{"gene":"DAPK1","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2010,"finding":"During cerebral ischemia, DAPK1 is recruited into the NMDA receptor NR2B protein complex in cortical neurons. DAPK1 directly binds the NR2B C-terminal tail (amino acids 1292–1304). A constitutively active DAPK1 phosphorylates NR2B at Ser-1303, enhancing NR1/NR2B receptor channel conductance and injurious Ca2+ influx. Genetic deletion of DAPK1 or peptide uncoupling of DAPK1 from NR2B blocks this Ca2+ influx and protects neurons against ischemic injury.","method":"Co-immunoprecipitation, direct binding assays, constitutively active DAPK1 overexpression, genetic knockout mice, in vivo peptide uncoupling (NR2B CT peptide), electrophysiology","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding demonstrated, genetic KO with defined phenotype, in vivo functional rescue, multiple orthogonal methods in a high-impact study","pmids":["20141836"],"is_preprint":false},{"year":2004,"finding":"DAPK interacts with ERK through a docking sequence within its death domain. ERK phosphorylates DAPK at Ser-735, increasing DAPK catalytic activity both in vitro and in vivo. Conversely, DAPK promotes cytoplasmic retention of ERK, inhibiting ERK nuclear signaling. This bidirectional regulation forms a positive feedback loop that promotes DAPK apoptotic activity.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, siRNA knockdown, subcellular fractionation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, reciprocal co-IP, multiple orthogonal methods demonstrating bidirectional regulation","pmids":["15616583"],"is_preprint":false},{"year":2010,"finding":"The BTB-Kelch protein KLHL20 binds DAPK via its Kelch-repeat domain and assembles with Cullin 3 (Cul3) and ROC1 to form an E3 ligase complex that promotes DAPK polyubiquitination and proteasomal degradation. IFN-α/γ sequesters KLHL20 into PML nuclear bodies, separating it from DAPK and stabilizing DAPK. This mechanism controls IFN-induced apoptosis and autophagy.","method":"Co-immunoprecipitation, ubiquitination assay in vitro and in vivo, siRNA depletion, immunofluorescence localization, functional cell death assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro and in vivo ubiquitination assays, localization experiments with functional consequence, multiple orthogonal methods","pmids":["20389280"],"is_preprint":false},{"year":2014,"finding":"DAPK1 directly binds the DNA-binding motif of p53 via its death domain (DAPK1DD), and phosphorylates p53 at Ser-23. This phosphorylation creates a functional form of p53 that drives both apoptotic (nuclear, inducing Bax expression) and necrotic (mitochondrial matrix, via CypD interaction) neuronal death pathways. Deletion of DAPK1DD or a Tat-p53DM uncoupling peptide blocks both pathways in cortical neurons and in vivo.","method":"Co-immunoprecipitation, in vitro kinase assay, deletion mutant analysis, Tat-peptide uncoupling in vivo, primary cortical neuron culture assays, mouse ischemia model","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding, in vitro phosphorylation, genetic deletion, in vivo peptide rescue with defined mechanistic readouts","pmids":["24806680"],"is_preprint":false},{"year":2007,"finding":"The tyrosine phosphatase LAR dephosphorylates DAPK at pY491/492, stimulating DAPK catalytic, proapoptotic, and anti-adhesion/migration activities. Conversely, Src phosphorylates DAPK at Y491/492, inducing DAPK intramolecular/intermolecular interaction and inactivation. EGF stimulation activates Src and downregulates LAR synergistically to inactivate DAPK, facilitating tumor cell migration.","method":"In vitro kinase/phosphatase assay, site-directed mutagenesis at Y491/492, co-immunoprecipitation, cell migration assays, immunostaining of human cancer samples","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro phosphorylation/dephosphorylation with mutagenesis, reciprocal co-IP, multiple functional readouts, validated in human cancer samples","pmids":["17803936"],"is_preprint":false},{"year":2006,"finding":"DAPK inhibits cell migration and polarization by blocking the association of the talin head domain with integrin, thereby suppressing the integrin-Cdc42 polarity pathway. This anti-migratory effect is apoptosis-independent and contributes to DAPK tumor suppression.","method":"Cell migration assays, cell polarization assays, co-immunoprecipitation of talin-integrin, overexpression and knockdown of DAPK, invasion assays with adenocarcinoma cell lines","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for talin-integrin interaction, multiple cell-based functional assays, mechanistic pathway dissection with defined epistasis","pmids":["16476779"],"is_preprint":false},{"year":2008,"finding":"DAPK-1 binds to the microtubule-associated protein MAP1B via a linear peptide motif in the MAP1B N-terminal domain (residues 1–126). Amino acid starvation induces formation of the endogenous DAPK-1/MAP1B complex. MAP1B is required for DAPK-1-stimulated autophagy and membrane blebbing; siRNA knockdown of MAP1B attenuates both activities.","method":"Peptide combinatorial library screening, co-immunoprecipitation, siRNA knockdown, clonogenic assay, confocal co-localization, autophagy inhibitor (3-MA) treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — peptide library identification plus co-IP validation plus siRNA functional rescue, single lab but multiple orthogonal methods","pmids":["18195017"],"is_preprint":false},{"year":2008,"finding":"DAPK's death domain binds the tumor suppressor TSC2 (tuberin). Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity (stimulated by growth factors) promotes TSC2 phosphorylation in vivo, leading to TSC1-TSC2 complex dissociation. DAPK thereby acts as a positive regulator of mTORC1 signaling downstream of RAS-MEK-ERK and PI3K-AKT pathways. DAPK+/- MEFs show attenuated mTORC1 signaling.","method":"Peptide aptamer library, in vitro binding with death domain miniproteins, in vitro kinase assay, co-immunoprecipitation, siRNA knockdown, DAPK+/- MEF analysis, epistasis with pathway inhibitors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution kinase assay, genetic hypomorph MEFs, epistasis experiments, multiple orthogonal binding and functional methods","pmids":["18974095"],"is_preprint":false},{"year":2011,"finding":"DAPK activates MARK1/2 kinases through its death domain (not its catalytic domain) by binding the MARK1/2 spacer region, disrupting an inhibitory intramolecular interaction in MARK. This leads to MARK-dependent phosphorylation of tau and MAP2/4, destabilizing microtubules. DAPK-/- mouse brains show reduced tau phosphorylation. In a Drosophila tauopathy model, DAPK enhances tau toxicity via PAR-1 (MARK ortholog) in a phosphorylation-dependent manner.","method":"Co-immunoprecipitation, deletion mutagenesis (catalytic vs. death domain), in vitro kinase assay, DAPK-/- mouse brain biochemistry, Drosophila genetic model, neuronal differentiation assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, domain-specific mutagenesis, genetic KO mouse, Drosophila in vivo model, multiple orthogonal methods across two organisms","pmids":["21311567"],"is_preprint":false},{"year":2002,"finding":"DIP-1, a multi-RING finger protein, binds DAPK and acts as an E3 ubiquitin ligase that ubiquitinates DAPK in vitro and in vivo, targeting DAPK for proteasomal degradation. DIP-1 expression antagonizes the anti-apoptotic function of DAPK and promotes caspase-dependent apoptosis.","method":"Yeast two-hybrid and co-immunoprecipitation (binding), in vitro ubiquitination assay, in vivo ubiquitination, transient transfection apoptosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstituted ubiquitination assay plus in vivo ubiquitination plus functional apoptosis assay, single lab but multiple orthogonal methods","pmids":["12351649"],"is_preprint":false},{"year":2006,"finding":"DAPK activity is controlled by phosphorylation status: dephosphorylation at Ser-308 activates DAPK by allowing calcium/calmodulin association. TNF or ceramide treatment induces rapid Ser-308 dephosphorylation and transient kinase activation, followed by proteasome-dependent DAPK degradation. Dephosphorylation and activation temporally precede degradation.","method":"In vitro kinase assay, phosphorylation site mutagenesis (Ser-308), proteasome inhibitor treatment, TNF/ceramide stimulation time course, Western blot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay with mutagenesis, pharmacological inhibitor rescue, single lab with multiple biochemical methods","pmids":["17056602"],"is_preprint":false},{"year":2017,"finding":"DAPK1 mediates long-term depression (LTD) by competitively blocking CaMKII binding to GluN2B. During LTD, calcineurin-dependent DAPK1 activation suppresses CaMKII synaptic accumulation. Ca2+/CaM negatively regulates DAPK1/GluN2B binding, causing DAPK1 removal from synapses during LTP but retention during LTD. A pharmacogenetic approach confirmed that DAPK1 suppression of CaMKII/GluN2B binding is required for LTD.","method":"Pharmacogenetic (chemical-genetic) approach, biochemical binding assays, synaptic fractionation, LTP/LTD electrophysiology, DAPK1 inhibition and knockdown","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacogenetic dissection of DAPK1-specific function, biochemical binding competition assays, electrophysiological LTP/LTD readouts, multiple orthogonal methods","pmids":["28614711"],"is_preprint":false},{"year":2020,"finding":"In ischemic neuronal death following cardiac arrest/resuscitation, a GluN2B mutation (L1298A/R1300Q) that selectively abolishes CaMKII—but not DAPK1—binding to GluN2B is neuroprotective. CaMKII accumulates at synaptic GluN2B during ischemia; extra-synaptic GluN2B decreases but its relative association with DAPK1 increases. This demonstrates that ischemic neuronal death requires CaMKII binding to synaptic GluN2B, while DAPK1 binding is restricted to a distinct extra-synaptic GluN2B population.","method":"GluN2B knock-in mutation (L1298A/R1300Q), cardiac arrest/resuscitation mouse model, synaptic fractionation, co-immunoprecipitation, cell death quantification","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — selective knock-in mutation differentiating DAPK1 vs CaMKII binding, in vivo cardiac arrest model, biochemical fractionation, functional cell death readout","pmids":["31914378"],"is_preprint":false},{"year":2019,"finding":"A crystal structure (1.1 Å) of the KLHL20 Kelch domain bound to a DAPK1 death domain peptide reveals an 'LPDLV' motif in the DAPK1 death domain that inserts as a loose helical turn deeply into the central pocket of the KLHL20 β-propeller, contacting all six blades and forming salt-bridge and hydrophobic interactions. This structural basis determines DAPK1 recruitment and ubiquitination by the KLHL20-CUL3-ROC1 E3 ligase.","method":"X-ray crystallography (1.1 Å resolution), peptide binding assays, mutagenesis of LPDLV motif","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic-resolution crystal structure with functional validation of binding motif, single lab but highest-tier structural method","pmids":["31279627"],"is_preprint":false},{"year":2007,"finding":"DAPK-1 forms a multiprotein survival complex with cathepsin B. TNFR-1 activation induces complex formation between DAPK-1 and cathepsin B. The minimal DAPK-1 binding region for cathepsin B was mapped to amino acids 836–947. A DAPK-1(836–947) miniprotein acts as a dominant negative, promoting endogenous DAPK-1 degradation in a TNFR-1-dependent manner. Cathepsin B depletion by siRNA stimulates TNFR-1-dependent apoptosis.","method":"Co-immunoprecipitation, domain mapping with miniproteins, siRNA knockdown, dominant-negative transfection, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP with domain mapping, dominant-negative approach, siRNA functional validation, single lab","pmids":["17324927"],"is_preprint":false},{"year":2013,"finding":"DAPK acts as a scaffold protein for the LIMK/cofilin complex during TNF-induced apoptosis. TNF treatment enhances LIMK phosphorylation at Thr508 and cofilin phosphorylation at Ser3, both dependent on DAPK activity and expression. DAPK, LIMK, and cofilin redistribute to the perinuclear compartment upon TNF stimulation. Phospho-cofilin(Ser3) accumulates in cells with apoptotic morphology.","method":"Peptide array screen (binding identification), DAPK inhibitor treatment, siRNA knockdown, overexpression, immunofluorescence, 3D structural modeling","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — peptide array plus pharmacological and siRNA modulation, localization data; no direct in vitro reconstitution of ternary complex, single lab","pmids":["23702034"],"is_preprint":false},{"year":2014,"finding":"DAPK phosphorylates HSF1 at Ser230 in response to low-concentration TNF. DAPK and HSF1 physically interact both in vitro and in vivo. HSF1 binds the DAPK promoter and transient HSF1 overexpression increases DAPK mRNA and apoptosis levels, defining a DAPK-HSF1 positive-feedback loop in TNF-induced apoptosis.","method":"In vitro binding assay, co-immunoprecipitation in vivo, ChIP (HSF1 on DAPK promoter), DAPK inhibitor treatment, siRNA knockdown, overexpression, immunohistochemistry on colorectal carcinoma","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, ChIP, pharmacological inhibitor rescue, single lab with multiple orthogonal methods","pmids":["25380824"],"is_preprint":false},{"year":2012,"finding":"DAPK possesses a ROC (Ras of complex proteins) domain that binds and hydrolyzes GTP. GTP binding regulates DAPK catalytic activity by enhancing autophosphorylation at the inhibitory Ser308, thereby promoting the kinase 'off' state. This represents a novel in cis regulatory mechanism of DAPK kinase activity by its distal ROC domain.","method":"GTP binding assay, GTPase activity assay, autophosphorylation assay at Ser308, domain mutagenesis","journal":"Biochemical Society transactions","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical reconstitution of GTP binding and hydrolysis with functional autophosphorylation readout, single lab review/summary paper with limited full experimental detail in abstract","pmids":["22988864"],"is_preprint":false},{"year":2017,"finding":"Bik dissociates the Bak/Bcl-2 complex, enriching ER-associated Bak, and interacts with the kinase domain of DAPK1 to form a Bik-DAPK1-ERK1/2-Bak complex. ER-associated Bak interacts with kinase and calmodulin domains of DAPK1 to increase ER-mitochondria contact sites, facilitating mitochondrial Ca2+ uptake from the ER. Reduced Bak levels block Bik-induced mitochondrial Ca2+ uptake.","method":"Co-immunoprecipitation, domain interaction mapping (kinase/calmodulin domains), siRNA knockdown of Bak, Ca2+ imaging, ER-mitochondria contact site measurement, in vivo mouse airway model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, functional Ca2+ transfer assay, siRNA rescue, single lab with multiple orthogonal methods","pmids":["28986568"],"is_preprint":false},{"year":2016,"finding":"In C. elegans, DAPK-1 maintains epidermal integrity through regulation of the microtubule (MT) cytoskeleton. DAPK-1 physically interacts with PTRN-1 (Patronin/CAMSAP), a MT minus-end binding protein. Loss of ptrn-1 suppresses dapk-1 epidermal and innate immunity phenotypes; overexpression of the PTRN-1 CKK domain mimics dapk-1 mutant phenotypes. PTRN-1 localization is regulated by DAPK-1, and DAPK-1 itself undergoes MT-dependent transport.","method":"Genetic suppressor screen, co-immunoprecipitation, pharmacological MT destabilization/stabilization, overexpression of CKK domain, confocal localization, C. elegans genetic epistasis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis by suppressor screen, reciprocal Co-IP, pharmacological and genetic validation, localization with functional consequence, multiple orthogonal methods","pmids":["27661253"],"is_preprint":false},{"year":2019,"finding":"DAPK1 directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and proteasomal turnover. This DAPK1-mediated phosphorylation of Pellino1 couples MyD88-dependent inflammatory signaling to tubular cell damage during septic acute kidney injury under hypoxia. DAPK1 kinase activity is essential for this process; pharmacological or genetic ablation of DAPK1 protects against septic AKI.","method":"In vitro protein kinase assay, co-immunoprecipitation, GST-pulldown, ubiquitination assay, immunofluorescence, CRISPR-based gene ablation, DAPK1-/- mouse model (CLP), pharmacological DAPK1 inhibition","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay identifying Ser39 site, GST-pulldown, reciprocal Co-IP, in vivo KO mouse model, CRISPR, multiple orthogonal methods","pmids":["33052227"],"is_preprint":false},{"year":2019,"finding":"TCR-induced calcineurin activation leads to DAPK1 activation, which then interacts with TSC2 via its death domain and phosphorylates TSC2, mediating mTORC1 activation in CD8+ T cells. Both the kinase domain and death domain of DAPK1 are required for maximal mTOR activation and CD8+ T-cell antiviral function.","method":"Co-immunoprecipitation, domain deletion analysis (kinase vs. death domain), in vitro phosphorylation of TSC2, LCMV infection model (in vivo), calcineurin pharmacological activation","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain analysis, in vitro phosphorylation, in vivo viral infection model, single lab","pmids":["31541182"],"is_preprint":false},{"year":2020,"finding":"Caytaxin (a brain-specific BNIP-2 family member) interacts with DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Caytaxin levels increase as early as 2 hours after MCAO in the penumbra. Silencing Caytaxin enhances DAPK1 activity and worsens neuronal apoptosis and brain injury both in vitro and in vivo.","method":"Co-immunoprecipitation, in vitro kinase activity assay, shRNA knockdown, mouse MCAO model, in vitro neuronal apoptosis assay","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro kinase assay, in vivo mouse model with functional outcome, single lab","pmids":["32277960"],"is_preprint":false},{"year":2022,"finding":"DAPK1 catalyzes matrix adhesion assembly on rigid surfaces and triggers anoikis on soft matrices through rigidity sensing. DAPK1 phosphorylates tropomyosin1.1 and the talin1 head domain, and is itself tyrosine-phosphorylated by Src. On soft surfaces, PTPN12 activity and talin1 head promote DAPK1 dissociation from adhesion complexes and activation of apoptosis. Inhibition of DAPK1 activity transforms cells for growth on soft matrices.","method":"DAPK1 kinase inhibition, phosphorylation assays (tropomyosin1.1, talin1), Src tyrosine phosphorylation of DAPK1, PTPN12 activity assay, rigidity-sensing matrix assays, anoikis assays","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple substrate phosphorylation assays, functional rigidity-sensing and anoikis readouts, single lab","pmids":["35927990"],"is_preprint":false},{"year":2009,"finding":"An alternatively spliced product of the DAPK1 locus (s-DAPK-1), which lacks the kinase domain but contains part of the ankyrin-repeat region, destabilizes full-length DAPK-1 protein in a proteasome-independent manner. The core ankyrin-repeat region of s-DAPK-1 is sufficient for this effect; the target minimal domain on full-length DAPK-1 is the kinase domain. s-DAPK-1 itself is rapidly degraded by a proteasome-dependent pathway.","method":"Transfection of s-DAPK-1 variants, domain mapping, proteasome inhibitor treatment, pulse-chase protein stability assays","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain deletion mapping, pharmacological rescue, multiple transfection experiments, single lab","pmids":["19267229"],"is_preprint":false},{"year":2021,"finding":"DAPK1 negatively regulates IKKβ; loss of DAPK1 allows IKKβ to activate CSN5, which upregulates PD-L1 expression, enabling immune evasion of gastric cancer cells. DAPK1 overexpression restores NK cell killing ability and reduces PD-L1-mediated immune evasion. Binding affinity among DAPK1, IKKβ, CSN5, and PD-L1 was characterized in vitro and in vivo.","method":"Co-immunoprecipitation/binding assays among DAPK1-IKKβ-CSN5-PD-L1, siRNA and overexpression in GC cell lines, NK cell co-culture killing assay, in vivo xenograft model","journal":"Cellular immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — protein interaction characterization, functional NK cell killing assay, in vivo xenograft, single lab","pmids":["35114597"],"is_preprint":false},{"year":2022,"finding":"ERK phosphorylates and activates DAPK1 in epilepsy (kainic acid model). DAPK1 activation increases seizure severity and neuronal death. Selective ERK antagonism, DAPK1 gene ablation, ERK-DAPK1 uncoupling peptides, or a DAPK1 phosphorylation-deficient mutant each exert anti-seizure and anti-apoptotic effects in vitro and in vivo.","method":"ERK inhibitors, DAPK1 KO mice, uncoupling peptides, DAPK1 phosphorylation-deficient knock-in mutant, EEG, KA-induced seizure model, neuronal cell death assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO, phosphorylation-deficient mutant, peptide uncoupling, multiple functional readouts, single lab","pmids":["35742817"],"is_preprint":false},{"year":2016,"finding":"ATF6 cleavage during ER stress upregulates DAPK1 transcription. DAPK1 then promotes MRLC (myosin regulatory light chain) phosphorylation, stimulating mAtg9a trafficking that is critical for autophagosome formation. Stable ATF6 and DAPK1 knockdown cell lines show decreased LC3 conversion in response to quinocetone.","method":"Western blotting, RT-PCR, GFP-LC3 transfection, immunofluorescence, stable shRNA knockdown cell lines, ER stress inhibitor treatments","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — stable KD cell lines with multiple biochemical readouts, single lab, mechanistic pathway placement by epistasis","pmids":["27085326"],"is_preprint":false},{"year":2018,"finding":"DHA (dihydroartemisinin) induces DAPK1 expression in cholangiocarcinoma cells, which reduces the interaction of BECLIN1 with BCL-2 and promotes BECLIN1 interaction with PI3KC3, triggering autophagy-dependent cell death. Genetic silencing of DAPK1 prevents DHA-induced autophagy, establishing DAPK1-BECLIN1 as a functional signaling pathway.","method":"DAPK1 siRNA knockdown, co-immunoprecipitation (BECLIN1/BCL-2, BECLIN1/PI3KC3), pharmacological autophagy inhibition, cell death assays in multiple CCA cell lines","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP demonstrating complex remodeling, siRNA functional rescue, multiple CCA cell lines, single lab","pmids":["30136419"],"is_preprint":false},{"year":2019,"finding":"DAPK1 interacts with ZEB1 and represses ZEB1 expression in prostate cancer CD133+ stem-like cells. DAPK1 suppresses the Hippo/YAP signaling pathway through this ZEB1 interaction. DAPK1 knockdown promotes stem cell-like characteristics, sphere formation, and migration/invasion, all reversed by ZEB1 knockdown.","method":"Co-immunoprecipitation (DAPK1-ZEB1), siRNA knockdown and overexpression, sphere formation assay, tumor xenograft model, flow cytometry for stem cell markers","journal":"Stem cells and development","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for DAPK1-ZEB1 interaction, pathway placement by knockdown rescue, single lab","pmids":["34289746"],"is_preprint":false}],"current_model":"DAPK1 is a Ca2+/calmodulin-regulated serine/threonine kinase that promotes apoptosis and autophagy through multiple mechanisms: it directly binds and phosphorylates the NMDA receptor GluN2B subunit at Ser-1303 to enhance excitotoxic Ca2+ influx in stroke; phosphorylates p53 at Ser-23 via its death domain to trigger both apoptotic and necrotic neuronal death; activates MARK1/2 kinases through a death-domain-mediated mechanism to regulate microtubule stability and tau phosphorylation; is reciprocally regulated by Src (inhibitory Y491/492 phosphorylation) and LAR phosphatase (activating dephosphorylation); undergoes proteasomal degradation via the KLHL20-CUL3-ROC1 E3 ligase (which binds an LPDLV motif in the DAPK1 death domain) and via DIP-1; interacts with ERK in a bidirectional feedback loop to amplify apoptotic signaling; acts as a scaffold for the LIMK/cofilin complex during TNF-induced cytoskeletal reorganization; phosphorylates TSC2 to promote mTORC1 activation downstream of growth factor and TCR signals; and inhibits cell migration by blocking talin–integrin association and the integrin-Cdc42 polarity pathway."},"narrative":{"mechanistic_narrative":"DAPK1 is a Ca2+/calmodulin-regulated serine/threonine kinase that functions as a central executioner of apoptotic, necrotic, and autophagic death programs and as a tumor and migration suppressor, integrating signals through both its catalytic domain and a protein-interaction death domain [PMID:20141836, PMID:24806680, PMID:16476779]. In neuronal injury, DAPK1 is recruited into the NMDA receptor GluN2B (NR2B) complex during ischemia, where it binds the NR2B C-terminal tail and phosphorylates Ser-1303 to amplify channel conductance and injurious Ca2+ influx; uncoupling DAPK1 from NR2B or deleting DAPK1 is neuroprotective [PMID:20141836], and a distinct extra-synaptic GluN2B pool engages DAPK1 separately from the synaptic CaMKII pool that drives ischemic death [PMID:28614711, PMID:31914378]. DAPK1 propagates death signaling by phosphorylating substrates through its death domain, including p53 at Ser-23 to drive parallel nuclear-apoptotic and mitochondrial-necrotic pathways [PMID:24806680], and by phosphorylating TSC2 to dissociate the TSC1-TSC2 complex and activate mTORC1 downstream of growth-factor and TCR/calcineurin signals [PMID:18974095, PMID:31541182]. Beyond canonical death substrates, the death domain serves as a non-catalytic activator of MARK1/2 kinases, driving tau and MAP2/4 phosphorylation and microtubule destabilization [PMID:21311567]. DAPK1 catalytic output is held in check by a multilayered regulatory system: dephosphorylation of the inhibitory Ser-308 permits calmodulin binding and activation [PMID:17056602], an intrinsic ROC GTPase domain promotes inhibitory Ser-308 autophosphorylation [PMID:22988864], reciprocal Src (inhibitory Y491/492) and LAR phosphatase (activating) control its anti-migratory and proapoptotic activity [PMID:17803936], and ERK phosphorylates DAPK1 at Ser-735 in a positive-feedback loop that amplifies death signaling in apoptosis and seizure-induced neuronal death [PMID:15616583, PMID:35742817]. DAPK1 abundance is set by ubiquitin-proteasome turnover via the KLHL20-CUL3-ROC1 E3 ligase, which recognizes an LPDLV motif buried in the death domain [PMID:20389280, PMID:31279627], and via the E3 ligase DIP-1 [PMID:12351649]. Functionally, DAPK1 suppresses cell migration and rigidity-dependent survival by blocking talin-integrin association and the integrin-Cdc42 polarity pathway [PMID:16476779, PMID:35927990], promotes autophagy through MAP1B and BECLIN1-complex remodeling [PMID:18195017, PMID:30136419], and acts as a tumor suppressor that restrains PD-L1-mediated immune evasion [PMID:35114597].","teleology":[{"year":2002,"claim":"Establishing that DAPK protein levels are actively controlled answered how its proapoptotic potential is restrained, identifying ubiquitin-mediated turnover as a brake.","evidence":"Yeast two-hybrid, in vitro and in vivo ubiquitination, and apoptosis assays identifying the E3 ligase DIP-1","pmids":["12351649"],"confidence":"High","gaps":["Physiological signals controlling DIP-1 activity unresolved","Relationship to other DAPK1 E3 ligases not addressed"]},{"year":2004,"claim":"Linking DAPK to ERK answered how upstream MAPK signaling tunes DAPK activity, revealing a bidirectional feedback loop that amplifies apoptosis.","evidence":"In vitro kinase assay with Ser-735 mutagenesis, reciprocal co-IP, and subcellular fractionation","pmids":["15616583"],"confidence":"High","gaps":["In vivo physiological contexts of the loop not defined at this stage","Structural basis of the death-domain ERK docking site unknown"]},{"year":2006,"claim":"Defining Ser-308 dephosphorylation as an activation switch and mapping DAPK's block of talin-integrin coupling answered how DAPK is rapidly activated and how it suppresses migration independent of apoptosis.","evidence":"Ser-308 mutagenesis with TNF/ceramide time course; talin-integrin co-IP and migration/polarization assays","pmids":["17056602","16476779"],"confidence":"Medium","gaps":["Phosphatase responsible for Ser-308 dephosphorylation not identified here","Direct kinase substrate among adhesion components not defined in the migration study"]},{"year":2007,"claim":"Identifying Src/LAR tyrosine control at Y491/492 and a cathepsin B survival complex answered how DAPK is reversibly inactivated and protected from degradation, connecting it to EGF-driven tumor cell migration.","evidence":"In vitro kinase/phosphatase assays with Y491/492 mutagenesis, co-IP, migration assays, and human cancer staining; co-IP domain mapping with cathepsin B and dominant-negative miniprotein","pmids":["17803936","17324927"],"confidence":"High","gaps":["Mechanism by which Y491/492 phosphorylation drives intramolecular inactivation only inferred","Cathepsin B interaction is Medium-confidence single-lab"]},{"year":2008,"claim":"Mapping DAPK death-domain interactions to TSC2 and MAP1B answered how DAPK couples to mTORC1 signaling and to the autophagy machinery, revealing roles beyond classical apoptosis.","evidence":"Peptide aptamer/library screens, in vitro kinase assays, death-domain miniproteins, siRNA, and DAPK+/- MEF analysis","pmids":["18974095","18195017"],"confidence":"High","gaps":["TSC2 phosphosite(s) not precisely mapped","MAP1B finding is Medium-confidence single-lab","How DAPK switches between proautophagic and promTORC1 outputs unclear"]},{"year":2009,"claim":"Identifying a kinase-deficient splice variant answered whether the DAPK1 locus encodes endogenous negative regulators of the full-length protein.","evidence":"Transfection of s-DAPK-1 variants, domain mapping, proteasome inhibitor and pulse-chase stability assays","pmids":["19267229"],"confidence":"Medium","gaps":["Mechanism of proteasome-independent destabilization undefined","Physiological abundance/relevance of s-DAPK-1 not established"]},{"year":2010,"claim":"Demonstrating that DAPK1 binds and phosphorylates GluN2B at Ser-1303 and that KLHL20-CUL3-ROC1 degrades it answered, respectively, the molecular cause of excitotoxic Ca2+ influx in stroke and how interferon stabilizes DAPK1 to license death.","evidence":"Co-IP, direct binding, constitutively active DAPK1, knockout mice, in vivo NR2B uncoupling peptide and electrophysiology; reciprocal co-IP, in vitro/in vivo ubiquitination, and PML body localization for KLHL20","pmids":["20141836","20389280"],"confidence":"High","gaps":["Whether the NR2B and degradation pathways intersect not addressed","Kinetics of DAPK1 recruitment to GluN2B during ischemia partially defined"]},{"year":2011,"claim":"Showing that the death domain (not the catalytic domain) activates MARK1/2 answered how DAPK1 destabilizes microtubules and drives tau phosphorylation, defining a non-catalytic effector mechanism.","evidence":"Co-IP, domain-specific mutagenesis, in vitro kinase assay, DAPK-/- mouse brain biochemistry, and a Drosophila tauopathy model","pmids":["21311567"],"confidence":"High","gaps":["Structural basis for MARK spacer-region engagement not resolved","Direct tauopathy relevance in mammals via this axis not established"]},{"year":2012,"claim":"Identifying an intrinsic ROC GTPase domain answered how DAPK1 catalytic activity is regulated in cis, linking GTP binding to inhibitory Ser-308 autophosphorylation.","evidence":"In vitro GTP binding/hydrolysis assays, autophosphorylation at Ser-308, and domain mutagenesis","pmids":["22988864"],"confidence":"Medium","gaps":["Cellular GTP/GDP states governing this switch not measured","Limited experimental detail; single-lab summary"]},{"year":2013,"claim":"Establishing DAPK as a scaffold for LIMK/cofilin answered how DAPK links TNF signaling to cytoskeletal reorganization during apoptotic morphology changes.","evidence":"Peptide array binding, DAPK inhibitor and siRNA, immunofluorescence redistribution, and structural modeling","pmids":["23702034"],"confidence":"Medium","gaps":["No in vitro reconstitution of the ternary complex","Whether DAPK directly phosphorylates LIMK or cofilin not demonstrated"]},{"year":2014,"claim":"Identifying p53 Ser-23 phosphorylation and an HSF1 feedback loop answered how DAPK1 commits neurons to dual apoptotic/necrotic death and how it transcriptionally amplifies its own expression.","evidence":"Death-domain p53 binding/kinase assays with in vivo Tat-peptide uncoupling and ischemia model; DAPK-HSF1 co-IP, ChIP on DAPK promoter, and inhibitor/siRNA","pmids":["24806680","25380824"],"confidence":"High","gaps":["HSF1 study is Medium-confidence single-lab","Determinants partitioning p53 to nuclear versus mitochondrial fates only partially defined"]},{"year":2016,"claim":"Linking ATF6/ER stress to DAPK1 transcription and identifying PTRN-1/microtubule control in C. elegans answered how DAPK1 is induced under stress to drive autophagosome formation and how it maintains cytoskeletal/epidermal integrity.","evidence":"Stable shRNA knockdowns with LC3/MRLC readouts; C. elegans suppressor screen, co-IP with PTRN-1, and pharmacological MT modulation","pmids":["27085326","27661253"],"confidence":"High","gaps":["ATF6-DAPK1 axis is Medium-confidence single-lab","Whether mammalian DAPK1 engages CAMSAP analogously not tested"]},{"year":2017,"claim":"Defining DAPK1's competitive block of CaMKII-GluN2B binding and a Bik-DAPK1-Bak ER-mitochondria complex answered how DAPK1 mediates synaptic LTD and how it facilitates ER-to-mitochondrial Ca2+ transfer.","evidence":"Pharmacogenetic dissection with LTP/LTD electrophysiology and binding competition; co-IP domain mapping, Ca2+ imaging, and ER-mito contact measurements","pmids":["28614711","28986568"],"confidence":"High","gaps":["Bik-DAPK1-Bak study is Medium-confidence single-lab","How Ca2+/CaM dynamically gates DAPK1/GluN2B during LTP versus LTD not fully resolved"]},{"year":2019,"claim":"Resolving the KLHL20 Kelch-DAPK1 LPDLV interface, the DAPK1-TSC2-mTORC1 axis in T cells, the DAPK1-Pellino1 inflammatory pathway, and a DAPK1-ZEB1 tumor axis expanded DAPK1's mechanistic and disease reach in degradation, immunity, and cancer.","evidence":"1.1 Å crystal structure with motif mutagenesis; TCR/calcineurin co-IP, domain analysis and LCMV model; in vitro Pellino1 Ser39 kinase assay with DAPK1-/- septic AKI model; DAPK1-ZEB1 co-IP and xenograft","pmids":["31279627","31541182","33052227","34289746"],"confidence":"High","gaps":["ZEB1 finding is Low-confidence single co-IP","Whether the TSC2 axis operates identically in neurons and T cells unclear"]},{"year":2020,"claim":"Genetic separation of synaptic CaMKII from extra-synaptic DAPK1 GluN2B pools, plus identification of caytaxin as a presynaptic DAPK1 inhibitor, refined which GluN2B-associated kinase drives ischemic death and how DAPK1 is restrained in injury.","evidence":"GluN2B L1298A/R1300Q knock-in with cardiac-arrest model and fractionation; caytaxin co-IP, in vitro kinase assay, and MCAO model","pmids":["31914378","32277960"],"confidence":"High","gaps":["Caytaxin study is Medium-confidence single-lab","Functional role of the extra-synaptic DAPK1-GluN2B pool not fully defined"]},{"year":2022,"claim":"Defining DAPK1 rigidity-sensing substrates (tropomyosin1.1, talin1 head), the ERK-DAPK1 epilepsy axis, and a DAPK1-IKKβ-CSN5-PD-L1 immune-evasion pathway extended DAPK1 mechanism into mechanotransduction, seizure-induced neuronal death, and anti-tumor immunity.","evidence":"Phosphorylation/anoikis rigidity assays with Src and PTPN12; ERK inhibitors, DAPK1 KO and phospho-deficient knock-in with EEG/seizure model; DAPK1-IKKβ-CSN5-PD-L1 binding, NK killing and xenograft assays","pmids":["35927990","35742817","35114597"],"confidence":"Medium","gaps":["Rigidity-sensing and PD-L1 axes are single-lab","Direct versus indirect DAPK1 regulation of IKKβ/CSN5 not fully dissected"]},{"year":null,"claim":"How DAPK1's many context-specific outputs (excitotoxicity, mTORC1 activation, autophagy, migration suppression, immune regulation) are selected by upstream signals and substrate availability in a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating catalytic versus death-domain effector branches","Tissue-specific substrate repertoire incompletely mapped","Relative in vivo contribution of competing E3 ligases (KLHL20 vs DIP-1) unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,7,8,20,23]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,7,20,23]},{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[17]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,10,22]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8,15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,19]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,3,9]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6,27,28]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,7,21]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,11,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[20,25]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,9,13]}],"complexes":["NMDA receptor GluN2B complex","KLHL20-CUL3-ROC1 E3 ligase","Bik-DAPK1-ERK1/2-Bak complex"],"partners":["GRIN2B","TP53","TSC2","MAPK1","KLHL20","SRC","MARK1","MAP1B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P53355","full_name":"Death-associated protein kinase 1","aliases":[],"length_aa":1430,"mass_kda":160.0,"function":"Calcium/calmodulin-dependent serine/threonine kinase involved in multiple cellular signaling pathways that trigger cell survival, apoptosis, and autophagy. Regulates both type I apoptotic and type II autophagic cell deaths signal, depending on the cellular setting. The former is caspase-dependent, while the latter is caspase-independent and is characterized by the accumulation of autophagic vesicles. Phosphorylates PIN1 resulting in inhibition of its catalytic activity, nuclear localization, and cellular function. Phosphorylates TPM1, enhancing stress fiber formation in endothelial cells. Phosphorylates STX1A and significantly decreases its binding to STXBP1. Phosphorylates PRKD1 and regulates JNK signaling by binding and activating PRKD1 under oxidative stress. Phosphorylates BECN1, reducing its interaction with BCL2 and BCL2L1 and promoting the induction of autophagy. Phosphorylates TSC2, disrupting the TSC1-TSC2 complex and stimulating mTORC1 activity in a growth factor-dependent pathway. Phosphorylates RPS6, MYL9 and DAPK3. Acts as a signaling amplifier of NMDA receptors at extrasynaptic sites for mediating brain damage in stroke. Cerebral ischemia recruits DAPK1 into the NMDA receptor complex and it phosphorylates GRINB at Ser-1303 inducing injurious Ca(2+) influx through NMDA receptor channels, resulting in an irreversible neuronal death. Required together with DAPK3 for phosphorylation of RPL13A upon interferon-gamma activation which is causing RPL13A involvement in transcript-selective translation inhibition Isoform 2 cannot induce apoptosis but can induce membrane blebbing","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/P53355/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DAPK1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DAPK1","total_profiled":1310},"omim":[{"mim_id":"621390","title":"NEURODEVELOPMENTAL DISORDER WITH EARLY-ONSET SEIZURES, FACIAL DYSMORPHISM, AND BEHAVIORAL ABNORMALITIES; NEDSZFB","url":"https://www.omim.org/entry/621390"},{"mim_id":"619225","title":"RIBOSOMAL PROTEIN L13A; RPL13A","url":"https://www.omim.org/entry/619225"},{"mim_id":"617679","title":"KELCH-LIKE 20; KLHL20","url":"https://www.omim.org/entry/617679"},{"mim_id":"616567","title":"DEATH-ASSOCIATED PROTEIN KINASE 2; DAPK2","url":"https://www.omim.org/entry/616567"},{"mim_id":"615950","title":"SPEG COMPLEX LOCUS; SPEG","url":"https://www.omim.org/entry/615950"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Centrosome","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DAPK1"},"hgnc":{"alias_symbol":["DAPK"],"prev_symbol":[]},"alphafold":{"accession":"P53355","domains":[{"cath_id":"3.30.200.20","chopping":"10-95","consensus_level":"medium","plddt":86.728,"start":10,"end":95},{"cath_id":"1.10.510.10","chopping":"100-308","consensus_level":"medium","plddt":84.9725,"start":100,"end":308},{"cath_id":"3.40.50.300","chopping":"689-715_793-959","consensus_level":"high","plddt":89.2726,"start":689,"end":959},{"cath_id":"3.30.310.200","chopping":"1111-1286","consensus_level":"high","plddt":85.0819,"start":1111,"end":1286}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P53355","model_url":"https://alphafold.ebi.ac.uk/files/AF-P53355-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P53355-F1-predicted_aligned_error_v6.png","plddt_mean":82.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DAPK1","jax_strain_url":"https://www.jax.org/strain/search?query=DAPK1"},"sequence":{"accession":"P53355","fasta_url":"https://rest.uniprot.org/uniprotkb/P53355.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P53355/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P53355"}},"corpus_meta":[{"pmid":"20141836","id":"PMC_20141836","title":"DAPK1 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\"finding\": \"During cerebral ischemia, DAPK1 is recruited into the NMDA receptor NR2B protein complex in cortical neurons. DAPK1 directly binds the NR2B C-terminal tail (amino acids 1292–1304). A constitutively active DAPK1 phosphorylates NR2B at Ser-1303, enhancing NR1/NR2B receptor channel conductance and injurious Ca2+ influx. Genetic deletion of DAPK1 or peptide uncoupling of DAPK1 from NR2B blocks this Ca2+ influx and protects neurons against ischemic injury.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assays, constitutively active DAPK1 overexpression, genetic knockout mice, in vivo peptide uncoupling (NR2B CT peptide), electrophysiology\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding demonstrated, genetic KO with defined phenotype, in vivo functional rescue, multiple orthogonal methods in a high-impact study\",\n      \"pmids\": [\"20141836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DAPK interacts with ERK through a docking sequence within its death domain. ERK phosphorylates DAPK at Ser-735, increasing DAPK catalytic activity both in vitro and in vivo. Conversely, DAPK promotes cytoplasmic retention of ERK, inhibiting ERK nuclear signaling. This bidirectional regulation forms a positive feedback loop that promotes DAPK apoptotic activity.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, siRNA knockdown, subcellular fractionation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, reciprocal co-IP, multiple orthogonal methods demonstrating bidirectional regulation\",\n      \"pmids\": [\"15616583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The BTB-Kelch protein KLHL20 binds DAPK via its Kelch-repeat domain and assembles with Cullin 3 (Cul3) and ROC1 to form an E3 ligase complex that promotes DAPK polyubiquitination and proteasomal degradation. IFN-α/γ sequesters KLHL20 into PML nuclear bodies, separating it from DAPK and stabilizing DAPK. This mechanism controls IFN-induced apoptosis and autophagy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay in vitro and in vivo, siRNA depletion, immunofluorescence localization, functional cell death assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro and in vivo ubiquitination assays, localization experiments with functional consequence, multiple orthogonal methods\",\n      \"pmids\": [\"20389280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DAPK1 directly binds the DNA-binding motif of p53 via its death domain (DAPK1DD), and phosphorylates p53 at Ser-23. This phosphorylation creates a functional form of p53 that drives both apoptotic (nuclear, inducing Bax expression) and necrotic (mitochondrial matrix, via CypD interaction) neuronal death pathways. Deletion of DAPK1DD or a Tat-p53DM uncoupling peptide blocks both pathways in cortical neurons and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, deletion mutant analysis, Tat-peptide uncoupling in vivo, primary cortical neuron culture assays, mouse ischemia model\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding, in vitro phosphorylation, genetic deletion, in vivo peptide rescue with defined mechanistic readouts\",\n      \"pmids\": [\"24806680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The tyrosine phosphatase LAR dephosphorylates DAPK at pY491/492, stimulating DAPK catalytic, proapoptotic, and anti-adhesion/migration activities. Conversely, Src phosphorylates DAPK at Y491/492, inducing DAPK intramolecular/intermolecular interaction and inactivation. EGF stimulation activates Src and downregulates LAR synergistically to inactivate DAPK, facilitating tumor cell migration.\",\n      \"method\": \"In vitro kinase/phosphatase assay, site-directed mutagenesis at Y491/492, co-immunoprecipitation, cell migration assays, immunostaining of human cancer samples\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro phosphorylation/dephosphorylation with mutagenesis, reciprocal co-IP, multiple functional readouts, validated in human cancer samples\",\n      \"pmids\": [\"17803936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DAPK inhibits cell migration and polarization by blocking the association of the talin head domain with integrin, thereby suppressing the integrin-Cdc42 polarity pathway. This anti-migratory effect is apoptosis-independent and contributes to DAPK tumor suppression.\",\n      \"method\": \"Cell migration assays, cell polarization assays, co-immunoprecipitation of talin-integrin, overexpression and knockdown of DAPK, invasion assays with adenocarcinoma cell lines\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for talin-integrin interaction, multiple cell-based functional assays, mechanistic pathway dissection with defined epistasis\",\n      \"pmids\": [\"16476779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DAPK-1 binds to the microtubule-associated protein MAP1B via a linear peptide motif in the MAP1B N-terminal domain (residues 1–126). Amino acid starvation induces formation of the endogenous DAPK-1/MAP1B complex. MAP1B is required for DAPK-1-stimulated autophagy and membrane blebbing; siRNA knockdown of MAP1B attenuates both activities.\",\n      \"method\": \"Peptide combinatorial library screening, co-immunoprecipitation, siRNA knockdown, clonogenic assay, confocal co-localization, autophagy inhibitor (3-MA) treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — peptide library identification plus co-IP validation plus siRNA functional rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18195017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DAPK's death domain binds the tumor suppressor TSC2 (tuberin). Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity (stimulated by growth factors) promotes TSC2 phosphorylation in vivo, leading to TSC1-TSC2 complex dissociation. DAPK thereby acts as a positive regulator of mTORC1 signaling downstream of RAS-MEK-ERK and PI3K-AKT pathways. DAPK+/- MEFs show attenuated mTORC1 signaling.\",\n      \"method\": \"Peptide aptamer library, in vitro binding with death domain miniproteins, in vitro kinase assay, co-immunoprecipitation, siRNA knockdown, DAPK+/- MEF analysis, epistasis with pathway inhibitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution kinase assay, genetic hypomorph MEFs, epistasis experiments, multiple orthogonal binding and functional methods\",\n      \"pmids\": [\"18974095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"DAPK activates MARK1/2 kinases through its death domain (not its catalytic domain) by binding the MARK1/2 spacer region, disrupting an inhibitory intramolecular interaction in MARK. This leads to MARK-dependent phosphorylation of tau and MAP2/4, destabilizing microtubules. DAPK-/- mouse brains show reduced tau phosphorylation. In a Drosophila tauopathy model, DAPK enhances tau toxicity via PAR-1 (MARK ortholog) in a phosphorylation-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, deletion mutagenesis (catalytic vs. death domain), in vitro kinase assay, DAPK-/- mouse brain biochemistry, Drosophila genetic model, neuronal differentiation assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, domain-specific mutagenesis, genetic KO mouse, Drosophila in vivo model, multiple orthogonal methods across two organisms\",\n      \"pmids\": [\"21311567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DIP-1, a multi-RING finger protein, binds DAPK and acts as an E3 ubiquitin ligase that ubiquitinates DAPK in vitro and in vivo, targeting DAPK for proteasomal degradation. DIP-1 expression antagonizes the anti-apoptotic function of DAPK and promotes caspase-dependent apoptosis.\",\n      \"method\": \"Yeast two-hybrid and co-immunoprecipitation (binding), in vitro ubiquitination assay, in vivo ubiquitination, transient transfection apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstituted ubiquitination assay plus in vivo ubiquitination plus functional apoptosis assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12351649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DAPK activity is controlled by phosphorylation status: dephosphorylation at Ser-308 activates DAPK by allowing calcium/calmodulin association. TNF or ceramide treatment induces rapid Ser-308 dephosphorylation and transient kinase activation, followed by proteasome-dependent DAPK degradation. Dephosphorylation and activation temporally precede degradation.\",\n      \"method\": \"In vitro kinase assay, phosphorylation site mutagenesis (Ser-308), proteasome inhibitor treatment, TNF/ceramide stimulation time course, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay with mutagenesis, pharmacological inhibitor rescue, single lab with multiple biochemical methods\",\n      \"pmids\": [\"17056602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DAPK1 mediates long-term depression (LTD) by competitively blocking CaMKII binding to GluN2B. During LTD, calcineurin-dependent DAPK1 activation suppresses CaMKII synaptic accumulation. Ca2+/CaM negatively regulates DAPK1/GluN2B binding, causing DAPK1 removal from synapses during LTP but retention during LTD. A pharmacogenetic approach confirmed that DAPK1 suppression of CaMKII/GluN2B binding is required for LTD.\",\n      \"method\": \"Pharmacogenetic (chemical-genetic) approach, biochemical binding assays, synaptic fractionation, LTP/LTD electrophysiology, DAPK1 inhibition and knockdown\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacogenetic dissection of DAPK1-specific function, biochemical binding competition assays, electrophysiological LTP/LTD readouts, multiple orthogonal methods\",\n      \"pmids\": [\"28614711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In ischemic neuronal death following cardiac arrest/resuscitation, a GluN2B mutation (L1298A/R1300Q) that selectively abolishes CaMKII—but not DAPK1—binding to GluN2B is neuroprotective. CaMKII accumulates at synaptic GluN2B during ischemia; extra-synaptic GluN2B decreases but its relative association with DAPK1 increases. This demonstrates that ischemic neuronal death requires CaMKII binding to synaptic GluN2B, while DAPK1 binding is restricted to a distinct extra-synaptic GluN2B population.\",\n      \"method\": \"GluN2B knock-in mutation (L1298A/R1300Q), cardiac arrest/resuscitation mouse model, synaptic fractionation, co-immunoprecipitation, cell death quantification\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — selective knock-in mutation differentiating DAPK1 vs CaMKII binding, in vivo cardiac arrest model, biochemical fractionation, functional cell death readout\",\n      \"pmids\": [\"31914378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A crystal structure (1.1 Å) of the KLHL20 Kelch domain bound to a DAPK1 death domain peptide reveals an 'LPDLV' motif in the DAPK1 death domain that inserts as a loose helical turn deeply into the central pocket of the KLHL20 β-propeller, contacting all six blades and forming salt-bridge and hydrophobic interactions. This structural basis determines DAPK1 recruitment and ubiquitination by the KLHL20-CUL3-ROC1 E3 ligase.\",\n      \"method\": \"X-ray crystallography (1.1 Å resolution), peptide binding assays, mutagenesis of LPDLV motif\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic-resolution crystal structure with functional validation of binding motif, single lab but highest-tier structural method\",\n      \"pmids\": [\"31279627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"DAPK-1 forms a multiprotein survival complex with cathepsin B. TNFR-1 activation induces complex formation between DAPK-1 and cathepsin B. The minimal DAPK-1 binding region for cathepsin B was mapped to amino acids 836–947. A DAPK-1(836–947) miniprotein acts as a dominant negative, promoting endogenous DAPK-1 degradation in a TNFR-1-dependent manner. Cathepsin B depletion by siRNA stimulates TNFR-1-dependent apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping with miniproteins, siRNA knockdown, dominant-negative transfection, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP with domain mapping, dominant-negative approach, siRNA functional validation, single lab\",\n      \"pmids\": [\"17324927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DAPK acts as a scaffold protein for the LIMK/cofilin complex during TNF-induced apoptosis. TNF treatment enhances LIMK phosphorylation at Thr508 and cofilin phosphorylation at Ser3, both dependent on DAPK activity and expression. DAPK, LIMK, and cofilin redistribute to the perinuclear compartment upon TNF stimulation. Phospho-cofilin(Ser3) accumulates in cells with apoptotic morphology.\",\n      \"method\": \"Peptide array screen (binding identification), DAPK inhibitor treatment, siRNA knockdown, overexpression, immunofluorescence, 3D structural modeling\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — peptide array plus pharmacological and siRNA modulation, localization data; no direct in vitro reconstitution of ternary complex, single lab\",\n      \"pmids\": [\"23702034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DAPK phosphorylates HSF1 at Ser230 in response to low-concentration TNF. DAPK and HSF1 physically interact both in vitro and in vivo. HSF1 binds the DAPK promoter and transient HSF1 overexpression increases DAPK mRNA and apoptosis levels, defining a DAPK-HSF1 positive-feedback loop in TNF-induced apoptosis.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation in vivo, ChIP (HSF1 on DAPK promoter), DAPK inhibitor treatment, siRNA knockdown, overexpression, immunohistochemistry on colorectal carcinoma\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, ChIP, pharmacological inhibitor rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25380824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DAPK possesses a ROC (Ras of complex proteins) domain that binds and hydrolyzes GTP. GTP binding regulates DAPK catalytic activity by enhancing autophosphorylation at the inhibitory Ser308, thereby promoting the kinase 'off' state. This represents a novel in cis regulatory mechanism of DAPK kinase activity by its distal ROC domain.\",\n      \"method\": \"GTP binding assay, GTPase activity assay, autophosphorylation assay at Ser308, domain mutagenesis\",\n      \"journal\": \"Biochemical Society transactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical reconstitution of GTP binding and hydrolysis with functional autophosphorylation readout, single lab review/summary paper with limited full experimental detail in abstract\",\n      \"pmids\": [\"22988864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Bik dissociates the Bak/Bcl-2 complex, enriching ER-associated Bak, and interacts with the kinase domain of DAPK1 to form a Bik-DAPK1-ERK1/2-Bak complex. ER-associated Bak interacts with kinase and calmodulin domains of DAPK1 to increase ER-mitochondria contact sites, facilitating mitochondrial Ca2+ uptake from the ER. Reduced Bak levels block Bik-induced mitochondrial Ca2+ uptake.\",\n      \"method\": \"Co-immunoprecipitation, domain interaction mapping (kinase/calmodulin domains), siRNA knockdown of Bak, Ca2+ imaging, ER-mitochondria contact site measurement, in vivo mouse airway model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, functional Ca2+ transfer assay, siRNA rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28986568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In C. elegans, DAPK-1 maintains epidermal integrity through regulation of the microtubule (MT) cytoskeleton. DAPK-1 physically interacts with PTRN-1 (Patronin/CAMSAP), a MT minus-end binding protein. Loss of ptrn-1 suppresses dapk-1 epidermal and innate immunity phenotypes; overexpression of the PTRN-1 CKK domain mimics dapk-1 mutant phenotypes. PTRN-1 localization is regulated by DAPK-1, and DAPK-1 itself undergoes MT-dependent transport.\",\n      \"method\": \"Genetic suppressor screen, co-immunoprecipitation, pharmacological MT destabilization/stabilization, overexpression of CKK domain, confocal localization, C. elegans genetic epistasis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis by suppressor screen, reciprocal Co-IP, pharmacological and genetic validation, localization with functional consequence, multiple orthogonal methods\",\n      \"pmids\": [\"27661253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DAPK1 directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and proteasomal turnover. This DAPK1-mediated phosphorylation of Pellino1 couples MyD88-dependent inflammatory signaling to tubular cell damage during septic acute kidney injury under hypoxia. DAPK1 kinase activity is essential for this process; pharmacological or genetic ablation of DAPK1 protects against septic AKI.\",\n      \"method\": \"In vitro protein kinase assay, co-immunoprecipitation, GST-pulldown, ubiquitination assay, immunofluorescence, CRISPR-based gene ablation, DAPK1-/- mouse model (CLP), pharmacological DAPK1 inhibition\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay identifying Ser39 site, GST-pulldown, reciprocal Co-IP, in vivo KO mouse model, CRISPR, multiple orthogonal methods\",\n      \"pmids\": [\"33052227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TCR-induced calcineurin activation leads to DAPK1 activation, which then interacts with TSC2 via its death domain and phosphorylates TSC2, mediating mTORC1 activation in CD8+ T cells. Both the kinase domain and death domain of DAPK1 are required for maximal mTOR activation and CD8+ T-cell antiviral function.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion analysis (kinase vs. death domain), in vitro phosphorylation of TSC2, LCMV infection model (in vivo), calcineurin pharmacological activation\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain analysis, in vitro phosphorylation, in vivo viral infection model, single lab\",\n      \"pmids\": [\"31541182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Caytaxin (a brain-specific BNIP-2 family member) interacts with DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Caytaxin levels increase as early as 2 hours after MCAO in the penumbra. Silencing Caytaxin enhances DAPK1 activity and worsens neuronal apoptosis and brain injury both in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase activity assay, shRNA knockdown, mouse MCAO model, in vitro neuronal apoptosis assay\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro kinase assay, in vivo mouse model with functional outcome, single lab\",\n      \"pmids\": [\"32277960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DAPK1 catalyzes matrix adhesion assembly on rigid surfaces and triggers anoikis on soft matrices through rigidity sensing. DAPK1 phosphorylates tropomyosin1.1 and the talin1 head domain, and is itself tyrosine-phosphorylated by Src. On soft surfaces, PTPN12 activity and talin1 head promote DAPK1 dissociation from adhesion complexes and activation of apoptosis. Inhibition of DAPK1 activity transforms cells for growth on soft matrices.\",\n      \"method\": \"DAPK1 kinase inhibition, phosphorylation assays (tropomyosin1.1, talin1), Src tyrosine phosphorylation of DAPK1, PTPN12 activity assay, rigidity-sensing matrix assays, anoikis assays\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple substrate phosphorylation assays, functional rigidity-sensing and anoikis readouts, single lab\",\n      \"pmids\": [\"35927990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"An alternatively spliced product of the DAPK1 locus (s-DAPK-1), which lacks the kinase domain but contains part of the ankyrin-repeat region, destabilizes full-length DAPK-1 protein in a proteasome-independent manner. The core ankyrin-repeat region of s-DAPK-1 is sufficient for this effect; the target minimal domain on full-length DAPK-1 is the kinase domain. s-DAPK-1 itself is rapidly degraded by a proteasome-dependent pathway.\",\n      \"method\": \"Transfection of s-DAPK-1 variants, domain mapping, proteasome inhibitor treatment, pulse-chase protein stability assays\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain deletion mapping, pharmacological rescue, multiple transfection experiments, single lab\",\n      \"pmids\": [\"19267229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DAPK1 negatively regulates IKKβ; loss of DAPK1 allows IKKβ to activate CSN5, which upregulates PD-L1 expression, enabling immune evasion of gastric cancer cells. DAPK1 overexpression restores NK cell killing ability and reduces PD-L1-mediated immune evasion. Binding affinity among DAPK1, IKKβ, CSN5, and PD-L1 was characterized in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation/binding assays among DAPK1-IKKβ-CSN5-PD-L1, siRNA and overexpression in GC cell lines, NK cell co-culture killing assay, in vivo xenograft model\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — protein interaction characterization, functional NK cell killing assay, in vivo xenograft, single lab\",\n      \"pmids\": [\"35114597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERK phosphorylates and activates DAPK1 in epilepsy (kainic acid model). DAPK1 activation increases seizure severity and neuronal death. Selective ERK antagonism, DAPK1 gene ablation, ERK-DAPK1 uncoupling peptides, or a DAPK1 phosphorylation-deficient mutant each exert anti-seizure and anti-apoptotic effects in vitro and in vivo.\",\n      \"method\": \"ERK inhibitors, DAPK1 KO mice, uncoupling peptides, DAPK1 phosphorylation-deficient knock-in mutant, EEG, KA-induced seizure model, neuronal cell death assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, phosphorylation-deficient mutant, peptide uncoupling, multiple functional readouts, single lab\",\n      \"pmids\": [\"35742817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATF6 cleavage during ER stress upregulates DAPK1 transcription. DAPK1 then promotes MRLC (myosin regulatory light chain) phosphorylation, stimulating mAtg9a trafficking that is critical for autophagosome formation. Stable ATF6 and DAPK1 knockdown cell lines show decreased LC3 conversion in response to quinocetone.\",\n      \"method\": \"Western blotting, RT-PCR, GFP-LC3 transfection, immunofluorescence, stable shRNA knockdown cell lines, ER stress inhibitor treatments\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — stable KD cell lines with multiple biochemical readouts, single lab, mechanistic pathway placement by epistasis\",\n      \"pmids\": [\"27085326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DHA (dihydroartemisinin) induces DAPK1 expression in cholangiocarcinoma cells, which reduces the interaction of BECLIN1 with BCL-2 and promotes BECLIN1 interaction with PI3KC3, triggering autophagy-dependent cell death. Genetic silencing of DAPK1 prevents DHA-induced autophagy, establishing DAPK1-BECLIN1 as a functional signaling pathway.\",\n      \"method\": \"DAPK1 siRNA knockdown, co-immunoprecipitation (BECLIN1/BCL-2, BECLIN1/PI3KC3), pharmacological autophagy inhibition, cell death assays in multiple CCA cell lines\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP demonstrating complex remodeling, siRNA functional rescue, multiple CCA cell lines, single lab\",\n      \"pmids\": [\"30136419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DAPK1 interacts with ZEB1 and represses ZEB1 expression in prostate cancer CD133+ stem-like cells. DAPK1 suppresses the Hippo/YAP signaling pathway through this ZEB1 interaction. DAPK1 knockdown promotes stem cell-like characteristics, sphere formation, and migration/invasion, all reversed by ZEB1 knockdown.\",\n      \"method\": \"Co-immunoprecipitation (DAPK1-ZEB1), siRNA knockdown and overexpression, sphere formation assay, tumor xenograft model, flow cytometry for stem cell markers\",\n      \"journal\": \"Stem cells and development\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for DAPK1-ZEB1 interaction, pathway placement by knockdown rescue, single lab\",\n      \"pmids\": [\"34289746\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DAPK1 is a Ca2+/calmodulin-regulated serine/threonine kinase that promotes apoptosis and autophagy through multiple mechanisms: it directly binds and phosphorylates the NMDA receptor GluN2B subunit at Ser-1303 to enhance excitotoxic Ca2+ influx in stroke; phosphorylates p53 at Ser-23 via its death domain to trigger both apoptotic and necrotic neuronal death; activates MARK1/2 kinases through a death-domain-mediated mechanism to regulate microtubule stability and tau phosphorylation; is reciprocally regulated by Src (inhibitory Y491/492 phosphorylation) and LAR phosphatase (activating dephosphorylation); undergoes proteasomal degradation via the KLHL20-CUL3-ROC1 E3 ligase (which binds an LPDLV motif in the DAPK1 death domain) and via DIP-1; interacts with ERK in a bidirectional feedback loop to amplify apoptotic signaling; acts as a scaffold for the LIMK/cofilin complex during TNF-induced cytoskeletal reorganization; phosphorylates TSC2 to promote mTORC1 activation downstream of growth factor and TCR signals; and inhibits cell migration by blocking talin–integrin association and the integrin-Cdc42 polarity pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DAPK1 is a Ca2+/calmodulin-regulated serine/threonine kinase that functions as a central executioner of apoptotic, necrotic, and autophagic death programs and as a tumor and migration suppressor, integrating signals through both its catalytic domain and a protein-interaction death domain [#0, #3, #5]. In neuronal injury, DAPK1 is recruited into the NMDA receptor GluN2B (NR2B) complex during ischemia, where it binds the NR2B C-terminal tail and phosphorylates Ser-1303 to amplify channel conductance and injurious Ca2+ influx; uncoupling DAPK1 from NR2B or deleting DAPK1 is neuroprotective [#0], and a distinct extra-synaptic GluN2B pool engages DAPK1 separately from the synaptic CaMKII pool that drives ischemic death [#11, #12]. DAPK1 propagates death signaling by phosphorylating substrates through its death domain, including p53 at Ser-23 to drive parallel nuclear-apoptotic and mitochondrial-necrotic pathways [#3], and by phosphorylating TSC2 to dissociate the TSC1-TSC2 complex and activate mTORC1 downstream of growth-factor and TCR/calcineurin signals [#7, #21]. Beyond canonical death substrates, the death domain serves as a non-catalytic activator of MARK1/2 kinases, driving tau and MAP2/4 phosphorylation and microtubule destabilization [#8]. DAPK1 catalytic output is held in check by a multilayered regulatory system: dephosphorylation of the inhibitory Ser-308 permits calmodulin binding and activation [#10], an intrinsic ROC GTPase domain promotes inhibitory Ser-308 autophosphorylation [#17], reciprocal Src (inhibitory Y491/492) and LAR phosphatase (activating) control its anti-migratory and proapoptotic activity [#4], and ERK phosphorylates DAPK1 at Ser-735 in a positive-feedback loop that amplifies death signaling in apoptosis and seizure-induced neuronal death [#1, #26]. DAPK1 abundance is set by ubiquitin-proteasome turnover via the KLHL20-CUL3-ROC1 E3 ligase, which recognizes an LPDLV motif buried in the death domain [#2, #13], and via the E3 ligase DIP-1 [#9]. Functionally, DAPK1 suppresses cell migration and rigidity-dependent survival by blocking talin-integrin association and the integrin-Cdc42 polarity pathway [#5, #23], promotes autophagy through MAP1B and BECLIN1-complex remodeling [#6, #28], and acts as a tumor suppressor that restrains PD-L1-mediated immune evasion [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that DAPK protein levels are actively controlled answered how its proapoptotic potential is restrained, identifying ubiquitin-mediated turnover as a brake.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro and in vivo ubiquitination, and apoptosis assays identifying the E3 ligase DIP-1\",\n      \"pmids\": [\"12351649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals controlling DIP-1 activity unresolved\", \"Relationship to other DAPK1 E3 ligases not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking DAPK to ERK answered how upstream MAPK signaling tunes DAPK activity, revealing a bidirectional feedback loop that amplifies apoptosis.\",\n      \"evidence\": \"In vitro kinase assay with Ser-735 mutagenesis, reciprocal co-IP, and subcellular fractionation\",\n      \"pmids\": [\"15616583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological contexts of the loop not defined at this stage\", \"Structural basis of the death-domain ERK docking site unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defining Ser-308 dephosphorylation as an activation switch and mapping DAPK's block of talin-integrin coupling answered how DAPK is rapidly activated and how it suppresses migration independent of apoptosis.\",\n      \"evidence\": \"Ser-308 mutagenesis with TNF/ceramide time course; talin-integrin co-IP and migration/polarization assays\",\n      \"pmids\": [\"17056602\", \"16476779\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphatase responsible for Ser-308 dephosphorylation not identified here\", \"Direct kinase substrate among adhesion components not defined in the migration study\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identifying Src/LAR tyrosine control at Y491/492 and a cathepsin B survival complex answered how DAPK is reversibly inactivated and protected from degradation, connecting it to EGF-driven tumor cell migration.\",\n      \"evidence\": \"In vitro kinase/phosphatase assays with Y491/492 mutagenesis, co-IP, migration assays, and human cancer staining; co-IP domain mapping with cathepsin B and dominant-negative miniprotein\",\n      \"pmids\": [\"17803936\", \"17324927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Y491/492 phosphorylation drives intramolecular inactivation only inferred\", \"Cathepsin B interaction is Medium-confidence single-lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapping DAPK death-domain interactions to TSC2 and MAP1B answered how DAPK couples to mTORC1 signaling and to the autophagy machinery, revealing roles beyond classical apoptosis.\",\n      \"evidence\": \"Peptide aptamer/library screens, in vitro kinase assays, death-domain miniproteins, siRNA, and DAPK+/- MEF analysis\",\n      \"pmids\": [\"18974095\", \"18195017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TSC2 phosphosite(s) not precisely mapped\", \"MAP1B finding is Medium-confidence single-lab\", \"How DAPK switches between proautophagic and promTORC1 outputs unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying a kinase-deficient splice variant answered whether the DAPK1 locus encodes endogenous negative regulators of the full-length protein.\",\n      \"evidence\": \"Transfection of s-DAPK-1 variants, domain mapping, proteasome inhibitor and pulse-chase stability assays\",\n      \"pmids\": [\"19267229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of proteasome-independent destabilization undefined\", \"Physiological abundance/relevance of s-DAPK-1 not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that DAPK1 binds and phosphorylates GluN2B at Ser-1303 and that KLHL20-CUL3-ROC1 degrades it answered, respectively, the molecular cause of excitotoxic Ca2+ influx in stroke and how interferon stabilizes DAPK1 to license death.\",\n      \"evidence\": \"Co-IP, direct binding, constitutively active DAPK1, knockout mice, in vivo NR2B uncoupling peptide and electrophysiology; reciprocal co-IP, in vitro/in vivo ubiquitination, and PML body localization for KLHL20\",\n      \"pmids\": [\"20141836\", \"20389280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the NR2B and degradation pathways intersect not addressed\", \"Kinetics of DAPK1 recruitment to GluN2B during ischemia partially defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showing that the death domain (not the catalytic domain) activates MARK1/2 answered how DAPK1 destabilizes microtubules and drives tau phosphorylation, defining a non-catalytic effector mechanism.\",\n      \"evidence\": \"Co-IP, domain-specific mutagenesis, in vitro kinase assay, DAPK-/- mouse brain biochemistry, and a Drosophila tauopathy model\",\n      \"pmids\": [\"21311567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for MARK spacer-region engagement not resolved\", \"Direct tauopathy relevance in mammals via this axis not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying an intrinsic ROC GTPase domain answered how DAPK1 catalytic activity is regulated in cis, linking GTP binding to inhibitory Ser-308 autophosphorylation.\",\n      \"evidence\": \"In vitro GTP binding/hydrolysis assays, autophosphorylation at Ser-308, and domain mutagenesis\",\n      \"pmids\": [\"22988864\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular GTP/GDP states governing this switch not measured\", \"Limited experimental detail; single-lab summary\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing DAPK as a scaffold for LIMK/cofilin answered how DAPK links TNF signaling to cytoskeletal reorganization during apoptotic morphology changes.\",\n      \"evidence\": \"Peptide array binding, DAPK inhibitor and siRNA, immunofluorescence redistribution, and structural modeling\",\n      \"pmids\": [\"23702034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of the ternary complex\", \"Whether DAPK directly phosphorylates LIMK or cofilin not demonstrated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying p53 Ser-23 phosphorylation and an HSF1 feedback loop answered how DAPK1 commits neurons to dual apoptotic/necrotic death and how it transcriptionally amplifies its own expression.\",\n      \"evidence\": \"Death-domain p53 binding/kinase assays with in vivo Tat-peptide uncoupling and ischemia model; DAPK-HSF1 co-IP, ChIP on DAPK promoter, and inhibitor/siRNA\",\n      \"pmids\": [\"24806680\", \"25380824\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"HSF1 study is Medium-confidence single-lab\", \"Determinants partitioning p53 to nuclear versus mitochondrial fates only partially defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linking ATF6/ER stress to DAPK1 transcription and identifying PTRN-1/microtubule control in C. elegans answered how DAPK1 is induced under stress to drive autophagosome formation and how it maintains cytoskeletal/epidermal integrity.\",\n      \"evidence\": \"Stable shRNA knockdowns with LC3/MRLC readouts; C. elegans suppressor screen, co-IP with PTRN-1, and pharmacological MT modulation\",\n      \"pmids\": [\"27085326\", \"27661253\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATF6-DAPK1 axis is Medium-confidence single-lab\", \"Whether mammalian DAPK1 engages CAMSAP analogously not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defining DAPK1's competitive block of CaMKII-GluN2B binding and a Bik-DAPK1-Bak ER-mitochondria complex answered how DAPK1 mediates synaptic LTD and how it facilitates ER-to-mitochondrial Ca2+ transfer.\",\n      \"evidence\": \"Pharmacogenetic dissection with LTP/LTD electrophysiology and binding competition; co-IP domain mapping, Ca2+ imaging, and ER-mito contact measurements\",\n      \"pmids\": [\"28614711\", \"28986568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Bik-DAPK1-Bak study is Medium-confidence single-lab\", \"How Ca2+/CaM dynamically gates DAPK1/GluN2B during LTP versus LTD not fully resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolving the KLHL20 Kelch-DAPK1 LPDLV interface, the DAPK1-TSC2-mTORC1 axis in T cells, the DAPK1-Pellino1 inflammatory pathway, and a DAPK1-ZEB1 tumor axis expanded DAPK1's mechanistic and disease reach in degradation, immunity, and cancer.\",\n      \"evidence\": \"1.1 Å crystal structure with motif mutagenesis; TCR/calcineurin co-IP, domain analysis and LCMV model; in vitro Pellino1 Ser39 kinase assay with DAPK1-/- septic AKI model; DAPK1-ZEB1 co-IP and xenograft\",\n      \"pmids\": [\"31279627\", \"31541182\", \"33052227\", \"34289746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ZEB1 finding is Low-confidence single co-IP\", \"Whether the TSC2 axis operates identically in neurons and T cells unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Genetic separation of synaptic CaMKII from extra-synaptic DAPK1 GluN2B pools, plus identification of caytaxin as a presynaptic DAPK1 inhibitor, refined which GluN2B-associated kinase drives ischemic death and how DAPK1 is restrained in injury.\",\n      \"evidence\": \"GluN2B L1298A/R1300Q knock-in with cardiac-arrest model and fractionation; caytaxin co-IP, in vitro kinase assay, and MCAO model\",\n      \"pmids\": [\"31914378\", \"32277960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Caytaxin study is Medium-confidence single-lab\", \"Functional role of the extra-synaptic DAPK1-GluN2B pool not fully defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defining DAPK1 rigidity-sensing substrates (tropomyosin1.1, talin1 head), the ERK-DAPK1 epilepsy axis, and a DAPK1-IKKβ-CSN5-PD-L1 immune-evasion pathway extended DAPK1 mechanism into mechanotransduction, seizure-induced neuronal death, and anti-tumor immunity.\",\n      \"evidence\": \"Phosphorylation/anoikis rigidity assays with Src and PTPN12; ERK inhibitors, DAPK1 KO and phospho-deficient knock-in with EEG/seizure model; DAPK1-IKKβ-CSN5-PD-L1 binding, NK killing and xenograft assays\",\n      \"pmids\": [\"35927990\", \"35742817\", \"35114597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rigidity-sensing and PD-L1 axes are single-lab\", \"Direct versus indirect DAPK1 regulation of IKKβ/CSN5 not fully dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DAPK1's many context-specific outputs (excitotoxicity, mTORC1 activation, autophagy, migration suppression, immune regulation) are selected by upstream signals and substrate availability in a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating catalytic versus death-domain effector branches\", \"Tissue-specific substrate repertoire incompletely mapped\", \"Relative in vivo contribution of competing E3 ligases (KLHL20 vs DIP-1) unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 7, 8, 20, 23]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 7, 20, 23]},\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 10, 22]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 3, 9]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6, 27, 28]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 7, 21]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 11, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [20, 25]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 9, 13]}\n    ],\n    \"complexes\": [\n      \"NMDA receptor GluN2B complex\",\n      \"KLHL20-CUL3-ROC1 E3 ligase\",\n      \"Bik-DAPK1-ERK1/2-Bak complex\"\n    ],\n    \"partners\": [\n      \"GRIN2B\",\n      \"TP53\",\n      \"TSC2\",\n      \"MAPK1\",\n      \"KLHL20\",\n      \"SRC\",\n      \"MARK1\",\n      \"MAP1B\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}