-abl) directly phosphorylates p62(dok) on C-terminal tyrosine residues, and this phosphorylation is required for RasGAP binding. Five C-terminal tyrosines are involved in RasGAP binding in vitro.","method":"In vitro kinase assay with recombinant p210(bcr-abl), site-directed mutagenesis, RasGAP activity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis demonstrating both phosphorylation sites and functional consequence on RasGAP activity","pmids":["10688886"],"is_preprint":false},{"year":1999,"finding":"Cell adhesion to extracellular matrix induces tyrosine phosphorylation of DOK1 via Src family kinases; the pleckstrin homology (PH) domain is required for membrane localization and adhesion-dependent phosphorylation. Insulin promotes DOK1 association with NCK (via Y361) and RasGAP, and overexpression of wild-type DOK1 (but not PH-domain or Y361F mutants) enhances insulin-stimulated cell migration.","method":"Site-directed mutagenesis (DeltaPH, Y361F), co-immunoprecipitation, cell migration assay, subcellular fractionation/localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including mutagenesis, co-IP, migration assay, and localization; multiple labs subsequently confirmed key findings","pmids":["10202139"],"is_preprint":false},{"year":2000,"finding":"DOK1 functions as a negative regulator of BCR-mediated MAP kinase signaling in B cells; upon BCR or BCR/FcgammaRIIB co-crosslinking, DOK1 is tyrosine phosphorylated in a Lyn-dependent manner. DOK1 knockout (homologous recombination) results in increased ERK activation and abolishes FcgammaRIIB-mediated negative regulation of cell proliferation.","method":"Gene knockout by homologous recombination, Western blot for phosphorylation, cell proliferation assay, genetic epistasis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean gene knockout with defined signaling and proliferation phenotype; Lyn-dependent phosphorylation established; independently confirmed","pmids":["10640270"],"is_preprint":false},{"year":2000,"finding":"FcgammaRIIB-BCR co-aggregation leads to increased tyrosine phosphorylation of DOK1 and increased RasGAP binding. SHIP (phosphatidylinositol 5-phosphatase) recruits DOK1 via DOK1's PTB domain after being recruited and phosphorylated by FcgammaRIIB. A chimeric FcgammaRIIB containing the RasGAP-binding domain of DOK1 mediates FcgammaRIIB inhibition of ERK activation, demonstrating DOK1's structural information is sufficient for this inhibitory effect.","method":"Co-immunoprecipitation, chimeric receptor reconstitution, signaling assays in B cells","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — chimeric receptor reconstitution combined with co-IP provides direct mechanistic evidence; independently replicated","pmids":["10755621"],"is_preprint":false},{"year":2000,"finding":"DOK1 is a major tyrosine-phosphorylated protein in v-Src-transformed cells; the PTB domain and C-terminal tail (residues 336-363) of DOK1 are both necessary for inhibition of Src-mediated cellular transformation. The PTB domain binds phosphopeptides with consensus motif Y/MXXNXL-pY. DOK1 homodimerizes through its PTB domain and Y146; mutations blocking homodimerization reduce DOK1's ability to inhibit Src transformation.","method":"Combinatorial peptide library, site-directed mutagenesis, cellular transformation assay, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — peptide library approach defining PTB binding consensus, mutagenesis of dimerization and functional domains, transformation assay","pmids":["11042170"],"is_preprint":false},{"year":2001,"finding":"Loss of p62(dok) in knockout mice results in increased cell proliferation and sustained Ras/MAPK activation after growth factor removal; p62(dok) acts as a negative regulator of growth factor-induced Ras/MAPK signaling and opposes p210(bcr-abl)-induced leukemogenesis by shortening latency of myeloproliferative disease.","method":"p62(dok)-/- mouse model, proliferation assay, Ras/MAPK activation assay, retroviral bone marrow transduction/transplantation","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with multiple phenotypic readouts including signaling and disease model; independently replicated by two groups simultaneously","pmids":["11489947"],"is_preprint":false},{"year":2001,"finding":"PI3-kinase-dependent membrane recruitment of p62(dok) via its PH domain binding 3'-phosphorylated phosphoinositides is essential for its negative effect on PDGFR/Ras/MAPK signaling; p62(dok) can exert its negative effect on the MAPK pathway independently of its ability to associate with RasGAP and Nck.","method":"p62(dok)-/- cell reconstitution, PI3-kinase inhibition, PH domain mutants, membrane fractionation, MAPK assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reconstitution in knockout cells, PI3K inhibitor + PH mutant, multiple orthogonal methods","pmids":["11489946"],"is_preprint":false},{"year":2001,"finding":"Insulin receptor (IR) directly phosphorylates p62(dok) at Y362 and Y398 in vitro; Y362 phosphorylation is required for Nck binding and Y362/Y398 phosphorylation are required for RasGAP binding. Mutations at Y362/Y398 decrease inhibitory effect of p62(dok) on insulin-stimulated Ras and Akt activation. DOK1 inhibitory effect on Akt is upstream of Ras (blocked by constitutively active Ras).","method":"In vitro kinase assay, site-directed mutagenesis (Y362F, Y398F), co-immunoprecipitation, Ras and Akt activation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus mutagenesis plus functional signaling assays in a single study","pmids":["11551902"],"is_preprint":false},{"year":2002,"finding":"DOK1 binds RET receptor tyrosine kinase (identified by yeast two-hybrid); Y361 of Dok1 is the binding site for Nck; Y295, Y314, Y361, Y376, Y397, and Y408 mediate RasGAP binding. Overexpression of Dok1 or Y361F mutant suppresses RET-induced Ras/Erk activation (RasGAP-dependent), while Nck binding to Y361 is required for JNK/c-Jun activation by RET.","method":"Yeast two-hybrid, site-directed mutagenesis, co-immunoprecipitation, Ras/Erk and JNK activation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by mutagenesis and multiple signaling assays; clear separation of Nck vs. RasGAP-mediated functions","pmids":["12087092"],"is_preprint":false},{"year":2002,"finding":"DOK1 acts as an adaptor that links activin (TGF-beta family) receptors with Smad3 and Smad4 in a tyrosine phosphorylation-independent manner. Activin stimulation triggers DOK1 association with Smad3 and association of Smad3 with Smad4. DOK1 is associated with both type I and type II activin receptors. Overexpression of DOK1 augments activin A-induced apoptosis and down-regulates bcl-XL via inhibition of the Ras/Erk pathway.","method":"Retroviral gene trap screening, co-immunoprecipitation, apoptosis assay, Western blot","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP demonstrates novel binding to Smad3 and activin receptors; single lab, single method for binding","pmids":["11927552"],"is_preprint":false},{"year":2002,"finding":"DOK1 associates with the juxtamembrane region and C-terminal tail of c-Kit; PI3-kinase activation by c-Kit ligand promotes DOK1 PH domain binding and membrane recruitment; Lyn is required for KL-dependent DOK1 tyrosine phosphorylation (demonstrated using Lyn-deficient bone marrow mast cells); both Lyn and Tec can phosphorylate DOK1 in vitro. Membrane localization is required for DOK1's function as a negative regulator of cell proliferation.","method":"PI3-kinase inhibition, constitutively membrane-targeted DOK1 mutant, Lyn-deficient primary cells, in vitro kinase assay, membrane fractionation, proliferation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches including primary knockout cells, membrane-targeted mutants, and in vitro kinase assay","pmids":["11825908"],"is_preprint":false},{"year":2003,"finding":"Crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase was solved. The PTB domain consists of a beta-sandwich of two antiparallel beta-sheets capped by a C-terminal alpha-helix; the RET phosphopeptide binds in a surface groove between strand beta5 and the C-terminal alpha-helix. Dok1 PTB does not recognize peptide sequences from TrkA or IL-4 (recognized by Shc and IRS1).","method":"X-ray crystallography, peptide binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with bound ligand providing atomic-level mechanistic understanding; structural basis for RET specificity directly revealed","pmids":["14607833"],"is_preprint":false},{"year":2004,"finding":"c-Abl tyrosine kinase phosphorylates DOK1 at Y361 during cell spreading, promoting DOK1 association with the Nck SH2/SH3 adaptor. Each component (c-Abl, DOK1, Nck) is required for filopodia formation during cell spreading; cells lacking c-Abl, DOK1, or Nck have fewer filopodia. DOK1 and c-Abl act in the same signaling pathway and are both detected in filopodia of spreading cells.","method":"Genetic knockout fibroblasts (c-Abl-/-, Dok1-/-, Nck-/-), rescue by re-expression, specific substrate identification assay, immunofluorescence/localization, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple knockout mouse cells with rescue, localization and co-IP; multiple labs, orthogonal methods","pmids":["15148308"],"is_preprint":false},{"year":2004,"finding":"Dok1 and Dok2 double-deficient mice spontaneously develop CML-like myeloproliferative disease; myeloid cells show hyperproliferation and hypo-apoptosis, with enhanced Erk and Akt activation upon cytokine stimulation. Single knockouts show normal steady-state hematopoiesis, establishing cooperative/redundant roles.","method":"Single and double knockout mouse generation, hematopoiesis analysis, proliferation/apoptosis assay, Erk/Akt signaling assay, bone marrow transplantation","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent groups (Yasuda et al. and Niki et al.) generated double-knockout mice with same phenotype at the same time","pmids":["15611294","15611295"],"is_preprint":false},{"year":2004,"finding":"IKKbeta associates with and phosphorylates DOK1 at S439, S443, S446, and S450 in response to TNF-alpha, IL-1, or gamma radiation. Recombinant IKKbeta phosphorylates DOK1 in vitro. DOK1 with serine-to-alanine mutations at these sites fails to inhibit PDGF-induced ERK activation or cell growth, and also fails to promote cell motility. Serine-phosphorylation-mimicking mutant (EEEE) further enhances cell motility.","method":"In vitro kinase assay with recombinant IKKbeta, site-directed mutagenesis (S-to-A and S-to-E), phospho-site-specific antisera, ERK assay, cell growth and motility assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay with recombinant enzyme plus mutagenesis plus functional cellular assays; phospho-specific antibodies confirm in vivo phosphorylation","pmids":["15574499"],"is_preprint":false},{"year":2004,"finding":"DOK1 tyrosine residues Y336 and Y340 are essential for the negative regulation of Ras-Erk signaling but dispensable for RasGAP binding, indicating DOK1 recruits an additional, unidentified molecule that cooperates with RasGAP to inhibit Ras-Erk signaling and cellular transformation.","method":"C-terminal deletion mutants, site-directed mutagenesis (Y336F, Y340F, Y295F, Y361F), Erk activation assay, v-Abl transformation assay","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — systematic mutagenesis study, single lab, but mechanistically informative for separating RasGAP-dependent and -independent inhibitory functions","pmids":["15189452"],"is_preprint":false},{"year":2004,"finding":"DOK1 mediates SHP-2 binding to the alphaVbeta3 integrin beta3 subunit in response to IGF-I in vascular smooth muscle cells. IGF-I induces DOK1 binding to beta3 and to SHP-2; blocking DOK1/beta3 or DOK1/SHP-2 interactions (with synthetic peptides or non-binding DOK1 mutant) inhibits SHP-2 recruitment to beta3 and impairs IGF-I-dependent Akt and MAPK phosphorylation, cell migration and proliferation.","method":"Co-immunoprecipitation, synthetic peptide blocking, DOK1 mutant expression, Akt/MAPK assays, cell migration/proliferation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP with peptide blocking and mutant studies; single lab, no in vitro reconstitution","pmids":["15546884"],"is_preprint":false},{"year":2004,"finding":"Only the Dok1 PTB domain (not Dok4 or Dok5) specifically binds to phosphorylated Y1086 and Y1148 in EGFR, as shown by yeast two-hybrid and biochemical binding assays; structure-based mutagenesis defines molecular determinants for two distinct Dok1 PTB domain/EGFR interactions.","method":"Yeast two-hybrid, biochemical binding assays, structure-based mutagenesis","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — binding assay with mutagenesis; single lab, no crystal structure of the EGFR-Dok1 complex","pmids":["15476828"],"is_preprint":false},{"year":2005,"finding":"LPS (via TLR4) rapidly induces tyrosine phosphorylation of DOK1 and Dok2 in macrophages. Macrophages lacking DOK1 or Dok2 show elevated Erk activation (but not NF-kappaB or other MAPKs) and hyperproduction of TNF-alpha and nitric oxide. A Tyr/Phe substitution mutant of DOK1 fails to inhibit LPS-induced Erk activation, establishing tyrosine phosphorylation as mechanistically required. Macrophages respond normally to TLR agonists that do not induce DOK1 tyrosine phosphorylation.","method":"Knockout macrophages, forced expression with Y/F mutant, Erk/NF-kappaB signaling assays, cytokine measurements, in vivo LPS challenge","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout plus Y/F mutant reconstitution; multiple signaling readouts; in vitro and in vivo validation","pmids":["15699069"],"is_preprint":false},{"year":2006,"finding":"DOK1 inhibits PDGF-stimulated mitogenesis by two independent pathways: (1) recruiting Csk to active Src kinases (via a specific domain), thereby attenuating c-myc induction; and (2) negatively regulating Ras/MAPK activation via RasGAP and at least one additional DOK1-interacting protein. Both pathways contribute to DOK1's inhibitory effect on mitogenesis.","method":"DOK1 knockout cells, DOK1 interaction-deficient mutants, c-myc induction assay, Src activity assay, Csk co-IP, Ras/MAPK assay, proliferation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knockout + binding-deficient mutants systematically dissecting two independent pathways; multiple orthogonal assays","pmids":["16537894"],"is_preprint":false},{"year":2006,"finding":"DOK1 shuttles between the nucleus and cytoplasm; a functional nuclear export signal (NES: 348LLKAKLTDPKED359) keeps DOK1 cytoplasmic. CRM1 inhibition (leptomycin B) causes nuclear accumulation. Src-induced tyrosine phosphorylation and IKKbeta both promote cytoplasmic localization. Serum starvation or cell suspension favors nuclear localization; serum/growth factor/adhesion promotes cytoplasmic localization. Nuclear NES-mutant DOK1 has impaired ability to inhibit cell proliferation, promote cell spreading and cell motility.","method":"Leptomycin B treatment, NES mutagenesis, subcellular fractionation, live-cell imaging, proliferation/spreading/migration assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — NES mutation plus CRM1 inhibitor plus multiple functional assays; direct localization tied to functional consequences","pmids":["16705178"],"is_preprint":false},{"year":2007,"finding":"NMR spectroscopy and X-ray crystallography show that phosphorylation of Y747 in the beta3 integrin tail acts as a switch: unphosphorylated beta3 tail binds talin more strongly than DOK1 PTB domain; upon Y747 phosphorylation, DOK1 PTB domain binds much more strongly than talin. DOK1 interacts exclusively with the canonical NPXY motif of beta3, unlike talin.","method":"NMR (15N-1H HSQC titrations, chemical shift mapping), X-ray crystallography, affinity quantification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus NMR with quantitative affinity measurements; two complementary structural methods in single study","pmids":["18156175"],"is_preprint":false},{"year":2007,"finding":"Dok-1 and Dok-2 negatively regulate TCR signaling; mice lacking both show augmented responses to thymus-dependent antigens and elevated ZAP-70 activation. Forced expression of Dok-1 or Dok-2 in CD3+CD4+ T cell clone inhibits ZAP-70 activation. The C-terminal SH2-target motifs are dispensable for this negative regulation, indicating a distinct, unidentified mechanism.","method":"Double knockout mice, forced expression in T cell clone, ZAP-70 phosphorylation assay, proliferation/cytokine assays, C-terminal truncation mutants","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knockout mice plus forced expression with domain-mapping; multiple orthogonal methods","pmids":["17329234"],"is_preprint":false},{"year":2008,"finding":"DOK1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPARgamma. In Dok1-deficient embryonic fibroblasts, ERK activity is elevated, leading to increased phosphorylation of PPARgamma on S112 (a negative regulatory site for PPARgamma transactivation). Mutation of PPARgamma S112 blocks the lean phenotype caused by Dok1 ablation in mice, establishing the DOK1-ERK-PPARgamma axis.","method":"Dok1-knockout mouse adipocytes and embryonic fibroblasts, PPARgamma S112 phosphorylation assay, PPARgamma-S112A genetic rescue, ERK inhibition, adipogenic differentiation assay","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout cells plus genetic epistasis (PPARgamma S112A rescue); multiple orthogonal approaches; in vitro and in vivo","pmids":["18204460"],"is_preprint":false},{"year":2009,"finding":"DOK1 PH domain binds phosphatidylinositol 5-phosphate (PtdIns5P) in vitro; DOK1 tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. PtdIns5P increase regulates DOK1 tyrosine phosphorylation in vivo. The PH domain is necessary for DOK1 tyrosine phosphorylation and negative functions in T cells.","method":"In vitro lipid binding assay, intracellular PtdIns5P modulation, phosphorylation assay in T cells, PH domain deletion mutants","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro binding combined with in vivo PtdIns5P modulation; single lab, novel lipid ligand for PH domain","pmids":["19299694"],"is_preprint":false},{"year":2009,"finding":"CD45 recruits DOK1 to the proximal plasma membrane; DOK1-Y296F mutant abrogates interaction with CD45. CD45 expression is required for DOK1 membrane targeting in response to anti-CD3 stimulation. Stable expression of DOK1 decreases JAK-2 and STAT-3/5 phosphorylation following IL-3 and IFN-alpha stimulation; silencing DOK1 rescues MAP kinase and JAK activities in CD45-positive cells.","method":"Co-immunoprecipitation, site-directed mutagenesis (Y296F), stable overexpression, siRNA knockdown, JAK/STAT phosphorylation assay, membrane localization assay","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP with mutagenesis plus functional signaling assays; single lab","pmids":["19481264"],"is_preprint":false},{"year":2011,"finding":"Oncogenic tyrosine kinases (p210(bcr-abl) and oncogenic Src) downregulate DOK1 by targeting it for ubiquitin-proteasome-mediated degradation; this process is tyrosine kinase activity-dependent and mediated primarily by lysine-dependent polyubiquitination. Restoration of DOK1 strongly suppresses transformation; a DOK1 mutant refractory to degradation shows stronger suppression.","method":"Proteasome inhibitor treatment, ubiquitination assay, DOK1 degradation-resistant mutant, transformation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ubiquitination assay and degradation-resistant mutant; single lab but mechanistically clear","pmids":["21536658"],"is_preprint":false},{"year":2014,"finding":"BRK (breast tumor kinase/PTK6) interacts with and phosphorylates DOK1 specifically on Y362; this phosphorylation significantly downregulates DOK1 through the ubiquitin-proteasome pathway, promoting cell proliferation and migration.","method":"Co-immunoprecipitation, in vitro kinase assay (Y362 site-directed mutagenesis), ubiquitination assay, DOK1 protein stability assay, cell proliferation and migration assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — kinase assay plus mutagenesis plus ubiquitination and functional assays; single lab","pmids":["24523872"],"is_preprint":false},{"year":2014,"finding":"DOK1 regulates PDGF-BB-stimulated glioma cell invasion through tyrosine phosphorylation of p130Cas and activation of Rap1. DOK1 becomes tyrosine phosphorylated at Y362 and Y398 upon PDGF-BB stimulation; DOK1 knockdown or Y362F/Y398F double mutant inhibits both PDGF-BB-induced p130Cas phosphorylation and Rap1 activation. DOK1 co-localizes with phospho-p130Cas at the cell membrane.","method":"siRNA knockdown, DOK1 phosphosite mutant (DOK1FF), co-localization (immunofluorescence), Rap1 activation assay, 3D spheroid invasion assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — knockdown plus phosphosite mutant plus multiple assays; single lab, mechanistically informative for invasion pathway","pmids":["24762811"],"is_preprint":false},{"year":2016,"finding":"DOK1 negatively regulates integrin alphaIIbbeta3 outside-in signaling in platelets (but not inside-out signaling). Dok-1-/- platelets show increased clot retraction, increased PLCgamma2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation. Dok-1-/- mice exhibit shortened bleeding times and accelerated carotid artery thrombosis.","method":"Dok-1-/- mouse, platelet functional assays (clot retraction, fibrinogen binding, P-selectin expression), PLCgamma2 phosphorylation assay, in vivo thrombosis model","journal":"Thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean knockout mouse with multiple in vitro and in vivo functional platelet readouts; mechanistic distinction of inside-out vs outside-in signaling established","pmids":["26790499"],"is_preprint":false},{"year":2023,"finding":"The two SH2 domains of RasGAP (p120RasGAP) generate distinct binding interactions with doubly phosphorylated DOK1; RasGAP binding to DOK1 is ~100-fold stronger than to EphB4, driven by dual SH2 engagement with dominant N-SH2 interaction. RasGAP-DOK1 interactions do not impact RasGAP catalytic activity, implying RasGAP utilizes its SH2 domains to achieve spatial-temporal regulation of Ras signaling by localizing to DOK1.","method":"Affinity measurements, small-angle X-ray scattering, in vitro RasGAP activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — biophysical measurements (SAXS, affinity) with functional GAP activity assay; single lab, provides structural insight but no cell-based validation","pmids":["37507023"],"is_preprint":false},{"year":2000,"finding":"SHIP1 forms a complex with DOK1 in BCR/ABL-transformed hematopoietic cells; this interaction requires the SH2 domain of SHIP1 and the PTB domain of DOK1. DOK1-SHIP1 association is mutually exclusive with DOK1-RasGAP association. The DOK1/SHIP1 complex is only detected in the cytosolic fraction.","method":"Co-immunoprecipitation, domain-deletion mutants, subcellular fractionation","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP with domain mutants; single lab, mechanistically informative for SHIP1/DOK1 interaction requirements","pmids":["10822173"],"is_preprint":false},{"year":2000,"finding":"SH2D1A (XLP gene product) associates with DOK1 via its SH2 domain; this interaction depends on DOK1 phospho-Y449 in the sequence ALYSQVQK. An XLP-associated SH2D1A mutant fails to associate with DOK1. Overexpression of SH2D1A activates NF-kappaB (independent of SH2 domain, via IKKbeta).","method":"Co-immunoprecipitation, site-directed mutagenesis (XLP mutant SH2D1A), NF-kappaB reporter assay, dominant-negative IKKbeta","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP with defined phosphosite (Y449) and disease-relevant mutant; single lab","pmids":["10852966"],"is_preprint":false},{"year":1999,"finding":"SHP-1 constitutively associates with p62(DOK) in bone marrow macrophages, and this association occurs independently of p62(DOK) tyrosine phosphorylation. The SHP-1 catalytic domain (identified via catalytically inert SHP-1 C453S trapping) directly binds tyrosine-phosphorylated p62(DOK). p62(DOK) is the major CSF-1R-associated tyrosine-phosphorylated protein in CSF-1-treated macrophages; hyperphosphorylation in SHP-1-deficient macrophages correlates with growth factor-independent survival.","method":"Co-immunoprecipitation, far Western analysis, SHP-1-deficient (motheaten) mice, CSF-1 stimulation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus far Western with catalytic mutant trap; single lab but well-controlled with primary knockout macrophages","pmids":["10585470"],"is_preprint":false},{"year":2015,"finding":"Phosphorylated serine residues S745 and S756 (adjacent to the NxxF motif) in the integrin beta2 tail are required for DOK1 PTB domain interaction, providing an alternative phosphorylation switch for Dok1 binding to beta2 integrins (which lack a phosphorylatable NxxY motif). NMR analyses show higher affinity for the pSer756-containing beta2 peptide; the phosphorylated beta2 peptide occupies the canonical Dok1 PTB ligand-binding pocket.","method":"NMR spectroscopy, docking/structural modelling, cell-based binding assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR-based structural analysis with cell-based validation; single lab, no X-ray crystal structure","pmids":["26108885"],"is_preprint":false},{"year":2018,"finding":"The scaffold protein 14-3-3zeta interacts with the PTB domain of DOK1 (mapped to beta-sheet region) independently of phosphorylated integrin beta CTs. The 14-3-3zeta/Dok1 binary complex can simultaneously bind cognate phosphorylated integrin beta CT sequences, with Thr-phosphorylated beta2 or beta3 CTs binding 14-3-3zeta and Ser-phosphorylated beta2 CT or Tyr-phosphorylated beta3 CT binding DOK1, suggesting a molecular switch for integrin regulation.","method":"NMR spectroscopy, co-immunoprecipitation, binding assays with phosphorylated peptides","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR mapping with co-IP; single lab; proposes but partially validates a molecular switch","pmids":["30243836"],"is_preprint":false},{"year":2004,"finding":"Frameshift mutation in DOK1 (GGCC deletion causing protein truncation) identified in CLL changes DOK1 from a cytoplasmic/membrane protein to a nuclear protein containing a functional bipartite nuclear localization signal. Mutant DOK1 fails to inhibit PDGF-induced MAP kinase activation and forms heterodimers with wild-type DOK1 (enhanced by Lck-mediated phosphorylation), indicating a dominant-negative mechanism.","method":"Heteroduplex analysis/sequencing, subcellular localization assay, MAP kinase assay, co-immunoprecipitation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — natural mutation study with localization and signaling assays; single case but mechanistically informative","pmids":["14730347"],"is_preprint":false},{"year":2014,"finding":"Dok1 and Dok2 are tyrosine phosphorylated upon NK cell activation; overexpression of Dok proteins in human NK cells reduces NK cell activation induced by activating receptors. Gene ablation of Dok1 and Dok2 in mice induces an NK cell maturation defect and leads to increased IFN-gamma production, establishing an intrinsic negative feedback loop downstream of NK cell activating receptors.","method":"Overexpression in human NK cells, Dok1/Dok2 double-knockout mice, NK cell maturation analysis, IFN-gamma production assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — combined human overexpression and mouse knockout with functional readouts; single lab","pmids":["24963146"],"is_preprint":false},{"year":2022,"finding":"Upon FcεRI stimulation, DOK1 is recruited to the plasma membrane and undergoes tyrosine phosphorylation. Phosphorylated DOK1 inhibits FcεRI-operated calcium influx and negatively regulates mast cell degranulation by inhibiting calcium-dependent disassembly of actin filaments (F-actin disassembly).","method":"DOK1 overexpression in RBL-2H3 cells, co-immunoprecipitation, calcium influx assay, degranulation assay, F-actin staining","journal":"Clinical immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — overexpression system with multiple functional readouts; single lab; proposes calcium-actin mechanism","pmids":["35421591"],"is_preprint":false}],"current_model":"DOK1 (p62dok) is a multi-domain adapter/docking protein (containing PH, PTB, and C-terminal tyrosine-rich domains) that, upon PI3K-dependent membrane recruitment via its PH domain binding 3'-phosphoinositides, is phosphorylated by multiple tyrosine kinases (including Bcr-Abl, c-Abl, Src/Lyn, RET, insulin receptor, BRK, and others) and by IKKbeta on serine residues; phosphorylated DOK1 recruits RasGAP (via C-terminal phosphotyrosines), Nck (via pY361), Csk, SHP-1, SHIP1, Smad3, and other effectors to the plasma membrane, where it acts as a negative regulator of Ras/ERK/MAPK and Akt signaling, integrin outside-in signaling (via competition with talin regulated by a phosphotyrosine switch), and immune receptor signaling in B cells, T cells, NK cells, and macrophages, while also promoting cell spreading/migration and filopodia formation through c-Abl-phosphorylated Y361-Nck recruitment, and is subject to inactivation by oncogenic tyrosine kinases via ubiquitin-proteasome-dependent degradation."},"narrative":{"mechanistic_narrative":"DOK1 (p62dok) is a multi-domain docking protein that acts predominantly as a negative regulator of Ras/ERK/MAPK and PI3K/Akt signaling downstream of growth factor, cytokine, integrin, and immune receptors [PMID:11489947, PMID:11489946, PMID:15611294, PMID:15611295]. Following PI3-kinase activation, DOK1 is recruited to the plasma membrane through its PH domain binding 3'-phosphoinositides (including PtdIns5P), a step essential for both its tyrosine phosphorylation and its inhibitory output [PMID:11489946, PMID:11825908, PMID:19299694]. Once membrane-localized, DOK1 is phosphorylated on C-terminal tyrosines by an array of kinases—Bcr-Abl, c-Abl, Src/Lyn, the insulin receptor, RET, and others—creating docking sites that recruit p120 RasGAP (whose tandem SH2 domains engage doubly phosphorylated DOK1 to spatially localize Ras inhibition rather than alter GAP catalysis), and the Nck adaptor via Y361 [PMID:9008160, PMID:10688886, PMID:11551902, PMID:12087092, PMID:37507023]. Its PTB domain binds phosphotyrosine NPXY motifs—structurally defined in complex with a RET phosphopeptide—and recruits SHIP1, SHP-1, and the phospho-Y747 β3 integrin tail in competition with talin, the latter constituting a phosphotyrosine switch that governs integrin outside-in signaling [PMID:11042170, PMID:14607833, PMID:18156175, PMID:10822173, PMID:10585470]. Through these activities DOK1 restrains cell proliferation while promoting cell spreading, migration, and filopodia formation via c-Abl–dependent Y361–Nck recruitment [PMID:15148308, PMID:16537894, PMID:16705178]. DOK1 and the paralog DOK2 cooperatively suppress myeloproliferation, and loss of both produces CML-like disease, while oncogenic tyrosine kinases inactivate DOK1 via ubiquitin-proteasome–dependent degradation [PMID:15611294, PMID:15611295, PMID:21536658]. DOK1 additionally serine-phosphorylated by IKKbeta couples inflammatory signaling to its regulatory function, shuttles between nucleus and cytoplasm under CRM1 control, and serves as a negative feedback brake across B, T, NK, mast, and macrophage immune responses [PMID:15574499, PMID:15699069, PMID:16705178, PMID:17329234, PMID:24963146, PMID:35421591].","teleology":[{"year":1997,"claim":"Establishing DOK1 as a constitutively phosphorylated RasGAP-associated protein in leukemic cells defined it as a candidate node linking aberrant tyrosine kinase activity to Ras pathway control.","evidence":"Biochemical purification and co-IP from CML progenitor cells and c-Kit-activated hematopoietic lines","pmids":["9008160"],"confidence":"High","gaps":["Did not establish whether the RasGAP association was functionally inhibitory or activating","No domain mapping of the interaction"]},{"year":2000,"claim":"Demonstrating that Bcr-Abl directly phosphorylates DOK1 C-terminal tyrosines required for RasGAP binding, and that phospho-DOK1 inhibits RasGAP activity, gave the first direct mechanism for DOK1-mediated Ras regulation.","evidence":"In vitro kinase assay with recombinant p210(bcr-abl), site-directed mutagenesis, RasGAP activity assay","pmids":["10688886"],"confidence":"High","gaps":["In vitro RasGAP inhibition not directly tied to cellular Ras output","Identity of additional cooperating effectors unknown"]},{"year":2000,"claim":"Genetic knockout and chimeric receptor reconstitution placed DOK1 as the negative regulatory module of FcgammaRIIB/BCR signaling, showing its structural information alone is sufficient to inhibit ERK.","evidence":"DOK1 knockout by homologous recombination, Lyn-dependent phosphorylation assays, chimeric FcgammaRIIB reconstitution, proliferation assays in B cells","pmids":["10640270","10755621","10822173"],"confidence":"High","gaps":["Mechanism downstream of RasGAP recruitment in B cells incompletely defined","SHIP1-DOK1 vs RasGAP-DOK1 complex switching not resolved spatially"]},{"year":2001,"claim":"Knockout mouse models and PI3K/PH-domain dissection established DOK1 as a physiological negative regulator of growth factor–induced Ras/MAPK signaling whose membrane recruitment, not solely RasGAP/Nck binding, drives inhibition and opposes leukemogenesis.","evidence":"p62(dok)-/- mice, retroviral bone marrow transplantation, PI3K inhibition with PH-domain mutants, membrane fractionation, insulin receptor in vitro kinase assays","pmids":["11489947","11489946","11551902","11825908"],"confidence":"High","gaps":["The RasGAP/Nck-independent inhibitory effector remained unidentified","Single-knockout phenotype mild, hinting at redundancy"]},{"year":2002,"claim":"Mapping DOK1's interactions with RET and activin receptors separated RasGAP-dependent ERK suppression from Nck-dependent JNK activation and revealed a phosphorylation-independent adaptor role linking TGF-beta family receptors to Smad3.","evidence":"Yeast two-hybrid, gene-trap screening, mutagenesis, co-IP, Ras/Erk, JNK and apoptosis assays","pmids":["12087092","11927552"],"confidence":"High","gaps":["Smad3 linkage based on single-lab co-IP","Physiological contribution of DOK1 to activin signaling in vivo untested"]},{"year":2003,"claim":"The crystal structure of the DOK1 PTB domain bound to a RET phosphopeptide provided the atomic basis for its phosphotyrosine recognition and selectivity against Shc/IRS1 ligands.","evidence":"X-ray crystallography of PTB domain alone and in complex, peptide binding assays","pmids":["14607833"],"confidence":"High","gaps":["No structure of full-length DOK1 or PH domain","Did not capture EGFR or integrin tail complexes"]},{"year":2004,"claim":"c-Abl-dependent Y361 phosphorylation and Nck recruitment were shown to drive filopodia formation, establishing a positive cytoskeletal role for DOK1 distinct from its inhibitory signaling functions.","evidence":"c-Abl-/-, Dok1-/-, Nck-/- fibroblasts with rescue, immunofluorescence, co-IP","pmids":["15148308"],"confidence":"High","gaps":["Downstream actin regulators of the Nck arm not defined","Reconciliation of pro-migratory and anti-proliferative roles not addressed"]},{"year":2004,"claim":"Double-knockout mice and IKKbeta serine phosphorylation studies revealed DOK1/DOK2 redundancy in suppressing myeloproliferation and a serine-phosphorylation input coupling inflammatory kinases to DOK1 function.","evidence":"Dok1/Dok2 single and double knockout mice, in vitro IKKbeta kinase assay, S-to-A/S-to-E mutagenesis, ERK/motility assays","pmids":["15611294","15611295","15574499"],"confidence":"High","gaps":["How serine phosphorylation mechanistically alters DOK1 activity unresolved","Division of labor between DOK1 and DOK2 not fully mapped"]},{"year":2004,"claim":"Systematic mutagenesis showed that Y336/Y340 are required for Ras-Erk inhibition independently of RasGAP binding, formalizing the existence of an unidentified cooperating effector.","evidence":"C-terminal deletion and point mutants, Erk and v-Abl transformation assays","pmids":["15189452"],"confidence":"Medium","gaps":["The cooperating Y336/Y340-recruited molecule was never identified","Single-lab mutagenesis without biochemical isolation of the partner"]},{"year":2005,"claim":"Knockout-plus-Y/F-mutant reconstitution defined DOK1/DOK2 as tyrosine-phosphorylation-dependent brakes on TLR4/ERK signaling and inflammatory cytokine output in macrophages.","evidence":"Knockout macrophages, Y/F mutant reconstitution, ERK/NF-kappaB assays, cytokine measurement, in vivo LPS challenge","pmids":["15699069"],"confidence":"High","gaps":["Kinase responsible for TLR4-induced DOK1 phosphorylation not pinned down","Selectivity for ERK over other MAPKs mechanistically unexplained"]},{"year":2006,"claim":"Pathway dissection revealed DOK1 inhibits mitogenesis through two arms—Csk recruitment to Src attenuating c-myc, and RasGAP plus an additional effector—and demonstrated CRM1-dependent nucleocytoplasmic shuttling tied to its anti-proliferative and pro-spreading functions.","evidence":"Knockout cells with binding-deficient mutants, Csk co-IP, c-myc/Src assays, leptomycin B treatment, NES mutagenesis, localization and functional assays","pmids":["16537894","16705178"],"confidence":"High","gaps":["Nuclear function of DOK1, if any, undefined","The additional RasGAP-cooperating effector still unidentified"]},{"year":2007,"claim":"Structural and genetic studies established the beta3 integrin Y747 phosphotyrosine switch between talin and DOK1 PTB binding, and defined DOK1/DOK2 as negative regulators of TCR/ZAP-70 signaling via a C-terminal-motif-independent mechanism.","evidence":"NMR and X-ray with affinity quantification of beta3 tail; double-knockout mice and forced expression in T cells with truncation mutants","pmids":["18156175","17329234"],"confidence":"High","gaps":["The C-terminal-independent mechanism inhibiting ZAP-70 not identified","Cellular trigger for beta3 Y747 phosphorylation not defined"]},{"year":2008,"claim":"Genetic epistasis through PPARgamma-S112A rescue established a DOK1-ERK-PPARgamma axis controlling adipocyte hypertrophy, extending DOK1's ERK-suppressive role to metabolic physiology.","evidence":"Dok1-knockout adipocytes and MEFs, PPARgamma S112 phosphorylation assay, PPARgamma-S112A genetic rescue in mice","pmids":["18204460"],"confidence":"High","gaps":["Direct molecular link from DOK1 to the relevant ERK pool in adipocytes not shown","Whether the same axis operates in human adipose tissue untested"]},{"year":2009,"claim":"Identification of PtdIns5P as a PH-domain ligand connected lipid second-messenger production to DOK1 tyrosine phosphorylation and negative function in T cells.","evidence":"In vitro lipid binding, intracellular PtdIns5P modulation, PH-deletion mutants, phosphorylation assays in T cells","pmids":["19299694"],"confidence":"Medium","gaps":["Single-lab lipid binding without structural validation of the PH-PtdIns5P interaction","Relative contributions of PtdIns5P versus PI3K products unclear"]},{"year":2011,"claim":"Demonstration that oncogenic Bcr-Abl and Src target DOK1 for ubiquitin-proteasome degradation showed that tumor kinases inactivate DOK1 by destruction, and that degradation-resistant DOK1 more strongly suppresses transformation.","evidence":"Proteasome inhibition, ubiquitination assay, degradation-resistant mutant, transformation assay","pmids":["21536658"],"confidence":"Medium","gaps":["E3 ligase responsible not identified","Single-lab study without in vivo confirmation"]},{"year":2014,"claim":"Studies of BRK and PDGF-BB/glioma signaling refined DOK1 as both a degradation substrate of pro-tumorigenic kinases and a Y362/Y398-dependent driver of p130Cas/Rap1-mediated invasion.","evidence":"Co-IP, in vitro kinase and ubiquitination assays, phosphosite mutants, Rap1 activation and 3D invasion assays","pmids":["24523872","24762811"],"confidence":"Medium","gaps":["Reconciliation of DOK1's tumor-suppressive versus pro-invasive roles unresolved","Both findings rest on single-lab knockdown/overexpression systems"]},{"year":2016,"claim":"A clean platelet knockout established DOK1 as a selective negative regulator of integrin alphaIIbbeta3 outside-in (not inside-out) signaling, with in vivo consequences for thrombosis and bleeding.","evidence":"Dok-1-/- mice, platelet clot retraction/spreading assays, PLCgamma2 phosphorylation, in vivo carotid thrombosis model","pmids":["26790499"],"confidence":"High","gaps":["Molecular basis for outside-in selectivity not resolved","Direct competition with talin in platelets not demonstrated in this system"]},{"year":2023,"claim":"Biophysical analysis of the RasGAP-DOK1 interface showed dual SH2 engagement confers ~100-fold selectivity without altering GAP catalysis, reframing RasGAP recruitment as spatial localization rather than direct enzymatic modulation.","evidence":"Affinity measurements, small-angle X-ray scattering, in vitro RasGAP activity assay","pmids":["37507023"],"confidence":"Medium","gaps":["No cell-based validation of the localization model","Apparent tension with earlier in vitro RasGAP-inhibition reports unreconciled"]},{"year":null,"claim":"The identity of the RasGAP-independent effector recruited via Y336/Y340 that cooperates to inhibit Ras-Erk signaling, and the unifying mechanism reconciling DOK1's anti-proliferative versus pro-migratory/pro-invasive roles, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No biochemical isolation of the Y336/Y340-binding partner","No single framework linking DOK1's tumor-suppressive and pro-invasive activities","Context determinants selecting RasGAP versus Nck versus SHIP1 complexes undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,9,13,17,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3,6,7]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[7,25]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[13,39]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,7,11,21]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[21,32]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[21,37]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6,7,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,19,23,38]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,14,27,37]}],"complexes":[],"partners":["RASA1","NCK1","CSK","INPP5D","PTPN6","SH2D1A","YWHAZ","ITGB3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99704","full_name":"Docking protein 1","aliases":["Downstream of tyrosine kinase 1","p62(dok)","pp62"],"length_aa":481,"mass_kda":52.4,"function":"DOK proteins are enzymatically inert adaptor or scaffolding proteins. They provide a docking platform for the assembly of multimolecular signaling complexes. DOK1 appears to be a negative regulator of the insulin signaling pathway. Modulates integrin activation by competing with talin for the same binding site on ITGB3","subcellular_location":"Cytoplasm, perinuclear region","url":"https://www.uniprot.org/uniprotkb/Q99704/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DOK1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DOK1","total_profiled":1310},"omim":[{"mim_id":"617797","title":"SRC-RELATED KINASE LACKING C-TERMINAL REGULATORY TYROSINE AND N-TERMINAL MYRISTYLATION SITES; SRMS","url":"https://www.omim.org/entry/617797"},{"mim_id":"611435","title":"DOCKING PROTEIN 3; DOK3","url":"https://www.omim.org/entry/611435"},{"mim_id":"611402","title":"DOCKING PROTEIN 6; DOK6","url":"https://www.omim.org/entry/611402"},{"mim_id":"604997","title":"DOCKING PROTEIN 2; DOK2","url":"https://www.omim.org/entry/604997"},{"mim_id":"604590","title":"Fc FRAGMENT OF IgG RECEPTOR IIb; FCGR2B","url":"https://www.omim.org/entry/604590"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DOK1"},"hgnc":{"alias_symbol":["p62dok"],"prev_symbol":[]},"alphafold":{"accession":"Q99704","domains":[{"cath_id":"2.30.29.30","chopping":"7-48_61-119","consensus_level":"high","plddt":89.8789,"start":7,"end":119},{"cath_id":"2.30.29.30","chopping":"154-257","consensus_level":"high","plddt":93.7483,"start":154,"end":257}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99704","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99704-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99704-F1-predicted_aligned_error_v6.png","plddt_mean":67.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DOK1","jax_strain_url":"https://www.jax.org/strain/search?query=DOK1"},"sequence":{"accession":"Q99704","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99704.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99704/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99704"}},"corpus_meta":[{"pmid":"9008160","id":"PMC_9008160","title":"p62(dok): a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells.","date":"1997","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/9008160","citation_count":331,"is_preprint":false},{"pmid":"10755621","id":"PMC_10755621","title":"The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells.","date":"2000","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/10755621","citation_count":187,"is_preprint":false},{"pmid":"11470823","id":"PMC_11470823","title":"Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11470823","citation_count":142,"is_preprint":false},{"pmid":"10640270","id":"PMC_10640270","title":"Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/11489947","citation_count":96,"is_preprint":false},{"pmid":"18204460","id":"PMC_18204460","title":"Dok1 mediates high-fat diet-induced adipocyte hypertrophy and obesity through modulation of PPAR-gamma phosphorylation.","date":"2008","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/18204460","citation_count":92,"is_preprint":false},{"pmid":"15611294","id":"PMC_15611294","title":"Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia.","date":"2004","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/15611294","citation_count":86,"is_preprint":false},{"pmid":"17329234","id":"PMC_17329234","title":"Dok-1 and Dok-2 are negative regulators of T cell receptor signaling.","date":"2007","source":"International immunology","url":"https://pubmed.ncbi.nlm.nih.gov/17329234","citation_count":76,"is_preprint":false},{"pmid":"15611295","id":"PMC_15611295","title":"Role of Dok-1 and Dok-2 in leukemia suppression.","date":"2004","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/15611295","citation_count":74,"is_preprint":false},{"pmid":"15699069","id":"PMC_15699069","title":"Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling.","date":"2005","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/15699069","citation_count":74,"is_preprint":false},{"pmid":"11970986","id":"PMC_11970986","title":"Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling.","date":"2002","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/11970986","citation_count":69,"is_preprint":false},{"pmid":"11042170","id":"PMC_11042170","title":"Domain-dependent function of the rasGAP-binding protein p62Dok in cell signaling.","date":"2000","source":"The Journal of biological 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1950)","url":"https://pubmed.ncbi.nlm.nih.gov/19299694","citation_count":52,"is_preprint":false},{"pmid":"11551902","id":"PMC_11551902","title":"Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11551902","citation_count":48,"is_preprint":false},{"pmid":"11031258","id":"PMC_11031258","title":"SHIP1, an SH2 domain containing polyinositol-5-phosphatase, regulates migration through two critical tyrosine residues and forms a novel signaling complex with DOK1 and CRKL.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11031258","citation_count":47,"is_preprint":false},{"pmid":"10852966","id":"PMC_10852966","title":"The X-linked lymphoproliferative syndrome gene 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Section D, Biological crystallography","url":"https://pubmed.ncbi.nlm.nih.gov/14747716","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46177,"output_tokens":10904,"usd":0.151045,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":21505,"output_tokens":6124,"usd":0.130312,"stage2_stop_reason":"end_turn"},"total_usd":0.281357,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"p62(dok)/DOK1 is a constitutively tyrosine-phosphorylated protein in CML progenitor cells that associates with the p120 RasGTPase-activating protein (RasGAP); association with RasGAP correlates with its tyrosine phosphorylation. DOK1 is also rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor tyrosine kinase.\",\n      \"method\": \"Biochemical purification, co-immunoprecipitation, SDS-PAGE/Western blot from CML progenitor cells and hematopoietic cell lines\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — original identification by biochemical purification and co-IP, foundational paper, replicated extensively in subsequent work\",\n      \"pmids\": [\"9008160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Tyrosine-phosphorylated p62(dok) directly inhibits RasGAP activity in vitro. p210(bcr-abl) directly phosphorylates p62(dok) on C-terminal tyrosine residues, and this phosphorylation is required for RasGAP binding. Five C-terminal tyrosines are involved in RasGAP binding in vitro.\",\n      \"method\": \"In vitro kinase assay with recombinant p210(bcr-abl), site-directed mutagenesis, RasGAP activity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis demonstrating both phosphorylation sites and functional consequence on RasGAP activity\",\n      \"pmids\": [\"10688886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Cell adhesion to extracellular matrix induces tyrosine phosphorylation of DOK1 via Src family kinases; the pleckstrin homology (PH) domain is required for membrane localization and adhesion-dependent phosphorylation. Insulin promotes DOK1 association with NCK (via Y361) and RasGAP, and overexpression of wild-type DOK1 (but not PH-domain or Y361F mutants) enhances insulin-stimulated cell migration.\",\n      \"method\": \"Site-directed mutagenesis (DeltaPH, Y361F), co-immunoprecipitation, cell migration assay, subcellular fractionation/localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including mutagenesis, co-IP, migration assay, and localization; multiple labs subsequently confirmed key findings\",\n      \"pmids\": [\"10202139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"DOK1 functions as a negative regulator of BCR-mediated MAP kinase signaling in B cells; upon BCR or BCR/FcgammaRIIB co-crosslinking, DOK1 is tyrosine phosphorylated in a Lyn-dependent manner. DOK1 knockout (homologous recombination) results in increased ERK activation and abolishes FcgammaRIIB-mediated negative regulation of cell proliferation.\",\n      \"method\": \"Gene knockout by homologous recombination, Western blot for phosphorylation, cell proliferation assay, genetic epistasis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean gene knockout with defined signaling and proliferation phenotype; Lyn-dependent phosphorylation established; independently confirmed\",\n      \"pmids\": [\"10640270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"FcgammaRIIB-BCR co-aggregation leads to increased tyrosine phosphorylation of DOK1 and increased RasGAP binding. SHIP (phosphatidylinositol 5-phosphatase) recruits DOK1 via DOK1's PTB domain after being recruited and phosphorylated by FcgammaRIIB. A chimeric FcgammaRIIB containing the RasGAP-binding domain of DOK1 mediates FcgammaRIIB inhibition of ERK activation, demonstrating DOK1's structural information is sufficient for this inhibitory effect.\",\n      \"method\": \"Co-immunoprecipitation, chimeric receptor reconstitution, signaling assays in B cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chimeric receptor reconstitution combined with co-IP provides direct mechanistic evidence; independently replicated\",\n      \"pmids\": [\"10755621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"DOK1 is a major tyrosine-phosphorylated protein in v-Src-transformed cells; the PTB domain and C-terminal tail (residues 336-363) of DOK1 are both necessary for inhibition of Src-mediated cellular transformation. The PTB domain binds phosphopeptides with consensus motif Y/MXXNXL-pY. DOK1 homodimerizes through its PTB domain and Y146; mutations blocking homodimerization reduce DOK1's ability to inhibit Src transformation.\",\n      \"method\": \"Combinatorial peptide library, site-directed mutagenesis, cellular transformation assay, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — peptide library approach defining PTB binding consensus, mutagenesis of dimerization and functional domains, transformation assay\",\n      \"pmids\": [\"11042170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Loss of p62(dok) in knockout mice results in increased cell proliferation and sustained Ras/MAPK activation after growth factor removal; p62(dok) acts as a negative regulator of growth factor-induced Ras/MAPK signaling and opposes p210(bcr-abl)-induced leukemogenesis by shortening latency of myeloproliferative disease.\",\n      \"method\": \"p62(dok)-/- mouse model, proliferation assay, Ras/MAPK activation assay, retroviral bone marrow transduction/transplantation\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with multiple phenotypic readouts including signaling and disease model; independently replicated by two groups simultaneously\",\n      \"pmids\": [\"11489947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PI3-kinase-dependent membrane recruitment of p62(dok) via its PH domain binding 3'-phosphorylated phosphoinositides is essential for its negative effect on PDGFR/Ras/MAPK signaling; p62(dok) can exert its negative effect on the MAPK pathway independently of its ability to associate with RasGAP and Nck.\",\n      \"method\": \"p62(dok)-/- cell reconstitution, PI3-kinase inhibition, PH domain mutants, membrane fractionation, MAPK assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reconstitution in knockout cells, PI3K inhibitor + PH mutant, multiple orthogonal methods\",\n      \"pmids\": [\"11489946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Insulin receptor (IR) directly phosphorylates p62(dok) at Y362 and Y398 in vitro; Y362 phosphorylation is required for Nck binding and Y362/Y398 phosphorylation are required for RasGAP binding. Mutations at Y362/Y398 decrease inhibitory effect of p62(dok) on insulin-stimulated Ras and Akt activation. DOK1 inhibitory effect on Akt is upstream of Ras (blocked by constitutively active Ras).\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (Y362F, Y398F), co-immunoprecipitation, Ras and Akt activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus mutagenesis plus functional signaling assays in a single study\",\n      \"pmids\": [\"11551902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DOK1 binds RET receptor tyrosine kinase (identified by yeast two-hybrid); Y361 of Dok1 is the binding site for Nck; Y295, Y314, Y361, Y376, Y397, and Y408 mediate RasGAP binding. Overexpression of Dok1 or Y361F mutant suppresses RET-induced Ras/Erk activation (RasGAP-dependent), while Nck binding to Y361 is required for JNK/c-Jun activation by RET.\",\n      \"method\": \"Yeast two-hybrid, site-directed mutagenesis, co-immunoprecipitation, Ras/Erk and JNK activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by mutagenesis and multiple signaling assays; clear separation of Nck vs. RasGAP-mediated functions\",\n      \"pmids\": [\"12087092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DOK1 acts as an adaptor that links activin (TGF-beta family) receptors with Smad3 and Smad4 in a tyrosine phosphorylation-independent manner. Activin stimulation triggers DOK1 association with Smad3 and association of Smad3 with Smad4. DOK1 is associated with both type I and type II activin receptors. Overexpression of DOK1 augments activin A-induced apoptosis and down-regulates bcl-XL via inhibition of the Ras/Erk pathway.\",\n      \"method\": \"Retroviral gene trap screening, co-immunoprecipitation, apoptosis assay, Western blot\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP demonstrates novel binding to Smad3 and activin receptors; single lab, single method for binding\",\n      \"pmids\": [\"11927552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DOK1 associates with the juxtamembrane region and C-terminal tail of c-Kit; PI3-kinase activation by c-Kit ligand promotes DOK1 PH domain binding and membrane recruitment; Lyn is required for KL-dependent DOK1 tyrosine phosphorylation (demonstrated using Lyn-deficient bone marrow mast cells); both Lyn and Tec can phosphorylate DOK1 in vitro. Membrane localization is required for DOK1's function as a negative regulator of cell proliferation.\",\n      \"method\": \"PI3-kinase inhibition, constitutively membrane-targeted DOK1 mutant, Lyn-deficient primary cells, in vitro kinase assay, membrane fractionation, proliferation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches including primary knockout cells, membrane-targeted mutants, and in vitro kinase assay\",\n      \"pmids\": [\"11825908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase was solved. The PTB domain consists of a beta-sandwich of two antiparallel beta-sheets capped by a C-terminal alpha-helix; the RET phosphopeptide binds in a surface groove between strand beta5 and the C-terminal alpha-helix. Dok1 PTB does not recognize peptide sequences from TrkA or IL-4 (recognized by Shc and IRS1).\",\n      \"method\": \"X-ray crystallography, peptide binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with bound ligand providing atomic-level mechanistic understanding; structural basis for RET specificity directly revealed\",\n      \"pmids\": [\"14607833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"c-Abl tyrosine kinase phosphorylates DOK1 at Y361 during cell spreading, promoting DOK1 association with the Nck SH2/SH3 adaptor. Each component (c-Abl, DOK1, Nck) is required for filopodia formation during cell spreading; cells lacking c-Abl, DOK1, or Nck have fewer filopodia. DOK1 and c-Abl act in the same signaling pathway and are both detected in filopodia of spreading cells.\",\n      \"method\": \"Genetic knockout fibroblasts (c-Abl-/-, Dok1-/-, Nck-/-), rescue by re-expression, specific substrate identification assay, immunofluorescence/localization, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple knockout mouse cells with rescue, localization and co-IP; multiple labs, orthogonal methods\",\n      \"pmids\": [\"15148308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Dok1 and Dok2 double-deficient mice spontaneously develop CML-like myeloproliferative disease; myeloid cells show hyperproliferation and hypo-apoptosis, with enhanced Erk and Akt activation upon cytokine stimulation. Single knockouts show normal steady-state hematopoiesis, establishing cooperative/redundant roles.\",\n      \"method\": \"Single and double knockout mouse generation, hematopoiesis analysis, proliferation/apoptosis assay, Erk/Akt signaling assay, bone marrow transplantation\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent groups (Yasuda et al. and Niki et al.) generated double-knockout mice with same phenotype at the same time\",\n      \"pmids\": [\"15611294\", \"15611295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"IKKbeta associates with and phosphorylates DOK1 at S439, S443, S446, and S450 in response to TNF-alpha, IL-1, or gamma radiation. Recombinant IKKbeta phosphorylates DOK1 in vitro. DOK1 with serine-to-alanine mutations at these sites fails to inhibit PDGF-induced ERK activation or cell growth, and also fails to promote cell motility. Serine-phosphorylation-mimicking mutant (EEEE) further enhances cell motility.\",\n      \"method\": \"In vitro kinase assay with recombinant IKKbeta, site-directed mutagenesis (S-to-A and S-to-E), phospho-site-specific antisera, ERK assay, cell growth and motility assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay with recombinant enzyme plus mutagenesis plus functional cellular assays; phospho-specific antibodies confirm in vivo phosphorylation\",\n      \"pmids\": [\"15574499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DOK1 tyrosine residues Y336 and Y340 are essential for the negative regulation of Ras-Erk signaling but dispensable for RasGAP binding, indicating DOK1 recruits an additional, unidentified molecule that cooperates with RasGAP to inhibit Ras-Erk signaling and cellular transformation.\",\n      \"method\": \"C-terminal deletion mutants, site-directed mutagenesis (Y336F, Y340F, Y295F, Y361F), Erk activation assay, v-Abl transformation assay\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — systematic mutagenesis study, single lab, but mechanistically informative for separating RasGAP-dependent and -independent inhibitory functions\",\n      \"pmids\": [\"15189452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DOK1 mediates SHP-2 binding to the alphaVbeta3 integrin beta3 subunit in response to IGF-I in vascular smooth muscle cells. IGF-I induces DOK1 binding to beta3 and to SHP-2; blocking DOK1/beta3 or DOK1/SHP-2 interactions (with synthetic peptides or non-binding DOK1 mutant) inhibits SHP-2 recruitment to beta3 and impairs IGF-I-dependent Akt and MAPK phosphorylation, cell migration and proliferation.\",\n      \"method\": \"Co-immunoprecipitation, synthetic peptide blocking, DOK1 mutant expression, Akt/MAPK assays, cell migration/proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP with peptide blocking and mutant studies; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"15546884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Only the Dok1 PTB domain (not Dok4 or Dok5) specifically binds to phosphorylated Y1086 and Y1148 in EGFR, as shown by yeast two-hybrid and biochemical binding assays; structure-based mutagenesis defines molecular determinants for two distinct Dok1 PTB domain/EGFR interactions.\",\n      \"method\": \"Yeast two-hybrid, biochemical binding assays, structure-based mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — binding assay with mutagenesis; single lab, no crystal structure of the EGFR-Dok1 complex\",\n      \"pmids\": [\"15476828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"LPS (via TLR4) rapidly induces tyrosine phosphorylation of DOK1 and Dok2 in macrophages. Macrophages lacking DOK1 or Dok2 show elevated Erk activation (but not NF-kappaB or other MAPKs) and hyperproduction of TNF-alpha and nitric oxide. A Tyr/Phe substitution mutant of DOK1 fails to inhibit LPS-induced Erk activation, establishing tyrosine phosphorylation as mechanistically required. Macrophages respond normally to TLR agonists that do not induce DOK1 tyrosine phosphorylation.\",\n      \"method\": \"Knockout macrophages, forced expression with Y/F mutant, Erk/NF-kappaB signaling assays, cytokine measurements, in vivo LPS challenge\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout plus Y/F mutant reconstitution; multiple signaling readouts; in vitro and in vivo validation\",\n      \"pmids\": [\"15699069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DOK1 inhibits PDGF-stimulated mitogenesis by two independent pathways: (1) recruiting Csk to active Src kinases (via a specific domain), thereby attenuating c-myc induction; and (2) negatively regulating Ras/MAPK activation via RasGAP and at least one additional DOK1-interacting protein. Both pathways contribute to DOK1's inhibitory effect on mitogenesis.\",\n      \"method\": \"DOK1 knockout cells, DOK1 interaction-deficient mutants, c-myc induction assay, Src activity assay, Csk co-IP, Ras/MAPK assay, proliferation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout + binding-deficient mutants systematically dissecting two independent pathways; multiple orthogonal assays\",\n      \"pmids\": [\"16537894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DOK1 shuttles between the nucleus and cytoplasm; a functional nuclear export signal (NES: 348LLKAKLTDPKED359) keeps DOK1 cytoplasmic. CRM1 inhibition (leptomycin B) causes nuclear accumulation. Src-induced tyrosine phosphorylation and IKKbeta both promote cytoplasmic localization. Serum starvation or cell suspension favors nuclear localization; serum/growth factor/adhesion promotes cytoplasmic localization. Nuclear NES-mutant DOK1 has impaired ability to inhibit cell proliferation, promote cell spreading and cell motility.\",\n      \"method\": \"Leptomycin B treatment, NES mutagenesis, subcellular fractionation, live-cell imaging, proliferation/spreading/migration assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NES mutation plus CRM1 inhibitor plus multiple functional assays; direct localization tied to functional consequences\",\n      \"pmids\": [\"16705178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NMR spectroscopy and X-ray crystallography show that phosphorylation of Y747 in the beta3 integrin tail acts as a switch: unphosphorylated beta3 tail binds talin more strongly than DOK1 PTB domain; upon Y747 phosphorylation, DOK1 PTB domain binds much more strongly than talin. DOK1 interacts exclusively with the canonical NPXY motif of beta3, unlike talin.\",\n      \"method\": \"NMR (15N-1H HSQC titrations, chemical shift mapping), X-ray crystallography, affinity quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus NMR with quantitative affinity measurements; two complementary structural methods in single study\",\n      \"pmids\": [\"18156175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Dok-1 and Dok-2 negatively regulate TCR signaling; mice lacking both show augmented responses to thymus-dependent antigens and elevated ZAP-70 activation. Forced expression of Dok-1 or Dok-2 in CD3+CD4+ T cell clone inhibits ZAP-70 activation. The C-terminal SH2-target motifs are dispensable for this negative regulation, indicating a distinct, unidentified mechanism.\",\n      \"method\": \"Double knockout mice, forced expression in T cell clone, ZAP-70 phosphorylation assay, proliferation/cytokine assays, C-terminal truncation mutants\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mice plus forced expression with domain-mapping; multiple orthogonal methods\",\n      \"pmids\": [\"17329234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DOK1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPARgamma. In Dok1-deficient embryonic fibroblasts, ERK activity is elevated, leading to increased phosphorylation of PPARgamma on S112 (a negative regulatory site for PPARgamma transactivation). Mutation of PPARgamma S112 blocks the lean phenotype caused by Dok1 ablation in mice, establishing the DOK1-ERK-PPARgamma axis.\",\n      \"method\": \"Dok1-knockout mouse adipocytes and embryonic fibroblasts, PPARgamma S112 phosphorylation assay, PPARgamma-S112A genetic rescue, ERK inhibition, adipogenic differentiation assay\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout cells plus genetic epistasis (PPARgamma S112A rescue); multiple orthogonal approaches; in vitro and in vivo\",\n      \"pmids\": [\"18204460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DOK1 PH domain binds phosphatidylinositol 5-phosphate (PtdIns5P) in vitro; DOK1 tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. PtdIns5P increase regulates DOK1 tyrosine phosphorylation in vivo. The PH domain is necessary for DOK1 tyrosine phosphorylation and negative functions in T cells.\",\n      \"method\": \"In vitro lipid binding assay, intracellular PtdIns5P modulation, phosphorylation assay in T cells, PH domain deletion mutants\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro binding combined with in vivo PtdIns5P modulation; single lab, novel lipid ligand for PH domain\",\n      \"pmids\": [\"19299694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD45 recruits DOK1 to the proximal plasma membrane; DOK1-Y296F mutant abrogates interaction with CD45. CD45 expression is required for DOK1 membrane targeting in response to anti-CD3 stimulation. Stable expression of DOK1 decreases JAK-2 and STAT-3/5 phosphorylation following IL-3 and IFN-alpha stimulation; silencing DOK1 rescues MAP kinase and JAK activities in CD45-positive cells.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Y296F), stable overexpression, siRNA knockdown, JAK/STAT phosphorylation assay, membrane localization assay\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP with mutagenesis plus functional signaling assays; single lab\",\n      \"pmids\": [\"19481264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Oncogenic tyrosine kinases (p210(bcr-abl) and oncogenic Src) downregulate DOK1 by targeting it for ubiquitin-proteasome-mediated degradation; this process is tyrosine kinase activity-dependent and mediated primarily by lysine-dependent polyubiquitination. Restoration of DOK1 strongly suppresses transformation; a DOK1 mutant refractory to degradation shows stronger suppression.\",\n      \"method\": \"Proteasome inhibitor treatment, ubiquitination assay, DOK1 degradation-resistant mutant, transformation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ubiquitination assay and degradation-resistant mutant; single lab but mechanistically clear\",\n      \"pmids\": [\"21536658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BRK (breast tumor kinase/PTK6) interacts with and phosphorylates DOK1 specifically on Y362; this phosphorylation significantly downregulates DOK1 through the ubiquitin-proteasome pathway, promoting cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay (Y362 site-directed mutagenesis), ubiquitination assay, DOK1 protein stability assay, cell proliferation and migration assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — kinase assay plus mutagenesis plus ubiquitination and functional assays; single lab\",\n      \"pmids\": [\"24523872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DOK1 regulates PDGF-BB-stimulated glioma cell invasion through tyrosine phosphorylation of p130Cas and activation of Rap1. DOK1 becomes tyrosine phosphorylated at Y362 and Y398 upon PDGF-BB stimulation; DOK1 knockdown or Y362F/Y398F double mutant inhibits both PDGF-BB-induced p130Cas phosphorylation and Rap1 activation. DOK1 co-localizes with phospho-p130Cas at the cell membrane.\",\n      \"method\": \"siRNA knockdown, DOK1 phosphosite mutant (DOK1FF), co-localization (immunofluorescence), Rap1 activation assay, 3D spheroid invasion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — knockdown plus phosphosite mutant plus multiple assays; single lab, mechanistically informative for invasion pathway\",\n      \"pmids\": [\"24762811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DOK1 negatively regulates integrin alphaIIbbeta3 outside-in signaling in platelets (but not inside-out signaling). Dok-1-/- platelets show increased clot retraction, increased PLCgamma2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation. Dok-1-/- mice exhibit shortened bleeding times and accelerated carotid artery thrombosis.\",\n      \"method\": \"Dok-1-/- mouse, platelet functional assays (clot retraction, fibrinogen binding, P-selectin expression), PLCgamma2 phosphorylation assay, in vivo thrombosis model\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout mouse with multiple in vitro and in vivo functional platelet readouts; mechanistic distinction of inside-out vs outside-in signaling established\",\n      \"pmids\": [\"26790499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The two SH2 domains of RasGAP (p120RasGAP) generate distinct binding interactions with doubly phosphorylated DOK1; RasGAP binding to DOK1 is ~100-fold stronger than to EphB4, driven by dual SH2 engagement with dominant N-SH2 interaction. RasGAP-DOK1 interactions do not impact RasGAP catalytic activity, implying RasGAP utilizes its SH2 domains to achieve spatial-temporal regulation of Ras signaling by localizing to DOK1.\",\n      \"method\": \"Affinity measurements, small-angle X-ray scattering, in vitro RasGAP activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — biophysical measurements (SAXS, affinity) with functional GAP activity assay; single lab, provides structural insight but no cell-based validation\",\n      \"pmids\": [\"37507023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SHIP1 forms a complex with DOK1 in BCR/ABL-transformed hematopoietic cells; this interaction requires the SH2 domain of SHIP1 and the PTB domain of DOK1. DOK1-SHIP1 association is mutually exclusive with DOK1-RasGAP association. The DOK1/SHIP1 complex is only detected in the cytosolic fraction.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion mutants, subcellular fractionation\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP with domain mutants; single lab, mechanistically informative for SHIP1/DOK1 interaction requirements\",\n      \"pmids\": [\"10822173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SH2D1A (XLP gene product) associates with DOK1 via its SH2 domain; this interaction depends on DOK1 phospho-Y449 in the sequence ALYSQVQK. An XLP-associated SH2D1A mutant fails to associate with DOK1. Overexpression of SH2D1A activates NF-kappaB (independent of SH2 domain, via IKKbeta).\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (XLP mutant SH2D1A), NF-kappaB reporter assay, dominant-negative IKKbeta\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP with defined phosphosite (Y449) and disease-relevant mutant; single lab\",\n      \"pmids\": [\"10852966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SHP-1 constitutively associates with p62(DOK) in bone marrow macrophages, and this association occurs independently of p62(DOK) tyrosine phosphorylation. The SHP-1 catalytic domain (identified via catalytically inert SHP-1 C453S trapping) directly binds tyrosine-phosphorylated p62(DOK). p62(DOK) is the major CSF-1R-associated tyrosine-phosphorylated protein in CSF-1-treated macrophages; hyperphosphorylation in SHP-1-deficient macrophages correlates with growth factor-independent survival.\",\n      \"method\": \"Co-immunoprecipitation, far Western analysis, SHP-1-deficient (motheaten) mice, CSF-1 stimulation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus far Western with catalytic mutant trap; single lab but well-controlled with primary knockout macrophages\",\n      \"pmids\": [\"10585470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Phosphorylated serine residues S745 and S756 (adjacent to the NxxF motif) in the integrin beta2 tail are required for DOK1 PTB domain interaction, providing an alternative phosphorylation switch for Dok1 binding to beta2 integrins (which lack a phosphorylatable NxxY motif). NMR analyses show higher affinity for the pSer756-containing beta2 peptide; the phosphorylated beta2 peptide occupies the canonical Dok1 PTB ligand-binding pocket.\",\n      \"method\": \"NMR spectroscopy, docking/structural modelling, cell-based binding assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR-based structural analysis with cell-based validation; single lab, no X-ray crystal structure\",\n      \"pmids\": [\"26108885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The scaffold protein 14-3-3zeta interacts with the PTB domain of DOK1 (mapped to beta-sheet region) independently of phosphorylated integrin beta CTs. The 14-3-3zeta/Dok1 binary complex can simultaneously bind cognate phosphorylated integrin beta CT sequences, with Thr-phosphorylated beta2 or beta3 CTs binding 14-3-3zeta and Ser-phosphorylated beta2 CT or Tyr-phosphorylated beta3 CT binding DOK1, suggesting a molecular switch for integrin regulation.\",\n      \"method\": \"NMR spectroscopy, co-immunoprecipitation, binding assays with phosphorylated peptides\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR mapping with co-IP; single lab; proposes but partially validates a molecular switch\",\n      \"pmids\": [\"30243836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Frameshift mutation in DOK1 (GGCC deletion causing protein truncation) identified in CLL changes DOK1 from a cytoplasmic/membrane protein to a nuclear protein containing a functional bipartite nuclear localization signal. Mutant DOK1 fails to inhibit PDGF-induced MAP kinase activation and forms heterodimers with wild-type DOK1 (enhanced by Lck-mediated phosphorylation), indicating a dominant-negative mechanism.\",\n      \"method\": \"Heteroduplex analysis/sequencing, subcellular localization assay, MAP kinase assay, co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — natural mutation study with localization and signaling assays; single case but mechanistically informative\",\n      \"pmids\": [\"14730347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Dok1 and Dok2 are tyrosine phosphorylated upon NK cell activation; overexpression of Dok proteins in human NK cells reduces NK cell activation induced by activating receptors. Gene ablation of Dok1 and Dok2 in mice induces an NK cell maturation defect and leads to increased IFN-gamma production, establishing an intrinsic negative feedback loop downstream of NK cell activating receptors.\",\n      \"method\": \"Overexpression in human NK cells, Dok1/Dok2 double-knockout mice, NK cell maturation analysis, IFN-gamma production assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — combined human overexpression and mouse knockout with functional readouts; single lab\",\n      \"pmids\": [\"24963146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Upon FcεRI stimulation, DOK1 is recruited to the plasma membrane and undergoes tyrosine phosphorylation. Phosphorylated DOK1 inhibits FcεRI-operated calcium influx and negatively regulates mast cell degranulation by inhibiting calcium-dependent disassembly of actin filaments (F-actin disassembly).\",\n      \"method\": \"DOK1 overexpression in RBL-2H3 cells, co-immunoprecipitation, calcium influx assay, degranulation assay, F-actin staining\",\n      \"journal\": \"Clinical immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — overexpression system with multiple functional readouts; single lab; proposes calcium-actin mechanism\",\n      \"pmids\": [\"35421591\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DOK1 (p62dok) is a multi-domain adapter/docking protein (containing PH, PTB, and C-terminal tyrosine-rich domains) that, upon PI3K-dependent membrane recruitment via its PH domain binding 3'-phosphoinositides, is phosphorylated by multiple tyrosine kinases (including Bcr-Abl, c-Abl, Src/Lyn, RET, insulin receptor, BRK, and others) and by IKKbeta on serine residues; phosphorylated DOK1 recruits RasGAP (via C-terminal phosphotyrosines), Nck (via pY361), Csk, SHP-1, SHIP1, Smad3, and other effectors to the plasma membrane, where it acts as a negative regulator of Ras/ERK/MAPK and Akt signaling, integrin outside-in signaling (via competition with talin regulated by a phosphotyrosine switch), and immune receptor signaling in B cells, T cells, NK cells, and macrophages, while also promoting cell spreading/migration and filopodia formation through c-Abl-phosphorylated Y361-Nck recruitment, and is subject to inactivation by oncogenic tyrosine kinases via ubiquitin-proteasome-dependent degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DOK1 (p62dok) is a multi-domain docking protein that acts predominantly as a negative regulator of Ras/ERK/MAPK and PI3K/Akt signaling downstream of growth factor, cytokine, integrin, and immune receptors [#6, #7, #14]. Following PI3-kinase activation, DOK1 is recruited to the plasma membrane through its PH domain binding 3'-phosphoinositides (including PtdIns5P), a step essential for both its tyrosine phosphorylation and its inhibitory output [#7, #11, #25]. Once membrane-localized, DOK1 is phosphorylated on C-terminal tyrosines by an array of kinases—Bcr-Abl, c-Abl, Src/Lyn, the insulin receptor, RET, and others—creating docking sites that recruit p120 RasGAP (whose tandem SH2 domains engage doubly phosphorylated DOK1 to spatially localize Ras inhibition rather than alter GAP catalysis), and the Nck adaptor via Y361 [#0, #1, #8, #9, #31]. Its PTB domain binds phosphotyrosine NPXY motifs—structurally defined in complex with a RET phosphopeptide—and recruits SHIP1, SHP-1, and the phospho-Y747 β3 integrin tail in competition with talin, the latter constituting a phosphotyrosine switch that governs integrin outside-in signaling [#5, #12, #22, #32, #34]. Through these activities DOK1 restrains cell proliferation while promoting cell spreading, migration, and filopodia formation via c-Abl–dependent Y361–Nck recruitment [#13, #20, #21]. DOK1 and the paralog DOK2 cooperatively suppress myeloproliferation, and loss of both produces CML-like disease, while oncogenic tyrosine kinases inactivate DOK1 via ubiquitin-proteasome–dependent degradation [#14, #27]. DOK1 additionally serine-phosphorylated by IKKbeta couples inflammatory signaling to its regulatory function, shuttles between nucleus and cytoplasm under CRM1 control, and serves as a negative feedback brake across B, T, NK, mast, and macrophage immune responses [#15, #19, #21, #23, #38, #39].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing DOK1 as a constitutively phosphorylated RasGAP-associated protein in leukemic cells defined it as a candidate node linking aberrant tyrosine kinase activity to Ras pathway control.\",\n      \"evidence\": \"Biochemical purification and co-IP from CML progenitor cells and c-Kit-activated hematopoietic lines\",\n      \"pmids\": [\"9008160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether the RasGAP association was functionally inhibitory or activating\", \"No domain mapping of the interaction\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that Bcr-Abl directly phosphorylates DOK1 C-terminal tyrosines required for RasGAP binding, and that phospho-DOK1 inhibits RasGAP activity, gave the first direct mechanism for DOK1-mediated Ras regulation.\",\n      \"evidence\": \"In vitro kinase assay with recombinant p210(bcr-abl), site-directed mutagenesis, RasGAP activity assay\",\n      \"pmids\": [\"10688886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro RasGAP inhibition not directly tied to cellular Ras output\", \"Identity of additional cooperating effectors unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetic knockout and chimeric receptor reconstitution placed DOK1 as the negative regulatory module of FcgammaRIIB/BCR signaling, showing its structural information alone is sufficient to inhibit ERK.\",\n      \"evidence\": \"DOK1 knockout by homologous recombination, Lyn-dependent phosphorylation assays, chimeric FcgammaRIIB reconstitution, proliferation assays in B cells\",\n      \"pmids\": [\"10640270\", \"10755621\", \"10822173\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism downstream of RasGAP recruitment in B cells incompletely defined\", \"SHIP1-DOK1 vs RasGAP-DOK1 complex switching not resolved spatially\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Knockout mouse models and PI3K/PH-domain dissection established DOK1 as a physiological negative regulator of growth factor–induced Ras/MAPK signaling whose membrane recruitment, not solely RasGAP/Nck binding, drives inhibition and opposes leukemogenesis.\",\n      \"evidence\": \"p62(dok)-/- mice, retroviral bone marrow transplantation, PI3K inhibition with PH-domain mutants, membrane fractionation, insulin receptor in vitro kinase assays\",\n      \"pmids\": [\"11489947\", \"11489946\", \"11551902\", \"11825908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The RasGAP/Nck-independent inhibitory effector remained unidentified\", \"Single-knockout phenotype mild, hinting at redundancy\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping DOK1's interactions with RET and activin receptors separated RasGAP-dependent ERK suppression from Nck-dependent JNK activation and revealed a phosphorylation-independent adaptor role linking TGF-beta family receptors to Smad3.\",\n      \"evidence\": \"Yeast two-hybrid, gene-trap screening, mutagenesis, co-IP, Ras/Erk, JNK and apoptosis assays\",\n      \"pmids\": [\"12087092\", \"11927552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Smad3 linkage based on single-lab co-IP\", \"Physiological contribution of DOK1 to activin signaling in vivo untested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The crystal structure of the DOK1 PTB domain bound to a RET phosphopeptide provided the atomic basis for its phosphotyrosine recognition and selectivity against Shc/IRS1 ligands.\",\n      \"evidence\": \"X-ray crystallography of PTB domain alone and in complex, peptide binding assays\",\n      \"pmids\": [\"14607833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of full-length DOK1 or PH domain\", \"Did not capture EGFR or integrin tail complexes\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"c-Abl-dependent Y361 phosphorylation and Nck recruitment were shown to drive filopodia formation, establishing a positive cytoskeletal role for DOK1 distinct from its inhibitory signaling functions.\",\n      \"evidence\": \"c-Abl-/-, Dok1-/-, Nck-/- fibroblasts with rescue, immunofluorescence, co-IP\",\n      \"pmids\": [\"15148308\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream actin regulators of the Nck arm not defined\", \"Reconciliation of pro-migratory and anti-proliferative roles not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Double-knockout mice and IKKbeta serine phosphorylation studies revealed DOK1/DOK2 redundancy in suppressing myeloproliferation and a serine-phosphorylation input coupling inflammatory kinases to DOK1 function.\",\n      \"evidence\": \"Dok1/Dok2 single and double knockout mice, in vitro IKKbeta kinase assay, S-to-A/S-to-E mutagenesis, ERK/motility assays\",\n      \"pmids\": [\"15611294\", \"15611295\", \"15574499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How serine phosphorylation mechanistically alters DOK1 activity unresolved\", \"Division of labor between DOK1 and DOK2 not fully mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Systematic mutagenesis showed that Y336/Y340 are required for Ras-Erk inhibition independently of RasGAP binding, formalizing the existence of an unidentified cooperating effector.\",\n      \"evidence\": \"C-terminal deletion and point mutants, Erk and v-Abl transformation assays\",\n      \"pmids\": [\"15189452\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The cooperating Y336/Y340-recruited molecule was never identified\", \"Single-lab mutagenesis without biochemical isolation of the partner\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Knockout-plus-Y/F-mutant reconstitution defined DOK1/DOK2 as tyrosine-phosphorylation-dependent brakes on TLR4/ERK signaling and inflammatory cytokine output in macrophages.\",\n      \"evidence\": \"Knockout macrophages, Y/F mutant reconstitution, ERK/NF-kappaB assays, cytokine measurement, in vivo LPS challenge\",\n      \"pmids\": [\"15699069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for TLR4-induced DOK1 phosphorylation not pinned down\", \"Selectivity for ERK over other MAPKs mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Pathway dissection revealed DOK1 inhibits mitogenesis through two arms—Csk recruitment to Src attenuating c-myc, and RasGAP plus an additional effector—and demonstrated CRM1-dependent nucleocytoplasmic shuttling tied to its anti-proliferative and pro-spreading functions.\",\n      \"evidence\": \"Knockout cells with binding-deficient mutants, Csk co-IP, c-myc/Src assays, leptomycin B treatment, NES mutagenesis, localization and functional assays\",\n      \"pmids\": [\"16537894\", \"16705178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear function of DOK1, if any, undefined\", \"The additional RasGAP-cooperating effector still unidentified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Structural and genetic studies established the beta3 integrin Y747 phosphotyrosine switch between talin and DOK1 PTB binding, and defined DOK1/DOK2 as negative regulators of TCR/ZAP-70 signaling via a C-terminal-motif-independent mechanism.\",\n      \"evidence\": \"NMR and X-ray with affinity quantification of beta3 tail; double-knockout mice and forced expression in T cells with truncation mutants\",\n      \"pmids\": [\"18156175\", \"17329234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The C-terminal-independent mechanism inhibiting ZAP-70 not identified\", \"Cellular trigger for beta3 Y747 phosphorylation not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genetic epistasis through PPARgamma-S112A rescue established a DOK1-ERK-PPARgamma axis controlling adipocyte hypertrophy, extending DOK1's ERK-suppressive role to metabolic physiology.\",\n      \"evidence\": \"Dok1-knockout adipocytes and MEFs, PPARgamma S112 phosphorylation assay, PPARgamma-S112A genetic rescue in mice\",\n      \"pmids\": [\"18204460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link from DOK1 to the relevant ERK pool in adipocytes not shown\", \"Whether the same axis operates in human adipose tissue untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of PtdIns5P as a PH-domain ligand connected lipid second-messenger production to DOK1 tyrosine phosphorylation and negative function in T cells.\",\n      \"evidence\": \"In vitro lipid binding, intracellular PtdIns5P modulation, PH-deletion mutants, phosphorylation assays in T cells\",\n      \"pmids\": [\"19299694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab lipid binding without structural validation of the PH-PtdIns5P interaction\", \"Relative contributions of PtdIns5P versus PI3K products unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that oncogenic Bcr-Abl and Src target DOK1 for ubiquitin-proteasome degradation showed that tumor kinases inactivate DOK1 by destruction, and that degradation-resistant DOK1 more strongly suppresses transformation.\",\n      \"evidence\": \"Proteasome inhibition, ubiquitination assay, degradation-resistant mutant, transformation assay\",\n      \"pmids\": [\"21536658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase responsible not identified\", \"Single-lab study without in vivo confirmation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Studies of BRK and PDGF-BB/glioma signaling refined DOK1 as both a degradation substrate of pro-tumorigenic kinases and a Y362/Y398-dependent driver of p130Cas/Rap1-mediated invasion.\",\n      \"evidence\": \"Co-IP, in vitro kinase and ubiquitination assays, phosphosite mutants, Rap1 activation and 3D invasion assays\",\n      \"pmids\": [\"24523872\", \"24762811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of DOK1's tumor-suppressive versus pro-invasive roles unresolved\", \"Both findings rest on single-lab knockdown/overexpression systems\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A clean platelet knockout established DOK1 as a selective negative regulator of integrin alphaIIbbeta3 outside-in (not inside-out) signaling, with in vivo consequences for thrombosis and bleeding.\",\n      \"evidence\": \"Dok-1-/- mice, platelet clot retraction/spreading assays, PLCgamma2 phosphorylation, in vivo carotid thrombosis model\",\n      \"pmids\": [\"26790499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for outside-in selectivity not resolved\", \"Direct competition with talin in platelets not demonstrated in this system\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Biophysical analysis of the RasGAP-DOK1 interface showed dual SH2 engagement confers ~100-fold selectivity without altering GAP catalysis, reframing RasGAP recruitment as spatial localization rather than direct enzymatic modulation.\",\n      \"evidence\": \"Affinity measurements, small-angle X-ray scattering, in vitro RasGAP activity assay\",\n      \"pmids\": [\"37507023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No cell-based validation of the localization model\", \"Apparent tension with earlier in vitro RasGAP-inhibition reports unreconciled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the RasGAP-independent effector recruited via Y336/Y340 that cooperates to inhibit Ras-Erk signaling, and the unifying mechanism reconciling DOK1's anti-proliferative versus pro-migratory/pro-invasive roles, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical isolation of the Y336/Y340-binding partner\", \"No single framework linking DOK1's tumor-suppressive and pro-invasive activities\", \"Context determinants selecting RasGAP versus Nck versus SHIP1 complexes undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 9, 13, 17, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3, 6, 7]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [7, 25]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [13, 39]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 7, 11, 21]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [21, 32]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [21, 37]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 19, 23, 38]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 14, 27, 37]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RASA1\", \"NCK1\", \"CSK\", \"INPP5D\", \"PTPN6\", \"SH2D1A\", \"YWHAZ\", \"ITGB3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}