{"gene":"ULK1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2011,"finding":"AMPK directly phosphorylates ULK1 at Ser317 and Ser777 to activate it under glucose starvation, promoting autophagy. Conversely, mTOR phosphorylates ULK1 at Ser757 under nutrient sufficiency, disrupting the interaction between ULK1 and AMPK and preventing ULK1 activation. This coordinated phosphorylation is required for autophagy induction.","method":"In vitro kinase assays, phospho-specific antibodies, mutagenesis, co-immunoprecipitation, genetic epistasis in cell lines and mouse models","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct in vitro phosphorylation assays with site-specific mutagenesis, replicated independently by two groups (PMID:21258367 and PMID:21205641)","pmids":["21258367"],"is_preprint":false},{"year":2010,"finding":"AMPK phosphorylates ULK1 (and ULK2), connecting energy sensing to autophagy and mitophagy. Loss of AMPK or ULK1 causes aberrant accumulation of the autophagy adaptor p62 and defective mitophagy; reconstitution with a ULK1 mutant that cannot be phosphorylated by AMPK reveals this phosphorylation is required for mitochondrial homeostasis and cell survival during starvation.","method":"Genetic analysis in mammalian liver and C. elegans, biochemical reconstitution with phospho-deficient mutants, co-immunoprecipitation","journal":"Science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution with phospho-deficient mutant, genetic epistasis across two organisms, replicated independently","pmids":["21205641"],"is_preprint":false},{"year":2013,"finding":"Upon amino-acid starvation or mTOR inhibition, activated ULK1 phosphorylates Beclin-1 on Ser14, enhancing the activity of the ATG14L-containing VPS34 complex and promoting autophagic flux. This phosphorylation is required for full autophagic induction in mammals and C. elegans.","method":"In vitro kinase assay, site-directed mutagenesis, genetic epistasis in C. elegans and mammalian cells, VPS34 lipid kinase activity assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, cross-species genetic epistasis, multiple orthogonal methods in one study","pmids":["23685627"],"is_preprint":false},{"year":2006,"finding":"siRNA-mediated depletion of ULK1 inhibits starvation-induced redistribution of mammalian Atg9 from the trans-Golgi network to peripheral endosomal/autophagosomal membranes, placing ULK1 upstream of Atg9 trafficking in autophagy.","method":"siRNA knockdown, live-cell fluorescence imaging, immunofluorescence colocalization","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA knockdown with clear cellular phenotype (Atg9 redistribution), single lab but well-controlled imaging experiment","pmids":["16940348"],"is_preprint":false},{"year":2014,"finding":"ULK1 translocates to fragmented mitochondria upon mitophagy induction by hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1 and phosphorylates it at Ser17, enhancing FUNDC1 binding to LC3. A ULK1-binding-deficient FUNDC1 mutant prevents ULK1 translocation and inhibits mitophagy; kinase-active ULK1 and phospho-mimicking FUNDC1 rescue mitophagy in ULK1-null cells.","method":"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, rescue experiments in ULK1-null cells, live imaging","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, genetic rescue in null cells, multiple orthogonal methods","pmids":["24671035"],"is_preprint":false},{"year":2011,"finding":"ULK1 binds raptor (a component of mTORC1) and induces its phosphorylation, inhibiting mTORC1 kinase activity and cell proliferation independently of Atg5 and TSC2. This identifies a negative feedback loop from ULK1 to mTORC1.","method":"Co-immunoprecipitation, in vitro kinase assay, ULK1 knockdown/overexpression, cell proliferation assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and in vitro kinase data from single lab, multiple orthogonal methods","pmids":["21795849"],"is_preprint":false},{"year":2019,"finding":"During selective autophagy, NDP52 localizes and activates the ULK1 complex at cargo through interaction with FIP200/ULK1, facilitated by TBK1. Ectopic tethering of ULK1 to cargo bypasses the need for autophagy receptors and TBK1. This focal activation of ULK1 occurs independently of AMPK and mTOR.","method":"Chemically inducible dimerization assays, CRISPR KO lines, genetic epistasis, co-immunoprecipitation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic CRISPR-based epistasis combined with CID assays and co-IP, multiple orthogonal methods in one study","pmids":["30853401"],"is_preprint":false},{"year":2019,"finding":"ULK1 and ULK2 localize to stress granules and phosphorylate VCP/p97, increasing VCP's ATPase activity and ability to disassemble stress granules. Disruption of ULK1/2 expression in mice causes a vacuolar myopathy with TDP-43-positive inclusions similar to that caused by VCP mutations.","method":"In vitro kinase assay, co-immunoprecipitation, mouse knockout model, biochemical VCP activity assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay, mouse KO phenotype, enzymatic activity assay, multiple orthogonal methods","pmids":["30979586"],"is_preprint":false},{"year":2016,"finding":"The E3 ubiquitin ligase NEDD4L ubiquitylates ULK1 for proteasomal degradation during autophagy progression, down-regulating ULK1 protein levels. ULK1 mRNA continues to be transcribed during this period, and upon reactivation of mTOR-dependent protein synthesis, ULK1 levels are restored. This NEDD4L-ULK1 axis controls oscillatory activation of autophagy.","method":"Co-immunoprecipitation, ubiquitination assays, cycloheximide chase, mTOR inhibitor experiments, pulse-chase","journal":"Journal of Cell Biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP with ubiquitination assay, single lab, multiple supporting methods","pmids":["27932573"],"is_preprint":false},{"year":2018,"finding":"ULK1 phosphorylates SEC23B at Ser186 upon starvation, preventing its interaction with FBXW5 (an F-box E3 ubiquitin ligase) and thus stabilizing SEC23B. Phosphorylated SEC23B associates with SEC24A/B and relocalizes to the ER-Golgi intermediate compartment, promoting autophagic flux.","method":"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry, site-directed mutagenesis, subcellular fractionation/localization","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, MS-identified sites, multiple orthogonal methods in one study","pmids":["30596474"],"is_preprint":false},{"year":2017,"finding":"ULK1 phosphorylates Sec23A, a COPII vesicle coat component, inhibiting its interaction with Sec31A. This reduces ER-to-Golgi trafficking during autophagy induction. Phosphorylation sites Ser207, Ser312, and Thr405 on Sec23A were identified; Ser207 most faithfully recapitulates ULK1's regulatory effects.","method":"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, live-cell imaging of ERES","journal":"BMC cell biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, single lab","pmids":["28486929"],"is_preprint":false},{"year":2021,"finding":"ULK1 phosphorylates BNIP3 at Ser17 adjacent to its LIR motif, promoting BNIP3 interaction with LC3 and mitophagy. ULK1 also similarly phosphorylates BNIP3L at Ser35. Additionally, ULK1 interaction promotes BNIP3 protein stability by limiting its proteasomal turnover, dependent on an intact BH3 domain.","method":"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, cycloheximide chase, mitophagy flux assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with site mutagenesis, single lab, multiple methods","pmids":["34654847"],"is_preprint":false},{"year":2020,"finding":"ULK1 phosphorylates Exo70, a component of the exocyst complex, inhibiting Exo70 homo-oligomerization and assembly into the exocyst complex, thereby suppressing breast cancer cell migration and invasion. ERK1/2 phosphorylates Exo70 upon growth factor stimulation, which in turn suppresses its phosphorylation by ULK1.","method":"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell migration/invasion assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with substrate identification, single lab, multiple methods","pmids":["31913283"],"is_preprint":false},{"year":2019,"finding":"GABARAP subfamily ATG8 proteins positively regulate ULK1 activity and autophagosome formation, while LC3 subfamily proteins negatively regulate ULK1 activity. Mutation of the LIR motif of endogenous ULK1 (disrupting ATG8-ULK1 interaction) drastically reduced ULK1 kinase activity, autophagic degradation, and phagophore formation. Similarly, disruption of ATG13-ATG8 interaction suppressed ULK1 activity.","method":"CRISPR/TALEN genome editing of LIR motif, ATG8 subfamily depletion, ULK1 kinase activity assays, autophagy flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous LIR mutagenesis by genome editing, multiple CRISPR KO lines, multiple orthogonal readouts","pmids":["31208283"],"is_preprint":false},{"year":2022,"finding":"PRMT5 catalyzes symmetrical dimethylation of ULK1 at Arg170 (R170me2s), a modification removed by KDM5C. Low oxygen decreases KDM5C activity, causing accumulation of ULK1 R170me2s. Dimethylation promotes ULK1 autophosphorylation at T180, a prerequisite for activation, leading to phosphorylation of Atg13 and Beclin1, autophagosome formation, and mitochondrial clearance.","method":"In vitro methylation assay, mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, autophagy flux assays, cell proliferation under hypoxia","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro assays with writer/eraser identification, mutagenesis, and functional validation through multiple downstream readouts","pmids":["35246531"],"is_preprint":false},{"year":2021,"finding":"GSK3B directly interacts with ULK1 and phosphorylates it at Ser405 and Ser415 within the GABARAP-interacting region upon insulin withdrawal. This phosphorylation facilitates ULK1 interaction with MAP1LC3B and GABARAPL1; phosphorylation-defective ULK1 mutants fail to interact with these ATG8 proteins and cannot induce autophagy flux.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, autophagy flux assays","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, single lab","pmids":["33654220"],"is_preprint":false},{"year":2020,"finding":"MAPK1/ERK2-MAPK3/ERK1 phosphorylates ULK1, triggering its interaction with the E3 ubiquitin ligase BTRC (β-TrCP) and subsequent K48-linked ubiquitination and proteasomal degradation of ULK1. This attenuates mitophagy and promotes breast cancer bone metastasis.","method":"Co-immunoprecipitation, ubiquitination assays, pharmacological inhibitors, in vitro kinase assay, cancer cell invasion assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP and ubiquitination assays, single lab, multiple orthogonal methods","pmids":["33213267"],"is_preprint":false},{"year":2023,"finding":"ULK1 directly interacts with LDHA and phosphorylates it at Ser196 under nutrient deprivation, promoting lactate production. Lactate in turn mediates Vps34 lactylation at Lys356 and Lys781 (via KAT5/TIP60), enhancing Vps34 association with Beclin1, Atg14L, and UVRAG and increasing Vps34 lipid kinase activity, thereby promoting autophagic flux.","method":"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry identification of lactylation sites, site-directed mutagenesis, Vps34 lipid kinase activity assay","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis and enzymatic activity readout, single lab, multiple methods","pmids":["37267363"],"is_preprint":false},{"year":2018,"finding":"USP20 (ubiquitin-specific protease 20) binds to and stabilizes ULK1 by removing ubiquitin moieties, preventing lysosomal degradation of ULK1 at basal state. Depletion of USP20 inhibits LC3 puncta formation and autophagy initiation. At later stages of autophagy, USP20 dissociates from ULK1, leading to ULK1 degradation.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, autophagy flux assays","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP and ubiquitination assay, single lab, multiple supporting methods","pmids":["29487085"],"is_preprint":false},{"year":2022,"finding":"TRIM27 directly polyubiquitinates ULK1 at Lys568 and Lys571 with K48-linked chains for proteasomal degradation, maintaining basal ULK1 levels. During starvation-induced autophagy, TRIM27 also catalyzes K6- and K11-linked ubiquitination of STK38L, activating STK38L which then phosphorylates ULK1 at Ser495, rendering ULK1 susceptible to TRIM27-mediated hyper-ubiquitination to restrain autophagy amplitude.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis, in vitro kinase assay, mouse knockout models","journal":"EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination and kinase assays with mutagenesis, mouse KO validation, multiple orthogonal methods","pmids":["35670107"],"is_preprint":false},{"year":2020,"finding":"ULK1-ATG13 complex is phosphorylated by CDK1/cyclin B in mitosis. CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Double knockout of ULK1 and ATG13 blocks cell cycle progression and decreases cancer cell proliferation.","method":"Mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, cell cycle analysis, CRISPR KO, mouse xenograft model","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified sites with mutagenesis, single lab, multiple methods including in vivo model","pmids":["32516310"],"is_preprint":false},{"year":2020,"finding":"ULK1 phosphorylates RIPK1 at multiple sites, including Ser357 within its intermediate domain, reducing complex IIb/necrosome assembly and inhibiting TNF-induced apoptosis and necroptosis. Loss of ULK1 enhances TNF-induced cell death.","method":"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation, cell death assays, ULK1 KO cells","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis and functional cell death readout, single lab","pmids":["32320653"],"is_preprint":false},{"year":2021,"finding":"ULK1 directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A. This creates a positive feedback loop where ULK1-activated PP2A promotes autophagy-dependent protein turnover. ULK1 is also itself a PP2A target (dephosphorylated by PP2A).","method":"Quantitative phosphoproteomics, in vitro kinase assay, co-immunoprecipitation, PP2A activity assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay combined with phosphoproteomics, single lab","pmids":["34592149"],"is_preprint":false},{"year":2018,"finding":"ULK1 is the principal kinase responsible for phosphorylation of the mineralocorticoid receptor (MR) at Ser843, as identified by a high-throughput screen of 197 kinases and confirmed by in vitro kinase assay, mass spectrometry, and siRNA knockdown. ULK1/2 double KO mouse embryonic fibroblasts show markedly reduced MRS843 phosphorylation. ULK1 activity is itself inhibited by angiotensin II-induced mTOR-mediated phosphorylation.","method":"High-throughput kinase screen, in vitro kinase assay, mass spectrometry, siRNA knockdown, ULK1/2 double KO MEFs","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — identified by kinase screen, confirmed by in vitro kinase assay, MS, and genetic KO — multiple orthogonal methods","pmids":["30021155"],"is_preprint":false},{"year":2024,"finding":"ULK1 is palmitoylated by the palmitoyltransferase ZDHHC13 upon autophagy induction, and this palmitoylation is required for ULK1 translocation to autophagosome formation sites. Palmitoylated ULK1 enhances phosphorylation of ATG14L, activating PI3-kinase activity and promoting phosphatidylinositol 3-phosphate production needed for autophagosome membrane formation.","method":"Acyl-RAC palmitoylation assay, ZDHHC13 knockout/knockdown, live-cell imaging, in vitro kinase assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct palmitoylation assay identifying the writer enzyme, functional mutagenesis, and downstream kinase readout — multiple orthogonal methods","pmids":["39169022"],"is_preprint":false},{"year":2015,"finding":"The chaperone-like protein p32 forms a complex with ULK1 and regulates its stability. p32 depletion promotes K48-linked polyubiquitination and proteasomal degradation of ULK1 while impairing K63-linked ubiquitination, resulting in impaired starvation-induced autophagic flux and mitophagy. Restoring ULK1 expression in p32-depleted cells rescues autophagy and mitophagy defects.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiments, mitophagy assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP with rescue experiment, single lab, multiple methods","pmids":["25909887"],"is_preprint":false},{"year":2019,"finding":"USP24, encoded by the PD-risk PARK10 locus, regulates ULK1 stability by affecting its ubiquitination. USP24 knockdown elevates ULK1 protein levels and increases autophagy flux in a manner independent of mTORC1 but dependent on class III PI3K activity.","method":"siRNA knockdown, co-immunoprecipitation, ubiquitination assay, autophagy flux assays, iPSC-derived dopaminergic neurons","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — genetic knockdown with ubiquitination assay, single lab","pmids":["30957634"],"is_preprint":false},{"year":2020,"finding":"TRAF3 forms a complex with TRAF2 and cIAP1 and mediates K48-linked ubiquitination and degradation of ULK1, promoting mitochondrial ROS production and inflammasome-mediated pyroptosis in macrophages. ULK1 knockdown promotes LPS/nigericin-induced inflammasome activation.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, ROS measurement, inflammasome activation assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP and ubiquitination assay, single lab, multiple cellular readouts","pmids":["32275117"],"is_preprint":false},{"year":2024,"finding":"DRAK2 directly phosphorylates ULK1 at Ser56, inducing ULK1 ubiquitylation and proteasomal degradation, thereby suppressing autophagy and impairing pancreatic β cell function. ULK1-S56A mutation or DRAK2 inhibition preserves mitochondrial function and insulin secretion against lipotoxicity.","method":"Phosphoproteomics, in vitro kinase assay, site-directed mutagenesis, conditional knockout mice, autophagy flux assays","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — phosphoproteomics-identified site confirmed by in vitro kinase assay with mutagenesis, validated in cKO mouse model","pmids":["38324636"],"is_preprint":false},{"year":2023,"finding":"ULK1 phosphorylates the SNARE protein YKT6, preventing premature bundling with vacuolar/lysosomal SNAREs (Vam3, Vti1) and inhibiting premature autophagosome-lysosome fusion. This regulation of YKT6 is conserved from yeast to mammals.","method":"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, SNARE bundling assay, genetic analysis in yeast and C. elegans","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, SNARE biochemistry, cross-species conservation demonstrated","pmids":["36644903"],"is_preprint":false},{"year":2023,"finding":"The ULK1 complex effector BAG2 regulates autophagy initiation: in growth conditions, unphosphorylated BAG2 sequesters AMBRA1 away from the ER, attenuating autophagy. Under starvation, ULK1 phosphorylates BAG2 at Ser31, supporting AMBRA1 recruitment to the ER membrane and promoting autophagy initiation.","method":"Affinity purification- and proximity labeling-MS interactome, co-immunoprecipitation, in vitro kinase assay, subcellular localization by imaging","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with site identification and localization readout, single lab, multiple methods","pmids":["39207901"],"is_preprint":false},{"year":2022,"finding":"ULK1 physically interacts with SARM1 (via SARM1's SAM domains) in vitro and in vivo; this interaction increases upon neurite damage. ULK1 inhibition or knockdown attenuates neurite fragmentation and SARM1 puncta accumulation in neurons, suggesting ULK1-SARM1 interaction contributes to axonal degeneration.","method":"Co-immunoprecipitation, domain mutagenesis, ULK1 siRNA knockdown, pharmacological inhibition, spinal cord injury mouse model","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal co-IP with domain mapping, single lab, in vitro and in vivo validation","pmids":["36375051"],"is_preprint":false},{"year":2023,"finding":"ULK1 forms large clusters of up to 161 molecules at the ER during amino acid starvation. Clusters with ≥30 molecules are required for autophagosome formation. ULK1 activity is dispensable for initial clustering but required for subsequent cluster expansion involving Atg14, Atg16, and LC3B; expansion also requires Vps34 activity.","method":"Quantitative photoactivated localization microscopy (PALM) with single-molecule sensitivity, CRISPR-based endogenous tagging, pharmacological inhibitors","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — single-molecule super-resolution imaging with quantitative analysis, genetic and pharmacological perturbations, multiple orthogonal methods","pmids":["37774021"],"is_preprint":false},{"year":2023,"finding":"ULK1 phosphorylates SH3PXD2A/TKS5 upon mTOR inactivation by starvation, leading to MMP14 upregulation and promoting cell migration in ovarian carcinoma through a starvation-induced pathway.","method":"In vitro kinase assay, site-directed mutagenesis, LC-MS/MS, cell migration assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with MS-identified sites, single lab","pmids":["37505094"],"is_preprint":false},{"year":2023,"finding":"ULK1/2 phosphorylates paxillin (PXN) at Ser32 and Ser119, weakening its homotypic interactions and liquid-liquid phase separation, thereby impairing focal adhesion assembly and inhibiting breast cancer cell migration. ULK1/2 and FAK/Src have opposing effects on PXN-dependent mechanotransduction and compete for adjacent serine/tyrosine residues.","method":"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, phase separation assay, focal adhesion imaging, cell migration assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, phase separation biochemistry, and functional migration readout — multiple orthogonal methods","pmids":["37846507"],"is_preprint":false},{"year":2021,"finding":"In fission yeast, Atg1 kinase activity requires Atg11 (FIP200 ortholog) but not Atg13. A 62-amino-acid region of Atg11 containing an Atg1-binding domain and a homodimerization domain is sufficient for Atg1 activation. Atg11 activates Atg1 by mediating its dimerization, enabling cis-autophosphorylation of Atg1.","method":"In vitro kinase assay, domain mutagenesis, artificial dimerization rescue experiments, genetic analysis in S. pombe","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro with mutagenesis and artificial dimerization rescue, mechanistically precise","pmids":["32909946"],"is_preprint":false},{"year":2019,"finding":"PP2C phosphatases Ptc2 and Ptc3 in yeast directly dephosphorylate Atg13 and Atg1, promoting macroautophagy induction. These phosphatases interact with the Atg1-Atg13 complex; in their absence, starvation-induced autophagy is inhibited and PAS recruitment of essential autophagy machinery is impaired.","method":"Genetic screens, in vitro phosphatase assay, co-immunoprecipitation, GFP-Atg8 flux assays, PAS imaging","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphatase assay and co-IP with genetic epistasis, single study","pmids":["30655342"],"is_preprint":false},{"year":2014,"finding":"In S. cerevisiae, the Atg1 complex assembly follows a hierarchical mechanism: Atg1 and Atg13 form a tight complex (~100 nM affinity) via their mutually interacting domains (EAT domain of Atg1 and central domain of Atg13), which then binds as a unit to the Atg17-Atg31-Atg29 scaffold (~10 µM affinity) via Atg13. The resulting complex forms primarily a dimer of pentamers in solution.","method":"Hydrogen-deuterium exchange coupled to mass spectrometry, binding affinity measurements, analytical ultracentrifugation","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 / Strong — biophysical characterization with HDX-MS and quantitative binding measurements defining complex architecture","pmids":["25139988"],"is_preprint":false},{"year":2010,"finding":"Atg1 activation requires phosphorylation of Thr226 and Ser230 in the activation loop of its kinase domain; mutation of either site abolishes kinase activity and function in autophagy and the Cvt pathway, without affecting Atg1 localization to the PAS or assembly of the Atg1 kinase complex.","method":"Quantitative mass spectrometry phosphoproteomics, site-directed mutagenesis, in vitro kinase assay, yeast genetic analysis","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — MS-identified activation loop phosphorylation confirmed by mutagenesis and in vitro kinase assay with mechanistic precision","pmids":["20953146"],"is_preprint":false},{"year":2012,"finding":"Atg8/LC3 directly binds Atg1/ULK1 via an LIR-dependent interaction, targeting Atg1/ULK1 to autophagosomes. This promotes autophagosome maturation and/or vacuolar fusion. Atg8 binding also triggers vacuolar degradation of the Atg1-Atg13 complex, coupling Atg1 activity to autophagic flux. Separately, Atg13 directly and constitutively binds Atg1 (not regulated by TORC1 in mammals), and mutations abolishing this interaction impair Atg1 function.","method":"Co-immunoprecipitation, LIR mutagenesis, in vivo autophagy assays in yeast and mammalian cells, fluorescence microscopy","journal":"EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — LIR mutagenesis with direct binding and functional autophagy readouts, cross-species validation","pmids":["22885598"],"is_preprint":false},{"year":2023,"finding":"Hsp90 forms a complex with Atg1/ULK1, suppressing its kinase activity. Upon autophagy-inducing stimuli, Atg1/ULK1 phosphorylates a conserved serine in the Hsp90 amino domain, inhibiting Hsp90 ATPase activity and altering chaperone dynamics, leading to dissociation of the Hsp90:Atg1/ULK1 complex and activation of Atg1/ULK1.","method":"Co-immunoprecipitation, in vitro kinase assay, ATPase activity assay, mutagenesis, NMR/HDX analysis of Hsp90 conformational dynamics","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro assays with mutagenesis and ATPase readout, single study, single lab","pmids":["37453059"],"is_preprint":false},{"year":2020,"finding":"In S. cerevisiae, Atg1 phosphorylates Ykt6 SNARE, keeping it in an inactive state by preventing its interaction with vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion.","method":"In vitro kinase assay, co-immunoprecipitation, SNARE bundling assay, yeast genetic analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with SNARE biochemical bundling assay and genetic validation, mechanistically precise","pmids":["33274589"],"is_preprint":false},{"year":2014,"finding":"In S. cerevisiae, Atg1 phosphorylates Atg9 and this phosphorylation is required for phagophore elongation. The Atg1 consensus phosphorylation sequence was determined using a peptide array approach.","method":"Peptide array-based phosphorylation consensus determination, in vitro kinase assay, yeast genetic analysis","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with peptide array, single lab, yeast model","pmids":["24905091"],"is_preprint":false},{"year":2021,"finding":"In S. cerevisiae, Atg1 phosphorylates Atg13, triggering Atg1 complex dissociation and enabling rapid turnover of Atg1 complex subunits at the PAS. Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1 also self-regulates Atg8-PE levels by phosphorylating and inhibiting the Atg8-specific E2 and E3 enzymes.","method":"In vitro reconstitution with lipidated Atg8, in vitro kinase assays, mutagenesis, live imaging of PAS dynamics","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with lipidated Atg8-PE, multiple substrate phosphorylation events characterized, mechanistically rigorous","pmids":["34798055"],"is_preprint":false},{"year":2022,"finding":"In S. cerevisiae, Atg1 phosphorylates Atg11 at Ser949, Ser1057, and Ser1064 in its CC4 domain; phosphorylation of these sites is required for Atg11 binding to selective autophagy receptors and for efficient cytoplasm-to-vacuole targeting, mitophagy, reticulophagy, and pexophagy.","method":"In vitro kinase assay, mass spectrometry, site-directed mutagenesis, selective autophagy substrate cleavage assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with MS-identified sites, mutagenesis validated across multiple selective autophagy pathways","pmids":["35427192"],"is_preprint":false},{"year":2023,"finding":"The PI3KCI complex in S. cerevisiae is recruited to the pre-autophagosomal structure via interaction of its Atg38 subunit with the Atg1 complex; this interaction is enhanced upon macroautophagy induction in an Atg1 kinase activity-dependent manner.","method":"Co-immunoprecipitation, domain mutagenesis, yeast genetic analysis, fluorescence microscopy","journal":"Journal of Cell Biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP with kinase-dead mutant validation, single lab","pmids":["37436710"],"is_preprint":false},{"year":2020,"finding":"Drosophila Atg1/ULK1 directly phosphorylates the transcriptional co-activator Yorkie at Ser74 and Ser97 (confirmed by in vitro kinase assay), preventing its binding to Scalloped and inhibiting Yorkie's growth-promoting activity. This regulation is independent of Atg13 and the Hippo-Warts pathway, representing a non-autophagy function of Atg1.","method":"In vitro kinase assay, Drosophila genetics (gain-of-function and loss-of-function), co-immunoprecipitation","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with Drosophila genetic validation, single lab","pmids":["32032548"],"is_preprint":false},{"year":2007,"finding":"In Drosophila, overexpression of Atg1 is sufficient to induce autophagy and this requires its kinase activity. Atg1-induced autophagy leads to caspase-dependent apoptotic cell death. Atg1 expression inhibits cell growth and causes negative feedback on TOR activity.","method":"Drosophila genetics, overexpression and loss-of-function studies, kinase-dead mutagenesis, caspase activity assays, TOR reporter assays","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutagenesis with multiple Drosophila genetic readouts, single lab","pmids":["17208179"],"is_preprint":false},{"year":2007,"finding":"In Drosophila, ATG1 inhibits TOR/S6K-dependent cell growth by blocking S6K phosphorylation at Thr389. ATG1 knockdown in mammalian cells induces ectopic S6K activation and S6 phosphorylation. This demonstrates cross-talk between autophagy and cell growth regulation through ATG1-mediated S6K inhibition.","method":"Drosophila genetics, siRNA knockdown in mammalian cells, in vitro kinase assay, immunoblotting for pS6K","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical data across two organisms, single lab","pmids":["17347671"],"is_preprint":false}],"current_model":"ULK1 is a serine/threonine kinase that acts as the master initiator of autophagy: it is activated by AMPK (via phosphorylation of Ser317/Ser777) upon energy stress and inhibited by mTORC1 (via phosphorylation of Ser757) under nutrient sufficiency; once active, ULK1 phosphorylates multiple downstream targets — including Beclin-1 (Ser14), FUNDC1 (Ser17), BNIP3 (Ser17), SEC23B (Ser186), Sec23A, Atg9, Atg11, Atg13, Ykt6/YKT6, VCP, RIPK1, Exo70, paxillin, LDHA, MR, striatin, BAG2, and SH3PXD2A — to regulate autophagosome biogenesis, selective mitophagy, ER-to-Golgi trafficking, stress granule disassembly, cell death, and mechanotransduction; ULK1 activity and stability are tightly controlled by palmitoylation (ZDHHC13), arginine methylation (PRMT5/KDM5C), and multiple ubiquitin E3 ligases/DUBs (NEDD4L, TRIM27, TRAF3, BTRC, USP20, USP24), as well as by binding to GABARAP/LC3 subfamily members via its LIR motif and by feedback phosphorylation of mTORC1 component raptor."},"narrative":{"mechanistic_narrative":"ULK1 is a serine/threonine kinase that functions as the apical initiator of autophagy, integrating nutrient and energy status to trigger autophagosome biogenesis [PMID:21258367, PMID:21205641]. Its activity is reciprocally gated by upstream kinases: AMPK phosphorylates ULK1 at Ser317/Ser777 under glucose starvation to promote autophagy and mitophagy, while mTOR phosphorylates Ser757 under nutrient sufficiency to disrupt the ULK1–AMPK interaction and block activation [PMID:21258367, PMID:21205641]. Once active, ULK1 nucleates and expands large ER-associated clusters required for autophagosome formation, with cluster expansion depending on ULK1 kinase activity and Vps34 [PMID:37774021]. ULK1 propagates the autophagy signal by phosphorylating a broad substrate set: Beclin-1 (Ser14) to stimulate the ATG14L–VPS34 lipid kinase complex [PMID:23685627], FUNDC1 (Ser17) and BNIP3 (Ser17) at the mitochondrial surface to enhance LC3 binding and selective mitophagy [PMID:24671035, PMID:34654847], and ATG13 [PMID:35246531]; it also controls Atg9 trafficking [PMID:16940348] and SNARE-dependent autophagosome–lysosome fusion by phosphorylating YKT6 to prevent premature bundling [PMID:36644903]. Beyond canonical autophagy, ULK1 phosphorylates COPII components SEC23B (Ser186) and Sec23A to remodel ER-to-Golgi trafficking during autophagy [PMID:30596474, PMID:28486929], phosphorylates VCP/p97 to drive stress-granule disassembly [PMID:30979586], and acts in cell death and migration pathways via RIPK1 [PMID:32320653] and paxillin [PMID:37846507]. ULK1 activity, localization, and abundance are tightly controlled by post-translational modification — arginine dimethylation by PRMT5 (reversed by KDM5C) that licenses activating autophosphorylation at Thr180 [PMID:35246531], palmitoylation by ZDHHC13 that drives translocation to autophagosome formation sites [PMID:39169022], and a network of E3 ligases and deubiquitinases (NEDD4L, TRIM27, TRAF3, BTRC, USP20, USP24) that set ULK1 protein levels [PMID:27932573, PMID:35670107, PMID:33213267, PMID:29487085]. ULK1 also feeds back to inhibit mTORC1 by phosphorylating raptor [PMID:21795849]. Mechanistic studies in yeast establish that the kinase (Atg1) is activated by FIP200/Atg11-mediated dimerization and activation-loop autophosphorylation, with ATG8/LC3-family binding through the LIR motif being required for full kinase activity [PMID:32909946, PMID:20953146, PMID:31208283].","teleology":[{"year":2010,"claim":"Established the upstream signal that switches ULK1 on, answering how energy status is transduced to autophagy and mitochondrial homeostasis.","evidence":"Genetic analysis in mammalian liver and C. elegans with phospho-deficient ULK1 reconstitution","pmids":["21205641"],"confidence":"High","gaps":["Did not map all activating sites","Did not resolve how phosphorylation alters ULK1 conformation or substrate selection"]},{"year":2011,"claim":"Resolved the reciprocal AMPK/mTOR phosphorylation logic and identified a ULK1-to-mTORC1 negative feedback loop, defining ULK1 as a bidirectional node coupling nutrient sensing to autophagy.","evidence":"In vitro kinase assays, site-specific mutagenesis, co-IP and epistasis in cells and mice; separate raptor phosphorylation study","pmids":["21258367","21795849"],"confidence":"High","gaps":["Stoichiometry and timing of competing AMPK/mTOR inputs in vivo not resolved","raptor feedback significance under physiological autophagy unclear"]},{"year":2013,"claim":"Identified Beclin-1 Ser14 as a direct ULK1 substrate linking ULK1 activation to VPS34 lipid kinase activation, establishing the downstream PI3K-activating step.","evidence":"In vitro kinase assay, mutagenesis, cross-species epistasis, VPS34 lipid kinase assay","pmids":["23685627"],"confidence":"High","gaps":["Did not establish spatial coordination of Beclin-1 phosphorylation with autophagosome nucleation"]},{"year":2014,"claim":"Placed ULK1 at the mitochondrial surface in selective mitophagy by identifying FUNDC1 Ser17 phosphorylation, explaining how ULK1 directs cargo-specific autophagy.","evidence":"Co-IP, in vitro kinase assay, rescue in ULK1-null cells, live imaging","pmids":["24671035"],"confidence":"High","gaps":["Trigger for ULK1 translocation to mitochondria not fully defined"]},{"year":2018,"claim":"Extended ULK1 substrate range to COPII machinery (SEC23B), showing ULK1 reroutes secretory trafficking to support autophagic flux.","evidence":"In vitro kinase assay, MS site mapping, mutagenesis, subcellular fractionation","pmids":["30596474"],"confidence":"High","gaps":["Reconciliation with opposing Sec23A regulation not addressed","Quantitative contribution to flux unclear"]},{"year":2019,"claim":"Defined cargo-localized, AMPK/mTOR-independent ULK1 activation via NDP52/FIP200/TBK1, and uncovered non-autophagy roles in stress-granule disassembly through VCP phosphorylation.","evidence":"CRISPR epistasis with chemically induced dimerization; in vitro kinase, VCP ATPase assay, mouse KO myopathy model","pmids":["30853401","30979586"],"confidence":"High","gaps":["How focal activation bypasses canonical upstream kinases mechanistically unclear","Direct disease causality of ULK1/2-VCP axis not established in humans"]},{"year":2022,"claim":"Revealed that ULK1 activation is licensed by arginine methylation and gated by competing E3 ligase networks, defining oxygen- and ubiquitin-dependent control of ULK1 abundance and activity.","evidence":"In vitro methylation/ubiquitination assays, writer/eraser identification, mutagenesis, mouse KO","pmids":["35246531","35670107"],"confidence":"High","gaps":["Crosstalk hierarchy among the many ULK1 PTMs not integrated","Physiological dominance of each modifier in different tissues unclear"]},{"year":2023,"claim":"Quantified ULK1 cluster formation at the ER as the physical basis of autophagosome initiation and showed kinase-dependent cluster expansion.","evidence":"Single-molecule PALM with endogenous tagging and pharmacological perturbation","pmids":["37774021"],"confidence":"High","gaps":["Molecular driver of initial clustering (kinase-independent) unidentified","Link between cluster size and downstream Atg14/LC3 recruitment kinetics incomplete"]},{"year":2024,"claim":"Identified palmitoylation by ZDHHC13 as the membrane-targeting signal for ULK1 translocation to autophagosome formation sites, connecting lipid modification to autophagy initiation.","evidence":"Acyl-RAC assay, ZDHHC13 KO/KD, live imaging, in vitro kinase readout on ATG14L","pmids":["39169022"],"confidence":"High","gaps":["Depalmitoylating enzyme and turnover dynamics not defined","Interplay with phosphorylation-dependent clustering unresolved"]},{"year":null,"claim":"How the dozens of ULK1 phosphorylation events, ubiquitin marks, methylation, and palmitoylation are temporally and spatially integrated to produce a single coherent autophagy decision remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model ordering competing PTMs in time","Substrate selectivity determinants across autophagy vs non-autophagy functions undefined","Structural basis of human ULK1 activation not fully resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,4,9,21,29,34,42,43,44]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,4,38]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[9,30,32]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[4,11]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[3,9,10]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,1,2,4,32,43]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,1,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[21,27]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[9,10,29]}],"complexes":["ULK1-ATG13-FIP200 complex","Atg1-Atg13-Atg17 complex (yeast)"],"partners":["ATG13","FIP200/ATG11","AMPK","RPTOR","FUNDC1","GABARAP","MAP1LC3B","VCP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75385","full_name":"Serine/threonine-protein kinase ULK1","aliases":["Autophagy-related protein 1 homolog","ATG1","hATG1","Unc-51-like kinase 1"],"length_aa":1050,"mass_kda":112.6,"function":"Serine/threonine-protein kinase involved in autophagy in response to starvation (PubMed:18936157, PubMed:21460634, PubMed:21795849, PubMed:23524951, PubMed:25040165, PubMed:29487085, PubMed:31123703). Acts upstream of phosphatidylinositol 3-kinase PIK3C3 to regulate the formation of autophagophores, the precursors of autophagosomes (PubMed:18936157, PubMed:21460634, PubMed:21795849, PubMed:25040165, PubMed:39384743). Part of regulatory feedback loops in autophagy: acts both as a downstream effector and negative regulator of mammalian target of rapamycin complex 1 (mTORC1) via interaction with RPTOR (PubMed:21795849). Activated via phosphorylation by AMPK and also acts as a regulator of AMPK by mediating phosphorylation of AMPK subunits PRKAA1, PRKAB2 and PRKAG1, leading to negatively regulate AMPK activity (PubMed:21460634). May phosphorylate ATG13/KIAA0652 and RPTOR; however such data need additional evidences (PubMed:18936157). Plays a role early in neuronal differentiation and is required for granule cell axon formation (PubMed:11146101). Also phosphorylates SESN2 and SQSTM1 to regulate autophagy (PubMed:25040165, PubMed:37306101). Phosphorylates FLCN, promoting autophagy (PubMed:25126726). Phosphorylates AMBRA1 in response to autophagy induction, releasing AMBRA1 from the cytoskeletal docking site to induce autophagosome nucleation (PubMed:20921139). Phosphorylates ATG4B, leading to inhibit autophagy by decreasing both proteolytic activation and delipidation activities of ATG4B (PubMed:28821708)","subcellular_location":"Cytoplasm, cytosol; Preautophagosomal structure","url":"https://www.uniprot.org/uniprotkb/O75385/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ULK1","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000177169","cell_line_id":"CID001300","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"cytoplasmic","grade":2},{"compartment":"er","grade":2}],"interactors":[{"gene":"ATG101","stoichiometry":10.0},{"gene":"RCBTB2","stoichiometry":10.0},{"gene":"RB1CC1","stoichiometry":10.0},{"gene":"ATG13","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001300","total_profiled":1310},"omim":[{"mim_id":"620175","title":"RUBICON-LIKE AUTOPHAGY ENHANCER; RUBCNL","url":"https://www.omim.org/entry/620175"},{"mim_id":"619705","title":"IMMUNODEFICIENCY 93 AND HYPERTROPHIC CARDIOMYOPATHY; IMD93","url":"https://www.omim.org/entry/619705"},{"mim_id":"618616","title":"MITOGEN-ACTIVATED PROTEIN KINASE 15; MAPK15","url":"https://www.omim.org/entry/618616"},{"mim_id":"617679","title":"KELCH-LIKE 20; KLHL20","url":"https://www.omim.org/entry/617679"},{"mim_id":"617502","title":"WD REPEAT-CONTAINING PROTEIN 41; WDR41","url":"https://www.omim.org/entry/617502"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ULK1"},"hgnc":{"alias_symbol":["ATG1","ATG1A"],"prev_symbol":[]},"alphafold":{"accession":"O75385","domains":[{"cath_id":"3.30.200.20","chopping":"9-95","consensus_level":"medium","plddt":87.0521,"start":9,"end":95},{"cath_id":"1.10.510.10","chopping":"98-280","consensus_level":"medium","plddt":91.8206,"start":98,"end":280},{"cath_id":"1.20.58.80","chopping":"976-1050","consensus_level":"medium","plddt":84.5585,"start":976,"end":1050},{"cath_id":"1.20.58","chopping":"837-868_887-972","consensus_level":"medium","plddt":85.5925,"start":837,"end":972}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75385","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75385-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75385-F1-predicted_aligned_error_v6.png","plddt_mean":59.41},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ULK1","jax_strain_url":"https://www.jax.org/strain/search?query=ULK1"},"sequence":{"accession":"O75385","fasta_url":"https://rest.uniprot.org/uniprotkb/O75385.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75385/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75385"}},"corpus_meta":[{"pmid":"21258367","id":"PMC_21258367","title":"AMPK 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and Ser777 to activate it under glucose starvation, promoting autophagy. Conversely, mTOR phosphorylates ULK1 at Ser757 under nutrient sufficiency, disrupting the interaction between ULK1 and AMPK and preventing ULK1 activation. This coordinated phosphorylation is required for autophagy induction.\",\n      \"method\": \"In vitro kinase assays, phospho-specific antibodies, mutagenesis, co-immunoprecipitation, genetic epistasis in cell lines and mouse models\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct in vitro phosphorylation assays with site-specific mutagenesis, replicated independently by two groups (PMID:21258367 and PMID:21205641)\",\n      \"pmids\": [\"21258367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AMPK phosphorylates ULK1 (and ULK2), connecting energy sensing to autophagy and mitophagy. Loss of AMPK or ULK1 causes aberrant accumulation of the autophagy adaptor p62 and defective mitophagy; reconstitution with a ULK1 mutant that cannot be phosphorylated by AMPK reveals this phosphorylation is required for mitochondrial homeostasis and cell survival during starvation.\",\n      \"method\": \"Genetic analysis in mammalian liver and C. elegans, biochemical reconstitution with phospho-deficient mutants, co-immunoprecipitation\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution with phospho-deficient mutant, genetic epistasis across two organisms, replicated independently\",\n      \"pmids\": [\"21205641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Upon amino-acid starvation or mTOR inhibition, activated ULK1 phosphorylates Beclin-1 on Ser14, enhancing the activity of the ATG14L-containing VPS34 complex and promoting autophagic flux. This phosphorylation is required for full autophagic induction in mammals and C. elegans.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, genetic epistasis in C. elegans and mammalian cells, VPS34 lipid kinase activity assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, cross-species genetic epistasis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"23685627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"siRNA-mediated depletion of ULK1 inhibits starvation-induced redistribution of mammalian Atg9 from the trans-Golgi network to peripheral endosomal/autophagosomal membranes, placing ULK1 upstream of Atg9 trafficking in autophagy.\",\n      \"method\": \"siRNA knockdown, live-cell fluorescence imaging, immunofluorescence colocalization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA knockdown with clear cellular phenotype (Atg9 redistribution), single lab but well-controlled imaging experiment\",\n      \"pmids\": [\"16940348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ULK1 translocates to fragmented mitochondria upon mitophagy induction by hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1 and phosphorylates it at Ser17, enhancing FUNDC1 binding to LC3. A ULK1-binding-deficient FUNDC1 mutant prevents ULK1 translocation and inhibits mitophagy; kinase-active ULK1 and phospho-mimicking FUNDC1 rescue mitophagy in ULK1-null cells.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, rescue experiments in ULK1-null cells, live imaging\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, genetic rescue in null cells, multiple orthogonal methods\",\n      \"pmids\": [\"24671035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ULK1 binds raptor (a component of mTORC1) and induces its phosphorylation, inhibiting mTORC1 kinase activity and cell proliferation independently of Atg5 and TSC2. This identifies a negative feedback loop from ULK1 to mTORC1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, ULK1 knockdown/overexpression, cell proliferation assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and in vitro kinase data from single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21795849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"During selective autophagy, NDP52 localizes and activates the ULK1 complex at cargo through interaction with FIP200/ULK1, facilitated by TBK1. Ectopic tethering of ULK1 to cargo bypasses the need for autophagy receptors and TBK1. This focal activation of ULK1 occurs independently of AMPK and mTOR.\",\n      \"method\": \"Chemically inducible dimerization assays, CRISPR KO lines, genetic epistasis, co-immunoprecipitation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic CRISPR-based epistasis combined with CID assays and co-IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"30853401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ULK1 and ULK2 localize to stress granules and phosphorylate VCP/p97, increasing VCP's ATPase activity and ability to disassemble stress granules. Disruption of ULK1/2 expression in mice causes a vacuolar myopathy with TDP-43-positive inclusions similar to that caused by VCP mutations.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, mouse knockout model, biochemical VCP activity assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay, mouse KO phenotype, enzymatic activity assay, multiple orthogonal methods\",\n      \"pmids\": [\"30979586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The E3 ubiquitin ligase NEDD4L ubiquitylates ULK1 for proteasomal degradation during autophagy progression, down-regulating ULK1 protein levels. ULK1 mRNA continues to be transcribed during this period, and upon reactivation of mTOR-dependent protein synthesis, ULK1 levels are restored. This NEDD4L-ULK1 axis controls oscillatory activation of autophagy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, cycloheximide chase, mTOR inhibitor experiments, pulse-chase\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP with ubiquitination assay, single lab, multiple supporting methods\",\n      \"pmids\": [\"27932573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ULK1 phosphorylates SEC23B at Ser186 upon starvation, preventing its interaction with FBXW5 (an F-box E3 ubiquitin ligase) and thus stabilizing SEC23B. Phosphorylated SEC23B associates with SEC24A/B and relocalizes to the ER-Golgi intermediate compartment, promoting autophagic flux.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry, site-directed mutagenesis, subcellular fractionation/localization\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, MS-identified sites, multiple orthogonal methods in one study\",\n      \"pmids\": [\"30596474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ULK1 phosphorylates Sec23A, a COPII vesicle coat component, inhibiting its interaction with Sec31A. This reduces ER-to-Golgi trafficking during autophagy induction. Phosphorylation sites Ser207, Ser312, and Thr405 on Sec23A were identified; Ser207 most faithfully recapitulates ULK1's regulatory effects.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, live-cell imaging of ERES\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, single lab\",\n      \"pmids\": [\"28486929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ULK1 phosphorylates BNIP3 at Ser17 adjacent to its LIR motif, promoting BNIP3 interaction with LC3 and mitophagy. ULK1 also similarly phosphorylates BNIP3L at Ser35. Additionally, ULK1 interaction promotes BNIP3 protein stability by limiting its proteasomal turnover, dependent on an intact BH3 domain.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, cycloheximide chase, mitophagy flux assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with site mutagenesis, single lab, multiple methods\",\n      \"pmids\": [\"34654847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ULK1 phosphorylates Exo70, a component of the exocyst complex, inhibiting Exo70 homo-oligomerization and assembly into the exocyst complex, thereby suppressing breast cancer cell migration and invasion. ERK1/2 phosphorylates Exo70 upon growth factor stimulation, which in turn suppresses its phosphorylation by ULK1.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell migration/invasion assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with substrate identification, single lab, multiple methods\",\n      \"pmids\": [\"31913283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GABARAP subfamily ATG8 proteins positively regulate ULK1 activity and autophagosome formation, while LC3 subfamily proteins negatively regulate ULK1 activity. Mutation of the LIR motif of endogenous ULK1 (disrupting ATG8-ULK1 interaction) drastically reduced ULK1 kinase activity, autophagic degradation, and phagophore formation. Similarly, disruption of ATG13-ATG8 interaction suppressed ULK1 activity.\",\n      \"method\": \"CRISPR/TALEN genome editing of LIR motif, ATG8 subfamily depletion, ULK1 kinase activity assays, autophagy flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous LIR mutagenesis by genome editing, multiple CRISPR KO lines, multiple orthogonal readouts\",\n      \"pmids\": [\"31208283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRMT5 catalyzes symmetrical dimethylation of ULK1 at Arg170 (R170me2s), a modification removed by KDM5C. Low oxygen decreases KDM5C activity, causing accumulation of ULK1 R170me2s. Dimethylation promotes ULK1 autophosphorylation at T180, a prerequisite for activation, leading to phosphorylation of Atg13 and Beclin1, autophagosome formation, and mitochondrial clearance.\",\n      \"method\": \"In vitro methylation assay, mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, autophagy flux assays, cell proliferation under hypoxia\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro assays with writer/eraser identification, mutagenesis, and functional validation through multiple downstream readouts\",\n      \"pmids\": [\"35246531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GSK3B directly interacts with ULK1 and phosphorylates it at Ser405 and Ser415 within the GABARAP-interacting region upon insulin withdrawal. This phosphorylation facilitates ULK1 interaction with MAP1LC3B and GABARAPL1; phosphorylation-defective ULK1 mutants fail to interact with these ATG8 proteins and cannot induce autophagy flux.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, autophagy flux assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, single lab\",\n      \"pmids\": [\"33654220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MAPK1/ERK2-MAPK3/ERK1 phosphorylates ULK1, triggering its interaction with the E3 ubiquitin ligase BTRC (β-TrCP) and subsequent K48-linked ubiquitination and proteasomal degradation of ULK1. This attenuates mitophagy and promotes breast cancer bone metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, pharmacological inhibitors, in vitro kinase assay, cancer cell invasion assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP and ubiquitination assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33213267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 directly interacts with LDHA and phosphorylates it at Ser196 under nutrient deprivation, promoting lactate production. Lactate in turn mediates Vps34 lactylation at Lys356 and Lys781 (via KAT5/TIP60), enhancing Vps34 association with Beclin1, Atg14L, and UVRAG and increasing Vps34 lipid kinase activity, thereby promoting autophagic flux.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry identification of lactylation sites, site-directed mutagenesis, Vps34 lipid kinase activity assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis and enzymatic activity readout, single lab, multiple methods\",\n      \"pmids\": [\"37267363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP20 (ubiquitin-specific protease 20) binds to and stabilizes ULK1 by removing ubiquitin moieties, preventing lysosomal degradation of ULK1 at basal state. Depletion of USP20 inhibits LC3 puncta formation and autophagy initiation. At later stages of autophagy, USP20 dissociates from ULK1, leading to ULK1 degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, autophagy flux assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP and ubiquitination assay, single lab, multiple supporting methods\",\n      \"pmids\": [\"29487085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM27 directly polyubiquitinates ULK1 at Lys568 and Lys571 with K48-linked chains for proteasomal degradation, maintaining basal ULK1 levels. During starvation-induced autophagy, TRIM27 also catalyzes K6- and K11-linked ubiquitination of STK38L, activating STK38L which then phosphorylates ULK1 at Ser495, rendering ULK1 susceptible to TRIM27-mediated hyper-ubiquitination to restrain autophagy amplitude.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis, in vitro kinase assay, mouse knockout models\",\n      \"journal\": \"EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination and kinase assays with mutagenesis, mouse KO validation, multiple orthogonal methods\",\n      \"pmids\": [\"35670107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ULK1-ATG13 complex is phosphorylated by CDK1/cyclin B in mitosis. CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Double knockout of ULK1 and ATG13 blocks cell cycle progression and decreases cancer cell proliferation.\",\n      \"method\": \"Mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, cell cycle analysis, CRISPR KO, mouse xenograft model\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified sites with mutagenesis, single lab, multiple methods including in vivo model\",\n      \"pmids\": [\"32516310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ULK1 phosphorylates RIPK1 at multiple sites, including Ser357 within its intermediate domain, reducing complex IIb/necrosome assembly and inhibiting TNF-induced apoptosis and necroptosis. Loss of ULK1 enhances TNF-induced cell death.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation, cell death assays, ULK1 KO cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis and functional cell death readout, single lab\",\n      \"pmids\": [\"32320653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ULK1 directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A. This creates a positive feedback loop where ULK1-activated PP2A promotes autophagy-dependent protein turnover. ULK1 is also itself a PP2A target (dephosphorylated by PP2A).\",\n      \"method\": \"Quantitative phosphoproteomics, in vitro kinase assay, co-immunoprecipitation, PP2A activity assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay combined with phosphoproteomics, single lab\",\n      \"pmids\": [\"34592149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ULK1 is the principal kinase responsible for phosphorylation of the mineralocorticoid receptor (MR) at Ser843, as identified by a high-throughput screen of 197 kinases and confirmed by in vitro kinase assay, mass spectrometry, and siRNA knockdown. ULK1/2 double KO mouse embryonic fibroblasts show markedly reduced MRS843 phosphorylation. ULK1 activity is itself inhibited by angiotensin II-induced mTOR-mediated phosphorylation.\",\n      \"method\": \"High-throughput kinase screen, in vitro kinase assay, mass spectrometry, siRNA knockdown, ULK1/2 double KO MEFs\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — identified by kinase screen, confirmed by in vitro kinase assay, MS, and genetic KO — multiple orthogonal methods\",\n      \"pmids\": [\"30021155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ULK1 is palmitoylated by the palmitoyltransferase ZDHHC13 upon autophagy induction, and this palmitoylation is required for ULK1 translocation to autophagosome formation sites. Palmitoylated ULK1 enhances phosphorylation of ATG14L, activating PI3-kinase activity and promoting phosphatidylinositol 3-phosphate production needed for autophagosome membrane formation.\",\n      \"method\": \"Acyl-RAC palmitoylation assay, ZDHHC13 knockout/knockdown, live-cell imaging, in vitro kinase assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct palmitoylation assay identifying the writer enzyme, functional mutagenesis, and downstream kinase readout — multiple orthogonal methods\",\n      \"pmids\": [\"39169022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The chaperone-like protein p32 forms a complex with ULK1 and regulates its stability. p32 depletion promotes K48-linked polyubiquitination and proteasomal degradation of ULK1 while impairing K63-linked ubiquitination, resulting in impaired starvation-induced autophagic flux and mitophagy. Restoring ULK1 expression in p32-depleted cells rescues autophagy and mitophagy defects.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiments, mitophagy assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP with rescue experiment, single lab, multiple methods\",\n      \"pmids\": [\"25909887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP24, encoded by the PD-risk PARK10 locus, regulates ULK1 stability by affecting its ubiquitination. USP24 knockdown elevates ULK1 protein levels and increases autophagy flux in a manner independent of mTORC1 but dependent on class III PI3K activity.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, ubiquitination assay, autophagy flux assays, iPSC-derived dopaminergic neurons\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — genetic knockdown with ubiquitination assay, single lab\",\n      \"pmids\": [\"30957634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRAF3 forms a complex with TRAF2 and cIAP1 and mediates K48-linked ubiquitination and degradation of ULK1, promoting mitochondrial ROS production and inflammasome-mediated pyroptosis in macrophages. ULK1 knockdown promotes LPS/nigericin-induced inflammasome activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, ROS measurement, inflammasome activation assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP and ubiquitination assay, single lab, multiple cellular readouts\",\n      \"pmids\": [\"32275117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DRAK2 directly phosphorylates ULK1 at Ser56, inducing ULK1 ubiquitylation and proteasomal degradation, thereby suppressing autophagy and impairing pancreatic β cell function. ULK1-S56A mutation or DRAK2 inhibition preserves mitochondrial function and insulin secretion against lipotoxicity.\",\n      \"method\": \"Phosphoproteomics, in vitro kinase assay, site-directed mutagenesis, conditional knockout mice, autophagy flux assays\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — phosphoproteomics-identified site confirmed by in vitro kinase assay with mutagenesis, validated in cKO mouse model\",\n      \"pmids\": [\"38324636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 phosphorylates the SNARE protein YKT6, preventing premature bundling with vacuolar/lysosomal SNAREs (Vam3, Vti1) and inhibiting premature autophagosome-lysosome fusion. This regulation of YKT6 is conserved from yeast to mammals.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, SNARE bundling assay, genetic analysis in yeast and C. elegans\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, SNARE biochemistry, cross-species conservation demonstrated\",\n      \"pmids\": [\"36644903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The ULK1 complex effector BAG2 regulates autophagy initiation: in growth conditions, unphosphorylated BAG2 sequesters AMBRA1 away from the ER, attenuating autophagy. Under starvation, ULK1 phosphorylates BAG2 at Ser31, supporting AMBRA1 recruitment to the ER membrane and promoting autophagy initiation.\",\n      \"method\": \"Affinity purification- and proximity labeling-MS interactome, co-immunoprecipitation, in vitro kinase assay, subcellular localization by imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with site identification and localization readout, single lab, multiple methods\",\n      \"pmids\": [\"39207901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ULK1 physically interacts with SARM1 (via SARM1's SAM domains) in vitro and in vivo; this interaction increases upon neurite damage. ULK1 inhibition or knockdown attenuates neurite fragmentation and SARM1 puncta accumulation in neurons, suggesting ULK1-SARM1 interaction contributes to axonal degeneration.\",\n      \"method\": \"Co-immunoprecipitation, domain mutagenesis, ULK1 siRNA knockdown, pharmacological inhibition, spinal cord injury mouse model\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal co-IP with domain mapping, single lab, in vitro and in vivo validation\",\n      \"pmids\": [\"36375051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 forms large clusters of up to 161 molecules at the ER during amino acid starvation. Clusters with ≥30 molecules are required for autophagosome formation. ULK1 activity is dispensable for initial clustering but required for subsequent cluster expansion involving Atg14, Atg16, and LC3B; expansion also requires Vps34 activity.\",\n      \"method\": \"Quantitative photoactivated localization microscopy (PALM) with single-molecule sensitivity, CRISPR-based endogenous tagging, pharmacological inhibitors\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — single-molecule super-resolution imaging with quantitative analysis, genetic and pharmacological perturbations, multiple orthogonal methods\",\n      \"pmids\": [\"37774021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 phosphorylates SH3PXD2A/TKS5 upon mTOR inactivation by starvation, leading to MMP14 upregulation and promoting cell migration in ovarian carcinoma through a starvation-induced pathway.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, LC-MS/MS, cell migration assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with MS-identified sites, single lab\",\n      \"pmids\": [\"37505094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1/2 phosphorylates paxillin (PXN) at Ser32 and Ser119, weakening its homotypic interactions and liquid-liquid phase separation, thereby impairing focal adhesion assembly and inhibiting breast cancer cell migration. ULK1/2 and FAK/Src have opposing effects on PXN-dependent mechanotransduction and compete for adjacent serine/tyrosine residues.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis, phase separation assay, focal adhesion imaging, cell migration assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis, phase separation biochemistry, and functional migration readout — multiple orthogonal methods\",\n      \"pmids\": [\"37846507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In fission yeast, Atg1 kinase activity requires Atg11 (FIP200 ortholog) but not Atg13. A 62-amino-acid region of Atg11 containing an Atg1-binding domain and a homodimerization domain is sufficient for Atg1 activation. Atg11 activates Atg1 by mediating its dimerization, enabling cis-autophosphorylation of Atg1.\",\n      \"method\": \"In vitro kinase assay, domain mutagenesis, artificial dimerization rescue experiments, genetic analysis in S. pombe\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro with mutagenesis and artificial dimerization rescue, mechanistically precise\",\n      \"pmids\": [\"32909946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PP2C phosphatases Ptc2 and Ptc3 in yeast directly dephosphorylate Atg13 and Atg1, promoting macroautophagy induction. These phosphatases interact with the Atg1-Atg13 complex; in their absence, starvation-induced autophagy is inhibited and PAS recruitment of essential autophagy machinery is impaired.\",\n      \"method\": \"Genetic screens, in vitro phosphatase assay, co-immunoprecipitation, GFP-Atg8 flux assays, PAS imaging\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphatase assay and co-IP with genetic epistasis, single study\",\n      \"pmids\": [\"30655342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In S. cerevisiae, the Atg1 complex assembly follows a hierarchical mechanism: Atg1 and Atg13 form a tight complex (~100 nM affinity) via their mutually interacting domains (EAT domain of Atg1 and central domain of Atg13), which then binds as a unit to the Atg17-Atg31-Atg29 scaffold (~10 µM affinity) via Atg13. The resulting complex forms primarily a dimer of pentamers in solution.\",\n      \"method\": \"Hydrogen-deuterium exchange coupled to mass spectrometry, binding affinity measurements, analytical ultracentrifugation\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biophysical characterization with HDX-MS and quantitative binding measurements defining complex architecture\",\n      \"pmids\": [\"25139988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Atg1 activation requires phosphorylation of Thr226 and Ser230 in the activation loop of its kinase domain; mutation of either site abolishes kinase activity and function in autophagy and the Cvt pathway, without affecting Atg1 localization to the PAS or assembly of the Atg1 kinase complex.\",\n      \"method\": \"Quantitative mass spectrometry phosphoproteomics, site-directed mutagenesis, in vitro kinase assay, yeast genetic analysis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — MS-identified activation loop phosphorylation confirmed by mutagenesis and in vitro kinase assay with mechanistic precision\",\n      \"pmids\": [\"20953146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Atg8/LC3 directly binds Atg1/ULK1 via an LIR-dependent interaction, targeting Atg1/ULK1 to autophagosomes. This promotes autophagosome maturation and/or vacuolar fusion. Atg8 binding also triggers vacuolar degradation of the Atg1-Atg13 complex, coupling Atg1 activity to autophagic flux. Separately, Atg13 directly and constitutively binds Atg1 (not regulated by TORC1 in mammals), and mutations abolishing this interaction impair Atg1 function.\",\n      \"method\": \"Co-immunoprecipitation, LIR mutagenesis, in vivo autophagy assays in yeast and mammalian cells, fluorescence microscopy\",\n      \"journal\": \"EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — LIR mutagenesis with direct binding and functional autophagy readouts, cross-species validation\",\n      \"pmids\": [\"22885598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Hsp90 forms a complex with Atg1/ULK1, suppressing its kinase activity. Upon autophagy-inducing stimuli, Atg1/ULK1 phosphorylates a conserved serine in the Hsp90 amino domain, inhibiting Hsp90 ATPase activity and altering chaperone dynamics, leading to dissociation of the Hsp90:Atg1/ULK1 complex and activation of Atg1/ULK1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, ATPase activity assay, mutagenesis, NMR/HDX analysis of Hsp90 conformational dynamics\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assays with mutagenesis and ATPase readout, single study, single lab\",\n      \"pmids\": [\"37453059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In S. cerevisiae, Atg1 phosphorylates Ykt6 SNARE, keeping it in an inactive state by preventing its interaction with vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, SNARE bundling assay, yeast genetic analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with SNARE biochemical bundling assay and genetic validation, mechanistically precise\",\n      \"pmids\": [\"33274589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In S. cerevisiae, Atg1 phosphorylates Atg9 and this phosphorylation is required for phagophore elongation. The Atg1 consensus phosphorylation sequence was determined using a peptide array approach.\",\n      \"method\": \"Peptide array-based phosphorylation consensus determination, in vitro kinase assay, yeast genetic analysis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with peptide array, single lab, yeast model\",\n      \"pmids\": [\"24905091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In S. cerevisiae, Atg1 phosphorylates Atg13, triggering Atg1 complex dissociation and enabling rapid turnover of Atg1 complex subunits at the PAS. Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1 also self-regulates Atg8-PE levels by phosphorylating and inhibiting the Atg8-specific E2 and E3 enzymes.\",\n      \"method\": \"In vitro reconstitution with lipidated Atg8, in vitro kinase assays, mutagenesis, live imaging of PAS dynamics\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with lipidated Atg8-PE, multiple substrate phosphorylation events characterized, mechanistically rigorous\",\n      \"pmids\": [\"34798055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In S. cerevisiae, Atg1 phosphorylates Atg11 at Ser949, Ser1057, and Ser1064 in its CC4 domain; phosphorylation of these sites is required for Atg11 binding to selective autophagy receptors and for efficient cytoplasm-to-vacuole targeting, mitophagy, reticulophagy, and pexophagy.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry, site-directed mutagenesis, selective autophagy substrate cleavage assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with MS-identified sites, mutagenesis validated across multiple selective autophagy pathways\",\n      \"pmids\": [\"35427192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The PI3KCI complex in S. cerevisiae is recruited to the pre-autophagosomal structure via interaction of its Atg38 subunit with the Atg1 complex; this interaction is enhanced upon macroautophagy induction in an Atg1 kinase activity-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, domain mutagenesis, yeast genetic analysis, fluorescence microscopy\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP with kinase-dead mutant validation, single lab\",\n      \"pmids\": [\"37436710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Drosophila Atg1/ULK1 directly phosphorylates the transcriptional co-activator Yorkie at Ser74 and Ser97 (confirmed by in vitro kinase assay), preventing its binding to Scalloped and inhibiting Yorkie's growth-promoting activity. This regulation is independent of Atg13 and the Hippo-Warts pathway, representing a non-autophagy function of Atg1.\",\n      \"method\": \"In vitro kinase assay, Drosophila genetics (gain-of-function and loss-of-function), co-immunoprecipitation\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with Drosophila genetic validation, single lab\",\n      \"pmids\": [\"32032548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Drosophila, overexpression of Atg1 is sufficient to induce autophagy and this requires its kinase activity. Atg1-induced autophagy leads to caspase-dependent apoptotic cell death. Atg1 expression inhibits cell growth and causes negative feedback on TOR activity.\",\n      \"method\": \"Drosophila genetics, overexpression and loss-of-function studies, kinase-dead mutagenesis, caspase activity assays, TOR reporter assays\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutagenesis with multiple Drosophila genetic readouts, single lab\",\n      \"pmids\": [\"17208179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Drosophila, ATG1 inhibits TOR/S6K-dependent cell growth by blocking S6K phosphorylation at Thr389. ATG1 knockdown in mammalian cells induces ectopic S6K activation and S6 phosphorylation. This demonstrates cross-talk between autophagy and cell growth regulation through ATG1-mediated S6K inhibition.\",\n      \"method\": \"Drosophila genetics, siRNA knockdown in mammalian cells, in vitro kinase assay, immunoblotting for pS6K\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical data across two organisms, single lab\",\n      \"pmids\": [\"17347671\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ULK1 is a serine/threonine kinase that acts as the master initiator of autophagy: it is activated by AMPK (via phosphorylation of Ser317/Ser777) upon energy stress and inhibited by mTORC1 (via phosphorylation of Ser757) under nutrient sufficiency; once active, ULK1 phosphorylates multiple downstream targets — including Beclin-1 (Ser14), FUNDC1 (Ser17), BNIP3 (Ser17), SEC23B (Ser186), Sec23A, Atg9, Atg11, Atg13, Ykt6/YKT6, VCP, RIPK1, Exo70, paxillin, LDHA, MR, striatin, BAG2, and SH3PXD2A — to regulate autophagosome biogenesis, selective mitophagy, ER-to-Golgi trafficking, stress granule disassembly, cell death, and mechanotransduction; ULK1 activity and stability are tightly controlled by palmitoylation (ZDHHC13), arginine methylation (PRMT5/KDM5C), and multiple ubiquitin E3 ligases/DUBs (NEDD4L, TRIM27, TRAF3, BTRC, USP20, USP24), as well as by binding to GABARAP/LC3 subfamily members via its LIR motif and by feedback phosphorylation of mTORC1 component raptor.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ULK1 is a serine/threonine kinase that functions as the apical initiator of autophagy, integrating nutrient and energy status to trigger autophagosome biogenesis [#0, #1]. Its activity is reciprocally gated by upstream kinases: AMPK phosphorylates ULK1 at Ser317/Ser777 under glucose starvation to promote autophagy and mitophagy, while mTOR phosphorylates Ser757 under nutrient sufficiency to disrupt the ULK1–AMPK interaction and block activation [#0, #1]. Once active, ULK1 nucleates and expands large ER-associated clusters required for autophagosome formation, with cluster expansion depending on ULK1 kinase activity and Vps34 [#32]. ULK1 propagates the autophagy signal by phosphorylating a broad substrate set: Beclin-1 (Ser14) to stimulate the ATG14L–VPS34 lipid kinase complex [#2], FUNDC1 (Ser17) and BNIP3 (Ser17) at the mitochondrial surface to enhance LC3 binding and selective mitophagy [#4, #11], and ATG13 [#14]; it also controls Atg9 trafficking [#3] and SNARE-dependent autophagosome–lysosome fusion by phosphorylating YKT6 to prevent premature bundling [#29]. Beyond canonical autophagy, ULK1 phosphorylates COPII components SEC23B (Ser186) and Sec23A to remodel ER-to-Golgi trafficking during autophagy [#9, #10], phosphorylates VCP/p97 to drive stress-granule disassembly [#7], and acts in cell death and migration pathways via RIPK1 [#21] and paxillin [#34]. ULK1 activity, localization, and abundance are tightly controlled by post-translational modification — arginine dimethylation by PRMT5 (reversed by KDM5C) that licenses activating autophosphorylation at Thr180 [#14], palmitoylation by ZDHHC13 that drives translocation to autophagosome formation sites [#24], and a network of E3 ligases and deubiquitinases (NEDD4L, TRIM27, TRAF3, BTRC, USP20, USP24) that set ULK1 protein levels [#8, #19, #16, #18]. ULK1 also feeds back to inhibit mTORC1 by phosphorylating raptor [#5]. Mechanistic studies in yeast establish that the kinase (Atg1) is activated by FIP200/Atg11-mediated dimerization and activation-loop autophosphorylation, with ATG8/LC3-family binding through the LIR motif being required for full kinase activity [#35, #38, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established the upstream signal that switches ULK1 on, answering how energy status is transduced to autophagy and mitochondrial homeostasis.\",\n      \"evidence\": \"Genetic analysis in mammalian liver and C. elegans with phospho-deficient ULK1 reconstitution\",\n      \"pmids\": [\"21205641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map all activating sites\", \"Did not resolve how phosphorylation alters ULK1 conformation or substrate selection\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the reciprocal AMPK/mTOR phosphorylation logic and identified a ULK1-to-mTORC1 negative feedback loop, defining ULK1 as a bidirectional node coupling nutrient sensing to autophagy.\",\n      \"evidence\": \"In vitro kinase assays, site-specific mutagenesis, co-IP and epistasis in cells and mice; separate raptor phosphorylation study\",\n      \"pmids\": [\"21258367\", \"21795849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and timing of competing AMPK/mTOR inputs in vivo not resolved\", \"raptor feedback significance under physiological autophagy unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified Beclin-1 Ser14 as a direct ULK1 substrate linking ULK1 activation to VPS34 lipid kinase activation, establishing the downstream PI3K-activating step.\",\n      \"evidence\": \"In vitro kinase assay, mutagenesis, cross-species epistasis, VPS34 lipid kinase assay\",\n      \"pmids\": [\"23685627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish spatial coordination of Beclin-1 phosphorylation with autophagosome nucleation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed ULK1 at the mitochondrial surface in selective mitophagy by identifying FUNDC1 Ser17 phosphorylation, explaining how ULK1 directs cargo-specific autophagy.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, rescue in ULK1-null cells, live imaging\",\n      \"pmids\": [\"24671035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for ULK1 translocation to mitochondria not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended ULK1 substrate range to COPII machinery (SEC23B), showing ULK1 reroutes secretory trafficking to support autophagic flux.\",\n      \"evidence\": \"In vitro kinase assay, MS site mapping, mutagenesis, subcellular fractionation\",\n      \"pmids\": [\"30596474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with opposing Sec23A regulation not addressed\", \"Quantitative contribution to flux unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined cargo-localized, AMPK/mTOR-independent ULK1 activation via NDP52/FIP200/TBK1, and uncovered non-autophagy roles in stress-granule disassembly through VCP phosphorylation.\",\n      \"evidence\": \"CRISPR epistasis with chemically induced dimerization; in vitro kinase, VCP ATPase assay, mouse KO myopathy model\",\n      \"pmids\": [\"30853401\", \"30979586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How focal activation bypasses canonical upstream kinases mechanistically unclear\", \"Direct disease causality of ULK1/2-VCP axis not established in humans\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed that ULK1 activation is licensed by arginine methylation and gated by competing E3 ligase networks, defining oxygen- and ubiquitin-dependent control of ULK1 abundance and activity.\",\n      \"evidence\": \"In vitro methylation/ubiquitination assays, writer/eraser identification, mutagenesis, mouse KO\",\n      \"pmids\": [\"35246531\", \"35670107\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk hierarchy among the many ULK1 PTMs not integrated\", \"Physiological dominance of each modifier in different tissues unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Quantified ULK1 cluster formation at the ER as the physical basis of autophagosome initiation and showed kinase-dependent cluster expansion.\",\n      \"evidence\": \"Single-molecule PALM with endogenous tagging and pharmacological perturbation\",\n      \"pmids\": [\"37774021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular driver of initial clustering (kinase-independent) unidentified\", \"Link between cluster size and downstream Atg14/LC3 recruitment kinetics incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified palmitoylation by ZDHHC13 as the membrane-targeting signal for ULK1 translocation to autophagosome formation sites, connecting lipid modification to autophagy initiation.\",\n      \"evidence\": \"Acyl-RAC assay, ZDHHC13 KO/KD, live imaging, in vitro kinase readout on ATG14L\",\n      \"pmids\": [\"39169022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Depalmitoylating enzyme and turnover dynamics not defined\", \"Interplay with phosphorylation-dependent clustering unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the dozens of ULK1 phosphorylation events, ubiquitin marks, methylation, and palmitoylation are temporally and spatially integrated to produce a single coherent autophagy decision remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model ordering competing PTMs in time\", \"Substrate selectivity determinants across autophagy vs non-autophagy functions undefined\", \"Structural basis of human ULK1 activation not fully resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 4, 9, 21, 29, 34, 42, 43, 44]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 4, 38]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [9, 30, 32]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [4, 11]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [3, 9, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 1, 2, 4, 32, 43]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 1, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [21, 27]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [9, 10, 29]}\n    ],\n    \"complexes\": [\"ULK1-ATG13-FIP200 complex\", \"Atg1-Atg13-Atg17 complex (yeast)\"],\n    \"partners\": [\"ATG13\", \"FIP200/Atg11\", \"AMPK\", \"RPTOR\", \"FUNDC1\", \"GABARAP\", \"MAP1LC3B\", \"VCP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}