{"gene":"DOK4","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2003,"finding":"DOK4/IRS5 is tyrosine-phosphorylated in response to insulin and IGF-1 receptor activation in transfected cells; phosphorylated DOK4 associates with RasGAP, Crk, Src, and Fyn (but not PI3K p85, Grb2, SHP-2, Nck, or PLCγ SH2 domains), and activates MAPK.","method":"Transfection in mammalian cells, tyrosine phosphorylation assay, SH2 domain pull-down/binding assay, MAPK activation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple SH2 domain binding assays and functional MAPK readout, single lab","pmids":["12730241"],"is_preprint":false},{"year":2001,"finding":"Dok-4 directly associates with phospho-Y1062 of the c-Ret receptor tyrosine kinase; unlike Dok-1/2, Dok-4 does not associate with rasGAP or Nck; Dok-4 enhances c-Ret-dependent MAPK activation and is sufficient to mediate ligand-dependent neurite outgrowth of PC12 cells when fused to c-Ret.","method":"Co-immunoprecipitation, c-Ret/dok fusion protein constructs expressed in PC12 cells, neurite outgrowth assay, MAPK activation assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional rescue with fusion proteins, single lab, two orthogonal methods","pmids":["11470823"],"is_preprint":false},{"year":2005,"finding":"Dok-4 localizes to mitochondria via an N-terminal mitochondrial targeting sequence (aa 11–29); mitochondrial Dok-4 acts as an anchoring protein for c-Src kinase in mitochondria, recruits c-Src to mitochondria, decreases mitochondrial complex I (39-kDa subunit) expression in a Src-dependent manner, enhances TNF-α-mediated ROS production, and promotes TNF-α-mediated NF-κB activation through a Dok-4–Src–complex I/ROS pathway.","method":"Immunofluorescence microscopy, subcellular fractionation, EGFP chimera live imaging, Co-immunoprecipitation, siRNA knockdown, dominant-negative c-Src, Src inhibitor PP2, NF-κB reporter assay, ROS measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (fractionation, Co-IP, KD, dominant-negative, pharmacological inhibition, functional reporter) in a single rigorous study","pmids":["15855164"],"is_preprint":false},{"year":2006,"finding":"Dok-4 is required downstream of GDNF/RET signaling for sustained ERK1/2 activation and neurite outgrowth; specific tyrosine residues (Y187, Y220, Y270) in Dok-4 are essential for these effects. Dok-4 activates the small GTPase Rap1, and dominant-active Rap1 rescues neurite outgrowth in Dok-4-depleted cells, placing Dok-4 upstream of a Rap1–ERK1/2 pathway.","method":"siRNA knockdown in TGW neuroblastoma cells, tyrosine-to-phenylalanine point mutants, dominant-active/dominant-negative Rap1 epistasis, ERK phosphorylation assay, neurite outgrowth assay, cultured rat hippocampal neurons","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis with Rap1 mutants plus site-directed mutagenesis plus KD rescue, multiple orthogonal methods in single study","pmids":["16820412"],"is_preprint":false},{"year":2004,"finding":"Dok-4 is constitutively localized at the plasma membrane in a manner requiring both its PH and PTB domains; it is phosphorylated by cytosolic kinases Src, Fyn, and Jak2, and by the receptor tyrosine kinase Ret (but not by PDGFR-β or IGF-IR); membrane localization (via PH domain) is required for its inhibitory effect on Elk-1 activation by Ret or Fyn.","method":"Transfection in COS cells and yeast, PH/PTB deletion mutants, myristoylation signal rescue, substrate kinase panel assay, Elk-1 reporter assay, membrane localization assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutagenesis with functional reporter readout, single lab","pmids":["14963042"],"is_preprint":false},{"year":2009,"finding":"In T cells, Dok-4 is phosphorylated after TCR engagement, shuttles within the cytoplasm, and is recruited to the polarized microtubule organizing center upon immunological synapse formation. Dok-4 negatively regulates ERK phosphorylation, IL-2 promoter activity, and T cell proliferation via Rap1 activation; the PH domain is required for cytoplasmic shuttling/relocalization and for inhibitory function.","method":"siRNA knockdown, wild-type Dok-4 overexpression, PH domain deletion mutant, TCR stimulation, immunofluorescence microscopy (MTOC localization), ERK phosphorylation assay, IL-2 promoter reporter, T cell proliferation assay, Rap1 activation assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function, domain mutant, and functional readouts, single lab","pmids":["19494292"],"is_preprint":false},{"year":2010,"finding":"Dok4 is required for Schwann cell myelination in vitro; siRNA-mediated knockdown severely impairs myelination, initial Schwann cell–axon interaction, Schwann cell migration, and proliferation, demonstrating a role at early stages of the myelination process.","method":"siRNA knockdown in Schwann cell–neuron co-culture, in vitro myelination assay, migration and proliferation assays","journal":"Glia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotype, single lab, single method type","pmids":["21264944"],"is_preprint":false},{"year":2007,"finding":"A splice variant Dok-4b containing a 39 aa C-terminal insert inhibits tyrosine kinase-induced ERK and Elk-1 activation more strongly than Dok-4; truncation of the Dok-4 C-terminal region also enhances inhibitory activity, while the isolated C-terminal domain enhances Elk-1 activation, revealing that the N- and C-termini of Dok-4 have opposing inhibitory and stimulatory properties whose balance is altered by alternative splicing.","method":"Identification of splice variant by genomic analysis, expression in epithelial cell lines, ERK activation assay, Elk-1 reporter assay, C-terminal truncation and isolated domain constructs","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain dissection with functional reporter readout, single lab","pmids":["17258175"],"is_preprint":false},{"year":2012,"finding":"The Dok-4 PTB domain requires residues C-terminal to its classically defined boundaries (up to aa 246, forming a C-terminal α-helix extension) for binding to Ret; the Dok-4 PTB domain binds phosphorylated NPXY motifs in Ship1 but not Ship2; a rare human SNP (R186H) in the PTB domain abolishes Ret-mediated tyrosine phosphorylation of Dok-4.","method":"PTB domain deletion/extension constructs, binding assays to Ret and Ship1/Ship2 NPXY peptides, R186H point mutant, tyrosine phosphorylation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — domain mutagenesis with binding and phosphorylation assays, single lab","pmids":["22982678"],"is_preprint":false},{"year":2017,"finding":"Dok-4 interacts with the transactivation domain of the transcription factor Elk-4/Sap1 through an atypical PTB domain-mediated interaction; Dok-4 contains a nuclear export signal and relocalizes Elk-4 from nucleus to cytosol; Dok-4 promotes proteasome-dependent destabilization of Elk-4 via both interaction-dependent and PH domain-dependent (but interaction-independent) mechanisms; Dok-4 overexpression potently inhibits Elk-4 transactivation and reduces basal/EGF-induced expression of Egr-1, Fos, and cyclin D1 mRNA and cell proliferation independently of ERK inhibition.","method":"Co-immunoprecipitation (PTB domain interaction), nuclear export signal mapping, subcellular localization imaging, proteasome inhibitor rescue, siRNA knockdown, lysine-to-arginine ubiquitination-site mutants, Elk-4 transactivation reporter, mRNA expression assay, cell proliferation assay in MDCK cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, localization, proteasome inhibition, KD, ubiquitination-site mutagenesis, functional reporter) in a single rigorous study","pmids":["28275114"],"is_preprint":false}],"current_model":"DOK4 is a membrane- and mitochondria-associated adapter protein with PH and PTB domains that is phosphorylated by multiple tyrosine kinases (Ret, Src, Fyn, Jak2, insulin/IGF-1 receptors) and acts primarily as a negative regulator of tyrosine kinase signaling: in neurons it couples Ret/GDNF signaling to a Rap1–ERK pathway to promote neurite outgrowth; in T cells it acts as a negative regulator of ERK, IL-2, and proliferation via Rap1; in endothelial cells it anchors c-Src to mitochondria to regulate complex I, ROS production, and NF-κB activation; and in epithelial cells it inhibits Elk-1/Elk-4 transcriptional activity through direct PTB-mediated binding, nuclear exclusion, and proteasome-dependent degradation of Elk-4, while a splice variant (Dok-4b) and the Ship1-binding PTB domain further tune its inhibitory output."},"narrative":{"mechanistic_narrative":"DOK4 (IRS5) is a PH- and PTB-domain adapter protein that couples activated tyrosine kinases to downstream signaling and predominantly restrains those pathways [PMID:16820412, PMID:28275114]. It is a substrate of multiple kinases, becoming tyrosine-phosphorylated downstream of the c-Ret receptor, the insulin/IGF-1 receptors, and cytosolic Src, Fyn and Jak2, and its PTB domain docks directly onto the phospho-Y1062 NPXY motif of c-Ret via a C-terminally extended α-helix and also binds Ship1 [PMID:12730241, PMID:11470823, PMID:14963042, PMID:22982678]. Through its PH domain it is constitutively positioned at the plasma membrane, a localization required for its inhibitory output [PMID:14963042]. In neuronal cells DOK4 acts downstream of GDNF/RET to activate the small GTPase Rap1, driving sustained ERK1/2 signaling and neurite outgrowth dependent on tyrosines Y187, Y220 and Y270 [PMID:11470823, PMID:16820412], and it is similarly required for early Schwann cell myelination [PMID:21264944]. In T cells the same Rap1 axis instead operates negatively, with DOK4 relocalizing to the immunological-synapse MTOC to dampen ERK phosphorylation, IL-2 promoter activity and proliferation [PMID:19494292]. DOK4 also localizes to mitochondria via an N-terminal targeting sequence where it anchors c-Src, lowers complex I expression, and promotes TNF-α-driven ROS production and NF-κB activation [PMID:15855164]. Finally, DOK4 inhibits the transcription factor Elk-4/Sap1 by direct PTB-mediated binding, nuclear export, and proteasome-dependent degradation, thereby reducing Egr-1, Fos and cyclin D1 expression and proliferation independently of ERK [PMID:28275114]; an alternatively spliced isoform (Dok-4b) and the opposing activities of its N- and C-termini further tune this inhibitory balance [PMID:17258175].","teleology":[{"year":2001,"claim":"Established DOK4 as a direct effector of an activated receptor tyrosine kinase by showing it docks onto c-Ret and can drive a differentiation output.","evidence":"Co-IP and c-Ret/Dok-4 fusion constructs with neurite outgrowth and MAPK assays in PC12 cells","pmids":["11470823"],"confidence":"Medium","gaps":["Did not define which DOK4 domain mediates Ret binding","Downstream effectors beyond MAPK not identified"]},{"year":2003,"claim":"Extended the kinase inputs to DOK4 to the insulin/IGF-1 receptors and mapped its phospho-dependent SH2 partners, framing it as a signaling adapter.","evidence":"Transfection, tyrosine phosphorylation, and SH2-domain pull-down/MAPK assays","pmids":["12730241"],"confidence":"Medium","gaps":["Binding partner panel from overexpression, not endogenous","Functional consequence beyond MAPK activation unclear"]},{"year":2004,"claim":"Defined the kinase substrate spectrum and showed PH-domain-dependent membrane localization is required for DOK4's inhibitory effect on Elk-1, recasting it as a negative regulator.","evidence":"PH/PTB deletion and myristoylation-rescue constructs, kinase panel, and Elk-1 reporter in COS cells","pmids":["14963042"],"confidence":"Medium","gaps":["Mechanism of Elk-1 inhibition not resolved","Single overexpression system"]},{"year":2005,"claim":"Revealed a distinct mitochondrial pool of DOK4 that anchors c-Src to regulate complex I, ROS and NF-κB, expanding its roles beyond plasma-membrane signaling.","evidence":"Mitochondrial targeting-sequence mapping, fractionation, Co-IP, siRNA, dominant-negative Src, PP2, ROS and NF-κB reporters","pmids":["15855164"],"confidence":"High","gaps":["How complex I expression is lowered mechanistically not defined","Physiological context of mitochondrial pool versus membrane pool unclear"]},{"year":2006,"claim":"Placed DOK4 upstream of a Rap1–ERK pathway required for GDNF/RET-driven neurite outgrowth and identified the essential phosphotyrosines.","evidence":"siRNA, Y-to-F mutants, dominant-active/negative Rap1 epistasis and rescue in neuroblastoma and hippocampal neurons","pmids":["16820412"],"confidence":"High","gaps":["Direct Rap1 GEF linkage not identified","How phosphotyrosines couple to Rap1 activation unknown"]},{"year":2007,"claim":"Showed alternative splicing and opposing N-/C-terminal activities tune DOK4's net inhibitory strength, introducing a regulatory dimension to its output.","evidence":"Identification of Dok-4b splice variant, truncation/isolated-domain constructs, ERK and Elk-1 reporters in epithelial cells","pmids":["17258175"],"confidence":"Medium","gaps":["Endogenous relevance of Dok-4b isoform not established","Molecular basis of C-terminal stimulatory effect unknown"]},{"year":2009,"claim":"Demonstrated DOK4 as a negative regulator in T cells, relocalizing to the immunological-synapse MTOC and suppressing ERK, IL-2 and proliferation via Rap1.","evidence":"siRNA, PH-domain mutant, TCR stimulation, MTOC imaging, ERK/IL-2 reporter and proliferation assays","pmids":["19494292"],"confidence":"Medium","gaps":["MTOC recruitment mechanism not defined","Reconciliation of positive (neuron) versus negative (T cell) Rap1 outputs unexplained"]},{"year":2010,"claim":"Identified a requirement for DOK4 in early Schwann cell myelination, extending its neuronal RTK roles to glial development.","evidence":"siRNA knockdown in Schwann cell–neuron co-cultures with myelination, migration and proliferation assays","pmids":["21264944"],"confidence":"Medium","gaps":["Upstream kinase driving the myelination role not identified","Single in vitro system"]},{"year":2012,"claim":"Defined the structural requirements of the DOK4 PTB domain for Ret and Ship1 binding and identified a human SNP that abolishes Ret-mediated phosphorylation.","evidence":"PTB deletion/extension constructs, NPXY peptide binding assays, R186H mutant and phosphorylation assay","pmids":["22982678"],"confidence":"Medium","gaps":["Functional/phenotypic consequence of R186H in vivo unknown","Selectivity for Ship1 over Ship2 not mechanistically explained"]},{"year":2017,"claim":"Established a direct, ERK-independent route by which DOK4 silences gene expression—binding, exporting and degrading the transcription factor Elk-4.","evidence":"Co-IP, NES mapping, localization imaging, proteasome inhibition, siRNA, ubiquitination-site mutants, transactivation/mRNA/proliferation assays in MDCK cells","pmids":["28275114"],"confidence":"High","gaps":["Identity of the E3 ligase mediating Elk-4 degradation unknown","Whether DOK4 enters the nucleus to capture Elk-4 not fully resolved"]},{"year":null,"claim":"How DOK4 switches between positive (neuronal differentiation) and negative (lymphocyte, epithelial) signaling outputs through the same Rap1/ERK machinery remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in vivo loss-of-function model defining physiological function","Context-determining cofactors that flip DOK4 output unidentified","No structural model of full-length DOK4 with its partners"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2,4,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,5,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,4]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,6]}],"complexes":[],"partners":["RET","SRC","FYN","JAK2","INSR","INPPL1","ELK4","RASA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TEW6","full_name":"Docking protein 4","aliases":["Downstream of tyrosine kinase 4","Insulin receptor substrate 5","IRS-5","IRS5"],"length_aa":326,"mass_kda":37.0,"function":"DOK proteins are enzymatically inert adaptor or scaffolding proteins. They provide a docking platform for the assembly of multimolecular signaling complexes. DOK4 functions in RET-mediated neurite outgrowth and plays a positive role in activation of the MAP kinase pathway (By similarity). Putative link with downstream effectors of RET in neuronal differentiation. May be involved in the regulation of the immune response induced by T-cells","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8TEW6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DOK4","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DOK4","total_profiled":1310},"omim":[{"mim_id":"611402","title":"DOCKING PROTEIN 6; DOK6","url":"https://www.omim.org/entry/611402"},{"mim_id":"608334","title":"DOCKING PROTEIN 5; DOK5","url":"https://www.omim.org/entry/608334"},{"mim_id":"608333","title":"DOCKING PROTEIN 4; DOK4","url":"https://www.omim.org/entry/608333"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DOK4"},"hgnc":{"alias_symbol":["FLJ10488"],"prev_symbol":[]},"alphafold":{"accession":"Q8TEW6","domains":[{"cath_id":"2.30.29.30","chopping":"1-111","consensus_level":"high","plddt":87.6775,"start":1,"end":111},{"cath_id":"2.30.29.30","chopping":"136-224","consensus_level":"high","plddt":91.4163,"start":136,"end":224}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEW6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEW6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEW6-F1-predicted_aligned_error_v6.png","plddt_mean":74.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DOK4","jax_strain_url":"https://www.jax.org/strain/search?query=DOK4"},"sequence":{"accession":"Q8TEW6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TEW6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TEW6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEW6"}},"corpus_meta":[{"pmid":"12730241","id":"PMC_12730241","title":"Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12730241","citation_count":152,"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":"15855164","id":"PMC_15855164","title":"Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15855164","citation_count":58,"is_preprint":false},{"pmid":"16820412","id":"PMC_16820412","title":"Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16820412","citation_count":45,"is_preprint":false},{"pmid":"12595900","id":"PMC_12595900","title":"DOK4 and DOK5: new Dok-related genes expressed in human T cells.","date":"2003","source":"Genes and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/12595900","citation_count":36,"is_preprint":false},{"pmid":"14963042","id":"PMC_14963042","title":"Pleckstrin homology and phosphotyrosine-binding domain-dependent membrane association and tyrosine phosphorylation of Dok-4, an inhibitory adapter molecule expressed in epithelial cells.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14963042","citation_count":32,"is_preprint":false},{"pmid":"19494292","id":"PMC_19494292","title":"Dok-4 is a novel negative regulator of T cell activation.","date":"2009","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/19494292","citation_count":20,"is_preprint":false},{"pmid":"21264944","id":"PMC_21264944","title":"Dok4 is involved in Schwann cell myelination and axonal interaction in vitro.","date":"2010","source":"Glia","url":"https://pubmed.ncbi.nlm.nih.gov/21264944","citation_count":10,"is_preprint":false},{"pmid":"19073520","id":"PMC_19073520","title":"Transcriptional regulation of IRS5/DOK4 expression in non-small-cell lung cancer cells.","date":"2008","source":"Clinical lung cancer","url":"https://pubmed.ncbi.nlm.nih.gov/19073520","citation_count":7,"is_preprint":false},{"pmid":"22982678","id":"PMC_22982678","title":"New insights into Dok-4 PTB domain structure and function.","date":"2012","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/22982678","citation_count":7,"is_preprint":false},{"pmid":"17443497","id":"PMC_17443497","title":"DOK4/IRS-5 expression is altered in clear cell renal cell carcinoma.","date":"2007","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/17443497","citation_count":6,"is_preprint":false},{"pmid":"17258175","id":"PMC_17258175","title":"Identification of Dok-4b, a Dok-4 splice variant with enhanced inhibitory properties.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17258175","citation_count":5,"is_preprint":false},{"pmid":"28275114","id":"PMC_28275114","title":"Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.","date":"2017","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/28275114","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8047,"output_tokens":3041,"usd":0.034878,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10436,"output_tokens":3575,"usd":0.070777,"stage2_stop_reason":"end_turn"},"total_usd":0.105655,"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\": 2003,\n      \"finding\": \"DOK4/IRS5 is tyrosine-phosphorylated in response to insulin and IGF-1 receptor activation in transfected cells; phosphorylated DOK4 associates with RasGAP, Crk, Src, and Fyn (but not PI3K p85, Grb2, SHP-2, Nck, or PLCγ SH2 domains), and activates MAPK.\",\n      \"method\": \"Transfection in mammalian cells, tyrosine phosphorylation assay, SH2 domain pull-down/binding assay, MAPK activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple SH2 domain binding assays and functional MAPK readout, single lab\",\n      \"pmids\": [\"12730241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Dok-4 directly associates with phospho-Y1062 of the c-Ret receptor tyrosine kinase; unlike Dok-1/2, Dok-4 does not associate with rasGAP or Nck; Dok-4 enhances c-Ret-dependent MAPK activation and is sufficient to mediate ligand-dependent neurite outgrowth of PC12 cells when fused to c-Ret.\",\n      \"method\": \"Co-immunoprecipitation, c-Ret/dok fusion protein constructs expressed in PC12 cells, neurite outgrowth assay, MAPK activation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional rescue with fusion proteins, single lab, two orthogonal methods\",\n      \"pmids\": [\"11470823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Dok-4 localizes to mitochondria via an N-terminal mitochondrial targeting sequence (aa 11–29); mitochondrial Dok-4 acts as an anchoring protein for c-Src kinase in mitochondria, recruits c-Src to mitochondria, decreases mitochondrial complex I (39-kDa subunit) expression in a Src-dependent manner, enhances TNF-α-mediated ROS production, and promotes TNF-α-mediated NF-κB activation through a Dok-4–Src–complex I/ROS pathway.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, EGFP chimera live imaging, Co-immunoprecipitation, siRNA knockdown, dominant-negative c-Src, Src inhibitor PP2, NF-κB reporter assay, ROS measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (fractionation, Co-IP, KD, dominant-negative, pharmacological inhibition, functional reporter) in a single rigorous study\",\n      \"pmids\": [\"15855164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Dok-4 is required downstream of GDNF/RET signaling for sustained ERK1/2 activation and neurite outgrowth; specific tyrosine residues (Y187, Y220, Y270) in Dok-4 are essential for these effects. Dok-4 activates the small GTPase Rap1, and dominant-active Rap1 rescues neurite outgrowth in Dok-4-depleted cells, placing Dok-4 upstream of a Rap1–ERK1/2 pathway.\",\n      \"method\": \"siRNA knockdown in TGW neuroblastoma cells, tyrosine-to-phenylalanine point mutants, dominant-active/dominant-negative Rap1 epistasis, ERK phosphorylation assay, neurite outgrowth assay, cultured rat hippocampal neurons\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis with Rap1 mutants plus site-directed mutagenesis plus KD rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"16820412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Dok-4 is constitutively localized at the plasma membrane in a manner requiring both its PH and PTB domains; it is phosphorylated by cytosolic kinases Src, Fyn, and Jak2, and by the receptor tyrosine kinase Ret (but not by PDGFR-β or IGF-IR); membrane localization (via PH domain) is required for its inhibitory effect on Elk-1 activation by Ret or Fyn.\",\n      \"method\": \"Transfection in COS cells and yeast, PH/PTB deletion mutants, myristoylation signal rescue, substrate kinase panel assay, Elk-1 reporter assay, membrane localization assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutagenesis with functional reporter readout, single lab\",\n      \"pmids\": [\"14963042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In T cells, Dok-4 is phosphorylated after TCR engagement, shuttles within the cytoplasm, and is recruited to the polarized microtubule organizing center upon immunological synapse formation. Dok-4 negatively regulates ERK phosphorylation, IL-2 promoter activity, and T cell proliferation via Rap1 activation; the PH domain is required for cytoplasmic shuttling/relocalization and for inhibitory function.\",\n      \"method\": \"siRNA knockdown, wild-type Dok-4 overexpression, PH domain deletion mutant, TCR stimulation, immunofluorescence microscopy (MTOC localization), ERK phosphorylation assay, IL-2 promoter reporter, T cell proliferation assay, Rap1 activation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function, domain mutant, and functional readouts, single lab\",\n      \"pmids\": [\"19494292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Dok4 is required for Schwann cell myelination in vitro; siRNA-mediated knockdown severely impairs myelination, initial Schwann cell–axon interaction, Schwann cell migration, and proliferation, demonstrating a role at early stages of the myelination process.\",\n      \"method\": \"siRNA knockdown in Schwann cell–neuron co-culture, in vitro myelination assay, migration and proliferation assays\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotype, single lab, single method type\",\n      \"pmids\": [\"21264944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A splice variant Dok-4b containing a 39 aa C-terminal insert inhibits tyrosine kinase-induced ERK and Elk-1 activation more strongly than Dok-4; truncation of the Dok-4 C-terminal region also enhances inhibitory activity, while the isolated C-terminal domain enhances Elk-1 activation, revealing that the N- and C-termini of Dok-4 have opposing inhibitory and stimulatory properties whose balance is altered by alternative splicing.\",\n      \"method\": \"Identification of splice variant by genomic analysis, expression in epithelial cell lines, ERK activation assay, Elk-1 reporter assay, C-terminal truncation and isolated domain constructs\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain dissection with functional reporter readout, single lab\",\n      \"pmids\": [\"17258175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The Dok-4 PTB domain requires residues C-terminal to its classically defined boundaries (up to aa 246, forming a C-terminal α-helix extension) for binding to Ret; the Dok-4 PTB domain binds phosphorylated NPXY motifs in Ship1 but not Ship2; a rare human SNP (R186H) in the PTB domain abolishes Ret-mediated tyrosine phosphorylation of Dok-4.\",\n      \"method\": \"PTB domain deletion/extension constructs, binding assays to Ret and Ship1/Ship2 NPXY peptides, R186H point mutant, tyrosine phosphorylation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — domain mutagenesis with binding and phosphorylation assays, single lab\",\n      \"pmids\": [\"22982678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Dok-4 interacts with the transactivation domain of the transcription factor Elk-4/Sap1 through an atypical PTB domain-mediated interaction; Dok-4 contains a nuclear export signal and relocalizes Elk-4 from nucleus to cytosol; Dok-4 promotes proteasome-dependent destabilization of Elk-4 via both interaction-dependent and PH domain-dependent (but interaction-independent) mechanisms; Dok-4 overexpression potently inhibits Elk-4 transactivation and reduces basal/EGF-induced expression of Egr-1, Fos, and cyclin D1 mRNA and cell proliferation independently of ERK inhibition.\",\n      \"method\": \"Co-immunoprecipitation (PTB domain interaction), nuclear export signal mapping, subcellular localization imaging, proteasome inhibitor rescue, siRNA knockdown, lysine-to-arginine ubiquitination-site mutants, Elk-4 transactivation reporter, mRNA expression assay, cell proliferation assay in MDCK cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, localization, proteasome inhibition, KD, ubiquitination-site mutagenesis, functional reporter) in a single rigorous study\",\n      \"pmids\": [\"28275114\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DOK4 is a membrane- and mitochondria-associated adapter protein with PH and PTB domains that is phosphorylated by multiple tyrosine kinases (Ret, Src, Fyn, Jak2, insulin/IGF-1 receptors) and acts primarily as a negative regulator of tyrosine kinase signaling: in neurons it couples Ret/GDNF signaling to a Rap1–ERK pathway to promote neurite outgrowth; in T cells it acts as a negative regulator of ERK, IL-2, and proliferation via Rap1; in endothelial cells it anchors c-Src to mitochondria to regulate complex I, ROS production, and NF-κB activation; and in epithelial cells it inhibits Elk-1/Elk-4 transcriptional activity through direct PTB-mediated binding, nuclear exclusion, and proteasome-dependent degradation of Elk-4, while a splice variant (Dok-4b) and the Ship1-binding PTB domain further tune its inhibitory output.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DOK4 (IRS5) is a PH- and PTB-domain adapter protein that couples activated tyrosine kinases to downstream signaling and predominantly restrains those pathways [#3, #9]. It is a substrate of multiple kinases, becoming tyrosine-phosphorylated downstream of the c-Ret receptor, the insulin/IGF-1 receptors, and cytosolic Src, Fyn and Jak2, and its PTB domain docks directly onto the phospho-Y1062 NPXY motif of c-Ret via a C-terminally extended α-helix and also binds Ship1 [#0, #1, #4, #8]. Through its PH domain it is constitutively positioned at the plasma membrane, a localization required for its inhibitory output [#4]. In neuronal cells DOK4 acts downstream of GDNF/RET to activate the small GTPase Rap1, driving sustained ERK1/2 signaling and neurite outgrowth dependent on tyrosines Y187, Y220 and Y270 [#1, #3], and it is similarly required for early Schwann cell myelination [#6]. In T cells the same Rap1 axis instead operates negatively, with DOK4 relocalizing to the immunological-synapse MTOC to dampen ERK phosphorylation, IL-2 promoter activity and proliferation [#5]. DOK4 also localizes to mitochondria via an N-terminal targeting sequence where it anchors c-Src, lowers complex I expression, and promotes TNF-α-driven ROS production and NF-κB activation [#2]. Finally, DOK4 inhibits the transcription factor Elk-4/Sap1 by direct PTB-mediated binding, nuclear export, and proteasome-dependent degradation, thereby reducing Egr-1, Fos and cyclin D1 expression and proliferation independently of ERK [#9]; an alternatively spliced isoform (Dok-4b) and the opposing activities of its N- and C-termini further tune this inhibitory balance [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established DOK4 as a direct effector of an activated receptor tyrosine kinase by showing it docks onto c-Ret and can drive a differentiation output.\",\n      \"evidence\": \"Co-IP and c-Ret/Dok-4 fusion constructs with neurite outgrowth and MAPK assays in PC12 cells\",\n      \"pmids\": [\"11470823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define which DOK4 domain mediates Ret binding\", \"Downstream effectors beyond MAPK not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended the kinase inputs to DOK4 to the insulin/IGF-1 receptors and mapped its phospho-dependent SH2 partners, framing it as a signaling adapter.\",\n      \"evidence\": \"Transfection, tyrosine phosphorylation, and SH2-domain pull-down/MAPK assays\",\n      \"pmids\": [\"12730241\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding partner panel from overexpression, not endogenous\", \"Functional consequence beyond MAPK activation unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the kinase substrate spectrum and showed PH-domain-dependent membrane localization is required for DOK4's inhibitory effect on Elk-1, recasting it as a negative regulator.\",\n      \"evidence\": \"PH/PTB deletion and myristoylation-rescue constructs, kinase panel, and Elk-1 reporter in COS cells\",\n      \"pmids\": [\"14963042\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Elk-1 inhibition not resolved\", \"Single overexpression system\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed a distinct mitochondrial pool of DOK4 that anchors c-Src to regulate complex I, ROS and NF-κB, expanding its roles beyond plasma-membrane signaling.\",\n      \"evidence\": \"Mitochondrial targeting-sequence mapping, fractionation, Co-IP, siRNA, dominant-negative Src, PP2, ROS and NF-κB reporters\",\n      \"pmids\": [\"15855164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How complex I expression is lowered mechanistically not defined\", \"Physiological context of mitochondrial pool versus membrane pool unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed DOK4 upstream of a Rap1–ERK pathway required for GDNF/RET-driven neurite outgrowth and identified the essential phosphotyrosines.\",\n      \"evidence\": \"siRNA, Y-to-F mutants, dominant-active/negative Rap1 epistasis and rescue in neuroblastoma and hippocampal neurons\",\n      \"pmids\": [\"16820412\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Rap1 GEF linkage not identified\", \"How phosphotyrosines couple to Rap1 activation unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed alternative splicing and opposing N-/C-terminal activities tune DOK4's net inhibitory strength, introducing a regulatory dimension to its output.\",\n      \"evidence\": \"Identification of Dok-4b splice variant, truncation/isolated-domain constructs, ERK and Elk-1 reporters in epithelial cells\",\n      \"pmids\": [\"17258175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous relevance of Dok-4b isoform not established\", \"Molecular basis of C-terminal stimulatory effect unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated DOK4 as a negative regulator in T cells, relocalizing to the immunological-synapse MTOC and suppressing ERK, IL-2 and proliferation via Rap1.\",\n      \"evidence\": \"siRNA, PH-domain mutant, TCR stimulation, MTOC imaging, ERK/IL-2 reporter and proliferation assays\",\n      \"pmids\": [\"19494292\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MTOC recruitment mechanism not defined\", \"Reconciliation of positive (neuron) versus negative (T cell) Rap1 outputs unexplained\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified a requirement for DOK4 in early Schwann cell myelination, extending its neuronal RTK roles to glial development.\",\n      \"evidence\": \"siRNA knockdown in Schwann cell–neuron co-cultures with myelination, migration and proliferation assays\",\n      \"pmids\": [\"21264944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream kinase driving the myelination role not identified\", \"Single in vitro system\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the structural requirements of the DOK4 PTB domain for Ret and Ship1 binding and identified a human SNP that abolishes Ret-mediated phosphorylation.\",\n      \"evidence\": \"PTB deletion/extension constructs, NPXY peptide binding assays, R186H mutant and phosphorylation assay\",\n      \"pmids\": [\"22982678\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional/phenotypic consequence of R186H in vivo unknown\", \"Selectivity for Ship1 over Ship2 not mechanistically explained\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established a direct, ERK-independent route by which DOK4 silences gene expression—binding, exporting and degrading the transcription factor Elk-4.\",\n      \"evidence\": \"Co-IP, NES mapping, localization imaging, proteasome inhibition, siRNA, ubiquitination-site mutants, transactivation/mRNA/proliferation assays in MDCK cells\",\n      \"pmids\": [\"28275114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the E3 ligase mediating Elk-4 degradation unknown\", \"Whether DOK4 enters the nucleus to capture Elk-4 not fully resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DOK4 switches between positive (neuronal differentiation) and negative (lymphocyte, epithelial) signaling outputs through the same Rap1/ERK machinery remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo loss-of-function model defining physiological function\", \"Context-determining cofactors that flip DOK4 output unidentified\", \"No structural model of full-length DOK4 with its partners\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2, 4, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RET\", \"SRC\", \"FYN\", \"JAK2\", \"INSR\", \"INPPL1\", \"ELK4\", \"RASA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}