{"gene":"CENPU","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2006,"finding":"PLK1 phosphorylates PBIP1 (CENPU) at Thr-78, creating a self-tethering phosphopeptide motif that specifically binds the polo-box domain (PBD) of PLK1 (but not PLK2 or PLK3), recruiting PLK1 to interphase and mitotic kinetochores. Later in mitosis, PLK1 also induces PBIP1 degradation in a T78-dependent manner, enabling PLK1 to interact with other kinetochore components required for proper chromosome segregation.","method":"In vitro kinase assays, mutagenesis of T78, PBD-binding assays, live-cell imaging, spindle checkpoint assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphorylation reconstitution combined with mutagenesis and functional rescue, replicated in multiple subsequent studies","pmids":["17081991"],"is_preprint":false},{"year":2005,"finding":"CENP-50 (CENPU) is a constitutive centromere component that colocalizes with CENP-A and CENP-H throughout the cell cycle; its centromere localization depends on CENP-H and CENP-I. CENP-50 physically interacts with the CENP-H/CENP-I complex (shown by co-immunoprecipitation). Loss of CENP-50 causes prolonged mitosis and premature sister chromatid separation upon spindle checkpoint activation, indicating a role in recovery from spindle damage.","method":"Knockout in chicken DT40 cells, co-immunoprecipitation, immunofluorescence","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus clean KO with defined mitotic phenotype; independently confirmed by multiple subsequent studies","pmids":["16287847"],"is_preprint":false},{"year":2010,"finding":"CENPU directly interacts with Hec1 (NDC80) at kinetochores. CENPU itself binds microtubules directly in vitro and displays cooperative microtubule-binding activity with Hec1. Aurora B phosphorylates CENPU, and this phosphorylation reduces kinetochore–microtubule interaction, contributing to Aurora B's error-correction function. shRNA-mediated knockdown of CENPU impairs kinetochore–microtubule attachment.","method":"Co-immunoprecipitation, in vitro microtubule-binding assays, in vitro Aurora B kinase assay, shRNA knockdown with mitotic phenotype analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution of microtubule binding, identification of Aurora B as writer with kinase assay, Co-IP for complex, single lab but multiple orthogonal methods","pmids":["21056971"],"is_preprint":false},{"year":2011,"finding":"PBIP1 (CENPU) directly interacts with CENP-Q, and this interaction is mutually required for their stability and centromere localization. PLK1 forms a ternary complex with PBIP1 and CENP-Q through the self-generated p-T78 motif on PBIP1, and PLK1-dependent phosphorylation of CENP-Q within this complex leads to delocalization of the PBIP1–CENP-Q complex from mitotic centromeres.","method":"Co-immunoprecipitation, in vitro PLK1 kinase assay, mutagenesis, immunofluorescence localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct interaction reconstituted, ternary complex identified, kinase assay with mutagenesis, single lab with multiple orthogonal methods","pmids":["21454580"],"is_preprint":false},{"year":2015,"finding":"PLK1 phosphorylates the CENP-Q subunit of the PBIP1–CENP-Q complex at multiple sites; phosphorylation of nine sites drives delocalization of the complex from kinetochores. Phospho-mimetic (9D/E) mutations prevent localization to interphase prekinetochores, while phospho-null (9A) mutations prolong kinetochore residence. Both mutants impair proper chromosome segregation, demonstrating that timely localization and delocalization of the PBIP1–CENP-Q complex are both critical for mitotic progression. PLK1-dependent delocalization indirectly leads to cytosolic degradation of PBIP1 and CENP-Q.","method":"In vitro PLK1 kinase assay, mutagenesis (9A and 9D/E), chromatin fractionation, immunofluorescence, chromosome segregation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with site-specific mutagenesis and functional validation, single lab with multiple orthogonal methods","pmids":["25670858"],"is_preprint":false},{"year":2020,"finding":"BUB1 (outer kinetochore) and CENP-U (inner kinetochore) are the main PLK1 receptors at kinetochores. Both share a constellation of sequence motifs (a PP2A-docking motif and two PLK1-docking sites). PLK1 recruitment to CENP-U requires priming phosphorylation by CDK1 followed by PLK1 itself. The two PLK1-docking sites on CENP-U promote PLK1 dimerization. This was demonstrated by ectopic localization assays, in vitro reconstitution, and kinetochore localization studies.","method":"Ectopic localization assay, in vitro reconstitution of PLK1–CENP-U interaction, kinetochore localization studies, mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution plus ectopic localization plus mutagenesis, replicated in subsequent independent study (PMID:34551298)","pmids":["33248027"],"is_preprint":false},{"year":2021,"finding":"BUB1 and CENP-U redundantly recruit PLK1 to kinetochores to stabilize kinetochore–microtubule attachments. CENP-U is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex. Depletion of both BUB1 and CENP-U causes chromosome mis-segregation; depletion of either alone is insufficient, demonstrating functional redundancy. Cells depleted of BUB1 or CENP-U are sensitized to PLK1 inhibition but not Aurora B inhibition. Unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B in human cells.","method":"Stable depletion by siRNA/shRNA, epistasis analysis (double depletion), chemical inhibitor sensitization assays, immunofluorescence","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double depletion, chemical-genetic sensitization, consistent with PMID:33248027 from independent lab","pmids":["34551298"],"is_preprint":false},{"year":2004,"finding":"CENPU (MLF1IP) was identified as a protein that specifically associates with MLF1 by yeast two-hybrid analysis and pulldown assays, and colocalizes with MLF1 in both nuclei and cytoplasm. The protein contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, and multiple potential phosphorylation sites.","method":"Yeast two-hybrid, pulldown assay, co-localization by immunofluorescence","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus pulldown from single lab; functional consequence of MLF1 interaction not fully characterized","pmids":["15116101"],"is_preprint":false},{"year":2021,"finding":"In budding yeast, Cdk1 phosphorylation of the CENPU ortholog Ame1 activates phospho-degrons that are recognized by the SCF-Cdc4 E3 ubiquitin ligase complex, targeting Ame1 for proteasomal degradation during M-phase. Binding of the Mtw1 (Mis12) complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from degradation. This mechanism ensures efficient centromere-dependent kinetochore assembly.","method":"Comprehensive phosphorylation analysis of native CCAN subunits, biochemical ubiquitination assays, genetic assays in budding yeast (cdc4 mutants, overexpression toxicity)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — phosphorylation mapping, biochemical reconstitution of degron recognition, genetic suppression/toxicity assays, single lab but multiple orthogonal methods; yeast ortholog","pmids":["34308839"],"is_preprint":false},{"year":2019,"finding":"CENPU promotes angiogenesis in triple-negative breast cancer by inhibiting the ubiquitination and proteasomal degradation of COX-2, leading to increased COX-2-p-ERK-HIF-1α-VEGFA signaling. CENPU knockdown reduced VEGFA production, tube formation by endothelial cells, and tumor microvessel density in xenograft models.","method":"shRNA knockdown, ubiquitination assay, western blot (COX-2 protein stability), endothelial tube formation assay, xenograft mouse model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — ubiquitination assay supports mechanism, single lab, limited orthogonal validation of direct interaction between CENPU and COX-2 degradation machinery","pmids":["31705927"],"is_preprint":false},{"year":2022,"finding":"CENPU physically interacts with the transcription factor E2F6 and promotes its ubiquitin-mediated degradation, thereby de-repressing E2F1 transcription. E2F1 in turn binds the CENPU promoter to increase CENPU transcription, forming a positive feedback loop that accelerates G1/S transition in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation (E2F1 binding to CENPU promoter), western blot, knockdown and overexpression","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP for interaction, ubiquitination assay, ChIP for transcriptional regulation, single lab with multiple methods","pmids":["35844791"],"is_preprint":false},{"year":2020,"finding":"MLF1-IP (CENPU) KO mice die by embryonic day 6.5 due to degeneration of epiblasts, establishing an essential role in early embryonic development. Heterozygous KO mice are viable and fertile with no apparent immune system defects, indicating that ~50% expression is sufficient for normal postnatal development.","method":"Germline knockout mouse (homozygous lethal, heterozygous viable), in situ hybridization, immune phenotyping","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined lethal phenotype; developmental role demonstrated but molecular mechanism in embryo not specified; single lab","pmids":["23724000"],"is_preprint":false},{"year":2025,"finding":"CENPU promotes furin activity by inhibiting lysosomal degradation of furin in triple-negative breast cancer cells. Furin, a precursor-processing enzyme, converts proNGF to NGF, which promotes breast cancer stem cell properties. Co-immunoprecipitation experiments demonstrated association between CENPU, furin, and NGF/proNGF. A furin inhibitor suppressed CENPU-promoted tumor growth in xenograft models.","method":"Co-immunoprecipitation, western blot (furin protein stability), ELISA (NGF), 4D-DIA quantitative proteomics, mammosphere formation, xenograft mouse model","journal":"International journal of molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP without full mechanistic dissection of how CENPU blocks lysosomal furin degradation; novel and not independently replicated","pmids":["41041859"],"is_preprint":false},{"year":2025,"finding":"CENP-U contains a single high-affinity master PLK1-docking motif that is generated by initial CDK1 priming and subsequent PLK1 phosphorylation. Biochemical and biophysical analyses showed this motif forms extensive interactions with multiple pockets on the PBD surface. Evidence did not support a requirement for PBD dimerization in CENP-U-mediated PLK1 docking.","method":"Biochemical binding assays, biophysical measurements, structural modelling","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — biophysical reconstitution with multiple methods, but preprint not yet peer-reviewed and from single lab","pmids":["bio_10.1101_2025.03.28.645803"],"is_preprint":true},{"year":2025,"finding":"In budding yeast, an N-terminal auto-inhibitory segment of Dsn1 (Mis12 complex subunit) occludes binding sites for both CENP-C/Mif2 and CENP-U/Ame1 on the Mis12 complex head domain. Aurora B/Ipl1 phosphorylation of this auto-inhibitory segment would release auto-inhibition and strengthen inner–outer kinetochore connections involving CENP-U/Ame1. This was established by cryo-EM structure combined with biochemical and genetic experiments.","method":"Cryo-EM, biochemical binding assays, genetic assays in budding yeast","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — high-quality cryo-EM with biochemical validation, but preprint not peer-reviewed; finding is for yeast ortholog Ame1","pmids":["bio_10.1101_2025.06.03.657598"],"is_preprint":true}],"current_model":"CENPU (PBIP1/MLF1IP/CENP-50/CENP-U) is a constitutive inner kinetochore scaffold protein that recruits PLK1 to kinetochores via a CDK1-primed, PLK1-amplified phospho-T78 docking motif on CENPU that binds the PLK1 polo-box domain; PLK1 then phosphorylates and delocalizes the CENPU–CENP-Q complex from kinetochores during mitotic progression. CENPU also directly binds microtubules and cooperates with Hec1/NDC80 for kinetochore–microtubule attachment, is phosphorylated by Aurora B to promote error correction, and is localized to the inner kinetochore through the CENP-O complex (CENP-P/Q). In budding yeast, the Ame1 ortholog is additionally regulated by CDK1-activated phospho-degrons recognized by SCF-Cdc4, with kinetochore-bound Ame1 protected from degradation by Mis12 complex shielding."},"narrative":{"mechanistic_narrative":"CENPU (PBIP1/MLF1IP/CENP-50/CENP-U) is a constitutive inner-kinetochore protein of the CENP-O/CCAN class that couples centromere architecture to mitotic kinase signaling and kinetochore–microtubule attachment [PMID:16287847, PMID:34551298]. Its centromere localization depends on CENP-H/CENP-I and on the CENP-P/CENP-Q subunits of the CENP-O complex, and CENPU loss causes prolonged mitosis and premature sister chromatid separation under spindle-checkpoint stress [PMID:16287847, PMID:34551298]. CENPU forms a mutually stabilizing complex with CENP-Q, binds microtubules directly, and cooperates with Hec1/NDC80 to support kinetochore–microtubule attachment, an interaction tuned by Aurora B phosphorylation that drives error correction [PMID:21056971, PMID:21454580]. CENPU is a principal kinetochore receptor for PLK1: CDK1 priming followed by PLK1 self-phosphorylation generates a phospho-Thr78 docking motif that binds the PLK1 polo-box domain, recruiting PLK1 to interphase and mitotic kinetochores, where it acts redundantly with BUB1 [PMID:17081991, PMID:33248027, PMID:34551298]. PLK1 then phosphorylates CENP-Q within the ternary complex at multiple sites, delocalizing the CENPU–CENP-Q complex and driving its degradation, with both timely localization and removal being required for faithful chromosome segregation [PMID:21454580, PMID:25670858]. Genetic ablation in mice is embryonic-lethal by E6.5 from epiblast degeneration, establishing an essential developmental role [PMID:23724000]. Beyond its mitotic function, CENPU has been implicated in cancer through stabilization of partner proteins, including E2F6-dependent control of the E2F1–CENPU feedback loop in hepatocellular carcinoma and COX-2 stabilization driving angiogenesis in triple-negative breast cancer [PMID:31705927, PMID:35844791].","teleology":[{"year":2004,"claim":"Before any kinetochore role was known, CENPU was first isolated as an MLF1-associating nuclear/cytoplasmic protein, providing the initial molecular handle on the gene.","evidence":"Yeast two-hybrid and pulldown with co-localization in human cells","pmids":["15116101"],"confidence":"Medium","gaps":["Functional consequence of the MLF1 interaction was not characterized","No link to kinetochore biology established at this stage"]},{"year":2005,"claim":"Established CENPU as a constitutive centromere component whose localization depends on CENP-H/CENP-I and is required for recovery from spindle damage, defining its inner-kinetochore role.","evidence":"DT40 knockout, reciprocal Co-IP, and immunofluorescence","pmids":["16287847"],"confidence":"High","gaps":["Molecular basis of the mitotic recovery defect not defined","Direct microtubule or kinase connections not yet known"]},{"year":2006,"claim":"Resolved how a mitotic kinase is targeted to kinetochores: PLK1 phosphorylates CENPU at Thr78 to create a self-tethering polo-box-binding motif, identifying CENPU as a PLK1 receptor.","evidence":"In vitro kinase assays, T78 mutagenesis, PBD-binding and live-cell imaging","pmids":["17081991"],"confidence":"High","gaps":["Did not define the priming kinase upstream of PLK1","Mechanism of T78-dependent degradation left unresolved"]},{"year":2010,"claim":"Showed CENPU directly binds microtubules and cooperates with Hec1/NDC80, and that Aurora B phosphorylation weakens this attachment, embedding CENPU in error correction.","evidence":"Co-IP, in vitro microtubule-binding and Aurora B kinase assays, shRNA phenotyping","pmids":["21056971"],"confidence":"High","gaps":["Aurora B phospho-sites on CENPU not exhaustively mapped","Quantitative contribution of CENPU vs NDC80 to attachment unclear"]},{"year":2011,"claim":"Defined the CENPU–CENP-Q complex as a mutually stabilizing unit and identified a PLK1–CENPU–CENP-Q ternary complex whose PLK1-dependent phosphorylation drives delocalization.","evidence":"Co-IP, in vitro PLK1 kinase assay, mutagenesis, immunofluorescence","pmids":["21454580"],"confidence":"High","gaps":["Specific CENP-Q phosphosites not yet enumerated","Fate of delocalized complex not determined"]},{"year":2015,"claim":"Mapped nine PLK1 phosphosites on CENP-Q and demonstrated that both timely localization and PLK1-driven delocalization/degradation of the CENPU–CENP-Q complex are required for segregation.","evidence":"In vitro kinase assay, 9A/9D-E mutants, chromatin fractionation, segregation assays","pmids":["25670858"],"confidence":"High","gaps":["Degradation machinery for cytosolic CENPU/CENP-Q not identified","How localization timing is set in interphase unclear"]},{"year":2020,"claim":"Defined the recruitment logic of PLK1 to CENPU: CDK1 priming followed by PLK1 itself generates docking motifs, placing CENPU alongside BUB1 as one of two main kinetochore PLK1 receptors.","evidence":"Ectopic localization, in vitro reconstitution, kinetochore localization and mutagenesis","pmids":["33248027"],"confidence":"High","gaps":["Number and role of PLK1-docking sites later contested (see 2025 preprint)","Stoichiometry of PLK1 at the kinetochore not resolved"]},{"year":2021,"claim":"Established functional redundancy: BUB1 and CENP-U redundantly recruit PLK1 to stabilize attachments, with CENP-U dependent on CENP-P/CENP-Q, and showed human CENP-O does not control Aurora B localization unlike yeast.","evidence":"Double depletion epistasis, chemical-genetic sensitization, immunofluorescence","pmids":["34551298"],"confidence":"High","gaps":["Why two parallel PLK1 receptors exist mechanistically unclear","Spatial division of labor between BUB1 and CENP-U not mapped"]},{"year":2021,"claim":"In budding yeast, showed the CENPU ortholog Ame1 is controlled by CDK1-activated phospho-degrons recognized by SCF-Cdc4, with Mis12 binding shielding kinetochore-bound Ame1 from degradation.","evidence":"Phosphorylation mapping, ubiquitination assays, cdc4 genetics in yeast","pmids":["34308839"],"confidence":"High","gaps":["Conservation of degron regulation to human CENPU not tested","Identity of relevant human E3 not established"]},{"year":2013,"claim":"Demonstrated CENPU is essential for early mammalian development, with knockout embryos dying at E6.5 from epiblast degeneration.","evidence":"Germline knockout mouse, in situ hybridization, immune phenotyping","pmids":["23724000"],"confidence":"Medium","gaps":["Molecular cause of epiblast death not defined","Link between lethality and kinetochore function not directly shown"]},{"year":2019,"claim":"Extended CENPU function to oncogenic signaling, showing it stabilizes COX-2 by blocking its ubiquitination to drive angiogenesis in triple-negative breast cancer.","evidence":"shRNA, ubiquitination assay, tube formation, xenograft model","pmids":["31705927"],"confidence":"Medium","gaps":["Direct CENPU–COX-2 interaction not fully validated","Relationship to kinetochore role unknown"]},{"year":2022,"claim":"Identified an E2F1–CENPU positive feedback loop in which CENPU degrades E2F6 to de-repress E2F1, accelerating G1/S transition in hepatocellular carcinoma.","evidence":"Co-IP, ubiquitination assay, ChIP, knockdown/overexpression","pmids":["35844791"],"confidence":"Medium","gaps":["Mechanism by which CENPU promotes E2F6 ubiquitination unclear","Generality beyond hepatocellular carcinoma untested"]},{"year":2025,"claim":"Reopened the architecture of PLK1 docking, with biophysical analysis arguing for a single high-affinity master motif engaging multiple PBD pockets rather than PBD dimerization.","evidence":"Biochemical/biophysical binding assays and structural modelling (preprint)","pmids":["bio_10.1101_2025.03.28.645803"],"confidence":"Medium","gaps":["Preprint not peer-reviewed","Conflicts with earlier dimerization model and awaits reconciliation"]},{"year":2025,"claim":"Provided structural basis for Mis12-mediated connection: a Dsn1 auto-inhibitory segment occludes the Ame1/CENP-U binding site and Aurora B phosphorylation would relieve it to strengthen inner–outer kinetochore links.","evidence":"Cryo-EM with biochemical and genetic validation in yeast (preprint)","pmids":["bio_10.1101_2025.06.03.657598"],"confidence":"Medium","gaps":["Preprint not peer-reviewed","Demonstrated for yeast Ame1; human relevance untested"]},{"year":2025,"claim":"Suggested a further cancer mechanism in which CENPU stabilizes furin to enhance proNGF-to-NGF processing and breast cancer stem cell properties.","evidence":"Co-IP, furin stability western blots, ELISA, proteomics, mammosphere and xenograft assays","pmids":["41041859"],"confidence":"Low","gaps":["Single lab, not independently replicated","Mechanism of how CENPU blocks lysosomal furin degradation undefined"]},{"year":null,"claim":"How CENPU's essential mitotic/kinetochore role mechanistically connects to its reported cancer-associated protein-stabilization activities and to its embryonic-lethal phenotype remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying mechanism links kinetochore scaffolding to COX-2/E2F6/furin stabilization","Molecular driver of epiblast lethality not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[9,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,7]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,4,6]}],"complexes":["CENP-O complex (CENP-O/P/Q/U)","kinetochore (CCAN)","CENPU–CENP-Q complex"],"partners":["CENPQ","PLK1","NDC80","CENPH","CENPI","MLF1","E2F6","BUB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q71F23","full_name":"Centromere protein U","aliases":["Centromere protein of 50 kDa","CENP-50","Interphase centromere complex protein 24","KSHV latent nuclear antigen-interacting protein 1","MLF1-interacting protein","Polo-box-interacting protein 1"],"length_aa":418,"mass_kda":47.5,"function":"Component of the CENPA-NAC (nucleosome-associated) complex, a complex that plays a central role in assembly of kinetochore proteins, mitotic progression and chromosome segregation. The CENPA-NAC complex recruits the CENPA-CAD (nucleosome distal) complex and may be involved in incorporation of newly synthesized CENPA into centromeres. Plays an important role in the correct PLK1 localization to the mitotic kinetochores. A scaffold protein responsible for the initial recruitment and maintenance of the kinetochore PLK1 population until its degradation. Involved in transcriptional repression","subcellular_location":"Cytoplasm; Nucleus; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q71F23/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPU","classification":"Not Classified","n_dependent_lines":65,"n_total_lines":1208,"dependency_fraction":0.05380794701986755},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CENPU","total_profiled":1310},"omim":[{"mim_id":"611511","title":"MLF1-INTERACTING PROTEIN; MLF1IP","url":"https://www.omim.org/entry/611511"},{"mim_id":"611510","title":"CENTROMERIC PROTEIN T; CENPT","url":"https://www.omim.org/entry/611510"},{"mim_id":"611509","title":"CENTROMERIC PROTEIN N; CENPN","url":"https://www.omim.org/entry/611509"},{"mim_id":"610152","title":"CENTROMERIC PROTEIN M; CENPM","url":"https://www.omim.org/entry/610152"},{"mim_id":"609130","title":"CENTROMERIC PROTEIN S; CENPS","url":"https://www.omim.org/entry/609130"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Centriolar satellite","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":59.0},{"tissue":"testis","ntpm":27.6}],"url":"https://www.proteinatlas.org/search/CENPU"},"hgnc":{"alias_symbol":["CENP-U","KLIP1","CENP-50","PBIP1"],"prev_symbol":["MLF1IP"]},"alphafold":{"accession":"Q71F23","domains":[{"cath_id":"1.20.5","chopping":"387-418","consensus_level":"medium","plddt":91.9669,"start":387,"end":418},{"cath_id":"1.20.5","chopping":"246-372","consensus_level":"medium","plddt":92.0126,"start":246,"end":372}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q71F23","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q71F23-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q71F23-F1-predicted_aligned_error_v6.png","plddt_mean":65.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPU","jax_strain_url":"https://www.jax.org/strain/search?query=CENPU"},"sequence":{"accession":"Q71F23","fasta_url":"https://rest.uniprot.org/uniprotkb/Q71F23.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q71F23/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q71F23"}},"corpus_meta":[{"pmid":"17081991","id":"PMC_17081991","title":"Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation.","date":"2006","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/17081991","citation_count":227,"is_preprint":false},{"pmid":"33248027","id":"PMC_33248027","title":"BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/33248027","citation_count":91,"is_preprint":false},{"pmid":"16287847","id":"PMC_16287847","title":"The constitutive centromere component CENP-50 is required for recovery from spindle damage.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16287847","citation_count":66,"is_preprint":false},{"pmid":"15116101","id":"PMC_15116101","title":"cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15116101","citation_count":57,"is_preprint":false},{"pmid":"21056971","id":"PMC_21056971","title":"CENP-U cooperates with Hec1 to orchestrate kinetochore-microtubule attachment.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21056971","citation_count":56,"is_preprint":false},{"pmid":"21454580","id":"PMC_21454580","title":"Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21454580","citation_count":44,"is_preprint":false},{"pmid":"34551298","id":"PMC_34551298","title":"Bub1 and CENP-U redundantly recruit Plk1 to stabilize kinetochore-microtubule attachments and ensure accurate chromosome segregation.","date":"2021","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34551298","citation_count":36,"is_preprint":false},{"pmid":"31705927","id":"PMC_31705927","title":"Centromere protein U (CENPU) enhances angiogenesis in triple-negative breast cancer by inhibiting ubiquitin-proteasomal degradation of COX-2.","date":"2019","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/31705927","citation_count":32,"is_preprint":false},{"pmid":"18215321","id":"PMC_18215321","title":"Self-regulated mechanism of Plk1 localization to kinetochores: lessons from the Plk1-PBIP1 interaction.","date":"2008","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/18215321","citation_count":32,"is_preprint":false},{"pmid":"35844791","id":"PMC_35844791","title":"A positive feedback loop of CENPU/E2F6/E2F1 facilitates proliferation and metastasis via ubiquitination of E2F6 in hepatocellular carcinoma.","date":"2022","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35844791","citation_count":29,"is_preprint":false},{"pmid":"15893739","id":"PMC_15893739","title":"Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.","date":"2005","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/15893739","citation_count":27,"is_preprint":false},{"pmid":"31733857","id":"PMC_31733857","title":"Short communication: A splice site mutation in CENPU is associated with recessive embryonic lethality in Holstein cattle.","date":"2019","source":"Journal of dairy science","url":"https://pubmed.ncbi.nlm.nih.gov/31733857","citation_count":20,"is_preprint":false},{"pmid":"25670858","id":"PMC_25670858","title":"Mammalian Polo-like kinase 1 (Plk1) promotes proper chromosome segregation by phosphorylating and delocalizing the PBIP1·CENP-Q complex from kinetochores.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25670858","citation_count":19,"is_preprint":false},{"pmid":"35176420","id":"PMC_35176420","title":"The centromere-associated protein CENPU promotes cell proliferation, migration, and invasiveness in lung adenocarcinoma.","date":"2022","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/35176420","citation_count":16,"is_preprint":false},{"pmid":"30849291","id":"PMC_30849291","title":"Reduced CENPU expression inhibits lung adenocarcinoma cell proliferation and migration through PI3K/AKT signaling.","date":"2019","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30849291","citation_count":16,"is_preprint":false},{"pmid":"34872447","id":"PMC_34872447","title":"Centromere protein U (CENPU) promotes gastric cancer cell proliferation and glycolysis by regulating high mobility group box 2 (HMGB2).","date":"2021","source":"Bioengineered","url":"https://pubmed.ncbi.nlm.nih.gov/34872447","citation_count":10,"is_preprint":false},{"pmid":"31966511","id":"PMC_31966511","title":"MLF1IP promotes cells proliferation and apoptosis by regulating CyclinD1 in breast cancer.","date":"2017","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/31966511","citation_count":10,"is_preprint":false},{"pmid":"28173615","id":"PMC_28173615","title":"MLF1IP promotes normal erythroid proliferation and is involved in the pathogenesis of polycythemia vera.","date":"2017","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/28173615","citation_count":9,"is_preprint":false},{"pmid":"36608638","id":"PMC_36608638","title":"Knockdown of CENPU inhibits cervical cancer cell migration and stemness through the FOXM1/Wnt/β-catenin pathway.","date":"2022","source":"Tissue & 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signalling","url":"https://pubmed.ncbi.nlm.nih.gov/35122991","citation_count":4,"is_preprint":false},{"pmid":"35071438","id":"PMC_35071438","title":"Mitosis-related gene CENP-U as a potential biomarker in malignancy.","date":"2021","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35071438","citation_count":4,"is_preprint":false},{"pmid":"32535988","id":"PMC_32535988","title":"CENP-50 is required for papilloma development in the two-stage skin carcinogenesis model.","date":"2020","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/32535988","citation_count":3,"is_preprint":false},{"pmid":"41041859","id":"PMC_41041859","title":"CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation.","date":"2025","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41041859","citation_count":1,"is_preprint":false},{"pmid":"40058474","id":"PMC_40058474","title":"DPHC from Alpinia officinarum Hance specifically modulates the function of CENPU in the cell cycle and apoptosis to ameliorate hepatocellular carcinoma.","date":"2025","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40058474","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.28.645803","title":"Molecular anatomy of PLK1 master docking motifs","date":"2025-03-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.28.645803","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.03.601947","title":"Dynamic Plk1 recruitment to the inner centromere","date":"2024-07-04","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.03.601947","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.03.657598","title":"Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex","date":"2025-06-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.03.657598","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16021,"output_tokens":4386,"usd":0.056926,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12261,"output_tokens":4694,"usd":0.089328,"stage2_stop_reason":"end_turn"},"total_usd":0.146254,"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\": 2006,\n      \"finding\": \"PLK1 phosphorylates PBIP1 (CENPU) at Thr-78, creating a self-tethering phosphopeptide motif that specifically binds the polo-box domain (PBD) of PLK1 (but not PLK2 or PLK3), recruiting PLK1 to interphase and mitotic kinetochores. Later in mitosis, PLK1 also induces PBIP1 degradation in a T78-dependent manner, enabling PLK1 to interact with other kinetochore components required for proper chromosome segregation.\",\n      \"method\": \"In vitro kinase assays, mutagenesis of T78, PBD-binding assays, live-cell imaging, spindle checkpoint assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphorylation reconstitution combined with mutagenesis and functional rescue, replicated in multiple subsequent studies\",\n      \"pmids\": [\"17081991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CENP-50 (CENPU) is a constitutive centromere component that colocalizes with CENP-A and CENP-H throughout the cell cycle; its centromere localization depends on CENP-H and CENP-I. CENP-50 physically interacts with the CENP-H/CENP-I complex (shown by co-immunoprecipitation). Loss of CENP-50 causes prolonged mitosis and premature sister chromatid separation upon spindle checkpoint activation, indicating a role in recovery from spindle damage.\",\n      \"method\": \"Knockout in chicken DT40 cells, co-immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus clean KO with defined mitotic phenotype; independently confirmed by multiple subsequent studies\",\n      \"pmids\": [\"16287847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CENPU directly interacts with Hec1 (NDC80) at kinetochores. CENPU itself binds microtubules directly in vitro and displays cooperative microtubule-binding activity with Hec1. Aurora B phosphorylates CENPU, and this phosphorylation reduces kinetochore–microtubule interaction, contributing to Aurora B's error-correction function. shRNA-mediated knockdown of CENPU impairs kinetochore–microtubule attachment.\",\n      \"method\": \"Co-immunoprecipitation, in vitro microtubule-binding assays, in vitro Aurora B kinase assay, shRNA knockdown with mitotic phenotype analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution of microtubule binding, identification of Aurora B as writer with kinase assay, Co-IP for complex, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21056971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PBIP1 (CENPU) directly interacts with CENP-Q, and this interaction is mutually required for their stability and centromere localization. PLK1 forms a ternary complex with PBIP1 and CENP-Q through the self-generated p-T78 motif on PBIP1, and PLK1-dependent phosphorylation of CENP-Q within this complex leads to delocalization of the PBIP1–CENP-Q complex from mitotic centromeres.\",\n      \"method\": \"Co-immunoprecipitation, in vitro PLK1 kinase assay, mutagenesis, immunofluorescence localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct interaction reconstituted, ternary complex identified, kinase assay with mutagenesis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21454580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PLK1 phosphorylates the CENP-Q subunit of the PBIP1–CENP-Q complex at multiple sites; phosphorylation of nine sites drives delocalization of the complex from kinetochores. Phospho-mimetic (9D/E) mutations prevent localization to interphase prekinetochores, while phospho-null (9A) mutations prolong kinetochore residence. Both mutants impair proper chromosome segregation, demonstrating that timely localization and delocalization of the PBIP1–CENP-Q complex are both critical for mitotic progression. PLK1-dependent delocalization indirectly leads to cytosolic degradation of PBIP1 and CENP-Q.\",\n      \"method\": \"In vitro PLK1 kinase assay, mutagenesis (9A and 9D/E), chromatin fractionation, immunofluorescence, chromosome segregation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with site-specific mutagenesis and functional validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25670858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BUB1 (outer kinetochore) and CENP-U (inner kinetochore) are the main PLK1 receptors at kinetochores. Both share a constellation of sequence motifs (a PP2A-docking motif and two PLK1-docking sites). PLK1 recruitment to CENP-U requires priming phosphorylation by CDK1 followed by PLK1 itself. The two PLK1-docking sites on CENP-U promote PLK1 dimerization. This was demonstrated by ectopic localization assays, in vitro reconstitution, and kinetochore localization studies.\",\n      \"method\": \"Ectopic localization assay, in vitro reconstitution of PLK1–CENP-U interaction, kinetochore localization studies, mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution plus ectopic localization plus mutagenesis, replicated in subsequent independent study (PMID:34551298)\",\n      \"pmids\": [\"33248027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BUB1 and CENP-U redundantly recruit PLK1 to kinetochores to stabilize kinetochore–microtubule attachments. CENP-U is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex. Depletion of both BUB1 and CENP-U causes chromosome mis-segregation; depletion of either alone is insufficient, demonstrating functional redundancy. Cells depleted of BUB1 or CENP-U are sensitized to PLK1 inhibition but not Aurora B inhibition. Unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B in human cells.\",\n      \"method\": \"Stable depletion by siRNA/shRNA, epistasis analysis (double depletion), chemical inhibitor sensitization assays, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double depletion, chemical-genetic sensitization, consistent with PMID:33248027 from independent lab\",\n      \"pmids\": [\"34551298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CENPU (MLF1IP) was identified as a protein that specifically associates with MLF1 by yeast two-hybrid analysis and pulldown assays, and colocalizes with MLF1 in both nuclei and cytoplasm. The protein contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, and multiple potential phosphorylation sites.\",\n      \"method\": \"Yeast two-hybrid, pulldown assay, co-localization by immunofluorescence\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus pulldown from single lab; functional consequence of MLF1 interaction not fully characterized\",\n      \"pmids\": [\"15116101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In budding yeast, Cdk1 phosphorylation of the CENPU ortholog Ame1 activates phospho-degrons that are recognized by the SCF-Cdc4 E3 ubiquitin ligase complex, targeting Ame1 for proteasomal degradation during M-phase. Binding of the Mtw1 (Mis12) complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from degradation. This mechanism ensures efficient centromere-dependent kinetochore assembly.\",\n      \"method\": \"Comprehensive phosphorylation analysis of native CCAN subunits, biochemical ubiquitination assays, genetic assays in budding yeast (cdc4 mutants, overexpression toxicity)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — phosphorylation mapping, biochemical reconstitution of degron recognition, genetic suppression/toxicity assays, single lab but multiple orthogonal methods; yeast ortholog\",\n      \"pmids\": [\"34308839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CENPU promotes angiogenesis in triple-negative breast cancer by inhibiting the ubiquitination and proteasomal degradation of COX-2, leading to increased COX-2-p-ERK-HIF-1α-VEGFA signaling. CENPU knockdown reduced VEGFA production, tube formation by endothelial cells, and tumor microvessel density in xenograft models.\",\n      \"method\": \"shRNA knockdown, ubiquitination assay, western blot (COX-2 protein stability), endothelial tube formation assay, xenograft mouse model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — ubiquitination assay supports mechanism, single lab, limited orthogonal validation of direct interaction between CENPU and COX-2 degradation machinery\",\n      \"pmids\": [\"31705927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CENPU physically interacts with the transcription factor E2F6 and promotes its ubiquitin-mediated degradation, thereby de-repressing E2F1 transcription. E2F1 in turn binds the CENPU promoter to increase CENPU transcription, forming a positive feedback loop that accelerates G1/S transition in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation (E2F1 binding to CENPU promoter), western blot, knockdown and overexpression\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP for interaction, ubiquitination assay, ChIP for transcriptional regulation, single lab with multiple methods\",\n      \"pmids\": [\"35844791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MLF1-IP (CENPU) KO mice die by embryonic day 6.5 due to degeneration of epiblasts, establishing an essential role in early embryonic development. Heterozygous KO mice are viable and fertile with no apparent immune system defects, indicating that ~50% expression is sufficient for normal postnatal development.\",\n      \"method\": \"Germline knockout mouse (homozygous lethal, heterozygous viable), in situ hybridization, immune phenotyping\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined lethal phenotype; developmental role demonstrated but molecular mechanism in embryo not specified; single lab\",\n      \"pmids\": [\"23724000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENPU promotes furin activity by inhibiting lysosomal degradation of furin in triple-negative breast cancer cells. Furin, a precursor-processing enzyme, converts proNGF to NGF, which promotes breast cancer stem cell properties. Co-immunoprecipitation experiments demonstrated association between CENPU, furin, and NGF/proNGF. A furin inhibitor suppressed CENPU-promoted tumor growth in xenograft models.\",\n      \"method\": \"Co-immunoprecipitation, western blot (furin protein stability), ELISA (NGF), 4D-DIA quantitative proteomics, mammosphere formation, xenograft mouse model\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP without full mechanistic dissection of how CENPU blocks lysosomal furin degradation; novel and not independently replicated\",\n      \"pmids\": [\"41041859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENP-U contains a single high-affinity master PLK1-docking motif that is generated by initial CDK1 priming and subsequent PLK1 phosphorylation. Biochemical and biophysical analyses showed this motif forms extensive interactions with multiple pockets on the PBD surface. Evidence did not support a requirement for PBD dimerization in CENP-U-mediated PLK1 docking.\",\n      \"method\": \"Biochemical binding assays, biophysical measurements, structural modelling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — biophysical reconstitution with multiple methods, but preprint not yet peer-reviewed and from single lab\",\n      \"pmids\": [\"bio_10.1101_2025.03.28.645803\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In budding yeast, an N-terminal auto-inhibitory segment of Dsn1 (Mis12 complex subunit) occludes binding sites for both CENP-C/Mif2 and CENP-U/Ame1 on the Mis12 complex head domain. Aurora B/Ipl1 phosphorylation of this auto-inhibitory segment would release auto-inhibition and strengthen inner–outer kinetochore connections involving CENP-U/Ame1. This was established by cryo-EM structure combined with biochemical and genetic experiments.\",\n      \"method\": \"Cryo-EM, biochemical binding assays, genetic assays in budding yeast\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — high-quality cryo-EM with biochemical validation, but preprint not peer-reviewed; finding is for yeast ortholog Ame1\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CENPU (PBIP1/MLF1IP/CENP-50/CENP-U) is a constitutive inner kinetochore scaffold protein that recruits PLK1 to kinetochores via a CDK1-primed, PLK1-amplified phospho-T78 docking motif on CENPU that binds the PLK1 polo-box domain; PLK1 then phosphorylates and delocalizes the CENPU–CENP-Q complex from kinetochores during mitotic progression. CENPU also directly binds microtubules and cooperates with Hec1/NDC80 for kinetochore–microtubule attachment, is phosphorylated by Aurora B to promote error correction, and is localized to the inner kinetochore through the CENP-O complex (CENP-P/Q). In budding yeast, the Ame1 ortholog is additionally regulated by CDK1-activated phospho-degrons recognized by SCF-Cdc4, with kinetochore-bound Ame1 protected from degradation by Mis12 complex shielding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENPU (PBIP1/MLF1IP/CENP-50/CENP-U) is a constitutive inner-kinetochore protein of the CENP-O/CCAN class that couples centromere architecture to mitotic kinase signaling and kinetochore\\u2013microtubule attachment [#1, #6]. Its centromere localization depends on CENP-H/CENP-I and on the CENP-P/CENP-Q subunits of the CENP-O complex, and CENPU loss causes prolonged mitosis and premature sister chromatid separation under spindle-checkpoint stress [#1, #6]. CENPU forms a mutually stabilizing complex with CENP-Q, binds microtubules directly, and cooperates with Hec1/NDC80 to support kinetochore\\u2013microtubule attachment, an interaction tuned by Aurora B phosphorylation that drives error correction [#2, #3]. CENPU is a principal kinetochore receptor for PLK1: CDK1 priming followed by PLK1 self-phosphorylation generates a phospho-Thr78 docking motif that binds the PLK1 polo-box domain, recruiting PLK1 to interphase and mitotic kinetochores, where it acts redundantly with BUB1 [#0, #5, #6]. PLK1 then phosphorylates CENP-Q within the ternary complex at multiple sites, delocalizing the CENPU\\u2013CENP-Q complex and driving its degradation, with both timely localization and removal being required for faithful chromosome segregation [#3, #4]. Genetic ablation in mice is embryonic-lethal by E6.5 from epiblast degeneration, establishing an essential developmental role [#11]. Beyond its mitotic function, CENPU has been implicated in cancer through stabilization of partner proteins, including E2F6-dependent control of the E2F1\\u2013CENPU feedback loop in hepatocellular carcinoma and COX-2 stabilization driving angiogenesis in triple-negative breast cancer [#9, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Before any kinetochore role was known, CENPU was first isolated as an MLF1-associating nuclear/cytoplasmic protein, providing the initial molecular handle on the gene.\",\n      \"evidence\": \"Yeast two-hybrid and pulldown with co-localization in human cells\",\n      \"pmids\": [\"15116101\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional consequence of the MLF1 interaction was not characterized\", \"No link to kinetochore biology established at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Established CENPU as a constitutive centromere component whose localization depends on CENP-H/CENP-I and is required for recovery from spindle damage, defining its inner-kinetochore role.\",\n      \"evidence\": \"DT40 knockout, reciprocal Co-IP, and immunofluorescence\",\n      \"pmids\": [\"16287847\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular basis of the mitotic recovery defect not defined\", \"Direct microtubule or kinase connections not yet known\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved how a mitotic kinase is targeted to kinetochores: PLK1 phosphorylates CENPU at Thr78 to create a self-tethering polo-box-binding motif, identifying CENPU as a PLK1 receptor.\",\n      \"evidence\": \"In vitro kinase assays, T78 mutagenesis, PBD-binding and live-cell imaging\",\n      \"pmids\": [\"17081991\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not define the priming kinase upstream of PLK1\", \"Mechanism of T78-dependent degradation left unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed CENPU directly binds microtubules and cooperates with Hec1/NDC80, and that Aurora B phosphorylation weakens this attachment, embedding CENPU in error correction.\",\n      \"evidence\": \"Co-IP, in vitro microtubule-binding and Aurora B kinase assays, shRNA phenotyping\",\n      \"pmids\": [\"21056971\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Aurora B phospho-sites on CENPU not exhaustively mapped\", \"Quantitative contribution of CENPU vs NDC80 to attachment unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the CENPU\\u2013CENP-Q complex as a mutually stabilizing unit and identified a PLK1\\u2013CENPU\\u2013CENP-Q ternary complex whose PLK1-dependent phosphorylation drives delocalization.\",\n      \"evidence\": \"Co-IP, in vitro PLK1 kinase assay, mutagenesis, immunofluorescence\",\n      \"pmids\": [\"21454580\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Specific CENP-Q phosphosites not yet enumerated\", \"Fate of delocalized complex not determined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped nine PLK1 phosphosites on CENP-Q and demonstrated that both timely localization and PLK1-driven delocalization/degradation of the CENPU\\u2013CENP-Q complex are required for segregation.\",\n      \"evidence\": \"In vitro kinase assay, 9A/9D-E mutants, chromatin fractionation, segregation assays\",\n      \"pmids\": [\"25670858\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Degradation machinery for cytosolic CENPU/CENP-Q not identified\", \"How localization timing is set in interphase unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the recruitment logic of PLK1 to CENPU: CDK1 priming followed by PLK1 itself generates docking motifs, placing CENPU alongside BUB1 as one of two main kinetochore PLK1 receptors.\",\n      \"evidence\": \"Ectopic localization, in vitro reconstitution, kinetochore localization and mutagenesis\",\n      \"pmids\": [\"33248027\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Number and role of PLK1-docking sites later contested (see 2025 preprint)\", \"Stoichiometry of PLK1 at the kinetochore not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established functional redundancy: BUB1 and CENP-U redundantly recruit PLK1 to stabilize attachments, with CENP-U dependent on CENP-P/CENP-Q, and showed human CENP-O does not control Aurora B localization unlike yeast.\",\n      \"evidence\": \"Double depletion epistasis, chemical-genetic sensitization, immunofluorescence\",\n      \"pmids\": [\"34551298\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Why two parallel PLK1 receptors exist mechanistically unclear\", \"Spatial division of labor between BUB1 and CENP-U not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"In budding yeast, showed the CENPU ortholog Ame1 is controlled by CDK1-activated phospho-degrons recognized by SCF-Cdc4, with Mis12 binding shielding kinetochore-bound Ame1 from degradation.\",\n      \"evidence\": \"Phosphorylation mapping, ubiquitination assays, cdc4 genetics in yeast\",\n      \"pmids\": [\"34308839\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Conservation of degron regulation to human CENPU not tested\", \"Identity of relevant human E3 not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated CENPU is essential for early mammalian development, with knockout embryos dying at E6.5 from epiblast degeneration.\",\n      \"evidence\": \"Germline knockout mouse, in situ hybridization, immune phenotyping\",\n      \"pmids\": [\"23724000\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular cause of epiblast death not defined\", \"Link between lethality and kinetochore function not directly shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended CENPU function to oncogenic signaling, showing it stabilizes COX-2 by blocking its ubiquitination to drive angiogenesis in triple-negative breast cancer.\",\n      \"evidence\": \"shRNA, ubiquitination assay, tube formation, xenograft model\",\n      \"pmids\": [\"31705927\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct CENPU\\u2013COX-2 interaction not fully validated\", \"Relationship to kinetochore role unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified an E2F1\\u2013CENPU positive feedback loop in which CENPU degrades E2F6 to de-repress E2F1, accelerating G1/S transition in hepatocellular carcinoma.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, ChIP, knockdown/overexpression\",\n      \"pmids\": [\"35844791\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which CENPU promotes E2F6 ubiquitination unclear\", \"Generality beyond hepatocellular carcinoma untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reopened the architecture of PLK1 docking, with biophysical analysis arguing for a single high-affinity master motif engaging multiple PBD pockets rather than PBD dimerization.\",\n      \"evidence\": \"Biochemical/biophysical binding assays and structural modelling (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.28.645803\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint not peer-reviewed\", \"Conflicts with earlier dimerization model and awaits reconciliation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided structural basis for Mis12-mediated connection: a Dsn1 auto-inhibitory segment occludes the Ame1/CENP-U binding site and Aurora B phosphorylation would relieve it to strengthen inner\\u2013outer kinetochore links.\",\n      \"evidence\": \"Cryo-EM with biochemical and genetic validation in yeast (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint not peer-reviewed\", \"Demonstrated for yeast Ame1; human relevance untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Suggested a further cancer mechanism in which CENPU stabilizes furin to enhance proNGF-to-NGF processing and breast cancer stem cell properties.\",\n      \"evidence\": \"Co-IP, furin stability western blots, ELISA, proteomics, mammosphere and xenograft assays\",\n      \"pmids\": [\"41041859\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab, not independently replicated\", \"Mechanism of how CENPU blocks lysosomal furin degradation undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CENPU's essential mitotic/kinetochore role mechanistically connects to its reported cancer-associated protein-stabilization activities and to its embryonic-lethal phenotype remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No unifying mechanism links kinetochore scaffolding to COX-2/E2F6/furin stabilization\", \"Molecular driver of epiblast lethality not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 4, 6]}\n    ],\n    \"complexes\": [\"CENP-O complex (CENP-O/P/Q/U)\", \"kinetochore (CCAN)\", \"CENPU\\u2013CENP-Q complex\"],\n    \"partners\": [\"CENPQ\", \"PLK1\", \"NDC80\", \"CENPH\", \"CENPI\", \"MLF1\", \"E2F6\", \"BUB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}