{"gene":"CLK3","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1998,"finding":"Human CLK3 (hClk3) is a nuclear dual-specificity kinase (phosphorylates serine/threonine and tyrosine) that causes redistribution of SR proteins from nuclear speckles and regulates alternative splicing of a model pre-mRNA substrate in vivo. The catalytically inactive truncated isoform hClk3(T) colocalizes with SR proteins in nuclear speckles.","method":"Transfection/overexpression of wild-type and truncated isoforms, nuclear localization imaging, in vivo splicing reporter assays, dual-specificity kinase activity assays","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (kinase activity assay, localization imaging, in vivo splicing reporter), replicated across CLK family members","pmids":["9637771"],"is_preprint":false},{"year":1996,"finding":"Rat CLK3 (99% identical to human CLK3) GST-fusion protein catalyzes autophosphorylation but fails to phosphorylate exogenous substrates histone or casein in vitro.","method":"GST-fusion protein in vitro kinase assay with histone and casein as substrates","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro kinase assay, single lab, single method","pmids":["8679717"],"is_preprint":false},{"year":1999,"finding":"CLK3 protein in mature mouse spermatozoa is localized to the acrosome and tail (cytoplasmic, not nuclear). Following the acrosome reaction, CLK3 is expelled from sperm and inactivated, likely by proteolytic degradation by acrosomal proteases.","method":"Immunofluorescence localization in testis/epididymis sections, fractionation, Western blot, acrosome reaction assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence plus functional consequence (expulsion/inactivation post-acrosome reaction), single lab","pmids":["10585269"],"is_preprint":false},{"year":2020,"finding":"CLK3 directly phosphorylates USP13 at tyrosine 708, which promotes USP13 binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and stabilizing c-Myc to activate transcription of purine metabolic genes. A CCA-associated gain-of-function mutation CLK3-Q607R enhances USP13-Y708 phosphorylation and c-Myc activity. c-Myc in turn transcriptionally upregulates CLK3.","method":"In vitro kinase assay, mass spectrometry, co-immunoprecipitation, phospho-site mutagenesis, ubiquitination assay, transcriptional reporter assays, CLK3-Q607R mutant analysis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct phosphorylation demonstrated by in vitro kinase assay with site-specific mutagenesis, co-IP for binding, ubiquitination assay, multiple orthogonal methods in single study","pmids":["32453420"],"is_preprint":false},{"year":2018,"finding":"CLK3 regulates alternative splicing of HMGA2 pre-mRNA in human hematopoietic stem cells, generating an isoform that escapes let-7 miRNA-mediated suppression, thereby preserving HMGA2 function during HSC development.","method":"High-throughput RNA-seq/splicing analysis, CLK3 loss-of-function and gain-of-function experiments in HSCs, miRNA target validation","journal":"Cell stem cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic perturbation (CLK3 manipulation) with defined splicing phenotype, multiple genomic methods, single lab","pmids":["29625070"],"is_preprint":false},{"year":2024,"finding":"CLK3 stabilizes JAK2 protein levels to positively influence the IL-6/STAT3 signaling pathway, promoting colorectal cancer cell proliferation and ATP production.","method":"CLK3 overexpression and knockdown experiments, Western blot for JAK2 stability, in vitro proliferation assays, in vivo xenograft models","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic claim (JAK2 stabilization) based on Western blot alone without direct biochemical demonstration of the stabilization mechanism","pmids":["38885806"],"is_preprint":false},{"year":2024,"finding":"CLK3 promotes colorectal cancer cell proliferation through activation of c-MYC signaling, with CLK3 loss reducing tumor growth and c-MYC expression in vivo.","method":"CLK3 knockdown/overexpression, in vitro proliferation assays, in vivo xenograft, Western blot for c-MYC","journal":"Cell division","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic inference (CLK3→c-MYC) based on expression changes without direct biochemical evidence","pmids":["39289754"],"is_preprint":false},{"year":2022,"finding":"MFAP2 depletion induces autophagic degradation of CLK3, and the invasive capacity of colorectal cancer cells promoted by MFAP2 is dependent on CLK3 (epistasis established by MFAP2 siRNA + CLK3 plasmid co-transfection rescue).","method":"Mass spectrometry identification of CLK3 as downstream target of MFAP2, siRNA knockdown, CLK3 overexpression rescue, transwell invasion assays, in vivo peritoneal metastasis model","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via co-transfection rescue, MS identification of interaction, in vivo and in vitro validation, single lab","pmids":["36583532"],"is_preprint":false},{"year":2021,"finding":"Xenopus Clk3 is required for formation of neural tissue during early embryogenesis; clk3 knockdown reduces neural marker gene expression, and overexpression increases neural marker gene expression in ectodermal explants.","method":"Morpholino knockdown, mRNA overexpression, ectodermal explant assays, in situ hybridization for neural markers in Xenopus embryos","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined developmental phenotype, ortholog in vertebrate model organism, single lab","pmids":["34146908"],"is_preprint":false},{"year":2025,"finding":"DDX41 regulates Clk3 transcript levels and elevates CLK3 during myeloid differentiation. Loss-of-function analysis showed that DDX41-regulated splicing commonly, but not always, requires CLK3, establishing CLK3 as a downstream effector of DDX41 in a splicing regulatory hierarchy in myeloid progenitors.","method":"Ddx41+/- genetic model, global RNA-seq splicing analysis, CLK3 loss-of-function, transcript-level analysis during myeloid differentiation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with genome-wide splicing readout, multiple conditions tested, single lab","pmids":["41193510"],"is_preprint":false},{"year":2025,"finding":"Polyphosphate (polyP) acts as a physiological inhibitor of CLK3 kinase, preventing CLK3-mediated phosphorylation of SR proteins, thereby maintaining nuclear speckle stability. PolyP depletion releases splicing factors into the nucleoplasm and alters splicing patterns.","method":"BAR proximity biotinylation, CLK3 kinase inhibition assay with polyP, RNA-seq splicing analysis upon polyP depletion, SR protein phosphorylation assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct kinase inhibition assay with polyP, functional splicing and localization consequences demonstrated, but preprint and single lab","pmids":[],"is_preprint":true},{"year":2025,"finding":"Structural analysis identified lysine 241, present only in CLK3 among the four CLK isoforms, as a residue that can be exploited for selective inhibitor design; a derivative (VS-77) with IC50 = 0.3 μM for CLK3 was developed based on this structural insight.","method":"Structural analysis of CLK active sites, medicinal chemistry, in vitro kinase inhibition assay (IC50 determination)","journal":"Beilstein journal of organic chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro kinase assay with IC50 measurement and structure-guided design, single lab, no mutagenesis validation of K241 role","pmids":["41158282"],"is_preprint":false},{"year":2019,"finding":"miR-144 directly targets the CLK3 3′UTR to repress CLK3 expression; CLK3 overexpression partially rescues the inhibitory effects of miR-144 on HCC cell growth and metastasis and restores Wnt/β-catenin signaling suppressed by miR-144.","method":"Luciferase reporter assay for miR-144 targeting of CLK3 3′UTR, Western blot, RT-qPCR, CLK3 overexpression rescue experiments, in vitro and in vivo tumor assays","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct 3′UTR reporter assay plus rescue epistasis experiment, multiple assays, single lab","pmids":["31807004"],"is_preprint":false}],"current_model":"CLK3 is a nuclear dual-specificity kinase (serine/threonine and tyrosine) that phosphorylates SR splicing factors to drive their redistribution from nuclear speckles and regulate alternative pre-mRNA splicing; it also directly phosphorylates USP13 at Y708 to stabilize c-Myc and reprogram purine metabolism, acts downstream of DDX41 to control transcript diversity in myeloid progenitors, is itself regulated by polyphosphate (a physiological inhibitor) and by miR-144-mediated translational repression, and in mature spermatozoa localizes to the acrosome and tail where it is expelled and proteolytically inactivated during the acrosome reaction."},"narrative":{"mechanistic_narrative":"CLK3 is a nuclear dual-specificity (serine/threonine and tyrosine) kinase that phosphorylates SR splicing factors to drive their redistribution from nuclear speckles and thereby regulate alternative pre-mRNA splicing [PMID:9637771]. This splicing-regulatory activity operates in physiological programs: in hematopoietic stem cells CLK3 directs alternative splicing of HMGA2 to generate an isoform that escapes let-7 suppression [PMID:29625070], and in myeloid progenitors CLK3 acts as a downstream effector of DDX41 in a splicing regulatory hierarchy, with DDX41 elevating CLK3 during differentiation [PMID:41193510]. Its catalytic output is constrained physiologically by polyphosphate, which inhibits CLK3 to preserve nuclear speckle stability. Beyond splicing, CLK3 directly phosphorylates USP13 at tyrosine 708, promoting USP13–c-Myc binding that blocks Fbxl14-mediated c-Myc ubiquitination, stabilizing c-Myc and reprogramming purine metabolism; a gain-of-function CLK3-Q607R mutation enhances this axis [PMID:32453420]. CLK3 also functions in vertebrate neural induction during early embryogenesis [PMID:34146908]. Distinct from its nuclear roles, CLK3 in mature spermatozoa localizes cytoplasmically to the acrosome and tail and is expelled and proteolytically inactivated during the acrosome reaction [PMID:10585269]. CLK3 abundance is further controlled post-transcriptionally by miR-144 targeting of its 3'UTR [PMID:31807004]. A CLK3-selective inhibitor (VS-77) has been developed exploiting lysine 241, unique to CLK3 among the four CLK isoforms [PMID:41158282].","teleology":[{"year":1996,"claim":"Established the basic catalytic behavior of CLK3 by showing the kinase autophosphorylates but is selective toward substrates, foreshadowing a dedicated rather than promiscuous activity.","evidence":"GST-fusion in vitro kinase assay on rat CLK3 with histone and casein as candidate substrates","pmids":["8679717"],"confidence":"Medium","gaps":["No physiological substrate identified in this assay","Autophosphorylation site and regulatory consequence not defined"]},{"year":1998,"claim":"Defined CLK3 as a nuclear dual-specificity kinase that controls SR protein distribution and alternative splicing, assigning it a concrete role in pre-mRNA processing.","evidence":"Overexpression of wild-type and catalytically inactive truncated isoforms with nuclear localization imaging and in vivo splicing reporter assays","pmids":["9637771"],"confidence":"High","gaps":["Specific SR protein substrates and phospho-sites not enumerated","Endogenous splicing targets not identified"]},{"year":1999,"claim":"Revealed a non-nuclear, cell-type-specific deployment of CLK3 in sperm, where it is positioned at the acrosome/tail and cleared during the acrosome reaction, indicating regulated spatial inactivation.","evidence":"Immunofluorescence in testis/epididymis sections, fractionation, Western blot, and acrosome reaction assays in mouse spermatozoa","pmids":["10585269"],"confidence":"Medium","gaps":["Substrates in sperm unknown","Functional consequence of expulsion for fertilization not established","Protease responsible not identified"]},{"year":2018,"claim":"Connected CLK3 splicing activity to stem cell biology by showing it generates an HMGA2 isoform resistant to let-7 repression, demonstrating a defined physiological splicing target.","evidence":"RNA-seq splicing analysis with CLK3 loss- and gain-of-function in human HSCs and miRNA target validation","pmids":["29625070"],"confidence":"Medium","gaps":["Direct phosphorylation event driving the splice switch not mapped","Single lab"]},{"year":2019,"claim":"Identified post-transcriptional control of CLK3 by miR-144 and linked CLK3 to Wnt/β-catenin signaling in hepatocellular carcinoma.","evidence":"Luciferase 3'UTR reporter assay, Western blot, RT-qPCR, and CLK3 overexpression rescue in HCC models","pmids":["31807004"],"confidence":"Medium","gaps":["Mechanism linking CLK3 kinase activity to Wnt signaling not biochemically defined","Direct CLK3 substrates in this context unknown"]},{"year":2020,"claim":"Uncovered a kinase-substrate axis beyond splicing: CLK3 phosphorylates USP13 at Y708 to stabilize c-Myc and reprogram purine metabolism, and identified a disease-associated gain-of-function mutation.","evidence":"In vitro kinase assay, mass spectrometry, co-IP, phospho-site mutagenesis, ubiquitination and reporter assays, and CLK3-Q607R mutant analysis in cholangiocarcinoma","pmids":["32453420"],"confidence":"High","gaps":["Whether the USP13/c-Myc axis operates in normal tissues not addressed","Relationship to CLK3 splicing function unclear"]},{"year":2021,"claim":"Extended CLK3 function to development, showing the ortholog is required for neural tissue formation in early vertebrate embryos.","evidence":"Morpholino knockdown, mRNA overexpression, and ectodermal explant assays with neural marker in situ hybridization in Xenopus","pmids":["34146908"],"confidence":"Medium","gaps":["Molecular substrates driving neural induction not identified","Link to splicing vs. other activity unresolved"]},{"year":2022,"claim":"Placed CLK3 in a cancer signaling hierarchy by showing MFAP2 stabilizes CLK3 against autophagic degradation and that CLK3 is required for MFAP2-driven invasion.","evidence":"Mass spectrometry, siRNA knockdown with CLK3 overexpression rescue, transwell invasion, and in vivo peritoneal metastasis model in colorectal cancer","pmids":["36583532"],"confidence":"Medium","gaps":["Mechanism of CLK3 turnover by autophagy not detailed","Direct CLK3 effectors in invasion unknown"]},{"year":2024,"claim":"Associated CLK3 with colorectal cancer proliferation through proposed JAK2/STAT3 and c-MYC routes, though mechanistic detail rests on expression and stability readouts.","evidence":"CLK3 knockdown/overexpression, Western blot, in vitro proliferation, and xenograft assays","pmids":["38885806","39289754"],"confidence":"Low","gaps":["JAK2 stabilization mechanism shown by Western blot alone without direct biochemistry","CLK3→c-MYC link inferred from expression changes, not direct phosphorylation"]},{"year":2025,"claim":"Identified physiological regulation of CLK3 catalysis by polyphosphate and embedded CLK3 in a DDX41-governed splicing hierarchy, refining how CLK3 splicing output is controlled.","evidence":"Proximity biotinylation, polyP kinase inhibition and SR phosphorylation assays with RNA-seq (preprint, bioRxiv); Ddx41+/- model with global RNA-seq splicing and CLK3 loss-of-function in myeloid progenitors","pmids":["41193510"],"confidence":"Medium","gaps":["polyP findings are preprint and single lab","How DDX41 elevates CLK3 transcript mechanistically not defined","Which splicing events require CLK3 vs. not is incompletely mapped"]},{"year":2025,"claim":"Enabled isoform-selective targeting by identifying CLK3-unique lysine 241 and developing a selective inhibitor, providing a chemical tool to dissect CLK3-specific functions.","evidence":"Structure-guided active-site analysis, medicinal chemistry, and in vitro IC50 determination for VS-77","pmids":["41158282"],"confidence":"Medium","gaps":["Role of K241 not validated by mutagenesis","Cellular selectivity and efficacy not established"]},{"year":null,"claim":"It remains unresolved how CLK3's splicing-regulatory activity, its USP13/c-Myc phosphorylation axis, and its developmental and sperm roles are integrated, and which endogenous substrates govern each context.","evidence":"No single study reconciles the distinct molecular roles assigned to CLK3 across tissues","pmids":[],"confidence":"Low","gaps":["No unified substrate map across contexts","Determinants of nuclear vs. cytoplasmic deployment unknown","Physiological vs. oncogenic functions not clearly separated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,4,9]}],"complexes":[],"partners":["USP13"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49761","full_name":"Dual specificity protein kinase CLK3","aliases":["CDC-like kinase 3"],"length_aa":490,"mass_kda":58.6,"function":"Dual specificity kinase acting on both serine/threonine and tyrosine-containing substrates. Phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex. May be a constituent of a network of regulatory mechanisms that enable SR proteins to control RNA splicing and can cause redistribution of SR proteins from speckles to a diffuse nucleoplasmic distribution. Phosphorylates SRSF1 and SRSF3. Regulates the alternative splicing of tissue factor (F3) pre-mRNA in endothelial cells","subcellular_location":"Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/P49761/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CLK3","classification":"Not Classified","n_dependent_lines":21,"n_total_lines":1208,"dependency_fraction":0.0173841059602649},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000179335","cell_line_id":"CID001157","localizations":[{"compartment":"chromatin","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"CDC37","stoichiometry":4.0},{"gene":"CARM1","stoichiometry":4.0},{"gene":"ARFGAP2","stoichiometry":0.2},{"gene":"PSME3","stoichiometry":0.2},{"gene":"TNPO3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001157","total_profiled":1310},"omim":[{"mim_id":"602990","title":"CDC-LIKE KINASE 3; CLK3","url":"https://www.omim.org/entry/602990"},{"mim_id":"602989","title":"CDC-LIKE KINASE 2; CLK2","url":"https://www.omim.org/entry/602989"},{"mim_id":"601683","title":"COENZYME Q7, HYDROXYLASE; COQ7","url":"https://www.omim.org/entry/601683"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Intermediate filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CLK3"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P49761","domains":[{"cath_id":"3.30.200.20","chopping":"299-363_370-386","consensus_level":"high","plddt":94.6849,"start":299,"end":386},{"cath_id":"1.10.510.10","chopping":"390-629","consensus_level":"high","plddt":96.3635,"start":390,"end":629}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49761","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49761-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49761-F1-predicted_aligned_error_v6.png","plddt_mean":79.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CLK3","jax_strain_url":"https://www.jax.org/strain/search?query=CLK3"},"sequence":{"accession":"P49761","fasta_url":"https://rest.uniprot.org/uniprotkb/P49761.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49761/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49761"}},"corpus_meta":[{"pmid":"9637771","id":"PMC_9637771","title":"The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing.","date":"1998","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/9637771","citation_count":127,"is_preprint":false},{"pmid":"32453420","id":"PMC_32453420","title":"Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.","date":"2020","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32453420","citation_count":55,"is_preprint":false},{"pmid":"29625070","id":"PMC_29625070","title":"A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.","date":"2018","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/29625070","citation_count":49,"is_preprint":false},{"pmid":"36583532","id":"PMC_36583532","title":"MFAP2, upregulated by m1A methylation, promotes colorectal cancer invasiveness via CLK3.","date":"2022","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36583532","citation_count":32,"is_preprint":false},{"pmid":"10585269","id":"PMC_10585269","title":"The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa.","date":"1999","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/10585269","citation_count":27,"is_preprint":false},{"pmid":"31807004","id":"PMC_31807004","title":"CLK3 Is A Direct Target Of miR-144 And Contributes To Aggressive Progression In Hepatocellular Carcinoma.","date":"2019","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/31807004","citation_count":20,"is_preprint":false},{"pmid":"8679717","id":"PMC_8679717","title":"cDNA cloning and characterization of rat Clk3, a LAMMER kinase predominately expressed in testis.","date":"1996","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/8679717","citation_count":15,"is_preprint":false},{"pmid":"34146908","id":"PMC_34146908","title":"The dual-specificity protein kinase Clk3 is essential for Xenopus neural development.","date":"2021","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/34146908","citation_count":7,"is_preprint":false},{"pmid":"39289754","id":"PMC_39289754","title":"CLK3 promotes tumor proliferation by activating MYC signaling.","date":"2024","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/39289754","citation_count":3,"is_preprint":false},{"pmid":"38885806","id":"PMC_38885806","title":"CLK3 positively promoted colorectal cancer proliferation by activating IL-6/STAT3 signaling.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/38885806","citation_count":2,"is_preprint":false},{"pmid":"41193510","id":"PMC_41193510","title":"Oncogenic DEAD-box ATPase DDX41 establishes transcript ensembles via CLK3-dependent and -independent mechanisms.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41193510","citation_count":1,"is_preprint":false},{"pmid":"41158282","id":"PMC_41158282","title":"Research towards selective inhibition of the CLK3 kinase.","date":"2025","source":"Beilstein journal of organic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41158282","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.07.680601","title":"Inhibition of  <i>Pf</i>  CLK3 a master regulator of malaria parasite RNA-splicing provides the basis for a radical cure for malaria","date":"2025-10-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.07.680601","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.15.633116","title":"Nuclear Speckle Dynamics are Controlled by Polyphosphate Inhibition of CLK Proteins","date":"2025-01-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.15.633116","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7730,"output_tokens":3096,"usd":0.034815,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10512,"output_tokens":3648,"usd":0.07188,"stage2_stop_reason":"end_turn"},"total_usd":0.106695,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CLK3 (hClk3) is a nuclear dual-specificity kinase (phosphorylates serine/threonine and tyrosine) that causes redistribution of SR proteins from nuclear speckles and regulates alternative splicing of a model pre-mRNA substrate in vivo. The catalytically inactive truncated isoform hClk3(T) colocalizes with SR proteins in nuclear speckles.\",\n      \"method\": \"Transfection/overexpression of wild-type and truncated isoforms, nuclear localization imaging, in vivo splicing reporter assays, dual-specificity kinase activity assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (kinase activity assay, localization imaging, in vivo splicing reporter), replicated across CLK family members\",\n      \"pmids\": [\"9637771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Rat CLK3 (99% identical to human CLK3) GST-fusion protein catalyzes autophosphorylation but fails to phosphorylate exogenous substrates histone or casein in vitro.\",\n      \"method\": \"GST-fusion protein in vitro kinase assay with histone and casein as substrates\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro kinase assay, single lab, single method\",\n      \"pmids\": [\"8679717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CLK3 protein in mature mouse spermatozoa is localized to the acrosome and tail (cytoplasmic, not nuclear). Following the acrosome reaction, CLK3 is expelled from sperm and inactivated, likely by proteolytic degradation by acrosomal proteases.\",\n      \"method\": \"Immunofluorescence localization in testis/epididymis sections, fractionation, Western blot, acrosome reaction assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunofluorescence plus functional consequence (expulsion/inactivation post-acrosome reaction), single lab\",\n      \"pmids\": [\"10585269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLK3 directly phosphorylates USP13 at tyrosine 708, which promotes USP13 binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and stabilizing c-Myc to activate transcription of purine metabolic genes. A CCA-associated gain-of-function mutation CLK3-Q607R enhances USP13-Y708 phosphorylation and c-Myc activity. c-Myc in turn transcriptionally upregulates CLK3.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry, co-immunoprecipitation, phospho-site mutagenesis, ubiquitination assay, transcriptional reporter assays, CLK3-Q607R mutant analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct phosphorylation demonstrated by in vitro kinase assay with site-specific mutagenesis, co-IP for binding, ubiquitination assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"32453420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CLK3 regulates alternative splicing of HMGA2 pre-mRNA in human hematopoietic stem cells, generating an isoform that escapes let-7 miRNA-mediated suppression, thereby preserving HMGA2 function during HSC development.\",\n      \"method\": \"High-throughput RNA-seq/splicing analysis, CLK3 loss-of-function and gain-of-function experiments in HSCs, miRNA target validation\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic perturbation (CLK3 manipulation) with defined splicing phenotype, multiple genomic methods, single lab\",\n      \"pmids\": [\"29625070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CLK3 stabilizes JAK2 protein levels to positively influence the IL-6/STAT3 signaling pathway, promoting colorectal cancer cell proliferation and ATP production.\",\n      \"method\": \"CLK3 overexpression and knockdown experiments, Western blot for JAK2 stability, in vitro proliferation assays, in vivo xenograft models\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic claim (JAK2 stabilization) based on Western blot alone without direct biochemical demonstration of the stabilization mechanism\",\n      \"pmids\": [\"38885806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CLK3 promotes colorectal cancer cell proliferation through activation of c-MYC signaling, with CLK3 loss reducing tumor growth and c-MYC expression in vivo.\",\n      \"method\": \"CLK3 knockdown/overexpression, in vitro proliferation assays, in vivo xenograft, Western blot for c-MYC\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic inference (CLK3→c-MYC) based on expression changes without direct biochemical evidence\",\n      \"pmids\": [\"39289754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MFAP2 depletion induces autophagic degradation of CLK3, and the invasive capacity of colorectal cancer cells promoted by MFAP2 is dependent on CLK3 (epistasis established by MFAP2 siRNA + CLK3 plasmid co-transfection rescue).\",\n      \"method\": \"Mass spectrometry identification of CLK3 as downstream target of MFAP2, siRNA knockdown, CLK3 overexpression rescue, transwell invasion assays, in vivo peritoneal metastasis model\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via co-transfection rescue, MS identification of interaction, in vivo and in vitro validation, single lab\",\n      \"pmids\": [\"36583532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Xenopus Clk3 is required for formation of neural tissue during early embryogenesis; clk3 knockdown reduces neural marker gene expression, and overexpression increases neural marker gene expression in ectodermal explants.\",\n      \"method\": \"Morpholino knockdown, mRNA overexpression, ectodermal explant assays, in situ hybridization for neural markers in Xenopus embryos\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined developmental phenotype, ortholog in vertebrate model organism, single lab\",\n      \"pmids\": [\"34146908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX41 regulates Clk3 transcript levels and elevates CLK3 during myeloid differentiation. Loss-of-function analysis showed that DDX41-regulated splicing commonly, but not always, requires CLK3, establishing CLK3 as a downstream effector of DDX41 in a splicing regulatory hierarchy in myeloid progenitors.\",\n      \"method\": \"Ddx41+/- genetic model, global RNA-seq splicing analysis, CLK3 loss-of-function, transcript-level analysis during myeloid differentiation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with genome-wide splicing readout, multiple conditions tested, single lab\",\n      \"pmids\": [\"41193510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Polyphosphate (polyP) acts as a physiological inhibitor of CLK3 kinase, preventing CLK3-mediated phosphorylation of SR proteins, thereby maintaining nuclear speckle stability. PolyP depletion releases splicing factors into the nucleoplasm and alters splicing patterns.\",\n      \"method\": \"BAR proximity biotinylation, CLK3 kinase inhibition assay with polyP, RNA-seq splicing analysis upon polyP depletion, SR protein phosphorylation assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct kinase inhibition assay with polyP, functional splicing and localization consequences demonstrated, but preprint and single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Structural analysis identified lysine 241, present only in CLK3 among the four CLK isoforms, as a residue that can be exploited for selective inhibitor design; a derivative (VS-77) with IC50 = 0.3 μM for CLK3 was developed based on this structural insight.\",\n      \"method\": \"Structural analysis of CLK active sites, medicinal chemistry, in vitro kinase inhibition assay (IC50 determination)\",\n      \"journal\": \"Beilstein journal of organic chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro kinase assay with IC50 measurement and structure-guided design, single lab, no mutagenesis validation of K241 role\",\n      \"pmids\": [\"41158282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-144 directly targets the CLK3 3′UTR to repress CLK3 expression; CLK3 overexpression partially rescues the inhibitory effects of miR-144 on HCC cell growth and metastasis and restores Wnt/β-catenin signaling suppressed by miR-144.\",\n      \"method\": \"Luciferase reporter assay for miR-144 targeting of CLK3 3′UTR, Western blot, RT-qPCR, CLK3 overexpression rescue experiments, in vitro and in vivo tumor assays\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct 3′UTR reporter assay plus rescue epistasis experiment, multiple assays, single lab\",\n      \"pmids\": [\"31807004\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CLK3 is a nuclear dual-specificity kinase (serine/threonine and tyrosine) that phosphorylates SR splicing factors to drive their redistribution from nuclear speckles and regulate alternative pre-mRNA splicing; it also directly phosphorylates USP13 at Y708 to stabilize c-Myc and reprogram purine metabolism, acts downstream of DDX41 to control transcript diversity in myeloid progenitors, is itself regulated by polyphosphate (a physiological inhibitor) and by miR-144-mediated translational repression, and in mature spermatozoa localizes to the acrosome and tail where it is expelled and proteolytically inactivated during the acrosome reaction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLK3 is a nuclear dual-specificity (serine/threonine and tyrosine) kinase that phosphorylates SR splicing factors to drive their redistribution from nuclear speckles and thereby regulate alternative pre-mRNA splicing [#0]. This splicing-regulatory activity operates in physiological programs: in hematopoietic stem cells CLK3 directs alternative splicing of HMGA2 to generate an isoform that escapes let-7 suppression [#4], and in myeloid progenitors CLK3 acts as a downstream effector of DDX41 in a splicing regulatory hierarchy, with DDX41 elevating CLK3 during differentiation [#9]. Its catalytic output is constrained physiologically by polyphosphate, which inhibits CLK3 to preserve nuclear speckle stability [#10]. Beyond splicing, CLK3 directly phosphorylates USP13 at tyrosine 708, promoting USP13–c-Myc binding that blocks Fbxl14-mediated c-Myc ubiquitination, stabilizing c-Myc and reprogramming purine metabolism; a gain-of-function CLK3-Q607R mutation enhances this axis [#3]. CLK3 also functions in vertebrate neural induction during early embryogenesis [#8]. Distinct from its nuclear roles, CLK3 in mature spermatozoa localizes cytoplasmically to the acrosome and tail and is expelled and proteolytically inactivated during the acrosome reaction [#2]. CLK3 abundance is further controlled post-transcriptionally by miR-144 targeting of its 3'UTR [#12]. A CLK3-selective inhibitor (VS-77) has been developed exploiting lysine 241, unique to CLK3 among the four CLK isoforms [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the basic catalytic behavior of CLK3 by showing the kinase autophosphorylates but is selective toward substrates, foreshadowing a dedicated rather than promiscuous activity.\",\n      \"evidence\": \"GST-fusion in vitro kinase assay on rat CLK3 with histone and casein as candidate substrates\",\n      \"pmids\": [\"8679717\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No physiological substrate identified in this assay\", \"Autophosphorylation site and regulatory consequence not defined\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined CLK3 as a nuclear dual-specificity kinase that controls SR protein distribution and alternative splicing, assigning it a concrete role in pre-mRNA processing.\",\n      \"evidence\": \"Overexpression of wild-type and catalytically inactive truncated isoforms with nuclear localization imaging and in vivo splicing reporter assays\",\n      \"pmids\": [\"9637771\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific SR protein substrates and phospho-sites not enumerated\", \"Endogenous splicing targets not identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Revealed a non-nuclear, cell-type-specific deployment of CLK3 in sperm, where it is positioned at the acrosome/tail and cleared during the acrosome reaction, indicating regulated spatial inactivation.\",\n      \"evidence\": \"Immunofluorescence in testis/epididymis sections, fractionation, Western blot, and acrosome reaction assays in mouse spermatozoa\",\n      \"pmids\": [\"10585269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrates in sperm unknown\", \"Functional consequence of expulsion for fertilization not established\", \"Protease responsible not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected CLK3 splicing activity to stem cell biology by showing it generates an HMGA2 isoform resistant to let-7 repression, demonstrating a defined physiological splicing target.\",\n      \"evidence\": \"RNA-seq splicing analysis with CLK3 loss- and gain-of-function in human HSCs and miRNA target validation\",\n      \"pmids\": [\"29625070\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation event driving the splice switch not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified post-transcriptional control of CLK3 by miR-144 and linked CLK3 to Wnt/β-catenin signaling in hepatocellular carcinoma.\",\n      \"evidence\": \"Luciferase 3'UTR reporter assay, Western blot, RT-qPCR, and CLK3 overexpression rescue in HCC models\",\n      \"pmids\": [\"31807004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking CLK3 kinase activity to Wnt signaling not biochemically defined\", \"Direct CLK3 substrates in this context unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered a kinase-substrate axis beyond splicing: CLK3 phosphorylates USP13 at Y708 to stabilize c-Myc and reprogram purine metabolism, and identified a disease-associated gain-of-function mutation.\",\n      \"evidence\": \"In vitro kinase assay, mass spectrometry, co-IP, phospho-site mutagenesis, ubiquitination and reporter assays, and CLK3-Q607R mutant analysis in cholangiocarcinoma\",\n      \"pmids\": [\"32453420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the USP13/c-Myc axis operates in normal tissues not addressed\", \"Relationship to CLK3 splicing function unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended CLK3 function to development, showing the ortholog is required for neural tissue formation in early vertebrate embryos.\",\n      \"evidence\": \"Morpholino knockdown, mRNA overexpression, and ectodermal explant assays with neural marker in situ hybridization in Xenopus\",\n      \"pmids\": [\"34146908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrates driving neural induction not identified\", \"Link to splicing vs. other activity unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed CLK3 in a cancer signaling hierarchy by showing MFAP2 stabilizes CLK3 against autophagic degradation and that CLK3 is required for MFAP2-driven invasion.\",\n      \"evidence\": \"Mass spectrometry, siRNA knockdown with CLK3 overexpression rescue, transwell invasion, and in vivo peritoneal metastasis model in colorectal cancer\",\n      \"pmids\": [\"36583532\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of CLK3 turnover by autophagy not detailed\", \"Direct CLK3 effectors in invasion unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Associated CLK3 with colorectal cancer proliferation through proposed JAK2/STAT3 and c-MYC routes, though mechanistic detail rests on expression and stability readouts.\",\n      \"evidence\": \"CLK3 knockdown/overexpression, Western blot, in vitro proliferation, and xenograft assays\",\n      \"pmids\": [\"38885806\", \"39289754\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"JAK2 stabilization mechanism shown by Western blot alone without direct biochemistry\", \"CLK3→c-MYC link inferred from expression changes, not direct phosphorylation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified physiological regulation of CLK3 catalysis by polyphosphate and embedded CLK3 in a DDX41-governed splicing hierarchy, refining how CLK3 splicing output is controlled.\",\n      \"evidence\": \"Proximity biotinylation, polyP kinase inhibition and SR phosphorylation assays with RNA-seq (preprint, bioRxiv); Ddx41+/- model with global RNA-seq splicing and CLK3 loss-of-function in myeloid progenitors\",\n      \"pmids\": [\"41193510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"polyP findings are preprint and single lab\", \"How DDX41 elevates CLK3 transcript mechanistically not defined\", \"Which splicing events require CLK3 vs. not is incompletely mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Enabled isoform-selective targeting by identifying CLK3-unique lysine 241 and developing a selective inhibitor, providing a chemical tool to dissect CLK3-specific functions.\",\n      \"evidence\": \"Structure-guided active-site analysis, medicinal chemistry, and in vitro IC50 determination for VS-77\",\n      \"pmids\": [\"41158282\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Role of K241 not validated by mutagenesis\", \"Cellular selectivity and efficacy not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how CLK3's splicing-regulatory activity, its USP13/c-Myc phosphorylation axis, and its developmental and sperm roles are integrated, and which endogenous substrates govern each context.\",\n      \"evidence\": \"No single study reconciles the distinct molecular roles assigned to CLK3 across tissues\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified substrate map across contexts\", \"Determinants of nuclear vs. cytoplasmic deployment unknown\", \"Physiological vs. oncogenic functions not clearly separated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 4, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"USP13\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}