{"gene":"CKAP2","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2004,"finding":"Mouse CKAP2 (mCKAP2) colocalizes with microtubules throughout the cell cycle and overexpression stabilizes microtubules against nocodazole-induced depolymerization, identifying CKAP2 as a microtubule-associated protein with microtubule-stabilizing properties.","method":"Immunocytochemistry, colocalization with microtubule markers, nocodazole resistance assay","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct colocalization and functional stabilization assay, single lab","pmids":["15504249"],"is_preprint":false},{"year":2005,"finding":"CKAP2 is a p53 target gene; DNA damage induces CKAP2 expression in a p53-dependent manner, p53 activates the Ckap2 promoter, overexpressed CKAP2 colocalizes with and stabilizes microtubules, and in p53-null cells CKAP2 overexpression induces tetraploidy with aberrant centrosome numbers, while in p53-competent cells it activates p53-mediated cell cycle arrest and apoptosis.","method":"DNA microarray, promoter assay, immunofluorescence colocalization, overexpression in p53-null vs p53-competent cells","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, p53-dependent regulation validated with promoter assay and cell biology","pmids":["16061649"],"is_preprint":false},{"year":2006,"finding":"CKAP2/TMAP is expressed in a cell-cycle-dependent manner (low in G0/G1, peaking at G2/M), and knockdown reduces pRB phosphorylation and increases p27 expression, reducing proliferation, while constitutive expression increases pRB phosphorylation and enhances proliferation.","method":"siRNA knockdown, cell cycle synchronization, Western blot for pRB and p27, proliferation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — clean KD/OE with defined molecular readouts, single lab","pmids":["16876122"],"is_preprint":false},{"year":2007,"finding":"CKAP2 is a substrate of APC/C-Cdh1 ubiquitin ligase; ubiquitination and degradation of CKAP2 in vitro requires a KEN-box motif and is mediated by Cdh1. CKAP2 levels fluctuate across the cell cycle (high in mitosis, low during mitotic exit); Cdh1 overexpression reduces CKAP2 in a KEN-box-dependent manner, and Cdh1 knockdown increases CKAP2 half-life. CKAP2 associates with centrosomal microtubules in late G2 and with spindle poles and spindle microtubules from prophase through anaphase. Overexpression causes mitotic arrest with monopolar spindles containing highly bundled microtubules.","method":"In vitro ubiquitination assay, KEN-box mutagenesis, Cdh1 overexpression/knockdown, immunofluorescence, cell cycle profiling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution of ubiquitination, mutagenesis, and cell biology, replicated by independent lab (PMID:17339342)","pmids":["17376772"],"is_preprint":false},{"year":2007,"finding":"APC/C-Cdh1-mediated degradation of CKAP2 during mitotic exit is required for proper cytokinesis and spindle bipolarity; the KEN box near the N-terminus is necessary for its destruction. Overexpression causes centrosome separation defects and monopolar spindle formation. A non-degradable CKAP2 mutant increases spindle defects and cytokinesis failure.","method":"KEN-box mutagenesis, non-degradable mutant expression, overexpression, time-lapse microscopy, immunofluorescence","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis and functional rescue, replicated findings from independent lab (PMID:17376772)","pmids":["17339342"],"is_preprint":false},{"year":2008,"finding":"CKAP2/TMAP is transiently phosphorylated at Thr-596 specifically during early phases of mitosis (prophase to metaphase); phosphorylation at T596 abolishes reactivity of monoclonal antibody D-12-3, and immunofluorescence shows loss of epitope from prophase to metaphase with recovery in anaphase.","method":"Epitope mapping, phospho-specific antibody reactivity, immunofluorescence across mitotic stages","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — epitope mapping and cell-cycle-specific immunofluorescence, single lab","pmids":["18779650"],"is_preprint":false},{"year":2009,"finding":"CKAP2/TMAP depletion by siRNA causes defects in chromosome segregation (chromatin bridges, abnormal nuclear morphology, abnormal nuclear lamina organization) without significant alteration of the spindle apparatus; spindle checkpoint activity is significantly reduced in CKAP2-depleted cells, leading to chromosomal instability and reduced cell viability.","method":"siRNA knockdown, time-lapse video microscopy, immunofluorescence for nuclear lamina, spindle checkpoint assay","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 — clean KD with multiple orthogonal readouts including live imaging and checkpoint assay","pmids":["19158495"],"is_preprint":false},{"year":2010,"finding":"Aurora B kinase phosphorylates CKAP2/TMAP at Ser627 both in vitro and in vivo; neighboring conserved residues are required for efficient phosphorylation. Mutations at the phosphorylation motif (RRSRRL) cause CKAP2 to remain associated with microtubules and centrosomes throughout mitosis instead of relocalizing to the chromosome region during late mitosis.","method":"In vitro kinase assay, in vivo phosphorylation, site-directed mutagenesis, immunofluorescence localization","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay combined with mutagenesis and localization studies","pmids":["20458174"],"is_preprint":false},{"year":2012,"finding":"Cell-cycle-dependent expression of CKAP2 is regulated at the transcriptional level by Cyclin A/Cdk2 phosphorylation of Sp1, which binds a GC box in the CKAP2 promoter (-41 to -32 bp). Mutation of the GC box abolishes CKAP2 promoter activity; Cyclin A depletion reduces Sp1 binding to the GC box, and adding back Cyclin A/Cdk2 complex restores it.","method":"Promoter deletion/mutation analysis, supershift assay, ChIP assay, Cyclin A depletion and add-back","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1-2 — promoter mutagenesis, ChIP, and biochemical rescue with multiple orthogonal methods","pmids":["22465120"],"is_preprint":false},{"year":2013,"finding":"CKAP2 depletion by RNAi causes multipolar mitoses, spindle pole defects, and dispersal of newly polymerized microtubules through the chromatin region (cage-like structure) in early mitosis, indicating CKAP2 maintains microtubule nucleation site integrity by focusing microtubule minus ends to spindle poles. CKAP2 depletion also increases merotelic attachments, anaphase lagging, and polyploidy.","method":"RNAi, immunofluorescence, microtubule regrowth assay, chromosome segregation analysis","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — clean KD with multiple orthogonal phenotypic readouts","pmids":["23737987"],"is_preprint":false},{"year":2016,"finding":"CKAP2 depletion in primary diploid hepatocytes leads to over-duplicated centrosomes, disjoined chromosomes from the metaphase plate, apoptosis or multipolar cell division, and markedly decreased liver regeneration efficiency in vivo, demonstrating a physiological role in spindle bipolarity and chromosomal stability.","method":"siRNA knockdown in primary hepatocytes, immunofluorescence, in vivo liver regeneration model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo knockdown with specific phenotypic readouts, single lab","pmids":["27055594"],"is_preprint":false},{"year":2017,"finding":"CKAP2 phosphorylation at T603 (and possibly S608) by CDK1-cyclin B1 during mitosis is critical for centrosome integrity; ectopic expression of T603A and S608A mutants fails to restore centrosomal abnormalities in CKAP2-depleted cells.","method":"Phosphorylation site identification, site-directed mutagenesis, rescue experiments in CKAP2-depleted cells, centrosome analysis","journal":"Experimental & molecular medicine","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with functional rescue, identifies CDK1-cyclin B1 as the kinase","pmids":["28706298"],"is_preprint":false},{"year":2017,"finding":"CKAP2 knockdown in cervical carcinoma cells inhibits FAK-ERK2 signaling (reduced p-ERK2), and FAK inhibitor and ERK2 inhibitor block CKAP2 overexpression-induced proliferation, migration, and invasion, placing CKAP2 upstream of FAK-ERK2 pathway.","method":"siRNA knockdown, FAK inhibitor (PF-562271), ERK2 inhibitor (VX-11e), Western blot for p-ERK2, in vivo tumor growth","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — KD and pharmacological inhibition with defined molecular readouts, single lab","pmids":["28522860"],"is_preprint":false},{"year":2022,"finding":"In vitro reconstitution shows CKAP2 lowers the critical tubulin concentration for microtubule nucleation 100-fold, increases the apparent rate constant k of microtubule growth by 50-fold, increases microtubule growth rates, and strongly suppresses catastrophes, identifying CKAP2 as the most potent microtubule growth factor known.","method":"In vitro reconstitution assay, TIRF microscopy, single-molecule microtubule dynamics measurements","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — rigorous in vitro reconstitution with quantitative kinetics","pmids":["35029146"],"is_preprint":false},{"year":2022,"finding":"DLEU1 lncRNA interacts with HIF-1α to collectively activate transcription of CKAP2; CKAP2 in turn activates ERK and STAT3 signaling to mediate DLEU1 pro-tumor activities in breast cancer.","method":"Co-IP for DLEU1-HIF-1α interaction, CKAP2 knockdown/overexpression, Western blot for ERK and STAT3 phosphorylation, xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP and functional rescue, single lab, positions CKAP2 as downstream of HIF-1α/DLEU1","pmids":["35853854"],"is_preprint":false},{"year":2024,"finding":"Endogenous CKAP2 (CRISPR knock-in) localizes to the mitotic spindle during mitosis and rapidly shifts to chromatin upon mitotic exit before degradation. CKAP2 KO cells display reduced microtubule growth rates, increased chromosome segregation errors, aneuploidy, and accumulation of kinetochore-microtubule misattachments; ectopic CKAP2 expression rescues microtubule growth rates and chromosome segregation fidelity.","method":"CRISPR-Cas9 knock-in/knock-out, live cell microtubule plus-end tracking imaging, chromosome segregation analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — CRISPR KO with quantitative live imaging rescue, multiple orthogonal methods","pmids":["38381793"],"is_preprint":false},{"year":2025,"finding":"Aurora Kinase A, together with its cofactor TPX2, interacts with CKAP2 throughout mitosis (identified by co-IP/MS of endogenous CKAP2 from mitotic cells). Aurora Kinase A phosphorylates CKAP2 both in vitro and in mitotic cells; phosphorylated CKAP2 has reduced affinity for microtubules in vitro. Expression of Aurora A phospho-mimic CKAP2 mutants results in smaller mitotic spindles and reduced microtubule stability.","method":"Endogenous co-IP/mass spectrometry, in vitro kinase assay, phospho-mimic mutagenesis, microtubule binding assay, live cell imaging","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay + mutagenesis + co-IP/MS + cell phenotype, multiple orthogonal methods","pmids":["bio_10.1101_2025.09.09.675182"],"is_preprint":true},{"year":2025,"finding":"A 284-amino acid intrinsically disordered domain of CKAP2 is sufficient to catalyze both microtubule polymerization and depolymerization in vitro. CKAP2 promotes tubulin incorporation without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from known microtubule polymerases.","method":"In vitro reconstitution with isolated CKAP2 domain, microtubule polymerization/depolymerization assays, tubulin binding assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstitution with isolated domain, single lab preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.09.02.673730"],"is_preprint":true},{"year":2023,"finding":"ZC3H13 mediates N6-methyladenosine (m6A) modification of CKAP2 mRNA to enhance CKAP2 expression in cervical cancer cells; CKAP2 overexpression partially restores malignant phenotypes suppressed by ZC3H13 inhibition.","method":"m6A modification assay, ZC3H13 knockdown/overexpression, CKAP2 rescue experiment, qRT-PCR","journal":"Critical reviews in immunology","confidence":"Medium","confidence_rationale":"Tier 3 — m6A modification documented and epistasis shown, single lab","pmids":["37943149"],"is_preprint":false},{"year":2021,"finding":"DARS-AS1 lncRNA functions as a ceRNA to competitively sponge miR-3200-5p, thereby upregulating CKAP2 expression; CKAP2 activates the FAK-ERK pathway. miR-3200-5p directly targets CKAP2 and inactivates FAK-ERK signaling.","method":"Dual-luciferase reporter assay, miR-3200-5p mimic/inhibitor, CKAP2 knockdown/overexpression, Western blot for FAK-ERK","journal":"Bioengineered","confidence":"Medium","confidence_rationale":"Tier 3 — dual-luciferase validation of miRNA-CKAP2 interaction and epistasis, single lab","pmids":["34596006"],"is_preprint":false}],"current_model":"CKAP2 is a microtubule-associated protein that functions as a potent microtubule polymerase — lowering the critical concentration for nucleation ~100-fold, accelerating growth rates, and suppressing catastrophes via an intrinsically disordered catalytic domain — and localizes to spindle poles and spindle microtubules during mitosis where it is regulated by multiple kinases (Aurora B phosphorylates Ser627 to control localization, CDK1/cyclin B1 phosphorylates T603/S608 to maintain centrosome integrity, and Aurora Kinase A/TPX2 phosphorylates CKAP2 to reduce its microtubule affinity and limit spindle size); its protein levels are cell-cycle-controlled through transcriptional activation by Cyclin A/Cdk2-mediated Sp1 phosphorylation and through proteasomal degradation by APC/C-Cdh1 via a KEN-box during mitotic exit, and loss of CKAP2 causes reduced microtubule growth rates, kinetochore-microtubule misattachments, chromosome segregation errors, and aneuploidy."},"narrative":{"teleology":[{"year":2004,"claim":"The initial question of whether CKAP2 interacts with the cytoskeleton was answered by demonstrating that it colocalizes with microtubules throughout the cell cycle and stabilizes them against nocodazole-induced depolymerization, establishing CKAP2 as a microtubule-associated protein.","evidence":"Immunocytochemistry and nocodazole resistance assay in mouse cells","pmids":["15504249"],"confidence":"Medium","gaps":["Single lab observation without biochemical demonstration of direct microtubule binding","Mechanism of stabilization unknown"]},{"year":2005,"claim":"CKAP2 was connected to the DNA damage response when it was identified as a p53 transcriptional target whose overexpression causes tetraploidy in p53-null cells but triggers p53-dependent arrest in p53-competent cells, linking microtubule stabilization to genomic integrity surveillance.","evidence":"DNA microarray, promoter assay, overexpression in p53-null vs p53-competent cells","pmids":["16061649"],"confidence":"High","gaps":["Whether p53 regulation of CKAP2 is relevant during normal cell division vs. stress only","Mechanism by which CKAP2 overexpression causes tetraploidy not defined"]},{"year":2007,"claim":"The question of how CKAP2 protein levels are cleared after mitosis was resolved by showing that APC/C-Cdh1 ubiquitinates CKAP2 through a KEN-box motif, and that failure to degrade CKAP2 causes monopolar spindles and cytokinesis failure, establishing cell-cycle-dependent proteolysis as essential for proper mitotic exit.","evidence":"In vitro ubiquitination assay, KEN-box mutagenesis, non-degradable mutant expression, time-lapse microscopy; replicated by two independent labs","pmids":["17376772","17339342"],"confidence":"High","gaps":["Whether other E3 ligases contribute to CKAP2 turnover","How excess CKAP2 mechanistically generates monopolar spindles"]},{"year":2009,"claim":"Depletion studies revealed that CKAP2 is required not only for spindle architecture but also for spindle checkpoint function, as CKAP2 knockdown causes chromosome segregation defects and reduced checkpoint activity, broadening its role beyond microtubule stabilization.","evidence":"siRNA knockdown with time-lapse video microscopy, nuclear lamina immunofluorescence, spindle checkpoint assay","pmids":["19158495"],"confidence":"High","gaps":["Mechanism by which CKAP2 influences the spindle assembly checkpoint is unknown","Whether checkpoint reduction is direct or secondary to kinetochore-microtubule defects"]},{"year":2010,"claim":"Aurora B was identified as a mitotic kinase that phosphorylates CKAP2 at Ser627 to control its subcellular relocalization from spindle microtubules to the chromosome region during late mitosis, providing the first mechanism for dynamic regulation of CKAP2 localization.","evidence":"In vitro kinase assay, site-directed mutagenesis, immunofluorescence localization across mitotic stages","pmids":["20458174"],"confidence":"High","gaps":["Functional consequence of Ser627 phosphorylation on microtubule dynamics not measured","Whether Aurora B-mediated relocalization is required for chromosome segregation fidelity"]},{"year":2012,"claim":"The transcriptional mechanism driving cell-cycle-dependent CKAP2 expression was defined: Cyclin A/Cdk2 phosphorylates the transcription factor Sp1, which binds a GC box in the CKAP2 promoter to activate transcription, explaining how CKAP2 accumulates as cells approach mitosis.","evidence":"Promoter deletion/mutation analysis, ChIP, Cyclin A depletion and add-back with Sp1 binding assay","pmids":["22465120"],"confidence":"High","gaps":["Whether additional transcription factors cooperate with Sp1","Post-transcriptional regulation at the mRNA level not fully explored at this point"]},{"year":2013,"claim":"CKAP2 was shown to maintain spindle pole integrity by focusing microtubule minus ends, as its depletion disperses newly nucleated microtubules, causes multipolar spindles, and increases merotelic kinetochore attachments leading to aneuploidy.","evidence":"RNAi, microtubule regrowth assay, immunofluorescence, chromosome segregation analysis","pmids":["23737987"],"confidence":"High","gaps":["Direct mechanism of minus-end focusing by CKAP2 not defined","Whether CKAP2 cooperates with NuMA or other pole-focusing factors"]},{"year":2017,"claim":"CDK1/cyclin B1 was identified as a second mitotic kinase regulating CKAP2 via phosphorylation at T603/S608, with phospho-dead mutants failing to rescue centrosomal abnormalities in depleted cells, demonstrating that multiple kinase inputs converge on CKAP2 to maintain centrosome integrity.","evidence":"Phosphorylation site identification, site-directed mutagenesis, rescue experiments in CKAP2-depleted cells","pmids":["28706298"],"confidence":"High","gaps":["How CDK1 and Aurora B phosphorylation are coordinated on CKAP2","Whether phosphorylation at T603/S608 affects microtubule binding affinity directly"]},{"year":2022,"claim":"In vitro reconstitution established that CKAP2 is the most potent microtubule growth factor known, lowering the nucleation critical concentration 100-fold, increasing the growth rate constant 50-fold, and suppressing catastrophes, transforming the understanding of CKAP2 from a passive stabilizer to an active polymerase.","evidence":"TIRF microscopy-based in vitro reconstitution with purified components and single-molecule dynamics measurements","pmids":["35029146"],"confidence":"High","gaps":["Structural basis for how CKAP2 catalyzes tubulin addition unknown","Whether polymerase activity is regulated by mitotic phosphorylation in vitro"]},{"year":2024,"claim":"CRISPR knockout of endogenous CKAP2 quantitatively demonstrated reduced microtubule growth rates and increased chromosome segregation errors rescued by ectopic CKAP2 expression, validating the in vitro polymerase activity as the physiologically relevant mechanism for ensuring mitotic fidelity.","evidence":"CRISPR-Cas9 knock-in/knock-out, live cell microtubule plus-end tracking, chromosome segregation analysis with rescue","pmids":["38381793"],"confidence":"High","gaps":["Relative contribution of CKAP2 polymerase vs. stabilizer activity in vivo not deconvolved","Whether CKAP2 loss triggers compensatory upregulation of other polymerases"]},{"year":2025,"claim":"Aurora Kinase A/TPX2 was identified as a third kinase complex regulating CKAP2, phosphorylating it to reduce microtubule affinity and limit spindle size, revealing a negative feedback mechanism that constrains CKAP2 polymerase activity during mitosis.","evidence":"(preprint) Endogenous co-IP/mass spectrometry, in vitro kinase assay, phospho-mimic mutagenesis, microtubule binding and live imaging","pmids":["bio_10.1101_2025.09.09.675182"],"confidence":"High","gaps":["Not yet peer-reviewed","Specific phosphorylation sites on CKAP2 targeted by Aurora A not all mapped","How Aurora A and CDK1 phosphorylation events are temporally coordinated"]},{"year":2025,"claim":"A 284-amino acid intrinsically disordered domain was identified as sufficient for both microtubule polymerization and depolymerization catalysis without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from classical polymerases like XMAP215.","evidence":"(preprint) In vitro reconstitution with isolated CKAP2 domain, polymerization/depolymerization and tubulin binding assays","pmids":["bio_10.1101_2025.09.02.673730"],"confidence":"Medium","gaps":["Not yet peer-reviewed","Structural basis of the catalytic domain remains undefined","Whether the depolymerization activity is physiologically relevant in cells"]},{"year":null,"claim":"Key unresolved questions include the structural basis for CKAP2's catalytic mechanism, how the three kinase inputs (Aurora B, CDK1, Aurora A) are temporally integrated to tune CKAP2 activity across mitotic stages, and whether CKAP2's spindle checkpoint role is direct or secondary to kinetochore-microtubule attachment defects.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of CKAP2 or its catalytic domain exists","Mechanism linking CKAP2 to spindle assembly checkpoint signaling undefined","Physiological relevance of CKAP2's depolymerase activity in cells untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,3,7,13,15]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[13,17]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,3,7,15]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[3,9,11]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,3,4,6,9,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["CDH1","AURKA","TPX2","AURKB","CDK1","CCNB1","SP1","CCNA2"],"other_free_text":[]},"mechanistic_narrative":"CKAP2 is a microtubule-associated protein that functions as a potent microtubule polymerase to promote spindle assembly, chromosome segregation fidelity, and centrosome integrity during mitosis. In vitro reconstitution demonstrates that CKAP2 lowers the critical tubulin concentration for microtubule nucleation ~100-fold, accelerates growth rates, and suppresses catastrophes through an intrinsically disordered catalytic domain, while CKAP2 knockout cells exhibit reduced microtubule growth rates, kinetochore-microtubule misattachments, chromosome segregation errors, and aneuploidy [PMID:35029146, PMID:38381793]. CKAP2 protein levels are cell-cycle-controlled through transcriptional activation via Cyclin A/Cdk2-mediated Sp1 phosphorylation and proteasomal degradation by APC/C-Cdh1 through a KEN-box motif during mitotic exit, and its mitotic localization and microtubule affinity are regulated by Aurora B phosphorylation at Ser627 and CDK1/cyclin B1 phosphorylation at T603/S608 [PMID:22465120, PMID:17376772, PMID:20458174, PMID:28706298]. Loss of CKAP2 causes multipolar spindles, centrosome over-duplication, reduced spindle checkpoint activity, and chromosomal instability, establishing it as essential for mitotic fidelity [PMID:19158495, PMID:23737987]."},"prefetch_data":{"uniprot":{"accession":"Q8WWK9","full_name":"Cytoskeleton-associated protein 2","aliases":["CTCL tumor antigen se20-10","Tumor- and microtubule-associated protein"],"length_aa":683,"mass_kda":77.0,"function":"Possesses microtubule stabilizing properties. Involved in regulating aneuploidy, cell cycling, and cell death in a p53/TP53-dependent manner (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton; Cytoplasm, cytoskeleton, spindle; Cytoplasm, cytoskeleton, spindle pole","url":"https://www.uniprot.org/uniprotkb/Q8WWK9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CKAP2","classification":"Not Classified","n_dependent_lines":102,"n_total_lines":1208,"dependency_fraction":0.08443708609271523},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000136108","cell_line_id":"CID001503","localizations":[{"compartment":"centrosome","grade":3},{"compartment":"cytoskeleton","grade":3}],"interactors":[{"gene":"MED18","stoichiometry":10.0},{"gene":"KIF22","stoichiometry":10.0},{"gene":"SSBP1","stoichiometry":10.0},{"gene":"HERC2","stoichiometry":4.0},{"gene":"ACTG1","stoichiometry":0.2},{"gene":"KIF2A","stoichiometry":0.2},{"gene":"SSSCA1","stoichiometry":0.2},{"gene":"HSPA5","stoichiometry":0.2},{"gene":"MYCBP2","stoichiometry":0.2},{"gene":"PEO1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001503","total_profiled":1310},"omim":[{"mim_id":"616174","title":"CYTOSKELETON-ASSOCIATED PROTEIN 2-LIKE; CKAP2L","url":"https://www.omim.org/entry/616174"},{"mim_id":"611569","title":"CYTOSKELETON-ASSOCIATED PROTEIN 2; CKAP2","url":"https://www.omim.org/entry/611569"},{"mim_id":"263450","title":"POLYDACTYLY, POSTAXIAL, TYPE A5; PAPA5","url":"https://www.omim.org/entry/263450"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Microtubules","reliability":"Supported"},{"location":"Mitotic spindle","reliability":"Supported"},{"location":"Primary cilium","reliability":"Supported"},{"location":"Centrosome","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":63.9}],"url":"https://www.proteinatlas.org/search/CKAP2"},"hgnc":{"alias_symbol":["LB1","FLJ10749","se20-10","TMAP"],"prev_symbol":[]},"alphafold":{"accession":"Q8WWK9","domains":[{"cath_id":"-","chopping":"589-674","consensus_level":"high","plddt":82.0262,"start":589,"end":674},{"cath_id":"1.25.40","chopping":"415-513","consensus_level":"high","plddt":88.6363,"start":415,"end":513}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WWK9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WWK9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WWK9-F1-predicted_aligned_error_v6.png","plddt_mean":58.78},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CKAP2","jax_strain_url":"https://www.jax.org/strain/search?query=CKAP2"},"sequence":{"accession":"Q8WWK9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WWK9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WWK9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WWK9"}},"corpus_meta":[{"pmid":"25189355","id":"PMC_25189355","title":"RSK promotes prostate cancer progression in bone through ING3, CKAP2, and PTK6-mediated cell survival.","date":"2014","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/25189355","citation_count":45,"is_preprint":false},{"pmid":"16061649","id":"PMC_16061649","title":"Ckap2 regulates aneuploidy, cell cycling, and cell death in a p53-dependent manner.","date":"2005","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/16061649","citation_count":43,"is_preprint":false},{"pmid":"17376772","id":"PMC_17376772","title":"CKAP2 is a spindle-associated protein degraded by APC/C-Cdh1 during mitotic exit.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17376772","citation_count":41,"is_preprint":false},{"pmid":"17339342","id":"PMC_17339342","title":"Functional importance of the anaphase-promoting complex-Cdh1-mediated degradation of TMAP/CKAP2 in regulation of spindle function and 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\"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct colocalization and functional stabilization assay, single lab\",\n      \"pmids\": [\"15504249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CKAP2 is a p53 target gene; DNA damage induces CKAP2 expression in a p53-dependent manner, p53 activates the Ckap2 promoter, overexpressed CKAP2 colocalizes with and stabilizes microtubules, and in p53-null cells CKAP2 overexpression induces tetraploidy with aberrant centrosome numbers, while in p53-competent cells it activates p53-mediated cell cycle arrest and apoptosis.\",\n      \"method\": \"DNA microarray, promoter assay, immunofluorescence colocalization, overexpression in p53-null vs p53-competent cells\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, p53-dependent regulation validated with promoter assay and cell biology\",\n      \"pmids\": [\"16061649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CKAP2/TMAP is expressed in a cell-cycle-dependent manner (low in G0/G1, peaking at G2/M), and knockdown reduces pRB phosphorylation and increases p27 expression, reducing proliferation, while constitutive expression increases pRB phosphorylation and enhances proliferation.\",\n      \"method\": \"siRNA knockdown, cell cycle synchronization, Western blot for pRB and p27, proliferation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — clean KD/OE with defined molecular readouts, single lab\",\n      \"pmids\": [\"16876122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CKAP2 is a substrate of APC/C-Cdh1 ubiquitin ligase; ubiquitination and degradation of CKAP2 in vitro requires a KEN-box motif and is mediated by Cdh1. CKAP2 levels fluctuate across the cell cycle (high in mitosis, low during mitotic exit); Cdh1 overexpression reduces CKAP2 in a KEN-box-dependent manner, and Cdh1 knockdown increases CKAP2 half-life. CKAP2 associates with centrosomal microtubules in late G2 and with spindle poles and spindle microtubules from prophase through anaphase. Overexpression causes mitotic arrest with monopolar spindles containing highly bundled microtubules.\",\n      \"method\": \"In vitro ubiquitination assay, KEN-box mutagenesis, Cdh1 overexpression/knockdown, immunofluorescence, cell cycle profiling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution of ubiquitination, mutagenesis, and cell biology, replicated by independent lab (PMID:17339342)\",\n      \"pmids\": [\"17376772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"APC/C-Cdh1-mediated degradation of CKAP2 during mitotic exit is required for proper cytokinesis and spindle bipolarity; the KEN box near the N-terminus is necessary for its destruction. Overexpression causes centrosome separation defects and monopolar spindle formation. A non-degradable CKAP2 mutant increases spindle defects and cytokinesis failure.\",\n      \"method\": \"KEN-box mutagenesis, non-degradable mutant expression, overexpression, time-lapse microscopy, immunofluorescence\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis and functional rescue, replicated findings from independent lab (PMID:17376772)\",\n      \"pmids\": [\"17339342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CKAP2/TMAP is transiently phosphorylated at Thr-596 specifically during early phases of mitosis (prophase to metaphase); phosphorylation at T596 abolishes reactivity of monoclonal antibody D-12-3, and immunofluorescence shows loss of epitope from prophase to metaphase with recovery in anaphase.\",\n      \"method\": \"Epitope mapping, phospho-specific antibody reactivity, immunofluorescence across mitotic stages\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epitope mapping and cell-cycle-specific immunofluorescence, single lab\",\n      \"pmids\": [\"18779650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CKAP2/TMAP depletion by siRNA causes defects in chromosome segregation (chromatin bridges, abnormal nuclear morphology, abnormal nuclear lamina organization) without significant alteration of the spindle apparatus; spindle checkpoint activity is significantly reduced in CKAP2-depleted cells, leading to chromosomal instability and reduced cell viability.\",\n      \"method\": \"siRNA knockdown, time-lapse video microscopy, immunofluorescence for nuclear lamina, spindle checkpoint assay\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with multiple orthogonal readouts including live imaging and checkpoint assay\",\n      \"pmids\": [\"19158495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Aurora B kinase phosphorylates CKAP2/TMAP at Ser627 both in vitro and in vivo; neighboring conserved residues are required for efficient phosphorylation. Mutations at the phosphorylation motif (RRSRRL) cause CKAP2 to remain associated with microtubules and centrosomes throughout mitosis instead of relocalizing to the chromosome region during late mitosis.\",\n      \"method\": \"In vitro kinase assay, in vivo phosphorylation, site-directed mutagenesis, immunofluorescence localization\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay combined with mutagenesis and localization studies\",\n      \"pmids\": [\"20458174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cell-cycle-dependent expression of CKAP2 is regulated at the transcriptional level by Cyclin A/Cdk2 phosphorylation of Sp1, which binds a GC box in the CKAP2 promoter (-41 to -32 bp). Mutation of the GC box abolishes CKAP2 promoter activity; Cyclin A depletion reduces Sp1 binding to the GC box, and adding back Cyclin A/Cdk2 complex restores it.\",\n      \"method\": \"Promoter deletion/mutation analysis, supershift assay, ChIP assay, Cyclin A depletion and add-back\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — promoter mutagenesis, ChIP, and biochemical rescue with multiple orthogonal methods\",\n      \"pmids\": [\"22465120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CKAP2 depletion by RNAi causes multipolar mitoses, spindle pole defects, and dispersal of newly polymerized microtubules through the chromatin region (cage-like structure) in early mitosis, indicating CKAP2 maintains microtubule nucleation site integrity by focusing microtubule minus ends to spindle poles. CKAP2 depletion also increases merotelic attachments, anaphase lagging, and polyploidy.\",\n      \"method\": \"RNAi, immunofluorescence, microtubule regrowth assay, chromosome segregation analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"23737987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CKAP2 depletion in primary diploid hepatocytes leads to over-duplicated centrosomes, disjoined chromosomes from the metaphase plate, apoptosis or multipolar cell division, and markedly decreased liver regeneration efficiency in vivo, demonstrating a physiological role in spindle bipolarity and chromosomal stability.\",\n      \"method\": \"siRNA knockdown in primary hepatocytes, immunofluorescence, in vivo liver regeneration model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo knockdown with specific phenotypic readouts, single lab\",\n      \"pmids\": [\"27055594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CKAP2 phosphorylation at T603 (and possibly S608) by CDK1-cyclin B1 during mitosis is critical for centrosome integrity; ectopic expression of T603A and S608A mutants fails to restore centrosomal abnormalities in CKAP2-depleted cells.\",\n      \"method\": \"Phosphorylation site identification, site-directed mutagenesis, rescue experiments in CKAP2-depleted cells, centrosome analysis\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with functional rescue, identifies CDK1-cyclin B1 as the kinase\",\n      \"pmids\": [\"28706298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CKAP2 knockdown in cervical carcinoma cells inhibits FAK-ERK2 signaling (reduced p-ERK2), and FAK inhibitor and ERK2 inhibitor block CKAP2 overexpression-induced proliferation, migration, and invasion, placing CKAP2 upstream of FAK-ERK2 pathway.\",\n      \"method\": \"siRNA knockdown, FAK inhibitor (PF-562271), ERK2 inhibitor (VX-11e), Western blot for p-ERK2, in vivo tumor growth\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — KD and pharmacological inhibition with defined molecular readouts, single lab\",\n      \"pmids\": [\"28522860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In vitro reconstitution shows CKAP2 lowers the critical tubulin concentration for microtubule nucleation 100-fold, increases the apparent rate constant k of microtubule growth by 50-fold, increases microtubule growth rates, and strongly suppresses catastrophes, identifying CKAP2 as the most potent microtubule growth factor known.\",\n      \"method\": \"In vitro reconstitution assay, TIRF microscopy, single-molecule microtubule dynamics measurements\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro reconstitution with quantitative kinetics\",\n      \"pmids\": [\"35029146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DLEU1 lncRNA interacts with HIF-1α to collectively activate transcription of CKAP2; CKAP2 in turn activates ERK and STAT3 signaling to mediate DLEU1 pro-tumor activities in breast cancer.\",\n      \"method\": \"Co-IP for DLEU1-HIF-1α interaction, CKAP2 knockdown/overexpression, Western blot for ERK and STAT3 phosphorylation, xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and functional rescue, single lab, positions CKAP2 as downstream of HIF-1α/DLEU1\",\n      \"pmids\": [\"35853854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Endogenous CKAP2 (CRISPR knock-in) localizes to the mitotic spindle during mitosis and rapidly shifts to chromatin upon mitotic exit before degradation. CKAP2 KO cells display reduced microtubule growth rates, increased chromosome segregation errors, aneuploidy, and accumulation of kinetochore-microtubule misattachments; ectopic CKAP2 expression rescues microtubule growth rates and chromosome segregation fidelity.\",\n      \"method\": \"CRISPR-Cas9 knock-in/knock-out, live cell microtubule plus-end tracking imaging, chromosome segregation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — CRISPR KO with quantitative live imaging rescue, multiple orthogonal methods\",\n      \"pmids\": [\"38381793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Aurora Kinase A, together with its cofactor TPX2, interacts with CKAP2 throughout mitosis (identified by co-IP/MS of endogenous CKAP2 from mitotic cells). Aurora Kinase A phosphorylates CKAP2 both in vitro and in mitotic cells; phosphorylated CKAP2 has reduced affinity for microtubules in vitro. Expression of Aurora A phospho-mimic CKAP2 mutants results in smaller mitotic spindles and reduced microtubule stability.\",\n      \"method\": \"Endogenous co-IP/mass spectrometry, in vitro kinase assay, phospho-mimic mutagenesis, microtubule binding assay, live cell imaging\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay + mutagenesis + co-IP/MS + cell phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"bio_10.1101_2025.09.09.675182\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A 284-amino acid intrinsically disordered domain of CKAP2 is sufficient to catalyze both microtubule polymerization and depolymerization in vitro. CKAP2 promotes tubulin incorporation without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from known microtubule polymerases.\",\n      \"method\": \"In vitro reconstitution with isolated CKAP2 domain, microtubule polymerization/depolymerization assays, tubulin binding assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with isolated domain, single lab preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.09.02.673730\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZC3H13 mediates N6-methyladenosine (m6A) modification of CKAP2 mRNA to enhance CKAP2 expression in cervical cancer cells; CKAP2 overexpression partially restores malignant phenotypes suppressed by ZC3H13 inhibition.\",\n      \"method\": \"m6A modification assay, ZC3H13 knockdown/overexpression, CKAP2 rescue experiment, qRT-PCR\",\n      \"journal\": \"Critical reviews in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — m6A modification documented and epistasis shown, single lab\",\n      \"pmids\": [\"37943149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DARS-AS1 lncRNA functions as a ceRNA to competitively sponge miR-3200-5p, thereby upregulating CKAP2 expression; CKAP2 activates the FAK-ERK pathway. miR-3200-5p directly targets CKAP2 and inactivates FAK-ERK signaling.\",\n      \"method\": \"Dual-luciferase reporter assay, miR-3200-5p mimic/inhibitor, CKAP2 knockdown/overexpression, Western blot for FAK-ERK\",\n      \"journal\": \"Bioengineered\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — dual-luciferase validation of miRNA-CKAP2 interaction and epistasis, single lab\",\n      \"pmids\": [\"34596006\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CKAP2 is a microtubule-associated protein that functions as a potent microtubule polymerase — lowering the critical concentration for nucleation ~100-fold, accelerating growth rates, and suppressing catastrophes via an intrinsically disordered catalytic domain — and localizes to spindle poles and spindle microtubules during mitosis where it is regulated by multiple kinases (Aurora B phosphorylates Ser627 to control localization, CDK1/cyclin B1 phosphorylates T603/S608 to maintain centrosome integrity, and Aurora Kinase A/TPX2 phosphorylates CKAP2 to reduce its microtubule affinity and limit spindle size); its protein levels are cell-cycle-controlled through transcriptional activation by Cyclin A/Cdk2-mediated Sp1 phosphorylation and through proteasomal degradation by APC/C-Cdh1 via a KEN-box during mitotic exit, and loss of CKAP2 causes reduced microtubule growth rates, kinetochore-microtubule misattachments, chromosome segregation errors, and aneuploidy.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CKAP2 is a microtubule-associated protein that functions as a potent microtubule polymerase to promote spindle assembly, chromosome segregation fidelity, and centrosome integrity during mitosis. In vitro reconstitution demonstrates that CKAP2 lowers the critical tubulin concentration for microtubule nucleation ~100-fold, accelerates growth rates, and suppresses catastrophes through an intrinsically disordered catalytic domain, while CKAP2 knockout cells exhibit reduced microtubule growth rates, kinetochore-microtubule misattachments, chromosome segregation errors, and aneuploidy [PMID:35029146, PMID:38381793]. CKAP2 protein levels are cell-cycle-controlled through transcriptional activation via Cyclin A/Cdk2-mediated Sp1 phosphorylation and proteasomal degradation by APC/C-Cdh1 through a KEN-box motif during mitotic exit, and its mitotic localization and microtubule affinity are regulated by Aurora B phosphorylation at Ser627 and CDK1/cyclin B1 phosphorylation at T603/S608 [PMID:22465120, PMID:17376772, PMID:20458174, PMID:28706298]. Loss of CKAP2 causes multipolar spindles, centrosome over-duplication, reduced spindle checkpoint activity, and chromosomal instability, establishing it as essential for mitotic fidelity [PMID:19158495, PMID:23737987].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"The initial question of whether CKAP2 interacts with the cytoskeleton was answered by demonstrating that it colocalizes with microtubules throughout the cell cycle and stabilizes them against nocodazole-induced depolymerization, establishing CKAP2 as a microtubule-associated protein.\",\n      \"evidence\": \"Immunocytochemistry and nocodazole resistance assay in mouse cells\",\n      \"pmids\": [\"15504249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab observation without biochemical demonstration of direct microtubule binding\", \"Mechanism of stabilization unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"CKAP2 was connected to the DNA damage response when it was identified as a p53 transcriptional target whose overexpression causes tetraploidy in p53-null cells but triggers p53-dependent arrest in p53-competent cells, linking microtubule stabilization to genomic integrity surveillance.\",\n      \"evidence\": \"DNA microarray, promoter assay, overexpression in p53-null vs p53-competent cells\",\n      \"pmids\": [\"16061649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p53 regulation of CKAP2 is relevant during normal cell division vs. stress only\", \"Mechanism by which CKAP2 overexpression causes tetraploidy not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The question of how CKAP2 protein levels are cleared after mitosis was resolved by showing that APC/C-Cdh1 ubiquitinates CKAP2 through a KEN-box motif, and that failure to degrade CKAP2 causes monopolar spindles and cytokinesis failure, establishing cell-cycle-dependent proteolysis as essential for proper mitotic exit.\",\n      \"evidence\": \"In vitro ubiquitination assay, KEN-box mutagenesis, non-degradable mutant expression, time-lapse microscopy; replicated by two independent labs\",\n      \"pmids\": [\"17376772\", \"17339342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other E3 ligases contribute to CKAP2 turnover\", \"How excess CKAP2 mechanistically generates monopolar spindles\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Depletion studies revealed that CKAP2 is required not only for spindle architecture but also for spindle checkpoint function, as CKAP2 knockdown causes chromosome segregation defects and reduced checkpoint activity, broadening its role beyond microtubule stabilization.\",\n      \"evidence\": \"siRNA knockdown with time-lapse video microscopy, nuclear lamina immunofluorescence, spindle checkpoint assay\",\n      \"pmids\": [\"19158495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CKAP2 influences the spindle assembly checkpoint is unknown\", \"Whether checkpoint reduction is direct or secondary to kinetochore-microtubule defects\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Aurora B was identified as a mitotic kinase that phosphorylates CKAP2 at Ser627 to control its subcellular relocalization from spindle microtubules to the chromosome region during late mitosis, providing the first mechanism for dynamic regulation of CKAP2 localization.\",\n      \"evidence\": \"In vitro kinase assay, site-directed mutagenesis, immunofluorescence localization across mitotic stages\",\n      \"pmids\": [\"20458174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ser627 phosphorylation on microtubule dynamics not measured\", \"Whether Aurora B-mediated relocalization is required for chromosome segregation fidelity\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The transcriptional mechanism driving cell-cycle-dependent CKAP2 expression was defined: Cyclin A/Cdk2 phosphorylates the transcription factor Sp1, which binds a GC box in the CKAP2 promoter to activate transcription, explaining how CKAP2 accumulates as cells approach mitosis.\",\n      \"evidence\": \"Promoter deletion/mutation analysis, ChIP, Cyclin A depletion and add-back with Sp1 binding assay\",\n      \"pmids\": [\"22465120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether additional transcription factors cooperate with Sp1\", \"Post-transcriptional regulation at the mRNA level not fully explored at this point\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"CKAP2 was shown to maintain spindle pole integrity by focusing microtubule minus ends, as its depletion disperses newly nucleated microtubules, causes multipolar spindles, and increases merotelic kinetochore attachments leading to aneuploidy.\",\n      \"evidence\": \"RNAi, microtubule regrowth assay, immunofluorescence, chromosome segregation analysis\",\n      \"pmids\": [\"23737987\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism of minus-end focusing by CKAP2 not defined\", \"Whether CKAP2 cooperates with NuMA or other pole-focusing factors\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"CDK1/cyclin B1 was identified as a second mitotic kinase regulating CKAP2 via phosphorylation at T603/S608, with phospho-dead mutants failing to rescue centrosomal abnormalities in depleted cells, demonstrating that multiple kinase inputs converge on CKAP2 to maintain centrosome integrity.\",\n      \"evidence\": \"Phosphorylation site identification, site-directed mutagenesis, rescue experiments in CKAP2-depleted cells\",\n      \"pmids\": [\"28706298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CDK1 and Aurora B phosphorylation are coordinated on CKAP2\", \"Whether phosphorylation at T603/S608 affects microtubule binding affinity directly\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"In vitro reconstitution established that CKAP2 is the most potent microtubule growth factor known, lowering the nucleation critical concentration 100-fold, increasing the growth rate constant 50-fold, and suppressing catastrophes, transforming the understanding of CKAP2 from a passive stabilizer to an active polymerase.\",\n      \"evidence\": \"TIRF microscopy-based in vitro reconstitution with purified components and single-molecule dynamics measurements\",\n      \"pmids\": [\"35029146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for how CKAP2 catalyzes tubulin addition unknown\", \"Whether polymerase activity is regulated by mitotic phosphorylation in vitro\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CRISPR knockout of endogenous CKAP2 quantitatively demonstrated reduced microtubule growth rates and increased chromosome segregation errors rescued by ectopic CKAP2 expression, validating the in vitro polymerase activity as the physiologically relevant mechanism for ensuring mitotic fidelity.\",\n      \"evidence\": \"CRISPR-Cas9 knock-in/knock-out, live cell microtubule plus-end tracking, chromosome segregation analysis with rescue\",\n      \"pmids\": [\"38381793\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of CKAP2 polymerase vs. stabilizer activity in vivo not deconvolved\", \"Whether CKAP2 loss triggers compensatory upregulation of other polymerases\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Aurora Kinase A/TPX2 was identified as a third kinase complex regulating CKAP2, phosphorylating it to reduce microtubule affinity and limit spindle size, revealing a negative feedback mechanism that constrains CKAP2 polymerase activity during mitosis.\",\n      \"evidence\": \"(preprint) Endogenous co-IP/mass spectrometry, in vitro kinase assay, phospho-mimic mutagenesis, microtubule binding and live imaging\",\n      \"pmids\": [\"bio_10.1101_2025.09.09.675182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Not yet peer-reviewed\", \"Specific phosphorylation sites on CKAP2 targeted by Aurora A not all mapped\", \"How Aurora A and CDK1 phosphorylation events are temporally coordinated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A 284-amino acid intrinsically disordered domain was identified as sufficient for both microtubule polymerization and depolymerization catalysis without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from classical polymerases like XMAP215.\",\n      \"evidence\": \"(preprint) In vitro reconstitution with isolated CKAP2 domain, polymerization/depolymerization and tubulin binding assays\",\n      \"pmids\": [\"bio_10.1101_2025.09.02.673730\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet peer-reviewed\", \"Structural basis of the catalytic domain remains undefined\", \"Whether the depolymerization activity is physiologically relevant in cells\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for CKAP2's catalytic mechanism, how the three kinase inputs (Aurora B, CDK1, Aurora A) are temporally integrated to tune CKAP2 activity across mitotic stages, and whether CKAP2's spindle checkpoint role is direct or secondary to kinetochore-microtubule attachment defects.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of CKAP2 or its catalytic domain exists\", \"Mechanism linking CKAP2 to spindle assembly checkpoint signaling undefined\", \"Physiological relevance of CKAP2's depolymerase activity in cells untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 3, 7, 13, 15]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [13, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 3, 7, 15]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [3, 9, 11]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 4, 6, 9, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CDH1\",\n      \"AURKA\",\n      \"TPX2\",\n      \"AURKB\",\n      \"CDK1\",\n      \"CCNB1\",\n      \"SP1\",\n      \"CCNA2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}