{"gene":"TNK2","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1999,"finding":"NMR solution structure of Cdc42 bound to the GTPase-binding domain of ACK1 revealed that both proteins undergo significant conformational changes on binding, forming a new type of G-protein/effector complex in which an extended strand from ACK intercalates into the beta-sheet of Cdc42; this defines the structural basis for selective Cdc42 (not Rac) binding.","method":"NMR structure determination with functional validation of binding specificity","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution NMR structure with functional validation of the binding interface, published in high-impact journal","pmids":["10360579"],"is_preprint":false},{"year":2003,"finding":"ACK1 autophosphorylates at Tyr284 in the activation loop (identified by mass spectrometry); this is the primary autophosphorylation site and its mutation (Y284F) dramatically reduces tyrosine phosphorylation in cells. ACK1 substrate specificity most closely resembles Abl. ACK1 interacts with Hck SH3 domains via its proline-rich C-terminal domain, and Hck can phosphorylate ACK1, suggesting Hck as an upstream regulator.","method":"In vitro kinase assay with purified baculovirus-expressed ACK1, mass spectrometry phosphosite mapping, site-directed mutagenesis, SH2/SH3 domain binding screens, co-expression in mammalian cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution + MS phosphosite mapping + mutagenesis + cell-based validation in one study","pmids":["14506255"],"is_preprint":false},{"year":2004,"finding":"Crystal structures of human ACK1 kinase domain in both unphosphorylated and phosphorylated states revealed that ACK1 adopts an activated conformation independent of phosphorylation, with the unphosphorylated activation loop structured and resembling that of activated tyrosine kinases. Inhibitor-bound co-crystal structures (with AMPPCP and debromohymenialdisine) defined the ATP-binding cleft.","method":"X-ray crystallography of phosphorylated and unphosphorylated kinase domain; inhibitor co-crystal structures","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures (apo, phosphorylated, inhibitor-bound) providing atomic-resolution mechanistic insight","pmids":["15308621"],"is_preprint":false},{"year":2001,"finding":"ACK1 associates directly with clathrin heavy chain via a central adaptor motif that competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain; ACK1 also interacts with the adaptor Nck via SH3 interactions; stable low-level GFP-ACK1 expression localizes to clathrin/AP-2-containing vesicles and increases receptor-mediated transferrin uptake.","method":"Direct binding assays, competition assays with beta-arrestin, GFP-ACK1 live-cell imaging, co-localization with clathrin and AP-2, transferrin uptake assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, live imaging, functional uptake assay, multiple orthogonal methods in one study","pmids":["11278436"],"is_preprint":false},{"year":2006,"finding":"ACK1 activation loop autophosphorylation requires both the amino-terminal SAM-like domain (for membrane targeting) and Cdc42 binding via the CRIB domain; the SH3 domain plays an autoinhibitory role. Cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of ACK1; EGF or PDGF stimulation recruits ACK1 to activated receptors; tyrosine-phosphorylated ACK1 forms a stable complex with adaptor Nck via its SH2 domain.","method":"Domain deletion mutant analysis, immunoprecipitation, kinase assays, EGF/PDGF stimulation of cells, fibronectin adhesion assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mutants, orthogonal stimulation conditions, reciprocal co-IP, from dedicated mechanistic study","pmids":["16777958"],"is_preprint":false},{"year":2005,"finding":"Activated ACK1 tyrosine-phosphorylates tumor suppressor Wwox at Tyr287 (identified by site-directed mutagenesis), leading to rapid Wwox polyubiquitination and proteasomal degradation. Hsp90beta associates with ACK1 and its inhibition (geldanamycin) blocks ACK1 kinase activity. A splice variant (WwoxΔ5-8) not phosphorylated by ACK1 is not ubiquitinated or degraded.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, ubiquitination assay, Hsp90 inhibitor treatment, xenograft tumor model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro phosphorylation + mutagenesis + ubiquitination assays + in vivo validation, multiple orthogonal methods","pmids":["16288044"],"is_preprint":false},{"year":2007,"finding":"Activated ACK1 directly phosphorylates androgen receptor (AR) at Tyr267 and Tyr363 within the transactivation domain. Mutation of Tyr267 completely abrogates, and Tyr363 mutation reduces, Ack1-induced AR reporter activation and AR recruitment to androgen-responsive enhancers. Heregulin-stimulated HER2 activates ACK1, which then phosphorylates AR to drive androgen-independent gene expression and tumor growth.","method":"Site-directed mutagenesis of AR, AR reporter assays, ChIP (AR recruitment to enhancers), ACK1 knockdown by siRNA, xenograft tumor models, phospho-specific antibodies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis of phosphosites + reporter assays + ChIP + in vivo xenograft, multiple orthogonal methods","pmids":["17494760"],"is_preprint":false},{"year":2005,"finding":"ACK1 phosphorylates WASP at both Tyr256 (tyrosine kinase activity) and Ser242 (serine kinase activity, demonstrating dual-specificity), with serine phosphorylation enhanced by Cdc42 or PIP2 (which releases WASP autoinhibition). Serine phosphorylation of WASP at Ser242 enhances WASP-stimulated actin polymerization in cell lysates. ACK1 expressed in bacteria retains serine kinase activity.","method":"In vitro kinase assay with purified proteins, phosphosite mapping by mutagenesis, bacterially expressed ACK1 kinase assay, actin polymerization assay in cell lysates","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, mutagenesis of phosphosites, bacterial expression controls, functional actin polymerization assay","pmids":["16257963"],"is_preprint":false},{"year":2010,"finding":"ACK1 directly phosphorylates AKT at the evolutionarily conserved Tyr176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473 phosphorylation leading to full AKT activation. This pathway operates downstream of RTK/growth factor signaling.","method":"In vitro kinase assay, phospho-specific antibody generation, plasma membrane fractionation, site-directed mutagenesis, transgenic mouse model (prostate-specific activated Ack1), co-immunoprecipitation","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay + phosphosite mutagenesis + subcellular fractionation + transgenic mouse validation","pmids":["20333297"],"is_preprint":false},{"year":2010,"finding":"HECT E3 ubiquitin ligase Nedd4-1 ubiquitinates ACK1 via a conserved PPXY WW-binding motif (WW3 domain of Nedd4-1 is critical); EGF-induced ACK1 degradation is processed by lysosomes, not proteasomes. The UBA domain of ACK1 suppresses Nedd4-1-mediated ubiquitination. Nedd4-1 (not Nedd4-2) knockdown inhibits degradation of both EGFR and ACK1, and ACK1 mutants deficient in Nedd4-1 binding block EGF-induced EGFR degradation.","method":"Co-immunoprecipitation, ubiquitination assay, RNAi knockdown, proteasome/lysosome inhibitors, EGFR degradation assay, deletion mutant analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, ubiquitination assay, RNAi rescue with multiple deletion mutants, inhibitor experiments, mechanistic dissection","pmids":["20086093"],"is_preprint":false},{"year":2009,"finding":"E3 ubiquitin ligase Nedd4-2 binds ACK1 via its PPXY motif, co-localizes with ACK1 in clathrin-rich vesicles, and strongly down-regulates ACK1 levels via proteasomal degradation that is driven by ACK1 kinase activity. Dominant-inhibitory Nedd4 blocks endogenous ACK1 turnover in response to EGF.","method":"Co-immunoprecipitation, co-localization imaging, proteasome inhibitor (MG132), ACK1 PPXY mutants, dominant-negative Nedd4","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP + imaging co-localization + proteasome inhibitor + dominant-negative + PPXY mutants, multiple orthogonal methods","pmids":["19144635"],"is_preprint":false},{"year":2012,"finding":"SIAH1 and SIAH2 ubiquitin ligases interact with ACK1 via a conserved SIAH-binding motif in the far C-terminus of ACK1 and induce proteasomal (not lysosomal) degradation of ACK1 in a manner independent of ACK1 kinase activity. SIAH2 expression induced by estrogen receptor activation decreases ACK1 levels in breast cancer cells.","method":"Yeast two-hybrid, co-immunoprecipitation, deletion/point mutants of ACK1, proteasome inhibitor, SIAH2 knockdown, ER activation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid + co-IP + mutagenesis + inhibitor + RNAi, multiple orthogonal methods","pmids":["23208506"],"is_preprint":false},{"year":2010,"finding":"ACK1 kinase activity is autoinhibited by an intramolecular interaction between the kinase domain C-lobe and the C-terminal Mig6 homology region (MHR, residues 802-990); cancer-associated mutation E346K prevents kinase-MHR binding and constitutively activates ACK1. The MHR-kinase domain interaction was demonstrated by direct binding of purified domains in vitro.","method":"In vitro pulldown with purified kinase domain and MHR fragments, immune complex kinase assays, cancer-associated mutant characterization (E346K, F820A), cell migration and proliferation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro domain interaction assays + mutagenesis + kinase activity assays + functional cell assays","pmids":["20110370"],"is_preprint":false},{"year":2012,"finding":"ACK1 activates AKT-mediated signaling in glioma cells downstream of PDGFR-β; PDGFR-β phosphorylates ACK1 at Y635, and this phosphorylation is required for sequential AKT activation. PDK1 interacts with ACK1 (via T325 of ACK1) during PDGF stimulation and is required for ACK1-PDGFR-β binding. Y635F or T325A ACK1 mutants abolish PDGFR-β-induced AKT activation and downstream β-catenin nuclear translocation.","method":"Co-immunoprecipitation, site-directed mutagenesis (Y635F, T325A), reporter and western blot assays, in vivo glioma model","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP + site-directed mutagenesis + functional cell/tumor assays, single lab","pmids":["25257795"],"is_preprint":false},{"year":2012,"finding":"ACK1 activation mechanism involves an autoinhibitory interaction between the SH3 domain and the EGFR-binding domain (EBD); release of this autoinhibition activates ACK1. Cell adhesion-mediated activation occurs through releasing this autoinhibition. Grb2 mediates ACK1 interaction with EGFR by binding the EBD and releasing autoinhibition. The N-terminal region (Leu10-Leu14) is essential for cell adhesion-mediated activation. Ser445Pro mutation causes constitutive ACK1 activation.","method":"SH3/EBD domain deletion and point mutants, kinase activity assays, co-immunoprecipitation with Grb2 and EGFR, cell adhesion assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutant panel + kinase assays + co-IP, but conflicting with another paper (PMID 21309750) on autoinhibition model; single lab","pmids":["22553920"],"is_preprint":false},{"year":2011,"finding":"Src kinase (not ACK1 autophosphorylation) is required for phosphorylation of ACK1 activation loop Tyr284 in vivo; Src SH2 and SH3 domains interact with ACK1 Tyr518 and residues 623-652, respectively. ACK1 fails to undergo significant Tyr284 autophosphorylation in vivo because its activation loop is basophilic (while Src is acidophilic). ACK1 activation downstream of EGFR/integrin requires Src; ACK1 turnover is blocked by Src inhibitors and is impaired in Src-deficient SYF cells.","method":"Co-immunoprecipitation of endogenous Src-ACK1, Src-deficient SYF cell line analysis, Src inhibitor treatment, domain mapping with SH2/SH3 domains","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + genetic (SYF cells) + pharmacological inhibition, single lab; contradicts autoinhibition model proposed by others","pmids":["21309750"],"is_preprint":false},{"year":2010,"finding":"ACK1 activity is required for N-terminal SAM domain-mediated plasma membrane localization and dimerization; the isolated kinase domain (without N-terminus) fails to autophosphorylate and shows cytosolic localization, while the N-terminus+kinase domain (NKD) localizes to plasma membrane and undergoes autophosphorylation. Increasing local concentration of purified ACK1 kinase domain at lipid vesicle surfaces stimulates autophosphorylation and activity, consistent with dimerization and trans-phosphorylation.","method":"Deletion mutant immunofluorescence, western blotting for autophosphorylation, co-immunoprecipitation (dimerization), lipid vesicle reconstitution assay","journal":"BMC biochemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro lipid vesicle reconstitution + cell-based deletion mutant analysis + co-IP for dimerization, single lab","pmids":["20979614"],"is_preprint":false},{"year":1999,"finding":"ACK-2 (the shorter Cdc42-associated kinase) is activated by cell adhesion via integrin beta1 in a Cdc42-dependent manner; ACK-2 co-immunoprecipitates with integrin beta1. Activation is F-actin-independent and does not require cell spreading. Overexpression of ACK-2 activates c-Jun kinase (not ERK). Anti-integrin beta1 antibodies and RGD peptides inhibit ACK-2 activation by cell adhesion.","method":"Co-immunoprecipitation with integrin beta1, RGD peptide/antibody inhibition, kinase assays, actin depolymerization controls","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + multiple inhibitor approaches + kinase assays, single lab","pmids":["10085085"],"is_preprint":false},{"year":1999,"finding":"MCSP stimulation recruits tyrosine-phosphorylated p130Cas and activates Cdc42, with MCSP-induced cell spreading dependent on active Cdc42, Ack-1, and tyrosine phosphorylation of p130Cas. Vectors inhibiting Ack-1 or Cdc42 abrogate MCSP-induced p130Cas tyrosine phosphorylation and recruitment.","method":"Dominant-negative/inhibitory vectors for Ack-1 and Cdc42, phospho-p130Cas immunoprecipitation, cell spreading assays","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative inhibition + co-IP phosphorylation assays + functional spreading readout, single lab","pmids":["10587647"],"is_preprint":false},{"year":2006,"finding":"Ack1 forms a signaling complex with Cdc42, p130Cas, and Crk, whose formation is regulated by collagen stimulation. Ack1 interaction with p130Cas occurs through their respective SH3 domains, while the substrate domain of p130Cas is the major site of Ack1-dependent phosphorylation. siRNA knockdown of either p130Cas or Ack1 blocks Cdc42-induced cell migration on collagen.","method":"Co-immunoprecipitation, SH3 domain interaction mapping, siRNA knockdown, p130Cas phosphorylation assay, cell migration assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP + siRNA knockdown + migration assay, single lab","pmids":["17038317"],"is_preprint":false},{"year":2000,"finding":"ACK1 tyrosine-phosphorylates and activates the guanine nucleotide exchange factor Dbl; in vitro GEF activity of Dbl toward Rho and Cdc42 is augmented after tyrosine phosphorylation. ACK1-dependent Dbl phosphorylation leads to accumulation of GTP-bound Rho and Rac in cells and enhanced JNK activation downstream.","method":"Co-expression in cells, in vitro GEF assay, GTP-bound Rho/Rac pull-down (RBD assay), JNK activation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro GEF assay + cell-based RBD pull-down + JNK assay, single lab","pmids":["10652228"],"is_preprint":false},{"year":2000,"finding":"ACK1 phosphorylates and activates the Ras GEF Ras-GRF1 at tyrosine residues, augmenting Ras-GEF activity (GDP release) specifically toward Ha-Ras (not Rac1). This results in increased GTP-Ras accumulation in cells and enhanced ERK2 activation downstream of Ras-GRF1 when co-expressed with activated ACK1.","method":"In vitro GEF assay (GDP binding/release), GTP-Ras pull-down (RBD assay), ERK2 activation assay, kinase-dead ACK1 control","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro GEF assay + cell-based RBD pull-down + ERK assay, kinase-dead control included, single lab","pmids":["10882715"],"is_preprint":false},{"year":2001,"finding":"Drosophila Ack (DAck) phosphorylates the sorting nexin DSH3PX1 in vivo and in vitro, with the major phosphorylation site at Tyr56 within the SH3 domain. Tyr56 phosphorylation by DAck diminishes DSH3PX1 SH3 domain binding to WASP while enabling association with Dock (Nck orthologue), targeting DSH3PX1 to a protein complex involved in axonal guidance.","method":"Co-immunoprecipitation from fly cell extracts, in vitro kinase assay, domain interaction mapping, site-directed mutagenesis (Y56D/E phosphomimetics), SH3 binding assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay + phosphosite mutagenesis + SH3 binding assays + co-IP, single lab studying Drosophila ortholog","pmids":["11773052"],"is_preprint":false},{"year":2005,"finding":"Sorting nexin 9 (SNX9/SH3PX1) acts as an adaptor linking ACK1 to synaptojanin-1; a single SNX9 binding site was identified in human ACK1 (residues 920-955). In the presence of SNX9, synaptojanin co-localizes with ACK1-containing vesicles, linking ACK1 to multiple endocytic trafficking components (clathrin, AP2, synaptojanin-1).","method":"In vivo biotinylation/blot overlay for SH3 domain interactions, synthetic peptide arrays for proline-rich binding sites, ACK1 truncation co-localization assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple biochemical binding methods + co-localization, single lab","pmids":["16137687"],"is_preprint":false},{"year":2008,"finding":"ACK1/TNK2 preserves EGFR at the cell surface by blocking its degradation; ACK1 associates with activated EGFR in a kinase-independent manner. TNK2 knockdown reduces cell-surface EGFR, decreasing migratory and invasive capacity of breast cancer cells.","method":"siRNA knockdown, flow cytometry for cell-surface EGFR, co-immunoprecipitation, invasion/migration assays, 125I-EGF internalization assay","journal":"Breast cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + FACS + functional migration/invasion, single lab","pmids":["18435854"],"is_preprint":false},{"year":2008,"finding":"ACK1 over-expression retains EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. ACK1 knockdown reduces EGFR internalization rate (but not transferrin internalization) and increases EGFR recycling while inhibiting its degradation, placing ACK1 at an early step in EGFR desensitization.","method":"siRNA knockdown, 125I-EGF internalization/recycling/degradation assays, 125I-transferrin assay (negative control), fluorescence co-localization in early endosomes","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative radioligand trafficking assays + imaging, single lab","pmids":["18262180"],"is_preprint":false},{"year":2009,"finding":"ACK1 interacts with multiple receptor tyrosine kinases (Axl, LTK, ALK, EGFR) via its C-terminal MIG6 homology region; interaction with Axl, LTK, and ALK (but not EGFR) requires Grb2 as adaptor, which binds conserved proline-rich regions. ACK1 controls Axl receptor levels; knockdown of endogenous ACK1 blocks GAS6-stimulated Axl downregulation and inhibits ruffling and migration.","method":"Co-immunoprecipitation, domain deletion mapping, ACK1 siRNA knockdown, receptor degradation assays, cell migration assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP domain mapping + siRNA + receptor degradation + migration assays, single lab","pmids":["19815557"],"is_preprint":false},{"year":2010,"finding":"Ack phosphorylation at endocytic clathrin-coated pits requires both clathrin assembly into pits and active Cdc42; in cells lacking dynamin (frozen deeply invaginated pits), ACK is constitutively phosphorylated and activated. ACK is concentrated at clathrin-coated pits and binds clathrin heavy chain via two clathrin boxes.","method":"Dynamin 1/2 double conditional knockout fibroblasts, mass spectrometry for phosphoproteomic changes, RNAi knockdown, pharmacological Cdc42 inhibition, clathrin box mutant analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO + MS + RNAi + pharmacology, multiple approaches, single lab","pmids":["21169560"],"is_preprint":false},{"year":2014,"finding":"Drosophila Ack (DAck) localizes to CTP synthase (CTPS) filaments in ovarian germline cells; DAck catalytic activity regulates CTPS filament architecture. Flies deficient in DAck or lacking DAck kinase activity exhibit disrupted CTPS filament architecture, morphological defects correlating with reduced fertility, and reduced total RNA levels.","method":"Genetic loss-of-function (DAck mutant flies), kinase-dead DAck transgenes, fluorescence localization to CTPS filaments, fertility and RNA level measurements","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic LOF + kinase-dead transgene + filament imaging + biochemical RNA measurement, single lab studying Drosophila ortholog","pmids":["25223282"],"is_preprint":false},{"year":2014,"finding":"ACK1 phosphorylates KDM3A (H3K9 demethylase) at Tyr1114 in a heregulin-dependent manner, decreasing H3K9me2 deposition. This ACK1-KDM3A-ER complex regulates HOXA1 transcription to promote tamoxifen resistance in breast cancer. Inhibition of ACK1 by AIM-100 or dasatinib restores H3K9me2 marks and suppresses HOXA1 expression.","method":"Co-immunoprecipitation of ACK1/ER/KDM3A complex, phospho-specific detection, histone methylation ChIP, ACK1 knockdown/inhibitor, HOXA1 expression analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous complex + ChIP for histone marks + inhibitor/KD rescue, single lab","pmids":["25148682"],"is_preprint":false},{"year":2012,"finding":"ACK1-mediated phosphorylation of AR at Tyr267 promotes AR recruitment to the ATM enhancer, up-regulating ATM expression and conferring radioresistance in castration-resistant prostate cancer. ACK1 inhibitor AIM-100 suppresses pTyr267-AR and reduces ATM expression, sensitizing CRPC tumors to radiotherapy.","method":"ChIP (AR recruitment to ATM enhancer), ACK1 transgenic mice (pTyr267-AR and ATM levels), ACK1 inhibitor AIM-100, primary human CRPC tissue analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP + transgenic mouse + pharmacological inhibition, single lab","pmids":["22566699"],"is_preprint":false},{"year":2013,"finding":"ACK1 interacts with Trk receptors and becomes tyrosine-phosphorylated in response to neurotrophins; ACK1 acts upstream of AKT and MAPK pathways in neurotrophin signaling. ACK1 overexpression induces neuritic outgrowth and branching in neurotrophin-treated neurons, while dominant-negative ACK1 or shRNA knockdown counteracts neurotrophin-stimulated differentiation.","method":"Co-immunoprecipitation with Trk receptors, kinase activity assays in response to neurotrophins, ACK1 overexpression/dominant-negative/shRNA in primary neurons and PC12 cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + kinase assay + loss/gain-of-function in primary neurons, single lab","pmids":["23598414"],"is_preprint":false},{"year":2015,"finding":"ACK1 (Ack1) is a DAT (dopamine transporter) endocytic brake that stabilizes DAT at the plasma membrane; both pharmacological and shRNA-mediated Ack1 silencing enhances basal DAT internalization. PKC activation and cdc42 activation converge on Ack1 to control DAT endocytic capacity; Ack1 inactivation is required for PKC-stimulated DAT internalization. Constitutively active Ack1 rescues the gain-of-function endocytic phenotype of the ADHD DAT coding variant R615C. Ack1 effects are specific for DAT (not SERT).","method":"shRNA knockdown, pharmacological Ack1 inhibition, DAT surface biotinylation, SERT internalization assay (specificity control), gain-of-function DAT variant rescue","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — shRNA + pharmacology + surface biotinylation + specificity controls + genetic variant rescue, multiple orthogonal approaches","pmids":["26621748"],"is_preprint":false},{"year":2017,"finding":"ACK1 (TNK2) phosphorylates STAT1 and STAT3, promoting their nuclear accumulation and STAT1-dependent gene expression. ACK1 physically interacts with endogenous STAT1. SIAH2 (which targets ACK1 for proteasomal degradation via Val909) attenuates the ACK1-STAT1 signaling node. HSP90 (HSP90α/β) is an upstream regulator of the ACK1-dependent STAT1/STAT3 phosphorylation axis; HSP90 inhibitor Onalespib suppresses this signaling.","method":"Co-immunoprecipitation (endogenous STAT1-ACK1), nuclear fractionation, reporter assays, SIAH2 degradation assay, HSP90 inhibitor treatment, global interactome analysis","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + nuclear fractionation + reporter + pharmacological inhibition, single lab","pmids":["28739485"],"is_preprint":false},{"year":2017,"finding":"ACK1 binds the SAM domain of adaptor SLP-76 and phosphorylates SLP-76 N-terminal tyrosines (Tyr113, Tyr128, Tyr145); interaction is induced by TCR ligation and requires the SLP-76 SAM domain. ACK1 promotes calcium flux and NFAT-AP1 promoter activity and decreases CD4+ T cell motility on ICAM-1, effects reversed by ACK1 inhibitor AIM-100.","method":"Co-precipitation, laser-scanning confocal microscopy, in situ proximity ligation assay, TCR stimulation, SAM domain deletion/3Y3F mutation, calcium flux, NFAT-AP1 reporter, T-cell motility assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + PLA + reporter + functional T-cell assays, single lab","pmids":["28188290"],"is_preprint":false},{"year":2018,"finding":"TNK2/ACK1 interacts directly with PTPN11; ACK1 phosphorylates PTPN11, which subsequently dephosphorylates ACK1 in a negative feedback loop. Mutations in PTPN11 increase basal PTPN11 activity such that TNK2-mediated activation is additive, synergistically increasing MAPK signaling. TNK2 inhibition blocks MAPK signaling and colony formation in PTPN11-mutant leukemia cells.","method":"Co-immunoprecipitation (direct interaction), phosphorylation assays, MAPK signaling assays, colony formation, TNK2 inhibitor treatment, patient dasatinib treatment","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + phosphorylation assays + functional colony/signaling assays + patient data, single lab","pmids":["30018082"],"is_preprint":false},{"year":2022,"finding":"ACK1 (TNK2) phosphorylates CSK (C-terminal Src kinase) at Tyr18 (pY18-CSK), enhancing CSK function and constraining T-cell activation. Tnk2 knockout mice exhibit diminished CSK Y18-phosphorylation and spontaneous activation of CD8+ and CD4+ T cells, inhibiting growth of ICB-resistant tumors. ACK1 inhibitor (R)-9b recapitulates tumor inhibition, identifying ACK1/pY18-CSK as a mechanism of immune checkpoint blockade resistance.","method":"Tnk2 knockout mice, phospho-specific antibodies for pY18-CSK, T-cell activation assays, transplanted ICB-resistant tumor models, ACK1 inhibitor (R)-9b treatment, ICB-treated CRPC patient samples","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO + phospho-specific antibody + in vivo tumor models + patient samples + pharmacological inhibitor, multiple orthogonal methods","pmids":["36376335"],"is_preprint":false},{"year":2022,"finding":"TNK2/ACK1 phosphorylates ATP5F1A (ATP synthase F1 alpha subunit) at Tyr243 and Tyr246, increasing complex V stability and mitochondrial energy output in cancer cells. Phospho-ATP5F1A prevents binding of its physiological inhibitor ATP5IF1, sustaining mitochondrial activity. ACK1 inhibitor (R)-9b reverses this, inducing mitophagy-based autophagy selectively in cancer cells.","method":"In vitro kinase assay (phosphosite identification), Y243/246A mutant analysis, co-immunoprecipitation of ATP5F1A and ATP5IF1, mitophagy assay, TNK2 transgenic mouse model, tumor xenograft, phospho-ATP5F1A antibody","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro phosphorylation + phosphosite mutagenesis + co-IP + mitophagy assay + transgenic mouse, multiple orthogonal methods","pmids":["35895804"],"is_preprint":false},{"year":2014,"finding":"ACK1 co-localizes and interacts with autophagy receptor p62/SQSTM1 via its UBA domain, and with NBR1 in a manner enhanced by p62 co-expression. ACK1 partially co-localizes with Atg16L-positive isolation membrane structures upon EGF stimulation. Ack1 knockdown accelerates EGFR localization to lysosomes, and the UBA domain is essential for p62/SQSTM1 co-localization, while the Mig6-homology domain and clathrin-binding domain contribute to EGFR co-localization.","method":"Co-immunoprecipitation, confocal co-localization, domain deletion mutant analysis, siRNA knockdown, EGF-stimulated EGFR trafficking assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + imaging + domain mutants + siRNA knockdown, single lab","pmids":["24413169"],"is_preprint":false},{"year":2012,"finding":"ACK1 directly binds and phosphorylates cortactin; the cortactin SH3 domain mediates binding to ACK1. ACK1 phosphorylates cortactin on key tyrosines that create docking sites for adaptor proteins enhancing Arp2/3 nucleation. ACK1 and cortactin co-localize on internalized EGF/EGFR vesicles. RNAi knockdown of ACK1 or the cortactin SH3 domain blocks EGF-induced EGFR internalization.","method":"Co-immunoprecipitation, in vitro kinase assay, phospho-specific antibodies, confocal co-localization, siRNA knockdown, EGFR internalization assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay + co-IP + imaging + siRNA functional assay, single lab","pmids":["22952966"],"is_preprint":false},{"year":2019,"finding":"TNK2, WASL (N-WASP), and NCK1 comprise a pathway required for entry of multiple picornaviruses (EMCV, CVB3, poliovirus, EV-D68); CRISPR deletion of TNK2 reduces viral internalization. Genetic epistasis analysis places all three genes in a common pathway. The actin nucleation activity of WASL is necessary for viral infection. Tnk2 knockout mice show reduced EMCV lethality.","method":"CRISPR gene deletion, genetic epistasis analysis, virus entry/internalization assays, TNK2/WASL chemical inhibitors, Tnk2 knockout mice, WASL domain mutants","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO + genetic epistasis + in vivo mouse model + domain mutant analysis + inhibitors, multiple orthogonal methods","pmids":["31769754"],"is_preprint":false},{"year":2013,"finding":"Structural and biochemical data indicate ACK1 kinase domain in its monomeric state is autoinhibited (parallel to EGFR and CDK); activation may require N-lobe-mediated symmetric dimerization facilitated by the N-terminal SAM domain. The SH3 domain does not directly control the activation state but may facilitate MIG6 homologous region binding to the kinase domain (allosteric model).","method":"X-ray crystallography of kinase domain and kinase+SH3 domain, biochemical activity assays, analytical ultracentrifugation for dimerization","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structures + biochemical activity assays but limited mutagenesis validation; mechanistic model partially speculative","pmids":["23342057"],"is_preprint":false},{"year":2004,"finding":"ACK-1 and ACK-2 undergo Cdc42-dependent nuclear translocation in semi-confluent glioblastoma cells (cytosolic in confluent cells); interaction of Cdc42 with ACKs is essential for their nuclear localization. A putative nuclear export signal was identified in both ACK-1 and ACK-2. Overexpression of the Cdc42-binding domain (ACK42) inhibits cell growth and movement.","method":"Immunocytochemistry, western blot of nuclear/cytosolic fractions, ACK42 overexpression functional assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-localization and fractionation study, limited mechanistic follow-up, single lab","pmids":["14733946"],"is_preprint":false},{"year":2023,"finding":"Activated ACK1 deposits pY88-H4 epigenetic marks at cell cycle gene promoters (CCNB1, CCNB2, CDC20), initiating their transcription; this is demonstrated by ChIP. ACK1 inhibitor (R)-9b dampens CCNB1/CCNB2/CDC20 expression, causes G2/M arrest, and suppresses CXCR4 receptor expression to impair breast cancer metastasis.","method":"Chromatin immunoprecipitation (ChIP) for pY88-H4 marks at cell cycle gene promoters, gene expression analysis, ACK1 inhibitor (R)-9b treatment, G2/M cell cycle analysis, xenograft metastasis model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for histone marks + pharmacological inhibition + functional tumor assays, single lab","pmids":["37330596"],"is_preprint":false},{"year":2017,"finding":"ACK1 specificity for Cdc42 over Rac binding requires a combination of at least 7 Cdc42 residues (S41A, A42V, N43T, D47G, N52T, W56F, R174L); hydrophobic interactions at both ends of the binding interface are critical for ACK1-Cdc42 affinity; ACK1 uses a 'dock and coalesce' binding mechanism driven by hydrophobic residues in its intrinsically disordered CRIB region.","method":"Panel of Rac gain-of-function mutants, equilibrium binding constant measurements (ITC/fluorescence), based on prior Cdc42-ACK NMR structure","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative binding measurements with large panel of mutants guided by structural data, single lab","pmids":["28539360"],"is_preprint":false}],"current_model":"TNK2/ACK1 is a non-receptor tyrosine kinase that functions as a cytoplasmic effector of activated Cdc42 (binding selectively to Cdc42-GTP via its CRIB domain), transduces signals from multiple receptor tyrosine kinases (EGFR, HER2, PDGFR, Axl, MerTK, Trk) and integrins by acting downstream of Src (which phosphorylates the ACK1 activation loop at Tyr284), and phosphorylates a diverse set of substrates including androgen receptor (Tyr267/363), AKT (Tyr176), Wwox (Tyr287), WASP (Tyr256/Ser242), cortactin, Dbl/Ras-GRF1 (activating Rho/Ras GEFs), KDM3A (Tyr1114, epigenetic regulation), CSK (Tyr18, immune checkpoint regulation), SLP-76 (T-cell activation), ATP5F1A (Tyr243/246, mitochondrial energy output), and PTPN11; ACK1 activity is regulated by intramolecular autoinhibition (kinase domain–MHR interaction), N-terminal SAM domain-dependent membrane localization and dimerization, and ubiquitin-mediated proteasomal (SIAH1/2, Nedd4-2) or lysosomal (Nedd4-1) degradation; ACK1 participates in clathrin-mediated endocytosis of EGFR by associating with clathrin heavy chain and stabilizing EGFR at the plasma membrane, and also acts as an endocytic brake for the dopamine transporter (DAT) at presynaptic terminals."},"narrative":{"mechanistic_narrative":"TNK2/ACK1 is a non-receptor tyrosine kinase that acts as a cytoplasmic effector of GTP-loaded Cdc42 and an integrator of signals from receptor tyrosine kinases, integrins, and adhesion cues to drive cytoskeletal remodeling, receptor trafficking, and growth-promoting transcriptional programs [PMID:10360579, PMID:17494760, PMID:10085085]. It binds selectively to Cdc42-GTP through an intercalated extended strand from its CRIB region, a 'dock and coalesce' recognition mode that discriminates Cdc42 from Rac [PMID:10360579, PMID:28539360]. The kinase domain adopts a constitutively activation-competent conformation [PMID:15308621], but full-length ACK1 is held in check by intramolecular autoinhibition involving the C-lobe and the C-terminal Mig6-homology region (MHR), which cancer mutations such as E346K disrupt to yield constitutive activity [PMID:20110370]; activation in cells requires N-terminal SAM-domain-dependent membrane targeting and dimerization together with Cdc42 binding, and Src-mediated phosphorylation of the activation-loop Tyr284 rather than autophosphorylation [PMID:20979614, PMID:21309750, PMID:17494760]. Once active, ACK1 phosphorylates a broad substrate set to amplify oncogenic signaling: androgen receptor at Tyr267/Tyr363 to drive androgen-independent and radioresistant transcription [PMID:17494760, PMID:22566699], AKT at Tyr176 to promote its membrane recruitment and activation [PMID:20333297], the tumor suppressor Wwox (triggering its degradation) [PMID:16288044], and the Rho/Ras GEFs Dbl and Ras-GRF1 to elevate GTP-loading of Rho/Rac and Ras [PMID:10652228, PMID:10882715]; it also reprograms chromatin via KDM3A Tyr1114 phosphorylation and pY88-H4 deposition at cell-cycle gene promoters [PMID:25148682, PMID:37330596]. In parallel, ACK1 organizes clathrin-mediated endocytosis by binding clathrin heavy chain through clathrin-box motifs (competing with beta-arrestin) and assembling with Nck, SNX9, cortactin, and WASP/N-WASP to couple receptor internalization to actin nucleation [PMID:11278436, PMID:16137687, PMID:22952966, PMID:16257963]; it stabilizes EGFR at the plasma membrane and early endosomes [PMID:18435854, PMID:18262180] and serves as an endocytic brake for the dopamine transporter [PMID:26621748]. ACK1 levels are tightly controlled by multiple E3 ligases—Nedd4-1 (lysosomal), Nedd4-2 and SIAH1/2 (proteasomal)—acting through PPXY and SIAH-binding motifs [PMID:20086093, PMID:19144635, PMID:23208506]. Beyond cancer, ACK1 functions in T-cell receptor signaling and immune checkpoint resistance via SLP-76 and CSK Tyr18 phosphorylation [PMID:28188290, PMID:36376335], supports mitochondrial energy output through ATP5F1A Tyr243/246 phosphorylation [PMID:35895804], and is required for picornavirus entry through a TNK2-WASL-NCK1 actin-nucleation pathway [PMID:31769754].","teleology":[{"year":1999,"claim":"Established the structural basis for how ACK1 reads out an activated Rho-family GTPase, defining it as a selective Cdc42 effector.","evidence":"NMR solution structure of the Cdc42-ACK1 GTPase-binding domain complex with binding-specificity validation","pmids":["10360579"],"confidence":"High","gaps":["Does not address how Cdc42 binding is coupled to kinase catalytic activation","Full-length ACK1 conformation not resolved"]},{"year":1999,"claim":"Linked ACK1 to upstream adhesion receptors, showing integrin/Cdc42 engagement and adhesion-driven signaling complexes activate the kinase.","evidence":"Co-IP with integrin beta1, RGD/antibody inhibition and kinase assays; dominant-negative analysis of MCSP-induced p130Cas/Cdc42 signaling","pmids":["10085085","10587647"],"confidence":"Medium","gaps":["Direct ACK1 substrates downstream of adhesion not defined here","Single-lab readouts"]},{"year":2000,"claim":"Identified GEF substrates as a mechanism by which ACK1 amplifies small-GTPase signaling, phosphorylating Dbl and Ras-GRF1 to elevate Rho/Rac and Ras-GTP.","evidence":"In vitro GEF assays, RBD pull-downs for GTP-loaded GTPases, and JNK/ERK activation readouts with kinase-dead controls","pmids":["10652228","10882715"],"confidence":"Medium","gaps":["Phosphosites on the GEFs not mapped","In vivo relevance of GEF phosphorylation not established"]},{"year":2001,"claim":"Placed ACK1 physically within the clathrin endocytic machinery via direct clathrin-heavy-chain and Nck binding, revealing a trafficking role distinct from kinase signaling.","evidence":"Direct binding/competition assays versus beta-arrestin, GFP-ACK1 imaging on clathrin/AP-2 vesicles, transferrin uptake","pmids":["11278436"],"confidence":"High","gaps":["Whether kinase activity is required for clathrin function not resolved here","Cargo specificity not addressed"]},{"year":2003,"claim":"Defined Tyr284 as the principal activation-loop autophosphorylation site and characterized ACK1 substrate specificity and an Hck regulatory interaction.","evidence":"In vitro kinase assays with purified ACK1, MS phosphosite mapping, Y284F mutagenesis, SH3 binding screens","pmids":["14506255"],"confidence":"High","gaps":["Whether Tyr284 is filled by autophosphorylation or a trans-kinase in vivo left open (later contested)","Physiological role of Hck unconfirmed in other systems"]},{"year":2004,"claim":"Showed the isolated kinase domain is intrinsically activation-competent independent of phosphorylation, reframing regulation as a problem of conformational/intramolecular control.","evidence":"X-ray crystallography of phosphorylated/unphosphorylated and inhibitor-bound kinase domains","pmids":["15308621"],"confidence":"High","gaps":["Full-length autoinhibitory architecture not captured","How cellular activation is gated not addressed"]},{"year":2005,"claim":"Demonstrated ACK1 drives degradation of the tumor suppressor Wwox and possesses unexpected dual-specificity (Ser as well as Tyr) toward WASP, coupling it to actin dynamics.","evidence":"In vitro kinase/ubiquitination assays, phosphosite mutagenesis (Wwox Y287, WASP Y256/S242), actin polymerization assays, xenograft","pmids":["16288044","16257963"],"confidence":"High","gaps":["Structural basis of serine kinase activity unexplained","Wwox degradation E3 ligase not identified here"]},{"year":2006,"claim":"Resolved the activation requirements—SAM-domain membrane targeting plus Cdc42 binding with an autoinhibitory SH3 role—and showed RTK/adhesion stimuli recruit ACK1 via Nck.","evidence":"Domain-deletion mutants, EGF/PDGF stimulation, fibronectin adhesion, reciprocal co-IP with Nck","pmids":["16777958"],"confidence":"High","gaps":["Quantitative contribution of each input to activation not dissected","SH3 autoinhibition mechanism later debated"]},{"year":2008,"claim":"Established ACK1 as a positive regulator of EGFR surface levels and trafficking, retaining receptor at the plasma membrane and early endosomes.","evidence":"siRNA knockdown, flow cytometry of surface EGFR, radioligand internalization/recycling/degradation assays, invasion/migration","pmids":["18435854","18262180"],"confidence":"Medium","gaps":["Reconciling EGFR stabilization with ACK1's pro-degradation role (via Nedd4) unresolved","Kinase dependence partially conflicting"]},{"year":2010,"claim":"Mapped the autoinhibition–degradation logic of ACK1: an MHR-kinase intramolecular clamp restrains activity (released by E346K), while Nedd4-1/Nedd4-2 ubiquitinate ACK1 for lysosomal/proteasomal turnover.","evidence":"In vitro domain pulldowns, cancer-mutant kinase assays, ubiquitination assays, RNAi rescue with PPXY/UBA mutants, inhibitor experiments","pmids":["20110370","20086093","19144635"],"confidence":"High","gaps":["Crosstalk between autoinhibition release and ligase recruitment not integrated","Which degradation route dominates in vivo unclear"]},{"year":2010,"claim":"Showed ACK1 phosphorylates AKT at Tyr176 to promote membrane localization and full activation, connecting ACK1 to a major oncogenic axis.","evidence":"In vitro kinase assay, phospho-specific antibody, membrane fractionation, mutagenesis, prostate-specific transgenic mouse","pmids":["20333297"],"confidence":"High","gaps":["Generality across tissues not established","Stoichiometry relative to PDK1/mTORC2 inputs unknown"]},{"year":2011,"claim":"Challenged the autophosphorylation model by showing Src, not ACK1 itself, phosphorylates Tyr284 in cells and is required for ACK1 activation and turnover.","evidence":"Endogenous Src-ACK1 co-IP, Src-deficient SYF cells, Src inhibitors, SH2/SH3 domain mapping","pmids":["21309750"],"confidence":"Medium","gaps":["Direct conflict with in vitro autophosphorylation data unresolved","Single lab"]},{"year":2012,"claim":"Extended ACK1's regulatory repertoire and disease links: SIAH1/2-mediated proteasomal degradation, SH3-EBD/Grb2 autoinhibition release, AR-driven radioresistance, PDGFR-beta/PDK1-AKT signaling, and cortactin phosphorylation for endocytic actin.","evidence":"Y2H/co-IP/mutagenesis (SIAH, Grb2, PDK1), ChIP at the ATM enhancer, transgenic mice, in vitro kinase assays and EGFR internalization assays","pmids":["23208506","22553920","22566699","25257795","22952966"],"confidence":"Medium","gaps":["Conflicting autoinhibition models (SH3-EBD vs MHR) not reconciled","Substrate hierarchy across contexts undefined"]},{"year":2014,"claim":"Identified epigenetic and autophagy/trafficking functions: KDM3A Tyr1114 phosphorylation reprogramming H3K9me2 for tamoxifen resistance, and UBA-domain engagement of p62/NBR1 linking ACK1 to EGFR autophagic handling.","evidence":"Co-IP of ACK1/ER/KDM3A, histone ChIP, inhibitor/knockdown rescue; p62/NBR1 co-IP, domain mutants, EGFR lysosomal trafficking","pmids":["25148682","24413169"],"confidence":"Medium","gaps":["Direct demethylase activity changes downstream of KDM3A phosphorylation not mechanistically resolved","Single-lab findings"]},{"year":2015,"claim":"Defined ACK1 as an endocytic brake for the dopamine transporter, integrating PKC and Cdc42 inputs and rescuing an ADHD-associated DAT variant.","evidence":"shRNA/pharmacological silencing, DAT surface biotinylation, SERT specificity control, R615C variant rescue","pmids":["26621748"],"confidence":"High","gaps":["Direct ACK1 substrate at the DAT endocytic step not identified","Neuronal in vivo relevance limited"]},{"year":2017,"claim":"Broadened ACK1 signaling to STAT activation and TCR-proximal adaptor SLP-76, implicating it in transcriptional and immune signaling.","evidence":"Endogenous co-IP (STAT1, SLP-76 SAM), nuclear fractionation, PLA, calcium flux/NFAT-AP1 reporters, T-cell motility, HSP90 inhibition","pmids":["28739485","28188290"],"confidence":"Medium","gaps":["Direct STAT phosphosites not mapped","In vivo immune consequences not tested here"]},{"year":2018,"claim":"Revealed an ACK1-PTPN11 reciprocal phosphorylation/feedback loop that synergizes with PTPN11 mutations to amplify MAPK signaling in leukemia.","evidence":"Direct-interaction co-IP, phosphorylation and MAPK/colony assays, TNK2 inhibitor and patient dasatinib data","pmids":["30018082"],"confidence":"Medium","gaps":["PTPN11 phosphosites and ACK1 dephosphorylation sites not mapped","Single lab"]},{"year":2019,"claim":"Placed TNK2 in a defined WASL-NCK1 actin-nucleation pathway required for picornavirus entry, with in vivo confirmation.","evidence":"CRISPR deletion, genetic epistasis, internalization assays, inhibitors, WASL domain mutants, Tnk2 knockout mice","pmids":["31769754"],"confidence":"High","gaps":["Direct viral or host substrate of TNK2 in entry not identified","Receptor specificity across viruses unresolved"]},{"year":2022,"claim":"Defined ACK1 as a node of immune checkpoint resistance and mitochondrial energy control via CSK Tyr18 and ATP5F1A Tyr243/246 phosphorylation.","evidence":"Tnk2 knockout mice, phospho-specific antibodies, in vitro kinase assays/mutagenesis, ATP5IF1 co-IP, mitophagy and tumor models, patient samples","pmids":["36376335","35895804"],"confidence":"High","gaps":["How ACK1 gains access to mitochondrial ATP synthase not defined","Tissue specificity of CSK pY18 control incomplete"]},{"year":2023,"claim":"Showed activated ACK1 deposits pY88-H4 marks at cell-cycle gene promoters to drive their transcription and metastasis, extending its direct chromatin role.","evidence":"ChIP for pY88-H4 at CCNB1/CCNB2/CDC20, expression/cell-cycle analysis, (R)-9b inhibitor, xenograft metastasis","pmids":["37330596"],"confidence":"Medium","gaps":["Direct enzymatic deposition of pY88-H4 by ACK1 versus an intermediary not distinguished","Single lab"]},{"year":null,"claim":"How the conflicting autoinhibition/activation models (Src-driven Tyr284, MHR-kinase clamp, SH3-EBD/Grb2 release, SAM-mediated dimerization) integrate into one quantitative scheme for cellular ACK1 activation remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified full-length structure reconciling autoinhibition and dimerization","Relative contribution of Src vs autophosphorylation in vivo undefined","Context-dependent substrate selection not mechanistically explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,5,6,7,8,37,39]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,6,8,36,37]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,6,13,31]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[20,21,35]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,23]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16,42]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,16,24,32]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,10,25,39]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[25,38]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[42]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,8,13,35]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,24,25,39]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[6,29,43]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,30,35,36]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[34,36]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[37,38]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9,10,11]}],"complexes":["clathrin coat / AP-2 endocytic complex","ACK1-Cdc42-p130Cas-Crk adhesion signaling complex","ACK1-ER-KDM3A transcriptional complex"],"partners":["CDC42","EGFR","SRC","NCK1","CLTC","GRB2","SNX9","PTPN11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q07912","full_name":"Activated CDC42 kinase 1","aliases":["Tyrosine kinase non-receptor protein 2"],"length_aa":1038,"mass_kda":114.6,"function":"Non-receptor tyrosine-protein and serine/threonine-protein kinase that is implicated in cell spreading and migration, cell survival, cell growth and proliferation. Transduces extracellular signals to cytosolic and nuclear effectors. Phosphorylates AKT1, AR, MCF2, WASL and WWOX. Implicated in trafficking and clathrin-mediated endocytosis through binding to epidermal growth factor receptor (EGFR) and clathrin. Binds to both poly- and mono-ubiquitin and regulates ligand-induced degradation of EGFR, thereby contributing to the accumulation of EGFR at the limiting membrane of early endosomes. Downstream effector of CDC42 which mediates CDC42-dependent cell migration via phosphorylation of BCAR1. May be involved both in adult synaptic function and plasticity and in brain development. Activates AKT1 by phosphorylating it on 'Tyr-176'. Phosphorylates AR on 'Tyr-267' and 'Tyr-363' thereby promoting its recruitment to androgen-responsive enhancers (AREs). Phosphorylates WWOX on 'Tyr-287'. Phosphorylates MCF2, thereby enhancing its activity as a guanine nucleotide exchange factor (GEF) toward Rho family proteins. Contributes to the control of AXL receptor levels. Confers metastatic properties on cancer cells and promotes tumor growth by negatively regulating tumor suppressor such as WWOX and positively regulating pro-survival factors such as AKT1 and AR. Phosphorylates WASP (PubMed:20110370)","subcellular_location":"Cell membrane; Nucleus; Endosome; Cell junction, adherens junction; Cytoplasmic vesicle membrane; Cytoplasmic vesicle, clathrin-coated vesicle; Membrane, clathrin-coated pit; Cytoplasm, perinuclear region; Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q07912/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TNK2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TNK2","total_profiled":1310},"omim":[{"mim_id":"606994","title":"TYROSINE KINASE, NONRECEPTOR, 2; TNK2","url":"https://www.omim.org/entry/606994"},{"mim_id":"138971","title":"COLONY-STIMULATING FACTOR 3 RECEPTOR, GRANULOCYTE; CSF3R","url":"https://www.omim.org/entry/138971"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TNK2"},"hgnc":{"alias_symbol":["p21cdc42Hs","ACK","ACK1"],"prev_symbol":[]},"alphafold":{"accession":"Q07912","domains":[{"cath_id":"1.10.150,1.10.150","chopping":"8-79","consensus_level":"high","plddt":85.8257,"start":8,"end":79},{"cath_id":"3.30.200.20","chopping":"118-208","consensus_level":"medium","plddt":85.4012,"start":118,"end":208},{"cath_id":"1.10.510.10","chopping":"211-390","consensus_level":"medium","plddt":92.3621,"start":211,"end":390},{"cath_id":"1.10.8","chopping":"969-1031","consensus_level":"high","plddt":85.1065,"start":969,"end":1031}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q07912","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q07912-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q07912-F1-predicted_aligned_error_v6.png","plddt_mean":61.28},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TNK2","jax_strain_url":"https://www.jax.org/strain/search?query=TNK2"},"sequence":{"accession":"Q07912","fasta_url":"https://rest.uniprot.org/uniprotkb/Q07912.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q07912/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q07912"}},"corpus_meta":[{"pmid":"17494760","id":"PMC_17494760","title":"Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17494760","citation_count":206,"is_preprint":false},{"pmid":"16288044","id":"PMC_16288044","title":"Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox.","date":"2005","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/16288044","citation_count":181,"is_preprint":false},{"pmid":"10587647","id":"PMC_10587647","title":"Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.","date":"1999","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10587647","citation_count":172,"is_preprint":false},{"pmid":"10360579","id":"PMC_10360579","title":"Structure of the small G protein Cdc42 bound to 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28539360","citation_count":14,"is_preprint":false},{"pmid":"3001033","id":"PMC_3001033","title":"Isolation and characterization of ack and pta mutations in Azotobacter vinelandii affecting acetate-glucose diauxie.","date":"1986","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/3001033","citation_count":14,"is_preprint":false},{"pmid":"27462444","id":"PMC_27462444","title":"Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded.","date":"2016","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/27462444","citation_count":13,"is_preprint":false},{"pmid":"33164271","id":"PMC_33164271","title":"TNK2-AS1 upregulated by YY1 boosts the course of osteosarcoma through targeting miR-4319/WDR1.","date":"2020","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/33164271","citation_count":13,"is_preprint":false},{"pmid":"28921811","id":"PMC_28921811","title":"A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.","date":"2017","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/28921811","citation_count":13,"is_preprint":false},{"pmid":"35669783","id":"PMC_35669783","title":"ACK1 Contributes to the Pathogenesis of Inflammation and Autoimmunity by Promoting the Activation of TLR Signaling Pathways.","date":"2022","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35669783","citation_count":13,"is_preprint":false},{"pmid":"30763529","id":"PMC_30763529","title":"Sugar and Fatty Acids Ack-celerate Prophage Induction.","date":"2019","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/30763529","citation_count":13,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49999,"output_tokens":12706,"usd":0.170293,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24005,"output_tokens":6922,"usd":0.146537,"stage2_stop_reason":"end_turn"},"total_usd":0.31683,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"NMR solution structure of Cdc42 bound to the GTPase-binding domain of ACK1 revealed that both proteins undergo significant conformational changes on binding, forming a new type of G-protein/effector complex in which an extended strand from ACK intercalates into the beta-sheet of Cdc42; this defines the structural basis for selective Cdc42 (not Rac) binding.\",\n      \"method\": \"NMR structure determination with functional validation of binding specificity\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution NMR structure with functional validation of the binding interface, published in high-impact journal\",\n      \"pmids\": [\"10360579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ACK1 autophosphorylates at Tyr284 in the activation loop (identified by mass spectrometry); this is the primary autophosphorylation site and its mutation (Y284F) dramatically reduces tyrosine phosphorylation in cells. ACK1 substrate specificity most closely resembles Abl. ACK1 interacts with Hck SH3 domains via its proline-rich C-terminal domain, and Hck can phosphorylate ACK1, suggesting Hck as an upstream regulator.\",\n      \"method\": \"In vitro kinase assay with purified baculovirus-expressed ACK1, mass spectrometry phosphosite mapping, site-directed mutagenesis, SH2/SH3 domain binding screens, co-expression in mammalian cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution + MS phosphosite mapping + mutagenesis + cell-based validation in one study\",\n      \"pmids\": [\"14506255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crystal structures of human ACK1 kinase domain in both unphosphorylated and phosphorylated states revealed that ACK1 adopts an activated conformation independent of phosphorylation, with the unphosphorylated activation loop structured and resembling that of activated tyrosine kinases. Inhibitor-bound co-crystal structures (with AMPPCP and debromohymenialdisine) defined the ATP-binding cleft.\",\n      \"method\": \"X-ray crystallography of phosphorylated and unphosphorylated kinase domain; inhibitor co-crystal structures\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures (apo, phosphorylated, inhibitor-bound) providing atomic-resolution mechanistic insight\",\n      \"pmids\": [\"15308621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ACK1 associates directly with clathrin heavy chain via a central adaptor motif that competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain; ACK1 also interacts with the adaptor Nck via SH3 interactions; stable low-level GFP-ACK1 expression localizes to clathrin/AP-2-containing vesicles and increases receptor-mediated transferrin uptake.\",\n      \"method\": \"Direct binding assays, competition assays with beta-arrestin, GFP-ACK1 live-cell imaging, co-localization with clathrin and AP-2, transferrin uptake assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, live imaging, functional uptake assay, multiple orthogonal methods in one study\",\n      \"pmids\": [\"11278436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ACK1 activation loop autophosphorylation requires both the amino-terminal SAM-like domain (for membrane targeting) and Cdc42 binding via the CRIB domain; the SH3 domain plays an autoinhibitory role. Cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of ACK1; EGF or PDGF stimulation recruits ACK1 to activated receptors; tyrosine-phosphorylated ACK1 forms a stable complex with adaptor Nck via its SH2 domain.\",\n      \"method\": \"Domain deletion mutant analysis, immunoprecipitation, kinase assays, EGF/PDGF stimulation of cells, fibronectin adhesion assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mutants, orthogonal stimulation conditions, reciprocal co-IP, from dedicated mechanistic study\",\n      \"pmids\": [\"16777958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Activated ACK1 tyrosine-phosphorylates tumor suppressor Wwox at Tyr287 (identified by site-directed mutagenesis), leading to rapid Wwox polyubiquitination and proteasomal degradation. Hsp90beta associates with ACK1 and its inhibition (geldanamycin) blocks ACK1 kinase activity. A splice variant (WwoxΔ5-8) not phosphorylated by ACK1 is not ubiquitinated or degraded.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, ubiquitination assay, Hsp90 inhibitor treatment, xenograft tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro phosphorylation + mutagenesis + ubiquitination assays + in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"16288044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Activated ACK1 directly phosphorylates androgen receptor (AR) at Tyr267 and Tyr363 within the transactivation domain. Mutation of Tyr267 completely abrogates, and Tyr363 mutation reduces, Ack1-induced AR reporter activation and AR recruitment to androgen-responsive enhancers. Heregulin-stimulated HER2 activates ACK1, which then phosphorylates AR to drive androgen-independent gene expression and tumor growth.\",\n      \"method\": \"Site-directed mutagenesis of AR, AR reporter assays, ChIP (AR recruitment to enhancers), ACK1 knockdown by siRNA, xenograft tumor models, phospho-specific antibodies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis of phosphosites + reporter assays + ChIP + in vivo xenograft, multiple orthogonal methods\",\n      \"pmids\": [\"17494760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ACK1 phosphorylates WASP at both Tyr256 (tyrosine kinase activity) and Ser242 (serine kinase activity, demonstrating dual-specificity), with serine phosphorylation enhanced by Cdc42 or PIP2 (which releases WASP autoinhibition). Serine phosphorylation of WASP at Ser242 enhances WASP-stimulated actin polymerization in cell lysates. ACK1 expressed in bacteria retains serine kinase activity.\",\n      \"method\": \"In vitro kinase assay with purified proteins, phosphosite mapping by mutagenesis, bacterially expressed ACK1 kinase assay, actin polymerization assay in cell lysates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, mutagenesis of phosphosites, bacterial expression controls, functional actin polymerization assay\",\n      \"pmids\": [\"16257963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ACK1 directly phosphorylates AKT at the evolutionarily conserved Tyr176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473 phosphorylation leading to full AKT activation. This pathway operates downstream of RTK/growth factor signaling.\",\n      \"method\": \"In vitro kinase assay, phospho-specific antibody generation, plasma membrane fractionation, site-directed mutagenesis, transgenic mouse model (prostate-specific activated Ack1), co-immunoprecipitation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay + phosphosite mutagenesis + subcellular fractionation + transgenic mouse validation\",\n      \"pmids\": [\"20333297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HECT E3 ubiquitin ligase Nedd4-1 ubiquitinates ACK1 via a conserved PPXY WW-binding motif (WW3 domain of Nedd4-1 is critical); EGF-induced ACK1 degradation is processed by lysosomes, not proteasomes. The UBA domain of ACK1 suppresses Nedd4-1-mediated ubiquitination. Nedd4-1 (not Nedd4-2) knockdown inhibits degradation of both EGFR and ACK1, and ACK1 mutants deficient in Nedd4-1 binding block EGF-induced EGFR degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNAi knockdown, proteasome/lysosome inhibitors, EGFR degradation assay, deletion mutant analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, ubiquitination assay, RNAi rescue with multiple deletion mutants, inhibitor experiments, mechanistic dissection\",\n      \"pmids\": [\"20086093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"E3 ubiquitin ligase Nedd4-2 binds ACK1 via its PPXY motif, co-localizes with ACK1 in clathrin-rich vesicles, and strongly down-regulates ACK1 levels via proteasomal degradation that is driven by ACK1 kinase activity. Dominant-inhibitory Nedd4 blocks endogenous ACK1 turnover in response to EGF.\",\n      \"method\": \"Co-immunoprecipitation, co-localization imaging, proteasome inhibitor (MG132), ACK1 PPXY mutants, dominant-negative Nedd4\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP + imaging co-localization + proteasome inhibitor + dominant-negative + PPXY mutants, multiple orthogonal methods\",\n      \"pmids\": [\"19144635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SIAH1 and SIAH2 ubiquitin ligases interact with ACK1 via a conserved SIAH-binding motif in the far C-terminus of ACK1 and induce proteasomal (not lysosomal) degradation of ACK1 in a manner independent of ACK1 kinase activity. SIAH2 expression induced by estrogen receptor activation decreases ACK1 levels in breast cancer cells.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, deletion/point mutants of ACK1, proteasome inhibitor, SIAH2 knockdown, ER activation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid + co-IP + mutagenesis + inhibitor + RNAi, multiple orthogonal methods\",\n      \"pmids\": [\"23208506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ACK1 kinase activity is autoinhibited by an intramolecular interaction between the kinase domain C-lobe and the C-terminal Mig6 homology region (MHR, residues 802-990); cancer-associated mutation E346K prevents kinase-MHR binding and constitutively activates ACK1. The MHR-kinase domain interaction was demonstrated by direct binding of purified domains in vitro.\",\n      \"method\": \"In vitro pulldown with purified kinase domain and MHR fragments, immune complex kinase assays, cancer-associated mutant characterization (E346K, F820A), cell migration and proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro domain interaction assays + mutagenesis + kinase activity assays + functional cell assays\",\n      \"pmids\": [\"20110370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ACK1 activates AKT-mediated signaling in glioma cells downstream of PDGFR-β; PDGFR-β phosphorylates ACK1 at Y635, and this phosphorylation is required for sequential AKT activation. PDK1 interacts with ACK1 (via T325 of ACK1) during PDGF stimulation and is required for ACK1-PDGFR-β binding. Y635F or T325A ACK1 mutants abolish PDGFR-β-induced AKT activation and downstream β-catenin nuclear translocation.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Y635F, T325A), reporter and western blot assays, in vivo glioma model\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP + site-directed mutagenesis + functional cell/tumor assays, single lab\",\n      \"pmids\": [\"25257795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ACK1 activation mechanism involves an autoinhibitory interaction between the SH3 domain and the EGFR-binding domain (EBD); release of this autoinhibition activates ACK1. Cell adhesion-mediated activation occurs through releasing this autoinhibition. Grb2 mediates ACK1 interaction with EGFR by binding the EBD and releasing autoinhibition. The N-terminal region (Leu10-Leu14) is essential for cell adhesion-mediated activation. Ser445Pro mutation causes constitutive ACK1 activation.\",\n      \"method\": \"SH3/EBD domain deletion and point mutants, kinase activity assays, co-immunoprecipitation with Grb2 and EGFR, cell adhesion assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutant panel + kinase assays + co-IP, but conflicting with another paper (PMID 21309750) on autoinhibition model; single lab\",\n      \"pmids\": [\"22553920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Src kinase (not ACK1 autophosphorylation) is required for phosphorylation of ACK1 activation loop Tyr284 in vivo; Src SH2 and SH3 domains interact with ACK1 Tyr518 and residues 623-652, respectively. ACK1 fails to undergo significant Tyr284 autophosphorylation in vivo because its activation loop is basophilic (while Src is acidophilic). ACK1 activation downstream of EGFR/integrin requires Src; ACK1 turnover is blocked by Src inhibitors and is impaired in Src-deficient SYF cells.\",\n      \"method\": \"Co-immunoprecipitation of endogenous Src-ACK1, Src-deficient SYF cell line analysis, Src inhibitor treatment, domain mapping with SH2/SH3 domains\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + genetic (SYF cells) + pharmacological inhibition, single lab; contradicts autoinhibition model proposed by others\",\n      \"pmids\": [\"21309750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ACK1 activity is required for N-terminal SAM domain-mediated plasma membrane localization and dimerization; the isolated kinase domain (without N-terminus) fails to autophosphorylate and shows cytosolic localization, while the N-terminus+kinase domain (NKD) localizes to plasma membrane and undergoes autophosphorylation. Increasing local concentration of purified ACK1 kinase domain at lipid vesicle surfaces stimulates autophosphorylation and activity, consistent with dimerization and trans-phosphorylation.\",\n      \"method\": \"Deletion mutant immunofluorescence, western blotting for autophosphorylation, co-immunoprecipitation (dimerization), lipid vesicle reconstitution assay\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro lipid vesicle reconstitution + cell-based deletion mutant analysis + co-IP for dimerization, single lab\",\n      \"pmids\": [\"20979614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ACK-2 (the shorter Cdc42-associated kinase) is activated by cell adhesion via integrin beta1 in a Cdc42-dependent manner; ACK-2 co-immunoprecipitates with integrin beta1. Activation is F-actin-independent and does not require cell spreading. Overexpression of ACK-2 activates c-Jun kinase (not ERK). Anti-integrin beta1 antibodies and RGD peptides inhibit ACK-2 activation by cell adhesion.\",\n      \"method\": \"Co-immunoprecipitation with integrin beta1, RGD peptide/antibody inhibition, kinase assays, actin depolymerization controls\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + multiple inhibitor approaches + kinase assays, single lab\",\n      \"pmids\": [\"10085085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MCSP stimulation recruits tyrosine-phosphorylated p130Cas and activates Cdc42, with MCSP-induced cell spreading dependent on active Cdc42, Ack-1, and tyrosine phosphorylation of p130Cas. Vectors inhibiting Ack-1 or Cdc42 abrogate MCSP-induced p130Cas tyrosine phosphorylation and recruitment.\",\n      \"method\": \"Dominant-negative/inhibitory vectors for Ack-1 and Cdc42, phospho-p130Cas immunoprecipitation, cell spreading assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative inhibition + co-IP phosphorylation assays + functional spreading readout, single lab\",\n      \"pmids\": [\"10587647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Ack1 forms a signaling complex with Cdc42, p130Cas, and Crk, whose formation is regulated by collagen stimulation. Ack1 interaction with p130Cas occurs through their respective SH3 domains, while the substrate domain of p130Cas is the major site of Ack1-dependent phosphorylation. siRNA knockdown of either p130Cas or Ack1 blocks Cdc42-induced cell migration on collagen.\",\n      \"method\": \"Co-immunoprecipitation, SH3 domain interaction mapping, siRNA knockdown, p130Cas phosphorylation assay, cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP + siRNA knockdown + migration assay, single lab\",\n      \"pmids\": [\"17038317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"ACK1 tyrosine-phosphorylates and activates the guanine nucleotide exchange factor Dbl; in vitro GEF activity of Dbl toward Rho and Cdc42 is augmented after tyrosine phosphorylation. ACK1-dependent Dbl phosphorylation leads to accumulation of GTP-bound Rho and Rac in cells and enhanced JNK activation downstream.\",\n      \"method\": \"Co-expression in cells, in vitro GEF assay, GTP-bound Rho/Rac pull-down (RBD assay), JNK activation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro GEF assay + cell-based RBD pull-down + JNK assay, single lab\",\n      \"pmids\": [\"10652228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"ACK1 phosphorylates and activates the Ras GEF Ras-GRF1 at tyrosine residues, augmenting Ras-GEF activity (GDP release) specifically toward Ha-Ras (not Rac1). This results in increased GTP-Ras accumulation in cells and enhanced ERK2 activation downstream of Ras-GRF1 when co-expressed with activated ACK1.\",\n      \"method\": \"In vitro GEF assay (GDP binding/release), GTP-Ras pull-down (RBD assay), ERK2 activation assay, kinase-dead ACK1 control\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro GEF assay + cell-based RBD pull-down + ERK assay, kinase-dead control included, single lab\",\n      \"pmids\": [\"10882715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Drosophila Ack (DAck) phosphorylates the sorting nexin DSH3PX1 in vivo and in vitro, with the major phosphorylation site at Tyr56 within the SH3 domain. Tyr56 phosphorylation by DAck diminishes DSH3PX1 SH3 domain binding to WASP while enabling association with Dock (Nck orthologue), targeting DSH3PX1 to a protein complex involved in axonal guidance.\",\n      \"method\": \"Co-immunoprecipitation from fly cell extracts, in vitro kinase assay, domain interaction mapping, site-directed mutagenesis (Y56D/E phosphomimetics), SH3 binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay + phosphosite mutagenesis + SH3 binding assays + co-IP, single lab studying Drosophila ortholog\",\n      \"pmids\": [\"11773052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Sorting nexin 9 (SNX9/SH3PX1) acts as an adaptor linking ACK1 to synaptojanin-1; a single SNX9 binding site was identified in human ACK1 (residues 920-955). In the presence of SNX9, synaptojanin co-localizes with ACK1-containing vesicles, linking ACK1 to multiple endocytic trafficking components (clathrin, AP2, synaptojanin-1).\",\n      \"method\": \"In vivo biotinylation/blot overlay for SH3 domain interactions, synthetic peptide arrays for proline-rich binding sites, ACK1 truncation co-localization assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple biochemical binding methods + co-localization, single lab\",\n      \"pmids\": [\"16137687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ACK1/TNK2 preserves EGFR at the cell surface by blocking its degradation; ACK1 associates with activated EGFR in a kinase-independent manner. TNK2 knockdown reduces cell-surface EGFR, decreasing migratory and invasive capacity of breast cancer cells.\",\n      \"method\": \"siRNA knockdown, flow cytometry for cell-surface EGFR, co-immunoprecipitation, invasion/migration assays, 125I-EGF internalization assay\",\n      \"journal\": \"Breast cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + FACS + functional migration/invasion, single lab\",\n      \"pmids\": [\"18435854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ACK1 over-expression retains EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. ACK1 knockdown reduces EGFR internalization rate (but not transferrin internalization) and increases EGFR recycling while inhibiting its degradation, placing ACK1 at an early step in EGFR desensitization.\",\n      \"method\": \"siRNA knockdown, 125I-EGF internalization/recycling/degradation assays, 125I-transferrin assay (negative control), fluorescence co-localization in early endosomes\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative radioligand trafficking assays + imaging, single lab\",\n      \"pmids\": [\"18262180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ACK1 interacts with multiple receptor tyrosine kinases (Axl, LTK, ALK, EGFR) via its C-terminal MIG6 homology region; interaction with Axl, LTK, and ALK (but not EGFR) requires Grb2 as adaptor, which binds conserved proline-rich regions. ACK1 controls Axl receptor levels; knockdown of endogenous ACK1 blocks GAS6-stimulated Axl downregulation and inhibits ruffling and migration.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion mapping, ACK1 siRNA knockdown, receptor degradation assays, cell migration assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP domain mapping + siRNA + receptor degradation + migration assays, single lab\",\n      \"pmids\": [\"19815557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Ack phosphorylation at endocytic clathrin-coated pits requires both clathrin assembly into pits and active Cdc42; in cells lacking dynamin (frozen deeply invaginated pits), ACK is constitutively phosphorylated and activated. ACK is concentrated at clathrin-coated pits and binds clathrin heavy chain via two clathrin boxes.\",\n      \"method\": \"Dynamin 1/2 double conditional knockout fibroblasts, mass spectrometry for phosphoproteomic changes, RNAi knockdown, pharmacological Cdc42 inhibition, clathrin box mutant analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO + MS + RNAi + pharmacology, multiple approaches, single lab\",\n      \"pmids\": [\"21169560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila Ack (DAck) localizes to CTP synthase (CTPS) filaments in ovarian germline cells; DAck catalytic activity regulates CTPS filament architecture. Flies deficient in DAck or lacking DAck kinase activity exhibit disrupted CTPS filament architecture, morphological defects correlating with reduced fertility, and reduced total RNA levels.\",\n      \"method\": \"Genetic loss-of-function (DAck mutant flies), kinase-dead DAck transgenes, fluorescence localization to CTPS filaments, fertility and RNA level measurements\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic LOF + kinase-dead transgene + filament imaging + biochemical RNA measurement, single lab studying Drosophila ortholog\",\n      \"pmids\": [\"25223282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ACK1 phosphorylates KDM3A (H3K9 demethylase) at Tyr1114 in a heregulin-dependent manner, decreasing H3K9me2 deposition. This ACK1-KDM3A-ER complex regulates HOXA1 transcription to promote tamoxifen resistance in breast cancer. Inhibition of ACK1 by AIM-100 or dasatinib restores H3K9me2 marks and suppresses HOXA1 expression.\",\n      \"method\": \"Co-immunoprecipitation of ACK1/ER/KDM3A complex, phospho-specific detection, histone methylation ChIP, ACK1 knockdown/inhibitor, HOXA1 expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous complex + ChIP for histone marks + inhibitor/KD rescue, single lab\",\n      \"pmids\": [\"25148682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ACK1-mediated phosphorylation of AR at Tyr267 promotes AR recruitment to the ATM enhancer, up-regulating ATM expression and conferring radioresistance in castration-resistant prostate cancer. ACK1 inhibitor AIM-100 suppresses pTyr267-AR and reduces ATM expression, sensitizing CRPC tumors to radiotherapy.\",\n      \"method\": \"ChIP (AR recruitment to ATM enhancer), ACK1 transgenic mice (pTyr267-AR and ATM levels), ACK1 inhibitor AIM-100, primary human CRPC tissue analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP + transgenic mouse + pharmacological inhibition, single lab\",\n      \"pmids\": [\"22566699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ACK1 interacts with Trk receptors and becomes tyrosine-phosphorylated in response to neurotrophins; ACK1 acts upstream of AKT and MAPK pathways in neurotrophin signaling. ACK1 overexpression induces neuritic outgrowth and branching in neurotrophin-treated neurons, while dominant-negative ACK1 or shRNA knockdown counteracts neurotrophin-stimulated differentiation.\",\n      \"method\": \"Co-immunoprecipitation with Trk receptors, kinase activity assays in response to neurotrophins, ACK1 overexpression/dominant-negative/shRNA in primary neurons and PC12 cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + kinase assay + loss/gain-of-function in primary neurons, single lab\",\n      \"pmids\": [\"23598414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ACK1 (Ack1) is a DAT (dopamine transporter) endocytic brake that stabilizes DAT at the plasma membrane; both pharmacological and shRNA-mediated Ack1 silencing enhances basal DAT internalization. PKC activation and cdc42 activation converge on Ack1 to control DAT endocytic capacity; Ack1 inactivation is required for PKC-stimulated DAT internalization. Constitutively active Ack1 rescues the gain-of-function endocytic phenotype of the ADHD DAT coding variant R615C. Ack1 effects are specific for DAT (not SERT).\",\n      \"method\": \"shRNA knockdown, pharmacological Ack1 inhibition, DAT surface biotinylation, SERT internalization assay (specificity control), gain-of-function DAT variant rescue\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — shRNA + pharmacology + surface biotinylation + specificity controls + genetic variant rescue, multiple orthogonal approaches\",\n      \"pmids\": [\"26621748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACK1 (TNK2) phosphorylates STAT1 and STAT3, promoting their nuclear accumulation and STAT1-dependent gene expression. ACK1 physically interacts with endogenous STAT1. SIAH2 (which targets ACK1 for proteasomal degradation via Val909) attenuates the ACK1-STAT1 signaling node. HSP90 (HSP90α/β) is an upstream regulator of the ACK1-dependent STAT1/STAT3 phosphorylation axis; HSP90 inhibitor Onalespib suppresses this signaling.\",\n      \"method\": \"Co-immunoprecipitation (endogenous STAT1-ACK1), nuclear fractionation, reporter assays, SIAH2 degradation assay, HSP90 inhibitor treatment, global interactome analysis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + nuclear fractionation + reporter + pharmacological inhibition, single lab\",\n      \"pmids\": [\"28739485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACK1 binds the SAM domain of adaptor SLP-76 and phosphorylates SLP-76 N-terminal tyrosines (Tyr113, Tyr128, Tyr145); interaction is induced by TCR ligation and requires the SLP-76 SAM domain. ACK1 promotes calcium flux and NFAT-AP1 promoter activity and decreases CD4+ T cell motility on ICAM-1, effects reversed by ACK1 inhibitor AIM-100.\",\n      \"method\": \"Co-precipitation, laser-scanning confocal microscopy, in situ proximity ligation assay, TCR stimulation, SAM domain deletion/3Y3F mutation, calcium flux, NFAT-AP1 reporter, T-cell motility assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + PLA + reporter + functional T-cell assays, single lab\",\n      \"pmids\": [\"28188290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TNK2/ACK1 interacts directly with PTPN11; ACK1 phosphorylates PTPN11, which subsequently dephosphorylates ACK1 in a negative feedback loop. Mutations in PTPN11 increase basal PTPN11 activity such that TNK2-mediated activation is additive, synergistically increasing MAPK signaling. TNK2 inhibition blocks MAPK signaling and colony formation in PTPN11-mutant leukemia cells.\",\n      \"method\": \"Co-immunoprecipitation (direct interaction), phosphorylation assays, MAPK signaling assays, colony formation, TNK2 inhibitor treatment, patient dasatinib treatment\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + phosphorylation assays + functional colony/signaling assays + patient data, single lab\",\n      \"pmids\": [\"30018082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ACK1 (TNK2) phosphorylates CSK (C-terminal Src kinase) at Tyr18 (pY18-CSK), enhancing CSK function and constraining T-cell activation. Tnk2 knockout mice exhibit diminished CSK Y18-phosphorylation and spontaneous activation of CD8+ and CD4+ T cells, inhibiting growth of ICB-resistant tumors. ACK1 inhibitor (R)-9b recapitulates tumor inhibition, identifying ACK1/pY18-CSK as a mechanism of immune checkpoint blockade resistance.\",\n      \"method\": \"Tnk2 knockout mice, phospho-specific antibodies for pY18-CSK, T-cell activation assays, transplanted ICB-resistant tumor models, ACK1 inhibitor (R)-9b treatment, ICB-treated CRPC patient samples\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO + phospho-specific antibody + in vivo tumor models + patient samples + pharmacological inhibitor, multiple orthogonal methods\",\n      \"pmids\": [\"36376335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TNK2/ACK1 phosphorylates ATP5F1A (ATP synthase F1 alpha subunit) at Tyr243 and Tyr246, increasing complex V stability and mitochondrial energy output in cancer cells. Phospho-ATP5F1A prevents binding of its physiological inhibitor ATP5IF1, sustaining mitochondrial activity. ACK1 inhibitor (R)-9b reverses this, inducing mitophagy-based autophagy selectively in cancer cells.\",\n      \"method\": \"In vitro kinase assay (phosphosite identification), Y243/246A mutant analysis, co-immunoprecipitation of ATP5F1A and ATP5IF1, mitophagy assay, TNK2 transgenic mouse model, tumor xenograft, phospho-ATP5F1A antibody\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro phosphorylation + phosphosite mutagenesis + co-IP + mitophagy assay + transgenic mouse, multiple orthogonal methods\",\n      \"pmids\": [\"35895804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ACK1 co-localizes and interacts with autophagy receptor p62/SQSTM1 via its UBA domain, and with NBR1 in a manner enhanced by p62 co-expression. ACK1 partially co-localizes with Atg16L-positive isolation membrane structures upon EGF stimulation. Ack1 knockdown accelerates EGFR localization to lysosomes, and the UBA domain is essential for p62/SQSTM1 co-localization, while the Mig6-homology domain and clathrin-binding domain contribute to EGFR co-localization.\",\n      \"method\": \"Co-immunoprecipitation, confocal co-localization, domain deletion mutant analysis, siRNA knockdown, EGF-stimulated EGFR trafficking assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + imaging + domain mutants + siRNA knockdown, single lab\",\n      \"pmids\": [\"24413169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ACK1 directly binds and phosphorylates cortactin; the cortactin SH3 domain mediates binding to ACK1. ACK1 phosphorylates cortactin on key tyrosines that create docking sites for adaptor proteins enhancing Arp2/3 nucleation. ACK1 and cortactin co-localize on internalized EGF/EGFR vesicles. RNAi knockdown of ACK1 or the cortactin SH3 domain blocks EGF-induced EGFR internalization.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, phospho-specific antibodies, confocal co-localization, siRNA knockdown, EGFR internalization assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay + co-IP + imaging + siRNA functional assay, single lab\",\n      \"pmids\": [\"22952966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TNK2, WASL (N-WASP), and NCK1 comprise a pathway required for entry of multiple picornaviruses (EMCV, CVB3, poliovirus, EV-D68); CRISPR deletion of TNK2 reduces viral internalization. Genetic epistasis analysis places all three genes in a common pathway. The actin nucleation activity of WASL is necessary for viral infection. Tnk2 knockout mice show reduced EMCV lethality.\",\n      \"method\": \"CRISPR gene deletion, genetic epistasis analysis, virus entry/internalization assays, TNK2/WASL chemical inhibitors, Tnk2 knockout mice, WASL domain mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO + genetic epistasis + in vivo mouse model + domain mutant analysis + inhibitors, multiple orthogonal methods\",\n      \"pmids\": [\"31769754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Structural and biochemical data indicate ACK1 kinase domain in its monomeric state is autoinhibited (parallel to EGFR and CDK); activation may require N-lobe-mediated symmetric dimerization facilitated by the N-terminal SAM domain. The SH3 domain does not directly control the activation state but may facilitate MIG6 homologous region binding to the kinase domain (allosteric model).\",\n      \"method\": \"X-ray crystallography of kinase domain and kinase+SH3 domain, biochemical activity assays, analytical ultracentrifugation for dimerization\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures + biochemical activity assays but limited mutagenesis validation; mechanistic model partially speculative\",\n      \"pmids\": [\"23342057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ACK-1 and ACK-2 undergo Cdc42-dependent nuclear translocation in semi-confluent glioblastoma cells (cytosolic in confluent cells); interaction of Cdc42 with ACKs is essential for their nuclear localization. A putative nuclear export signal was identified in both ACK-1 and ACK-2. Overexpression of the Cdc42-binding domain (ACK42) inhibits cell growth and movement.\",\n      \"method\": \"Immunocytochemistry, western blot of nuclear/cytosolic fractions, ACK42 overexpression functional assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-localization and fractionation study, limited mechanistic follow-up, single lab\",\n      \"pmids\": [\"14733946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Activated ACK1 deposits pY88-H4 epigenetic marks at cell cycle gene promoters (CCNB1, CCNB2, CDC20), initiating their transcription; this is demonstrated by ChIP. ACK1 inhibitor (R)-9b dampens CCNB1/CCNB2/CDC20 expression, causes G2/M arrest, and suppresses CXCR4 receptor expression to impair breast cancer metastasis.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for pY88-H4 marks at cell cycle gene promoters, gene expression analysis, ACK1 inhibitor (R)-9b treatment, G2/M cell cycle analysis, xenograft metastasis model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for histone marks + pharmacological inhibition + functional tumor assays, single lab\",\n      \"pmids\": [\"37330596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACK1 specificity for Cdc42 over Rac binding requires a combination of at least 7 Cdc42 residues (S41A, A42V, N43T, D47G, N52T, W56F, R174L); hydrophobic interactions at both ends of the binding interface are critical for ACK1-Cdc42 affinity; ACK1 uses a 'dock and coalesce' binding mechanism driven by hydrophobic residues in its intrinsically disordered CRIB region.\",\n      \"method\": \"Panel of Rac gain-of-function mutants, equilibrium binding constant measurements (ITC/fluorescence), based on prior Cdc42-ACK NMR structure\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative binding measurements with large panel of mutants guided by structural data, single lab\",\n      \"pmids\": [\"28539360\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TNK2/ACK1 is a non-receptor tyrosine kinase that functions as a cytoplasmic effector of activated Cdc42 (binding selectively to Cdc42-GTP via its CRIB domain), transduces signals from multiple receptor tyrosine kinases (EGFR, HER2, PDGFR, Axl, MerTK, Trk) and integrins by acting downstream of Src (which phosphorylates the ACK1 activation loop at Tyr284), and phosphorylates a diverse set of substrates including androgen receptor (Tyr267/363), AKT (Tyr176), Wwox (Tyr287), WASP (Tyr256/Ser242), cortactin, Dbl/Ras-GRF1 (activating Rho/Ras GEFs), KDM3A (Tyr1114, epigenetic regulation), CSK (Tyr18, immune checkpoint regulation), SLP-76 (T-cell activation), ATP5F1A (Tyr243/246, mitochondrial energy output), and PTPN11; ACK1 activity is regulated by intramolecular autoinhibition (kinase domain–MHR interaction), N-terminal SAM domain-dependent membrane localization and dimerization, and ubiquitin-mediated proteasomal (SIAH1/2, Nedd4-2) or lysosomal (Nedd4-1) degradation; ACK1 participates in clathrin-mediated endocytosis of EGFR by associating with clathrin heavy chain and stabilizing EGFR at the plasma membrane, and also acts as an endocytic brake for the dopamine transporter (DAT) at presynaptic terminals.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TNK2/ACK1 is a non-receptor tyrosine kinase that acts as a cytoplasmic effector of GTP-loaded Cdc42 and an integrator of signals from receptor tyrosine kinases, integrins, and adhesion cues to drive cytoskeletal remodeling, receptor trafficking, and growth-promoting transcriptional programs [#0, #6, #17]. It binds selectively to Cdc42-GTP through an intercalated extended strand from its CRIB region, a 'dock and coalesce' recognition mode that discriminates Cdc42 from Rac [#0, #44]. The kinase domain adopts a constitutively activation-competent conformation [#2], but full-length ACK1 is held in check by intramolecular autoinhibition involving the C-lobe and the C-terminal Mig6-homology region (MHR), which cancer mutations such as E346K disrupt to yield constitutive activity [#12]; activation in cells requires N-terminal SAM-domain-dependent membrane targeting and dimerization together with Cdc42 binding, and Src-mediated phosphorylation of the activation-loop Tyr284 rather than autophosphorylation [#16, #15, #6]. Once active, ACK1 phosphorylates a broad substrate set to amplify oncogenic signaling: androgen receptor at Tyr267/Tyr363 to drive androgen-independent and radioresistant transcription [#6, #30], AKT at Tyr176 to promote its membrane recruitment and activation [#8], the tumor suppressor Wwox (triggering its degradation) [#5], and the Rho/Ras GEFs Dbl and Ras-GRF1 to elevate GTP-loading of Rho/Rac and Ras [#20, #21]; it also reprograms chromatin via KDM3A Tyr1114 phosphorylation and pY88-H4 deposition at cell-cycle gene promoters [#29, #43]. In parallel, ACK1 organizes clathrin-mediated endocytosis by binding clathrin heavy chain through clathrin-box motifs (competing with beta-arrestin) and assembling with Nck, SNX9, cortactin, and WASP/N-WASP to couple receptor internalization to actin nucleation [#3, #23, #39, #7]; it stabilizes EGFR at the plasma membrane and early endosomes [#24, #25] and serves as an endocytic brake for the dopamine transporter [#32]. ACK1 levels are tightly controlled by multiple E3 ligases—Nedd4-1 (lysosomal), Nedd4-2 and SIAH1/2 (proteasomal)—acting through PPXY and SIAH-binding motifs [#9, #10, #11]. Beyond cancer, ACK1 functions in T-cell receptor signaling and immune checkpoint resistance via SLP-76 and CSK Tyr18 phosphorylation [#34, #36], supports mitochondrial energy output through ATP5F1A Tyr243/246 phosphorylation [#37], and is required for picornavirus entry through a TNK2-WASL-NCK1 actin-nucleation pathway [#40].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the structural basis for how ACK1 reads out an activated Rho-family GTPase, defining it as a selective Cdc42 effector.\",\n      \"evidence\": \"NMR solution structure of the Cdc42-ACK1 GTPase-binding domain complex with binding-specificity validation\",\n      \"pmids\": [\"10360579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address how Cdc42 binding is coupled to kinase catalytic activation\", \"Full-length ACK1 conformation not resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Linked ACK1 to upstream adhesion receptors, showing integrin/Cdc42 engagement and adhesion-driven signaling complexes activate the kinase.\",\n      \"evidence\": \"Co-IP with integrin beta1, RGD/antibody inhibition and kinase assays; dominant-negative analysis of MCSP-induced p130Cas/Cdc42 signaling\",\n      \"pmids\": [\"10085085\", \"10587647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ACK1 substrates downstream of adhesion not defined here\", \"Single-lab readouts\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identified GEF substrates as a mechanism by which ACK1 amplifies small-GTPase signaling, phosphorylating Dbl and Ras-GRF1 to elevate Rho/Rac and Ras-GTP.\",\n      \"evidence\": \"In vitro GEF assays, RBD pull-downs for GTP-loaded GTPases, and JNK/ERK activation readouts with kinase-dead controls\",\n      \"pmids\": [\"10652228\", \"10882715\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites on the GEFs not mapped\", \"In vivo relevance of GEF phosphorylation not established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placed ACK1 physically within the clathrin endocytic machinery via direct clathrin-heavy-chain and Nck binding, revealing a trafficking role distinct from kinase signaling.\",\n      \"evidence\": \"Direct binding/competition assays versus beta-arrestin, GFP-ACK1 imaging on clathrin/AP-2 vesicles, transferrin uptake\",\n      \"pmids\": [\"11278436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether kinase activity is required for clathrin function not resolved here\", \"Cargo specificity not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined Tyr284 as the principal activation-loop autophosphorylation site and characterized ACK1 substrate specificity and an Hck regulatory interaction.\",\n      \"evidence\": \"In vitro kinase assays with purified ACK1, MS phosphosite mapping, Y284F mutagenesis, SH3 binding screens\",\n      \"pmids\": [\"14506255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Tyr284 is filled by autophosphorylation or a trans-kinase in vivo left open (later contested)\", \"Physiological role of Hck unconfirmed in other systems\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed the isolated kinase domain is intrinsically activation-competent independent of phosphorylation, reframing regulation as a problem of conformational/intramolecular control.\",\n      \"evidence\": \"X-ray crystallography of phosphorylated/unphosphorylated and inhibitor-bound kinase domains\",\n      \"pmids\": [\"15308621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length autoinhibitory architecture not captured\", \"How cellular activation is gated not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated ACK1 drives degradation of the tumor suppressor Wwox and possesses unexpected dual-specificity (Ser as well as Tyr) toward WASP, coupling it to actin dynamics.\",\n      \"evidence\": \"In vitro kinase/ubiquitination assays, phosphosite mutagenesis (Wwox Y287, WASP Y256/S242), actin polymerization assays, xenograft\",\n      \"pmids\": [\"16288044\", \"16257963\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of serine kinase activity unexplained\", \"Wwox degradation E3 ligase not identified here\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the activation requirements—SAM-domain membrane targeting plus Cdc42 binding with an autoinhibitory SH3 role—and showed RTK/adhesion stimuli recruit ACK1 via Nck.\",\n      \"evidence\": \"Domain-deletion mutants, EGF/PDGF stimulation, fibronectin adhesion, reciprocal co-IP with Nck\",\n      \"pmids\": [\"16777958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each input to activation not dissected\", \"SH3 autoinhibition mechanism later debated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Established ACK1 as a positive regulator of EGFR surface levels and trafficking, retaining receptor at the plasma membrane and early endosomes.\",\n      \"evidence\": \"siRNA knockdown, flow cytometry of surface EGFR, radioligand internalization/recycling/degradation assays, invasion/migration\",\n      \"pmids\": [\"18435854\", \"18262180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciling EGFR stabilization with ACK1's pro-degradation role (via Nedd4) unresolved\", \"Kinase dependence partially conflicting\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped the autoinhibition–degradation logic of ACK1: an MHR-kinase intramolecular clamp restrains activity (released by E346K), while Nedd4-1/Nedd4-2 ubiquitinate ACK1 for lysosomal/proteasomal turnover.\",\n      \"evidence\": \"In vitro domain pulldowns, cancer-mutant kinase assays, ubiquitination assays, RNAi rescue with PPXY/UBA mutants, inhibitor experiments\",\n      \"pmids\": [\"20110370\", \"20086093\", \"19144635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between autoinhibition release and ligase recruitment not integrated\", \"Which degradation route dominates in vivo unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed ACK1 phosphorylates AKT at Tyr176 to promote membrane localization and full activation, connecting ACK1 to a major oncogenic axis.\",\n      \"evidence\": \"In vitro kinase assay, phospho-specific antibody, membrane fractionation, mutagenesis, prostate-specific transgenic mouse\",\n      \"pmids\": [\"20333297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality across tissues not established\", \"Stoichiometry relative to PDK1/mTORC2 inputs unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Challenged the autophosphorylation model by showing Src, not ACK1 itself, phosphorylates Tyr284 in cells and is required for ACK1 activation and turnover.\",\n      \"evidence\": \"Endogenous Src-ACK1 co-IP, Src-deficient SYF cells, Src inhibitors, SH2/SH3 domain mapping\",\n      \"pmids\": [\"21309750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct conflict with in vitro autophosphorylation data unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended ACK1's regulatory repertoire and disease links: SIAH1/2-mediated proteasomal degradation, SH3-EBD/Grb2 autoinhibition release, AR-driven radioresistance, PDGFR-beta/PDK1-AKT signaling, and cortactin phosphorylation for endocytic actin.\",\n      \"evidence\": \"Y2H/co-IP/mutagenesis (SIAH, Grb2, PDK1), ChIP at the ATM enhancer, transgenic mice, in vitro kinase assays and EGFR internalization assays\",\n      \"pmids\": [\"23208506\", \"22553920\", \"22566699\", \"25257795\", \"22952966\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conflicting autoinhibition models (SH3-EBD vs MHR) not reconciled\", \"Substrate hierarchy across contexts undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified epigenetic and autophagy/trafficking functions: KDM3A Tyr1114 phosphorylation reprogramming H3K9me2 for tamoxifen resistance, and UBA-domain engagement of p62/NBR1 linking ACK1 to EGFR autophagic handling.\",\n      \"evidence\": \"Co-IP of ACK1/ER/KDM3A, histone ChIP, inhibitor/knockdown rescue; p62/NBR1 co-IP, domain mutants, EGFR lysosomal trafficking\",\n      \"pmids\": [\"25148682\", \"24413169\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demethylase activity changes downstream of KDM3A phosphorylation not mechanistically resolved\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined ACK1 as an endocytic brake for the dopamine transporter, integrating PKC and Cdc42 inputs and rescuing an ADHD-associated DAT variant.\",\n      \"evidence\": \"shRNA/pharmacological silencing, DAT surface biotinylation, SERT specificity control, R615C variant rescue\",\n      \"pmids\": [\"26621748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ACK1 substrate at the DAT endocytic step not identified\", \"Neuronal in vivo relevance limited\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Broadened ACK1 signaling to STAT activation and TCR-proximal adaptor SLP-76, implicating it in transcriptional and immune signaling.\",\n      \"evidence\": \"Endogenous co-IP (STAT1, SLP-76 SAM), nuclear fractionation, PLA, calcium flux/NFAT-AP1 reporters, T-cell motility, HSP90 inhibition\",\n      \"pmids\": [\"28739485\", \"28188290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct STAT phosphosites not mapped\", \"In vivo immune consequences not tested here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed an ACK1-PTPN11 reciprocal phosphorylation/feedback loop that synergizes with PTPN11 mutations to amplify MAPK signaling in leukemia.\",\n      \"evidence\": \"Direct-interaction co-IP, phosphorylation and MAPK/colony assays, TNK2 inhibitor and patient dasatinib data\",\n      \"pmids\": [\"30018082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PTPN11 phosphosites and ACK1 dephosphorylation sites not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Placed TNK2 in a defined WASL-NCK1 actin-nucleation pathway required for picornavirus entry, with in vivo confirmation.\",\n      \"evidence\": \"CRISPR deletion, genetic epistasis, internalization assays, inhibitors, WASL domain mutants, Tnk2 knockout mice\",\n      \"pmids\": [\"31769754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct viral or host substrate of TNK2 in entry not identified\", \"Receptor specificity across viruses unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined ACK1 as a node of immune checkpoint resistance and mitochondrial energy control via CSK Tyr18 and ATP5F1A Tyr243/246 phosphorylation.\",\n      \"evidence\": \"Tnk2 knockout mice, phospho-specific antibodies, in vitro kinase assays/mutagenesis, ATP5IF1 co-IP, mitophagy and tumor models, patient samples\",\n      \"pmids\": [\"36376335\", \"35895804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ACK1 gains access to mitochondrial ATP synthase not defined\", \"Tissue specificity of CSK pY18 control incomplete\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed activated ACK1 deposits pY88-H4 marks at cell-cycle gene promoters to drive their transcription and metastasis, extending its direct chromatin role.\",\n      \"evidence\": \"ChIP for pY88-H4 at CCNB1/CCNB2/CDC20, expression/cell-cycle analysis, (R)-9b inhibitor, xenograft metastasis\",\n      \"pmids\": [\"37330596\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic deposition of pY88-H4 by ACK1 versus an intermediary not distinguished\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the conflicting autoinhibition/activation models (Src-driven Tyr284, MHR-kinase clamp, SH3-EBD/Grb2 release, SAM-mediated dimerization) integrate into one quantitative scheme for cellular ACK1 activation remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified full-length structure reconciling autoinhibition and dimerization\", \"Relative contribution of Src vs autophosphorylation in vivo undefined\", \"Context-dependent substrate selection not mechanistically explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 5, 6, 7, 8, 37, 39]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 6, 8, 36, 37]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 6, 13, 31]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [20, 21, 35]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16, 42]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 16, 24, 32]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 10, 25, 39]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [25, 38]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [42]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 8, 13, 35]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 24, 25, 39]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [6, 29, 43]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 30, 35, 36]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [34, 36]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [37, 38]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 10, 11]}\n    ],\n    \"complexes\": [\n      \"clathrin coat / AP-2 endocytic complex\",\n      \"ACK1-Cdc42-p130Cas-Crk adhesion signaling complex\",\n      \"ACK1-ER-KDM3A transcriptional complex\"\n    ],\n    \"partners\": [\n      \"CDC42\",\n      \"EGFR\",\n      \"SRC\",\n      \"NCK1\",\n      \"CLTC\",\n      \"GRB2\",\n      \"SNX9\",\n      \"PTPN11\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}