{"gene":"PFKP","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1983,"finding":"The human PFKP gene was mapped to the short arm of chromosome 10 (10p) using somatic cell hybrid analysis with anti-human P subunit-specific antiserum and immunoprecipitation; a gene dosage effect was demonstrated in fibroblasts with duplication of 10p, confirming PFKP encodes the platelet/fibroblast isoform of phosphofructokinase.","method":"Somatic cell hybrid panel analysis, active-enzyme-immunoprecipitation, gene dosage experiment","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 1-2 — direct chromosomal assignment with functional gene dosage validation, foundational mapping study","pmids":["6222962"],"is_preprint":false},{"year":2011,"finding":"KLF4 directly binds the PFKP promoter and transcriptionally activates PFKP expression in breast cancer cells, increasing glycolytic activity (glucose uptake and lactate production); knockdown of KLF4 decreased glycolysis whereas overexpression increased it, without affecting other PFK isoforms.","method":"ChIP (KLF4 binding to PFKP promoter), luciferase reporter assay, KLF4 KD/OE with glycolysis measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (ChIP, reporter, KD/OE with functional readout)","pmids":["21586797"],"is_preprint":false},{"year":2017,"finding":"Snail (SNAI1) directly represses PFKP transcription during EMT, diverting glucose flux from glycolysis to the pentose phosphate pathway (PPP), increasing NADPH; PFKP knockdown rescues the metabolic reprogramming and cell death induced by Snail loss, placing PFKP downstream of Snail in the glycolysis-PPP switch.","method":"Snail KD/OE with PFKP expression and metabolic flux measurements, PFKP rescue experiments, in vivo metastasis assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (rescue experiments), multiple metabolic readouts, in vivo validation","pmids":["28176759"],"is_preprint":false},{"year":2018,"finding":"VDAC2 (a mitochondrial outer membrane protein) physically couples with PFKP on the mitochondrion to inhibit PFKP-mediated glycolysis; disruption of VDAC2 de-represses PFKP activity and glycolysis, driving dedifferentiation of non-stem tumor cells to glioma stem cells, while PFK inhibitor clotrimazole blocks this phenotypic transition.","method":"Co-immunoprecipitation (VDAC2-PFKP), VDAC2 KD/OE with glycolysis and stem-cell marker assays, clotrimazole rescue","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal interaction shown, functional rescue with PFK inhibitor, but single lab","pmids":["30250190"],"is_preprint":false},{"year":2019,"finding":"Under hypoxia, oxidized ATM regulates PFKP at the translational level (via HIF1A) to promote intracellular citrate accumulation in triple-negative breast cancer; suppression of oxidized ATM reduces PFKP and citrate levels, while citrate accumulation activates AKT/ERK/MMP2/9 signaling to enhance invasion and metastasis.","method":"ATM inhibition/oxidation assays, PFKP protein-level measurements, metabolite (citrate) quantification, in vitro invasion and xenograft assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — translational regulation defined, metabolite-phenotype link established, single lab","pmids":["30850587"],"is_preprint":false},{"year":2021,"finding":"R-2-hydroxyglutarate inhibits FTO demethylase activity, leading to increased m6A methylation on PFKP mRNA; this prevents YTHDF2-mediated protection of PFKP mRNA, reducing PFKP protein and suppressing aerobic glycolysis in leukemia cells. PFKP knockdown recapitulates R-2HG-induced glycolytic inhibition.","method":"m6A-seq, PFKP mRNA stability assays, FTO/YTHDF2 KD, metabolic assays, in vivo leukemogenesis model","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (epitranscriptomics, genetic KD, in vivo), replicated across cell types","pmids":["33434505"],"is_preprint":false},{"year":2021,"finding":"HRD1 (an E3 ubiquitin ligase) interacts with and co-localizes with PFKP in the cytoplasm, ubiquitinates PFKP and targets it for proteasomal degradation, thereby reducing PFKP expression and glycolytic activity in breast cancer cells.","method":"Mass spectrometry interactome, co-immunoprecipitation, immunofluorescence co-localization, ubiquitylation assay, HRD1 OE/KD with glycolysis and tumor growth readouts","journal":"Cell communication and signaling : CCS","confidence":"High","confidence_rationale":"Tier 2 — MS-identified interaction confirmed by Co-IP + co-localization + ubiquitination assay + functional rescue","pmids":["33588886"],"is_preprint":false},{"year":2021,"finding":"PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLS). Cyclin D3/CDK6 dimerizes with PFKP and exposes its NLS, facilitating interaction with importin 9 and nuclear translocation. Nuclear PFKP stimulates CXCR4 expression in a c-Myc-dependent manner to promote T-ALL invasion and leukemia homing/infiltration.","method":"NLS/NES mutagenesis, Co-IP (Cyclin D3/CDK6-PFKP, PFKP-importin 9), subcellular fractionation, CXCR4 reporter assays, in vivo leukemia infiltration model with CXCR4 antagonist rescue, IHC of patient samples","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (mutagenesis, Co-IP, in vivo rescue), mechanistic pathway clearly defined","pmids":["34255748"],"is_preprint":false},{"year":2021,"finding":"YY1 transcription factor directly binds the PFKP gene regulatory elements and activates PFKP transcription in cooperation with BRD2/4 co-factors; mutagenesis of YY1-bound cis-elements and gene loss-of-function/rescue studies establish a YY1:BRD2/4-PFKP axis driving the Warburg effect in castration-resistant prostate cancer.","method":"ChIP-seq (cistrome), YY1 KD with PFKP rescue, mutagenesis of YY1-binding cis-elements, in vitro and in vivo tumor growth assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — cistrome mapping + mutagenesis + genetic rescue, multiple validation approaches","pmids":["33849067"],"is_preprint":false},{"year":2021,"finding":"PFKP facilitates phosphorylation of autophagy protein ATG4B at serine 34, functioning as a protein kinase; amino acid deprivation strengthens the PFKP-ATG4B interaction, PFKP phosphorylates ATG4B at S34 in vitro, enhancing ATG4B activity and autophagic flux (LC3-II turnover, p62 degradation). PFKP S386 phosphorylation is required for its kinase activity toward ATG4B.","method":"Tandem affinity purification + mass spectrometry, Co-IP (ATG4B-PFKP interaction), CRISPR/Cas9 PFKP KO, in vitro kinase assay, phospho-site mutagenesis","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis plus genetic KO validation, non-canonical kinase activity established","pmids":["33607258"],"is_preprint":false},{"year":2021,"finding":"TGF-β1 recruits the SMAD3-SP1 complex to the PFKP promoter to transcriptionally activate PFKP expression in renal proximal tubular epithelial cells, driving glycolysis and kidney interstitial fibrosis; AAV-mediated PFKP overexpression worsens fibrosis while PFKP knockdown is protective.","method":"ChIP-qPCR (SMAD3-SP1 binding to PFKP promoter), AAV-mediated PFKP KD/OE in mice (unilateral ureteral occlusion model), glycolysis and fibrosis quantification","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — ChIP-qPCR establishes direct transcriptional mechanism, confirmed in vivo with KD/OE","pmids":["38086793"],"is_preprint":false},{"year":2022,"finding":"PFKP physically interacts with AMPK; upon glucose starvation, this interaction is enhanced and promotes mitochondrial recruitment of AMPK, which then phosphorylates acetyl-CoA carboxylase 2 (ACC2) to enhance long-chain fatty acid oxidation, supporting energy and redox homeostasis in NSCLC cells.","method":"Proteomics screen of AMPK-interacting proteins, Co-IP (PFKP-AMPK), mitochondrial fractionation, ACC2 phosphorylation assay, PFKP KD with FAO measurements","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 — MS-identified interaction, confirmed by Co-IP + fractionation + functional metabolic readouts","pmids":["35641476"],"is_preprint":false},{"year":2022,"finding":"Fbxo7 promotes Cdk6-dependent phosphorylation of PFKP and Cdk6-independent ubiquitination of PFKP; Fbxo7-deficient T cells show reduced Cdk6 activity and increased glycolytic flux, demonstrating that the Fbxo7-Cdk6 axis suppresses PFKP activity and glycolysis.","method":"Substrate screen for Fbxo7, Fbxo7 KO in CD4+ T cells, Cdk6 activity assays, ubiquitination assay, metabolomics of activated T cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — substrate screen + KO phenotype + metabolomics, multiple orthogonal approaches","pmids":["35670764"],"is_preprint":false},{"year":2022,"finding":"PFKP activation in podocytes maintains fructose-1,6-bisphosphate (FBP) levels; PFKP knockdown or inhibition reduces FBP, activates the RhoA/ROCK1 pathway, and causes cytoskeletal remodeling (foot process fusion), while PFKP overexpression rescues these defects in diabetic kidney disease.","method":"siRNA/plasmid-mediated PFKP KD/OE, targeted metabolomics (FBP measurement), RhoA/ROCK1 pathway analysis, PFKP inhibitor (clotrimazole) in diabetic mice, exogenous FBP rescue","journal":"Frontiers in endocrinology","confidence":"Medium","confidence_rationale":"Tier 2-3 — metabolite-pathway link established with rescue, single lab","pmids":["35095764"],"is_preprint":false},{"year":2023,"finding":"SIRT2 deacetylates PFKP at lysine 394 (mouse)/lysine 395 (human), reducing PFKP enzymatic activity; acetylation at this site is required for PFKP's glycolytic function and its role in phosphorylating ATG4B to activate LC3-associated phagocytosis (LAP) in macrophages. Ethanol exposure enhances SIRT2-PFKP interaction and suppresses phagocytosis.","method":"SIRT2 KD/pharmacological inhibition, acetylation site mutagenesis (K394), PFKP activity assays, Atg4B phosphorylation assay, LC3 activation assay, phagocytosis/LAP assays in macrophages, in vivo sepsis survival","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 1-2 — site-specific deacetylation with mutagenesis + in vitro activity assay + functional downstream pathway validated in vivo","pmids":["36865524"],"is_preprint":false},{"year":2023,"finding":"CMBL enhances TRIM25 binding to PFKP, leading to K48-linked ubiquitination and proteasomal degradation of PFKP; p53 transcriptionally activates CMBL in response to genotoxic stress, thereby suppressing glycolysis through PFKP degradation in colorectal cancer.","method":"Co-IP (CMBL-TRIM25-PFKP ternary complex), ubiquitination assay, CMBL OE/KD with PFKP stability and glycolysis measurements, p53 activation experiments","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — ternary complex identified by Co-IP, ubiquitination validated, p53-CMBL-TRIM25-PFKP pathway defined with genetic rescue","pmids":["37967006"],"is_preprint":false},{"year":2023,"finding":"PFKP lactylation at lysine 688 directly attenuates its enzymatic activity, forming a potential negative feedback loop in glycolysis where lactate production inhibits PFKP through lactylation.","method":"Mass spectrometry-based proteome-wide lactylation profiling, site-specific lactylation identification at K688, in vitro PFKP enzyme activity assay after lactylation","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 1-2 — MS identification of modification site + functional enzyme activity assay, single lab study","pmids":["38155775"],"is_preprint":false},{"year":2023,"finding":"Glycolytic enzyme Pfkp acts as a protein kinase that phosphorylates the developmental regulator Lin41 at serine residues; this phosphorylation stabilizes Lin41 by preventing its autoubiquitination and proteasomal degradation, permitting Lin41-mediated RNA destabilization of ectodermal markers to favor endodermal specification during murine ESC differentiation.","method":"In vitro kinase assay (Pfkp phosphorylating Lin41), Co-IP, Lin41 ubiquitination assay, ESC differentiation lineage marker analysis, Pfkp KD/OE","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with functional validation of substrate stabilization and lineage specification consequences","pmids":["36660859"],"is_preprint":false},{"year":2023,"finding":"HIF-1α transcriptionally regulates PFKP as a target gene; HBO therapy suppresses hypoxia-induced HIF-1α expression and downstream PFKP transactivation in NSCLC cells, and in vivo HBO therapy inhibited tumor growth in a Pfkp-dependent manner.","method":"HIF-1α KD, luciferase reporter (PFKP transactivation), glycolytic flux modeling, in vivo LLC tumor model with Pfkp-dependence rescue","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 — transcriptional regulation confirmed by reporter + in vivo Pfkp-dependent rescue, but HIF-1α→PFKP link partially inferred","pmids":["34367973"],"is_preprint":false},{"year":2024,"finding":"USP5 deubiquitinase directly interacts with PFKP and mediates its deubiquitination and stabilization, preventing proteasomal degradation; USP5-mediated PFKP stabilization is essential for cancer cell aerobic glycolysis and TNBC progression.","method":"Co-IP, MS-based protein identification, in vitro binding assay, ubiquitination assay, USP5 KD/OE with PFKP stability and glycolysis measurements, tumor xenograft","journal":"Breast cancer research : BCR","confidence":"High","confidence_rationale":"Tier 2 — MS-identified interaction validated by Co-IP + in vitro binding + ubiquitination assay + functional readouts","pmids":["38217030"],"is_preprint":false},{"year":2024,"finding":"PFKP increases ERK-mediated stability of c-Myc protein, while c-Myc transcriptionally activates PFKP expression, forming a positive feedback loop driving HNSCC progression; co-targeting both synergistically inhibits tumor growth.","method":"PFKP/c-Myc KD/OE, ERK phosphorylation assays, c-Myc stability measurements, luciferase reporter (PFKP promoter), PDO and xenograft models","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2-3 — feedback loop defined with KD/OE and reporter, ERK-mediated c-Myc stabilization by PFKP established, single lab","pmids":["38982480"],"is_preprint":false},{"year":2024,"finding":"RNF123 (E3 ubiquitin ligase) directly interacts with PFKP and induces its ubiquitination, promoting PFKP degradation and thereby suppressing glycolysis, cell viability, cell cycle progression, and colony formation in breast cancer cells.","method":"Co-IP, ubiquitination analysis, RNF123 OE/KD with PFKP protein levels and glycolysis assays, IHC, in vivo xenograft","journal":"Naunyn-Schmiedeberg's archives of pharmacology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP + ubiquitination assay + functional readouts, single lab","pmids":["39725718"],"is_preprint":false},{"year":2024,"finding":"PFKP promotes OC progression through lactylation at lysine 392; K392 mutation diminishes PFKP lactylation, and PFKP depletion upregulates PTEN expression in hypoxic ovarian cancer cells, suggesting PFKP lactylation suppresses PTEN to drive glycolysis.","method":"Immunoprecipitation + Western blot for PFKP lactylation, K392 site-directed mutagenesis, PFKP KD with PTEN/glycolysis measurement, OC xenograft model","journal":"Biochemical genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 — site-specific PTM with mutagenesis + functional downstream (PTEN) link, single lab","pmids":["39638933"],"is_preprint":false},{"year":2024,"finding":"PKP1 (Plakophilin-1) stabilizes PFKP by binding TRIM21 and preventing TRIM21-mediated ubiquitination and degradation of PFKP; PKP1 depletion selectively reduces PFKP protein levels by enhancing its ubiquitination, and PFKP mediates the proliferative and metabolic role of PKP1 in lung squamous cell carcinoma.","method":"CRISPR KO screen, metabolic assays (OCR/ECAR), ubiquitination assay, Co-IP (PKP1-TRIM21-PFKP), PFKP functional rescue experiments","journal":"Biomarker research","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR screen + Co-IP + ubiquitination + functional rescue, single lab","pmids":["40890861"],"is_preprint":false},{"year":2024,"finding":"PFKP interacts with AMOTL1 in HNSCC cells, inhibiting AMOTL1's ubiquitin-mediated degradation; PFKP-driven glycolysis and EMT are AMOTL1-dependent, and PFKP promotes YAP nuclear translocation via AMOTL1, suppressing Hippo pathway activity to amplify glycolytic flux.","method":"Co-IP (PFKP-AMOTL1), ubiquitination analysis, PFKP/AMOTL1 KD/OE, YAP nuclear localization assays, in vivo xenograft","journal":"Journal of translational internal medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP + ubiquitination + YAP localization assay + functional rescue, single lab","pmids":["41727965"],"is_preprint":false},{"year":2024,"finding":"PFKP binds AXL and promotes its phosphorylation at Y779, activating AXL signaling and subsequently promoting MET phosphorylation in NSCLC cells, representing a non-metabolic, oncogenic signaling function of PFKP.","method":"Co-IP (PFKP-AXL), phosphorylation assays (AXL Y779, MET), PFKP KD with AXL/MET signaling readouts, nanoparticle-mediated in vivo PFKP silencing","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP + phosphorylation site defined + KD phenotype, single lab","pmids":["39664584"],"is_preprint":false},{"year":2024,"finding":"PFKP interacts with EIF2S2 (eukaryotic translation initiation factor 2 subunit beta); in cardiac hypertrophy models, PFKP overexpression increases protein synthesis through EIF2S2, and EIF2S2 knockdown after PFKP overexpression reduces new protein synthesis and alleviates hypertrophy.","method":"IP-MS/MS (PFKP interactome), PFKP KO/OE in mice (TAC model) and NRCMs, EIF2S2 KD rescue, protein synthesis assay","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 — IP-MS identifies interaction, rescue experiments confirm EIF2S2 as downstream mediator","pmids":["39419453"],"is_preprint":false},{"year":2025,"finding":"O-linked N-acetylglucosamine (O-GlcNAc) transferase interaction with PFKP is enhanced by TCEP exposure, decreasing PFKP enzymatic activity and impairing glycolysis and pentose phosphate pathway function in platelets, thereby suppressing platelet activation processes.","method":"Proteomic analysis of platelets, OGT-PFKP interaction assay, PFKP activity measurement, ex vivo platelet activation assays, in vivo TCEP exposure model","journal":"Environmental pollution","confidence":"Medium","confidence_rationale":"Tier 2-3 — OGT-PFKP interaction linked to enzyme activity change + functional platelet readouts, single study","pmids":["39828204"],"is_preprint":false},{"year":2025,"finding":"Stat1 transcription factor directly binds the Pfkp promoter (demonstrated by ChIP-qPCR) and transcriptionally activates Pfkp expression in macrophages downstream of dsHMGB1/Jak2/Stat1 signaling, promoting glycolysis and M1 macrophage polarization.","method":"ChIP-qPCR (Stat1 binding to Pfkp promoter), dual-luciferase assay, Stat1 inhibitor (fludarabine) rescue, macrophage polarization assays","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-qPCR + luciferase reporter confirm direct transcriptional regulation, functional rescue, single lab","pmids":["41079919"],"is_preprint":false},{"year":2025,"finding":"HIF-1α transcriptionally controls PFKP mRNA in macrophages; in ethanol-exposed macrophages, oxidative stress (ROS) impairs HIF-1α function, reducing PFKP transcription and dampening glycolysis and phagocytosis. MitoQ restores HIF-1α function, PFKP expression, glycolysis, and phagocytosis, validated by ChIP-qPCR.","method":"ChIP-qPCR (HIF-1α binding to PFKP locus), PFKP mRNA/protein measurements, MitoQ treatment, phagocytosis and glycolysis assays, in vivo sepsis survival","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-qPCR establishes direct transcriptional link, functional MitoQ rescue in vitro and in vivo, single lab","pmids":["40356076"],"is_preprint":false},{"year":2026,"finding":"ATM kinase phosphorylates PFKP at threonine 278 (T278) in response to ionizing radiation or high glucose, promoting transition of PFKP from tetramers to dimers and nuclear translocation of the dimeric form. Nuclear dimeric PFKP recruits casein kinase 2 (CK2), which phosphorylates RAD51 at T13, enhancing RAD51-BRCA2 interaction and homologous recombination repair efficiency.","method":"In vitro phosphorylation assay (ATM→PFKP T278), site-directed mutagenesis, native gel electrophoresis (tetramer/dimer shift), Co-IP (PFKP-CK2, RAD51-BRCA2), CK2 substrate assay (RAD51 T13), nuclear fractionation, HR repair assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay + mutagenesis + biochemical complex validation + HR functional readout, mechanistically rigorous","pmids":["42011782"],"is_preprint":false},{"year":2026,"finding":"PFKP-K688 lactylation (PFKP-K688la) is induced by hypoxia/ischemia; this modification increases PFKP enzymatic activity and glycolytic flux while suppressing mitochondrial respiration. A K688E mutation mimicking hyper-lactylation amplifies glycolysis, and this modification provides cardioprotection in ischemic cardiomyocytes.","method":"Lactylome proteomics, PFKP-K688E mutagenesis, PFKP enzyme activity assay, Seahorse (ECAR/OCR), hypoxic cardiomyocyte and LAD mouse models","journal":"Frontiers in pharmacology","confidence":"High","confidence_rationale":"Tier 1-2 — lactylome MS + mutagenesis + enzyme activity assay + functional metabolic readouts in vitro and in vivo","pmids":["41919225"],"is_preprint":false},{"year":2026,"finding":"USP14 deubiquitinase stabilizes PFKP through K48-linked deubiquitination, and c-Myc transcriptionally upregulates USP14; PFKP in turn enhances ERK-dependent c-Myc protein stability, forming a c-Myc-USP14-PFKP feed-forward circuit that sustains chemoresistance in pancreatic ductal adenocarcinoma.","method":"Ubiquitination assays (K48-linked), USP14 KD/OE with PFKP stability, c-Myc ChIP/reporter for USP14, ERK phosphorylation assay, patient-derived organoids, syngeneic/xenograft models","journal":"Drug resistance updates","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination specificity defined + multiple functional models, circuit validated in PDOs and in vivo, single lab","pmids":["41812332"],"is_preprint":false}],"current_model":"PFKP is the platelet isoform of phosphofructokinase-1 that catalyzes the rate-limiting step of glycolysis, but also functions as a non-canonical protein kinase (phosphorylating ATG4B and Lin41), undergoes nucleocytoplasmic shuttling regulated by Cyclin D3/CDK6-importin 9, and is subject to extensive post-translational regulation including ubiquitination (by HRD1, TRIM25, RNF123, and reversed by USP5/USP14), deacetylation (by SIRT2 at K394/K395), lactylation (at K688 and K392 with opposing effects on enzymatic activity), and phosphorylation (by ATM at T278 driving nuclear translocation and HR repair); in the nucleus, dimeric PFKP recruits CK2 to phosphorylate RAD51, while its cytoplasmic functions include interaction with AMPK to promote fatty acid oxidation, with VDAC2 on mitochondria to suppress glycolysis, and transcriptional activation of CXCR4 and PFKP itself through c-Myc and other transcription factors (KLF4, YY1, Snail, HIF-1α, SMAD3-SP1, Stat1)."},"narrative":{"teleology":[{"year":1983,"claim":"Establishing the chromosomal identity of PFKP resolved which locus encodes the platelet/fibroblast PFK-1 isoform, enabling all subsequent isoform-specific studies.","evidence":"Somatic cell hybrid analysis with anti-P subunit antiserum and gene dosage experiments mapping PFKP to chromosome 10p","pmids":["6222962"],"confidence":"High","gaps":["No coding sequence or protein structure determined at this stage","Regulatory elements uncharacterized"]},{"year":2011,"claim":"Identification of KLF4 as a direct transcriptional activator of PFKP established that PFKP expression is selectively regulated among PFK isoforms to control glycolytic flux in cancer.","evidence":"ChIP showing KLF4 binding to PFKP promoter, luciferase reporter, KD/OE with glycolysis readouts in breast cancer cells","pmids":["21586797"],"confidence":"High","gaps":["Whether KLF4-PFKP axis operates in non-cancer contexts unknown","Other transcription factors not yet mapped"]},{"year":2017,"claim":"Demonstrating that Snail represses PFKP to reroute glucose from glycolysis to the pentose phosphate pathway revealed PFKP as a metabolic switch point during EMT.","evidence":"Snail KD/OE with metabolic flux measurements and PFKP rescue experiments plus in vivo metastasis assays","pmids":["28176759"],"confidence":"High","gaps":["Mechanism of Snail-mediated PFKP repression (direct binding vs. indirect) not fully resolved","Contribution of other PFK isoforms during EMT unclear"]},{"year":2018,"claim":"Discovery that VDAC2 physically sequesters PFKP on the mitochondrial outer membrane to suppress glycolysis provided the first evidence of a mitochondrial interaction restraining PFKP activity.","evidence":"Co-IP of VDAC2-PFKP, VDAC2 KD/OE with glycolysis and stemness markers, clotrimazole rescue in glioma cells","pmids":["30250190"],"confidence":"Medium","gaps":["Structural basis of VDAC2-PFKP interaction unknown","Not independently replicated outside glioma context","Whether VDAC2 regulation is isoform-specific for PFKP unclear"]},{"year":2021,"claim":"Multiple studies in 2021 collectively revealed that PFKP is a nucleocytoplasmic shuttling protein with non-canonical protein kinase activity, fundamentally expanding its role beyond glycolysis: Cyclin D3/CDK6 exposes an NLS to drive importin 9-dependent nuclear entry where PFKP activates CXCR4 via c-Myc; separately, PFKP directly phosphorylates ATG4B at S34 to promote autophagy.","evidence":"NLS/NES mutagenesis, Co-IP of CDK6-PFKP and PFKP-importin 9, CXCR4 reporter, in vivo leukemia model (PMID:34255748); in vitro kinase assay with phospho-site mutagenesis, CRISPR KO, LC3 turnover (PMID:33607258)","pmids":["34255748","33607258"],"confidence":"High","gaps":["Kinase domain or catalytic residues responsible for PFKP's protein kinase activity not structurally defined","Full spectrum of PFKP kinase substrates unknown","Whether nuclear PFKP retains glycolytic activity unclear"]},{"year":2021,"claim":"Identification of HRD1 as an E3 ligase targeting PFKP for proteasomal degradation, together with the R-2HG/FTO/m6A epitranscriptomic axis controlling PFKP mRNA stability, established that PFKP abundance is tightly regulated at both protein and mRNA levels.","evidence":"MS interactome, Co-IP, ubiquitination assay for HRD1 (PMID:33588886); m6A-seq, FTO/YTHDF2 KD, mRNA decay assays in leukemia (PMID:33434505)","pmids":["33588886","33434505"],"confidence":"High","gaps":["Interplay between transcriptional, epitranscriptomic, and ubiquitin-mediated regulation not integrated","Whether HRD1 targets PFKP from the ER membrane or in the cytosol not fully clarified"]},{"year":2021,"claim":"Mapping additional transcriptional inputs — YY1/BRD2/4 in prostate cancer and SMAD3-SP1 downstream of TGF-β1 in kidney fibrosis — demonstrated that PFKP transcription is controlled by context-specific transcription factor combinations.","evidence":"ChIP-seq and cis-element mutagenesis for YY1 (PMID:33849067); ChIP-qPCR for SMAD3-SP1 binding, AAV-mediated PFKP KD/OE in mice (PMID:38086793)","pmids":["33849067","38086793"],"confidence":"High","gaps":["How multiple TFs are coordinated at the PFKP promoter in a single cell type is unknown","Epigenetic regulation of the PFKP locus beyond BRD2/4 not explored"]},{"year":2022,"claim":"The finding that PFKP physically interacts with AMPK and promotes mitochondrial AMPK-dependent fatty acid oxidation during glucose starvation revealed a metabolic adaptor function independent of PFK-1 catalytic activity.","evidence":"Proteomics screen, Co-IP, mitochondrial fractionation, ACC2 phosphorylation, FAO measurements in NSCLC cells","pmids":["35641476"],"confidence":"High","gaps":["Whether PFKP's glycolytic versus AMPK-scaffolding functions are mutually exclusive not determined","PFKP domains mediating AMPK interaction not mapped"]},{"year":2022,"claim":"Fbxo7 was shown to coordinate CDK6-dependent phosphorylation and CDK6-independent ubiquitination of PFKP, providing an additional regulatory node linking cell cycle machinery to glycolytic control in T cells.","evidence":"Fbxo7 KO in CD4+ T cells, CDK6 activity assays, ubiquitination assay, metabolomics","pmids":["35670764"],"confidence":"High","gaps":["Specific phosphorylation site(s) on PFKP targeted by CDK6 via Fbxo7 not mapped","Whether Fbxo7-mediated PFKP ubiquitination uses K48 or other linkage types not defined"]},{"year":2023,"claim":"SIRT2-mediated deacetylation at K394/K395 established that acetylation is an activating mark for PFKP enzymatic activity and its kinase activity toward ATG4B, linking metabolic regulation to LC3-associated phagocytosis in macrophages.","evidence":"SIRT2 inhibition, K394 mutagenesis, PFKP activity assays, ATG4B phosphorylation, LAP/phagocytosis assays, in vivo sepsis model","pmids":["36865524"],"confidence":"High","gaps":["Acetyltransferase that deposits K394/K395 acetylation not identified","Whether deacetylation also affects PFKP's kinase activity toward Lin41 unknown"]},{"year":2023,"claim":"Discovery that p53 activates CMBL to enhance TRIM25-mediated K48 ubiquitination of PFKP connected genotoxic stress signaling to glycolytic suppression, adding TRIM25 to the growing list of PFKP E3 ligases.","evidence":"Co-IP of CMBL-TRIM25-PFKP ternary complex, ubiquitination assay, p53 activation, glycolysis measurements in colorectal cancer","pmids":["37967006"],"confidence":"High","gaps":["Specific lysine(s) on PFKP targeted by TRIM25 not mapped","Whether CMBL adaptor function is specific to TRIM25 or generalizable to other E3s unknown"]},{"year":2023,"claim":"PFKP's non-canonical kinase activity was extended to a second substrate, Lin41, where phosphorylation prevents Lin41 autoubiquitination, stabilizing it to direct endodermal differentiation of mouse ESCs.","evidence":"In vitro kinase assay, Co-IP, Lin41 ubiquitination assay, ESC lineage marker analysis","pmids":["36660859"],"confidence":"High","gaps":["Consensus motif or structural basis for PFKP substrate recognition as a kinase not defined","Whether PFKP kinase activity is relevant in adult tissues beyond ESCs not tested"]},{"year":2023,"claim":"Identification of lactylation at K688 as an inhibitory modification on PFKP enzymatic activity suggested a product-feedback mechanism where glycolysis-derived lactate attenuates PFKP.","evidence":"Proteome-wide lactylation profiling by MS, site-specific identification, in vitro enzyme activity assay","pmids":["38155775"],"confidence":"Medium","gaps":["Lactyltransferase and delactylase for K688 not identified","In vivo relevance of K688 lactylation not tested in genetic models","Single study without independent replication"]},{"year":2024,"claim":"Multiple E3 ligases (RNF123, TRIM21 counteracted by PKP1) and the deubiquitinase USP5 were added to the PFKP ubiquitin regulatory network, reinforcing that PFKP protein turnover is a major control point for glycolysis across cancer types.","evidence":"Co-IP, ubiquitination assays, KD/OE with stability and glycolysis readouts for RNF123 (PMID:39725718), USP5 (PMID:38217030), and PKP1-TRIM21 (PMID:40890861)","pmids":["39725718","38217030","40890861"],"confidence":"Medium","gaps":["Relative contributions of each E3/DUB pair in a single cell type not compared","Lysine specificity for most E3 ligase–PFKP pairs not mapped"]},{"year":2024,"claim":"PFKP was shown to participate in non-metabolic signaling by binding AXL to promote its Y779 phosphorylation and downstream MET activation, and by interacting with AMOTL1 to suppress Hippo pathway activity via YAP nuclear translocation.","evidence":"Co-IP and phosphorylation assays for PFKP-AXL (PMID:39664584); Co-IP, ubiquitination, YAP localization for PFKP-AMOTL1 (PMID:41727965)","pmids":["39664584","41727965"],"confidence":"Medium","gaps":["Whether PFKP's kinase activity mediates AXL phosphorylation or acts via scaffolding not distinguished","PFKP-AMOTL1 interaction domain not mapped","Both findings from single labs"]},{"year":2024,"claim":"A PFKP–c-Myc positive feedback loop was defined in which c-Myc transcriptionally activates PFKP, while PFKP stabilizes c-Myc protein via ERK signaling; USP14-mediated deubiquitination feeds into this circuit.","evidence":"KD/OE, ERK assays, luciferase reporter in HNSCC (PMID:38982480); K48-deubiquitination assays, c-Myc ChIP for USP14, PDOs in pancreatic cancer (PMID:41812332)","pmids":["38982480","41812332"],"confidence":"Medium","gaps":["How PFKP activates ERK (direct or indirect) is mechanistically undefined","Whether USP14 and USP5 are redundant for PFKP stabilization not tested"]},{"year":2025,"claim":"HIF-1α and Stat1 were confirmed as direct transcriptional activators of PFKP in macrophages, establishing PFKP as a convergence point for hypoxia and inflammatory signaling that drives M1 polarization and phagocytic glycolysis.","evidence":"ChIP-qPCR for HIF-1α binding to PFKP locus, MitoQ rescue (PMID:40356076); ChIP-qPCR + luciferase for Stat1 (PMID:41079919)","pmids":["40356076","41079919"],"confidence":"Medium","gaps":["Whether HIF-1α and Stat1 act combinatorially or independently at the PFKP promoter not resolved","Contribution of PFKP versus other glycolytic enzymes to macrophage polarization not dissected"]},{"year":2026,"claim":"ATM-dependent phosphorylation at T278 was shown to convert PFKP tetramers to dimers that translocate to the nucleus and recruit CK2 to phosphorylate RAD51 at T13, directly linking PFKP to homologous recombination DNA repair.","evidence":"In vitro kinase assay (ATM→PFKP T278), native gel oligomeric shift, Co-IP of PFKP-CK2 and RAD51-BRCA2, HR repair assay","pmids":["42011782"],"confidence":"High","gaps":["Whether nuclear dimeric PFKP retains glycolytic or kinase activity not tested","Structural basis of tetramer-to-dimer switch upon T278 phosphorylation not resolved"]},{"year":2026,"claim":"The opposing functional effects of lactylation at different sites were clarified: K688 lactylation activates PFKP enzymatic activity and confers cardioprotection under ischemia, contrasting with earlier evidence of K688 lactylation as inhibitory.","evidence":"Lactylome MS, K688E mutagenesis, enzyme activity assay, Seahorse metabolic profiling, LAD ischemia mouse model","pmids":["41919225"],"confidence":"High","gaps":["Discrepancy between K688 lactylation being activating (PMID:41919225) versus inhibiting (PMID:38155775) needs resolution across cell types","Enzymes catalyzing K688 lactylation not identified"]},{"year":null,"claim":"The structural basis for PFKP's non-canonical protein kinase activity — including the catalytic site, substrate recognition motif, and how it coexists with PFK-1 enzymatic function — remains undefined.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure of PFKP in its dimeric nuclear form","Full catalog of PFKP kinase substrates unknown","Whether PFKP's glycolytic, kinase, and scaffolding activities are simultaneously active or mutually exclusive not determined","Integrated quantitative model of PFKP post-translational regulation (ubiquitination, acetylation, lactylation, phosphorylation, O-GlcNAcylation) lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[9,14,17,30]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[9,17,25,30]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,11,24,26]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,11,13]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,30]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3,11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,2,5,6,13,16,31]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[9,14]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,20,24,25]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,2,7,8,10,18,28,29]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,12]}],"complexes":[],"partners":["ATG4B","AMPK","VDAC2","CCND3","CDK6","CK2","USP5","TRIM25"],"other_free_text":[]},"mechanistic_narrative":"PFKP encodes the platelet/fibroblast isoform of phosphofructokinase-1, the rate-limiting glycolytic enzyme that converts fructose-6-phosphate to fructose-1,6-bisphosphate, and serves as a central node integrating metabolic flux with signal transduction, autophagy, DNA repair, and cell fate decisions. Beyond its canonical glycolytic function, PFKP possesses non-canonical protein kinase activity, directly phosphorylating ATG4B at S34 to activate autophagic flux and Lin41 to stabilize it during endodermal specification [PMID:33607258, PMID:36660859]. PFKP protein stability is tightly controlled by multiple E3 ubiquitin ligases (HRD1, TRIM25/CMBL, RNF123, TRIM21/PKP1) and counterbalanced by deubiquitinases (USP5, USP14), while its enzymatic activity is modulated by SIRT2-mediated deacetylation at K394/K395, lactylation at K688 and K392 with opposing effects, and O-GlcNAcylation [PMID:33588886, PMID:37967006, PMID:36865524, PMID:41919225]. ATM-dependent phosphorylation at T278 converts PFKP tetramers to dimers that translocate to the nucleus via Cyclin D3/CDK6–importin 9, where dimeric PFKP recruits CK2 to phosphorylate RAD51 for homologous recombination repair and activates CXCR4 transcription through c-Myc [PMID:42011782, PMID:34255748]."},"prefetch_data":{"uniprot":{"accession":"Q01813","full_name":"ATP-dependent 6-phosphofructokinase, platelet type","aliases":["6-phosphofructokinase type C","Phosphofructo-1-kinase isozyme C","PFK-C","Phosphohexokinase"],"length_aa":784,"mass_kda":85.6,"function":"Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q01813/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PFKP","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HSP90B1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PFKP","total_profiled":1310},"omim":[{"mim_id":"610681","title":"PHOSPHOFRUCTOKINASE, MUSCLE TYPE; PFKM","url":"https://www.omim.org/entry/610681"},{"mim_id":"603368","title":"CYCLIN-DEPENDENT KINASE 6; CDK6","url":"https://www.omim.org/entry/603368"},{"mim_id":"171860","title":"PHOSPHOFRUCTOKINASE, LIVER TYPE; PFKL","url":"https://www.omim.org/entry/171860"},{"mim_id":"171840","title":"PHOSPHOFRUCTOKINASE, PLATELET TYPE; PFKP","url":"https://www.omim.org/entry/171840"},{"mim_id":"123834","title":"CYCLIN D3; CCND3","url":"https://www.omim.org/entry/123834"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"retina","ntpm":250.0}],"url":"https://www.proteinatlas.org/search/PFKP"},"hgnc":{"alias_symbol":["PFK-C","PFKF"],"prev_symbol":[]},"alphafold":{"accession":"Q01813","domains":[{"cath_id":"3.40.50.450","chopping":"18-185_315-369","consensus_level":"high","plddt":93.4372,"start":18,"end":369},{"cath_id":"3.40.50.460","chopping":"188-311","consensus_level":"high","plddt":96.124,"start":188,"end":311},{"cath_id":"3.40.50.450","chopping":"406-539_686-736","consensus_level":"high","plddt":94.8501,"start":406,"end":736},{"cath_id":"3.40.50.460","chopping":"553-675","consensus_level":"medium","plddt":95.8321,"start":553,"end":675}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01813","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q01813-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q01813-F1-predicted_aligned_error_v6.png","plddt_mean":92.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PFKP","jax_strain_url":"https://www.jax.org/strain/search?query=PFKP"},"sequence":{"accession":"Q01813","fasta_url":"https://rest.uniprot.org/uniprotkb/Q01813.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q01813/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01813"}},"corpus_meta":[{"pmid":"33434505","id":"PMC_33434505","title":"R-2-hydroxyglutarate 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37326348","citation_count":18,"is_preprint":false},{"pmid":"35095764","id":"PMC_35095764","title":"PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease.","date":"2022","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/35095764","citation_count":15,"is_preprint":false},{"pmid":"34087332","id":"PMC_34087332","title":"Triptolide impairs glycolysis by suppressing GATA4/Sp1/PFKP signaling axis in mouse Sertoli cells.","date":"2021","source":"Toxicology and applied pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34087332","citation_count":14,"is_preprint":false},{"pmid":"37704285","id":"PMC_37704285","title":"PFKP: More than phosphofructokinase.","date":"2023","source":"Advances in cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/37704285","citation_count":13,"is_preprint":false},{"pmid":"36792847","id":"PMC_36792847","title":"Glycolysis regulator PFKP induces human 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Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/41448552","citation_count":0,"is_preprint":false},{"pmid":"40651151","id":"PMC_40651151","title":"TRIM25 potentiates innate immune response to Senecavirus A by enhancing K63-linked ubiquitination of RIG-I and K48-linked ubiquitin-facilitated degradation of PFKP.","date":"2025","source":"Veterinary microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/40651151","citation_count":0,"is_preprint":false},{"pmid":"40762768","id":"PMC_40762768","title":"P4HA2 promotes the progression of thyroid cancer by regulating PFKP-mediated glycolysis.","date":"2025","source":"Journal of physiology and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40762768","citation_count":0,"is_preprint":false},{"pmid":"41919225","id":"PMC_41919225","title":"Lactylation of PFKP-K688 enhances glycolytic flux and confers cardioprotection in myocardial ischemia.","date":"2026","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41919225","citation_count":0,"is_preprint":false},{"pmid":"42011782","id":"PMC_42011782","title":"ATM promotes PFKP nuclear translocation to balance glycolysis and homologous recombination repair.","date":"2026","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/42011782","citation_count":0,"is_preprint":false},{"pmid":"42044203","id":"PMC_42044203","title":"PFKP Drives Immune Checkpoint Co-Expression and Metabolic Pathway Activation in Liver Cancer: TCGA-Based and Experimental Validation.","date":"2026","source":"Clinical and translational gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/42044203","citation_count":0,"is_preprint":false},{"pmid":"40821334","id":"PMC_40821334","title":"PFKP Mediates Breast Cancer Metastasis Through Altered Glycolysis.","date":"2025","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/40821334","citation_count":0,"is_preprint":false},{"pmid":"41727965","id":"PMC_41727965","title":"PFKP binding AMOTL1 promotes tumor aerobic glycolysis and epithelial-mesenchymal transition by modulating Hippo pathway in head and neck cancer.","date":"2026","source":"Journal of translational internal medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41727965","citation_count":0,"is_preprint":false},{"pmid":"42024199","id":"PMC_42024199","title":"PFKP is required for chemoresistant phenotype of breast cancer through modulating the formation of CD133+ cancer stem like cells.","date":"2026","source":"Molecular biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/42024199","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.19.25324051","title":"Multi-level Regulatory Roles of Lactate Metabolism Gene Network in Oral Cancer: Machine Learning Insights","date":"2025-03-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.19.25324051","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.22.655446","title":"Pneumococcal H<sub>2</sub>O<sub>2</sub> Reshapes Mitochondrial Function and Reprograms Host Cell Metabolism","date":"2025-05-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.22.655446","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.30.656396","title":"Molecular subtyping based on hippocampal cryptic exon burden reveals proteome-wide changes associated with TDP-43 pathology across the spectrum of LATE and Alzheimer’s Disease","date":"2025-06-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.30.656396","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.09.651611","title":"The Establishment of Prostate-specific, SKP2 Humanized Mice by CRISPR Knock-in Method Reveals Neoplastic Initiation and Microenvironmental Reprogramming","date":"2025-05-10","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.09.651611","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":33665,"output_tokens":8488,"usd":0.114157},"stage2":{"model":"claude-opus-4-6","input_tokens":12261,"output_tokens":5205,"usd":0.287145},"total_usd":0.401302,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1983,\n      \"finding\": \"The human PFKP gene was mapped to the short arm of chromosome 10 (10p) using somatic cell hybrid analysis with anti-human P subunit-specific antiserum and immunoprecipitation; a gene dosage effect was demonstrated in fibroblasts with duplication of 10p, confirming PFKP encodes the platelet/fibroblast isoform of phosphofructokinase.\",\n      \"method\": \"Somatic cell hybrid panel analysis, active-enzyme-immunoprecipitation, gene dosage experiment\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct chromosomal assignment with functional gene dosage validation, foundational mapping study\",\n      \"pmids\": [\"6222962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KLF4 directly binds the PFKP promoter and transcriptionally activates PFKP expression in breast cancer cells, increasing glycolytic activity (glucose uptake and lactate production); knockdown of KLF4 decreased glycolysis whereas overexpression increased it, without affecting other PFK isoforms.\",\n      \"method\": \"ChIP (KLF4 binding to PFKP promoter), luciferase reporter assay, KLF4 KD/OE with glycolysis measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, reporter, KD/OE with functional readout)\",\n      \"pmids\": [\"21586797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Snail (SNAI1) directly represses PFKP transcription during EMT, diverting glucose flux from glycolysis to the pentose phosphate pathway (PPP), increasing NADPH; PFKP knockdown rescues the metabolic reprogramming and cell death induced by Snail loss, placing PFKP downstream of Snail in the glycolysis-PPP switch.\",\n      \"method\": \"Snail KD/OE with PFKP expression and metabolic flux measurements, PFKP rescue experiments, in vivo metastasis assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (rescue experiments), multiple metabolic readouts, in vivo validation\",\n      \"pmids\": [\"28176759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"VDAC2 (a mitochondrial outer membrane protein) physically couples with PFKP on the mitochondrion to inhibit PFKP-mediated glycolysis; disruption of VDAC2 de-represses PFKP activity and glycolysis, driving dedifferentiation of non-stem tumor cells to glioma stem cells, while PFK inhibitor clotrimazole blocks this phenotypic transition.\",\n      \"method\": \"Co-immunoprecipitation (VDAC2-PFKP), VDAC2 KD/OE with glycolysis and stem-cell marker assays, clotrimazole rescue\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal interaction shown, functional rescue with PFK inhibitor, but single lab\",\n      \"pmids\": [\"30250190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Under hypoxia, oxidized ATM regulates PFKP at the translational level (via HIF1A) to promote intracellular citrate accumulation in triple-negative breast cancer; suppression of oxidized ATM reduces PFKP and citrate levels, while citrate accumulation activates AKT/ERK/MMP2/9 signaling to enhance invasion and metastasis.\",\n      \"method\": \"ATM inhibition/oxidation assays, PFKP protein-level measurements, metabolite (citrate) quantification, in vitro invasion and xenograft assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — translational regulation defined, metabolite-phenotype link established, single lab\",\n      \"pmids\": [\"30850587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"R-2-hydroxyglutarate inhibits FTO demethylase activity, leading to increased m6A methylation on PFKP mRNA; this prevents YTHDF2-mediated protection of PFKP mRNA, reducing PFKP protein and suppressing aerobic glycolysis in leukemia cells. PFKP knockdown recapitulates R-2HG-induced glycolytic inhibition.\",\n      \"method\": \"m6A-seq, PFKP mRNA stability assays, FTO/YTHDF2 KD, metabolic assays, in vivo leukemogenesis model\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (epitranscriptomics, genetic KD, in vivo), replicated across cell types\",\n      \"pmids\": [\"33434505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HRD1 (an E3 ubiquitin ligase) interacts with and co-localizes with PFKP in the cytoplasm, ubiquitinates PFKP and targets it for proteasomal degradation, thereby reducing PFKP expression and glycolytic activity in breast cancer cells.\",\n      \"method\": \"Mass spectrometry interactome, co-immunoprecipitation, immunofluorescence co-localization, ubiquitylation assay, HRD1 OE/KD with glycolysis and tumor growth readouts\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interaction confirmed by Co-IP + co-localization + ubiquitination assay + functional rescue\",\n      \"pmids\": [\"33588886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLS). Cyclin D3/CDK6 dimerizes with PFKP and exposes its NLS, facilitating interaction with importin 9 and nuclear translocation. Nuclear PFKP stimulates CXCR4 expression in a c-Myc-dependent manner to promote T-ALL invasion and leukemia homing/infiltration.\",\n      \"method\": \"NLS/NES mutagenesis, Co-IP (Cyclin D3/CDK6-PFKP, PFKP-importin 9), subcellular fractionation, CXCR4 reporter assays, in vivo leukemia infiltration model with CXCR4 antagonist rescue, IHC of patient samples\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (mutagenesis, Co-IP, in vivo rescue), mechanistic pathway clearly defined\",\n      \"pmids\": [\"34255748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"YY1 transcription factor directly binds the PFKP gene regulatory elements and activates PFKP transcription in cooperation with BRD2/4 co-factors; mutagenesis of YY1-bound cis-elements and gene loss-of-function/rescue studies establish a YY1:BRD2/4-PFKP axis driving the Warburg effect in castration-resistant prostate cancer.\",\n      \"method\": \"ChIP-seq (cistrome), YY1 KD with PFKP rescue, mutagenesis of YY1-binding cis-elements, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cistrome mapping + mutagenesis + genetic rescue, multiple validation approaches\",\n      \"pmids\": [\"33849067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PFKP facilitates phosphorylation of autophagy protein ATG4B at serine 34, functioning as a protein kinase; amino acid deprivation strengthens the PFKP-ATG4B interaction, PFKP phosphorylates ATG4B at S34 in vitro, enhancing ATG4B activity and autophagic flux (LC3-II turnover, p62 degradation). PFKP S386 phosphorylation is required for its kinase activity toward ATG4B.\",\n      \"method\": \"Tandem affinity purification + mass spectrometry, Co-IP (ATG4B-PFKP interaction), CRISPR/Cas9 PFKP KO, in vitro kinase assay, phospho-site mutagenesis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis plus genetic KO validation, non-canonical kinase activity established\",\n      \"pmids\": [\"33607258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TGF-β1 recruits the SMAD3-SP1 complex to the PFKP promoter to transcriptionally activate PFKP expression in renal proximal tubular epithelial cells, driving glycolysis and kidney interstitial fibrosis; AAV-mediated PFKP overexpression worsens fibrosis while PFKP knockdown is protective.\",\n      \"method\": \"ChIP-qPCR (SMAD3-SP1 binding to PFKP promoter), AAV-mediated PFKP KD/OE in mice (unilateral ureteral occlusion model), glycolysis and fibrosis quantification\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-qPCR establishes direct transcriptional mechanism, confirmed in vivo with KD/OE\",\n      \"pmids\": [\"38086793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PFKP physically interacts with AMPK; upon glucose starvation, this interaction is enhanced and promotes mitochondrial recruitment of AMPK, which then phosphorylates acetyl-CoA carboxylase 2 (ACC2) to enhance long-chain fatty acid oxidation, supporting energy and redox homeostasis in NSCLC cells.\",\n      \"method\": \"Proteomics screen of AMPK-interacting proteins, Co-IP (PFKP-AMPK), mitochondrial fractionation, ACC2 phosphorylation assay, PFKP KD with FAO measurements\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interaction, confirmed by Co-IP + fractionation + functional metabolic readouts\",\n      \"pmids\": [\"35641476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Fbxo7 promotes Cdk6-dependent phosphorylation of PFKP and Cdk6-independent ubiquitination of PFKP; Fbxo7-deficient T cells show reduced Cdk6 activity and increased glycolytic flux, demonstrating that the Fbxo7-Cdk6 axis suppresses PFKP activity and glycolysis.\",\n      \"method\": \"Substrate screen for Fbxo7, Fbxo7 KO in CD4+ T cells, Cdk6 activity assays, ubiquitination assay, metabolomics of activated T cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate screen + KO phenotype + metabolomics, multiple orthogonal approaches\",\n      \"pmids\": [\"35670764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PFKP activation in podocytes maintains fructose-1,6-bisphosphate (FBP) levels; PFKP knockdown or inhibition reduces FBP, activates the RhoA/ROCK1 pathway, and causes cytoskeletal remodeling (foot process fusion), while PFKP overexpression rescues these defects in diabetic kidney disease.\",\n      \"method\": \"siRNA/plasmid-mediated PFKP KD/OE, targeted metabolomics (FBP measurement), RhoA/ROCK1 pathway analysis, PFKP inhibitor (clotrimazole) in diabetic mice, exogenous FBP rescue\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — metabolite-pathway link established with rescue, single lab\",\n      \"pmids\": [\"35095764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SIRT2 deacetylates PFKP at lysine 394 (mouse)/lysine 395 (human), reducing PFKP enzymatic activity; acetylation at this site is required for PFKP's glycolytic function and its role in phosphorylating ATG4B to activate LC3-associated phagocytosis (LAP) in macrophages. Ethanol exposure enhances SIRT2-PFKP interaction and suppresses phagocytosis.\",\n      \"method\": \"SIRT2 KD/pharmacological inhibition, acetylation site mutagenesis (K394), PFKP activity assays, Atg4B phosphorylation assay, LC3 activation assay, phagocytosis/LAP assays in macrophages, in vivo sepsis survival\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — site-specific deacetylation with mutagenesis + in vitro activity assay + functional downstream pathway validated in vivo\",\n      \"pmids\": [\"36865524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CMBL enhances TRIM25 binding to PFKP, leading to K48-linked ubiquitination and proteasomal degradation of PFKP; p53 transcriptionally activates CMBL in response to genotoxic stress, thereby suppressing glycolysis through PFKP degradation in colorectal cancer.\",\n      \"method\": \"Co-IP (CMBL-TRIM25-PFKP ternary complex), ubiquitination assay, CMBL OE/KD with PFKP stability and glycolysis measurements, p53 activation experiments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ternary complex identified by Co-IP, ubiquitination validated, p53-CMBL-TRIM25-PFKP pathway defined with genetic rescue\",\n      \"pmids\": [\"37967006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PFKP lactylation at lysine 688 directly attenuates its enzymatic activity, forming a potential negative feedback loop in glycolysis where lactate production inhibits PFKP through lactylation.\",\n      \"method\": \"Mass spectrometry-based proteome-wide lactylation profiling, site-specific lactylation identification at K688, in vitro PFKP enzyme activity assay after lactylation\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — MS identification of modification site + functional enzyme activity assay, single lab study\",\n      \"pmids\": [\"38155775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Glycolytic enzyme Pfkp acts as a protein kinase that phosphorylates the developmental regulator Lin41 at serine residues; this phosphorylation stabilizes Lin41 by preventing its autoubiquitination and proteasomal degradation, permitting Lin41-mediated RNA destabilization of ectodermal markers to favor endodermal specification during murine ESC differentiation.\",\n      \"method\": \"In vitro kinase assay (Pfkp phosphorylating Lin41), Co-IP, Lin41 ubiquitination assay, ESC differentiation lineage marker analysis, Pfkp KD/OE\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with functional validation of substrate stabilization and lineage specification consequences\",\n      \"pmids\": [\"36660859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIF-1α transcriptionally regulates PFKP as a target gene; HBO therapy suppresses hypoxia-induced HIF-1α expression and downstream PFKP transactivation in NSCLC cells, and in vivo HBO therapy inhibited tumor growth in a Pfkp-dependent manner.\",\n      \"method\": \"HIF-1α KD, luciferase reporter (PFKP transactivation), glycolytic flux modeling, in vivo LLC tumor model with Pfkp-dependence rescue\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — transcriptional regulation confirmed by reporter + in vivo Pfkp-dependent rescue, but HIF-1α→PFKP link partially inferred\",\n      \"pmids\": [\"34367973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP5 deubiquitinase directly interacts with PFKP and mediates its deubiquitination and stabilization, preventing proteasomal degradation; USP5-mediated PFKP stabilization is essential for cancer cell aerobic glycolysis and TNBC progression.\",\n      \"method\": \"Co-IP, MS-based protein identification, in vitro binding assay, ubiquitination assay, USP5 KD/OE with PFKP stability and glycolysis measurements, tumor xenograft\",\n      \"journal\": \"Breast cancer research : BCR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interaction validated by Co-IP + in vitro binding + ubiquitination assay + functional readouts\",\n      \"pmids\": [\"38217030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKP increases ERK-mediated stability of c-Myc protein, while c-Myc transcriptionally activates PFKP expression, forming a positive feedback loop driving HNSCC progression; co-targeting both synergistically inhibits tumor growth.\",\n      \"method\": \"PFKP/c-Myc KD/OE, ERK phosphorylation assays, c-Myc stability measurements, luciferase reporter (PFKP promoter), PDO and xenograft models\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — feedback loop defined with KD/OE and reporter, ERK-mediated c-Myc stabilization by PFKP established, single lab\",\n      \"pmids\": [\"38982480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF123 (E3 ubiquitin ligase) directly interacts with PFKP and induces its ubiquitination, promoting PFKP degradation and thereby suppressing glycolysis, cell viability, cell cycle progression, and colony formation in breast cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination analysis, RNF123 OE/KD with PFKP protein levels and glycolysis assays, IHC, in vivo xenograft\",\n      \"journal\": \"Naunyn-Schmiedeberg's archives of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP + ubiquitination assay + functional readouts, single lab\",\n      \"pmids\": [\"39725718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKP promotes OC progression through lactylation at lysine 392; K392 mutation diminishes PFKP lactylation, and PFKP depletion upregulates PTEN expression in hypoxic ovarian cancer cells, suggesting PFKP lactylation suppresses PTEN to drive glycolysis.\",\n      \"method\": \"Immunoprecipitation + Western blot for PFKP lactylation, K392 site-directed mutagenesis, PFKP KD with PTEN/glycolysis measurement, OC xenograft model\",\n      \"journal\": \"Biochemical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — site-specific PTM with mutagenesis + functional downstream (PTEN) link, single lab\",\n      \"pmids\": [\"39638933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKP1 (Plakophilin-1) stabilizes PFKP by binding TRIM21 and preventing TRIM21-mediated ubiquitination and degradation of PFKP; PKP1 depletion selectively reduces PFKP protein levels by enhancing its ubiquitination, and PFKP mediates the proliferative and metabolic role of PKP1 in lung squamous cell carcinoma.\",\n      \"method\": \"CRISPR KO screen, metabolic assays (OCR/ECAR), ubiquitination assay, Co-IP (PKP1-TRIM21-PFKP), PFKP functional rescue experiments\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR screen + Co-IP + ubiquitination + functional rescue, single lab\",\n      \"pmids\": [\"40890861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKP interacts with AMOTL1 in HNSCC cells, inhibiting AMOTL1's ubiquitin-mediated degradation; PFKP-driven glycolysis and EMT are AMOTL1-dependent, and PFKP promotes YAP nuclear translocation via AMOTL1, suppressing Hippo pathway activity to amplify glycolytic flux.\",\n      \"method\": \"Co-IP (PFKP-AMOTL1), ubiquitination analysis, PFKP/AMOTL1 KD/OE, YAP nuclear localization assays, in vivo xenograft\",\n      \"journal\": \"Journal of translational internal medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP + ubiquitination + YAP localization assay + functional rescue, single lab\",\n      \"pmids\": [\"41727965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKP binds AXL and promotes its phosphorylation at Y779, activating AXL signaling and subsequently promoting MET phosphorylation in NSCLC cells, representing a non-metabolic, oncogenic signaling function of PFKP.\",\n      \"method\": \"Co-IP (PFKP-AXL), phosphorylation assays (AXL Y779, MET), PFKP KD with AXL/MET signaling readouts, nanoparticle-mediated in vivo PFKP silencing\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP + phosphorylation site defined + KD phenotype, single lab\",\n      \"pmids\": [\"39664584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PFKP interacts with EIF2S2 (eukaryotic translation initiation factor 2 subunit beta); in cardiac hypertrophy models, PFKP overexpression increases protein synthesis through EIF2S2, and EIF2S2 knockdown after PFKP overexpression reduces new protein synthesis and alleviates hypertrophy.\",\n      \"method\": \"IP-MS/MS (PFKP interactome), PFKP KO/OE in mice (TAC model) and NRCMs, EIF2S2 KD rescue, protein synthesis assay\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS identifies interaction, rescue experiments confirm EIF2S2 as downstream mediator\",\n      \"pmids\": [\"39419453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"O-linked N-acetylglucosamine (O-GlcNAc) transferase interaction with PFKP is enhanced by TCEP exposure, decreasing PFKP enzymatic activity and impairing glycolysis and pentose phosphate pathway function in platelets, thereby suppressing platelet activation processes.\",\n      \"method\": \"Proteomic analysis of platelets, OGT-PFKP interaction assay, PFKP activity measurement, ex vivo platelet activation assays, in vivo TCEP exposure model\",\n      \"journal\": \"Environmental pollution\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — OGT-PFKP interaction linked to enzyme activity change + functional platelet readouts, single study\",\n      \"pmids\": [\"39828204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Stat1 transcription factor directly binds the Pfkp promoter (demonstrated by ChIP-qPCR) and transcriptionally activates Pfkp expression in macrophages downstream of dsHMGB1/Jak2/Stat1 signaling, promoting glycolysis and M1 macrophage polarization.\",\n      \"method\": \"ChIP-qPCR (Stat1 binding to Pfkp promoter), dual-luciferase assay, Stat1 inhibitor (fludarabine) rescue, macrophage polarization assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-qPCR + luciferase reporter confirm direct transcriptional regulation, functional rescue, single lab\",\n      \"pmids\": [\"41079919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HIF-1α transcriptionally controls PFKP mRNA in macrophages; in ethanol-exposed macrophages, oxidative stress (ROS) impairs HIF-1α function, reducing PFKP transcription and dampening glycolysis and phagocytosis. MitoQ restores HIF-1α function, PFKP expression, glycolysis, and phagocytosis, validated by ChIP-qPCR.\",\n      \"method\": \"ChIP-qPCR (HIF-1α binding to PFKP locus), PFKP mRNA/protein measurements, MitoQ treatment, phagocytosis and glycolysis assays, in vivo sepsis survival\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-qPCR establishes direct transcriptional link, functional MitoQ rescue in vitro and in vivo, single lab\",\n      \"pmids\": [\"40356076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ATM kinase phosphorylates PFKP at threonine 278 (T278) in response to ionizing radiation or high glucose, promoting transition of PFKP from tetramers to dimers and nuclear translocation of the dimeric form. Nuclear dimeric PFKP recruits casein kinase 2 (CK2), which phosphorylates RAD51 at T13, enhancing RAD51-BRCA2 interaction and homologous recombination repair efficiency.\",\n      \"method\": \"In vitro phosphorylation assay (ATM→PFKP T278), site-directed mutagenesis, native gel electrophoresis (tetramer/dimer shift), Co-IP (PFKP-CK2, RAD51-BRCA2), CK2 substrate assay (RAD51 T13), nuclear fractionation, HR repair assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay + mutagenesis + biochemical complex validation + HR functional readout, mechanistically rigorous\",\n      \"pmids\": [\"42011782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PFKP-K688 lactylation (PFKP-K688la) is induced by hypoxia/ischemia; this modification increases PFKP enzymatic activity and glycolytic flux while suppressing mitochondrial respiration. A K688E mutation mimicking hyper-lactylation amplifies glycolysis, and this modification provides cardioprotection in ischemic cardiomyocytes.\",\n      \"method\": \"Lactylome proteomics, PFKP-K688E mutagenesis, PFKP enzyme activity assay, Seahorse (ECAR/OCR), hypoxic cardiomyocyte and LAD mouse models\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — lactylome MS + mutagenesis + enzyme activity assay + functional metabolic readouts in vitro and in vivo\",\n      \"pmids\": [\"41919225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP14 deubiquitinase stabilizes PFKP through K48-linked deubiquitination, and c-Myc transcriptionally upregulates USP14; PFKP in turn enhances ERK-dependent c-Myc protein stability, forming a c-Myc-USP14-PFKP feed-forward circuit that sustains chemoresistance in pancreatic ductal adenocarcinoma.\",\n      \"method\": \"Ubiquitination assays (K48-linked), USP14 KD/OE with PFKP stability, c-Myc ChIP/reporter for USP14, ERK phosphorylation assay, patient-derived organoids, syngeneic/xenograft models\",\n      \"journal\": \"Drug resistance updates\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination specificity defined + multiple functional models, circuit validated in PDOs and in vivo, single lab\",\n      \"pmids\": [\"41812332\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PFKP is the platelet isoform of phosphofructokinase-1 that catalyzes the rate-limiting step of glycolysis, but also functions as a non-canonical protein kinase (phosphorylating ATG4B and Lin41), undergoes nucleocytoplasmic shuttling regulated by Cyclin D3/CDK6-importin 9, and is subject to extensive post-translational regulation including ubiquitination (by HRD1, TRIM25, RNF123, and reversed by USP5/USP14), deacetylation (by SIRT2 at K394/K395), lactylation (at K688 and K392 with opposing effects on enzymatic activity), and phosphorylation (by ATM at T278 driving nuclear translocation and HR repair); in the nucleus, dimeric PFKP recruits CK2 to phosphorylate RAD51, while its cytoplasmic functions include interaction with AMPK to promote fatty acid oxidation, with VDAC2 on mitochondria to suppress glycolysis, and transcriptional activation of CXCR4 and PFKP itself through c-Myc and other transcription factors (KLF4, YY1, Snail, HIF-1α, SMAD3-SP1, Stat1).\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PFKP encodes the platelet/fibroblast isoform of phosphofructokinase-1, the rate-limiting glycolytic enzyme that converts fructose-6-phosphate to fructose-1,6-bisphosphate, and serves as a central node integrating metabolic flux with signal transduction, autophagy, DNA repair, and cell fate decisions. Beyond its canonical glycolytic function, PFKP possesses non-canonical protein kinase activity, directly phosphorylating ATG4B at S34 to activate autophagic flux and Lin41 to stabilize it during endodermal specification [PMID:33607258, PMID:36660859]. PFKP protein stability is tightly controlled by multiple E3 ubiquitin ligases (HRD1, TRIM25/CMBL, RNF123, TRIM21/PKP1) and counterbalanced by deubiquitinases (USP5, USP14), while its enzymatic activity is modulated by SIRT2-mediated deacetylation at K394/K395, lactylation at K688 and K392 with opposing effects, and O-GlcNAcylation [PMID:33588886, PMID:37967006, PMID:36865524, PMID:41919225]. ATM-dependent phosphorylation at T278 converts PFKP tetramers to dimers that translocate to the nucleus via Cyclin D3/CDK6–importin 9, where dimeric PFKP recruits CK2 to phosphorylate RAD51 for homologous recombination repair and activates CXCR4 transcription through c-Myc [PMID:42011782, PMID:34255748].\",\n  \"teleology\": [\n    {\n      \"year\": 1983,\n      \"claim\": \"Establishing the chromosomal identity of PFKP resolved which locus encodes the platelet/fibroblast PFK-1 isoform, enabling all subsequent isoform-specific studies.\",\n      \"evidence\": \"Somatic cell hybrid analysis with anti-P subunit antiserum and gene dosage experiments mapping PFKP to chromosome 10p\",\n      \"pmids\": [\"6222962\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No coding sequence or protein structure determined at this stage\", \"Regulatory elements uncharacterized\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of KLF4 as a direct transcriptional activator of PFKP established that PFKP expression is selectively regulated among PFK isoforms to control glycolytic flux in cancer.\",\n      \"evidence\": \"ChIP showing KLF4 binding to PFKP promoter, luciferase reporter, KD/OE with glycolysis readouts in breast cancer cells\",\n      \"pmids\": [\"21586797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether KLF4-PFKP axis operates in non-cancer contexts unknown\", \"Other transcription factors not yet mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating that Snail represses PFKP to reroute glucose from glycolysis to the pentose phosphate pathway revealed PFKP as a metabolic switch point during EMT.\",\n      \"evidence\": \"Snail KD/OE with metabolic flux measurements and PFKP rescue experiments plus in vivo metastasis assays\",\n      \"pmids\": [\"28176759\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Snail-mediated PFKP repression (direct binding vs. indirect) not fully resolved\", \"Contribution of other PFK isoforms during EMT unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that VDAC2 physically sequesters PFKP on the mitochondrial outer membrane to suppress glycolysis provided the first evidence of a mitochondrial interaction restraining PFKP activity.\",\n      \"evidence\": \"Co-IP of VDAC2-PFKP, VDAC2 KD/OE with glycolysis and stemness markers, clotrimazole rescue in glioma cells\",\n      \"pmids\": [\"30250190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of VDAC2-PFKP interaction unknown\", \"Not independently replicated outside glioma context\", \"Whether VDAC2 regulation is isoform-specific for PFKP unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Multiple studies in 2021 collectively revealed that PFKP is a nucleocytoplasmic shuttling protein with non-canonical protein kinase activity, fundamentally expanding its role beyond glycolysis: Cyclin D3/CDK6 exposes an NLS to drive importin 9-dependent nuclear entry where PFKP activates CXCR4 via c-Myc; separately, PFKP directly phosphorylates ATG4B at S34 to promote autophagy.\",\n      \"evidence\": \"NLS/NES mutagenesis, Co-IP of CDK6-PFKP and PFKP-importin 9, CXCR4 reporter, in vivo leukemia model (PMID:34255748); in vitro kinase assay with phospho-site mutagenesis, CRISPR KO, LC3 turnover (PMID:33607258)\",\n      \"pmids\": [\"34255748\", \"33607258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase domain or catalytic residues responsible for PFKP's protein kinase activity not structurally defined\", \"Full spectrum of PFKP kinase substrates unknown\", \"Whether nuclear PFKP retains glycolytic activity unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of HRD1 as an E3 ligase targeting PFKP for proteasomal degradation, together with the R-2HG/FTO/m6A epitranscriptomic axis controlling PFKP mRNA stability, established that PFKP abundance is tightly regulated at both protein and mRNA levels.\",\n      \"evidence\": \"MS interactome, Co-IP, ubiquitination assay for HRD1 (PMID:33588886); m6A-seq, FTO/YTHDF2 KD, mRNA decay assays in leukemia (PMID:33434505)\",\n      \"pmids\": [\"33588886\", \"33434505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between transcriptional, epitranscriptomic, and ubiquitin-mediated regulation not integrated\", \"Whether HRD1 targets PFKP from the ER membrane or in the cytosol not fully clarified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping additional transcriptional inputs — YY1/BRD2/4 in prostate cancer and SMAD3-SP1 downstream of TGF-β1 in kidney fibrosis — demonstrated that PFKP transcription is controlled by context-specific transcription factor combinations.\",\n      \"evidence\": \"ChIP-seq and cis-element mutagenesis for YY1 (PMID:33849067); ChIP-qPCR for SMAD3-SP1 binding, AAV-mediated PFKP KD/OE in mice (PMID:38086793)\",\n      \"pmids\": [\"33849067\", \"38086793\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How multiple TFs are coordinated at the PFKP promoter in a single cell type is unknown\", \"Epigenetic regulation of the PFKP locus beyond BRD2/4 not explored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The finding that PFKP physically interacts with AMPK and promotes mitochondrial AMPK-dependent fatty acid oxidation during glucose starvation revealed a metabolic adaptor function independent of PFK-1 catalytic activity.\",\n      \"evidence\": \"Proteomics screen, Co-IP, mitochondrial fractionation, ACC2 phosphorylation, FAO measurements in NSCLC cells\",\n      \"pmids\": [\"35641476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PFKP's glycolytic versus AMPK-scaffolding functions are mutually exclusive not determined\", \"PFKP domains mediating AMPK interaction not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Fbxo7 was shown to coordinate CDK6-dependent phosphorylation and CDK6-independent ubiquitination of PFKP, providing an additional regulatory node linking cell cycle machinery to glycolytic control in T cells.\",\n      \"evidence\": \"Fbxo7 KO in CD4+ T cells, CDK6 activity assays, ubiquitination assay, metabolomics\",\n      \"pmids\": [\"35670764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylation site(s) on PFKP targeted by CDK6 via Fbxo7 not mapped\", \"Whether Fbxo7-mediated PFKP ubiquitination uses K48 or other linkage types not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"SIRT2-mediated deacetylation at K394/K395 established that acetylation is an activating mark for PFKP enzymatic activity and its kinase activity toward ATG4B, linking metabolic regulation to LC3-associated phagocytosis in macrophages.\",\n      \"evidence\": \"SIRT2 inhibition, K394 mutagenesis, PFKP activity assays, ATG4B phosphorylation, LAP/phagocytosis assays, in vivo sepsis model\",\n      \"pmids\": [\"36865524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Acetyltransferase that deposits K394/K395 acetylation not identified\", \"Whether deacetylation also affects PFKP's kinase activity toward Lin41 unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that p53 activates CMBL to enhance TRIM25-mediated K48 ubiquitination of PFKP connected genotoxic stress signaling to glycolytic suppression, adding TRIM25 to the growing list of PFKP E3 ligases.\",\n      \"evidence\": \"Co-IP of CMBL-TRIM25-PFKP ternary complex, ubiquitination assay, p53 activation, glycolysis measurements in colorectal cancer\",\n      \"pmids\": [\"37967006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific lysine(s) on PFKP targeted by TRIM25 not mapped\", \"Whether CMBL adaptor function is specific to TRIM25 or generalizable to other E3s unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"PFKP's non-canonical kinase activity was extended to a second substrate, Lin41, where phosphorylation prevents Lin41 autoubiquitination, stabilizing it to direct endodermal differentiation of mouse ESCs.\",\n      \"evidence\": \"In vitro kinase assay, Co-IP, Lin41 ubiquitination assay, ESC lineage marker analysis\",\n      \"pmids\": [\"36660859\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Consensus motif or structural basis for PFKP substrate recognition as a kinase not defined\", \"Whether PFKP kinase activity is relevant in adult tissues beyond ESCs not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of lactylation at K688 as an inhibitory modification on PFKP enzymatic activity suggested a product-feedback mechanism where glycolysis-derived lactate attenuates PFKP.\",\n      \"evidence\": \"Proteome-wide lactylation profiling by MS, site-specific identification, in vitro enzyme activity assay\",\n      \"pmids\": [\"38155775\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lactyltransferase and delactylase for K688 not identified\", \"In vivo relevance of K688 lactylation not tested in genetic models\", \"Single study without independent replication\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple E3 ligases (RNF123, TRIM21 counteracted by PKP1) and the deubiquitinase USP5 were added to the PFKP ubiquitin regulatory network, reinforcing that PFKP protein turnover is a major control point for glycolysis across cancer types.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, KD/OE with stability and glycolysis readouts for RNF123 (PMID:39725718), USP5 (PMID:38217030), and PKP1-TRIM21 (PMID:40890861)\",\n      \"pmids\": [\"39725718\", \"38217030\", \"40890861\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contributions of each E3/DUB pair in a single cell type not compared\", \"Lysine specificity for most E3 ligase–PFKP pairs not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PFKP was shown to participate in non-metabolic signaling by binding AXL to promote its Y779 phosphorylation and downstream MET activation, and by interacting with AMOTL1 to suppress Hippo pathway activity via YAP nuclear translocation.\",\n      \"evidence\": \"Co-IP and phosphorylation assays for PFKP-AXL (PMID:39664584); Co-IP, ubiquitination, YAP localization for PFKP-AMOTL1 (PMID:41727965)\",\n      \"pmids\": [\"39664584\", \"41727965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PFKP's kinase activity mediates AXL phosphorylation or acts via scaffolding not distinguished\", \"PFKP-AMOTL1 interaction domain not mapped\", \"Both findings from single labs\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A PFKP–c-Myc positive feedback loop was defined in which c-Myc transcriptionally activates PFKP, while PFKP stabilizes c-Myc protein via ERK signaling; USP14-mediated deubiquitination feeds into this circuit.\",\n      \"evidence\": \"KD/OE, ERK assays, luciferase reporter in HNSCC (PMID:38982480); K48-deubiquitination assays, c-Myc ChIP for USP14, PDOs in pancreatic cancer (PMID:41812332)\",\n      \"pmids\": [\"38982480\", \"41812332\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PFKP activates ERK (direct or indirect) is mechanistically undefined\", \"Whether USP14 and USP5 are redundant for PFKP stabilization not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HIF-1α and Stat1 were confirmed as direct transcriptional activators of PFKP in macrophages, establishing PFKP as a convergence point for hypoxia and inflammatory signaling that drives M1 polarization and phagocytic glycolysis.\",\n      \"evidence\": \"ChIP-qPCR for HIF-1α binding to PFKP locus, MitoQ rescue (PMID:40356076); ChIP-qPCR + luciferase for Stat1 (PMID:41079919)\",\n      \"pmids\": [\"40356076\", \"41079919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HIF-1α and Stat1 act combinatorially or independently at the PFKP promoter not resolved\", \"Contribution of PFKP versus other glycolytic enzymes to macrophage polarization not dissected\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"ATM-dependent phosphorylation at T278 was shown to convert PFKP tetramers to dimers that translocate to the nucleus and recruit CK2 to phosphorylate RAD51 at T13, directly linking PFKP to homologous recombination DNA repair.\",\n      \"evidence\": \"In vitro kinase assay (ATM→PFKP T278), native gel oligomeric shift, Co-IP of PFKP-CK2 and RAD51-BRCA2, HR repair assay\",\n      \"pmids\": [\"42011782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether nuclear dimeric PFKP retains glycolytic or kinase activity not tested\", \"Structural basis of tetramer-to-dimer switch upon T278 phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The opposing functional effects of lactylation at different sites were clarified: K688 lactylation activates PFKP enzymatic activity and confers cardioprotection under ischemia, contrasting with earlier evidence of K688 lactylation as inhibitory.\",\n      \"evidence\": \"Lactylome MS, K688E mutagenesis, enzyme activity assay, Seahorse metabolic profiling, LAD ischemia mouse model\",\n      \"pmids\": [\"41919225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Discrepancy between K688 lactylation being activating (PMID:41919225) versus inhibiting (PMID:38155775) needs resolution across cell types\", \"Enzymes catalyzing K688 lactylation not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for PFKP's non-canonical protein kinase activity — including the catalytic site, substrate recognition motif, and how it coexists with PFK-1 enzymatic function — remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal or cryo-EM structure of PFKP in its dimeric nuclear form\", \"Full catalog of PFKP kinase substrates unknown\", \"Whether PFKP's glycolytic, kinase, and scaffolding activities are simultaneously active or mutually exclusive not determined\", \"Integrated quantitative model of PFKP post-translational regulation (ubiquitination, acetylation, lactylation, phosphorylation, O-GlcNAcylation) lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [9, 14, 17, 30]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [9, 17, 25, 30]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 11, 24, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 11, 13]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 30]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 2, 5, 6, 13, 16, 31]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [9, 14]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 20, 24, 25]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 2, 7, 8, 10, 18, 28, 29]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ATG4B\",\n      \"AMPK\",\n      \"VDAC2\",\n      \"CCND3\",\n      \"CDK6\",\n      \"CK2\",\n      \"USP5\",\n      \"TRIM25\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}