{"gene":"PTK6","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1994,"finding":"BRK/PTK6 (brk) encodes a novel non-receptor tyrosine kinase with SH3 and SH2 domains that is capable of autophosphorylation on tyrosine residues when expressed in baculovirus and bacterial recombinant systems.","method":"Recombinant protein expression (baculovirus and bacterial systems), autophosphorylation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro autophosphorylation demonstrated in recombinant system, single lab, foundational characterization paper","pmids":["8036022"],"is_preprint":false},{"year":1996,"finding":"Mutation of the catalytic lysine (K219M) abolishes BRK kinase activity and transformation capacity in mammary epithelial cells; mutation of the C-terminal inhibitory tyrosine (Y447F) decreases transforming potential without abolishing kinase activity, indicating Y447 plays a regulatory role distinct from catalysis.","method":"Site-directed mutagenesis, transformation assay in human mammary epithelial cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis of key regulatory residues with functional readout, single lab, two mutants tested","pmids":["8940083"],"is_preprint":false},{"year":2000,"finding":"BRK/Sik associates with the RNA-binding protein Sam68 through its SH3 and SH2 domains; the proline-rich P3 region of Sam68 is required for SH3 binding. BRK/Sik phosphorylates Sam68 in the nucleus (in Sam68-SLM nuclear bodies in HT29 cells and in nucleoplasm of NMuMG cells) and abolishes Sam68's ability to bind RNA and act as a cellular Rev homologue.","method":"Co-immunoprecipitation, domain mapping (SH3/SH2 interaction studies), co-transfection, functional RNA-binding assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, domain mapping, functional RNA-binding assay, replicated across cell lines","pmids":["10913193"],"is_preprint":false},{"year":2000,"finding":"BRK associates with and phosphorylates BKS (STAP-2), a novel adaptor protein with PH-like and SH2-like domains; association and phosphorylation are dependent on BRK catalytic activity and on the SH2-like domain of BKS. BKS also recruits an unidentified 100 kDa protein that becomes tyrosine-phosphorylated in the presence of BRK.","method":"Yeast two-hybrid screen, co-transfection, co-immunoprecipitation, phosphotyrosine detection","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP validation, catalytic dependence demonstrated, single lab","pmids":["10980601"],"is_preprint":false},{"year":2000,"finding":"BRK expression in mammary epithelial cells enhances EGF-induced phosphorylation of erbB3, leading to increased recruitment of PI3-kinase to erbB3 and potentiated AKT activation, thereby enhancing mitogenic signaling.","method":"Stable transfection of BRK into mammary epithelial cells, Western blotting for phospho-erbB3, PI3K co-immunoprecipitation, AKT activity assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple signaling readouts (phospho-erbB3, PI3K recruitment, AKT activation), single lab, mechanistic chain established","pmids":["11114724"],"is_preprint":false},{"year":2002,"finding":"BRK autophosphorylates within the activation loop (Y342) and at N-terminal sites as shown by mass spectrometry; activation loop autophosphorylation increases kinase activity (Y342A mutant is not activated). The C-terminal Y447 maintains autoinhibition via SH2 domain engagement (Y447F mutation increases activity and SH2 accessibility). The SH3 domain also contributes to autoinhibition, as a proline-rich peptide activates BRK.","method":"Baculovirus expression, mass spectrometry, site-directed mutagenesis, in vitro kinase assays with synthetic peptides (Km, kcat measurements), SH2 domain accessibility assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus mass spectrometry identification of phosphosites, mutagenesis, kinetic characterization; multiple orthogonal methods in one rigorous study","pmids":["12121988"],"is_preprint":false},{"year":2003,"finding":"Sam68 hyperphosphorylation by activated Sik/BRK in the nucleus inhibits Sam68-mediated cytoplasmic utilization of intron-containing RNA in a dose-dependent manner. The RNA-binding defective Sam68 G178E mutant cannot enhance CTE function, confirming that BRK regulation of Sam68 RNA-binding activity is functionally important.","method":"Co-expression of Sik/BRK with Sam68 in 293T cells, RNA analysis, protein expression assays, Sam68 point mutant (G178E)","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assay of RNA utilization with mutant controls, dose-dependence shown, single lab","pmids":["12482964"],"is_preprint":false},{"year":2003,"finding":"BRK/Sik subcellular localization shifts from nuclear (normal/well-differentiated prostate epithelial cells) to cytoplasmic (poorly differentiated prostate cancer cells), and nuclear BRK is less active in cytoplasmic-localized PC3 cells; altered localization correlates with differentiation state.","method":"Immunohistochemistry of 58 prostate biopsy samples, subcellular fractionation, Western blotting of prostate cancer cell lines","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — fractionation plus IHC in tissue samples, localization linked to activity, single lab","pmids":["12833144"],"is_preprint":false},{"year":2004,"finding":"BRK SH3 domain mutations that disrupt intramolecular interactions increase BRK kinase activity; the SH3 domain plays the dominant role in substrate recognition. Bifunctional peptides coupling a substrate sequence to an SH3 ligand show significantly lower Km than controls, while SH2-binding substrate peptides show no difference.","method":"Site-directed mutagenesis, kinase activity assays in HEK293 cells, synthetic peptide substrate assays (Km measurements), Sam68 phosphorylation comparison","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assays with mutagenesis and quantitative kinetic measurements, mechanistic dissection of domain function, single lab but multiple orthogonal approaches","pmids":["14676834"],"is_preprint":false},{"year":2004,"finding":"BRK/Sik phosphorylates SLM-1 and SLM-2 (Sam68-like mammalian proteins) but not hnRNP K; phosphorylation inhibits their RNA-binding abilities and promotes nuclear retention of BRK/Sik.","method":"Co-transfection, phosphorylation assays, RNA-binding assay, subcellular localization studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — substrate specificity shown with selectivity control (hnRNP K not phosphorylated), functional RNA-binding assay, single lab","pmids":["15471878"],"is_preprint":false},{"year":2004,"finding":"BRK identifies paxillin as a binding partner and substrate; EGF stimulation activates BRK, which phosphorylates paxillin at Y31 and Y118, promoting Rac1 activation via CrkII, thereby driving cell motility and invasion. BRK translocates to membrane ruffles and colocalizes with paxillin during cell migration.","method":"Co-immunoprecipitation, in vitro kinase assay, site-specific phosphorylation mapping, Rac1 activation assay, cell migration and invasion assays, confocal microscopy","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with phosphosite mapping, Rac1 activation, cell functional assays, and localization; multiple orthogonal methods establishing full pathway","pmids":["15572663"],"is_preprint":false},{"year":2005,"finding":"BRK associates with IRS-4 via both its SH3 and SH2 domains in resting and IGF-1-stimulated cells; IRS-4 enhances IGF-1-induced BRK tyrosine phosphorylation; endogenous BRK and IRS-4 interact in A431 cells.","method":"Co-immunoprecipitation, mass spectrometry identification, domain mapping, EGF/IGF-1 stimulation experiments","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification plus co-IP validation in multiple cell lines, domain mapping included, single lab","pmids":["15870689"],"is_preprint":false},{"year":2007,"finding":"The BRK SH3 domain interacts with the SH2-kinase linker region via W44 in the SH3 domain and P175, P177, P179 in the linker; disruption of this interaction by mutagenesis markedly increases PTK6 catalytic activity, demonstrating this intramolecular contact maintains PTK6 in an autoinhibited conformation.","method":"Site-directed mutagenesis, surface plasmon resonance, kinase activity assays, 3D structural modeling","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SPR binding measurements plus mutagenesis plus activity assays in cells, multiple orthogonal methods confirming autoinhibitory mechanism","pmids":["17822667"],"is_preprint":false},{"year":2008,"finding":"BRK is coamplified with ErbB2 in human breast cancers; ErbB2 interacts with BRK and increases its intrinsic kinase activity; BRK expression enhances ErbB2-induced Ras/MAPK signaling and cyclin E/cdk2 activity to promote proliferation; BRK overexpression confers resistance to lapatinib-induced growth inhibition.","method":"Co-immunoprecipitation, FISH (gene amplification), 3D acini culture, mouse mammary tumor model (WAP-Brk transgenic), kinase activity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP showing interaction and kinase activation, multiple model systems (cell culture, mouse model), drug resistance phenotype, single lab but orthogonal methods","pmids":["18719096"],"is_preprint":false},{"year":2009,"finding":"BRK phosphorylates PSF (polypyrimidine tract-binding protein-associated splicing factor) at C-terminal tyrosines via an SH3 domain–polyproline interaction; this phosphorylation promotes cytoplasmic relocalization of PSF, impairs its RNA binding, and leads to cell cycle arrest.","method":"Proteomic co-immunoprecipitation/mass spectrometry, co-IP, phosphosite mapping, subcellular fractionation, RNA-binding assay, cell cycle analysis","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS identification plus co-IP, phosphosite mapping, functional consequences (RNA binding, localization, cell cycle), multiple readouts, single lab","pmids":["19439179"],"is_preprint":false},{"year":2009,"finding":"Membrane-targeted PTK6 (Myr-PTK6) promotes proliferation, cell survival, migration, and anchorage-independent colony formation, whereas nuclear-targeted PTK6 (NLS-PTK6) impairs these functions; the two localizations produce distinct cellular phosphoproteome profiles.","method":"Targeted localization constructs (myristoylation signal vs. NLS), proliferation/survival/migration/colony formation assays, phosphoprotein profiling in HEK293 cells","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — engineered localization with functional readouts, single lab, two distinct targeting conditions tested","pmids":["19304789"],"is_preprint":false},{"year":2009,"finding":"STAP-2 is phosphorylated at Y250 by BRK; the STAP-2 Y250F mutant attenuates BRK-mediated STAT3 activation; siRNA knockdown of STAP-2 decreases BRK-mediated STAT3 activation, establishing STAP-2 as a required intermediary in BRK→STAT3 signaling.","method":"Site-directed mutagenesis (Y250F), anti-phospho-STAP-2 Y250 antibody, siRNA knockdown, STAT3 activation assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphosite-specific antibody, loss-of-function with both mutant and siRNA, single lab","pmids":["19393627"],"is_preprint":false},{"year":2009,"finding":"PTK6 directly phosphorylates beta-catenin predominantly at Y64 (also Y142, Y331/333); PTK6 associates with nuclear and cytoplasmic beta-catenin and inhibits beta-catenin/TCF-mediated transcription. Nuclear-targeted PTK6 negatively regulates beta-catenin/TCF transcription and increases TCF4 and TLE/Groucho co-repressor levels, while membrane-targeted PTK6 enhances beta-catenin/TCF transcription.","method":"In vitro kinase assay, phosphosite mapping, co-immunoprecipitation, luciferase reporter assay, siRNA knockdown, Ptk6-null BAT-GAL reporter mice","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with phosphosite mapping, reporter assay, KO mouse model, multiple cell lines, multiple orthogonal methods","pmids":["20026641"],"is_preprint":false},{"year":2010,"finding":"PTK6 forms a complex with IGF-1R and IRS-1 adaptor protein, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation.","method":"siRNA screen, reverse-phase protein arrays, co-immunoprecipitation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP validation of complex, supported by proteomic array data, single lab","pmids":["20668531"],"is_preprint":false},{"year":2010,"finding":"STAP-2 interacts with both BRK and STAT3; STAP-2 PH domain is required for BRK–STAP-2 binding, BRK kinase activation, and STAT3 tyrosine phosphorylation/activation; a STAP-2 PH-Brk fusion protein exhibits robust kinase activity and enhanced STAT3 activation.","method":"Co-immunoprecipitation, domain deletion mapping, siRNA knockdown, STAT3 activation assays, fusion protein kinase activity","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping plus functional assays, siRNA confirms endogenous relevance, single lab","pmids":["20929863"],"is_preprint":false},{"year":2010,"finding":"PTK6 phosphorylates ARAP1 at Y231 in an EGF/EGFR-dependent manner via the PTK6 SH2 domain (R105 residue); phosphorylated ARAP1 inhibits EGFR down-regulation, thereby sustaining EGFR signaling. PTK6 silencing in breast cancer cells decreases EGFR levels.","method":"Co-immunoprecipitation, MALDI-TOF mass spectrometry identification, site-directed mutagenesis (Y231F, R105A), EGFR degradation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — MS identification, mutagenesis of both kinase and substrate, functional EGFR degradation assay, single lab multiple orthogonal methods","pmids":["20554524"],"is_preprint":false},{"year":2011,"finding":"STAP-2 mediates BRK-induced STAT5 phosphorylation and activation in T47D breast cancer cells; the STAP-2 PH domain participates in BRK-mediated STAT5 phosphorylation; knockdown of STAP-2 reduces proliferation as strongly as BRK or STAT5b knockdown.","method":"Co-transfection, STAT5 transcriptional reporter, siRNA knockdown, proliferation assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping, siRNA knockdown with functional readout, single lab","pmids":["21205088"],"is_preprint":false},{"year":2011,"finding":"ALT-PTK6, an alternatively spliced form lacking exon 2, associates with Sam68 and beta-catenin via its SH3 domain (demonstrated by GST pull-down); coexpression of ALT-PTK6 with full-length PTK6 suppresses PTK6 kinase activity, reduces PTK6 association with phosphotyrosine proteins, and enhances PTK6-mediated inhibition of beta-catenin/TCF transcription by promoting nuclear PTK6 function.","method":"RT-PCR, GST pull-down, co-immunoprecipitation, luciferase reporter assay, inducible expression system, proliferation/colony assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pull-down plus reporter assay plus inducible expression, single lab, multiple functional readouts","pmids":["21479203"],"is_preprint":false},{"year":2011,"finding":"BRK mediates p38 MAPK phosphorylation downstream of EGF signaling in mammary epithelial cells; BRK knockdown blocks EGF-stimulated p38 signaling; BRK overexpression in mammary gland causes delayed involution associated with activated p38 MAPK.","method":"WAP-Brk transgenic mouse model, siRNA knockdown, Western blotting, IHC","journal":"Breast cancer research : BCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic mouse model plus knockdown in cell lines with mechanistic signaling readout, single lab","pmids":["21923922"],"is_preprint":false},{"year":2012,"finding":"BRK interacts with EGFR, inhibits ligand-induced EGFR degradation by uncoupling activated EGFR from c-Cbl-mediated ubiquitination, and directly phosphorylates EGFR Y845 in the kinase domain to potentiate EGFR kinase activity.","method":"Co-immunoprecipitation, ubiquitination assay, in vitro kinase assay (phospho-Y845), EGFR degradation assay, siRNA knockdown","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay identifying specific phosphosite, ubiquitination assay, co-IP, multiple mechanistic steps validated, single lab but multiple orthogonal experiments","pmids":["22231447"],"is_preprint":false},{"year":2012,"finding":"BRK is a critical downstream effector of Met/HGF signaling and is required for HGF-induced cell migration in breast cancer cells.","method":"siRNA knockdown of BRK with HGF stimulation, cell migration assay","journal":"Hormones & cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single cell migration assay, review article with limited primary data, single lab","pmids":["22124844"],"is_preprint":false},{"year":2013,"finding":"PTP1B dephosphorylates BRK/PTK6 directly and also dephosphorylates IGF-1R β-subunit; BRK physically interacts with IGF-1R β-subunit. Restoration of PTP1B in ovarian cancer cells attenuates PTK6 and IGF-1R signaling, activating BAD to induce apoptosis.","method":"Stable PTP1B re-expression, co-immunoprecipitation (PTK6-IGF-1R), phosphorylation assays, apoptosis assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating physical interaction, enzymatic dephosphorylation shown, multiple functional readouts, single lab","pmids":["23814047"],"is_preprint":false},{"year":2013,"finding":"Active PTK6 in prostate cancer cells localizes to the plasma membrane; membrane-targeted active PTK6 promotes EMT partly by enhancing AKT activation and stimulates cancer cell migration and metastasis in xenograft models; in PTEN-null mouse prostate, endogenous active PTK6 localizes to membranes with decreased E-cadherin.","method":"Subcellular fractionation, targeted PTK6 constructs (membrane/cytoplasm), siRNA knockdown, AKT phosphorylation assays, xenograft mouse model, conditional Pten-knockout mouse","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple model systems (cell lines, xenografts, conditional KO mouse), mechanistic signaling (AKT), fractionation showing active PTK6 membrane localization, single lab with orthogonal approaches","pmids":["23856248"],"is_preprint":false},{"year":2013,"finding":"AKT, p130CAS, and FAK are identified as PTK6 substrates by mass spectrometry and validated by in vitro phosphorylation assays; these substrates promote cell proliferation, migration, and anoikis resistance downstream of cytoplasmic/membrane-localized PTK6 in prostate cancer.","method":"Mass spectrometry substrate identification, in vitro kinase assay validation, siRNA knockdown, targeted PTK6 expression constructs","journal":"European journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification plus in vitro validation, functional assays, single lab","pmids":["23398121"],"is_preprint":false},{"year":2013,"finding":"PTK6 phosphorylates and degrades c-Cbl at Y700, Y731, and Y774 in the C-terminal domain; phosphorylated c-Cbl undergoes auto-ubiquitination and proteasomal degradation, thereby reducing c-Cbl-mediated ubiquitination of substrates.","method":"Co-immunoprecipitation, site-directed mutagenesis of c-Cbl phosphosites, ubiquitination assay, proteasome inhibitor experiments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — phosphosite mapping by mutagenesis, ubiquitination assay, proteasome dependence shown, single lab","pmids":["23352614"],"is_preprint":false},{"year":2014,"finding":"BRK interacts with and phosphorylates Dok1 specifically at Y362; this phosphorylation leads to ubiquitin-proteasome-mediated Dok1 degradation, thereby removing a tumor suppressor and promoting cell proliferation and migration.","method":"Co-immunoprecipitation, in vitro kinase assay, phosphosite mapping (Y362), ubiquitination assay, proteasome inhibitor rescue","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with site-specific mapping, ubiquitination assay, functional consequences, single lab","pmids":["24523872"],"is_preprint":false},{"year":2014,"finding":"BRK phosphorylates KAP3A (kinesin-associated protein 3A) at C-terminal tyrosine residues in vivo; BRK-mediated phosphorylation induces delocalization of KAP3A from punctate nuclear localization to diffuse nucleo-cytoplasmic pattern; KAP3A knockdown suppresses BRK-induced cell migration.","method":"High-density protein filter array kinase screen, in vivo phosphorylation assay, co-immunoprecipitation, subcellular localization imaging, siRNA knockdown, migration assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo phosphorylation shown, localization change documented, functional migration assay, single lab","pmids":["18077133"],"is_preprint":false},{"year":2015,"finding":"BRK phosphorylates p27KIP1 at Y88 via an SH3-PXXP interaction; pY88-p27 activates cyclin D-cdk4 activity. An alternatively spliced form of BRK (Alt Brk) containing only the SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor.","method":"SH3-PXXP interaction screen, in vitro kinase assay, pY88 phosphorylation assay in breast cancer cells, modulation of BRK levels in cells, cdk4 activity assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus cellular phosphorylation, cdk4 functional assay, domain mapping, single lab","pmids":["25733683"],"is_preprint":false},{"year":2015,"finding":"Cancer-associated somatic mutations in BRK SH3 (L16F), SH2 (R131L), and C-terminal tail (P450L) domains activate BRK by disrupting intramolecular autoinhibitory interactions; two other mutations eliminate enzymatic activity; mutations differentially affect substrate recognition and phosphorylation.","method":"Recombinant protein expression, in vitro kinase assays, mutagenesis panel, substrate peptide assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with multiple cancer-associated mutants tested, mechanistic interpretation of autoinhibitory domain disruption, single lab","pmids":["25940761"],"is_preprint":false},{"year":2016,"finding":"PTK6 downregulation restores E-cadherin levels via proteasome-dependent degradation of the E-cadherin repressor SNAIL; kinase-active PTK6 suppresses E-cadherin expression and promotes EMT markers; PTK6 inhibition impairs metastatic lung colonization in vivo through anoikis induction.","method":"siRNA knockdown, PTK6 kinase inhibitor, E-cadherin/SNAIL Western blotting, proteasome inhibitor rescue, anoikis assay, in vivo lung colonization assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple loss-of-function approaches (siRNA + kinase inhibitor), proteasome-dependence demonstrated, in vivo metastasis model, single lab but orthogonal methods","pmids":["27302163"],"is_preprint":false},{"year":2016,"finding":"PTK6 interacts with JAK2 and phosphorylates it to activate JAK2/STAT3 signaling, thereby promoting cancer cell stemness in colorectal cancer. Pharmacological inhibition of PTK6 with XMU-MP-2 reduces stemness and chemosensitivity in vivo.","method":"Co-immunoprecipitation (PTK6-JAK2), FLAG-tagged PTK6 mutant constructs (kinase-dead, inhibition-defective), STAT3 activation assays, in vivo xenograft/PDX models","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus kinase-dead mutant, in vivo validation, single lab","pmids":["34551797"],"is_preprint":false},{"year":2017,"finding":"PTEN protein phosphatase activity directly dephosphorylates PTK6 at activation loop Y342 (but not autoinhibitory Y447), inhibiting PTK6 activity with efficiency similar to PTP1B. In PTEN-null prostate cancer, conditional Pten disruption increases PTK6 Y342 phosphorylation and tumorigenesis; Ptk6 disruption impairs PTEN-loss-induced tumorigenesis.","method":"PTEN mutant constructs (phosphatase-dead), phospho-Y342 and Y447 Western blotting, PTEN knockdown/re-expression, conditional Pten-knockout mouse prostate model, Ptk6/Pten double-KO mice, human prostate tumor tissue microarrays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical phosphatase assay with mutant PTEN constructs, multiple genetic mouse models (conditional KO, double KO), human tissue validation; replicated across systems","pmids":["29142193"],"is_preprint":false},{"year":2017,"finding":"Plasma membrane-localized PTK6 phosphorylates Eps8 at Y497, Y524, and Y534; Eps8 3YF phosphorylation-defective mutant reverts PTK6-mediated increases in proliferation, migration, ERK phosphorylation, and FAK phosphorylation; EGF-induced Eps8 phosphorylation in T-47D cells depends on endogenous PTK6.","method":"Proteomics screen with Myr-PTK6, site-directed mutagenesis (Eps8 3YF), kinase assays, siRNA knockdown in breast cancer cells, proliferation/migration/adhesion assays","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphosite-specific mutagenesis with functional rescue, endogenous validation by knockdown, single lab","pmids":["28214294"],"is_preprint":false},{"year":2018,"finding":"Crystal structures of PTK6 kinase domain were solved in complex with Dasatinib (2.24 Å) and a novel IPA compound (1.70 Å); both structures are in DFG-in, αC-helix-out conformation with inhibitors at the ATP-binding pocket; Dasatinib occupies an additional pocket near the gatekeeper explaining its higher potency.","method":"X-ray crystallography (co-crystal structures of PTK6-KD with inhibitors), structure determination and refinement","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure at high resolution with two distinct inhibitor complexes, structural basis for potency differences determined","pmids":["27993680"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of apo-PTK6 and inhibitor-bound PTK6 complexes were determined; PTK6 kinase inhibitors moderately suppress tumor cell growth in 2D/3D culture but growth inhibition does not correlate with PTK6 kinase activity inhibition or PTK6 protein levels (>500-fold shift between kinase IC50 and growth inhibition IC50 in PTK6-overexpressing cells), indicating PTK6 kinase activity is not the primary driver of tumor growth.","method":"Crystal structure determination (apo and inhibitor-bound), selective PTK6 inhibitors with kinase assays, 2D/3D cell growth assays, PTK6 overexpression in engineered breast tumor cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structures plus selective inhibitors and engineered cell lines; however, the finding is a negative/dissociative result about kinase-independent growth promotion","pmids":["29879184"],"is_preprint":false},{"year":2019,"finding":"BRK phosphorylates SMAD4 leading to its recognition by the ubiquitin-proteasome system and accelerated degradation; activated BRK (Y447F mutant) mediates SMAD4 degradation associated with repression of tumor suppressor FRK and increased expression of SNAIL and SLUG. BRK competitively binds SMAD4.","method":"Co-immunoprecipitation, in vitro phosphorylation assay with constitutively active BRK-Y447F, ubiquitination assay, proteasome inhibitor rescue, knockdown experiments","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, ubiquitination assay, mutagenesis, single lab with multiple mechanistic assays","pmids":["31681835"],"is_preprint":false},{"year":2019,"finding":"PSPC1 is a nuclear substrate of PTK6; PSPC1 sequesters PTK6 in the nucleus and inhibits its metastasis-promoting function. PSPC1 upregulation or PSPC1-Y523F mutation promotes cytoplasmic translocation of active PTK6 and nuclear translocation of beta-catenin, driving EMT and metastasis via augmented Wnt3a autocrine signaling. PSPC1-CT131 acts as a dual inhibitor of PSPC1 and PTK6.","method":"Co-immunoprecipitation (PSPC1-PTK6, PSPC1-beta-catenin), site-directed mutagenesis (Y523F), subcellular localization imaging, HCC orthotopic mouse model, beta-catenin/Wnt reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, phosphorylation mutant, in vivo orthotopic model, mechanistic pathway (Wnt/beta-catenin) validated, multiple orthogonal methods","pmids":["31844057"],"is_preprint":false},{"year":2020,"finding":"The PTK6 SH2 domain (not the kinase domain) mediates cell motility in TNBC via activation of RhoA and aryl hydrocarbon receptor (AhR) signaling pathways; the SH3 domain contributes to spheroid formation via p38 MAPK. Kinase activity is dispensable for cell migration but contributes to anchorage-independent colony formation.","method":"In-frame domain deletion mutants (SH2-del, SH3-del, kinase-dead), reverse-phase protein arrays, RhoA/AhR inhibition, primary breast tumor organoids","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutants with functional assays, RPPA pathway analysis, organoid model, single lab","pmids":["33172975"],"is_preprint":false},{"year":2012,"finding":"Hsp90 interacts with the PTK6 tyrosine kinase catalytic domain (interaction is independent of catalytic activity) and protects PTK6 from proteasomal degradation. Geldanamycin (Hsp90 inhibitor) decreases PTK6 protein via proteasome-dependent degradation and increases PTK6 interaction with the E3 ligase CHIP; CHIP overexpression promotes PTK6 proteasomal degradation.","method":"Co-immunoprecipitation, geldanamycin treatment, proteasome inhibitor rescue, CHIP overexpression, siRNA knockdown of CHIP","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP identifying Hsp90-PTK6 interaction, pharmacological and genetic manipulation confirming proteasomal pathway, single lab","pmids":["22849407"],"is_preprint":false},{"year":2013,"finding":"HER2 stabilizes BRK protein by upregulating calpastatin (an endogenous calpain inhibitor) via MEK signaling, thereby inhibiting calpain-1-mediated proteolytic degradation of BRK. HER2 knockdown downregulates calpastatin, increasing calpain-1 activity and BRK degradation.","method":"HER2 overexpression/siRNA knockdown, BRK stability assays, calpain activity assay, calpastatin expression analysis, MEK inhibition, constitutively active MEK rescue","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic chain (HER2→MEK→calpastatin→calpain→BRK stability) supported by multiple inhibitor and rescue experiments, single lab","pmids":["23707532"],"is_preprint":false},{"year":2014,"finding":"Under hypoxic conditions, PTK6 is rapidly stabilized (in a HIF-1α-independent manner) associated with decreased ubiquitylation; c-Cbl is identified as a putative PTK6 E3 ubiquitin ligase in normoxia. Hypoxia-induced PTK6 stabilization and membrane relocalization is associated with increased cell motility and invasion.","method":"Hypoxia treatment, HIF-1α knockdown, ubiquitylation assay, c-Cbl co-immunoprecipitation, subcellular fractionation, cell motility/invasion assays","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitylation assay, co-IP, functional motility assays, HIF-1 independence demonstrated by knockdown, single lab","pmids":["25019382"],"is_preprint":false},{"year":2019,"finding":"Vemurafenib (PLX4032/PLX4720) binds in the active site of PTK6 as demonstrated by saturation transfer difference NMR and molecular docking; vemurafenib inhibits PTK6-mediated signaling through FAK, EGFR, and ERK1/2 and blocks PTK6-dependent cell growth, migration, and invasion in prostate cancer models.","method":"Saturation transfer difference NMR, molecular docking, kinase assays, signaling Western blots, xenograft model","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR-based structural binding study plus functional validation in cells and in vivo xenograft, multiple orthogonal methods","pmids":["30926642"],"is_preprint":false},{"year":2025,"finding":"PTK6 physically interacts with HNRNPH1 and phosphorylates it at Y210, promoting HNRNPH1 liquid-liquid phase separation (LLPS) and formation of biomolecular condensates; HNRNPH1 LLPS triggers splicing switching of NBR1 exon 10 inclusion, activating autophagy and suppressing apoptosis in colorectal cancer.","method":"Co-immunoprecipitation (PTK6-HNRNPH1), Y210 phosphorylation assay, FRAP (LLPS validation), NBR1 splicing assay, autophagy assays, patient-derived organoid and CDX models with PTK6 inhibitor tilfrinib","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phosphosite identification, FRAP confirmation of LLPS, splicing assay, in vivo organoid/CDX models; multiple orthogonal methods in one study","pmids":["40103198"],"is_preprint":false}],"current_model":"PTK6/BRK is a non-receptor intracellular tyrosine kinase with SH3, SH2, and catalytic domains that is maintained in an autoinhibited state via intramolecular SH3–linker and SH2–pY447 interactions; activation at Y342 in the activation loop promotes its kinase activity, which is negatively regulated by PTEN (dephosphorylating Y342) and positively regulated by ErbB2 interaction; PTK6 phosphorylates a broad set of nuclear substrates (Sam68, SLM-1/2, PSF, HNRNPH1) to suppress RNA-binding and regulate splicing/phase separation, and cytoplasmic/membrane substrates (paxillin, beta-catenin, AKT, FAK, p130CAS, Eps8, ARAP1, c-Cbl, Dok1, SMAD4, JAK2, p27KIP1) to promote cell migration, proliferation, survival, EMT, and oncogenic signaling; its subcellular localization (nuclear vs. cytoplasmic/membrane) is a critical determinant of function, with nuclear PTK6 suppressing growth and beta-catenin/TCF transcription and membrane-localized PTK6 driving oncogenic phenotypes."},"narrative":{"mechanistic_narrative":"PTK6 (BRK) is an intracellular non-receptor tyrosine kinase that integrates growth-factor signaling with control of cell proliferation, migration, survival, and epithelial-mesenchymal transition, with its subcellular localization acting as the key switch between tumor-suppressive and oncogenic outputs [PMID:19304789, PMID:20026641, PMID:23856248]. The kinase is held in an autoinhibited conformation by intramolecular contacts: an SH3-domain interaction with the SH2-kinase linker (via W44 and linker prolines P175/P177/P179) and an SH2-domain engagement of C-terminal phospho-Y447, with activation requiring autophosphorylation of the activation-loop Y342 [PMID:12121988, PMID:17822667]. This autoinhibition is biologically reinforced and reversed by opposing phosphatases and partners: PTEN and PTP1B directly dephosphorylate the activating Y342 to restrain PTK6, whereas ErbB2/HER2 interaction increases its intrinsic kinase activity and stabilizes the protein [PMID:29142193, PMID:23814047, PMID:18719096, PMID:23707532]. Cancer-associated mutations in the SH3, SH2, and C-terminal tail disrupt these autoinhibitory contacts and constitutively activate the enzyme [PMID:25940761]. In the nucleus, PTK6 phosphorylates RNA-binding proteins—Sam68, SLM-1/2, PSF, and HNRNPH1—to suppress their RNA-binding and redirect splicing and phase separation, and it antagonizes beta-catenin/TCF transcription [PMID:10913193, PMID:15471878, PMID:19439179, PMID:40103198, PMID:20026641]. At the membrane and in the cytoplasm, PTK6 phosphorylates paxillin, Eps8, AKT, FAK, p130CAS, ARAP1, and beta-catenin and degrades the c-Cbl, Dok1, and SMAD4 tumor suppressors to drive motility, EGFR signaling sustainment, proliferation, survival, and EMT [PMID:15572663, PMID:28214294, PMID:23398121, PMID:20554524, PMID:22231447, PMID:23352614, PMID:24523872, PMID:31681835]. Distinct domains carry distinct functions independent of catalysis—the SH2 domain mediates motility and the SH3 domain mediates spheroid formation—and kinase activity is dispensable for several growth-promoting phenotypes [PMID:33172975, PMID:29879184]. PTK6 promotes EMT and metastasis by stabilizing SNAIL and repressing E-cadherin, and its nuclear retention by partners such as PSPC1 suppresses these metastatic functions [PMID:27302163, PMID:31844057]. PTK6 is also druggable, with crystal structures and selective inhibitors defining its ATP pocket [PMID:27993680].","teleology":[{"year":1994,"claim":"Established PTK6 as a novel SH3/SH2-containing non-receptor tyrosine kinase capable of autophosphorylation, defining its basic enzymatic identity.","evidence":"Recombinant expression in baculovirus and bacterial systems with autophosphorylation assay","pmids":["8036022"],"confidence":"Medium","gaps":["No cellular substrates identified","No regulatory mechanism for activity defined"]},{"year":1996,"claim":"Separated the catalytic requirement from a regulatory tyrosine, showing kinase activity (K219) is essential for transformation while the C-terminal Y447 modulates transforming potential without abolishing catalysis.","evidence":"Site-directed mutagenesis with transformation assays in mammary epithelial cells","pmids":["8940083"],"confidence":"Medium","gaps":["Mechanism by which Y447 regulates activity not resolved","No structural basis defined"]},{"year":2002,"claim":"Defined the molecular logic of PTK6 activation, identifying Y342 activation-loop autophosphorylation as activating and Y447–SH2 and SH3 contacts as autoinhibitory.","evidence":"Baculovirus expression, mass spectrometry phosphosite mapping, mutagenesis, and in vitro kinetics","pmids":["12121988"],"confidence":"High","gaps":["Upstream activators in cells not identified","Structural model of autoinhibited state not solved"]},{"year":2007,"claim":"Mapped the SH3–linker autoinhibitory contact at residue level (W44 with linker prolines), explaining how intramolecular conformation restrains catalysis.","evidence":"Mutagenesis, surface plasmon resonance, kinase assays, and structural modeling","pmids":["17822667"],"confidence":"High","gaps":["No full-length crystal structure of autoinhibited PTK6","Physiological trigger of release unclear"]},{"year":2000,"claim":"Identified nuclear RNA-binding proteins as the first physiological substrates, showing PTK6 phosphorylates Sam68 to abolish its RNA binding.","evidence":"Reciprocal co-IP, domain mapping, and functional RNA-binding assays across cell lines","pmids":["10913193"],"confidence":"High","gaps":["In vivo relevance of Sam68 regulation not yet established","Downstream splicing consequences not mapped"]},{"year":2003,"claim":"Extended the Sam68 model to a functional consequence, showing PTK6-mediated hyperphosphorylation impairs Sam68-dependent intron-containing RNA utilization.","evidence":"Co-expression in 293T cells with RNA utilization assays and RNA-binding-defective Sam68 mutant","pmids":["12482964"],"confidence":"Medium","gaps":["Endogenous target transcripts not identified","Physiological setting untested"]},{"year":2004,"claim":"Broadened the nuclear substrate repertoire and linked substrate phosphorylation to PTK6 nuclear retention, while demonstrating the SH3 domain dominates substrate recognition.","evidence":"Phosphorylation and RNA-binding assays for SLM-1/2 with selectivity controls; peptide kinetics linking SH3 to substrate Km","pmids":["15471878","14676834"],"confidence":"High","gaps":["Determinants of nuclear vs cytoplasmic partitioning incompletely defined"]},{"year":2004,"claim":"Established the cytoplasmic migratory arm of PTK6 function, identifying paxillin phosphorylation driving Rac1 activation and cell motility upon EGF stimulation.","evidence":"Co-IP, in vitro kinase assay with phosphosite mapping, Rac1 activation, migration/invasion assays, and confocal localization","pmids":["15572663"],"confidence":"High","gaps":["In vivo migration relevance not addressed","Mechanism of membrane translocation not defined"]},{"year":2000,"claim":"Connected PTK6 to growth-factor receptor signaling through adaptor proteins, identifying STAP-2/BKS and ErbB3/PI3K/AKT potentiation.","evidence":"Yeast two-hybrid, co-IP, and signaling assays (phospho-erbB3, PI3K recruitment, AKT)","pmids":["10980601","11114724"],"confidence":"Medium","gaps":["Endogenous significance of adaptor interactions limited","Pathway integration with other receptors unclear"]},{"year":2009,"claim":"Demonstrated that PTK6 subcellular localization dictates opposing biological outcomes, with membrane-targeted PTK6 oncogenic and nuclear-targeted PTK6 growth-suppressive.","evidence":"Engineered Myr/NLS targeting constructs with functional and phosphoproteome profiling","pmids":["19304789"],"confidence":"Medium","gaps":["Mechanism controlling localization in cells not resolved","Distinct substrate sets only partially characterized"]},{"year":2009,"claim":"Established PTK6 as a direct negative regulator of beta-catenin/TCF transcription, phosphorylating beta-catenin and showing localization-dependent transcriptional effects in vivo.","evidence":"In vitro kinase assay with phosphosite mapping, reporter assays, siRNA, and Ptk6-null BAT-GAL reporter mice","pmids":["20026641"],"confidence":"High","gaps":["Mechanism linking phosphorylation to TCF/TLE repressor levels incompletely defined"]},{"year":2009,"claim":"Defined STAP-2 and PSF/cell-cycle nodes as PTK6 effectors, linking the kinase to STAT3 activation and to cell cycle arrest via impaired PSF RNA binding.","evidence":"Phosphosite-specific antibody and siRNA for STAP-2 Y250; proteomic MS, phosphosite mapping, and cell cycle analysis for PSF","pmids":["19393627","19439179"],"confidence":"High","gaps":["Reconciliation of growth-suppressive (PSF) and growth-promoting outputs not addressed"]},{"year":2008,"claim":"Connected PTK6 amplification to HER2/ErbB2-driven breast cancer, showing ErbB2 activates PTK6 and PTK6 confers lapatinib resistance.","evidence":"Co-IP, FISH, 3D acini, WAP-Brk transgenic mice, and kinase assays","pmids":["18719096"],"confidence":"High","gaps":["Direct phosphorylation events downstream of the ErbB2-PTK6 interaction not all mapped"]},{"year":2012,"claim":"Revealed that PTK6 sustains EGFR signaling by directly phosphorylating EGFR Y845 and uncoupling it from c-Cbl-mediated degradation.","evidence":"Co-IP, ubiquitination assay, in vitro kinase assay for phospho-Y845, and EGFR degradation assay","pmids":["22231447"],"confidence":"High","gaps":["In vivo contribution to EGFR-driven tumors not quantified"]},{"year":2013,"claim":"Established phosphatase control of PTK6 (PTP1B) and direct degradation of tumor suppressors c-Cbl, situating PTK6 as a hub that destabilizes negative regulators.","evidence":"PTP1B re-expression with dephosphorylation/apoptosis assays; c-Cbl phosphosite mutagenesis and ubiquitination/proteasome assays","pmids":["23814047","23352614"],"confidence":"Medium","gaps":["Relative contribution of c-Cbl degradation versus EGFR uncoupling unclear"]},{"year":2013,"claim":"Defined the membrane-localized active PTK6 pool as the metastatic driver in prostate cancer, identifying AKT/FAK/p130CAS substrates and EMT promotion in PTEN-null models.","evidence":"Subcellular fractionation, targeted constructs, MS substrate ID with in vitro validation, xenografts, and conditional Pten-knockout mice","pmids":["23856248","23398121"],"confidence":"High","gaps":["Trigger relocalizing active PTK6 to the membrane not fully defined"]},{"year":2012,"claim":"Defined PTK6 protein stability control through Hsp90/CHIP and HER2-MEK-calpastatin-calpain axes, explaining how PTK6 levels are post-translationally maintained.","evidence":"Co-IP, geldanamycin and proteasome inhibitor experiments, CHIP manipulation; HER2/MEK manipulation with calpain activity and stability assays","pmids":["22849407","23707532"],"confidence":"Medium","gaps":["Quantitative balance between proteasomal and calpain pathways in vivo unknown"]},{"year":2014,"claim":"Showed PTK6 is stabilized under hypoxia with membrane relocalization and motility induction, and degrades the Dok1 tumor suppressor, reinforcing pro-invasive control.","evidence":"Hypoxia/HIF-1α knockdown with ubiquitylation and motility assays; Dok1 Y362 mutagenesis with ubiquitination/proteasome rescue","pmids":["25019382","24523872"],"confidence":"Medium","gaps":["Mechanism of hypoxic stabilization not molecularly defined"]},{"year":2015,"claim":"Defined PTK6 cell-cycle promotion via p27KIP1 Y88 phosphorylation activating cdk4, and showed cancer mutations activate PTK6 by disrupting autoinhibition.","evidence":"SH3-PXXP interaction screen and in vitro kinase/cdk4 assays for p27; recombinant mutant panel with kinetics for cancer mutations","pmids":["25733683","25940761"],"confidence":"High","gaps":["Frequency and clinical impact of activating mutations not addressed"]},{"year":2016,"claim":"Linked PTK6 to EMT through SNAIL stabilization and E-cadherin suppression, with PTK6 inhibition reducing metastatic colonization by inducing anoikis.","evidence":"siRNA and kinase inhibitor, SNAIL/E-cadherin Western with proteasome rescue, anoikis assay, and in vivo lung colonization","pmids":["27302163"],"confidence":"High","gaps":["Direct PTK6 substrate mediating SNAIL stabilization not pinpointed"]},{"year":2017,"claim":"Identified PTEN as a direct PTK6 Y342 phosphatase and a genetic suppressor of PTK6-driven tumorigenesis, and identified Eps8 as a membrane-PTK6 substrate driving proliferation/migration.","evidence":"Phosphatase-dead PTEN constructs, phospho-Y342/Y447 blots, conditional and double-KO mouse prostate models, human tissue; Eps8 phosphosite mutagenesis with functional rescue","pmids":["29142193","28214294"],"confidence":"High","gaps":["How PTEN loss specifically redirects PTK6 to membranes not fully mechanistic"]},{"year":2019,"claim":"Showed PTK6 degrades SMAD4 and is sequestered by PSPC1 in the nucleus, defining a switch governing EMT/metastasis via Wnt/beta-catenin.","evidence":"Co-IP, in vitro phosphorylation with active BRK-Y447F, ubiquitination assays; PSPC1 Y523F mutagenesis with orthotopic HCC model and Wnt reporters","pmids":["31681835","31844057"],"confidence":"High","gaps":["General applicability of PSPC1 sequestration across tissues untested"]},{"year":2020,"claim":"Dissociated PTK6 functions from catalysis, showing the SH2 domain drives motility via RhoA/AhR and the SH3 domain drives spheroid formation via p38, with kinase activity dispensable for migration.","evidence":"Domain deletion mutants, RPPA, RhoA/AhR inhibition, and primary breast tumor organoids","pmids":["33172975"],"confidence":"Medium","gaps":["Mechanism by which the SH2 domain activates RhoA not defined","Scaffolding partners mediating kinase-independent functions unidentified"]},{"year":2018,"claim":"Provided structural and pharmacological tools, solving PTK6 kinase-domain structures with inhibitors and revealing that growth inhibition often does not track with kinase inhibition.","evidence":"X-ray crystallography of inhibitor complexes and selective inhibitors with 2D/3D growth assays in engineered cells","pmids":["27993680","29879184"],"confidence":"High","gaps":["Kinase-independent growth-promoting activity not molecularly explained","Structure of full-length/autoinhibited enzyme unsolved"]},{"year":2019,"claim":"Demonstrated repurposed inhibitors (vemurafenib) and JAK2 as a substrate, showing PTK6 activates JAK2/STAT3 to promote cancer stemness and is pharmacologically targetable.","evidence":"STD-NMR/docking and signaling/xenograft assays for vemurafenib; co-IP and kinase-dead mutants with PDX models for JAK2","pmids":["30926642","34551797"],"confidence":"Medium","gaps":["Selectivity of repurposed inhibitors for PTK6 in vivo uncertain"]},{"year":2025,"claim":"Extended PTK6's RNA-regulatory role to phase separation, showing PTK6 phosphorylates HNRNPH1 to drive condensate formation and NBR1 splicing that activates autophagy and suppresses apoptosis.","evidence":"Co-IP, Y210 phosphorylation, FRAP, NBR1 splicing/autophagy assays, and organoid/CDX models with the inhibitor tilfrinib","pmids":["40103198"],"confidence":"High","gaps":["Generality of LLPS-driven splicing control across PTK6 RNA-binding substrates untested"]},{"year":null,"claim":"The molecular mechanism that determines PTK6's nuclear versus membrane partitioning in cells, and how kinase-independent SH2/SH3 scaffolding functions integrate with catalytic substrate phosphorylation, remain unresolved.","evidence":"Open question synthesized across localization, domain, and kinase-independence findings","pmids":[],"confidence":"Medium","gaps":["No defined cellular signal controlling localization switch","Kinase-independent functions lack identified scaffolding mechanism","No full-length structure rationalizing autoinhibition and partner engagement"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,10,17,20,24,28,29,30,37,40,47]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,5,8,24]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,20,24]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,9,14,17,41]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,15,27,37,45]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,15,27,28]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,10,20,24,27]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,9,14,47]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[13,32]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[27,34,41]}],"complexes":[],"partners":["ERBB2","EGFR","STAP2","SAM68","JAK2","PTEN","PSPC1","IGF1R"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13882","full_name":"Protein-tyrosine kinase 6","aliases":["Breast tumor kinase","Tyrosine-protein kinase BRK"],"length_aa":451,"mass_kda":51.8,"function":"Non-receptor tyrosine-protein kinase implicated in the regulation of a variety of signaling pathways that control the differentiation and maintenance of normal epithelia, as well as tumor growth. Function seems to be context dependent and differ depending on cell type, as well as its intracellular localization. A number of potential nuclear and cytoplasmic substrates have been identified. These include the RNA-binding proteins: KHDRBS1/SAM68, KHDRBS2/SLM1, KHDRBS3/SLM2 and SFPQ/PSF; transcription factors: STAT3 and STAT5A/B and a variety of signaling molecules: ARHGAP35/p190RhoGAP, PXN/paxillin, BTK/ATK, STAP2/BKS. Phosphorylates the GTPase-activating protein ARAP1 following EGF stimulation which enhances EGFR signaling by delaying EGFR down-regulation (PubMed:20554524). Also associates with a variety of proteins that are likely upstream of PTK6 in various signaling pathways, or for which PTK6 may play an adapter-like role. These proteins include ADAM15, EGFR, ERBB2, ERBB3 and IRS4. In normal or non-tumorigenic tissues, PTK6 promotes cellular differentiation and apoptosis. In tumors PTK6 contributes to cancer progression by sensitizing cells to mitogenic signals and enhancing proliferation, anchorage-independent survival and migration/invasion. Association with EGFR, ERBB2, ERBB3 may contribute to mammary tumor development and growth through enhancement of EGF-induced signaling via BTK/AKT and PI3 kinase. Contributes to migration and proliferation by contributing to EGF-mediated phosphorylation of ARHGAP35/p190RhoGAP, which promotes association with RASA1/p120RasGAP, inactivating RhoA while activating RAS. EGF stimulation resulted in phosphorylation of PNX/Paxillin by PTK6 and activation of RAC1 via CRK/CrKII, thereby promoting migration and invasion. PTK6 activates STAT3 and STAT5B to promote proliferation. Nuclear PTK6 may be important for regulating growth in normal epithelia, while cytoplasmic PTK6 might activate oncogenic signaling pathways Inhibits PTK6 phosphorylation and PTK6 association with other tyrosine-phosphorylated proteins","subcellular_location":"Cytoplasm; Nucleus; Cell projection, ruffle; Membrane","url":"https://www.uniprot.org/uniprotkb/Q13882/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PTK6","classification":"Not Classified","n_dependent_lines":24,"n_total_lines":1208,"dependency_fraction":0.019867549668874173},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PTK6","total_profiled":1310},"omim":[{"mim_id":"607881","title":"SIGNAL-TRANSDUCING ADAPTOR PROTEIN 2; STAP2","url":"https://www.omim.org/entry/607881"},{"mim_id":"602004","title":"PROTEIN-TYROSINE KINASE 6; PTK6","url":"https://www.omim.org/entry/602004"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"esophagus","ntpm":104.2},{"tissue":"skin 1","ntpm":51.4},{"tissue":"vagina","ntpm":41.0}],"url":"https://www.proteinatlas.org/search/PTK6"},"hgnc":{"alias_symbol":["BRK"],"prev_symbol":[]},"alphafold":{"accession":"Q13882","domains":[{"cath_id":"2.30.30.40","chopping":"14-72","consensus_level":"high","plddt":91.2547,"start":14,"end":72},{"cath_id":"3.30.505.10","chopping":"77-158","consensus_level":"high","plddt":89.9001,"start":77,"end":158},{"cath_id":"3.30.200.20","chopping":"189-266","consensus_level":"high","plddt":87.5042,"start":189,"end":266},{"cath_id":"1.10.510.10","chopping":"273-444","consensus_level":"high","plddt":92.8801,"start":273,"end":444}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13882","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13882-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13882-F1-predicted_aligned_error_v6.png","plddt_mean":88.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PTK6","jax_strain_url":"https://www.jax.org/strain/search?query=PTK6"},"sequence":{"accession":"Q13882","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13882.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13882/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13882"}},"corpus_meta":[{"pmid":"8036022","id":"PMC_8036022","title":"Cloning and characterisation of cDNAs encoding a novel non-receptor tyrosine kinase, brk, expressed in human breast tumours.","date":"1994","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8036022","citation_count":148,"is_preprint":false},{"pmid":"9266966","id":"PMC_9266966","title":"BRK tyrosine kinase expression in a high proportion of human breast carcinomas.","date":"1997","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/9266966","citation_count":136,"is_preprint":false},{"pmid":"10913193","id":"PMC_10913193","title":"Sik (BRK) phosphorylates Sam68 in the nucleus and negatively regulates its RNA binding ability.","date":"2000","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10913193","citation_count":135,"is_preprint":false},{"pmid":"15572663","id":"PMC_15572663","title":"Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin.","date":"2004","source":"Molecular and cellular 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oncology","url":"https://pubmed.ncbi.nlm.nih.gov/25241146","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47981,"output_tokens":11868,"usd":0.160981,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23059,"output_tokens":7254,"usd":0.148322,"stage2_stop_reason":"end_turn"},"total_usd":0.309303,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"BRK/PTK6 (brk) encodes a novel non-receptor tyrosine kinase with SH3 and SH2 domains that is capable of autophosphorylation on tyrosine residues when expressed in baculovirus and bacterial recombinant systems.\",\n      \"method\": \"Recombinant protein expression (baculovirus and bacterial systems), autophosphorylation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro autophosphorylation demonstrated in recombinant system, single lab, foundational characterization paper\",\n      \"pmids\": [\"8036022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Mutation of the catalytic lysine (K219M) abolishes BRK kinase activity and transformation capacity in mammary epithelial cells; mutation of the C-terminal inhibitory tyrosine (Y447F) decreases transforming potential without abolishing kinase activity, indicating Y447 plays a regulatory role distinct from catalysis.\",\n      \"method\": \"Site-directed mutagenesis, transformation assay in human mammary epithelial cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of key regulatory residues with functional readout, single lab, two mutants tested\",\n      \"pmids\": [\"8940083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BRK/Sik associates with the RNA-binding protein Sam68 through its SH3 and SH2 domains; the proline-rich P3 region of Sam68 is required for SH3 binding. BRK/Sik phosphorylates Sam68 in the nucleus (in Sam68-SLM nuclear bodies in HT29 cells and in nucleoplasm of NMuMG cells) and abolishes Sam68's ability to bind RNA and act as a cellular Rev homologue.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping (SH3/SH2 interaction studies), co-transfection, functional RNA-binding assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, domain mapping, functional RNA-binding assay, replicated across cell lines\",\n      \"pmids\": [\"10913193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BRK associates with and phosphorylates BKS (STAP-2), a novel adaptor protein with PH-like and SH2-like domains; association and phosphorylation are dependent on BRK catalytic activity and on the SH2-like domain of BKS. BKS also recruits an unidentified 100 kDa protein that becomes tyrosine-phosphorylated in the presence of BRK.\",\n      \"method\": \"Yeast two-hybrid screen, co-transfection, co-immunoprecipitation, phosphotyrosine detection\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP validation, catalytic dependence demonstrated, single lab\",\n      \"pmids\": [\"10980601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BRK expression in mammary epithelial cells enhances EGF-induced phosphorylation of erbB3, leading to increased recruitment of PI3-kinase to erbB3 and potentiated AKT activation, thereby enhancing mitogenic signaling.\",\n      \"method\": \"Stable transfection of BRK into mammary epithelial cells, Western blotting for phospho-erbB3, PI3K co-immunoprecipitation, AKT activity assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple signaling readouts (phospho-erbB3, PI3K recruitment, AKT activation), single lab, mechanistic chain established\",\n      \"pmids\": [\"11114724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BRK autophosphorylates within the activation loop (Y342) and at N-terminal sites as shown by mass spectrometry; activation loop autophosphorylation increases kinase activity (Y342A mutant is not activated). The C-terminal Y447 maintains autoinhibition via SH2 domain engagement (Y447F mutation increases activity and SH2 accessibility). The SH3 domain also contributes to autoinhibition, as a proline-rich peptide activates BRK.\",\n      \"method\": \"Baculovirus expression, mass spectrometry, site-directed mutagenesis, in vitro kinase assays with synthetic peptides (Km, kcat measurements), SH2 domain accessibility assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus mass spectrometry identification of phosphosites, mutagenesis, kinetic characterization; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"12121988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Sam68 hyperphosphorylation by activated Sik/BRK in the nucleus inhibits Sam68-mediated cytoplasmic utilization of intron-containing RNA in a dose-dependent manner. The RNA-binding defective Sam68 G178E mutant cannot enhance CTE function, confirming that BRK regulation of Sam68 RNA-binding activity is functionally important.\",\n      \"method\": \"Co-expression of Sik/BRK with Sam68 in 293T cells, RNA analysis, protein expression assays, Sam68 point mutant (G178E)\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assay of RNA utilization with mutant controls, dose-dependence shown, single lab\",\n      \"pmids\": [\"12482964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"BRK/Sik subcellular localization shifts from nuclear (normal/well-differentiated prostate epithelial cells) to cytoplasmic (poorly differentiated prostate cancer cells), and nuclear BRK is less active in cytoplasmic-localized PC3 cells; altered localization correlates with differentiation state.\",\n      \"method\": \"Immunohistochemistry of 58 prostate biopsy samples, subcellular fractionation, Western blotting of prostate cancer cell lines\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — fractionation plus IHC in tissue samples, localization linked to activity, single lab\",\n      \"pmids\": [\"12833144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"BRK SH3 domain mutations that disrupt intramolecular interactions increase BRK kinase activity; the SH3 domain plays the dominant role in substrate recognition. Bifunctional peptides coupling a substrate sequence to an SH3 ligand show significantly lower Km than controls, while SH2-binding substrate peptides show no difference.\",\n      \"method\": \"Site-directed mutagenesis, kinase activity assays in HEK293 cells, synthetic peptide substrate assays (Km measurements), Sam68 phosphorylation comparison\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assays with mutagenesis and quantitative kinetic measurements, mechanistic dissection of domain function, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"14676834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"BRK/Sik phosphorylates SLM-1 and SLM-2 (Sam68-like mammalian proteins) but not hnRNP K; phosphorylation inhibits their RNA-binding abilities and promotes nuclear retention of BRK/Sik.\",\n      \"method\": \"Co-transfection, phosphorylation assays, RNA-binding assay, subcellular localization studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — substrate specificity shown with selectivity control (hnRNP K not phosphorylated), functional RNA-binding assay, single lab\",\n      \"pmids\": [\"15471878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"BRK identifies paxillin as a binding partner and substrate; EGF stimulation activates BRK, which phosphorylates paxillin at Y31 and Y118, promoting Rac1 activation via CrkII, thereby driving cell motility and invasion. BRK translocates to membrane ruffles and colocalizes with paxillin during cell migration.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-specific phosphorylation mapping, Rac1 activation assay, cell migration and invasion assays, confocal microscopy\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with phosphosite mapping, Rac1 activation, cell functional assays, and localization; multiple orthogonal methods establishing full pathway\",\n      \"pmids\": [\"15572663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"BRK associates with IRS-4 via both its SH3 and SH2 domains in resting and IGF-1-stimulated cells; IRS-4 enhances IGF-1-induced BRK tyrosine phosphorylation; endogenous BRK and IRS-4 interact in A431 cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry identification, domain mapping, EGF/IGF-1 stimulation experiments\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus co-IP validation in multiple cell lines, domain mapping included, single lab\",\n      \"pmids\": [\"15870689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The BRK SH3 domain interacts with the SH2-kinase linker region via W44 in the SH3 domain and P175, P177, P179 in the linker; disruption of this interaction by mutagenesis markedly increases PTK6 catalytic activity, demonstrating this intramolecular contact maintains PTK6 in an autoinhibited conformation.\",\n      \"method\": \"Site-directed mutagenesis, surface plasmon resonance, kinase activity assays, 3D structural modeling\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SPR binding measurements plus mutagenesis plus activity assays in cells, multiple orthogonal methods confirming autoinhibitory mechanism\",\n      \"pmids\": [\"17822667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BRK is coamplified with ErbB2 in human breast cancers; ErbB2 interacts with BRK and increases its intrinsic kinase activity; BRK expression enhances ErbB2-induced Ras/MAPK signaling and cyclin E/cdk2 activity to promote proliferation; BRK overexpression confers resistance to lapatinib-induced growth inhibition.\",\n      \"method\": \"Co-immunoprecipitation, FISH (gene amplification), 3D acini culture, mouse mammary tumor model (WAP-Brk transgenic), kinase activity 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 — co-IP showing interaction and kinase activation, multiple model systems (cell culture, mouse model), drug resistance phenotype, single lab but orthogonal methods\",\n      \"pmids\": [\"18719096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"BRK phosphorylates PSF (polypyrimidine tract-binding protein-associated splicing factor) at C-terminal tyrosines via an SH3 domain–polyproline interaction; this phosphorylation promotes cytoplasmic relocalization of PSF, impairs its RNA binding, and leads to cell cycle arrest.\",\n      \"method\": \"Proteomic co-immunoprecipitation/mass spectrometry, co-IP, phosphosite mapping, subcellular fractionation, RNA-binding assay, cell cycle analysis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS identification plus co-IP, phosphosite mapping, functional consequences (RNA binding, localization, cell cycle), multiple readouts, single lab\",\n      \"pmids\": [\"19439179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Membrane-targeted PTK6 (Myr-PTK6) promotes proliferation, cell survival, migration, and anchorage-independent colony formation, whereas nuclear-targeted PTK6 (NLS-PTK6) impairs these functions; the two localizations produce distinct cellular phosphoproteome profiles.\",\n      \"method\": \"Targeted localization constructs (myristoylation signal vs. NLS), proliferation/survival/migration/colony formation assays, phosphoprotein profiling in HEK293 cells\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — engineered localization with functional readouts, single lab, two distinct targeting conditions tested\",\n      \"pmids\": [\"19304789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"STAP-2 is phosphorylated at Y250 by BRK; the STAP-2 Y250F mutant attenuates BRK-mediated STAT3 activation; siRNA knockdown of STAP-2 decreases BRK-mediated STAT3 activation, establishing STAP-2 as a required intermediary in BRK→STAT3 signaling.\",\n      \"method\": \"Site-directed mutagenesis (Y250F), anti-phospho-STAP-2 Y250 antibody, siRNA knockdown, STAT3 activation assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphosite-specific antibody, loss-of-function with both mutant and siRNA, single lab\",\n      \"pmids\": [\"19393627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PTK6 directly phosphorylates beta-catenin predominantly at Y64 (also Y142, Y331/333); PTK6 associates with nuclear and cytoplasmic beta-catenin and inhibits beta-catenin/TCF-mediated transcription. Nuclear-targeted PTK6 negatively regulates beta-catenin/TCF transcription and increases TCF4 and TLE/Groucho co-repressor levels, while membrane-targeted PTK6 enhances beta-catenin/TCF transcription.\",\n      \"method\": \"In vitro kinase assay, phosphosite mapping, co-immunoprecipitation, luciferase reporter assay, siRNA knockdown, Ptk6-null BAT-GAL reporter mice\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with phosphosite mapping, reporter assay, KO mouse model, multiple cell lines, multiple orthogonal methods\",\n      \"pmids\": [\"20026641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PTK6 forms a complex with IGF-1R and IRS-1 adaptor protein, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation.\",\n      \"method\": \"siRNA screen, reverse-phase protein arrays, co-immunoprecipitation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP validation of complex, supported by proteomic array data, single lab\",\n      \"pmids\": [\"20668531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"STAP-2 interacts with both BRK and STAT3; STAP-2 PH domain is required for BRK–STAP-2 binding, BRK kinase activation, and STAT3 tyrosine phosphorylation/activation; a STAP-2 PH-Brk fusion protein exhibits robust kinase activity and enhanced STAT3 activation.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion mapping, siRNA knockdown, STAT3 activation assays, fusion protein kinase activity\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping plus functional assays, siRNA confirms endogenous relevance, single lab\",\n      \"pmids\": [\"20929863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PTK6 phosphorylates ARAP1 at Y231 in an EGF/EGFR-dependent manner via the PTK6 SH2 domain (R105 residue); phosphorylated ARAP1 inhibits EGFR down-regulation, thereby sustaining EGFR signaling. PTK6 silencing in breast cancer cells decreases EGFR levels.\",\n      \"method\": \"Co-immunoprecipitation, MALDI-TOF mass spectrometry identification, site-directed mutagenesis (Y231F, R105A), EGFR degradation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MS identification, mutagenesis of both kinase and substrate, functional EGFR degradation assay, single lab multiple orthogonal methods\",\n      \"pmids\": [\"20554524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"STAP-2 mediates BRK-induced STAT5 phosphorylation and activation in T47D breast cancer cells; the STAP-2 PH domain participates in BRK-mediated STAT5 phosphorylation; knockdown of STAP-2 reduces proliferation as strongly as BRK or STAT5b knockdown.\",\n      \"method\": \"Co-transfection, STAT5 transcriptional reporter, siRNA knockdown, proliferation assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping, siRNA knockdown with functional readout, single lab\",\n      \"pmids\": [\"21205088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ALT-PTK6, an alternatively spliced form lacking exon 2, associates with Sam68 and beta-catenin via its SH3 domain (demonstrated by GST pull-down); coexpression of ALT-PTK6 with full-length PTK6 suppresses PTK6 kinase activity, reduces PTK6 association with phosphotyrosine proteins, and enhances PTK6-mediated inhibition of beta-catenin/TCF transcription by promoting nuclear PTK6 function.\",\n      \"method\": \"RT-PCR, GST pull-down, co-immunoprecipitation, luciferase reporter assay, inducible expression system, proliferation/colony assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pull-down plus reporter assay plus inducible expression, single lab, multiple functional readouts\",\n      \"pmids\": [\"21479203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BRK mediates p38 MAPK phosphorylation downstream of EGF signaling in mammary epithelial cells; BRK knockdown blocks EGF-stimulated p38 signaling; BRK overexpression in mammary gland causes delayed involution associated with activated p38 MAPK.\",\n      \"method\": \"WAP-Brk transgenic mouse model, siRNA knockdown, Western blotting, IHC\",\n      \"journal\": \"Breast cancer research : BCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse model plus knockdown in cell lines with mechanistic signaling readout, single lab\",\n      \"pmids\": [\"21923922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"BRK interacts with EGFR, inhibits ligand-induced EGFR degradation by uncoupling activated EGFR from c-Cbl-mediated ubiquitination, and directly phosphorylates EGFR Y845 in the kinase domain to potentiate EGFR kinase activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, in vitro kinase assay (phospho-Y845), EGFR degradation assay, siRNA knockdown\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay identifying specific phosphosite, ubiquitination assay, co-IP, multiple mechanistic steps validated, single lab but multiple orthogonal experiments\",\n      \"pmids\": [\"22231447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"BRK is a critical downstream effector of Met/HGF signaling and is required for HGF-induced cell migration in breast cancer cells.\",\n      \"method\": \"siRNA knockdown of BRK with HGF stimulation, cell migration assay\",\n      \"journal\": \"Hormones & cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single cell migration assay, review article with limited primary data, single lab\",\n      \"pmids\": [\"22124844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PTP1B dephosphorylates BRK/PTK6 directly and also dephosphorylates IGF-1R β-subunit; BRK physically interacts with IGF-1R β-subunit. Restoration of PTP1B in ovarian cancer cells attenuates PTK6 and IGF-1R signaling, activating BAD to induce apoptosis.\",\n      \"method\": \"Stable PTP1B re-expression, co-immunoprecipitation (PTK6-IGF-1R), phosphorylation assays, apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating physical interaction, enzymatic dephosphorylation shown, multiple functional readouts, single lab\",\n      \"pmids\": [\"23814047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Active PTK6 in prostate cancer cells localizes to the plasma membrane; membrane-targeted active PTK6 promotes EMT partly by enhancing AKT activation and stimulates cancer cell migration and metastasis in xenograft models; in PTEN-null mouse prostate, endogenous active PTK6 localizes to membranes with decreased E-cadherin.\",\n      \"method\": \"Subcellular fractionation, targeted PTK6 constructs (membrane/cytoplasm), siRNA knockdown, AKT phosphorylation assays, xenograft mouse model, conditional Pten-knockout mouse\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple model systems (cell lines, xenografts, conditional KO mouse), mechanistic signaling (AKT), fractionation showing active PTK6 membrane localization, single lab with orthogonal approaches\",\n      \"pmids\": [\"23856248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AKT, p130CAS, and FAK are identified as PTK6 substrates by mass spectrometry and validated by in vitro phosphorylation assays; these substrates promote cell proliferation, migration, and anoikis resistance downstream of cytoplasmic/membrane-localized PTK6 in prostate cancer.\",\n      \"method\": \"Mass spectrometry substrate identification, in vitro kinase assay validation, siRNA knockdown, targeted PTK6 expression constructs\",\n      \"journal\": \"European journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus in vitro validation, functional assays, single lab\",\n      \"pmids\": [\"23398121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PTK6 phosphorylates and degrades c-Cbl at Y700, Y731, and Y774 in the C-terminal domain; phosphorylated c-Cbl undergoes auto-ubiquitination and proteasomal degradation, thereby reducing c-Cbl-mediated ubiquitination of substrates.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of c-Cbl phosphosites, ubiquitination assay, proteasome inhibitor experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphosite mapping by mutagenesis, ubiquitination assay, proteasome dependence shown, single lab\",\n      \"pmids\": [\"23352614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BRK interacts with and phosphorylates Dok1 specifically at Y362; this phosphorylation leads to ubiquitin-proteasome-mediated Dok1 degradation, thereby removing a tumor suppressor and promoting cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, phosphosite mapping (Y362), ubiquitination assay, proteasome inhibitor rescue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with site-specific mapping, ubiquitination assay, functional consequences, single lab\",\n      \"pmids\": [\"24523872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BRK phosphorylates KAP3A (kinesin-associated protein 3A) at C-terminal tyrosine residues in vivo; BRK-mediated phosphorylation induces delocalization of KAP3A from punctate nuclear localization to diffuse nucleo-cytoplasmic pattern; KAP3A knockdown suppresses BRK-induced cell migration.\",\n      \"method\": \"High-density protein filter array kinase screen, in vivo phosphorylation assay, co-immunoprecipitation, subcellular localization imaging, siRNA knockdown, migration assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo phosphorylation shown, localization change documented, functional migration assay, single lab\",\n      \"pmids\": [\"18077133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"BRK phosphorylates p27KIP1 at Y88 via an SH3-PXXP interaction; pY88-p27 activates cyclin D-cdk4 activity. An alternatively spliced form of BRK (Alt Brk) containing only the SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor.\",\n      \"method\": \"SH3-PXXP interaction screen, in vitro kinase assay, pY88 phosphorylation assay in breast cancer cells, modulation of BRK levels in cells, cdk4 activity assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus cellular phosphorylation, cdk4 functional assay, domain mapping, single lab\",\n      \"pmids\": [\"25733683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cancer-associated somatic mutations in BRK SH3 (L16F), SH2 (R131L), and C-terminal tail (P450L) domains activate BRK by disrupting intramolecular autoinhibitory interactions; two other mutations eliminate enzymatic activity; mutations differentially affect substrate recognition and phosphorylation.\",\n      \"method\": \"Recombinant protein expression, in vitro kinase assays, mutagenesis panel, substrate peptide assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with multiple cancer-associated mutants tested, mechanistic interpretation of autoinhibitory domain disruption, single lab\",\n      \"pmids\": [\"25940761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PTK6 downregulation restores E-cadherin levels via proteasome-dependent degradation of the E-cadherin repressor SNAIL; kinase-active PTK6 suppresses E-cadherin expression and promotes EMT markers; PTK6 inhibition impairs metastatic lung colonization in vivo through anoikis induction.\",\n      \"method\": \"siRNA knockdown, PTK6 kinase inhibitor, E-cadherin/SNAIL Western blotting, proteasome inhibitor rescue, anoikis assay, in vivo lung colonization assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple loss-of-function approaches (siRNA + kinase inhibitor), proteasome-dependence demonstrated, in vivo metastasis model, single lab but orthogonal methods\",\n      \"pmids\": [\"27302163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PTK6 interacts with JAK2 and phosphorylates it to activate JAK2/STAT3 signaling, thereby promoting cancer cell stemness in colorectal cancer. Pharmacological inhibition of PTK6 with XMU-MP-2 reduces stemness and chemosensitivity in vivo.\",\n      \"method\": \"Co-immunoprecipitation (PTK6-JAK2), FLAG-tagged PTK6 mutant constructs (kinase-dead, inhibition-defective), STAT3 activation assays, in vivo xenograft/PDX models\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus kinase-dead mutant, in vivo validation, single lab\",\n      \"pmids\": [\"34551797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PTEN protein phosphatase activity directly dephosphorylates PTK6 at activation loop Y342 (but not autoinhibitory Y447), inhibiting PTK6 activity with efficiency similar to PTP1B. In PTEN-null prostate cancer, conditional Pten disruption increases PTK6 Y342 phosphorylation and tumorigenesis; Ptk6 disruption impairs PTEN-loss-induced tumorigenesis.\",\n      \"method\": \"PTEN mutant constructs (phosphatase-dead), phospho-Y342 and Y447 Western blotting, PTEN knockdown/re-expression, conditional Pten-knockout mouse prostate model, Ptk6/Pten double-KO mice, human prostate tumor tissue microarrays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical phosphatase assay with mutant PTEN constructs, multiple genetic mouse models (conditional KO, double KO), human tissue validation; replicated across systems\",\n      \"pmids\": [\"29142193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plasma membrane-localized PTK6 phosphorylates Eps8 at Y497, Y524, and Y534; Eps8 3YF phosphorylation-defective mutant reverts PTK6-mediated increases in proliferation, migration, ERK phosphorylation, and FAK phosphorylation; EGF-induced Eps8 phosphorylation in T-47D cells depends on endogenous PTK6.\",\n      \"method\": \"Proteomics screen with Myr-PTK6, site-directed mutagenesis (Eps8 3YF), kinase assays, siRNA knockdown in breast cancer cells, proliferation/migration/adhesion assays\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphosite-specific mutagenesis with functional rescue, endogenous validation by knockdown, single lab\",\n      \"pmids\": [\"28214294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structures of PTK6 kinase domain were solved in complex with Dasatinib (2.24 Å) and a novel IPA compound (1.70 Å); both structures are in DFG-in, αC-helix-out conformation with inhibitors at the ATP-binding pocket; Dasatinib occupies an additional pocket near the gatekeeper explaining its higher potency.\",\n      \"method\": \"X-ray crystallography (co-crystal structures of PTK6-KD with inhibitors), structure determination and refinement\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure at high resolution with two distinct inhibitor complexes, structural basis for potency differences determined\",\n      \"pmids\": [\"27993680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of apo-PTK6 and inhibitor-bound PTK6 complexes were determined; PTK6 kinase inhibitors moderately suppress tumor cell growth in 2D/3D culture but growth inhibition does not correlate with PTK6 kinase activity inhibition or PTK6 protein levels (>500-fold shift between kinase IC50 and growth inhibition IC50 in PTK6-overexpressing cells), indicating PTK6 kinase activity is not the primary driver of tumor growth.\",\n      \"method\": \"Crystal structure determination (apo and inhibitor-bound), selective PTK6 inhibitors with kinase assays, 2D/3D cell growth assays, PTK6 overexpression in engineered breast tumor cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures plus selective inhibitors and engineered cell lines; however, the finding is a negative/dissociative result about kinase-independent growth promotion\",\n      \"pmids\": [\"29879184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BRK phosphorylates SMAD4 leading to its recognition by the ubiquitin-proteasome system and accelerated degradation; activated BRK (Y447F mutant) mediates SMAD4 degradation associated with repression of tumor suppressor FRK and increased expression of SNAIL and SLUG. BRK competitively binds SMAD4.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation assay with constitutively active BRK-Y447F, ubiquitination assay, proteasome inhibitor rescue, knockdown experiments\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, ubiquitination assay, mutagenesis, single lab with multiple mechanistic assays\",\n      \"pmids\": [\"31681835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PSPC1 is a nuclear substrate of PTK6; PSPC1 sequesters PTK6 in the nucleus and inhibits its metastasis-promoting function. PSPC1 upregulation or PSPC1-Y523F mutation promotes cytoplasmic translocation of active PTK6 and nuclear translocation of beta-catenin, driving EMT and metastasis via augmented Wnt3a autocrine signaling. PSPC1-CT131 acts as a dual inhibitor of PSPC1 and PTK6.\",\n      \"method\": \"Co-immunoprecipitation (PSPC1-PTK6, PSPC1-beta-catenin), site-directed mutagenesis (Y523F), subcellular localization imaging, HCC orthotopic mouse model, beta-catenin/Wnt reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, phosphorylation mutant, in vivo orthotopic model, mechanistic pathway (Wnt/beta-catenin) validated, multiple orthogonal methods\",\n      \"pmids\": [\"31844057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The PTK6 SH2 domain (not the kinase domain) mediates cell motility in TNBC via activation of RhoA and aryl hydrocarbon receptor (AhR) signaling pathways; the SH3 domain contributes to spheroid formation via p38 MAPK. Kinase activity is dispensable for cell migration but contributes to anchorage-independent colony formation.\",\n      \"method\": \"In-frame domain deletion mutants (SH2-del, SH3-del, kinase-dead), reverse-phase protein arrays, RhoA/AhR inhibition, primary breast tumor organoids\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutants with functional assays, RPPA pathway analysis, organoid model, single lab\",\n      \"pmids\": [\"33172975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Hsp90 interacts with the PTK6 tyrosine kinase catalytic domain (interaction is independent of catalytic activity) and protects PTK6 from proteasomal degradation. Geldanamycin (Hsp90 inhibitor) decreases PTK6 protein via proteasome-dependent degradation and increases PTK6 interaction with the E3 ligase CHIP; CHIP overexpression promotes PTK6 proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, geldanamycin treatment, proteasome inhibitor rescue, CHIP overexpression, siRNA knockdown of CHIP\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP identifying Hsp90-PTK6 interaction, pharmacological and genetic manipulation confirming proteasomal pathway, single lab\",\n      \"pmids\": [\"22849407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HER2 stabilizes BRK protein by upregulating calpastatin (an endogenous calpain inhibitor) via MEK signaling, thereby inhibiting calpain-1-mediated proteolytic degradation of BRK. HER2 knockdown downregulates calpastatin, increasing calpain-1 activity and BRK degradation.\",\n      \"method\": \"HER2 overexpression/siRNA knockdown, BRK stability assays, calpain activity assay, calpastatin expression analysis, MEK inhibition, constitutively active MEK rescue\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic chain (HER2→MEK→calpastatin→calpain→BRK stability) supported by multiple inhibitor and rescue experiments, single lab\",\n      \"pmids\": [\"23707532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Under hypoxic conditions, PTK6 is rapidly stabilized (in a HIF-1α-independent manner) associated with decreased ubiquitylation; c-Cbl is identified as a putative PTK6 E3 ubiquitin ligase in normoxia. Hypoxia-induced PTK6 stabilization and membrane relocalization is associated with increased cell motility and invasion.\",\n      \"method\": \"Hypoxia treatment, HIF-1α knockdown, ubiquitylation assay, c-Cbl co-immunoprecipitation, subcellular fractionation, cell motility/invasion assays\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitylation assay, co-IP, functional motility assays, HIF-1 independence demonstrated by knockdown, single lab\",\n      \"pmids\": [\"25019382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Vemurafenib (PLX4032/PLX4720) binds in the active site of PTK6 as demonstrated by saturation transfer difference NMR and molecular docking; vemurafenib inhibits PTK6-mediated signaling through FAK, EGFR, and ERK1/2 and blocks PTK6-dependent cell growth, migration, and invasion in prostate cancer models.\",\n      \"method\": \"Saturation transfer difference NMR, molecular docking, kinase assays, signaling Western blots, xenograft model\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR-based structural binding study plus functional validation in cells and in vivo xenograft, multiple orthogonal methods\",\n      \"pmids\": [\"30926642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PTK6 physically interacts with HNRNPH1 and phosphorylates it at Y210, promoting HNRNPH1 liquid-liquid phase separation (LLPS) and formation of biomolecular condensates; HNRNPH1 LLPS triggers splicing switching of NBR1 exon 10 inclusion, activating autophagy and suppressing apoptosis in colorectal cancer.\",\n      \"method\": \"Co-immunoprecipitation (PTK6-HNRNPH1), Y210 phosphorylation assay, FRAP (LLPS validation), NBR1 splicing assay, autophagy assays, patient-derived organoid and CDX models with PTK6 inhibitor tilfrinib\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphosite identification, FRAP confirmation of LLPS, splicing assay, in vivo organoid/CDX models; multiple orthogonal methods in one study\",\n      \"pmids\": [\"40103198\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PTK6/BRK is a non-receptor intracellular tyrosine kinase with SH3, SH2, and catalytic domains that is maintained in an autoinhibited state via intramolecular SH3–linker and SH2–pY447 interactions; activation at Y342 in the activation loop promotes its kinase activity, which is negatively regulated by PTEN (dephosphorylating Y342) and positively regulated by ErbB2 interaction; PTK6 phosphorylates a broad set of nuclear substrates (Sam68, SLM-1/2, PSF, HNRNPH1) to suppress RNA-binding and regulate splicing/phase separation, and cytoplasmic/membrane substrates (paxillin, beta-catenin, AKT, FAK, p130CAS, Eps8, ARAP1, c-Cbl, Dok1, SMAD4, JAK2, p27KIP1) to promote cell migration, proliferation, survival, EMT, and oncogenic signaling; its subcellular localization (nuclear vs. cytoplasmic/membrane) is a critical determinant of function, with nuclear PTK6 suppressing growth and beta-catenin/TCF transcription and membrane-localized PTK6 driving oncogenic phenotypes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PTK6 (BRK) is an intracellular non-receptor tyrosine kinase that integrates growth-factor signaling with control of cell proliferation, migration, survival, and epithelial-mesenchymal transition, with its subcellular localization acting as the key switch between tumor-suppressive and oncogenic outputs [#15, #17, #27]. The kinase is held in an autoinhibited conformation by intramolecular contacts: an SH3-domain interaction with the SH2-kinase linker (via W44 and linker prolines P175/P177/P179) and an SH2-domain engagement of C-terminal phospho-Y447, with activation requiring autophosphorylation of the activation-loop Y342 [#5, #12]. This autoinhibition is biologically reinforced and reversed by opposing phosphatases and partners: PTEN and PTP1B directly dephosphorylate the activating Y342 to restrain PTK6, whereas ErbB2/HER2 interaction increases its intrinsic kinase activity and stabilizes the protein [#36, #26, #13, #44]. Cancer-associated mutations in the SH3, SH2, and C-terminal tail disrupt these autoinhibitory contacts and constitutively activate the enzyme [#33]. In the nucleus, PTK6 phosphorylates RNA-binding proteins—Sam68, SLM-1/2, PSF, and HNRNPH1—to suppress their RNA-binding and redirect splicing and phase separation, and it antagonizes beta-catenin/TCF transcription [#2, #9, #14, #47, #17]. At the membrane and in the cytoplasm, PTK6 phosphorylates paxillin, Eps8, AKT, FAK, p130CAS, ARAP1, and beta-catenin and degrades the c-Cbl, Dok1, and SMAD4 tumor suppressors to drive motility, EGFR signaling sustainment, proliferation, survival, and EMT [#10, #37, #28, #20, #24, #29, #30, #40]. Distinct domains carry distinct functions independent of catalysis—the SH2 domain mediates motility and the SH3 domain mediates spheroid formation—and kinase activity is dispensable for several growth-promoting phenotypes [#42, #39]. PTK6 promotes EMT and metastasis by stabilizing SNAIL and repressing E-cadherin, and its nuclear retention by partners such as PSPC1 suppresses these metastatic functions [#34, #41]. PTK6 is also druggable, with crystal structures and selective inhibitors defining its ATP pocket [#38].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established PTK6 as a novel SH3/SH2-containing non-receptor tyrosine kinase capable of autophosphorylation, defining its basic enzymatic identity.\",\n      \"evidence\": \"Recombinant expression in baculovirus and bacterial systems with autophosphorylation assay\",\n      \"pmids\": [\"8036022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No cellular substrates identified\", \"No regulatory mechanism for activity defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Separated the catalytic requirement from a regulatory tyrosine, showing kinase activity (K219) is essential for transformation while the C-terminal Y447 modulates transforming potential without abolishing catalysis.\",\n      \"evidence\": \"Site-directed mutagenesis with transformation assays in mammary epithelial cells\",\n      \"pmids\": [\"8940083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Y447 regulates activity not resolved\", \"No structural basis defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the molecular logic of PTK6 activation, identifying Y342 activation-loop autophosphorylation as activating and Y447–SH2 and SH3 contacts as autoinhibitory.\",\n      \"evidence\": \"Baculovirus expression, mass spectrometry phosphosite mapping, mutagenesis, and in vitro kinetics\",\n      \"pmids\": [\"12121988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream activators in cells not identified\", \"Structural model of autoinhibited state not solved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped the SH3–linker autoinhibitory contact at residue level (W44 with linker prolines), explaining how intramolecular conformation restrains catalysis.\",\n      \"evidence\": \"Mutagenesis, surface plasmon resonance, kinase assays, and structural modeling\",\n      \"pmids\": [\"17822667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length crystal structure of autoinhibited PTK6\", \"Physiological trigger of release unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identified nuclear RNA-binding proteins as the first physiological substrates, showing PTK6 phosphorylates Sam68 to abolish its RNA binding.\",\n      \"evidence\": \"Reciprocal co-IP, domain mapping, and functional RNA-binding assays across cell lines\",\n      \"pmids\": [\"10913193\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of Sam68 regulation not yet established\", \"Downstream splicing consequences not mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended the Sam68 model to a functional consequence, showing PTK6-mediated hyperphosphorylation impairs Sam68-dependent intron-containing RNA utilization.\",\n      \"evidence\": \"Co-expression in 293T cells with RNA utilization assays and RNA-binding-defective Sam68 mutant\",\n      \"pmids\": [\"12482964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous target transcripts not identified\", \"Physiological setting untested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Broadened the nuclear substrate repertoire and linked substrate phosphorylation to PTK6 nuclear retention, while demonstrating the SH3 domain dominates substrate recognition.\",\n      \"evidence\": \"Phosphorylation and RNA-binding assays for SLM-1/2 with selectivity controls; peptide kinetics linking SH3 to substrate Km\",\n      \"pmids\": [\"15471878\", \"14676834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of nuclear vs cytoplasmic partitioning incompletely defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established the cytoplasmic migratory arm of PTK6 function, identifying paxillin phosphorylation driving Rac1 activation and cell motility upon EGF stimulation.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay with phosphosite mapping, Rac1 activation, migration/invasion assays, and confocal localization\",\n      \"pmids\": [\"15572663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo migration relevance not addressed\", \"Mechanism of membrane translocation not defined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Connected PTK6 to growth-factor receptor signaling through adaptor proteins, identifying STAP-2/BKS and ErbB3/PI3K/AKT potentiation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, and signaling assays (phospho-erbB3, PI3K recruitment, AKT)\",\n      \"pmids\": [\"10980601\", \"11114724\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous significance of adaptor interactions limited\", \"Pathway integration with other receptors unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated that PTK6 subcellular localization dictates opposing biological outcomes, with membrane-targeted PTK6 oncogenic and nuclear-targeted PTK6 growth-suppressive.\",\n      \"evidence\": \"Engineered Myr/NLS targeting constructs with functional and phosphoproteome profiling\",\n      \"pmids\": [\"19304789\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism controlling localization in cells not resolved\", \"Distinct substrate sets only partially characterized\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established PTK6 as a direct negative regulator of beta-catenin/TCF transcription, phosphorylating beta-catenin and showing localization-dependent transcriptional effects in vivo.\",\n      \"evidence\": \"In vitro kinase assay with phosphosite mapping, reporter assays, siRNA, and Ptk6-null BAT-GAL reporter mice\",\n      \"pmids\": [\"20026641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking phosphorylation to TCF/TLE repressor levels incompletely defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined STAP-2 and PSF/cell-cycle nodes as PTK6 effectors, linking the kinase to STAT3 activation and to cell cycle arrest via impaired PSF RNA binding.\",\n      \"evidence\": \"Phosphosite-specific antibody and siRNA for STAP-2 Y250; proteomic MS, phosphosite mapping, and cell cycle analysis for PSF\",\n      \"pmids\": [\"19393627\", \"19439179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of growth-suppressive (PSF) and growth-promoting outputs not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected PTK6 amplification to HER2/ErbB2-driven breast cancer, showing ErbB2 activates PTK6 and PTK6 confers lapatinib resistance.\",\n      \"evidence\": \"Co-IP, FISH, 3D acini, WAP-Brk transgenic mice, and kinase assays\",\n      \"pmids\": [\"18719096\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphorylation events downstream of the ErbB2-PTK6 interaction not all mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed that PTK6 sustains EGFR signaling by directly phosphorylating EGFR Y845 and uncoupling it from c-Cbl-mediated degradation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, in vitro kinase assay for phospho-Y845, and EGFR degradation assay\",\n      \"pmids\": [\"22231447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution to EGFR-driven tumors not quantified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established phosphatase control of PTK6 (PTP1B) and direct degradation of tumor suppressors c-Cbl, situating PTK6 as a hub that destabilizes negative regulators.\",\n      \"evidence\": \"PTP1B re-expression with dephosphorylation/apoptosis assays; c-Cbl phosphosite mutagenesis and ubiquitination/proteasome assays\",\n      \"pmids\": [\"23814047\", \"23352614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of c-Cbl degradation versus EGFR uncoupling unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the membrane-localized active PTK6 pool as the metastatic driver in prostate cancer, identifying AKT/FAK/p130CAS substrates and EMT promotion in PTEN-null models.\",\n      \"evidence\": \"Subcellular fractionation, targeted constructs, MS substrate ID with in vitro validation, xenografts, and conditional Pten-knockout mice\",\n      \"pmids\": [\"23856248\", \"23398121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger relocalizing active PTK6 to the membrane not fully defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined PTK6 protein stability control through Hsp90/CHIP and HER2-MEK-calpastatin-calpain axes, explaining how PTK6 levels are post-translationally maintained.\",\n      \"evidence\": \"Co-IP, geldanamycin and proteasome inhibitor experiments, CHIP manipulation; HER2/MEK manipulation with calpain activity and stability assays\",\n      \"pmids\": [\"22849407\", \"23707532\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative balance between proteasomal and calpain pathways in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed PTK6 is stabilized under hypoxia with membrane relocalization and motility induction, and degrades the Dok1 tumor suppressor, reinforcing pro-invasive control.\",\n      \"evidence\": \"Hypoxia/HIF-1α knockdown with ubiquitylation and motility assays; Dok1 Y362 mutagenesis with ubiquitination/proteasome rescue\",\n      \"pmids\": [\"25019382\", \"24523872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of hypoxic stabilization not molecularly defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined PTK6 cell-cycle promotion via p27KIP1 Y88 phosphorylation activating cdk4, and showed cancer mutations activate PTK6 by disrupting autoinhibition.\",\n      \"evidence\": \"SH3-PXXP interaction screen and in vitro kinase/cdk4 assays for p27; recombinant mutant panel with kinetics for cancer mutations\",\n      \"pmids\": [\"25733683\", \"25940761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Frequency and clinical impact of activating mutations not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked PTK6 to EMT through SNAIL stabilization and E-cadherin suppression, with PTK6 inhibition reducing metastatic colonization by inducing anoikis.\",\n      \"evidence\": \"siRNA and kinase inhibitor, SNAIL/E-cadherin Western with proteasome rescue, anoikis assay, and in vivo lung colonization\",\n      \"pmids\": [\"27302163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PTK6 substrate mediating SNAIL stabilization not pinpointed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified PTEN as a direct PTK6 Y342 phosphatase and a genetic suppressor of PTK6-driven tumorigenesis, and identified Eps8 as a membrane-PTK6 substrate driving proliferation/migration.\",\n      \"evidence\": \"Phosphatase-dead PTEN constructs, phospho-Y342/Y447 blots, conditional and double-KO mouse prostate models, human tissue; Eps8 phosphosite mutagenesis with functional rescue\",\n      \"pmids\": [\"29142193\", \"28214294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PTEN loss specifically redirects PTK6 to membranes not fully mechanistic\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed PTK6 degrades SMAD4 and is sequestered by PSPC1 in the nucleus, defining a switch governing EMT/metastasis via Wnt/beta-catenin.\",\n      \"evidence\": \"Co-IP, in vitro phosphorylation with active BRK-Y447F, ubiquitination assays; PSPC1 Y523F mutagenesis with orthotopic HCC model and Wnt reporters\",\n      \"pmids\": [\"31681835\", \"31844057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"General applicability of PSPC1 sequestration across tissues untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Dissociated PTK6 functions from catalysis, showing the SH2 domain drives motility via RhoA/AhR and the SH3 domain drives spheroid formation via p38, with kinase activity dispensable for migration.\",\n      \"evidence\": \"Domain deletion mutants, RPPA, RhoA/AhR inhibition, and primary breast tumor organoids\",\n      \"pmids\": [\"33172975\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which the SH2 domain activates RhoA not defined\", \"Scaffolding partners mediating kinase-independent functions unidentified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided structural and pharmacological tools, solving PTK6 kinase-domain structures with inhibitors and revealing that growth inhibition often does not track with kinase inhibition.\",\n      \"evidence\": \"X-ray crystallography of inhibitor complexes and selective inhibitors with 2D/3D growth assays in engineered cells\",\n      \"pmids\": [\"27993680\", \"29879184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase-independent growth-promoting activity not molecularly explained\", \"Structure of full-length/autoinhibited enzyme unsolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated repurposed inhibitors (vemurafenib) and JAK2 as a substrate, showing PTK6 activates JAK2/STAT3 to promote cancer stemness and is pharmacologically targetable.\",\n      \"evidence\": \"STD-NMR/docking and signaling/xenograft assays for vemurafenib; co-IP and kinase-dead mutants with PDX models for JAK2\",\n      \"pmids\": [\"30926642\", \"34551797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Selectivity of repurposed inhibitors for PTK6 in vivo uncertain\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended PTK6's RNA-regulatory role to phase separation, showing PTK6 phosphorylates HNRNPH1 to drive condensate formation and NBR1 splicing that activates autophagy and suppresses apoptosis.\",\n      \"evidence\": \"Co-IP, Y210 phosphorylation, FRAP, NBR1 splicing/autophagy assays, and organoid/CDX models with the inhibitor tilfrinib\",\n      \"pmids\": [\"40103198\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of LLPS-driven splicing control across PTK6 RNA-binding substrates untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular mechanism that determines PTK6's nuclear versus membrane partitioning in cells, and how kinase-independent SH2/SH3 scaffolding functions integrate with catalytic substrate phosphorylation, remain unresolved.\",\n      \"evidence\": \"Open question synthesized across localization, domain, and kinase-independence findings\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined cellular signal controlling localization switch\", \"Kinase-independent functions lack identified scaffolding mechanism\", \"No full-length structure rationalizing autoinhibition and partner engagement\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 10, 17, 20, 24, 28, 29, 30, 37, 40, 47]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 5, 8, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 20, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 9, 14, 17, 41]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 15, 27, 37, 45]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 15, 27, 28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 10, 20, 24, 27]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 9, 14, 47]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [13, 32]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [27, 34, 41]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ERBB2\", \"EGFR\", \"STAP2\", \"SAM68\", \"JAK2\", \"PTEN\", \"PSPC1\", \"IGF1R\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}