{"gene":"DAPK2","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2011,"finding":"Crystal and solution structures of murine DAPK2 revealed that DAPK2 forms dimers through apposed catalytic domains in an autoinhibited conformation that precludes protein substrate binding; the 'basic loop' fingerprint of the DAPK family plays a central role in kinase domain dimerization, and two distinct active-site conformations are observed upon nucleotide binding.","method":"X-ray crystallography and solution scattering (SAXS) of recombinant murine DAPK2 with and without nucleotide ligands","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus solution structure, multiple conditions tested, single rigorous structural study with functional model","pmids":["21497605"],"is_preprint":false},{"year":2014,"finding":"DAPK2 directly interacts with and phosphorylates components of mTORC1; co-immunoprecipitation showed DAPK2 associates with mTOR, raptor, and ULK1, recombinant protein-binding assay confirmed direct DAPK2–raptor interaction, and in vitro kinase assay showed DAPK2 phosphorylates raptor at Ser721. DAPK2 knockdown increased mTORC1 kinase activity and reduced autophagy induced by amino acid deprivation or elevated intracellular Ca²⁺.","method":"Co-immunoprecipitation, recombinant protein-binding assay, in vitro kinase assay, siRNA knockdown with mTORC1 substrate phosphorylation readout","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay + recombinant binding + reciprocal Co-IP + functional KD, single lab but multiple orthogonal methods","pmids":["25361081"],"is_preprint":false},{"year":2018,"finding":"AMPK phosphorylates DAPK2 at a site between the catalytic and calmodulin-binding domains (Ser289), activating DAPK2 by functionally mimicking calmodulin binding and mitigating inhibitory autophosphorylation. Activated DAPK2 in turn phosphorylates Beclin-1, causing dissociation of its inhibitor Bcl-XL and promoting autophagy; DAPK2 depletion reduced autophagy in response to AMPK activation.","method":"In vitro kinase assays, phosphosite mapping, co-immunoprecipitation, siRNA knockdown, autophagy flux assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assays with phosphosite identification, Co-IP, and functional KD with multiple readouts, single lab with multiple orthogonal methods","pmids":["29717115"],"is_preprint":false},{"year":2021,"finding":"14-3-3 proteins bind DAPK2 via a canonical mode III motif created by phosphorylation of Thr369 at the DAPK2 C-terminus. X-ray crystallographic analysis of the full-length human DAPK2:14-3-3 complex showed that 14-3-3 binding stabilizes DAPK2 dimers, protects the inhibitory autophosphorylation site Ser318 from dephosphorylation, and prevents Ca²⁺/CaM binding, thereby inactivating DAPK2. The interaction is further enhanced by the diterpene glycoside Fusicoccin A.","method":"X-ray crystallography, biophysical binding assays (ITC, SAXS), phosphosite mapping, mutagenesis","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of complex plus multiple biophysical methods in single rigorous study, single lab","pmids":["34413451"],"is_preprint":false},{"year":2019,"finding":"Ser289 phosphorylation enhances DAPK2 catalytic activity (analogous to its effect on DAPK1); AMPK-mediated Ser289 phosphorylation of DAPK2 was compared to RSK-mediated phosphorylation of DAPK1 in the same cells, and the signaling pathways leading to Ser289 phosphorylation are mutually exclusive between DAPK1 and DAPK2.","method":"In-cell kinase activity assays, phospho-specific immunoblotting, pathway inhibitor treatments","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional kinase activity comparison in cells, single lab, single study","pmids":["31116076"],"is_preprint":false},{"year":2009,"finding":"DAPK2 promoter hypermethylation silences DAPK2 expression in Hodgkin lymphoma cell lines; a constitutively active calmodulin-deletion mutant of DAPK2 fused to CD30 ligand (immunokinase DAPK2'-CD30L) selectively induced apoptosis and inhibited proliferation in CD30-positive/DAPK2-negative tumor cells, demonstrating that restoration of DAPK2 catalytic activity is sufficient for pro-apoptotic function.","method":"Promoter methylation analysis, recombinant fusion protein construction, cell viability and apoptosis assays in tumor cell lines","journal":"Journal of immunotherapy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional reconstitution with constitutively active mutant in cells, methylation mechanism identified, single lab","pmids":["19609235"],"is_preprint":false},{"year":2013,"finding":"DAPK2 positively regulates motility of human neutrophils and eosinophils in response to intermediary chemoattractants; pharmacological inhibition of DAPK activity abolished granulocyte migration associated with reduced MLC phosphorylation and loss of cell polarization with radial F-actin, and reduced neutrophil recruitment to the peritoneum in a mouse peritonitis model.","method":"Pharmacological DAPK inhibition, ex vivo chemotaxis assays, F-actin/MLC phosphorylation analysis, in vivo peritonitis model","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo functional assays with mechanistic readout (MLC phosphorylation), single lab, two orthogonal systems","pmids":["24163421"],"is_preprint":false},{"year":2013,"finding":"DAPK2 expression is transcriptionally activated by the myeloid transcription factors PU.1 and C/EBPα during granulocytic differentiation; PML-RARα binds the DAPK2 promoter and represses its transcription in APL. Restoration of DAPK2 expression in PU.1-knockdown APL cells partially rescued neutrophil differentiation, establishing DAPK2 as a relevant PU.1 downstream effector.","method":"ChIP (PML-RARA and PU.1 binding at DAPK2 promoter), ectopic expression, PU.1 knockdown, differentiation assays","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for transcription factor binding plus functional rescue experiment, single lab, two orthogonal methods","pmids":["24038216"],"is_preprint":false},{"year":2008,"finding":"E2F1 and KLF6 transcriptionally activate the DAPK2 promoter via a GC-rich region upstream of exon 1 in an Sp1-dependent manner (no canonical E2F binding sites); ChIP confirmed Sp1 recruitment (and lesser E2F1/KLF6 recruitment) to the DAPK2 promoter. DAPK2 induction mediates the pro-apoptotic effects of both E2F1 and KLF6, as DAPK2 knockdown significantly reduced cell death upon activation of either transcription factor.","method":"Promoter reporter assays, chromatin immunoprecipitation (ChIP), Sp1-deficient insect cells, mithramycin A treatment, siRNA knockdown, cell death assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional KD rescue and multiple reporter assays, single lab, orthogonal methods","pmids":["18521079"],"is_preprint":false},{"year":2014,"finding":"Genetic ablation of DAPK2 by RNAi causes NF-κB phosphorylation and transcriptional activation, leading to upregulation of DR4 and DR5 (TRAIL receptors) on the cell surface and sensitizing resistant cancer cells to TRAIL-induced apoptosis in a p53-independent manner.","method":"siRNA knockdown, NF-κB reporter assay, cell surface flow cytometry for DR4/DR5, TRAIL apoptosis assays in multiple cancer cell lines","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with multiple orthogonal pathway readouts in several cell lines, single lab","pmids":["25012503"],"is_preprint":false},{"year":2015,"finding":"Adenoviral DAPK2 overexpression in 3T3-L1 adipocytes increased autophagic clearance in nutrient-rich conditions in a kinase activity-dependent manner; conversely, siRNA-mediated DAPK2 inhibition in human preadipocytes decreased LC3-II accumulation rates measured with lysosome inhibitors, demonstrating DAPK2 kinase activity is required for autophagic flux in adipocytes.","method":"Adenoviral overexpression (kinase-active and kinase-dead), siRNA knockdown, LC3-II accumulation assay with lysosome inhibitors in 3T3-L1 and human preadipocytes","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutant controls plus gain- and loss-of-function in two cell systems, single lab","pmids":["26038578"],"is_preprint":false},{"year":2016,"finding":"DAPK2 transcriptional activation by thyroid hormone (TH) enhances phosphorylation of SQSTM1/p62, promoting selective autophagic clearance of protein aggregates; ectopic DAPK2 expression attenuated DEN-induced hepatotoxicity and DNA damage via enhanced autophagy, while DAPK2 knockdown had the opposite effect.","method":"Adeno-associated virus knockdown, ectopic DAPK2 expression, phospho-SQSTM1 immunoblot, autophagy flux assays, murine hepatocarcinogenesis model","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro gain/loss-of-function with mechanistic phosphorylation readout, single lab","pmids":["27653365"],"is_preprint":false},{"year":2017,"finding":"TAp73 isoform binds to and activates the DAPK2 promoter, whereas ΔNp73 inhibits DAPK2 transcription; DAPK2 is an important downstream effector of p73 in ATO-induced apoptosis. Conversely, the p73-DAPK2 pathway is essential for ATRA-induced autophagy mediated by a direct interaction between DAPK2 and ATG5. DAPK2 also binds and stabilizes p73 protein, forming a positive feedback loop.","method":"Promoter reporter assay (TAp73/ΔNp73), siRNA knockdown of TP73 and DAPK2, co-immunoprecipitation of DAPK2-ATG5, apoptosis and autophagy assays in APL cells","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — promoter assay + Co-IP + functional KD with multiple readouts, single lab","pmids":["28978663"],"is_preprint":false},{"year":2015,"finding":"RNA interference-mediated depletion of endogenous DAPK2 in cancer cells causes decreased oxidative phosphorylation, destabilized mitochondrial membrane potential, increased mitochondrial superoxide anion production, and activation of ERK, JNK, and p38 stress kinases; overexpression of a kinase-dead DAPK2 mutant further enhanced oxidative stress, indicating that DAPK2 kinase activity is required to maintain mitochondrial integrity.","method":"siRNA knockdown, kinase-dead mutant overexpression, Seahorse metabolic flux analysis, mitochondrial membrane potential assay, superoxide detection, stress kinase immunoblotting","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD plus kinase-dead mutant with multiple orthogonal metabolic readouts, single lab","pmids":["25741596"],"is_preprint":false},{"year":2021,"finding":"Cigarette smoking-induced METTL3-mediated m6A modification of DAPK2 mRNA is read by YTHDF2, resulting in decreased DAPK2 mRNA stability and reduced DAPK2 expression; downregulation of DAPK2 activates NF-κB signaling to promote NSCLC proliferation and migration.","method":"m6A sequencing/MeRIP, METTL3/YTHDF2 knockdown, mRNA stability assay, NF-κB pathway inhibitor (BAY 11-7085), in vitro and in vivo functional assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A writer and reader identified by KD with mRNA stability readout, NF-κB pathway functionally validated, single lab","pmids":["34298122"],"is_preprint":false},{"year":2025,"finding":"DAPK2 dysfunction alters Mic60 (mitofilin) protein in mitochondrial cristae by promoting its lactylation, increasing cristae abundance and compactness and activating mitochondrial metabolism, leading to anoikis resistance and enhanced metastasis in EGFR-TKI-resistant lung cancer cells.","method":"Mass spectrometry, immunoprecipitation, anoikis-resistant cell model, mouse tail vein metastasis model, Mic60 lactylation analysis, mitochondrial metabolic assays","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification of lactylation + in vivo metastasis model + Co-IP, single lab, multiple methods","pmids":["40812308"],"is_preprint":false},{"year":2026,"finding":"DAPK2 directly phosphorylates PKM2 at threonine 45, causing PKM2 dimerization and nuclear translocation; nuclear PKM2 activates VCAM-1 and ICAM-1 expression by interacting with STAT1, promoting endothelial inflammation in response to oscillatory shear stress. KLF2 suppresses DAPK2 transcription, and OSS-induced KLF2 downregulation leads to DAPK2 upregulation. EC-specific Dapk2 deficiency reduced atherogenesis in Apoe-/- mice, and Pkm2T45A overexpression mitigated disturbed flow-induced atherogenesis.","method":"Mass spectrometry phosphosite identification, immunoprecipitation, proximity ligation assay, dominant-negative DAPK2 mutant, EC-specific Dapk2 knockout in Apoe-/- mice (carotid ligation and Western diet models), Pkm2T45A and Pkm2T45E knock-in overexpression mice","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mass spec phosphosite mapping + multiple in vivo genetic models (KO + phospho-mimetic/refractory mutants) + multiple orthogonal biochemical methods","pmids":["41614276"],"is_preprint":false},{"year":2019,"finding":"PCB118 enhances binding between Tubb3 (tubulin beta 3) and DAPK2 in thyroid cells; siRNA knockdown of Tubb3 suppressed DAPK2 protein expression and PKD phosphorylation, while DAPK2 (downstream of Tubb3) activated PKD which in turn phosphorylated VPS34, mediating autophagy. DAPK2 overexpression following PCB118 exposure upregulated PKD and VPS34 in the DAPK2/PKD/VPS34 pathway.","method":"Co-immunoprecipitation (Tubb3-DAPK2 interaction), siRNA knockdown, overexpression, immunofluorescence, rat in vivo model","journal":"Archives of toxicology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and KD with pathway readout, single lab, single study, no direct DAPK2 kinase assay on PKD","pmids":["31020377"],"is_preprint":false},{"year":2026,"finding":"In osteoblasts, VGLL3 regulates DAPK2 expression (identified by RNA-seq of Vgll3-KD cells); DAPK2 knockdown phenocopied Vgll3 loss, reducing autophagic flux and impairing osteoblast differentiation. DAPK2 overexpression partially rescued autophagy and osteogenic differentiation in Vgll3-deficient cells, and FOXM1 was implicated as a potential transcriptional regulator of DAPK2 in this context.","method":"RNA-seq, shRNA knockdown (Vgll3 and Dapk2), DAPK2 overexpression rescue, LC3-II/p62 immunoblot, transmission electron microscopy, alkaline phosphatase/Alizarin Red staining","journal":"BioFactors","confidence":"Low","confidence_rationale":"Tier 3 / Weak — KD and rescue with autophagy/differentiation readouts, single lab, FOXM1 link is preliminary","pmids":["42052791"],"is_preprint":false},{"year":2026,"finding":"YTHDF3 directly recognizes m6A-modified Dapk2 transcripts and promotes their decay, thereby suppressing DAPK2 protein levels; melatonin rescues YTHDF3 expression under mechanical unloading, leading to DAPK2 mRNA degradation that supports osteoblast differentiation and survival. DAPK2 was characterized as a negative regulator of osteoblast differentiation and survival.","method":"m6A-seq/MeRIP, YTHDF3 KD/OE, Dapk2 KD, hindlimb unloading mouse model, western blot, qPCR, mineralization assays","journal":"Journal of pineal research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — m6A reader-mRNA decay link and functional osteoblast phenotype, single lab, mechanistic link between YTHDF3 and DAPK2 decay shown by KD but no direct binding validated in this study","pmids":["42149012"],"is_preprint":false}],"current_model":"DAPK2 is a Ca²⁺/calmodulin-regulated serine/threonine kinase that exists in an autoinhibited dimer conformation stabilized by 14-3-3 protein binding (via pThr369) and inhibitory autophosphorylation (Ser318); it is activated by Ca²⁺/CaM binding or by AMPK-mediated phosphorylation at Ser289 (which mimics CaM binding), and once active it phosphorylates mTORC1 component raptor at Ser721 to suppress mTORC1 and induce autophagy, phosphorylates Beclin-1 to displace Bcl-XL and promote autophagy initiation, phosphorylates SQSTM1/p62 to drive selective autophagy, phosphorylates PKM2 at Thr45 to drive its nuclear translocation and endothelial inflammation, and regulates mitochondrial integrity and MLC phosphorylation for granulocyte motility; its transcription is activated by PU.1, C/EBPα, E2F1, KLF6 (via Sp1), and TAp73, and repressed by PML-RARα and ΔNp73, while post-transcriptional silencing occurs via METTL3/YTHDF2-mediated m6A-dependent mRNA decay."},"narrative":{"mechanistic_narrative":"DAPK2 is a Ca²⁺/calmodulin-regulated serine/threonine kinase that functions as a central pro-autophagic and pro-apoptotic effector across granulocyte differentiation, metabolic stress responses, and vascular and tumor biology [PMID:21497605, PMID:29717115, PMID:26038578]. The kinase forms autoinhibited dimers through apposed catalytic domains, a conformation that precludes substrate binding [PMID:21497605] and is stabilized by 14-3-3 binding via phospho-Thr369, which protects the inhibitory autophosphorylation site Ser318 and blocks Ca²⁺/CaM access [PMID:34413451]; activation is achieved through Ca²⁺/CaM or through AMPK-mediated phosphorylation at Ser289, which mimics CaM binding and relieves autoinhibition [PMID:29717115, PMID:31116076]. Once active, DAPK2 suppresses mTORC1 by directly binding raptor and phosphorylating it at Ser721 [PMID:25361081] and promotes autophagy initiation by phosphorylating Beclin-1 to displace Bcl-XL [PMID:29717115], by phosphorylating SQSTM1/p62 to drive selective clearance of protein aggregates [PMID:27653365], and by supporting autophagic flux in adipocytes in a kinase-dependent manner [PMID:26038578]. DAPK2 kinase activity also maintains mitochondrial integrity and oxidative phosphorylation [PMID:25741596] and drives MLC phosphorylation required for granulocyte motility [PMID:24163421]. In the vasculature, DAPK2 phosphorylates PKM2 at Thr45 to drive its nuclear translocation and STAT1-dependent induction of adhesion molecules, promoting endothelial inflammation and atherogenesis [PMID:41614276]. Its expression is transcriptionally activated by PU.1, C/EBPα, E2F1/KLF6 (via Sp1), and TAp73 and repressed by PML-RARα and KLF2 [PMID:24038216, PMID:18521079, PMID:28978663, PMID:41614276], while m6A modification deposited by METTL3 and read by YTHDF2 destabilizes its mRNA [PMID:34298122].","teleology":[{"year":2008,"claim":"Established how DAPK2 transcription is wired to apoptotic transcription factors, defining DAPK2 as an obligate downstream effector of E2F1/KLF6-induced cell death.","evidence":"Promoter reporter assays, ChIP, Sp1-deficient cells, and siRNA knockdown with cell-death readout","pmids":["18521079"],"confidence":"Medium","gaps":["Did not address kinase-substrate events downstream of induced DAPK2","Sp1-dependence rather than direct E2F1 binding leaves mechanism of promoter selection partial"]},{"year":2009,"claim":"Showed that restoring DAPK2 catalytic activity is sufficient to drive apoptosis in tumors that silence it by promoter methylation, validating DAPK2 as a tumor suppressor effector.","evidence":"Promoter methylation analysis and constitutively active CaM-deletion DAPK2 fusion in CD30-positive lymphoma cells","pmids":["19609235"],"confidence":"Medium","gaps":["Pro-apoptotic substrates not identified","Constitutively active mutant bypasses physiological activation"]},{"year":2011,"claim":"Resolved the structural basis of DAPK2 autoinhibition, showing catalytic-domain dimerization occludes substrate binding.","evidence":"X-ray crystallography and SAXS of recombinant murine DAPK2 ± nucleotide","pmids":["21497605"],"confidence":"High","gaps":["Did not capture activated/substrate-bound state","Did not address regulators that shift the dimer equilibrium in cells"]},{"year":2013,"claim":"Placed DAPK2 within myeloid differentiation by identifying its activating (PU.1, C/EBPα) and repressing (PML-RARα) transcriptional inputs and demonstrating functional rescue of neutrophil differentiation.","evidence":"ChIP for transcription-factor binding plus rescue in PU.1-knockdown APL cells","pmids":["24038216"],"confidence":"Medium","gaps":["Did not define DAPK2 catalytic targets in differentiation"]},{"year":2013,"claim":"Demonstrated a cytoskeletal/motility function by linking DAPK activity to MLC phosphorylation and granulocyte chemotaxis in vitro and in vivo.","evidence":"Pharmacological DAPK inhibition, chemotaxis and F-actin/MLC assays, and a mouse peritonitis model","pmids":["24163421"],"confidence":"Medium","gaps":["DAPK inhibitor is not DAPK2-selective","Direct MLC phosphorylation by DAPK2 not isolated from DAPK1"]},{"year":2014,"claim":"Identified the first direct DAPK2 phosphorylation substrate in autophagy control, showing DAPK2 represses mTORC1 via raptor Ser721.","evidence":"Reciprocal Co-IP, recombinant binding, in vitro kinase assay, and knockdown with mTORC1 activity readout","pmids":["25361081"],"confidence":"High","gaps":["In vivo relevance of raptor Ser721 not established","Single lab"]},{"year":2014,"claim":"Revealed that loss of DAPK2 derepresses NF-κB to upregulate TRAIL death receptors, sensitizing resistant cancer cells to apoptosis independently of p53.","evidence":"siRNA knockdown, NF-κB reporter, surface DR4/DR5 flow cytometry, and TRAIL apoptosis assays in multiple lines","pmids":["25012503"],"confidence":"Medium","gaps":["Mechanism connecting DAPK2 to NF-κB not molecularly defined","No kinase-substrate link shown"]},{"year":2015,"claim":"Showed DAPK2 kinase activity is required for autophagic flux in adipocytes and for maintaining mitochondrial integrity and oxidative phosphorylation.","evidence":"Gain/loss-of-function with kinase-dead controls; LC3-II flux, Seahorse, membrane potential and superoxide assays","pmids":["26038578","25741596"],"confidence":"Medium","gaps":["Mitochondrial substrates of DAPK2 not identified","Whether mitochondrial defects are autophagy-dependent unresolved"]},{"year":2016,"claim":"Linked DAPK2 to selective autophagy by showing it phosphorylates SQSTM1/p62 to clear protein aggregates and protect against hepatotoxicity.","evidence":"AAV knockdown and ectopic expression with phospho-SQSTM1 immunoblot and a murine hepatocarcinogenesis model","pmids":["27653365"],"confidence":"Medium","gaps":["Direct in vitro kinase assay on p62 not shown","Phosphosite not mapped"]},{"year":2017,"claim":"Established a p73-DAPK2 axis with feedback, where TAp73 activates and ΔNp73 represses DAPK2, and DAPK2 binds ATG5 and stabilizes p73.","evidence":"Promoter reporter, TP73/DAPK2 knockdown, DAPK2-ATG5 Co-IP, and apoptosis/autophagy assays in APL cells","pmids":["28978663"],"confidence":"Medium","gaps":["DAPK2-ATG5 interaction not shown to be catalytic","Feedback stoichiometry undefined"]},{"year":2018,"claim":"Defined the activating upstream kinase by showing AMPK phosphorylates DAPK2 at Ser289 to mimic CaM binding and relieve autoinhibition, driving Beclin-1 phosphorylation and autophagy.","evidence":"In vitro kinase assays, phosphosite mapping, Co-IP, and autophagy flux assays","pmids":["29717115","31116076"],"confidence":"High","gaps":["Quantitative contribution of Ser289 versus Ca²⁺/CaM in cells unresolved"]},{"year":2021,"claim":"Provided the structural mechanism of negative regulation, showing 14-3-3 binding at pThr369 locks DAPK2 in an inactive dimer and shields Ser318.","evidence":"Crystal structure of the human DAPK2:14-3-3 complex with ITC, SAXS, and mutagenesis","pmids":["34413451"],"confidence":"High","gaps":["Kinase that generates pThr369 not identified","Cellular conditions that release 14-3-3 not defined"]},{"year":2021,"claim":"Identified an epitranscriptomic silencing route, with smoking-induced METTL3/YTHDF2 m6A decay of DAPK2 mRNA derepressing NF-κB to promote lung cancer progression.","evidence":"MeRIP, METTL3/YTHDF2 knockdown, mRNA stability assays, and in vitro/in vivo NSCLC assays","pmids":["34298122"],"confidence":"Medium","gaps":["Direct m6A sites on DAPK2 not pinpointed mechanistically"]},{"year":2026,"claim":"Established DAPK2 as a pro-inflammatory vascular kinase by mapping its direct PKM2 Thr45 phosphorylation driving nuclear PKM2-STAT1 adhesion-molecule induction and atherogenesis in vivo.","evidence":"Mass spec phosphosite mapping, PLA, EC-specific Dapk2 knockout in Apoe-/- mice, and Pkm2T45A/T45E knock-in mice","pmids":["41614276"],"confidence":"High","gaps":["How disturbed flow activates DAPK2 catalytically not fully resolved"]},{"year":2025,"claim":"Connected DAPK2 dysfunction to mitochondrial cristae remodeling via Mic60 lactylation, promoting anoikis resistance and metastasis in TKI-resistant lung cancer.","evidence":"Mass spectrometry, IP, anoikis-resistant models, and tail-vein metastasis assays","pmids":["40812308"],"confidence":"Medium","gaps":["Whether DAPK2 directly controls Mic60 lactylation is not established","Kinase activity dependence unclear"]},{"year":null,"claim":"The identity of the kinase generating the 14-3-3-binding pThr369 and the in vivo physiological hierarchy among DAPK2's many substrates (raptor, Beclin-1, p62, PKM2) remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No upstream Thr369 kinase identified","No unified accounting of which substrate dominates in a given tissue context"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,11,16]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,2,16]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[13,15]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1,2,10,11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,8,9,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,16]}],"complexes":[],"partners":["RPTOR","MTOR","ULK1","BECN1","PKM2","ATG5","14-3-3","AMPK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UIK4","full_name":"Death-associated protein kinase 2","aliases":["DAP-kinase-related protein 1","DRP-1"],"length_aa":370,"mass_kda":42.9,"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 death signals, 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. Acts as a mediator of anoikis and a suppressor of beta-catenin-dependent anchorage-independent growth of malignant epithelial cells. May play a role in granulocytic maturation (PubMed:17347302). Regulates granulocytic motility by controlling cell spreading and polarization (PubMed:24163421) Isoform 2 is not regulated by calmodulin. It can phosphorylate MYL9. It can induce membrane blebbing and autophagic cell death","subcellular_location":"Cytoplasm; Cytoplasmic vesicle, autophagosome lumen","url":"https://www.uniprot.org/uniprotkb/Q9UIK4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DAPK2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DAPK2","total_profiled":1310},"omim":[{"mim_id":"616567","title":"DEATH-ASSOCIATED PROTEIN KINASE 2; DAPK2","url":"https://www.omim.org/entry/616567"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":45.4},{"tissue":"thyroid gland","ntpm":39.7}],"url":"https://www.proteinatlas.org/search/DAPK2"},"hgnc":{"alias_symbol":["DRP-1","MGC119312"],"prev_symbol":[]},"alphafold":{"accession":"Q9UIK4","domains":[{"cath_id":"3.30.200.20","chopping":"18-103","consensus_level":"high","plddt":94.3728,"start":18,"end":103},{"cath_id":"1.10.510.10","chopping":"108-316","consensus_level":"high","plddt":95.1469,"start":108,"end":316}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UIK4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UIK4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UIK4-F1-predicted_aligned_error_v6.png","plddt_mean":86.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DAPK2","jax_strain_url":"https://www.jax.org/strain/search?query=DAPK2"},"sequence":{"accession":"Q9UIK4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UIK4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UIK4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UIK4"}},"corpus_meta":[{"pmid":"28703801","id":"PMC_28703801","title":"lncRNA MIAT functions as a competing endogenous RNA to upregulate DAPK2 by sponging miR-22-3p in diabetic cardiomyopathy.","date":"2017","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/28703801","citation_count":176,"is_preprint":false},{"pmid":"26038578","id":"PMC_26038578","title":"DAPK2 Downregulation Associates With Attenuated Adipocyte Autophagic Clearance in Human Obesity.","date":"2015","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/26038578","citation_count":72,"is_preprint":false},{"pmid":"25361081","id":"PMC_25361081","title":"DAPK2 is a novel regulator of mTORC1 activity and autophagy.","date":"2014","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/25361081","citation_count":59,"is_preprint":false},{"pmid":"27653365","id":"PMC_27653365","title":"Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective 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smoking induces aberrant N6-methyladenosine of DAPK2 to promote non-small cell lung cancer progression by activating NF-κB pathway.","date":"2021","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/34298122","citation_count":32,"is_preprint":false},{"pmid":"21497605","id":"PMC_21497605","title":"Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase.","date":"2011","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21497605","citation_count":29,"is_preprint":false},{"pmid":"27049921","id":"PMC_27049921","title":"MicroRNA-520g promotes epithelial ovarian cancer progression and chemoresistance via DAPK2 repression.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27049921","citation_count":28,"is_preprint":false},{"pmid":"34413451","id":"PMC_34413451","title":"14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory 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metastasis.","date":"2025","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/40812308","citation_count":19,"is_preprint":false},{"pmid":"24038216","id":"PMC_24038216","title":"The tumor suppressor gene DAPK2 is induced by the myeloid transcription factors PU.1 and C/EBPα during granulocytic differentiation but repressed by PML-RARα in APL.","date":"2013","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/24038216","citation_count":19,"is_preprint":false},{"pmid":"25741596","id":"PMC_25741596","title":"DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function.","date":"2015","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/25741596","citation_count":18,"is_preprint":false},{"pmid":"36827114","id":"PMC_36827114","title":"Stellettin B Induces Cell Death in Bladder Cancer Via Activating the Autophagy/DAPK2/Apoptosis Signaling Cascade.","date":"2023","source":"Marine 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DAPK2 forms dimers through apposed catalytic domains in an autoinhibited conformation that precludes protein substrate binding; the 'basic loop' fingerprint of the DAPK family plays a central role in kinase domain dimerization, and two distinct active-site conformations are observed upon nucleotide binding.\",\n      \"method\": \"X-ray crystallography and solution scattering (SAXS) of recombinant murine DAPK2 with and without nucleotide ligands\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus solution structure, multiple conditions tested, single rigorous structural study with functional model\",\n      \"pmids\": [\"21497605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DAPK2 directly interacts with and phosphorylates components of mTORC1; co-immunoprecipitation showed DAPK2 associates with mTOR, raptor, and ULK1, recombinant protein-binding assay confirmed direct DAPK2–raptor interaction, and in vitro kinase assay showed DAPK2 phosphorylates raptor at Ser721. DAPK2 knockdown increased mTORC1 kinase activity and reduced autophagy induced by amino acid deprivation or elevated intracellular Ca²⁺.\",\n      \"method\": \"Co-immunoprecipitation, recombinant protein-binding assay, in vitro kinase assay, siRNA knockdown with mTORC1 substrate phosphorylation readout\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay + recombinant binding + reciprocal Co-IP + functional KD, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"25361081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMPK phosphorylates DAPK2 at a site between the catalytic and calmodulin-binding domains (Ser289), activating DAPK2 by functionally mimicking calmodulin binding and mitigating inhibitory autophosphorylation. Activated DAPK2 in turn phosphorylates Beclin-1, causing dissociation of its inhibitor Bcl-XL and promoting autophagy; DAPK2 depletion reduced autophagy in response to AMPK activation.\",\n      \"method\": \"In vitro kinase assays, phosphosite mapping, co-immunoprecipitation, siRNA knockdown, autophagy flux assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assays with phosphosite identification, Co-IP, and functional KD with multiple readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29717115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"14-3-3 proteins bind DAPK2 via a canonical mode III motif created by phosphorylation of Thr369 at the DAPK2 C-terminus. X-ray crystallographic analysis of the full-length human DAPK2:14-3-3 complex showed that 14-3-3 binding stabilizes DAPK2 dimers, protects the inhibitory autophosphorylation site Ser318 from dephosphorylation, and prevents Ca²⁺/CaM binding, thereby inactivating DAPK2. The interaction is further enhanced by the diterpene glycoside Fusicoccin A.\",\n      \"method\": \"X-ray crystallography, biophysical binding assays (ITC, SAXS), phosphosite mapping, mutagenesis\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of complex plus multiple biophysical methods in single rigorous study, single lab\",\n      \"pmids\": [\"34413451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Ser289 phosphorylation enhances DAPK2 catalytic activity (analogous to its effect on DAPK1); AMPK-mediated Ser289 phosphorylation of DAPK2 was compared to RSK-mediated phosphorylation of DAPK1 in the same cells, and the signaling pathways leading to Ser289 phosphorylation are mutually exclusive between DAPK1 and DAPK2.\",\n      \"method\": \"In-cell kinase activity assays, phospho-specific immunoblotting, pathway inhibitor treatments\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional kinase activity comparison in cells, single lab, single study\",\n      \"pmids\": [\"31116076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DAPK2 promoter hypermethylation silences DAPK2 expression in Hodgkin lymphoma cell lines; a constitutively active calmodulin-deletion mutant of DAPK2 fused to CD30 ligand (immunokinase DAPK2'-CD30L) selectively induced apoptosis and inhibited proliferation in CD30-positive/DAPK2-negative tumor cells, demonstrating that restoration of DAPK2 catalytic activity is sufficient for pro-apoptotic function.\",\n      \"method\": \"Promoter methylation analysis, recombinant fusion protein construction, cell viability and apoptosis assays in tumor cell lines\",\n      \"journal\": \"Journal of immunotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional reconstitution with constitutively active mutant in cells, methylation mechanism identified, single lab\",\n      \"pmids\": [\"19609235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DAPK2 positively regulates motility of human neutrophils and eosinophils in response to intermediary chemoattractants; pharmacological inhibition of DAPK activity abolished granulocyte migration associated with reduced MLC phosphorylation and loss of cell polarization with radial F-actin, and reduced neutrophil recruitment to the peritoneum in a mouse peritonitis model.\",\n      \"method\": \"Pharmacological DAPK inhibition, ex vivo chemotaxis assays, F-actin/MLC phosphorylation analysis, in vivo peritonitis model\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo functional assays with mechanistic readout (MLC phosphorylation), single lab, two orthogonal systems\",\n      \"pmids\": [\"24163421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DAPK2 expression is transcriptionally activated by the myeloid transcription factors PU.1 and C/EBPα during granulocytic differentiation; PML-RARα binds the DAPK2 promoter and represses its transcription in APL. Restoration of DAPK2 expression in PU.1-knockdown APL cells partially rescued neutrophil differentiation, establishing DAPK2 as a relevant PU.1 downstream effector.\",\n      \"method\": \"ChIP (PML-RARA and PU.1 binding at DAPK2 promoter), ectopic expression, PU.1 knockdown, differentiation assays\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for transcription factor binding plus functional rescue experiment, single lab, two orthogonal methods\",\n      \"pmids\": [\"24038216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"E2F1 and KLF6 transcriptionally activate the DAPK2 promoter via a GC-rich region upstream of exon 1 in an Sp1-dependent manner (no canonical E2F binding sites); ChIP confirmed Sp1 recruitment (and lesser E2F1/KLF6 recruitment) to the DAPK2 promoter. DAPK2 induction mediates the pro-apoptotic effects of both E2F1 and KLF6, as DAPK2 knockdown significantly reduced cell death upon activation of either transcription factor.\",\n      \"method\": \"Promoter reporter assays, chromatin immunoprecipitation (ChIP), Sp1-deficient insect cells, mithramycin A treatment, siRNA knockdown, cell death assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional KD rescue and multiple reporter assays, single lab, orthogonal methods\",\n      \"pmids\": [\"18521079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Genetic ablation of DAPK2 by RNAi causes NF-κB phosphorylation and transcriptional activation, leading to upregulation of DR4 and DR5 (TRAIL receptors) on the cell surface and sensitizing resistant cancer cells to TRAIL-induced apoptosis in a p53-independent manner.\",\n      \"method\": \"siRNA knockdown, NF-κB reporter assay, cell surface flow cytometry for DR4/DR5, TRAIL apoptosis assays in multiple cancer cell lines\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with multiple orthogonal pathway readouts in several cell lines, single lab\",\n      \"pmids\": [\"25012503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Adenoviral DAPK2 overexpression in 3T3-L1 adipocytes increased autophagic clearance in nutrient-rich conditions in a kinase activity-dependent manner; conversely, siRNA-mediated DAPK2 inhibition in human preadipocytes decreased LC3-II accumulation rates measured with lysosome inhibitors, demonstrating DAPK2 kinase activity is required for autophagic flux in adipocytes.\",\n      \"method\": \"Adenoviral overexpression (kinase-active and kinase-dead), siRNA knockdown, LC3-II accumulation assay with lysosome inhibitors in 3T3-L1 and human preadipocytes\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutant controls plus gain- and loss-of-function in two cell systems, single lab\",\n      \"pmids\": [\"26038578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DAPK2 transcriptional activation by thyroid hormone (TH) enhances phosphorylation of SQSTM1/p62, promoting selective autophagic clearance of protein aggregates; ectopic DAPK2 expression attenuated DEN-induced hepatotoxicity and DNA damage via enhanced autophagy, while DAPK2 knockdown had the opposite effect.\",\n      \"method\": \"Adeno-associated virus knockdown, ectopic DAPK2 expression, phospho-SQSTM1 immunoblot, autophagy flux assays, murine hepatocarcinogenesis model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro gain/loss-of-function with mechanistic phosphorylation readout, single lab\",\n      \"pmids\": [\"27653365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TAp73 isoform binds to and activates the DAPK2 promoter, whereas ΔNp73 inhibits DAPK2 transcription; DAPK2 is an important downstream effector of p73 in ATO-induced apoptosis. Conversely, the p73-DAPK2 pathway is essential for ATRA-induced autophagy mediated by a direct interaction between DAPK2 and ATG5. DAPK2 also binds and stabilizes p73 protein, forming a positive feedback loop.\",\n      \"method\": \"Promoter reporter assay (TAp73/ΔNp73), siRNA knockdown of TP73 and DAPK2, co-immunoprecipitation of DAPK2-ATG5, apoptosis and autophagy assays in APL cells\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — promoter assay + Co-IP + functional KD with multiple readouts, single lab\",\n      \"pmids\": [\"28978663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNA interference-mediated depletion of endogenous DAPK2 in cancer cells causes decreased oxidative phosphorylation, destabilized mitochondrial membrane potential, increased mitochondrial superoxide anion production, and activation of ERK, JNK, and p38 stress kinases; overexpression of a kinase-dead DAPK2 mutant further enhanced oxidative stress, indicating that DAPK2 kinase activity is required to maintain mitochondrial integrity.\",\n      \"method\": \"siRNA knockdown, kinase-dead mutant overexpression, Seahorse metabolic flux analysis, mitochondrial membrane potential assay, superoxide detection, stress kinase immunoblotting\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD plus kinase-dead mutant with multiple orthogonal metabolic readouts, single lab\",\n      \"pmids\": [\"25741596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cigarette smoking-induced METTL3-mediated m6A modification of DAPK2 mRNA is read by YTHDF2, resulting in decreased DAPK2 mRNA stability and reduced DAPK2 expression; downregulation of DAPK2 activates NF-κB signaling to promote NSCLC proliferation and migration.\",\n      \"method\": \"m6A sequencing/MeRIP, METTL3/YTHDF2 knockdown, mRNA stability assay, NF-κB pathway inhibitor (BAY 11-7085), in vitro and in vivo functional assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A writer and reader identified by KD with mRNA stability readout, NF-κB pathway functionally validated, single lab\",\n      \"pmids\": [\"34298122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DAPK2 dysfunction alters Mic60 (mitofilin) protein in mitochondrial cristae by promoting its lactylation, increasing cristae abundance and compactness and activating mitochondrial metabolism, leading to anoikis resistance and enhanced metastasis in EGFR-TKI-resistant lung cancer cells.\",\n      \"method\": \"Mass spectrometry, immunoprecipitation, anoikis-resistant cell model, mouse tail vein metastasis model, Mic60 lactylation analysis, mitochondrial metabolic assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification of lactylation + in vivo metastasis model + Co-IP, single lab, multiple methods\",\n      \"pmids\": [\"40812308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"DAPK2 directly phosphorylates PKM2 at threonine 45, causing PKM2 dimerization and nuclear translocation; nuclear PKM2 activates VCAM-1 and ICAM-1 expression by interacting with STAT1, promoting endothelial inflammation in response to oscillatory shear stress. KLF2 suppresses DAPK2 transcription, and OSS-induced KLF2 downregulation leads to DAPK2 upregulation. EC-specific Dapk2 deficiency reduced atherogenesis in Apoe-/- mice, and Pkm2T45A overexpression mitigated disturbed flow-induced atherogenesis.\",\n      \"method\": \"Mass spectrometry phosphosite identification, immunoprecipitation, proximity ligation assay, dominant-negative DAPK2 mutant, EC-specific Dapk2 knockout in Apoe-/- mice (carotid ligation and Western diet models), Pkm2T45A and Pkm2T45E knock-in overexpression mice\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mass spec phosphosite mapping + multiple in vivo genetic models (KO + phospho-mimetic/refractory mutants) + multiple orthogonal biochemical methods\",\n      \"pmids\": [\"41614276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PCB118 enhances binding between Tubb3 (tubulin beta 3) and DAPK2 in thyroid cells; siRNA knockdown of Tubb3 suppressed DAPK2 protein expression and PKD phosphorylation, while DAPK2 (downstream of Tubb3) activated PKD which in turn phosphorylated VPS34, mediating autophagy. DAPK2 overexpression following PCB118 exposure upregulated PKD and VPS34 in the DAPK2/PKD/VPS34 pathway.\",\n      \"method\": \"Co-immunoprecipitation (Tubb3-DAPK2 interaction), siRNA knockdown, overexpression, immunofluorescence, rat in vivo model\",\n      \"journal\": \"Archives of toxicology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and KD with pathway readout, single lab, single study, no direct DAPK2 kinase assay on PKD\",\n      \"pmids\": [\"31020377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In osteoblasts, VGLL3 regulates DAPK2 expression (identified by RNA-seq of Vgll3-KD cells); DAPK2 knockdown phenocopied Vgll3 loss, reducing autophagic flux and impairing osteoblast differentiation. DAPK2 overexpression partially rescued autophagy and osteogenic differentiation in Vgll3-deficient cells, and FOXM1 was implicated as a potential transcriptional regulator of DAPK2 in this context.\",\n      \"method\": \"RNA-seq, shRNA knockdown (Vgll3 and Dapk2), DAPK2 overexpression rescue, LC3-II/p62 immunoblot, transmission electron microscopy, alkaline phosphatase/Alizarin Red staining\",\n      \"journal\": \"BioFactors\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — KD and rescue with autophagy/differentiation readouts, single lab, FOXM1 link is preliminary\",\n      \"pmids\": [\"42052791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"YTHDF3 directly recognizes m6A-modified Dapk2 transcripts and promotes their decay, thereby suppressing DAPK2 protein levels; melatonin rescues YTHDF3 expression under mechanical unloading, leading to DAPK2 mRNA degradation that supports osteoblast differentiation and survival. DAPK2 was characterized as a negative regulator of osteoblast differentiation and survival.\",\n      \"method\": \"m6A-seq/MeRIP, YTHDF3 KD/OE, Dapk2 KD, hindlimb unloading mouse model, western blot, qPCR, mineralization assays\",\n      \"journal\": \"Journal of pineal research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — m6A reader-mRNA decay link and functional osteoblast phenotype, single lab, mechanistic link between YTHDF3 and DAPK2 decay shown by KD but no direct binding validated in this study\",\n      \"pmids\": [\"42149012\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DAPK2 is a Ca²⁺/calmodulin-regulated serine/threonine kinase that exists in an autoinhibited dimer conformation stabilized by 14-3-3 protein binding (via pThr369) and inhibitory autophosphorylation (Ser318); it is activated by Ca²⁺/CaM binding or by AMPK-mediated phosphorylation at Ser289 (which mimics CaM binding), and once active it phosphorylates mTORC1 component raptor at Ser721 to suppress mTORC1 and induce autophagy, phosphorylates Beclin-1 to displace Bcl-XL and promote autophagy initiation, phosphorylates SQSTM1/p62 to drive selective autophagy, phosphorylates PKM2 at Thr45 to drive its nuclear translocation and endothelial inflammation, and regulates mitochondrial integrity and MLC phosphorylation for granulocyte motility; its transcription is activated by PU.1, C/EBPα, E2F1, KLF6 (via Sp1), and TAp73, and repressed by PML-RARα and ΔNp73, while post-transcriptional silencing occurs via METTL3/YTHDF2-mediated m6A-dependent mRNA decay.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DAPK2 is a Ca²⁺/calmodulin-regulated serine/threonine kinase that functions as a central pro-autophagic and pro-apoptotic effector across granulocyte differentiation, metabolic stress responses, and vascular and tumor biology [#0, #2, #10]. The kinase forms autoinhibited dimers through apposed catalytic domains, a conformation that precludes substrate binding [#0] and is stabilized by 14-3-3 binding via phospho-Thr369, which protects the inhibitory autophosphorylation site Ser318 and blocks Ca²⁺/CaM access [#3]; activation is achieved through Ca²⁺/CaM or through AMPK-mediated phosphorylation at Ser289, which mimics CaM binding and relieves autoinhibition [#2, #4]. Once active, DAPK2 suppresses mTORC1 by directly binding raptor and phosphorylating it at Ser721 [#1] and promotes autophagy initiation by phosphorylating Beclin-1 to displace Bcl-XL [#2], by phosphorylating SQSTM1/p62 to drive selective clearance of protein aggregates [#11], and by supporting autophagic flux in adipocytes in a kinase-dependent manner [#10]. DAPK2 kinase activity also maintains mitochondrial integrity and oxidative phosphorylation [#13] and drives MLC phosphorylation required for granulocyte motility [#6]. In the vasculature, DAPK2 phosphorylates PKM2 at Thr45 to drive its nuclear translocation and STAT1-dependent induction of adhesion molecules, promoting endothelial inflammation and atherogenesis [#16]. Its expression is transcriptionally activated by PU.1, C/EBPα, E2F1/KLF6 (via Sp1), and TAp73 and repressed by PML-RARα and KLF2 [#7, #8, #12, #16], while m6A modification deposited by METTL3 and read by YTHDF2 destabilizes its mRNA [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established how DAPK2 transcription is wired to apoptotic transcription factors, defining DAPK2 as an obligate downstream effector of E2F1/KLF6-induced cell death.\",\n      \"evidence\": \"Promoter reporter assays, ChIP, Sp1-deficient cells, and siRNA knockdown with cell-death readout\",\n      \"pmids\": [\"18521079\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address kinase-substrate events downstream of induced DAPK2\", \"Sp1-dependence rather than direct E2F1 binding leaves mechanism of promoter selection partial\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed that restoring DAPK2 catalytic activity is sufficient to drive apoptosis in tumors that silence it by promoter methylation, validating DAPK2 as a tumor suppressor effector.\",\n      \"evidence\": \"Promoter methylation analysis and constitutively active CaM-deletion DAPK2 fusion in CD30-positive lymphoma cells\",\n      \"pmids\": [\"19609235\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pro-apoptotic substrates not identified\", \"Constitutively active mutant bypasses physiological activation\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the structural basis of DAPK2 autoinhibition, showing catalytic-domain dimerization occludes substrate binding.\",\n      \"evidence\": \"X-ray crystallography and SAXS of recombinant murine DAPK2 ± nucleotide\",\n      \"pmids\": [\"21497605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture activated/substrate-bound state\", \"Did not address regulators that shift the dimer equilibrium in cells\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed DAPK2 within myeloid differentiation by identifying its activating (PU.1, C/EBPα) and repressing (PML-RARα) transcriptional inputs and demonstrating functional rescue of neutrophil differentiation.\",\n      \"evidence\": \"ChIP for transcription-factor binding plus rescue in PU.1-knockdown APL cells\",\n      \"pmids\": [\"24038216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define DAPK2 catalytic targets in differentiation\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated a cytoskeletal/motility function by linking DAPK activity to MLC phosphorylation and granulocyte chemotaxis in vitro and in vivo.\",\n      \"evidence\": \"Pharmacological DAPK inhibition, chemotaxis and F-actin/MLC assays, and a mouse peritonitis model\",\n      \"pmids\": [\"24163421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DAPK inhibitor is not DAPK2-selective\", \"Direct MLC phosphorylation by DAPK2 not isolated from DAPK1\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified the first direct DAPK2 phosphorylation substrate in autophagy control, showing DAPK2 represses mTORC1 via raptor Ser721.\",\n      \"evidence\": \"Reciprocal Co-IP, recombinant binding, in vitro kinase assay, and knockdown with mTORC1 activity readout\",\n      \"pmids\": [\"25361081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of raptor Ser721 not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed that loss of DAPK2 derepresses NF-κB to upregulate TRAIL death receptors, sensitizing resistant cancer cells to apoptosis independently of p53.\",\n      \"evidence\": \"siRNA knockdown, NF-κB reporter, surface DR4/DR5 flow cytometry, and TRAIL apoptosis assays in multiple lines\",\n      \"pmids\": [\"25012503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting DAPK2 to NF-κB not molecularly defined\", \"No kinase-substrate link shown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed DAPK2 kinase activity is required for autophagic flux in adipocytes and for maintaining mitochondrial integrity and oxidative phosphorylation.\",\n      \"evidence\": \"Gain/loss-of-function with kinase-dead controls; LC3-II flux, Seahorse, membrane potential and superoxide assays\",\n      \"pmids\": [\"26038578\", \"25741596\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mitochondrial substrates of DAPK2 not identified\", \"Whether mitochondrial defects are autophagy-dependent unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked DAPK2 to selective autophagy by showing it phosphorylates SQSTM1/p62 to clear protein aggregates and protect against hepatotoxicity.\",\n      \"evidence\": \"AAV knockdown and ectopic expression with phospho-SQSTM1 immunoblot and a murine hepatocarcinogenesis model\",\n      \"pmids\": [\"27653365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro kinase assay on p62 not shown\", \"Phosphosite not mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established a p73-DAPK2 axis with feedback, where TAp73 activates and ΔNp73 represses DAPK2, and DAPK2 binds ATG5 and stabilizes p73.\",\n      \"evidence\": \"Promoter reporter, TP73/DAPK2 knockdown, DAPK2-ATG5 Co-IP, and apoptosis/autophagy assays in APL cells\",\n      \"pmids\": [\"28978663\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DAPK2-ATG5 interaction not shown to be catalytic\", \"Feedback stoichiometry undefined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the activating upstream kinase by showing AMPK phosphorylates DAPK2 at Ser289 to mimic CaM binding and relieve autoinhibition, driving Beclin-1 phosphorylation and autophagy.\",\n      \"evidence\": \"In vitro kinase assays, phosphosite mapping, Co-IP, and autophagy flux assays\",\n      \"pmids\": [\"29717115\", \"31116076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of Ser289 versus Ca²⁺/CaM in cells unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the structural mechanism of negative regulation, showing 14-3-3 binding at pThr369 locks DAPK2 in an inactive dimer and shields Ser318.\",\n      \"evidence\": \"Crystal structure of the human DAPK2:14-3-3 complex with ITC, SAXS, and mutagenesis\",\n      \"pmids\": [\"34413451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase that generates pThr369 not identified\", \"Cellular conditions that release 14-3-3 not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified an epitranscriptomic silencing route, with smoking-induced METTL3/YTHDF2 m6A decay of DAPK2 mRNA derepressing NF-κB to promote lung cancer progression.\",\n      \"evidence\": \"MeRIP, METTL3/YTHDF2 knockdown, mRNA stability assays, and in vitro/in vivo NSCLC assays\",\n      \"pmids\": [\"34298122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct m6A sites on DAPK2 not pinpointed mechanistically\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established DAPK2 as a pro-inflammatory vascular kinase by mapping its direct PKM2 Thr45 phosphorylation driving nuclear PKM2-STAT1 adhesion-molecule induction and atherogenesis in vivo.\",\n      \"evidence\": \"Mass spec phosphosite mapping, PLA, EC-specific Dapk2 knockout in Apoe-/- mice, and Pkm2T45A/T45E knock-in mice\",\n      \"pmids\": [\"41614276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How disturbed flow activates DAPK2 catalytically not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected DAPK2 dysfunction to mitochondrial cristae remodeling via Mic60 lactylation, promoting anoikis resistance and metastasis in TKI-resistant lung cancer.\",\n      \"evidence\": \"Mass spectrometry, IP, anoikis-resistant models, and tail-vein metastasis assays\",\n      \"pmids\": [\"40812308\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DAPK2 directly controls Mic60 lactylation is not established\", \"Kinase activity dependence unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the kinase generating the 14-3-3-binding pThr369 and the in vivo physiological hierarchy among DAPK2's many substrates (raptor, Beclin-1, p62, PKM2) remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No upstream Thr369 kinase identified\", \"No unified accounting of which substrate dominates in a given tissue context\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 11, 16]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 2, 16]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [13, 15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1, 2, 10, 11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 8, 9, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 16]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RPTOR\", \"MTOR\", \"ULK1\", \"BECN1\", \"PKM2\", \"ATG5\", \"14-3-3\", \"AMPK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}