{"gene":"UCK1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2001,"finding":"UCK1 was cloned and expressed in E. coli as an ~30-kDa protein that catalyzes phosphorylation of uridine and cytidine (but not deoxyribonucleosides or purine ribonucleosides) to their monophosphates. UCK1 also phosphorylates several pyrimidine ribonucleoside analogs including 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N4-acetylcytidine, N4-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N4-anisoylcytidine.","method":"Recombinant protein expression in E. coli; in vitro enzyme activity assays with 28 nucleoside analogs","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution with recombinant protein, replicated across substrate panel, foundational cloning paper","pmids":["11306702"],"is_preprint":false},{"year":2001,"finding":"UCK1 mRNA is expressed as two isoforms (~1.8 kb and ~2.7 kb); the 2.7-kb isoform is ubiquitously expressed across tissues, while the 1.8-kb isoform is restricted to skeletal muscle, heart, liver, and kidney. UCK1 gene maps to chromosome 9q34.2-9q34.3.","method":"Northern blot analysis; chromosomal mapping","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Northern blot and chromosomal mapping, single lab but clear experimental readouts","pmids":["11306702"],"is_preprint":false},{"year":2006,"finding":"UCK1 does NOT phosphorylate PSI-6130 (beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine); this compound is a substrate for dCK but not UCK1, as measured by coupled spectrophotometric assay with purified recombinant UCK1.","method":"In vitro coupled spectrophotometric kinase assay with purified recombinant UCK1","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified enzyme, direct negative result established for UCK1 substrate specificity","pmids":["17101674"],"is_preprint":false},{"year":2009,"finding":"Purified recombinant UCK1 does NOT phosphorylate 2'-C-methyl-cytidine (2'-MeC) in vitro, establishing that UCK1 lacks activity toward this HCV nucleoside analog.","method":"In vitro enzyme kinase assay with purified recombinant UCK1","journal":"Antiviral research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified recombinant enzyme, negative result independently consistent with PMID 17101674","pmids":["19883694"],"is_preprint":false},{"year":2007,"finding":"UCK1 phosphorylates ribavirin to its monophosphate form, identifying ribavirin as a substrate of UCK1.","method":"In vitro phosphorylation assay using human UCK1 with ribavirin as substrate","journal":"Journal of biomedical science","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay, single lab, single method","pmids":["18080217"],"is_preprint":false},{"year":2016,"finding":"UCK1-GFP localizes to the cell nucleus, while UCK2-GFP localizes to the cytosol. Co-expression of UCK1 with UCK2 causes UCK2 to relocalize from the cytosol to the nucleus, impairing UCK2 function. Physical association of UCK1 and UCK2 was demonstrated by pull-down with his-tagged UCK.","method":"Fluorescence microscopy of GFP-tagged proteins; his-tag pull-down assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization imaging plus pulldown for physical interaction, functional consequence (UCK2 impairment) demonstrated, single lab","pmids":["27239701"],"is_preprint":false},{"year":2016,"finding":"Overexpression of UCK1 in neuroblastoma cells decreased metabolism of uridine and cytidine and protected cells from 3-deazauridine-induced toxicity, indicating UCK1 acts as a negative regulator of pyrimidine salvage in this context (likely via sequestration of UCK2 into the nucleus).","method":"Stable overexpression of UCK1 in neuroblastoma cell lines; metabolic assays; cytotoxicity assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression with defined metabolic and cytotoxicity phenotypes, mechanistic link to UCK2 relocalization established in same study","pmids":["27239701"],"is_preprint":false},{"year":2020,"finding":"The E3 ubiquitin ligase KLHL2 interacts with UCK1, mediates its polyubiquitination at lysine K81, and promotes UCK1 degradation. The deubiquitinase USP28 antagonizes KLHL2-mediated polyubiquitination of UCK1, stabilizing it. ATM-mediated phosphorylation of USP28 causes disassociation of USP28 from KLHL2 and UCK1, leading to UCK1 destabilization. UCK1 phosphorylation by 5'-AZA-activated ATM enhances KLHL2-UCK1 complex formation.","method":"Mass spectrometry; Co-immunoprecipitation; molecular biochemistry; mutagenesis (K81 ubiquitination site); AML cell lines and murine AML model","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (MS, Co-IP, site-specific mutagenesis, in vivo model), key PTM sites identified, writer/eraser defined","pmids":["31938050"],"is_preprint":false},{"year":2025,"finding":"UCK1 affects the turnover of UCK2 by influencing its cellular localization; when mTORC1 is inhibited, UCK2 is degraded via the CTLH-WDR26 E3 complex, and UCK1 modulates this process by altering UCK2 localization.","method":"Genetic/pharmacological mTORC1 inhibition; protein stability assays; localization studies","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional pathway placement with defined E3 ligase complex and localization mechanism; UCK1 role described as modulatory, single lab","pmids":["39808525"],"is_preprint":false},{"year":2026,"finding":"Both UCK1 and UCK2 effectively phosphorylate N4-hydroxycytidine (NHC, the active compound of molnupiravir), with UCK2 showing 9-fold higher catalytic efficiency than UCK1. The structural basis of UCK1 catalysis was resolved by the first complete substrate-bound co-crystal structure: UCK1-NHC-AMPPNP.","method":"In vitro enzyme kinetic assays with recombinant UCK1 and UCK2; co-crystal structure of UCK1-NHC-AMPPNP; siRNA knockdown of UCK2 in SARS-CoV-2-infected cells; pan-UCK inhibitor treatment","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with kinetic parameters, crystal structure with substrate, cell-based siRNA validation, multiple orthogonal methods in one study","pmids":["42213735"],"is_preprint":false},{"year":2025,"finding":"Recombinant UCK1 binds and phosphorylates NHC with measurable catalytic efficiency (9-fold lower than UCK2); UCK2 is the dominant isoform for NHC phosphorylation in cells as shown by siRNA knockdown reducing intracellular NHC triphosphate accumulation 10-fold.","method":"Target engagement assays; enzyme kinetic assays with recombinant UCK1 and UCK2; siRNA knockdown in SARS-CoV-2-infected cells","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution plus cell-based validation, preprint, overlapping with peer-reviewed PMID 42213735","pmids":["bio_10.1101_2025.05.13.653844"],"is_preprint":true},{"year":2010,"finding":"siRNA-mediated loss of UCK1 was found to impair surface accumulation of EGFR and subsequent EGFR pathway activation in multiple cancer cell lines, placing UCK1 as required for EGFR signaling.","method":"Large-scale siRNA loss-of-function screen; reverse phase protein lysate arrays; phosphoprotein detection","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — identified in a kinome-wide siRNA screen, no direct mechanistic follow-up for UCK1 specifically, single screening method","pmids":["20421302"],"is_preprint":false},{"year":2013,"finding":"Silencing of UCK1 by siRNA in MDS cell lines leads to a blunted response to azacitidine (AZA) in vitro, establishing UCK1 activity as required for AZA activation/phosphorylation in human leukemia cells.","method":"siRNA knockdown of UCK1; in vitro AZA sensitivity assay","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean siRNA KD with defined pharmacological phenotype, single lab, single method","pmids":["24192812"],"is_preprint":false}],"current_model":"UCK1 is a ~30-kDa pyrimidine salvage enzyme that phosphorylates uridine, cytidine, and various pyrimidine ribonucleoside analogs (including ribavirin and NHC/molnupiravir) to their 5'-monophosphates using ATP as phosphate donor, but lacks activity toward 2'-deoxy analogs or certain modified nucleosides (e.g., PSI-6130, 2'-MeC); its protein stability is regulated by KLHL2-mediated polyubiquitination at K81 (opposed by USP28 deubiquitination under ATM signaling) and by mTORC1-CTLH pathway-dependent control of its isoform partner UCK2; UCK1 localizes to the cell nucleus and physically interacts with cytosolic UCK2, causing UCK2 nuclear relocalization and impaired pyrimidine salvage activity, thereby acting as a dominant-negative modulator of the salvage pathway; the first substrate-bound co-crystal structure (UCK1-NHC-AMPPNP) has defined the structural basis of catalysis."},"narrative":{"mechanistic_narrative":"UCK1 is a ~30-kDa pyrimidine ribonucleoside kinase of the salvage pathway that uses ATP to phosphorylate uridine and cytidine to their 5'-monophosphates, with no activity toward deoxyribonucleosides or purine ribonucleosides [PMID:11306702]. It accepts a broad panel of pyrimidine ribonucleoside analogs, including 5-fluorouridine, 5-fluorocytidine, and other modified uridine/cytidine derivatives [PMID:11306702], as well as the antiviral ribavirin [PMID:18080217] and N4-hydroxycytidine (NHC, the active form of molnupiravir), which it phosphorylates with ~9-fold lower catalytic efficiency than its isoform UCK2 [PMID:42213735]; the substrate-bound UCK1-NHC-AMPPNP co-crystal structure defines the structural basis of this catalysis [PMID:42213735]. Its specificity is restricted to ribonucleosides: UCK1 does not phosphorylate the 2'-deoxy/2'-fluoro/2'-methyl analogs PSI-6130 or 2'-C-methyl-cytidine [PMID:17101674, PMID:19883694]. Beyond direct catalysis, UCK1 functions as a negative regulator of pyrimidine salvage by localizing to the nucleus and physically binding cytosolic UCK2, driving UCK2 nuclear relocalization, impairing salvage flux, and protecting cells from cytidine-analog toxicity [PMID:27239701]. UCK1 abundance is controlled by ubiquitin-dependent turnover: the E3 ligase KLHL2 polyubiquitinates UCK1 at K81 to promote its degradation, opposed by the deubiquitinase USP28, with ATM signaling shifting this balance toward UCK1 destabilization [PMID:31938050]. Functionally, UCK1 activity is required for activation of the hypomethylating agent azacitidine in leukemia cells [PMID:24192812].","teleology":[{"year":2001,"claim":"Established the core biochemical identity of UCK1 as a pyrimidine ribonucleoside monophosphate kinase, answering what reaction the gene product catalyzes and on which substrates.","evidence":"Recombinant expression in E. coli and in vitro activity assays against a 28-analog panel","pmids":["11306702"],"confidence":"High","gaps":["Kinetic parameters for natural substrates not quantified","Physiological vs. analog substrate preference in cells not resolved"]},{"year":2006,"claim":"Defined the boundary of UCK1 substrate specificity by showing it cannot phosphorylate 2'-modified deoxy analogs, distinguishing it from dCK and clarifying which antiviral prodrugs it can activate.","evidence":"Coupled spectrophotometric kinase assay with purified recombinant UCK1 on PSI-6130; extended to 2'-C-methyl-cytidine in 2009","pmids":["17101674","19883694"],"confidence":"High","gaps":["Structural reason for exclusion of 2'-modified sugars not addressed at the time"]},{"year":2007,"claim":"Extended the analog substrate repertoire by identifying ribavirin as a UCK1 substrate, relevant to antiviral prodrug activation.","evidence":"In vitro phosphorylation assay with human UCK1","pmids":["18080217"],"confidence":"Medium","gaps":["Single method, single lab","Relative contribution of UCK1 vs other kinases to ribavirin activation in cells unknown"]},{"year":2016,"claim":"Revealed a non-catalytic regulatory role: UCK1 localizes to the nucleus and binds UCK2 to relocalize and inhibit it, recasting UCK1 as a dominant-negative modulator of salvage rather than purely a catalyst.","evidence":"GFP localization microscopy and his-tag pull-down; UCK1 overexpression with metabolic and cytotoxicity phenotypes in neuroblastoma cells","pmids":["27239701"],"confidence":"Medium","gaps":["Reciprocal/endogenous interaction validation limited","Structural basis of UCK1-UCK2 association not defined","Generality beyond neuroblastoma cells untested"]},{"year":2020,"claim":"Defined how UCK1 protein levels are set, identifying a writer (KLHL2), eraser (USP28), specific ubiquitination site (K81), and ATM-linked signaling control of its stability.","evidence":"Mass spectrometry, Co-IP, K81 site mutagenesis, and AML cell/murine models","pmids":["31938050"],"confidence":"High","gaps":["Downstream metabolic consequence of UCK1 turnover not quantified","How ATM-driven destabilization affects salvage flux in vivo unclear"]},{"year":2025,"claim":"Placed UCK1 within a nutrient-sensing regulatory axis by showing it modulates mTORC1-/CTLH-WDR26-dependent turnover of UCK2 through control of UCK2 localization.","evidence":"mTORC1 inhibition with protein stability and localization assays","pmids":["39808525"],"confidence":"Medium","gaps":["UCK1 role described as modulatory, not mechanistically dissected","Single lab","Direct effect of UCK1 on CTLH complex not shown"]},{"year":2026,"claim":"Provided atomic-resolution understanding of catalysis and quantified UCK1's contribution to molnupiravir activation relative to UCK2.","evidence":"Enzyme kinetics with recombinant UCK1/UCK2, UCK1-NHC-AMPPNP co-crystal structure, and siRNA knockdown in SARS-CoV-2-infected cells","pmids":["42213735"],"confidence":"High","gaps":["In vivo relevance of UCK1 vs UCK2 to molnupiravir efficacy not established","Structure of UCK1 bound to natural substrates not reported"]},{"year":null,"claim":"How UCK1's catalytic and dominant-negative regulatory functions are balanced under physiological and stress conditions, and the mechanistic basis of its reported role in EGFR signaling, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["EGFR-signaling requirement rests on a single siRNA screen with no mechanistic follow-up","Integration of nuclear sequestration and direct catalysis in normal physiology unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[7]}],"complexes":[],"partners":["UCK2","KLHL2","USP28"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9HA47","full_name":"Uridine-cytidine kinase 1","aliases":["Cytidine monophosphokinase 1","Uridine monophosphokinase 1"],"length_aa":277,"mass_kda":31.4,"function":"Phosphorylates uridine and cytidine to uridine monophosphate and cytidine monophosphate (PubMed:11306702). Does not phosphorylate deoxyribonucleosides or purine ribonucleosides (PubMed:11306702). Can use ATP or GTP as a phosphate donor (PubMed:11306702). Can also phosphorylate cytidine and uridine nucleoside analogs such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-anisoylcytidine (PubMed:11306702)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9HA47/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UCK1","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":[],"url":"https://opencell.sf.czbiohub.org/search/UCK1","total_profiled":1310},"omim":[{"mim_id":"610866","title":"URIDINE/CYTIDINE KINASE-LIKE 1; UCKL1","url":"https://www.omim.org/entry/610866"},{"mim_id":"609329","title":"URIDINE/CYTIDINE KINASE 2; UCK2","url":"https://www.omim.org/entry/609329"},{"mim_id":"609328","title":"URIDINE/CYTIDINE KINASE 1; UCK1","url":"https://www.omim.org/entry/609328"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UCK1"},"hgnc":{"alias_symbol":["URK1","FLJ12255"],"prev_symbol":[]},"alphafold":{"accession":"Q9HA47","domains":[{"cath_id":"3.40.50.300","chopping":"35-231","consensus_level":"medium","plddt":96.4523,"start":35,"end":231}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HA47","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HA47-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HA47-F1-predicted_aligned_error_v6.png","plddt_mean":83.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UCK1","jax_strain_url":"https://www.jax.org/strain/search?query=UCK1"},"sequence":{"accession":"Q9HA47","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HA47.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HA47/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HA47"}},"corpus_meta":[{"pmid":"11306702","id":"PMC_11306702","title":"Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases.","date":"2001","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/11306702","citation_count":132,"is_preprint":false},{"pmid":"24192812","id":"PMC_24192812","title":"Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine.","date":"2013","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/24192812","citation_count":79,"is_preprint":false},{"pmid":"17101674","id":"PMC_17101674","title":"Mechanism of activation of beta-D-2'-deoxy-2'-fluoro-2'-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase.","date":"2006","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17101674","citation_count":76,"is_preprint":false},{"pmid":"12149149","id":"PMC_12149149","title":"Sensitivity of human cancer cells to the new anticancer ribo-nucleoside TAS-106 is correlated with expression of uridine-cytidine kinase 2.","date":"2002","source":"Japanese journal of cancer research : Gann","url":"https://pubmed.ncbi.nlm.nih.gov/12149149","citation_count":51,"is_preprint":false},{"pmid":"15280220","id":"PMC_15280220","title":"A crucial role of uridine/cytidine kinase 2 in antitumor activity of 3'-ethynyl nucleosides.","date":"2004","source":"Drug metabolism and disposition: the biological fate of chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/15280220","citation_count":48,"is_preprint":false},{"pmid":"11872485","id":"PMC_11872485","title":"Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase.","date":"2002","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/11872485","citation_count":33,"is_preprint":false},{"pmid":"27239701","id":"PMC_27239701","title":"The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma.","date":"2016","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/27239701","citation_count":32,"is_preprint":false},{"pmid":"20421302","id":"PMC_20421302","title":"Comprehensive mapping of the human kinome to epidermal growth factor receptor signaling.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20421302","citation_count":31,"is_preprint":false},{"pmid":"12570998","id":"PMC_12570998","title":"A sensitive and inexpensive yeast bioassay for the mycotoxin zearalenone and other compounds with estrogenic activity.","date":"2003","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/12570998","citation_count":27,"is_preprint":false},{"pmid":"27612203","id":"PMC_27612203","title":"The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27612203","citation_count":24,"is_preprint":false},{"pmid":"9602145","id":"PMC_9602145","title":"Cloning, sequence analysis and expression of the basidiomycete Lentinus edodes gene uck1, encoding UMP-CMP kinase, the homologue of Saccharomyces cerevisae URA6 gene.","date":"1998","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9602145","citation_count":20,"is_preprint":false},{"pmid":"25160914","id":"PMC_25160914","title":"Phosphorylation of uridine and cytidine by uridine-cytidine kinase.","date":"2014","source":"Journal of biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/25160914","citation_count":20,"is_preprint":false},{"pmid":"15388965","id":"PMC_15388965","title":"Target genes of the developmental regulator PRIB of the mushroom Lentinula edodes.","date":"2004","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15388965","citation_count":16,"is_preprint":false},{"pmid":"7900425","id":"PMC_7900425","title":"Twelve open reading frames revealed in the 23.6 kb segment flanking the centromere on the Saccharomyces cerevisiae chromosome XIV right arm.","date":"1994","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/7900425","citation_count":16,"is_preprint":false},{"pmid":"11179666","id":"PMC_11179666","title":"Cloning and sequence analysis of the basidiomycete Lentinus edodes ribonucleotide reductase small subunit cDNA and expression of a corresponding gene in L. edodes.","date":"2001","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/11179666","citation_count":15,"is_preprint":false},{"pmid":"31938050","id":"PMC_31938050","title":"Deubiquitinase USP28 inhibits ubiquitin ligase KLHL2-mediated uridine-cytidine kinase 1 degradation and confers sensitivity to 5'-azacytidine-resistant human leukemia cells.","date":"2020","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/31938050","citation_count":14,"is_preprint":false},{"pmid":"39808525","id":"PMC_39808525","title":"mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/39808525","citation_count":13,"is_preprint":false},{"pmid":"19883694","id":"PMC_19883694","title":"Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite.","date":"2009","source":"Antiviral research","url":"https://pubmed.ncbi.nlm.nih.gov/19883694","citation_count":13,"is_preprint":false},{"pmid":"35583288","id":"PMC_35583288","title":"Characterization of uridine-cytidine kinase like-1 nucleoside kinase activity and its role in tumor growth.","date":"2022","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/35583288","citation_count":11,"is_preprint":false},{"pmid":"38204987","id":"PMC_38204987","title":"Integrative Analyses of Pyrimidine Salvage Pathway-Related Genes Revealing the Associations Between UPP1 and Tumor Microenvironment.","date":"2024","source":"Journal of inflammation research","url":"https://pubmed.ncbi.nlm.nih.gov/38204987","citation_count":7,"is_preprint":false},{"pmid":"12876357","id":"PMC_12876357","title":"Crystallization and preliminary X-ray analysis of human uridine-cytidine kinase 2.","date":"2003","source":"Acta crystallographica. Section D, Biological crystallography","url":"https://pubmed.ncbi.nlm.nih.gov/12876357","citation_count":7,"is_preprint":false},{"pmid":"19837748","id":"PMC_19837748","title":"Synthesis and biologic study of IV-14, a new ribonucleoside radiotracer for tumor visualization.","date":"2009","source":"Journal of nuclear medicine : official publication, Society of Nuclear Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/19837748","citation_count":5,"is_preprint":false},{"pmid":"37297025","id":"PMC_37297025","title":"Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37297025","citation_count":5,"is_preprint":false},{"pmid":"18080217","id":"PMC_18080217","title":"Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins.","date":"2007","source":"Journal of biomedical science","url":"https://pubmed.ncbi.nlm.nih.gov/18080217","citation_count":2,"is_preprint":false},{"pmid":"41461775","id":"PMC_41461775","title":"The kinases Hsl101 and Urk1 regulate metabolism of Cryptococcus neoformans.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41461775","citation_count":0,"is_preprint":false},{"pmid":"42213735","id":"PMC_42213735","title":"Uridine cytidine kinases govern molnupiravir bioactivation and anti-SARS-CoV-2 activity.","date":"2026","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/42213735","citation_count":0,"is_preprint":false},{"pmid":"38054921","id":"PMC_38054921","title":"Development and evaluation of a risk score model based on a WNT score gene-associated signature for predicting the clinical outcome and the tumour microenvironment of hepatocellular carcinoma.","date":"2023","source":"International journal of immunopathology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38054921","citation_count":0,"is_preprint":false},{"pmid":"42100918","id":"PMC_42100918","title":"Functional characterization of the 9q34.13 locus identifies RAPGEF1 as modulating risk for melanoma and nevi via RAS activation.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42100918","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.13.653844","title":"Uridine cytidine kinases dictate the therapeutic response of molnupiravir via its bioactivation","date":"2025-05-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.13.653844","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17294,"output_tokens":3283,"usd":0.050563,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10661,"output_tokens":2729,"usd":0.060765,"stage2_stop_reason":"end_turn"},"total_usd":0.111328,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"UCK1 was cloned and expressed in E. coli as an ~30-kDa protein that catalyzes phosphorylation of uridine and cytidine (but not deoxyribonucleosides or purine ribonucleosides) to their monophosphates. UCK1 also phosphorylates several pyrimidine ribonucleoside analogs including 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N4-acetylcytidine, N4-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N4-anisoylcytidine.\",\n      \"method\": \"Recombinant protein expression in E. coli; in vitro enzyme activity assays with 28 nucleoside analogs\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution with recombinant protein, replicated across substrate panel, foundational cloning paper\",\n      \"pmids\": [\"11306702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"UCK1 mRNA is expressed as two isoforms (~1.8 kb and ~2.7 kb); the 2.7-kb isoform is ubiquitously expressed across tissues, while the 1.8-kb isoform is restricted to skeletal muscle, heart, liver, and kidney. UCK1 gene maps to chromosome 9q34.2-9q34.3.\",\n      \"method\": \"Northern blot analysis; chromosomal mapping\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Northern blot and chromosomal mapping, single lab but clear experimental readouts\",\n      \"pmids\": [\"11306702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"UCK1 does NOT phosphorylate PSI-6130 (beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine); this compound is a substrate for dCK but not UCK1, as measured by coupled spectrophotometric assay with purified recombinant UCK1.\",\n      \"method\": \"In vitro coupled spectrophotometric kinase assay with purified recombinant UCK1\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified enzyme, direct negative result established for UCK1 substrate specificity\",\n      \"pmids\": [\"17101674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Purified recombinant UCK1 does NOT phosphorylate 2'-C-methyl-cytidine (2'-MeC) in vitro, establishing that UCK1 lacks activity toward this HCV nucleoside analog.\",\n      \"method\": \"In vitro enzyme kinase assay with purified recombinant UCK1\",\n      \"journal\": \"Antiviral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified recombinant enzyme, negative result independently consistent with PMID 17101674\",\n      \"pmids\": [\"19883694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"UCK1 phosphorylates ribavirin to its monophosphate form, identifying ribavirin as a substrate of UCK1.\",\n      \"method\": \"In vitro phosphorylation assay using human UCK1 with ribavirin as substrate\",\n      \"journal\": \"Journal of biomedical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay, single lab, single method\",\n      \"pmids\": [\"18080217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"UCK1-GFP localizes to the cell nucleus, while UCK2-GFP localizes to the cytosol. Co-expression of UCK1 with UCK2 causes UCK2 to relocalize from the cytosol to the nucleus, impairing UCK2 function. Physical association of UCK1 and UCK2 was demonstrated by pull-down with his-tagged UCK.\",\n      \"method\": \"Fluorescence microscopy of GFP-tagged proteins; his-tag pull-down assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization imaging plus pulldown for physical interaction, functional consequence (UCK2 impairment) demonstrated, single lab\",\n      \"pmids\": [\"27239701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of UCK1 in neuroblastoma cells decreased metabolism of uridine and cytidine and protected cells from 3-deazauridine-induced toxicity, indicating UCK1 acts as a negative regulator of pyrimidine salvage in this context (likely via sequestration of UCK2 into the nucleus).\",\n      \"method\": \"Stable overexpression of UCK1 in neuroblastoma cell lines; metabolic assays; cytotoxicity assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression with defined metabolic and cytotoxicity phenotypes, mechanistic link to UCK2 relocalization established in same study\",\n      \"pmids\": [\"27239701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The E3 ubiquitin ligase KLHL2 interacts with UCK1, mediates its polyubiquitination at lysine K81, and promotes UCK1 degradation. The deubiquitinase USP28 antagonizes KLHL2-mediated polyubiquitination of UCK1, stabilizing it. ATM-mediated phosphorylation of USP28 causes disassociation of USP28 from KLHL2 and UCK1, leading to UCK1 destabilization. UCK1 phosphorylation by 5'-AZA-activated ATM enhances KLHL2-UCK1 complex formation.\",\n      \"method\": \"Mass spectrometry; Co-immunoprecipitation; molecular biochemistry; mutagenesis (K81 ubiquitination site); AML cell lines and murine AML model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (MS, Co-IP, site-specific mutagenesis, in vivo model), key PTM sites identified, writer/eraser defined\",\n      \"pmids\": [\"31938050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UCK1 affects the turnover of UCK2 by influencing its cellular localization; when mTORC1 is inhibited, UCK2 is degraded via the CTLH-WDR26 E3 complex, and UCK1 modulates this process by altering UCK2 localization.\",\n      \"method\": \"Genetic/pharmacological mTORC1 inhibition; protein stability assays; localization studies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional pathway placement with defined E3 ligase complex and localization mechanism; UCK1 role described as modulatory, single lab\",\n      \"pmids\": [\"39808525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Both UCK1 and UCK2 effectively phosphorylate N4-hydroxycytidine (NHC, the active compound of molnupiravir), with UCK2 showing 9-fold higher catalytic efficiency than UCK1. The structural basis of UCK1 catalysis was resolved by the first complete substrate-bound co-crystal structure: UCK1-NHC-AMPPNP.\",\n      \"method\": \"In vitro enzyme kinetic assays with recombinant UCK1 and UCK2; co-crystal structure of UCK1-NHC-AMPPNP; siRNA knockdown of UCK2 in SARS-CoV-2-infected cells; pan-UCK inhibitor treatment\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with kinetic parameters, crystal structure with substrate, cell-based siRNA validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"42213735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Recombinant UCK1 binds and phosphorylates NHC with measurable catalytic efficiency (9-fold lower than UCK2); UCK2 is the dominant isoform for NHC phosphorylation in cells as shown by siRNA knockdown reducing intracellular NHC triphosphate accumulation 10-fold.\",\n      \"method\": \"Target engagement assays; enzyme kinetic assays with recombinant UCK1 and UCK2; siRNA knockdown in SARS-CoV-2-infected cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution plus cell-based validation, preprint, overlapping with peer-reviewed PMID 42213735\",\n      \"pmids\": [\"bio_10.1101_2025.05.13.653844\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"siRNA-mediated loss of UCK1 was found to impair surface accumulation of EGFR and subsequent EGFR pathway activation in multiple cancer cell lines, placing UCK1 as required for EGFR signaling.\",\n      \"method\": \"Large-scale siRNA loss-of-function screen; reverse phase protein lysate arrays; phosphoprotein detection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — identified in a kinome-wide siRNA screen, no direct mechanistic follow-up for UCK1 specifically, single screening method\",\n      \"pmids\": [\"20421302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Silencing of UCK1 by siRNA in MDS cell lines leads to a blunted response to azacitidine (AZA) in vitro, establishing UCK1 activity as required for AZA activation/phosphorylation in human leukemia cells.\",\n      \"method\": \"siRNA knockdown of UCK1; in vitro AZA sensitivity assay\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean siRNA KD with defined pharmacological phenotype, single lab, single method\",\n      \"pmids\": [\"24192812\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UCK1 is a ~30-kDa pyrimidine salvage enzyme that phosphorylates uridine, cytidine, and various pyrimidine ribonucleoside analogs (including ribavirin and NHC/molnupiravir) to their 5'-monophosphates using ATP as phosphate donor, but lacks activity toward 2'-deoxy analogs or certain modified nucleosides (e.g., PSI-6130, 2'-MeC); its protein stability is regulated by KLHL2-mediated polyubiquitination at K81 (opposed by USP28 deubiquitination under ATM signaling) and by mTORC1-CTLH pathway-dependent control of its isoform partner UCK2; UCK1 localizes to the cell nucleus and physically interacts with cytosolic UCK2, causing UCK2 nuclear relocalization and impaired pyrimidine salvage activity, thereby acting as a dominant-negative modulator of the salvage pathway; the first substrate-bound co-crystal structure (UCK1-NHC-AMPPNP) has defined the structural basis of catalysis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UCK1 is a ~30-kDa pyrimidine ribonucleoside kinase of the salvage pathway that uses ATP to phosphorylate uridine and cytidine to their 5'-monophosphates, with no activity toward deoxyribonucleosides or purine ribonucleosides [#0]. It accepts a broad panel of pyrimidine ribonucleoside analogs, including 5-fluorouridine, 5-fluorocytidine, and other modified uridine/cytidine derivatives [#0], as well as the antiviral ribavirin [#4] and N4-hydroxycytidine (NHC, the active form of molnupiravir), which it phosphorylates with ~9-fold lower catalytic efficiency than its isoform UCK2 [#9]; the substrate-bound UCK1-NHC-AMPPNP co-crystal structure defines the structural basis of this catalysis [#9]. Its specificity is restricted to ribonucleosides: UCK1 does not phosphorylate the 2'-deoxy/2'-fluoro/2'-methyl analogs PSI-6130 or 2'-C-methyl-cytidine [#2, #3]. Beyond direct catalysis, UCK1 functions as a negative regulator of pyrimidine salvage by localizing to the nucleus and physically binding cytosolic UCK2, driving UCK2 nuclear relocalization, impairing salvage flux, and protecting cells from cytidine-analog toxicity [#5, #6]. UCK1 abundance is controlled by ubiquitin-dependent turnover: the E3 ligase KLHL2 polyubiquitinates UCK1 at K81 to promote its degradation, opposed by the deubiquitinase USP28, with ATM signaling shifting this balance toward UCK1 destabilization [#7]. Functionally, UCK1 activity is required for activation of the hypomethylating agent azacitidine in leukemia cells [#12].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established the core biochemical identity of UCK1 as a pyrimidine ribonucleoside monophosphate kinase, answering what reaction the gene product catalyzes and on which substrates.\",\n      \"evidence\": \"Recombinant expression in E. coli and in vitro activity assays against a 28-analog panel\",\n      \"pmids\": [\"11306702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic parameters for natural substrates not quantified\", \"Physiological vs. analog substrate preference in cells not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the boundary of UCK1 substrate specificity by showing it cannot phosphorylate 2'-modified deoxy analogs, distinguishing it from dCK and clarifying which antiviral prodrugs it can activate.\",\n      \"evidence\": \"Coupled spectrophotometric kinase assay with purified recombinant UCK1 on PSI-6130; extended to 2'-C-methyl-cytidine in 2009\",\n      \"pmids\": [\"17101674\", \"19883694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural reason for exclusion of 2'-modified sugars not addressed at the time\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended the analog substrate repertoire by identifying ribavirin as a UCK1 substrate, relevant to antiviral prodrug activation.\",\n      \"evidence\": \"In vitro phosphorylation assay with human UCK1\",\n      \"pmids\": [\"18080217\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method, single lab\", \"Relative contribution of UCK1 vs other kinases to ribavirin activation in cells unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a non-catalytic regulatory role: UCK1 localizes to the nucleus and binds UCK2 to relocalize and inhibit it, recasting UCK1 as a dominant-negative modulator of salvage rather than purely a catalyst.\",\n      \"evidence\": \"GFP localization microscopy and his-tag pull-down; UCK1 overexpression with metabolic and cytotoxicity phenotypes in neuroblastoma cells\",\n      \"pmids\": [\"27239701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal/endogenous interaction validation limited\", \"Structural basis of UCK1-UCK2 association not defined\", \"Generality beyond neuroblastoma cells untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined how UCK1 protein levels are set, identifying a writer (KLHL2), eraser (USP28), specific ubiquitination site (K81), and ATM-linked signaling control of its stability.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, K81 site mutagenesis, and AML cell/murine models\",\n      \"pmids\": [\"31938050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream metabolic consequence of UCK1 turnover not quantified\", \"How ATM-driven destabilization affects salvage flux in vivo unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed UCK1 within a nutrient-sensing regulatory axis by showing it modulates mTORC1-/CTLH-WDR26-dependent turnover of UCK2 through control of UCK2 localization.\",\n      \"evidence\": \"mTORC1 inhibition with protein stability and localization assays\",\n      \"pmids\": [\"39808525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"UCK1 role described as modulatory, not mechanistically dissected\", \"Single lab\", \"Direct effect of UCK1 on CTLH complex not shown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided atomic-resolution understanding of catalysis and quantified UCK1's contribution to molnupiravir activation relative to UCK2.\",\n      \"evidence\": \"Enzyme kinetics with recombinant UCK1/UCK2, UCK1-NHC-AMPPNP co-crystal structure, and siRNA knockdown in SARS-CoV-2-infected cells\",\n      \"pmids\": [\"42213735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of UCK1 vs UCK2 to molnupiravir efficacy not established\", \"Structure of UCK1 bound to natural substrates not reported\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UCK1's catalytic and dominant-negative regulatory functions are balanced under physiological and stress conditions, and the mechanistic basis of its reported role in EGFR signaling, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"EGFR-signaling requirement rests on a single siRNA screen with no mechanistic follow-up\", \"Integration of nuclear sequestration and direct catalysis in normal physiology unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UCK2\", \"KLHL2\", \"USP28\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}