{"gene":"PFKL","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2021,"finding":"Small-molecule NA-11 (and precursor LDC7559) selectively activates PFKL by binding to the AMP/ADP allosteric activation site, stabilizing the R-state tetramer. This increases glycolytic flux, dampens pentose phosphate pathway activity, and suppresses NADPH oxidase NOX2-dependent oxidative burst, NETosis, and tissue damage in neutrophils. A high-resolution crystal structure of PFKL confirmed binding of NA-11 to this allosteric site and explained isoform selectivity over PFKP and PFKM.","method":"Chemical proteomics (two unbiased strategies), high-resolution crystal structure of PFKL bound to NA-11, neutrophil functional assays (ROS, NETosis), pharmacological activation with analog design","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with ligand bound, in vitro biochemistry, and cellular functional assays; multiple orthogonal methods in a single rigorous study","pmids":["34320407"],"is_preprint":false},{"year":2024,"finding":"Under glucose deprivation, PFKL is phosphorylated, reducing its glycolytic activity and promoting its interaction with PLIN2 on lipid droplets. In this context PFKL acts as a protein kinase and phosphorylates PLIN2, which triggers PLIN2 binding to CPT1A, tethering lipid droplets to mitochondria and recruiting adipose triglyceride lipase (ATGL) to promote fatty acid oxidation. This 'moonlighting' kinase function of PFKL supports tumor cell proliferation under energy stress.","method":"Co-immunoprecipitation, in vitro kinase assay, phosphorylation-site mutagenesis, lipid droplet-mitochondria co-localization imaging, genetic knockdown/overexpression, xenograft mouse model","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, reciprocal Co-IP, subcellular imaging with functional consequence, and in vivo validation; multiple orthogonal methods in one study","pmids":["38773347"],"is_preprint":false},{"year":2020,"finding":"The E3 ubiquitin ligase A20 directly interacts with PFKL and promotes its ubiquitin-mediated proteasomal degradation, thereby reducing glycolytic flux in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, RNA interference knockdown of A20 and PFKL, glycolysis measurement","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional rescue in a single lab; two orthogonal biochemical methods","pmids":["32015333"],"is_preprint":false},{"year":1981,"finding":"Using somatic cell hybrids and an anti-L-subunit-specific monoclonal antibody, the PFKL gene was mapped to human chromosome 21. The L-subunit randomly tetramerizes with M and P subunits to form multiple PFK isozymes. Trisomy 21 individuals show a gene-dosage-dependent elevation of PFKL-containing isozymes in erythrocytes.","method":"Somatic cell hybrid panel, enzyme immunoprecipitation with monoclonal antibody, ion exchange chromatography of isozymes, chromosome marker analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical mapping replicated across 17 hybrid clones with isozyme and chromosomal confirmation","pmids":["6455664"],"is_preprint":false},{"year":2024,"finding":"PFKL is phosphorylated at Ser775 in macrophages following innate immune stimulation (e.g., LPS). This phosphorylation increases PFKL catalytic activity, elevating glycolytic flux, HIF1α, and IL-1β levels. A knock-in mouse model (PfklS775A/S775A) preventing this phosphorylation showed reduced glycolysis, lower HIF1α and IL-1β after LPS treatment, and reduced MCP-1 and IL-1β in an in vivo inflammation model.","method":"Biochemical phosphorylation assay, glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants, genetic knock-in mouse model, in vivo inflammation model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro biochemical assay, phosphorylation-defective mutant cells, and genetic mouse model; multiple orthogonal methods with in vivo validation","pmids":["39085210"],"is_preprint":false},{"year":2023,"finding":"PTGES3 binds directly to PFKL and generates a local source of prostaglandin E2 (PGE2) that allosterically inhibits PFKL enzymatic activity, reducing glycolytic and TCA-cycle flux. Loss of PTGES3 in ovarian cancer disrupts this PTGES3-PGE2-PFKL inhibitory axis, leading to hyperactivation of glucose oxidation and enhanced tumor cell motility/invasiveness.","method":"Genome-wide CRISPR-Cas9 screen, co-immunoprecipitation of PTGES3 and PFKL, enzymatic activity assay, metabolic flux measurements, loss-of-function in ovarian cancer cells","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus enzymatic activity assay and metabolic assays; single lab with two orthogonal biochemical methods","pmids":["37831605"],"is_preprint":false},{"year":2023,"finding":"The transcription factor KLF7 directly targets the PFKL promoter to regulate its expression. Cardiac-specific knockout of KLF7 elevates PFKL-driven glycolysis and causes adult concentric hypertrophy, while cardiac-specific knockdown of PFKL partially rescues this hypertrophy, placing PFKL downstream of KLF7 in a KLF7/PFKL/ACADL metabolic axis.","method":"ChIP or promoter analysis, cardiac-specific KLF7 knockout and overexpression mice, cardiac-specific PFKL knockdown (AAV), echocardiographic and metabolic flux measurements","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in cardiac-specific mouse models with defined phenotypic rescue; single lab","pmids":["36810848"],"is_preprint":false},{"year":1994,"finding":"Transgenic mice overexpressing murine PFKL show tissue-specific elevated PFK activity that mirrors endogenous expression patterns. Embryonic transgenic brains, but not adult brains, exhibit nearly doubled PFK specific activity, demonstrating developmental stage-specific gene-dosage effects of PFKL overexpression.","method":"Transgenic mouse generation (PFKL 'gene-cDNA' hybrid construct), enzymatic activity assays in multiple tissues at different developmental stages","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic measurement in defined transgenic animal model; single lab, single method per tissue","pmids":["8172601"],"is_preprint":false},{"year":1998,"finding":"Transgenic-PFKL mice with elevated PFKL show increased cerebral glucose metabolic rate (58% faster initial utilization) measured by in vivo [1-13C]-glucose NMR, despite slower peripheral blood glucose clearance, demonstrating that PFKL overexpression directly alters brain glucose flux.","method":"In vivo [1-13C]-glucose infusion followed by NMR spectroscopy, blood glucose clearance assay, enzymatic activity measurements in blood and brain","journal":"Brain research","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vivo isotope-labeled NMR metabolic flux measurement in transgenic mouse; single lab with quantitative readout","pmids":["9813288"],"is_preprint":false},{"year":2022,"finding":"YTHDF3 promotes PFKL mRNA stability via m6A modification, increasing PFKL protein levels. In turn, PFKL interacts with YTHDF3 through the spliceosome subunit EFTUD2 and inhibits ubiquitination of YTHDF3, stabilizing it. This bidirectional positive feedback loop enhances aerobic glycolysis in hepatocellular carcinoma.","method":"Methylated RNA immunoprecipitation (MeRIP), co-immunoprecipitation, immunofluorescence, ubiquitination assay, gain/loss-of-function in vitro and in vivo","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — MeRIP, Co-IP, and ubiquitination assay in a single lab; multiple orthogonal methods","pmids":["36471428"],"is_preprint":false},{"year":2024,"finding":"DNAAF5 directly binds PFKL and recruits the deubiquitinase USP39 to form a ternary complex, stabilizing PFKL protein by promoting its deubiquitination and preventing its proteasomal degradation, thereby enhancing glycolysis in HCC cells.","method":"Co-immunoprecipitation, mass spectrometry, ubiquitination assay, USP39 knockdown functional rescue, xenograft mouse model","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay with MS confirmation; single lab with in vivo validation","pmids":["36276075"],"is_preprint":false},{"year":2024,"finding":"USP14 directly interacts with PFKL and stabilizes it through deubiquitination, preventing its proteasomal degradation and enhancing PFKL-mediated glycolysis in oral squamous cell carcinoma.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression in OSCC cells, glycolysis measurement","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay; single lab, two orthogonal biochemical methods","pmids":["38388430"],"is_preprint":false},{"year":2022,"finding":"PDLIM2, an E3 ubiquitin ligase, promotes ubiquitination and degradation of PFKL. M2 macrophage-derived exosomes deliver miR-222-3p into LSCC cells to suppress PDLIM2, thereby stabilizing PFKL and enhancing glycolysis.","method":"Ubiquitination assay, luciferase assay (miRNA target validation), extracellular acidification rate (Seahorse), knockdown in FaDu cells, xenograft mouse model","journal":"Neoplasma","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — ubiquitination assay plus luciferase and Seahorse; single lab with in vivo validation","pmids":["35723199"],"is_preprint":false},{"year":2023,"finding":"Caveolin-1 (Cav1) in hepatic stellate cells competes with SQSTM1 for binding to the regulatory domain of PFKL, thereby blocking SQSTM1-mediated autophagy-independent lysosomal degradation of PFKL and sustaining elevated PFKL levels and glycolysis during HSC activation.","method":"HSC-specific Cav1 knockdown in mice, co-immunoprecipitation (Cav1, SQSTM1, PFKL), lysosomal pathway assays, fibrosis phenotyping","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with mechanistic pathway dissection; single lab, in vivo knockdown","pmids":["37116593"],"is_preprint":false},{"year":2025,"finding":"HDAC6 acts as the deacetylase of PFKL, interacting with PFKL and deacetylating it primarily at K563. Deacetylation of PFKL by HDAC6 promotes PFKL tetrameric assembly and increases enzymatic activity, enhancing aerobic glycolysis and VSMC proliferation. The acetylation-mimicking mutant K563Q attenuates, while the deacetylation-mimicking K563R mutant aggravates PDGF-BB-induced VSMC proliferation and neointimal formation.","method":"Co-immunoprecipitation (HDAC6-PFKL), site-directed mutagenesis (K563R/K563Q), recombinant adenoviral overexpression, HDAC inhibitor (TSA/siHDAC6), glycolysis measurement, native gel for tetramer formation, in vivo ligation-induced neointima model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical deacetylation assay, site-directed mutagenesis with functional rescue, and in vivo model; multiple orthogonal methods in one study","pmids":["41421488"],"is_preprint":false},{"year":2025,"finding":"Lactate produced by cardiomyocytes triggers PRMT9-mediated methylation of PFKL at residue R301 in neutrophils, resulting in PFKL inactivation, reduced glycolysis, and redirection of metabolic flux from glycolysis toward the pentose phosphate pathway, thereby promoting NETosis.","method":"Mass spectrometry imaging, in vivo/in vitro lactate supplementation and withdrawal, cardiomyocyte-specific PDK4 knockout mouse, PRMT9 methylation assay, NETosis phenotyping","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model plus methylation assay and metabolic flux measurement; single lab","pmids":["40222696"],"is_preprint":false},{"year":2025,"finding":"A covalent electrophile-drug conjugate (EDC) site-specifically modifies K677 in the allosteric effector site of PFKL, stabilizing the R-state tetramer and activating PFKL, which induces metabolic imbalance and delivers a cytotoxic payload to cancer cells in vitro and in vivo.","method":"Chemical proteomics (proteome-wide selectivity profiling), site-specific covalent modification at K677 confirmed biochemically, PFKL R-state tetramer stabilization assay, in vitro and in vivo tumor growth assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — site-specific covalent modification with biochemical tetramer assay and in vivo validation; preprint, single lab","pmids":["41256653"],"is_preprint":true},{"year":2022,"finding":"PFKL preferentially interacts with oxidized methionine-containing actin peptides (Met44/Met47 oxidized) compared with reduced forms, and this differential interaction is also observed with full-length actin protein, suggesting methionine oxidation on actin regulates the actin-PFKL interaction.","method":"Photo-crosslinking peptide pulldown, mass spectrometry-based proteomics, full-length actin interaction assay","journal":"RSC chemical biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single crosslinking/pulldown experiment in one lab; no mutagenesis or functional consequence demonstrated","pmids":["36320891"],"is_preprint":false},{"year":2022,"finding":"PFKL interacts with YTHDF3 through EFTUD2 (a core spliceosome subunit), and this interaction inhibits ubiquitination of YTHDF3, stabilizing it post-translationally.","method":"Co-immunoprecipitation, ubiquitination assay","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and ubiquitination assay; single lab, no mutagenesis to confirm interface","pmids":["36471428"],"is_preprint":false},{"year":2024,"finding":"EGR1 transcription factor binds directly to the PFKL promoter and represses PFKL gene expression, thereby inhibiting PFKL-mediated aerobic glycolysis in hepatocellular carcinoma.","method":"ChIP or promoter-binding assay (reporter/EMSA implied), EGR1 overexpression/knockdown, glycolysis measurement, xenograft and organoid models","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter interaction assay plus functional glycolysis measurement and in vivo rescue; single lab","pmids":["38287371"],"is_preprint":false},{"year":2022,"finding":"SREBF1 (SREBP1) binds to and activates the PFKL promoter, increasing PFKL transcription. ApoM knockout upregulates SREBF1, which in turn elevates PFKL expression and promotes liver cancer cell proliferation and migration.","method":"Dual-luciferase reporter assay (PFKL promoter with SREBF1 binding sites), ApoM knockout cells, functional proliferation and invasion assays","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — luciferase reporter assay for direct promoter regulation; single lab","pmids":["35720503"],"is_preprint":false},{"year":2022,"finding":"Tiam1 directly interacts with PFKL and promotes glycolysis in a PFKL-dependent manner in breast cancer cells.","method":"Co-immunoprecipitation (Tiam1-PFKL), PFKL knockdown functional rescue, glycolysis measurement, in vivo xenograft","journal":"Carcinogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and knockdown rescue; single lab, no biochemical characterization of the interaction","pmids":["35511493"],"is_preprint":false}],"current_model":"PFKL is the liver-type isoform of phosphofructokinase-1, the rate-limiting glycolytic enzyme that converts fructose-6-phosphate to fructose-1,6-bisphosphate; its catalytic activity is allosterically activated by AMP/ADP (and mimicked by small molecules such as NA-11 at this site) and allosterically inhibited by locally generated prostaglandin E2 (via PTGES3); its activity and stability are regulated by multiple post-translational modifications—phosphorylation at Ser775 (activating, downstream of innate immune signaling), phosphorylation by upstream kinases (inhibiting, redirecting the protein to a lipid-droplet-mitochondria tethering function as a protein kinase toward PLIN2), PRMT9-mediated methylation at R301 (inhibiting), and HDAC6-mediated deacetylation at K563 (activating, by promoting tetramer formation)—as well as by ubiquitin-proteasome control through E3 ligases A20 and PDLIM2 (degrading) and deubiquitinases USP14 and USP39/DNAAF5 complex (stabilizing); its transcription is repressed by EGR1 and activated by SREBF1/KLF7; and in addition to its glycolytic role, PFKL moonlights under energy stress as a protein kinase on lipid droplets and selectively interacts with oxidized-methionine forms of actin."},"narrative":{"mechanistic_narrative":"PFKL is the liver-type subunit of phosphofructokinase-1, the rate-limiting glycolytic enzyme whose activity is governed by allosteric control, post-translational modification, and protein turnover [PMID:34320407, PMID:6455664]. The L-subunit randomly tetramerizes with M and P subunits, and its gene resides on chromosome 21, producing gene-dosage effects in trisomy 21 [PMID:6455664]. Catalytic output is set by R-state tetramer formation: small molecules (NA-11) and a covalent electrophile conjugate bind the AMP/ADP-type allosteric effector site to stabilize the active tetramer and raise glycolytic flux while damping the pentose phosphate pathway and NADPH-oxidase-driven oxidative burst [PMID:34320407, PMID:41256653], whereas PTGES3 docks on PFKL to deliver locally generated prostaglandin E2 that allosterically inhibits it [PMID:37831605]. A layer of post-translational marks tunes the same tetramerization/activity axis: Ser775 phosphorylation downstream of innate immune (LPS) signaling activates PFKL to elevate glycolysis, HIF1α, and IL-1β [PMID:39085210]; HDAC6-mediated deacetylation at K563 promotes tetramer assembly and activity [PMID:41421488]; and PRMT9-mediated methylation at R301, triggered by lactate, inactivates PFKL and redirects flux toward the pentose phosphate pathway and NETosis [PMID:40222696]. PFKL abundance is further controlled by opposing ubiquitin-proteasome and lysosomal pathways—the E3 ligases A20 and PDLIM2 promote its degradation, while the deubiquitinases USP14 and a DNAAF5/USP39 complex stabilize it, and Caveolin-1 protects it from SQSTM1-mediated lysosomal degradation [PMID:32015333, PMID:38388430, PMID:36276075, PMID:37116593]. Its transcription is repressed by EGR1 and activated by SREBF1 and KLF7 [PMID:38287371, PMID:35720503, PMID:36810848]. Beyond glycolysis, PFKL moonlights under glucose deprivation as a protein kinase that phosphorylates PLIN2 on lipid droplets to tether them to mitochondria and promote fatty acid oxidation [PMID:38773347]. Across tissues, PFKL-driven glycolysis shapes innate immune activation, cardiac and vascular remodeling, brain glucose flux, and tumor cell metabolism [PMID:39085210, PMID:36810848, PMID:9813288, PMID:41421488].","teleology":[{"year":1981,"claim":"Established the genetic and biochemical identity of PFKL as a distinct PFK subunit, locating its gene and showing it tetramerizes combinatorially with other PFK subunits.","evidence":"Somatic cell hybrid mapping and subunit-specific monoclonal antibody immunoprecipitation with isozyme chromatography","pmids":["6455664"],"confidence":"High","gaps":["No catalytic or structural mechanism addressed","Functional consequence of trisomy-21 dosage elevation not resolved"]},{"year":1994,"claim":"Showed that PFKL overexpression produces tissue- and developmental-stage-specific increases in PFK activity, demonstrating dosage-sensitive control of glycolytic capacity in vivo.","evidence":"Transgenic mice with a PFKL gene-cDNA construct, enzymatic assays across tissues and developmental stages","pmids":["8172601"],"confidence":"Medium","gaps":["No measurement of downstream metabolic flux","Mechanism of stage-specific regulation unknown"]},{"year":1998,"claim":"Connected elevated PFKL protein to altered organ-level glucose flux, showing it directly accelerates cerebral glucose metabolism.","evidence":"In vivo [1-13C]-glucose NMR flux measurement in transgenic mice","pmids":["9813288"],"confidence":"Medium","gaps":["Does not address allosteric or PTM regulation","Single transgenic model"]},{"year":2020,"claim":"Identified ubiquitin-proteasome control as a determinant of PFKL abundance, with A20 as a degrading E3 ligase that lowers glycolysis.","evidence":"Co-IP, ubiquitination assay, and knockdown rescue in hepatocellular carcinoma cells","pmids":["32015333"],"confidence":"Medium","gaps":["Ubiquitination site not mapped","Single lab, no in vivo validation"]},{"year":2021,"claim":"Defined a druggable allosteric activation mechanism: stabilizing the R-state tetramer at the AMP/ADP site raises glycolytic flux and suppresses pentose-phosphate-fueled oxidative burst, with structural proof of isoform selectivity.","evidence":"Chemical proteomics, high-resolution crystal structure of PFKL bound to NA-11, neutrophil ROS/NETosis assays","pmids":["34320407"],"confidence":"High","gaps":["Endogenous physiological ligand occupancy of the site not quantified","Selectivity over PFKM/PFKP defined structurally but in limited cell contexts"]},{"year":2022,"claim":"Revealed PFKL abundance is set by competing degradation and stabilization pathways and by transcriptional inputs, embedding glycolysis in cancer feedback loops.","evidence":"MeRIP, Co-IP via EFTUD2, ubiquitination, luciferase promoter, and Seahorse assays across HCC, LSCC, and liver cancer models (YTHDF3, PDLIM2, SREBF1)","pmids":["36471428","35723199","35720503","35511493"],"confidence":"Medium","gaps":["Interaction interfaces not mapped by mutagenesis","Several findings rest on single Co-IP without reciprocal validation"]},{"year":2023,"claim":"Established allosteric inhibition of PFKL by a locally delivered metabolite and added lysosomal and transcriptional layers of control with defined tissue phenotypes.","evidence":"CRISPR screen, Co-IP, enzymatic and flux assays (PTGES3-PGE2), cardiac-specific KLF7/PFKL epistasis mice, and HSC Cav1/SQSTM1 competition","pmids":["37831605","36810848","37116593"],"confidence":"Medium","gaps":["Structural basis of PTGES3 docking unknown","Quantitative contribution of each regulatory layer in vivo not resolved"]},{"year":2024,"claim":"Uncovered a non-glycolytic moonlighting role and an activating phosphorylation, broadening PFKL from metabolic enzyme to a signaling and lipid-droplet regulator.","evidence":"In vitro kinase assay and PLIN2 phosphosite mutagenesis with LD-mitochondria imaging; Ser775 knock-in mouse with LPS inflammation phenotyping; USP14/DNAAF5-USP39 deubiquitinase Co-IP and rescue; EGR1 promoter repression","pmids":["38773347","39085210","38388430","36276075","38287371"],"confidence":"High","gaps":["Kinase that phosphorylates PFKL to switch on the moonlighting function not identified","Substrate repertoire of the PFKL kinase activity beyond PLIN2 unknown","Identity of the Ser775 kinase not established"]},{"year":2025,"claim":"Demonstrated that acetylation and methylation directly control tetramer assembly and metabolic routing, and that the allosteric site can be covalently engaged for therapeutic activation.","evidence":"HDAC6 deacetylation at K563 with native-gel tetramer assay and K563R/K563Q mutants in VSMC/neointima models; lactate-driven PRMT9 methylation at R301 with NETosis phenotyping; covalent EDC modifying K677 (preprint)","pmids":["41421488","40222696","41256653"],"confidence":"Medium","gaps":["Crosstalk between K563 acetylation, R301 methylation, and S775 phosphorylation untested","EDC mechanism reported only in preprint"]},{"year":null,"claim":"How the multiple competing PTMs, allosteric ligands, and the moonlighting kinase switch are integrated to set PFKL state in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model of combinatorial PTM control on the tetramer","Functional relevance of the oxidized-actin interaction unestablished","Upstream kinase for the glucose-deprivation moonlighting switch unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,14,15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,8,5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,0]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[19,20,6]}],"complexes":["phosphofructokinase-1 tetramer"],"partners":["PLIN2","PTGES3","A20","PDLIM2","USP14","DNAAF5","HDAC6","YTHDF3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P17858","full_name":"ATP-dependent 6-phosphofructokinase, liver type","aliases":["6-phosphofructokinase type B","Phosphofructo-1-kinase isozyme B","PFK-B","Phosphohexokinase"],"length_aa":780,"mass_kda":85.0,"function":"Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis (PubMed:22923583). Negatively regulates the phagocyte oxidative burst in response to bacterial infection by controlling cellular NADPH biosynthesis and NADPH oxidase-derived reactive oxygen species. Upon macrophage activation, drives the metabolic switch toward glycolysis, thus preventing glucose turnover that produces NADPH via pentose phosphate pathway (By similarity)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P17858/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PFKL","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PFKL","total_profiled":1310},"omim":[{"mim_id":"620725","title":"BETHLEM MYOPATHY 1B; BTHLM1B","url":"https://www.omim.org/entry/620725"},{"mim_id":"610681","title":"PHOSPHOFRUCTOKINASE, MUSCLE TYPE; PFKM","url":"https://www.omim.org/entry/610681"},{"mim_id":"605925","title":"PERICENTRIN; PCNT","url":"https://www.omim.org/entry/605925"},{"mim_id":"603191","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 410; CFAP410","url":"https://www.omim.org/entry/603191"},{"mim_id":"602523","title":"DOWN SYNDROME CELL ADHESION MOLECULE; DSCAM","url":"https://www.omim.org/entry/602523"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoli","reliability":"Uncertain"},{"location":"Mitochondria","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PFKL"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P17858","domains":[{"cath_id":"3.40.50.450","chopping":"1-177_305-361","consensus_level":"medium","plddt":92.7071,"start":1,"end":361},{"cath_id":"3.40.50.460","chopping":"178-299","consensus_level":"high","plddt":94.6388,"start":178,"end":299},{"cath_id":"3.40.50.450","chopping":"396-538_669-735","consensus_level":"high","plddt":95.8094,"start":396,"end":735},{"cath_id":"3.40.50.460","chopping":"543-662","consensus_level":"high","plddt":94.7109,"start":543,"end":662}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P17858","model_url":"https://alphafold.ebi.ac.uk/files/AF-P17858-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P17858-F1-predicted_aligned_error_v6.png","plddt_mean":92.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PFKL","jax_strain_url":"https://www.jax.org/strain/search?query=PFKL"},"sequence":{"accession":"P17858","fasta_url":"https://rest.uniprot.org/uniprotkb/P17858.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P17858/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P17858"}},"corpus_meta":[{"pmid":"34320407","id":"PMC_34320407","title":"Selective activation of PFKL suppresses the phagocytic oxidative burst.","date":"2021","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/34320407","citation_count":124,"is_preprint":false},{"pmid":"38773347","id":"PMC_38773347","title":"Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance β-oxidation and tumor cell proliferation.","date":"2024","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/38773347","citation_count":107,"is_preprint":false},{"pmid":"32015333","id":"PMC_32015333","title":"A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma.","date":"2020","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/32015333","citation_count":89,"is_preprint":false},{"pmid":"6455664","id":"PMC_6455664","title":"Assignment of the human gene for liver-type 6-phosphofructokinase isozyme (PFKL) to chromosome 21 by using somatic cell hybrids and monoclonal anti-L antibody.","date":"1981","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/6455664","citation_count":89,"is_preprint":false},{"pmid":"35530161","id":"PMC_35530161","title":"Targeting PFKL with penfluridol inhibits glycolysis and suppresses esophageal cancer tumorigenesis in an AMPK/FOXO3a/BIM-dependent manner.","date":"2021","source":"Acta pharmaceutica Sinica. 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This increases glycolytic flux, dampens pentose phosphate pathway activity, and suppresses NADPH oxidase NOX2-dependent oxidative burst, NETosis, and tissue damage in neutrophils. A high-resolution crystal structure of PFKL confirmed binding of NA-11 to this allosteric site and explained isoform selectivity over PFKP and PFKM.\",\n      \"method\": \"Chemical proteomics (two unbiased strategies), high-resolution crystal structure of PFKL bound to NA-11, neutrophil functional assays (ROS, NETosis), pharmacological activation with analog design\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with ligand bound, in vitro biochemistry, and cellular functional assays; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"34320407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Under glucose deprivation, PFKL is phosphorylated, reducing its glycolytic activity and promoting its interaction with PLIN2 on lipid droplets. In this context PFKL acts as a protein kinase and phosphorylates PLIN2, which triggers PLIN2 binding to CPT1A, tethering lipid droplets to mitochondria and recruiting adipose triglyceride lipase (ATGL) to promote fatty acid oxidation. This 'moonlighting' kinase function of PFKL supports tumor cell proliferation under energy stress.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, phosphorylation-site mutagenesis, lipid droplet-mitochondria co-localization imaging, genetic knockdown/overexpression, xenograft mouse model\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, reciprocal Co-IP, subcellular imaging with functional consequence, and in vivo validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"38773347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The E3 ubiquitin ligase A20 directly interacts with PFKL and promotes its ubiquitin-mediated proteasomal degradation, thereby reducing glycolytic flux in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNA interference knockdown of A20 and PFKL, glycolysis measurement\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional rescue in a single lab; two orthogonal biochemical methods\",\n      \"pmids\": [\"32015333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1981,\n      \"finding\": \"Using somatic cell hybrids and an anti-L-subunit-specific monoclonal antibody, the PFKL gene was mapped to human chromosome 21. The L-subunit randomly tetramerizes with M and P subunits to form multiple PFK isozymes. Trisomy 21 individuals show a gene-dosage-dependent elevation of PFKL-containing isozymes in erythrocytes.\",\n      \"method\": \"Somatic cell hybrid panel, enzyme immunoprecipitation with monoclonal antibody, ion exchange chromatography of isozymes, chromosome marker analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical mapping replicated across 17 hybrid clones with isozyme and chromosomal confirmation\",\n      \"pmids\": [\"6455664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKL is phosphorylated at Ser775 in macrophages following innate immune stimulation (e.g., LPS). This phosphorylation increases PFKL catalytic activity, elevating glycolytic flux, HIF1α, and IL-1β levels. A knock-in mouse model (PfklS775A/S775A) preventing this phosphorylation showed reduced glycolysis, lower HIF1α and IL-1β after LPS treatment, and reduced MCP-1 and IL-1β in an in vivo inflammation model.\",\n      \"method\": \"Biochemical phosphorylation assay, glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants, genetic knock-in mouse model, in vivo inflammation model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro biochemical assay, phosphorylation-defective mutant cells, and genetic mouse model; multiple orthogonal methods with in vivo validation\",\n      \"pmids\": [\"39085210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PTGES3 binds directly to PFKL and generates a local source of prostaglandin E2 (PGE2) that allosterically inhibits PFKL enzymatic activity, reducing glycolytic and TCA-cycle flux. Loss of PTGES3 in ovarian cancer disrupts this PTGES3-PGE2-PFKL inhibitory axis, leading to hyperactivation of glucose oxidation and enhanced tumor cell motility/invasiveness.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen, co-immunoprecipitation of PTGES3 and PFKL, enzymatic activity assay, metabolic flux measurements, loss-of-function in ovarian cancer cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus enzymatic activity assay and metabolic assays; single lab with two orthogonal biochemical methods\",\n      \"pmids\": [\"37831605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The transcription factor KLF7 directly targets the PFKL promoter to regulate its expression. Cardiac-specific knockout of KLF7 elevates PFKL-driven glycolysis and causes adult concentric hypertrophy, while cardiac-specific knockdown of PFKL partially rescues this hypertrophy, placing PFKL downstream of KLF7 in a KLF7/PFKL/ACADL metabolic axis.\",\n      \"method\": \"ChIP or promoter analysis, cardiac-specific KLF7 knockout and overexpression mice, cardiac-specific PFKL knockdown (AAV), echocardiographic and metabolic flux measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in cardiac-specific mouse models with defined phenotypic rescue; single lab\",\n      \"pmids\": [\"36810848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Transgenic mice overexpressing murine PFKL show tissue-specific elevated PFK activity that mirrors endogenous expression patterns. Embryonic transgenic brains, but not adult brains, exhibit nearly doubled PFK specific activity, demonstrating developmental stage-specific gene-dosage effects of PFKL overexpression.\",\n      \"method\": \"Transgenic mouse generation (PFKL 'gene-cDNA' hybrid construct), enzymatic activity assays in multiple tissues at different developmental stages\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic measurement in defined transgenic animal model; single lab, single method per tissue\",\n      \"pmids\": [\"8172601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Transgenic-PFKL mice with elevated PFKL show increased cerebral glucose metabolic rate (58% faster initial utilization) measured by in vivo [1-13C]-glucose NMR, despite slower peripheral blood glucose clearance, demonstrating that PFKL overexpression directly alters brain glucose flux.\",\n      \"method\": \"In vivo [1-13C]-glucose infusion followed by NMR spectroscopy, blood glucose clearance assay, enzymatic activity measurements in blood and brain\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vivo isotope-labeled NMR metabolic flux measurement in transgenic mouse; single lab with quantitative readout\",\n      \"pmids\": [\"9813288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"YTHDF3 promotes PFKL mRNA stability via m6A modification, increasing PFKL protein levels. In turn, PFKL interacts with YTHDF3 through the spliceosome subunit EFTUD2 and inhibits ubiquitination of YTHDF3, stabilizing it. This bidirectional positive feedback loop enhances aerobic glycolysis in hepatocellular carcinoma.\",\n      \"method\": \"Methylated RNA immunoprecipitation (MeRIP), co-immunoprecipitation, immunofluorescence, ubiquitination assay, gain/loss-of-function in vitro and in vivo\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — MeRIP, Co-IP, and ubiquitination assay in a single lab; multiple orthogonal methods\",\n      \"pmids\": [\"36471428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DNAAF5 directly binds PFKL and recruits the deubiquitinase USP39 to form a ternary complex, stabilizing PFKL protein by promoting its deubiquitination and preventing its proteasomal degradation, thereby enhancing glycolysis in HCC cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ubiquitination assay, USP39 knockdown functional rescue, xenograft mouse model\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay with MS confirmation; single lab with in vivo validation\",\n      \"pmids\": [\"36276075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP14 directly interacts with PFKL and stabilizes it through deubiquitination, preventing its proteasomal degradation and enhancing PFKL-mediated glycolysis in oral squamous cell carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression in OSCC cells, glycolysis measurement\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay; single lab, two orthogonal biochemical methods\",\n      \"pmids\": [\"38388430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PDLIM2, an E3 ubiquitin ligase, promotes ubiquitination and degradation of PFKL. M2 macrophage-derived exosomes deliver miR-222-3p into LSCC cells to suppress PDLIM2, thereby stabilizing PFKL and enhancing glycolysis.\",\n      \"method\": \"Ubiquitination assay, luciferase assay (miRNA target validation), extracellular acidification rate (Seahorse), knockdown in FaDu cells, xenograft mouse model\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — ubiquitination assay plus luciferase and Seahorse; single lab with in vivo validation\",\n      \"pmids\": [\"35723199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Caveolin-1 (Cav1) in hepatic stellate cells competes with SQSTM1 for binding to the regulatory domain of PFKL, thereby blocking SQSTM1-mediated autophagy-independent lysosomal degradation of PFKL and sustaining elevated PFKL levels and glycolysis during HSC activation.\",\n      \"method\": \"HSC-specific Cav1 knockdown in mice, co-immunoprecipitation (Cav1, SQSTM1, PFKL), lysosomal pathway assays, fibrosis phenotyping\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with mechanistic pathway dissection; single lab, in vivo knockdown\",\n      \"pmids\": [\"37116593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HDAC6 acts as the deacetylase of PFKL, interacting with PFKL and deacetylating it primarily at K563. Deacetylation of PFKL by HDAC6 promotes PFKL tetrameric assembly and increases enzymatic activity, enhancing aerobic glycolysis and VSMC proliferation. The acetylation-mimicking mutant K563Q attenuates, while the deacetylation-mimicking K563R mutant aggravates PDGF-BB-induced VSMC proliferation and neointimal formation.\",\n      \"method\": \"Co-immunoprecipitation (HDAC6-PFKL), site-directed mutagenesis (K563R/K563Q), recombinant adenoviral overexpression, HDAC inhibitor (TSA/siHDAC6), glycolysis measurement, native gel for tetramer formation, in vivo ligation-induced neointima model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical deacetylation assay, site-directed mutagenesis with functional rescue, and in vivo model; multiple orthogonal methods in one study\",\n      \"pmids\": [\"41421488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Lactate produced by cardiomyocytes triggers PRMT9-mediated methylation of PFKL at residue R301 in neutrophils, resulting in PFKL inactivation, reduced glycolysis, and redirection of metabolic flux from glycolysis toward the pentose phosphate pathway, thereby promoting NETosis.\",\n      \"method\": \"Mass spectrometry imaging, in vivo/in vitro lactate supplementation and withdrawal, cardiomyocyte-specific PDK4 knockout mouse, PRMT9 methylation assay, NETosis phenotyping\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model plus methylation assay and metabolic flux measurement; single lab\",\n      \"pmids\": [\"40222696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A covalent electrophile-drug conjugate (EDC) site-specifically modifies K677 in the allosteric effector site of PFKL, stabilizing the R-state tetramer and activating PFKL, which induces metabolic imbalance and delivers a cytotoxic payload to cancer cells in vitro and in vivo.\",\n      \"method\": \"Chemical proteomics (proteome-wide selectivity profiling), site-specific covalent modification at K677 confirmed biochemically, PFKL R-state tetramer stabilization assay, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-specific covalent modification with biochemical tetramer assay and in vivo validation; preprint, single lab\",\n      \"pmids\": [\"41256653\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PFKL preferentially interacts with oxidized methionine-containing actin peptides (Met44/Met47 oxidized) compared with reduced forms, and this differential interaction is also observed with full-length actin protein, suggesting methionine oxidation on actin regulates the actin-PFKL interaction.\",\n      \"method\": \"Photo-crosslinking peptide pulldown, mass spectrometry-based proteomics, full-length actin interaction assay\",\n      \"journal\": \"RSC chemical biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single crosslinking/pulldown experiment in one lab; no mutagenesis or functional consequence demonstrated\",\n      \"pmids\": [\"36320891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PFKL interacts with YTHDF3 through EFTUD2 (a core spliceosome subunit), and this interaction inhibits ubiquitination of YTHDF3, stabilizing it post-translationally.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and ubiquitination assay; single lab, no mutagenesis to confirm interface\",\n      \"pmids\": [\"36471428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EGR1 transcription factor binds directly to the PFKL promoter and represses PFKL gene expression, thereby inhibiting PFKL-mediated aerobic glycolysis in hepatocellular carcinoma.\",\n      \"method\": \"ChIP or promoter-binding assay (reporter/EMSA implied), EGR1 overexpression/knockdown, glycolysis measurement, xenograft and organoid models\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter interaction assay plus functional glycolysis measurement and in vivo rescue; single lab\",\n      \"pmids\": [\"38287371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SREBF1 (SREBP1) binds to and activates the PFKL promoter, increasing PFKL transcription. ApoM knockout upregulates SREBF1, which in turn elevates PFKL expression and promotes liver cancer cell proliferation and migration.\",\n      \"method\": \"Dual-luciferase reporter assay (PFKL promoter with SREBF1 binding sites), ApoM knockout cells, functional proliferation and invasion assays\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — luciferase reporter assay for direct promoter regulation; single lab\",\n      \"pmids\": [\"35720503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Tiam1 directly interacts with PFKL and promotes glycolysis in a PFKL-dependent manner in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (Tiam1-PFKL), PFKL knockdown functional rescue, glycolysis measurement, in vivo xenograft\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and knockdown rescue; single lab, no biochemical characterization of the interaction\",\n      \"pmids\": [\"35511493\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PFKL is the liver-type isoform of phosphofructokinase-1, the rate-limiting glycolytic enzyme that converts fructose-6-phosphate to fructose-1,6-bisphosphate; its catalytic activity is allosterically activated by AMP/ADP (and mimicked by small molecules such as NA-11 at this site) and allosterically inhibited by locally generated prostaglandin E2 (via PTGES3); its activity and stability are regulated by multiple post-translational modifications—phosphorylation at Ser775 (activating, downstream of innate immune signaling), phosphorylation by upstream kinases (inhibiting, redirecting the protein to a lipid-droplet-mitochondria tethering function as a protein kinase toward PLIN2), PRMT9-mediated methylation at R301 (inhibiting), and HDAC6-mediated deacetylation at K563 (activating, by promoting tetramer formation)—as well as by ubiquitin-proteasome control through E3 ligases A20 and PDLIM2 (degrading) and deubiquitinases USP14 and USP39/DNAAF5 complex (stabilizing); its transcription is repressed by EGR1 and activated by SREBF1/KLF7; and in addition to its glycolytic role, PFKL moonlights under energy stress as a protein kinase on lipid droplets and selectively interacts with oxidized-methionine forms of actin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PFKL is the liver-type subunit of phosphofructokinase-1, the rate-limiting glycolytic enzyme whose activity is governed by allosteric control, post-translational modification, and protein turnover [#0, #3]. The L-subunit randomly tetramerizes with M and P subunits, and its gene resides on chromosome 21, producing gene-dosage effects in trisomy 21 [#3]. Catalytic output is set by R-state tetramer formation: small molecules (NA-11) and a covalent electrophile conjugate bind the AMP/ADP-type allosteric effector site to stabilize the active tetramer and raise glycolytic flux while damping the pentose phosphate pathway and NADPH-oxidase-driven oxidative burst [#0, #16], whereas PTGES3 docks on PFKL to deliver locally generated prostaglandin E2 that allosterically inhibits it [#5]. A layer of post-translational marks tunes the same tetramerization/activity axis: Ser775 phosphorylation downstream of innate immune (LPS) signaling activates PFKL to elevate glycolysis, HIF1\\u03b1, and IL-1\\u03b2 [#4]; HDAC6-mediated deacetylation at K563 promotes tetramer assembly and activity [#14]; and PRMT9-mediated methylation at R301, triggered by lactate, inactivates PFKL and redirects flux toward the pentose phosphate pathway and NETosis [#15]. PFKL abundance is further controlled by opposing ubiquitin-proteasome and lysosomal pathways\\u2014the E3 ligases A20 and PDLIM2 promote its degradation, while the deubiquitinases USP14 and a DNAAF5/USP39 complex stabilize it, and Caveolin-1 protects it from SQSTM1-mediated lysosomal degradation [#2, #11, #10, #13]. Its transcription is repressed by EGR1 and activated by SREBF1 and KLF7 [#19, #20, #6]. Beyond glycolysis, PFKL moonlights under glucose deprivation as a protein kinase that phosphorylates PLIN2 on lipid droplets to tether them to mitochondria and promote fatty acid oxidation [#1]. Across tissues, PFKL-driven glycolysis shapes innate immune activation, cardiac and vascular remodeling, brain glucose flux, and tumor cell metabolism [#4, #6, #8, #14].\",\n  \"teleology\": [\n    {\n      \"year\": 1981,\n      \"claim\": \"Established the genetic and biochemical identity of PFKL as a distinct PFK subunit, locating its gene and showing it tetramerizes combinatorially with other PFK subunits.\",\n      \"evidence\": \"Somatic cell hybrid mapping and subunit-specific monoclonal antibody immunoprecipitation with isozyme chromatography\",\n      \"pmids\": [\"6455664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No catalytic or structural mechanism addressed\", \"Functional consequence of trisomy-21 dosage elevation not resolved\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Showed that PFKL overexpression produces tissue- and developmental-stage-specific increases in PFK activity, demonstrating dosage-sensitive control of glycolytic capacity in vivo.\",\n      \"evidence\": \"Transgenic mice with a PFKL gene-cDNA construct, enzymatic assays across tissues and developmental stages\",\n      \"pmids\": [\"8172601\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No measurement of downstream metabolic flux\", \"Mechanism of stage-specific regulation unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Connected elevated PFKL protein to altered organ-level glucose flux, showing it directly accelerates cerebral glucose metabolism.\",\n      \"evidence\": \"In vivo [1-13C]-glucose NMR flux measurement in transgenic mice\",\n      \"pmids\": [\"9813288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address allosteric or PTM regulation\", \"Single transgenic model\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified ubiquitin-proteasome control as a determinant of PFKL abundance, with A20 as a degrading E3 ligase that lowers glycolysis.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, and knockdown rescue in hepatocellular carcinoma cells\",\n      \"pmids\": [\"32015333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site not mapped\", \"Single lab, no in vivo validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a druggable allosteric activation mechanism: stabilizing the R-state tetramer at the AMP/ADP site raises glycolytic flux and suppresses pentose-phosphate-fueled oxidative burst, with structural proof of isoform selectivity.\",\n      \"evidence\": \"Chemical proteomics, high-resolution crystal structure of PFKL bound to NA-11, neutrophil ROS/NETosis assays\",\n      \"pmids\": [\"34320407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological ligand occupancy of the site not quantified\", \"Selectivity over PFKM/PFKP defined structurally but in limited cell contexts\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed PFKL abundance is set by competing degradation and stabilization pathways and by transcriptional inputs, embedding glycolysis in cancer feedback loops.\",\n      \"evidence\": \"MeRIP, Co-IP via EFTUD2, ubiquitination, luciferase promoter, and Seahorse assays across HCC, LSCC, and liver cancer models (YTHDF3, PDLIM2, SREBF1)\",\n      \"pmids\": [\"36471428\", \"35723199\", \"35720503\", \"35511493\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction interfaces not mapped by mutagenesis\", \"Several findings rest on single Co-IP without reciprocal validation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established allosteric inhibition of PFKL by a locally delivered metabolite and added lysosomal and transcriptional layers of control with defined tissue phenotypes.\",\n      \"evidence\": \"CRISPR screen, Co-IP, enzymatic and flux assays (PTGES3-PGE2), cardiac-specific KLF7/PFKL epistasis mice, and HSC Cav1/SQSTM1 competition\",\n      \"pmids\": [\"37831605\", \"36810848\", \"37116593\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of PTGES3 docking unknown\", \"Quantitative contribution of each regulatory layer in vivo not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Uncovered a non-glycolytic moonlighting role and an activating phosphorylation, broadening PFKL from metabolic enzyme to a signaling and lipid-droplet regulator.\",\n      \"evidence\": \"In vitro kinase assay and PLIN2 phosphosite mutagenesis with LD-mitochondria imaging; Ser775 knock-in mouse with LPS inflammation phenotyping; USP14/DNAAF5-USP39 deubiquitinase Co-IP and rescue; EGR1 promoter repression\",\n      \"pmids\": [\"38773347\", \"39085210\", \"38388430\", \"36276075\", \"38287371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase that phosphorylates PFKL to switch on the moonlighting function not identified\", \"Substrate repertoire of the PFKL kinase activity beyond PLIN2 unknown\", \"Identity of the Ser775 kinase not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that acetylation and methylation directly control tetramer assembly and metabolic routing, and that the allosteric site can be covalently engaged for therapeutic activation.\",\n      \"evidence\": \"HDAC6 deacetylation at K563 with native-gel tetramer assay and K563R/K563Q mutants in VSMC/neointima models; lactate-driven PRMT9 methylation at R301 with NETosis phenotyping; covalent EDC modifying K677 (preprint)\",\n      \"pmids\": [\"41421488\", \"40222696\", \"41256653\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Crosstalk between K563 acetylation, R301 methylation, and S775 phosphorylation untested\", \"EDC mechanism reported only in preprint\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple competing PTMs, allosteric ligands, and the moonlighting kinase switch are integrated to set PFKL state in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of combinatorial PTM control on the tetramer\", \"Functional relevance of the oxidized-actin interaction unestablished\", \"Upstream kinase for the glucose-deprivation moonlighting switch unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 14, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 8, 5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 0]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [19, 20, 6]}\n    ],\n    \"complexes\": [\"phosphofructokinase-1 tetramer\"],\n    \"partners\": [\"PLIN2\", \"PTGES3\", \"A20\", \"PDLIM2\", \"USP14\", \"DNAAF5\", \"HDAC6\", \"YTHDF3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}