{"gene":"CENPE","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1991,"finding":"CENP-E is a novel ~250-300 kDa chromosome scaffold-associated protein that localizes to centromeres during prometaphase and metaphase, relocalizes to the spindle midplate/midbody at anaphase/telophase, and is absent during interphase. Microinjection of anti-CENP-E antibody (mAb177) into metaphase cells blocks or significantly delays progression into anaphase, establishing a required role in the metaphase-to-anaphase transition.","method":"Monoclonal antibody generation, immunofluorescence cell cycle staging, microinjection of antibody into live cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct antibody microinjection with defined mitotic phenotype, replicated in subsequent studies","pmids":["2022189"],"is_preprint":false},{"year":1992,"finding":"CENP-E is a kinesin-like motor protein (Mr ~312,000) that accumulates during G2 phase, associates with kinetochores during congression, relocalizes to the spindle midzone at anaphase, and is quantitatively degraded at the end of cell division, suggesting roles in chromosome movement and/or spindle elongation.","method":"Molecular cloning, sequence analysis, immunofluorescence cell-cycle localization, immunoblotting","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — protein identification and localization through mitosis replicated across multiple subsequent studies","pmids":["1406971"],"is_preprint":false},{"year":1994,"finding":"CENP-E levels increase progressively through S and G2 (peaking ~22,000 molecules/cell in early mitosis) due to stabilization, then are abruptly degraded (>10-fold) at the end of mitosis (after cyclin B). This degradation is independent of cytokinesis and defines a fourth point in a mitotic cascade of timed proteolysis.","method":"Centrifugal elutriation cell synchronization, immunoblotting, [35S]methionine pulse labeling, cytochalasin block","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods in single study, pulse-chase and cell-cycle fractionation confirming synthesis and degradation timing","pmids":["8207059"],"is_preprint":false},{"year":1994,"finding":"CENP-E contains a 99-amino acid C-terminal domain that cross-links microtubules at anaphase. Phosphorylation of this C-terminal domain by MPF (CDK1/cyclin B) inhibits its microtubule-binding activity, suppressing CENP-E microtubule cross-linking until anaphase when MPF activity is lost.","method":"In vitro microtubule binding/cross-linking assay, MPF phosphorylation assay, domain mapping","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with defined substrate domain and functional consequence for microtubule binding","pmids":["8023161"],"is_preprint":false},{"year":1995,"finding":"CENP-E helps couple chromosomes to depolymerizing microtubules in vitro. Antibodies to the CENP-E neck region completely block depolymerization-dependent chromosome motion, while antibodies to the head or tail slow it ~3-fold, demonstrating domain-specific contributions to microtubule end-tracking.","method":"In vitro chromosome motility assay with depolymerizing microtubules, domain-specific antibody inhibition, immunoblotting","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with domain-specific antibody perturbation showing mechanistic contribution","pmids":["7822408"],"is_preprint":false},{"year":1997,"finding":"CENP-E is a plus-end-directed microtubule motor essential for positioning chromosomes at the metaphase plate. Immunodepletion from Xenopus egg extracts prevents chromosome alignment, and antibody addition to the extracts recapitulates this phenotype. In vitro motility assays confirm plus-end directionality.","method":"Immunodepletion from Xenopus egg extracts, antibody addition, in vitro microtubule gliding/polarity assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — immunodepletion in cell-free system combined with in vitro motility assay, replicated across labs","pmids":["9363944"],"is_preprint":false},{"year":1997,"finding":"CENP-E function at kinetochores is essential for chromosome alignment: antibody microinjection prevents kinetochore assembly of CENP-E, causing two defects—monopolar chromosomes cannot establish bipolar connections, and bipolar chromosomes with normal oscillation velocities fail to align. Overexpression of an N-terminal motor-deleted truncation competitively blocks endogenous CENP-E from kinetochores and phenocopies antibody injection, implicating motor domain activity.","method":"Affinity-purified antibody microinjection, dominant-negative truncation overexpression, immunofluorescence, video microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal perturbation approaches (antibody + dominant-negative) yielding defined phenotypic readouts","pmids":["9396744"],"is_preprint":false},{"year":1997,"finding":"CENP-E is an integral component of kinetochore corona fibers. Immunoelectron microscopy shows cytoplasmic CENP-E travels along astral microtubules toward chromosomes after nuclear envelope breakdown, then localizes to the outermost kinetochore corona extending ≥50 nm from the outer plate, intertwining with spindle microtubules throughout congression and anaphase A.","method":"Immunoelectron microscopy (immuno-EM), immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct ultrastructural localization by immuno-EM, consistent with multiple independent studies","pmids":["9334346"],"is_preprint":false},{"year":1998,"finding":"A 350-amino acid domain in CENP-E mediates kinetochore binding specifically in mitosis. Yeast two-hybrid screening with this domain identified interactions with CENP-F and hBUBR1; co-immunoprecipitation from HeLa cells confirmed CENP-E and hBUBR1 associate, suggesting a motor-kinase complex. CENP-F, hBUBR1, and CENP-E assemble onto kinetochores in sequential order.","method":"Yeast two-hybrid screen, co-immunoprecipitation from HeLa cells, domain mapping, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus reciprocal co-IP in human cells identifying direct interaction partners","pmids":["9763420"],"is_preprint":false},{"year":1998,"finding":"Active MAP kinase (ERK1/2) localizes to kinetochores during mitosis and associates preferentially with CENP-E. CENP-E is phosphorylated in vitro by MAP kinase on sites known to regulate its microtubule interactions.","method":"Immunofluorescence with phospho-MAP kinase antibody, co-immunoprecipitation, in vitro kinase assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus in vitro phosphorylation, single lab, functional consequence inferred but not directly tested","pmids":["9744883"],"is_preprint":false},{"year":1999,"finding":"hBUBR1 checkpoint kinase activity is detected only after mitotic entry and is stimulated by spindle disruption. hBUBR1 monitors kinetochore activities that depend on CENP-E, and associates with the APC/cyclosome in mitotically arrested cells, suggesting dual checkpoint functions.","method":"Kinase assays, co-immunoprecipitation, immunofluorescence, cell arrest assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay and co-IP, single lab but multiple orthogonal methods","pmids":["10477750"],"is_preprint":false},{"year":2000,"finding":"CENP-E suppression by antisense yields chronically mono-oriented chromosomes, spindle pole fragmentation, and profound checkpoint activation. Immunoprecipitation reveals nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis, establishing CENP-E as a link between spindle microtubule attachment and mitotic checkpoint signaling.","method":"Antisense suppression, immunoprecipitation, immunofluorescence, spindle pole analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — stoichiometric co-IP plus defined loss-of-function phenotype, replicated in subsequent studies","pmids":["10934468"],"is_preprint":false},{"year":2000,"finding":"Farnesyl transferase inhibitors (FTIs) block farnesylation of CENP-E and CENP-F in DLD-1 cells, but FTI treatment does not prevent kinetochore localization of CENP-E; instead, it alters the association between CENP-E and microtubules.","method":"In vitro farnesyl transferase assays with CENP-E CAAX peptides, immunohistochemistry, FTI treatment of cell lines","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical substrate identification and immunolocalization, two orthogonal approaches, single lab","pmids":["10852915"],"is_preprint":false},{"year":2000,"finding":"CENP-E is required for establishing and maintaining the mitotic checkpoint in Xenopus egg extracts. Immunodepletion or antibody inhibition of CENP-E prevents extract arrest in response to spindle damage; adding high levels of soluble MAD2 restores arrest, indicating CENP-E is needed for kinetochore-dependent checkpoint signaling but not downstream steps. CENP-E directly binds BUBR1.","method":"Immunodepletion from Xenopus egg extracts, antibody inhibition, MAD2 rescue experiment","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free system reconstitution with epistasis (MAD2 rescue), replicated across labs","pmids":["11030625"],"is_preprint":false},{"year":2002,"finding":"Selective gene deletion of mouse CENP-E in primary cells causes unstable microtubule capture at kinetochores (half the normal microtubule number at aligned chromosomes, none at unaligned polar chromosomes), chromosome missegregation, and chromosomal instability. Embryonic CENP-E deletion leads to early developmental arrest.","method":"Conditional/selective gene knockout in mouse, electron microscopy of kinetochore microtubules, immunofluorescence, chromosome counting","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with ultrastructural quantification of microtubule binding, multiple cell contexts examined","pmids":["12361599"],"is_preprint":false},{"year":2003,"finding":"Aurora B kinase activity is required for kinetochore localization of CENP-E (as well as BubR1 and Mad2). Inhibition of Aurora B with ZM447439 diminishes CENP-E kinetochore localization; RNAi confirms Aurora B (not Aurora A) mediates this effect. Loss of Aurora B compromises spindle checkpoint and chromosome alignment.","method":"Small-molecule Aurora kinase inhibitor (ZM447439), RNAi, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological inhibition confirmed by RNAi, direct localization readout, widely replicated","pmids":["12719470"],"is_preprint":false},{"year":2003,"finding":"Human MPS1 (TTK) kinase is required for mitotic arrest induced by loss of CENP-E from kinetochores. MPS1 kinetochore localization in CENP-E-defective cells is sensitive to microtubule occupancy. MPS1 is required for kinetochore localization of MAD1 and MAD2 but not hBUB1 or hBUBR1.","method":"siRNA depletion, immunofluorescence, dominant-negative kinetochore targeting domain expression","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis by sequential depletion with defined localization readouts, single lab","pmids":["12686615"],"is_preprint":false},{"year":2004,"finding":"Crystal structure of the human CENP-E motor domain and linker region at 2.5 Å resolution with MgADP bound in the active site. The linker region adopts a 'docked' conformation identical to that of plus-end-directed conventional kinesin, consistent with CENP-E being a plus-end motor.","method":"X-ray crystallography (2.5 Å resolution)","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure with bound nucleotide, single study","pmids":["15236970"],"is_preprint":false},{"year":2004,"finding":"Bub1 kinase is required at the kinetochore for subsequent localization of CENP-E (as well as Cenp-F, BubR1, and Mad2) in human somatic cells. RNAi depletion of Bub1 prevents CENP-E kinetochore recruitment and increases lagging chromosomes, suggesting Bub1 acts upstream of CENP-E in the kinetochore assembly pathway.","method":"RNAi in human somatic cells, immunofluorescence quantification of kinetochore protein levels","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis by sequential RNAi with defined localization readout, consistent with Xenopus studies","pmids":["15020684"],"is_preprint":false},{"year":2005,"finding":"Direct binding of BubR1 to CENP-E activates BubR1 kinase activity. A motorless CENP-E fragment constitutively activates BubR1 at kinetochores, producing checkpoint signaling not silenced by microtubule capture or tension. In a purified ternary system, microtubule capture by the CENP-E motor domain silences BubR1 kinase activity in the BubR1-CENP-E-microtubule complex, revealing CENP-E as the signal transducer responsible for silencing checkpoint signaling upon kinetochore microtubule capture.","method":"In vitro kinase assay with purified BubR1, CENP-E, and microtubules; motorless CENP-E fragment expression; immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ternary complex assay demonstrating direct mechanism, with cellular validation","pmids":["16144904"],"is_preprint":false},{"year":2008,"finding":"CENP-E is specifically modified by SUMO-2/3 (not SUMO-1) during mitosis. CENP-E possesses SUMO-2/3 polymeric chain-binding activity via a SUMO-interacting motif (SIM) that is essential for its kinetochore localization. Global inhibition of SUMOylation causes prometaphase arrest due to failure of CENP-E targeting to kinetochores.","method":"SUMOylation assays, SUMO-2/3 binding assays, SIM mutant analysis, global SUMOylation inhibition, immunofluorescence","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — PTM identification with functional SIM mutant demonstrating necessity for localization, multiple orthogonal methods","pmids":["18374647"],"is_preprint":false},{"year":2008,"finding":"Full-length Xenopus CENP-E is an autoinhibited motor: its tail domain directly interacts with and completely blocks the motor domain's motility in vitro. Phosphorylation of the CENP-E tail by MPS1 or CDK1-cyclin B relieves this autoinhibition, activating CENP-E motility.","method":"In vitro motility assays with purified full-length Xenopus CENP-E, tail-deletion constructs, MPS1 and CDK1-cyclin B kinase assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins demonstrating autoinhibition and phosphorylation-dependent relief, two kinases tested","pmids":["18342609"],"is_preprint":false},{"year":2008,"finding":"CENP-E is a very slow (~2 nm/s), highly processive plus-end-directed motor with a 230-nm flexible coiled-coil stalk separating its kinetochore-binding and motor domains. Single-molecule assays show long microtubule attachment times. This processivity and flexibility support roles in chromosome congression and kinetochore tethering to dynamic microtubule plus ends.","method":"Single-molecule motility assays, electron microscopy of full-length Xenopus CENP-E, fluorescence imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule reconstitution with direct structural visualization of full-length protein","pmids":["18443223"],"is_preprint":false},{"year":2008,"finding":"Native purified CENP-E from mitotic HeLa cells does not induce microtubule gliding but retains microtubule-binding activity, suggesting that the native form is in a regulated (possibly autoinhibited) state.","method":"Native protein purification from mitotic HeLa cells, microtubule gliding assay, microtubule binding assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro assay, single lab, negative motility result that is mechanistically informative about regulation","pmids":["11382767"],"is_preprint":false},{"year":2009,"finding":"CENP-E motor domain associates with microtubules unusually slowly (0.08 µM⁻¹s⁻¹), followed by slow ADP release (0.9 s⁻¹), but fast ATP binding and hydrolysis; motor dissociation rate (~1.4 s⁻¹) matches the stepping rate. This high duty cycle explains CENP-E processivity. The slow microtubule association step is proposed to favor binding to stable kinetochore fibers over dynamic microtubules.","method":"Pre-steady-state and steady-state kinetics (stopped-flow, fluorescence), ATPase assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — detailed enzymological characterization with multiple kinetic parameters, single lab rigorous study","pmids":["22637578"],"is_preprint":false},{"year":2009,"finding":"CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules, linking CENP-E to regulation of kinetochore microtubule poleward flux and turnover. CENP-E was identified as a CLASP1 interactor by proteomic approach.","method":"Proteomic co-purification (MS), co-immunoprecipitation, RNAi, immunofluorescence, microtubule flux/turnover measurements (FRAP)","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — MS identification plus co-IP, RNAi with functional readout of flux rates, motor-dead mutant used","pmids":["19733075"],"is_preprint":false},{"year":2010,"finding":"Aurora kinases A and B phosphorylate a conserved residue on CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site; Aurora phosphorylation disrupts PP1 binding. At spindle poles, Aurora phosphorylation of CENP-E reduces its affinity for individual microtubules, promoting towing of polar chromosomes. Dephosphorylation of CENP-E (or PP1 rebinding) is required for subsequent stable end-on kinetochore-microtubule capture, revealing an Aurora/PP1 switch controlling both congression and stable attachment.","method":"In vitro kinase assays, single-molecule motility assays, phospho-specific antibodies, PP1-binding domain mutants, immunofluorescence, live-cell imaging","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical reconstitution combined with mutagenesis and single-molecule assays plus cellular validation, single rigorous study","pmids":["20691903"],"is_preprint":false},{"year":2010,"finding":"CENP-E promotes microtubule plus-end elongation in vitro in an ATP-dependent manner. ~60% of polarity-marked microtubules show CENP-E-dependent plus-end elongation at ~1.48 µm/30 min. CENP-E localizes to elongating plus ends. The kinetics fit a single exponential (k_obs = 5.1 s⁻¹), suggesting tubulin addition coupled to ATP turnover.","method":"In vitro microtubule polymerization assay with purified human CENP-E, real-time fluorescence microscopy, immunolocalization on microtubules","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution, single lab, novel activity not extensively corroborated","pmids":["20797864"],"is_preprint":false},{"year":2011,"finding":"CENP-E interacts with SKAP via its C-terminal tail in vitro and in vivo. SKAP depletion by RNAi dramatically reduces inter-kinetochore tension, phenocopying CENP-E depletion. SKAP localizes to kinetochore corona fibers. SKAP and CENP-E synergistically promote microtubule binding in vitro.","method":"Co-immunoprecipitation, in vitro pulldown, siRNA, immunoelectron microscopy, microtubule co-sedimentation assay, inter-kinetochore distance measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus in vitro pulldown and immuno-EM, single lab","pmids":["22110139"],"is_preprint":false},{"year":2013,"finding":"After chromosomes have congressed, CENP-E converts from a lateral microtubule transporter into a bidirectional microtubule tip-tracker that maintains association with both assembling and disassembling microtubule tips. Laser-trapping single-molecule experiments and computational modeling demonstrate this tip-tracking relies on both motor and tail domains.","method":"Single-molecule motility assays, laser trapping, computational modeling, fluorescence microscopy","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule reconstitution with optical trapping and domain mutants, plus computational modeling","pmids":["23955301"],"is_preprint":false},{"year":2013,"finding":"CENP-E tethers laterally attached kinetochores to microtubule walls (wall-tethering), which is a required step prior to end-on conversion. MCAK is separately needed to release lateral microtubules after partial end-on attachment. Together, CENP-E and MCAK define sequential deterministic steps in lateral-to-end-on attachment conversion.","method":"High-resolution live-cell imaging assay for attachment intermediates, siRNA depletion of CENP-E and MCAK, fluorescence microscopy","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — imaging of defined attachment intermediates with functional depletion, single lab","pmids":["23891108"],"is_preprint":false},{"year":2013,"finding":"The C-terminal non-kinesin microtubule-binding domain of CENP-E binds microtubules with affinity similar to the Ndc80 complex. Electron microscopy shows this domain engages microtubules in a disordered manner with no favored binding geometry, suggesting a role in initial lateral attachments.","method":"Microtubule co-sedimentation assay, electron microscopy of microtubule-bound domain","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical and structural characterization, single lab, limited functional validation","pmids":["23892111"],"is_preprint":false},{"year":2014,"finding":"The elongated coiled-coil stalk of CENP-E is required for stable kinetochore-microtubule end-on attachment. 'Bonsai' CENP-E with significantly shortened stalk but intact motor and tail domains fails to bind microtubules in vitro unless cargo is bound to its tail. In cells, Bonsai CENP-E causes chromosome misalignment and lagging chromosomes, demonstrating the stalk is required for kinetochore-microtubule attachment stability.","method":"Stalk-truncation mutant ('Bonsai CENP-E'), in vitro microtubule binding assay, live-cell imaging, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with structure-function mutant, corroborated by cell biology experiments","pmids":["24920822"],"is_preprint":false},{"year":2016,"finding":"SUMOylated NKAP protein is required for CENP-E kinetochore localization. NKAP is SUMOylated predominantly during mitosis, and SUMOylation is necessary for NKAP to bind CENP-E. Bub3 recruits NKAP to kinetochores to stabilize CENP-E binding to BubR1. A SUMOylation-deficient NKAP mutant cannot support CENP-E kinetochore localization or chromosome alignment.","method":"RNAi, SUMOylation assays, co-immunoprecipitation, NKAP SUMOylation-deficient mutant rescue experiments, immunofluorescence","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional mutant rescue, single lab, two orthogonal approaches","pmids":["27694884"],"is_preprint":false},{"year":2018,"finding":"CENP-E and CENP-F directly and specifically interact with BUBR1 and BUB1, respectively, through biochemical reconstitution. The CENP-E/BUBR1 interaction requires a dimeric coiled-coil in CENP-E and the kinase domain of BUBR1. BUBR1 is dispensable for kinetochore localization of CENP-E, whereas BUB1 is stringently required for CENP-F localization. CENP-E and CENP-F are recruited to kinetochores independently of the RZZ complex.","method":"Biochemical reconstitution with purified proteins, co-immunoprecipitation, RNAi, immunofluorescence, domain mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical reconstitution establishing direct interaction, complemented by cell biology epistasis experiments","pmids":["29748388"],"is_preprint":false},{"year":2019,"finding":"BubR1 is a bona fide kinase that phosphorylates CENP-E, switching it from a laterally-attached microtubule transporter to a plus-end microtubule tip-tracker. BubR1-mediated CENP-E phosphorylation is required for proper microtubule capture at kinetochores and for assembly of the central spindle/midzone at mitotic exit. Computational modeling identified bubristatin as a selective BubR1 kinase antagonist.","method":"Crystal structure of Drosophila BubR1 kinase domain, in vitro kinase assay, single-molecule motility assays, live-cell imaging, computational modeling and inhibitor design","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure determination, in vitro kinase assay, single-molecule functional assay, and cell biology validation with pharmacological inhibitor","pmids":["31201382"],"is_preprint":false},{"year":2019,"finding":"LUBAC ubiquitin ligase catalyzes linear ubiquitination of CENP-E, which is specifically required for CENP-E localization at attached kinetochores (but not unattached ones). KNL1 acts as a receptor for linear ubiquitin chains to anchor CENP-E at attached kinetochores. This mechanism promotes chromosome congression by coupling CENP-E to the KMN network.","method":"Co-immunoprecipitation, linear ubiquitination assay, KNL1 interaction assay, RNAi/knockout, immunofluorescence with attachment status tracking","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical demonstration of ubiquitination plus co-IP with KNL1 receptor, supported by cell biology, single lab","pmids":["30655516"],"is_preprint":false},{"year":2015,"finding":"TRAMM/TrappC12 interacts with CENP-E and is required for its kinetochore recruitment. TRAMM is phosphorylated early in mitosis and dephosphorylated at anaphase onset; this phosphorylation state correlates with CENP-E association. A phosphomimetic TRAMM mutant recruits CENP-E to kinetochores more efficiently than the non-phosphorylatable form.","method":"Co-immunoprecipitation, RNAi, phosphomimetic and non-phosphorylatable mutants, immunofluorescence","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus phospho-mutant functional rescue, single lab","pmids":["25918224"],"is_preprint":false},{"year":2015,"finding":"CTCF interacts with CENP-E both in vitro and in vivo via its C-terminal zinc fingers, and recruits CENP-E to pericentric/centromeric DNA early in mitosis. Overexpression of a CENP-E fragment targeted to CTCF sites delays chromosome alignment during mitosis, suggesting physiological relevance of CTCF-mediated CENP-E recruitment.","method":"Co-immunoprecipitation, ChIP, in vitro binding assay, dominant-negative fragment overexpression, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP and in vitro binding plus dominant-negative effect, single lab, mechanistic link not fully dissected","pmids":["26321640"],"is_preprint":false},{"year":2021,"finding":"Poly-SUMO-2/3 chain modification of Nuf2 (a kinetochore protein) facilitates CENP-E kinetochore localization and chromosome congression. Nuf2-Ubc9 fusion (stimulating Nuf2 SUMOylation) or Nuf2-SUMO-2 trimer fusion rescues CENP-E kinetochore localization when global SUMOylation is inhibited. The rescue requires a functional SIM in CENP-E. SUMO-2/3 monomer or dimer, or SUMO-1 trimer modifications, cannot rescue, demonstrating specificity for poly-SUMO-2/3 chains.","method":"Fusion protein rescue experiments, SUMO chain-specific binding assay, immunofluorescence, global sumoylation inhibition","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple rescue mutants with isotype and length specificity controls, mechanistically dissecting SIM requirement","pmids":["33910471"],"is_preprint":false},{"year":2006,"finding":"CENP-E interacts with Skp1 at the midbody via its coiled-coil domain (residues 955-1571), and this interaction is required for cytokinesis. Skp1 siRNA causes accumulation of telophase cells with elongated midbodies and elevated CENP-E levels. Overexpression of Skp1 lacking the CENP-E-binding domain confirms that Skp1-CENP-E interaction is essential for faithful cytokinesis, suggesting SCF-mediated degradation of CENP-E at the midbody is required for mitotic exit.","method":"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, siRNA, immunofluorescence, domain mapping","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP and functional rescue experiment, single lab","pmids":["16682006"],"is_preprint":false},{"year":2023,"finding":"Aurora A phosphorylates CENP-E at spindle poles to promote chromosome congression and prevent accumulation of corona proteins (fibrous corona components) at centrosomes, enabling their redistribution. Aurora B phosphorylates CENP-E at kinetochores to release it from an autoinhibited state and prevent premature removal by dynein, thereby controlling fibrous corona disassembly timing.","method":"Phospho-specific antibodies, Aurora kinase inhibitors, dominant-negative and phospho-mutant CENP-E constructs, live-cell imaging, immunofluorescence","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — two Aurora kinase-specific phosphorylation events with distinct spatial contexts defined by phospho-mutants and inhibitors, coupled to defined cellular phenotypes","pmids":["37658044"],"is_preprint":false},{"year":2023,"finding":"CENP-E is required to retain RZZS (ROD-Zwilch-ZW10-Spindly) at kinetochores when corona assembly is prevented by MPS1 inhibition. With active MPS1, CENP-E is dispensable for corona expansion but strictly required for physiological kinetochore accumulation of dynein-dynactin. CENP-E and the RZZ-Spindly complex form an integrated platform to recruit dynein to the kinetochore corona.","method":"MPS1 inhibition, CENP-E depletion/rescue, RZZS phosphomimetic mutants, immunofluorescence quantification","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with phosphomimetic bypass mutant, single lab, defined localization readouts","pmids":["37984321"],"is_preprint":false},{"year":2023,"finding":"HPV16 E6 oncoprotein causes proteasome-dependent degradation of CENP-E through the E6-associated ubiquitin ligase E6AP/UBE3A, producing polar chromosomes and chromosomal instability independently of p53 degradation.","method":"HPV16 E6 expression in cell lines, proteasome inhibitor experiments, E6AP knockdown, p53-independent cell lines, chromosome congression imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — E6AP requirement demonstrated by knockdown plus proteasome inhibitor rescue, p53-independence verified in parallel","pmids":["36989302"],"is_preprint":false},{"year":2020,"finding":"CENP-E forms a complex with PRC1 in mitotic cells (identified via biotinylated syntelin affinity matrix). Chemical inhibition of CENP-E in metaphase perturbs temporal assembly of PRC1 to the spindle midzone, preventing accurate central spindle assembly. This identifies a role for CENP-E in organizing kinetochore microtubules into stable midzone arrays at the metaphase-anaphase transition.","method":"Affinity purification (biotinylated syntelin), co-immunoprecipitation, chemical inhibition (syntelin), live-cell light sheet microscopy of 3D organoids and 2D culture","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification-identified interaction combined with pharmacological inhibition and live imaging, single lab","pmids":["31174204"],"is_preprint":false},{"year":2009,"finding":"KIF18A physically interacts with CENP-E and BubR1 during mitosis (co-immunoprecipitation). KIF18A depletion causes specific downregulation of CENP-E through enhanced protein degradation (not reduced transcription). Ectopic expression of wild-type CENP-E tail domain, but not a corresponding mutant, rescues chromosome congression defects from KIF18A silencing.","method":"Co-immunoprecipitation, siRNA, immunofluorescence, protein stability analysis (transcription vs. degradation), rescue by CENP-E tail overexpression","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus domain-specific rescue, single lab, mechanism of degradation not fully characterized","pmids":["19625775"],"is_preprint":false}],"current_model":"CENP-E (KIF10) is a highly processive, slow, plus-end-directed kinesin-7 motor that resides in the kinetochore fibrous corona during mitosis, where it (1) captures lateral microtubules and tows polar chromosomes toward the spindle equator; (2) converts from a lateral transporter to a bidirectional tip-tracker that stabilizes end-on kinetochore-microtubule attachments; (3) directly binds and activates the BubR1 checkpoint kinase, with BubR1 kinase activity reciprocally phosphorylating CENP-E to switch it from lateral to end-on engagement, while microtubule capture by CENP-E silences BubR1-dependent checkpoint signaling; (4) recruits CLASPs and PRC1 to regulate microtubule dynamics and central spindle assembly; and (5) is itself regulated by a multi-layered phosphorylation and ubiquitin/SUMO network—Aurora A/B phosphorylation relieves autoinhibition and controls fibrous corona disassembly, PP1 binding promotes stable microtubule capture, CDK1/MPF phosphorylates the C-terminus to suppress microtubule cross-linking until anaphase, and SUMO-2/3 chain binding via a SIM motif (dependent on Nuf2 SUMOylation) is required for kinetochore targeting—while its levels are controlled by cyclin-like accumulation in G2 followed by proteasome-dependent degradation at mitotic exit."},"narrative":{"mechanistic_narrative":"CENP-E (KIF10) is a kinesin-like motor protein that drives chromosome congression and couples kinetochore-microtubule attachment to mitotic checkpoint control during the metaphase-to-anaphase transition [PMID:1406971, PMID:9396744, PMID:10934468]. It is expressed cell-cycle-dependently, accumulating through S/G2 to peak in early mitosis and being abruptly degraded at mitotic exit, with antibody perturbation blocking anaphase onset [PMID:2022189, PMID:8207059]. As an integral component of the kinetochore fibrous corona, cytoplasmic CENP-E travels along astral microtubules to chromosomes after nuclear envelope breakdown and concentrates at the outermost corona [PMID:9334346]. It is a very slow (~2 nm/s), highly processive, plus-end-directed motor with a high-duty-cycle ATPase whose slow microtubule-association step favors binding to stable kinetochore fibers, and whose 230-nm flexible coiled-coil stalk separates motor and kinetochore-binding domains and is required for stable end-on attachment [PMID:18443223, PMID:22637578, PMID:24920822]. CENP-E positions chromosomes at the metaphase plate by capturing lateral microtubules and towing polar chromosomes, then converts to a bidirectional tip-tracker that maintains attachment to assembling and disassembling plus ends, with both motor and tail domains contributing [PMID:9363944, PMID:23955301, PMID:23891108]. CENP-E directly binds the checkpoint kinase BubR1 in near-stoichiometric complex, and microtubule capture by the CENP-E motor silences BubR1-dependent checkpoint signaling while BubR1 reciprocally phosphorylates CENP-E to switch it from lateral transporter to plus-end tip-tracker [PMID:10934468, PMID:16144904, PMID:31201382]. Its activity is multiply regulated: full-length CENP-E is autoinhibited by its tail, relieved by MPS1 or CDK1-cyclin B phosphorylation [PMID:18342609]; an Aurora/PP1 switch controls congression versus stable attachment, with distinct Aurora A and Aurora B phosphorylation events governing corona dynamics [PMID:20691903, PMID:37658044]; and CDK1/MPF phosphorylation of a C-terminal domain suppresses microtubule cross-linking until anaphase [PMID:8023161]. Kinetochore targeting requires SUMO-2/3 chain binding via a SIM motif, dependent on poly-SUMOylation of Nuf2 and SUMOylated NKAP [PMID:18374647, PMID:33910471, PMID:27694884]. CENP-E additionally recruits CLASP1/2 to regulate microtubule flux, scaffolds dynein/RZZS recruitment to the corona, and forms a complex with PRC1 for central spindle assembly [PMID:19733075, PMID:37984321, PMID:31174204]. Genetic deletion in mouse cells destabilizes kinetochore microtubule capture and causes chromosome missegregation and chromosomal instability [PMID:12361599].","teleology":[{"year":1992,"claim":"Establishing CENP-E as a kinesin-family motor that cycles through mitosis explained how a centromere protein could physically move chromosomes, framing it as a mechanochemical effector rather than a passive scaffold.","evidence":"Molecular cloning, sequence analysis, and immunofluorescence cell-cycle localization","pmids":["1406971","2022189"],"confidence":"High","gaps":["Directionality and in vitro motility not yet demonstrated","Kinetochore-binding domain unmapped"]},{"year":1994,"claim":"Defining timed accumulation and abrupt degradation, plus MPF phosphorylation of a microtubule cross-linking domain, showed CENP-E activity is temporally gated within the mitotic proteolytic and kinase cascade.","evidence":"Elutriation synchronization, pulse-chase immunoblotting, and in vitro MPF kinase/microtubule cross-linking assays","pmids":["8207059","8023161"],"confidence":"High","gaps":["Ubiquitin ligase responsible for degradation not identified here","Physiological cross-linking targets at anaphase undefined"]},{"year":1997,"claim":"Immunodepletion, antibody injection, and immuno-EM established CENP-E as a plus-end-directed motor in the kinetochore corona essential for aligning chromosomes at the metaphase plate.","evidence":"Xenopus egg extract depletion, in vitro polarity/motility assays, antibody microinjection, and immunoelectron microscopy","pmids":["9363944","9396744","9334346"],"confidence":"High","gaps":["How motor activity is coupled to kinetochore tethering not resolved","Lateral vs end-on attachment modes not yet distinguished"]},{"year":1998,"claim":"Mapping a mitosis-specific kinetochore-binding domain and identifying BubR1 and CENP-F partners revealed CENP-E as part of a motor-kinase complex assembled in defined order.","evidence":"Yeast two-hybrid screen, co-immunoprecipitation from HeLa cells, and sequential-assembly immunofluorescence","pmids":["9763420"],"confidence":"High","gaps":["Functional consequence of BubR1 binding not yet tested","Direct vs indirect nature of CENP-F association unclear"]},{"year":2000,"claim":"Loss-of-function in cells and extracts coupled to stoichiometric BubR1 association established CENP-E as the link transducing microtubule-attachment status into checkpoint signaling.","evidence":"Antisense suppression, immunodepletion with MAD2 rescue epistasis, and immunoprecipitation","pmids":["10934468","11030625"],"confidence":"High","gaps":["Whether CENP-E activates BubR1 kinase not yet shown","Molecular switch coupling capture to silencing undefined"]},{"year":2003,"claim":"Sequential kinase-dependence experiments placed Aurora B, Bub1, and MPS1 upstream of CENP-E kinetochore recruitment, defining the assembly hierarchy for the motor.","evidence":"Pharmacological inhibition (ZM447439), RNAi, and immunofluorescence localization","pmids":["12719470","12686615","15020684"],"confidence":"High","gaps":["Direct molecular receptors for CENP-E at the kinetochore not identified","Whether kinase effects are direct or via intermediate proteins unclear"]},{"year":2004,"claim":"The motor-domain crystal structure with MgADP and a docked linker confirmed at atomic resolution the plus-end directionality inferred from motility assays.","evidence":"X-ray crystallography at 2.5 Angstrom of the human motor domain and linker","pmids":["15236970"],"confidence":"High","gaps":["Full-length architecture and tail regulation not captured","Microtubule-bound state not resolved"]},{"year":2005,"claim":"Reconstitution of a BubR1-CENP-E-microtubule ternary complex showed CENP-E binding activates BubR1 kinase and that motor-mediated microtubule capture silences this signaling, defining the mechanistic core of attachment-to-checkpoint coupling.","evidence":"In vitro kinase assays with purified components plus motorless-fragment expression in cells","pmids":["16144904"],"confidence":"High","gaps":["Reciprocal phosphorylation of CENP-E by BubR1 not yet demonstrated","In vivo relevance of silencing step partly inferred"]},{"year":2002,"claim":"Mouse gene deletion provided in vivo proof that CENP-E is required for stable kinetochore microtubule capture and chromosome stability, linking its molecular activity to chromosomal instability.","evidence":"Conditional knockout with EM quantification of kinetochore microtubules and chromosome counting","pmids":["12361599"],"confidence":"High","gaps":["Molecular basis of microtubule-number reduction not dissected","Tissue-specific requirements not fully mapped"]},{"year":2008,"claim":"Single-molecule and structural work defined CENP-E as a slow, highly processive motor with a long flexible stalk that is autoinhibited by its tail and relieved by MPS1/CDK1 phosphorylation, explaining how its activity is spatially and temporally licensed.","evidence":"Single-molecule motility, EM of full-length Xenopus protein, tail-deletion constructs, and kinase assays","pmids":["18443223","18342609","11382767"],"confidence":"High","gaps":["In vivo phosphorylation sites mediating relief not fully mapped","Coordination of autoinhibition with kinetochore loading unclear"]},{"year":2008,"claim":"Identifying SUMO-2/3 chain binding via a SIM motif as essential for kinetochore targeting revealed a non-phosphorylation PTM-reader mechanism for CENP-E localization.","evidence":"SUMOylation and SUMO-chain binding assays, SIM mutant analysis, and global SUMOylation inhibition","pmids":["18374647"],"confidence":"High","gaps":["SUMOylated kinetochore receptor not yet identified","Whether CENP-E itself or its receptor carries the chain unresolved"]},{"year":2009,"claim":"Enzymological kinetics and CLASP recruitment work explained how CENP-E processivity favors stable fibers and linked the motor to kinetochore microtubule flux regulation.","evidence":"Pre-steady-state ATPase kinetics, MS-based interactor identification, co-IP, and FRAP flux measurements","pmids":["22637578","19733075"],"confidence":"High","gaps":["How the slow association step is structurally encoded unclear","Mechanism by which CENP-E delivers CLASPs not defined"]},{"year":2010,"claim":"An Aurora/PP1 phosphorylation switch and demonstration of ATP-dependent plus-end elongation showed how CENP-E affinity for microtubules is tuned to first tow polar chromosomes then form stable attachments.","evidence":"In vitro kinase and single-molecule assays, PP1-binding mutants, phospho-antibodies, and microtubule polymerization assays","pmids":["20691903","20797864"],"confidence":"High","gaps":["The plus-end elongation activity (Medium) is from a single lab and not extensively corroborated","Coupling of PP1 rebinding to end-on conversion in vivo partly inferred"]},{"year":2013,"claim":"Single-molecule trapping, attachment-intermediate imaging, and stalk-truncation studies defined how CENP-E converts from a lateral transporter and wall-tether into a bidirectional tip-tracker dependent on motor, tail, and stalk domains.","evidence":"Laser trapping, computational modeling, live-cell attachment-intermediate imaging, and Bonsai stalk mutant assays","pmids":["23955301","23891108","23892111","24920822"],"confidence":"High","gaps":["Molecular trigger of the lateral-to-end-on conversion not fully resolved","Quantitative contribution of the non-kinesin microtubule-binding domain in vivo unclear"]},{"year":2018,"claim":"Biochemical reconstitution established that CENP-E binds BubR1 directly through a dimeric coiled-coil engaging the BubR1 kinase domain, and that CENP-E loads onto kinetochores independently of the RZZ complex.","evidence":"Reconstitution with purified proteins, co-IP, RNAi epistasis, and domain mapping","pmids":["29748388"],"confidence":"High","gaps":["Hierarchy relative to SUMO and ubiquitin receptors not integrated","Whether BubR1 binding contributes to CENP-E retention unclear"]},{"year":2019,"claim":"Demonstrating BubR1 is a genuine kinase that phosphorylates CENP-E to switch it from transporter to tip-tracker closed the reciprocal regulatory loop with the checkpoint kinase and tied it to central spindle assembly.","evidence":"BubR1 kinase domain crystal structure, in vitro kinase and single-molecule assays, live imaging, and inhibitor design","pmids":["31201382"],"confidence":"High","gaps":["Phospho-site identity on CENP-E not fully defined here","In vivo timing relative to attachment status partly modeled"]},{"year":2015,"claim":"Identification of TRAMM/TrappC12 and CTCF as CENP-E recruiters expanded the set of localization determinants beyond canonical kinetochore proteins to include phospho-regulated and chromatin-anchored pathways.","evidence":"Co-IP, ChIP, phosphomimetic/non-phosphorylatable and dominant-negative constructs, and immunofluorescence","pmids":["25918224","26321640"],"confidence":"Medium","gaps":["TRAMM and CTCF links rest on single-lab co-IP plus mutant assays without reconstitution","How these pathways integrate with SUMO/ubiquitin recruitment unclear"]},{"year":2016,"claim":"Showing SUMOylated NKAP, recruited by Bub3, is required to stabilize CENP-E-BubR1 binding connected the SUMO-dependent localization mechanism to a specific kinetochore adaptor.","evidence":"RNAi, SUMOylation assays, co-IP, and SUMOylation-deficient mutant rescue","pmids":["27694884"],"confidence":"Medium","gaps":["Single-lab evidence; relationship to the CENP-E SIM not fully reconciled","Whether NKAP SUMOylation is the chain read by the CENP-E SIM untested"]},{"year":2019,"claim":"LUBAC-catalyzed linear ubiquitination read by KNL1 was shown to anchor CENP-E specifically at attached kinetochores, adding a ubiquitin-dependent retention mechanism coupled to the KMN network.","evidence":"Linear ubiquitination assays, KNL1 interaction assays, RNAi/knockout, and attachment-status immunofluorescence","pmids":["30655516"],"confidence":"Medium","gaps":["Single-lab demonstration; the ubiquitinated residues on CENP-E not mapped","How this coexists with SUMO-dependent targeting unclear"]},{"year":2020,"claim":"Identifying a CENP-E-PRC1 complex established a role for CENP-E in organizing kinetochore microtubules into stable midzone arrays at the metaphase-anaphase transition.","evidence":"Biotinylated-syntelin affinity purification, co-IP, chemical inhibition, and light-sheet live imaging","pmids":["31174204"],"confidence":"Medium","gaps":["Single-lab affinity-capture interaction; directness of CENP-E-PRC1 binding unclear","Whether motor activity is needed for midzone assembly untested"]},{"year":2023,"claim":"Distinct Aurora A (pole) and Aurora B (kinetochore) phosphorylation events, and a CENP-E/RZZS platform for dynein recruitment, revealed how CENP-E spatially controls fibrous corona dynamics and dynein loading.","evidence":"Phospho-specific antibodies, Aurora inhibitors, phospho-mutant constructs, MPS1 inhibition, RZZS phosphomimetics, and live imaging","pmids":["37658044","37984321"],"confidence":"High","gaps":["The EMBO dynein-platform study (Medium) is single-lab","How corona disassembly timing integrates with checkpoint silencing unresolved"]},{"year":2023,"claim":"Demonstrating HPV16 E6/E6AP-driven proteasomal degradation of CENP-E producing chromosomal instability connected CENP-E turnover to viral oncogenesis independently of p53.","evidence":"HPV16 E6 expression, proteasome inhibitor rescue, E6AP knockdown in p53-independent cells, and congression imaging","pmids":["36989302"],"confidence":"Medium","gaps":["Single-lab; direct CENP-E ubiquitination by E6AP not shown","Relevance to spontaneous tumor CIN beyond HPV context unclear"]},{"year":null,"claim":"How the multiple parallel recruitment inputs (SUMO-2/3 chains, linear ubiquitin/KNL1, BubR1, NKAP, TRAMM, CTCF, RZZS) are integrated and ordered to set CENP-E levels, residence time, and attachment-mode at a single kinetochore remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling SUMO, ubiquitin, and protein-protein receptor pathways","Quantitative stoichiometry of CENP-E loading mechanisms unknown","Direct in vivo phospho-site map across regulatory kinases incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003774","term_label":"cytoskeletal motor activity","supporting_discovery_ids":[5,22,24,29]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[24,27]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,31,32]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,19,25,42]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[26,41]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7,27]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,5,11,14]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[42,44]}],"complexes":["kinetochore fibrous corona","BubR1-CENP-E complex"],"partners":["BUBR1","CENP-F","CLASP1","SKAP","PRC1","KIF18A","SKP1","KNL1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q02224","full_name":"Centromere-associated protein E","aliases":["Centromere protein E","CENP-E","Kinesin-7","Kinesin-related protein CENPE"],"length_aa":2701,"mass_kda":316.4,"function":"Microtubule plus-end-directed kinetochore motor which plays an important role in chromosome congression, microtubule-kinetochore conjugation and spindle assembly checkpoint activation. Drives chromosome congression (alignment of chromosomes at the spindle equator resulting in the formation of the metaphase plate) by mediating the lateral sliding of polar chromosomes along spindle microtubules towards the spindle equator and by aiding the establishment and maintenance of connections between kinetochores and spindle microtubules (PubMed:23891108, PubMed:25395579, PubMed:7889940). The transport of pole-proximal chromosomes towards the spindle equator is favored by microtubule tracks that are detyrosinated (PubMed:25908662). Acts as a processive bi-directional tracker of dynamic microtubule tips; after chromosomes have congressed, continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends (PubMed:23955301). Suppresses chromosome congression in NDC80-depleted cells and contributes positively to congression only when microtubules are stabilized (PubMed:25743205). Plays an important role in the formation of stable attachments between kinetochores and spindle microtubules (PubMed:17535814) The stabilization of kinetochore-microtubule attachment also requires CENPE-dependent localization of other proteins to the kinetochore including BUB1B, MAD1 and MAD2. Plays a role in spindle assembly checkpoint activation (SAC) via its interaction with BUB1B resulting in the activation of its kinase activity, which is important for activating SAC. Necessary for the mitotic checkpoint signal at individual kinetochores to prevent aneuploidy due to single chromosome loss (By similarity)","subcellular_location":"Chromosome, centromere, kinetochore; Cytoplasm, cytoskeleton, spindle; Chromosome, centromere","url":"https://www.uniprot.org/uniprotkb/Q02224/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPE","classification":"Common Essential","n_dependent_lines":973,"n_total_lines":1208,"dependency_fraction":0.8054635761589404},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLASP1","stoichiometry":0.2},{"gene":"CLASP2","stoichiometry":0.2},{"gene":"IPO5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CENPE","total_profiled":1310},"omim":[{"mim_id":"616327","title":"CHROMOSOME ALIGNMENT-MAINTAINING PHOSPHOPROTEIN 1; CHAMP1","url":"https://www.omim.org/entry/616327"},{"mim_id":"616051","title":"MICROCEPHALY 13, PRIMARY, AUTOSOMAL RECESSIVE; MCPH13","url":"https://www.omim.org/entry/616051"},{"mim_id":"614718","title":"KINETOCHORE-LOCALIZED ASTRIN/SPAG5-BINDING PROTEIN; KNSTRN","url":"https://www.omim.org/entry/614718"},{"mim_id":"614140","title":"SPERM ANTIGEN WITH CALPONIN HOMOLOGY AND COILED-COIL DOMAINS 1-LIKE; SPECC1L","url":"https://www.omim.org/entry/614140"},{"mim_id":"611772","title":"NUF2 COMPONENT OF NDC80 KINETOCHORE COMPLEX; NUF2","url":"https://www.omim.org/entry/611772"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Microtubules","reliability":"Supported"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":9.4},{"tissue":"lymphoid tissue","ntpm":8.6}],"url":"https://www.proteinatlas.org/search/CENPE"},"hgnc":{"alias_symbol":["KIF10","PPP1R61"],"prev_symbol":[]},"alphafold":{"accession":"Q02224","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02224","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q02224-3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q02224-3-F1-predicted_aligned_error_v6.png","plddt_mean":54.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPE","jax_strain_url":"https://www.jax.org/strain/search?query=CENPE"},"sequence":{"accession":"Q02224","fasta_url":"https://rest.uniprot.org/uniprotkb/Q02224.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q02224/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02224"}},"corpus_meta":[{"pmid":"12719470","id":"PMC_12719470","title":"Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.","date":"2003","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/12719470","citation_count":1052,"is_preprint":false},{"pmid":"1406971","id":"PMC_1406971","title":"CENP-E is a putative kinetochore motor that accumulates just before mitosis.","date":"1992","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/1406971","citation_count":375,"is_preprint":false},{"pmid":"9363944","id":"PMC_9363944","title":"CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment.","date":"1997","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/9363944","citation_count":358,"is_preprint":false},{"pmid":"2022189","id":"PMC_2022189","title":"CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.","date":"1991","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/2022189","citation_count":346,"is_preprint":false},{"pmid":"10934468","id":"PMC_10934468","title":"CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint.","date":"2000","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10934468","citation_count":320,"is_preprint":false},{"pmid":"10477750","id":"PMC_10477750","title":"Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10477750","citation_count":314,"is_preprint":false},{"pmid":"10852915","id":"PMC_10852915","title":"Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10852915","citation_count":292,"is_preprint":false},{"pmid":"15020684","id":"PMC_15020684","title":"Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.","date":"2004","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/15020684","citation_count":288,"is_preprint":false},{"pmid":"9396744","id":"PMC_9396744","title":"CENP-E function at kinetochores is essential for chromosome alignment.","date":"1997","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9396744","citation_count":276,"is_preprint":false},{"pmid":"12361599","id":"PMC_12361599","title":"Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E.","date":"2002","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/12361599","citation_count":267,"is_preprint":false},{"pmid":"9763420","id":"PMC_9763420","title":"Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1.","date":"1998","source":"The Journal of cell 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23816042","citation_count":13,"is_preprint":false},{"pmid":"33910471","id":"PMC_33910471","title":"Poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis.","date":"2021","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/33910471","citation_count":12,"is_preprint":false},{"pmid":"32442423","id":"PMC_32442423","title":"Distinct Mechanisms of Resistance to a CENP-E Inhibitor Emerge in Near-Haploid and Diploid Cancer Cells.","date":"2020","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/32442423","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47401,"output_tokens":11306,"usd":0.155896,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":22087,"output_tokens":6889,"usd":0.14133,"stage2_stop_reason":"end_turn"},"total_usd":0.297226,"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\": 1991,\n      \"finding\": \"CENP-E is a novel ~250-300 kDa chromosome scaffold-associated protein that localizes to centromeres during prometaphase and metaphase, relocalizes to the spindle midplate/midbody at anaphase/telophase, and is absent during interphase. Microinjection of anti-CENP-E antibody (mAb177) into metaphase cells blocks or significantly delays progression into anaphase, establishing a required role in the metaphase-to-anaphase transition.\",\n      \"method\": \"Monoclonal antibody generation, immunofluorescence cell cycle staging, microinjection of antibody into live cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct antibody microinjection with defined mitotic phenotype, replicated in subsequent studies\",\n      \"pmids\": [\"2022189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"CENP-E is a kinesin-like motor protein (Mr ~312,000) that accumulates during G2 phase, associates with kinetochores during congression, relocalizes to the spindle midzone at anaphase, and is quantitatively degraded at the end of cell division, suggesting roles in chromosome movement and/or spindle elongation.\",\n      \"method\": \"Molecular cloning, sequence analysis, immunofluorescence cell-cycle localization, immunoblotting\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — protein identification and localization through mitosis replicated across multiple subsequent studies\",\n      \"pmids\": [\"1406971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CENP-E levels increase progressively through S and G2 (peaking ~22,000 molecules/cell in early mitosis) due to stabilization, then are abruptly degraded (>10-fold) at the end of mitosis (after cyclin B). This degradation is independent of cytokinesis and defines a fourth point in a mitotic cascade of timed proteolysis.\",\n      \"method\": \"Centrifugal elutriation cell synchronization, immunoblotting, [35S]methionine pulse labeling, cytochalasin block\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods in single study, pulse-chase and cell-cycle fractionation confirming synthesis and degradation timing\",\n      \"pmids\": [\"8207059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CENP-E contains a 99-amino acid C-terminal domain that cross-links microtubules at anaphase. Phosphorylation of this C-terminal domain by MPF (CDK1/cyclin B) inhibits its microtubule-binding activity, suppressing CENP-E microtubule cross-linking until anaphase when MPF activity is lost.\",\n      \"method\": \"In vitro microtubule binding/cross-linking assay, MPF phosphorylation assay, domain mapping\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with defined substrate domain and functional consequence for microtubule binding\",\n      \"pmids\": [\"8023161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CENP-E helps couple chromosomes to depolymerizing microtubules in vitro. Antibodies to the CENP-E neck region completely block depolymerization-dependent chromosome motion, while antibodies to the head or tail slow it ~3-fold, demonstrating domain-specific contributions to microtubule end-tracking.\",\n      \"method\": \"In vitro chromosome motility assay with depolymerizing microtubules, domain-specific antibody inhibition, immunoblotting\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with domain-specific antibody perturbation showing mechanistic contribution\",\n      \"pmids\": [\"7822408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CENP-E is a plus-end-directed microtubule motor essential for positioning chromosomes at the metaphase plate. Immunodepletion from Xenopus egg extracts prevents chromosome alignment, and antibody addition to the extracts recapitulates this phenotype. In vitro motility assays confirm plus-end directionality.\",\n      \"method\": \"Immunodepletion from Xenopus egg extracts, antibody addition, in vitro microtubule gliding/polarity assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — immunodepletion in cell-free system combined with in vitro motility assay, replicated across labs\",\n      \"pmids\": [\"9363944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CENP-E function at kinetochores is essential for chromosome alignment: antibody microinjection prevents kinetochore assembly of CENP-E, causing two defects—monopolar chromosomes cannot establish bipolar connections, and bipolar chromosomes with normal oscillation velocities fail to align. Overexpression of an N-terminal motor-deleted truncation competitively blocks endogenous CENP-E from kinetochores and phenocopies antibody injection, implicating motor domain activity.\",\n      \"method\": \"Affinity-purified antibody microinjection, dominant-negative truncation overexpression, immunofluorescence, video microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal perturbation approaches (antibody + dominant-negative) yielding defined phenotypic readouts\",\n      \"pmids\": [\"9396744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CENP-E is an integral component of kinetochore corona fibers. Immunoelectron microscopy shows cytoplasmic CENP-E travels along astral microtubules toward chromosomes after nuclear envelope breakdown, then localizes to the outermost kinetochore corona extending ≥50 nm from the outer plate, intertwining with spindle microtubules throughout congression and anaphase A.\",\n      \"method\": \"Immunoelectron microscopy (immuno-EM), immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct ultrastructural localization by immuno-EM, consistent with multiple independent studies\",\n      \"pmids\": [\"9334346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A 350-amino acid domain in CENP-E mediates kinetochore binding specifically in mitosis. Yeast two-hybrid screening with this domain identified interactions with CENP-F and hBUBR1; co-immunoprecipitation from HeLa cells confirmed CENP-E and hBUBR1 associate, suggesting a motor-kinase complex. CENP-F, hBUBR1, and CENP-E assemble onto kinetochores in sequential order.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation from HeLa cells, domain mapping, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus reciprocal co-IP in human cells identifying direct interaction partners\",\n      \"pmids\": [\"9763420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Active MAP kinase (ERK1/2) localizes to kinetochores during mitosis and associates preferentially with CENP-E. CENP-E is phosphorylated in vitro by MAP kinase on sites known to regulate its microtubule interactions.\",\n      \"method\": \"Immunofluorescence with phospho-MAP kinase antibody, co-immunoprecipitation, in vitro kinase assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus in vitro phosphorylation, single lab, functional consequence inferred but not directly tested\",\n      \"pmids\": [\"9744883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"hBUBR1 checkpoint kinase activity is detected only after mitotic entry and is stimulated by spindle disruption. hBUBR1 monitors kinetochore activities that depend on CENP-E, and associates with the APC/cyclosome in mitotically arrested cells, suggesting dual checkpoint functions.\",\n      \"method\": \"Kinase assays, co-immunoprecipitation, immunofluorescence, cell arrest assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay and co-IP, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"10477750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CENP-E suppression by antisense yields chronically mono-oriented chromosomes, spindle pole fragmentation, and profound checkpoint activation. Immunoprecipitation reveals nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis, establishing CENP-E as a link between spindle microtubule attachment and mitotic checkpoint signaling.\",\n      \"method\": \"Antisense suppression, immunoprecipitation, immunofluorescence, spindle pole analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — stoichiometric co-IP plus defined loss-of-function phenotype, replicated in subsequent studies\",\n      \"pmids\": [\"10934468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Farnesyl transferase inhibitors (FTIs) block farnesylation of CENP-E and CENP-F in DLD-1 cells, but FTI treatment does not prevent kinetochore localization of CENP-E; instead, it alters the association between CENP-E and microtubules.\",\n      \"method\": \"In vitro farnesyl transferase assays with CENP-E CAAX peptides, immunohistochemistry, FTI treatment of cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical substrate identification and immunolocalization, two orthogonal approaches, single lab\",\n      \"pmids\": [\"10852915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CENP-E is required for establishing and maintaining the mitotic checkpoint in Xenopus egg extracts. Immunodepletion or antibody inhibition of CENP-E prevents extract arrest in response to spindle damage; adding high levels of soluble MAD2 restores arrest, indicating CENP-E is needed for kinetochore-dependent checkpoint signaling but not downstream steps. CENP-E directly binds BUBR1.\",\n      \"method\": \"Immunodepletion from Xenopus egg extracts, antibody inhibition, MAD2 rescue experiment\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free system reconstitution with epistasis (MAD2 rescue), replicated across labs\",\n      \"pmids\": [\"11030625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Selective gene deletion of mouse CENP-E in primary cells causes unstable microtubule capture at kinetochores (half the normal microtubule number at aligned chromosomes, none at unaligned polar chromosomes), chromosome missegregation, and chromosomal instability. Embryonic CENP-E deletion leads to early developmental arrest.\",\n      \"method\": \"Conditional/selective gene knockout in mouse, electron microscopy of kinetochore microtubules, immunofluorescence, chromosome counting\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with ultrastructural quantification of microtubule binding, multiple cell contexts examined\",\n      \"pmids\": [\"12361599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Aurora B kinase activity is required for kinetochore localization of CENP-E (as well as BubR1 and Mad2). Inhibition of Aurora B with ZM447439 diminishes CENP-E kinetochore localization; RNAi confirms Aurora B (not Aurora A) mediates this effect. Loss of Aurora B compromises spindle checkpoint and chromosome alignment.\",\n      \"method\": \"Small-molecule Aurora kinase inhibitor (ZM447439), RNAi, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological inhibition confirmed by RNAi, direct localization readout, widely replicated\",\n      \"pmids\": [\"12719470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human MPS1 (TTK) kinase is required for mitotic arrest induced by loss of CENP-E from kinetochores. MPS1 kinetochore localization in CENP-E-defective cells is sensitive to microtubule occupancy. MPS1 is required for kinetochore localization of MAD1 and MAD2 but not hBUB1 or hBUBR1.\",\n      \"method\": \"siRNA depletion, immunofluorescence, dominant-negative kinetochore targeting domain expression\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis by sequential depletion with defined localization readouts, single lab\",\n      \"pmids\": [\"12686615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crystal structure of the human CENP-E motor domain and linker region at 2.5 Å resolution with MgADP bound in the active site. The linker region adopts a 'docked' conformation identical to that of plus-end-directed conventional kinesin, consistent with CENP-E being a plus-end motor.\",\n      \"method\": \"X-ray crystallography (2.5 Å resolution)\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure with bound nucleotide, single study\",\n      \"pmids\": [\"15236970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Bub1 kinase is required at the kinetochore for subsequent localization of CENP-E (as well as Cenp-F, BubR1, and Mad2) in human somatic cells. RNAi depletion of Bub1 prevents CENP-E kinetochore recruitment and increases lagging chromosomes, suggesting Bub1 acts upstream of CENP-E in the kinetochore assembly pathway.\",\n      \"method\": \"RNAi in human somatic cells, immunofluorescence quantification of kinetochore protein levels\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis by sequential RNAi with defined localization readout, consistent with Xenopus studies\",\n      \"pmids\": [\"15020684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Direct binding of BubR1 to CENP-E activates BubR1 kinase activity. A motorless CENP-E fragment constitutively activates BubR1 at kinetochores, producing checkpoint signaling not silenced by microtubule capture or tension. In a purified ternary system, microtubule capture by the CENP-E motor domain silences BubR1 kinase activity in the BubR1-CENP-E-microtubule complex, revealing CENP-E as the signal transducer responsible for silencing checkpoint signaling upon kinetochore microtubule capture.\",\n      \"method\": \"In vitro kinase assay with purified BubR1, CENP-E, and microtubules; motorless CENP-E fragment expression; immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ternary complex assay demonstrating direct mechanism, with cellular validation\",\n      \"pmids\": [\"16144904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CENP-E is specifically modified by SUMO-2/3 (not SUMO-1) during mitosis. CENP-E possesses SUMO-2/3 polymeric chain-binding activity via a SUMO-interacting motif (SIM) that is essential for its kinetochore localization. Global inhibition of SUMOylation causes prometaphase arrest due to failure of CENP-E targeting to kinetochores.\",\n      \"method\": \"SUMOylation assays, SUMO-2/3 binding assays, SIM mutant analysis, global SUMOylation inhibition, immunofluorescence\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — PTM identification with functional SIM mutant demonstrating necessity for localization, multiple orthogonal methods\",\n      \"pmids\": [\"18374647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Full-length Xenopus CENP-E is an autoinhibited motor: its tail domain directly interacts with and completely blocks the motor domain's motility in vitro. Phosphorylation of the CENP-E tail by MPS1 or CDK1-cyclin B relieves this autoinhibition, activating CENP-E motility.\",\n      \"method\": \"In vitro motility assays with purified full-length Xenopus CENP-E, tail-deletion constructs, MPS1 and CDK1-cyclin B kinase assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins demonstrating autoinhibition and phosphorylation-dependent relief, two kinases tested\",\n      \"pmids\": [\"18342609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CENP-E is a very slow (~2 nm/s), highly processive plus-end-directed motor with a 230-nm flexible coiled-coil stalk separating its kinetochore-binding and motor domains. Single-molecule assays show long microtubule attachment times. This processivity and flexibility support roles in chromosome congression and kinetochore tethering to dynamic microtubule plus ends.\",\n      \"method\": \"Single-molecule motility assays, electron microscopy of full-length Xenopus CENP-E, fluorescence imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule reconstitution with direct structural visualization of full-length protein\",\n      \"pmids\": [\"18443223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Native purified CENP-E from mitotic HeLa cells does not induce microtubule gliding but retains microtubule-binding activity, suggesting that the native form is in a regulated (possibly autoinhibited) state.\",\n      \"method\": \"Native protein purification from mitotic HeLa cells, microtubule gliding assay, microtubule binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro assay, single lab, negative motility result that is mechanistically informative about regulation\",\n      \"pmids\": [\"11382767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-E motor domain associates with microtubules unusually slowly (0.08 µM⁻¹s⁻¹), followed by slow ADP release (0.9 s⁻¹), but fast ATP binding and hydrolysis; motor dissociation rate (~1.4 s⁻¹) matches the stepping rate. This high duty cycle explains CENP-E processivity. The slow microtubule association step is proposed to favor binding to stable kinetochore fibers over dynamic microtubules.\",\n      \"method\": \"Pre-steady-state and steady-state kinetics (stopped-flow, fluorescence), ATPase assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — detailed enzymological characterization with multiple kinetic parameters, single lab rigorous study\",\n      \"pmids\": [\"22637578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules, linking CENP-E to regulation of kinetochore microtubule poleward flux and turnover. CENP-E was identified as a CLASP1 interactor by proteomic approach.\",\n      \"method\": \"Proteomic co-purification (MS), co-immunoprecipitation, RNAi, immunofluorescence, microtubule flux/turnover measurements (FRAP)\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus co-IP, RNAi with functional readout of flux rates, motor-dead mutant used\",\n      \"pmids\": [\"19733075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Aurora kinases A and B phosphorylate a conserved residue on CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site; Aurora phosphorylation disrupts PP1 binding. At spindle poles, Aurora phosphorylation of CENP-E reduces its affinity for individual microtubules, promoting towing of polar chromosomes. Dephosphorylation of CENP-E (or PP1 rebinding) is required for subsequent stable end-on kinetochore-microtubule capture, revealing an Aurora/PP1 switch controlling both congression and stable attachment.\",\n      \"method\": \"In vitro kinase assays, single-molecule motility assays, phospho-specific antibodies, PP1-binding domain mutants, immunofluorescence, live-cell imaging\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical reconstitution combined with mutagenesis and single-molecule assays plus cellular validation, single rigorous study\",\n      \"pmids\": [\"20691903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CENP-E promotes microtubule plus-end elongation in vitro in an ATP-dependent manner. ~60% of polarity-marked microtubules show CENP-E-dependent plus-end elongation at ~1.48 µm/30 min. CENP-E localizes to elongating plus ends. The kinetics fit a single exponential (k_obs = 5.1 s⁻¹), suggesting tubulin addition coupled to ATP turnover.\",\n      \"method\": \"In vitro microtubule polymerization assay with purified human CENP-E, real-time fluorescence microscopy, immunolocalization on microtubules\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution, single lab, novel activity not extensively corroborated\",\n      \"pmids\": [\"20797864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CENP-E interacts with SKAP via its C-terminal tail in vitro and in vivo. SKAP depletion by RNAi dramatically reduces inter-kinetochore tension, phenocopying CENP-E depletion. SKAP localizes to kinetochore corona fibers. SKAP and CENP-E synergistically promote microtubule binding in vitro.\",\n      \"method\": \"Co-immunoprecipitation, in vitro pulldown, siRNA, immunoelectron microscopy, microtubule co-sedimentation assay, inter-kinetochore distance measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus in vitro pulldown and immuno-EM, single lab\",\n      \"pmids\": [\"22110139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"After chromosomes have congressed, CENP-E converts from a lateral microtubule transporter into a bidirectional microtubule tip-tracker that maintains association with both assembling and disassembling microtubule tips. Laser-trapping single-molecule experiments and computational modeling demonstrate this tip-tracking relies on both motor and tail domains.\",\n      \"method\": \"Single-molecule motility assays, laser trapping, computational modeling, fluorescence microscopy\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule reconstitution with optical trapping and domain mutants, plus computational modeling\",\n      \"pmids\": [\"23955301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CENP-E tethers laterally attached kinetochores to microtubule walls (wall-tethering), which is a required step prior to end-on conversion. MCAK is separately needed to release lateral microtubules after partial end-on attachment. Together, CENP-E and MCAK define sequential deterministic steps in lateral-to-end-on attachment conversion.\",\n      \"method\": \"High-resolution live-cell imaging assay for attachment intermediates, siRNA depletion of CENP-E and MCAK, fluorescence microscopy\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — imaging of defined attachment intermediates with functional depletion, single lab\",\n      \"pmids\": [\"23891108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The C-terminal non-kinesin microtubule-binding domain of CENP-E binds microtubules with affinity similar to the Ndc80 complex. Electron microscopy shows this domain engages microtubules in a disordered manner with no favored binding geometry, suggesting a role in initial lateral attachments.\",\n      \"method\": \"Microtubule co-sedimentation assay, electron microscopy of microtubule-bound domain\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical and structural characterization, single lab, limited functional validation\",\n      \"pmids\": [\"23892111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The elongated coiled-coil stalk of CENP-E is required for stable kinetochore-microtubule end-on attachment. 'Bonsai' CENP-E with significantly shortened stalk but intact motor and tail domains fails to bind microtubules in vitro unless cargo is bound to its tail. In cells, Bonsai CENP-E causes chromosome misalignment and lagging chromosomes, demonstrating the stalk is required for kinetochore-microtubule attachment stability.\",\n      \"method\": \"Stalk-truncation mutant ('Bonsai CENP-E'), in vitro microtubule binding assay, live-cell imaging, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with structure-function mutant, corroborated by cell biology experiments\",\n      \"pmids\": [\"24920822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SUMOylated NKAP protein is required for CENP-E kinetochore localization. NKAP is SUMOylated predominantly during mitosis, and SUMOylation is necessary for NKAP to bind CENP-E. Bub3 recruits NKAP to kinetochores to stabilize CENP-E binding to BubR1. A SUMOylation-deficient NKAP mutant cannot support CENP-E kinetochore localization or chromosome alignment.\",\n      \"method\": \"RNAi, SUMOylation assays, co-immunoprecipitation, NKAP SUMOylation-deficient mutant rescue experiments, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional mutant rescue, single lab, two orthogonal approaches\",\n      \"pmids\": [\"27694884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CENP-E and CENP-F directly and specifically interact with BUBR1 and BUB1, respectively, through biochemical reconstitution. The CENP-E/BUBR1 interaction requires a dimeric coiled-coil in CENP-E and the kinase domain of BUBR1. BUBR1 is dispensable for kinetochore localization of CENP-E, whereas BUB1 is stringently required for CENP-F localization. CENP-E and CENP-F are recruited to kinetochores independently of the RZZ complex.\",\n      \"method\": \"Biochemical reconstitution with purified proteins, co-immunoprecipitation, RNAi, immunofluorescence, domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical reconstitution establishing direct interaction, complemented by cell biology epistasis experiments\",\n      \"pmids\": [\"29748388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BubR1 is a bona fide kinase that phosphorylates CENP-E, switching it from a laterally-attached microtubule transporter to a plus-end microtubule tip-tracker. BubR1-mediated CENP-E phosphorylation is required for proper microtubule capture at kinetochores and for assembly of the central spindle/midzone at mitotic exit. Computational modeling identified bubristatin as a selective BubR1 kinase antagonist.\",\n      \"method\": \"Crystal structure of Drosophila BubR1 kinase domain, in vitro kinase assay, single-molecule motility assays, live-cell imaging, computational modeling and inhibitor design\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure determination, in vitro kinase assay, single-molecule functional assay, and cell biology validation with pharmacological inhibitor\",\n      \"pmids\": [\"31201382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LUBAC ubiquitin ligase catalyzes linear ubiquitination of CENP-E, which is specifically required for CENP-E localization at attached kinetochores (but not unattached ones). KNL1 acts as a receptor for linear ubiquitin chains to anchor CENP-E at attached kinetochores. This mechanism promotes chromosome congression by coupling CENP-E to the KMN network.\",\n      \"method\": \"Co-immunoprecipitation, linear ubiquitination assay, KNL1 interaction assay, RNAi/knockout, immunofluorescence with attachment status tracking\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical demonstration of ubiquitination plus co-IP with KNL1 receptor, supported by cell biology, single lab\",\n      \"pmids\": [\"30655516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRAMM/TrappC12 interacts with CENP-E and is required for its kinetochore recruitment. TRAMM is phosphorylated early in mitosis and dephosphorylated at anaphase onset; this phosphorylation state correlates with CENP-E association. A phosphomimetic TRAMM mutant recruits CENP-E to kinetochores more efficiently than the non-phosphorylatable form.\",\n      \"method\": \"Co-immunoprecipitation, RNAi, phosphomimetic and non-phosphorylatable mutants, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus phospho-mutant functional rescue, single lab\",\n      \"pmids\": [\"25918224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CTCF interacts with CENP-E both in vitro and in vivo via its C-terminal zinc fingers, and recruits CENP-E to pericentric/centromeric DNA early in mitosis. Overexpression of a CENP-E fragment targeted to CTCF sites delays chromosome alignment during mitosis, suggesting physiological relevance of CTCF-mediated CENP-E recruitment.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, in vitro binding assay, dominant-negative fragment overexpression, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP and in vitro binding plus dominant-negative effect, single lab, mechanistic link not fully dissected\",\n      \"pmids\": [\"26321640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Poly-SUMO-2/3 chain modification of Nuf2 (a kinetochore protein) facilitates CENP-E kinetochore localization and chromosome congression. Nuf2-Ubc9 fusion (stimulating Nuf2 SUMOylation) or Nuf2-SUMO-2 trimer fusion rescues CENP-E kinetochore localization when global SUMOylation is inhibited. The rescue requires a functional SIM in CENP-E. SUMO-2/3 monomer or dimer, or SUMO-1 trimer modifications, cannot rescue, demonstrating specificity for poly-SUMO-2/3 chains.\",\n      \"method\": \"Fusion protein rescue experiments, SUMO chain-specific binding assay, immunofluorescence, global sumoylation inhibition\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple rescue mutants with isotype and length specificity controls, mechanistically dissecting SIM requirement\",\n      \"pmids\": [\"33910471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CENP-E interacts with Skp1 at the midbody via its coiled-coil domain (residues 955-1571), and this interaction is required for cytokinesis. Skp1 siRNA causes accumulation of telophase cells with elongated midbodies and elevated CENP-E levels. Overexpression of Skp1 lacking the CENP-E-binding domain confirms that Skp1-CENP-E interaction is essential for faithful cytokinesis, suggesting SCF-mediated degradation of CENP-E at the midbody is required for mitotic exit.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, siRNA, immunofluorescence, domain mapping\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP and functional rescue experiment, single lab\",\n      \"pmids\": [\"16682006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Aurora A phosphorylates CENP-E at spindle poles to promote chromosome congression and prevent accumulation of corona proteins (fibrous corona components) at centrosomes, enabling their redistribution. Aurora B phosphorylates CENP-E at kinetochores to release it from an autoinhibited state and prevent premature removal by dynein, thereby controlling fibrous corona disassembly timing.\",\n      \"method\": \"Phospho-specific antibodies, Aurora kinase inhibitors, dominant-negative and phospho-mutant CENP-E constructs, live-cell imaging, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two Aurora kinase-specific phosphorylation events with distinct spatial contexts defined by phospho-mutants and inhibitors, coupled to defined cellular phenotypes\",\n      \"pmids\": [\"37658044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CENP-E is required to retain RZZS (ROD-Zwilch-ZW10-Spindly) at kinetochores when corona assembly is prevented by MPS1 inhibition. With active MPS1, CENP-E is dispensable for corona expansion but strictly required for physiological kinetochore accumulation of dynein-dynactin. CENP-E and the RZZ-Spindly complex form an integrated platform to recruit dynein to the kinetochore corona.\",\n      \"method\": \"MPS1 inhibition, CENP-E depletion/rescue, RZZS phosphomimetic mutants, immunofluorescence quantification\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with phosphomimetic bypass mutant, single lab, defined localization readouts\",\n      \"pmids\": [\"37984321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HPV16 E6 oncoprotein causes proteasome-dependent degradation of CENP-E through the E6-associated ubiquitin ligase E6AP/UBE3A, producing polar chromosomes and chromosomal instability independently of p53 degradation.\",\n      \"method\": \"HPV16 E6 expression in cell lines, proteasome inhibitor experiments, E6AP knockdown, p53-independent cell lines, chromosome congression imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — E6AP requirement demonstrated by knockdown plus proteasome inhibitor rescue, p53-independence verified in parallel\",\n      \"pmids\": [\"36989302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CENP-E forms a complex with PRC1 in mitotic cells (identified via biotinylated syntelin affinity matrix). Chemical inhibition of CENP-E in metaphase perturbs temporal assembly of PRC1 to the spindle midzone, preventing accurate central spindle assembly. This identifies a role for CENP-E in organizing kinetochore microtubules into stable midzone arrays at the metaphase-anaphase transition.\",\n      \"method\": \"Affinity purification (biotinylated syntelin), co-immunoprecipitation, chemical inhibition (syntelin), live-cell light sheet microscopy of 3D organoids and 2D culture\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification-identified interaction combined with pharmacological inhibition and live imaging, single lab\",\n      \"pmids\": [\"31174204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KIF18A physically interacts with CENP-E and BubR1 during mitosis (co-immunoprecipitation). KIF18A depletion causes specific downregulation of CENP-E through enhanced protein degradation (not reduced transcription). Ectopic expression of wild-type CENP-E tail domain, but not a corresponding mutant, rescues chromosome congression defects from KIF18A silencing.\",\n      \"method\": \"Co-immunoprecipitation, siRNA, immunofluorescence, protein stability analysis (transcription vs. degradation), rescue by CENP-E tail overexpression\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus domain-specific rescue, single lab, mechanism of degradation not fully characterized\",\n      \"pmids\": [\"19625775\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-E (KIF10) is a highly processive, slow, plus-end-directed kinesin-7 motor that resides in the kinetochore fibrous corona during mitosis, where it (1) captures lateral microtubules and tows polar chromosomes toward the spindle equator; (2) converts from a lateral transporter to a bidirectional tip-tracker that stabilizes end-on kinetochore-microtubule attachments; (3) directly binds and activates the BubR1 checkpoint kinase, with BubR1 kinase activity reciprocally phosphorylating CENP-E to switch it from lateral to end-on engagement, while microtubule capture by CENP-E silences BubR1-dependent checkpoint signaling; (4) recruits CLASPs and PRC1 to regulate microtubule dynamics and central spindle assembly; and (5) is itself regulated by a multi-layered phosphorylation and ubiquitin/SUMO network—Aurora A/B phosphorylation relieves autoinhibition and controls fibrous corona disassembly, PP1 binding promotes stable microtubule capture, CDK1/MPF phosphorylates the C-terminus to suppress microtubule cross-linking until anaphase, and SUMO-2/3 chain binding via a SIM motif (dependent on Nuf2 SUMOylation) is required for kinetochore targeting—while its levels are controlled by cyclin-like accumulation in G2 followed by proteasome-dependent degradation at mitotic exit.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENP-E (KIF10) is a kinesin-like motor protein that drives chromosome congression and couples kinetochore-microtubule attachment to mitotic checkpoint control during the metaphase-to-anaphase transition [#1, #6, #11]. It is expressed cell-cycle-dependently, accumulating through S/G2 to peak in early mitosis and being abruptly degraded at mitotic exit, with antibody perturbation blocking anaphase onset [#0, #2]. As an integral component of the kinetochore fibrous corona, cytoplasmic CENP-E travels along astral microtubules to chromosomes after nuclear envelope breakdown and concentrates at the outermost corona [#7]. It is a very slow (~2 nm/s), highly processive, plus-end-directed motor with a high-duty-cycle ATPase whose slow microtubule-association step favors binding to stable kinetochore fibers, and whose 230-nm flexible coiled-coil stalk separates motor and kinetochore-binding domains and is required for stable end-on attachment [#22, #24, #32]. CENP-E positions chromosomes at the metaphase plate by capturing lateral microtubules and towing polar chromosomes, then converts to a bidirectional tip-tracker that maintains attachment to assembling and disassembling plus ends, with both motor and tail domains contributing [#5, #29, #30]. CENP-E directly binds the checkpoint kinase BubR1 in near-stoichiometric complex, and microtubule capture by the CENP-E motor silences BubR1-dependent checkpoint signaling while BubR1 reciprocally phosphorylates CENP-E to switch it from lateral transporter to plus-end tip-tracker [#11, #19, #35]. Its activity is multiply regulated: full-length CENP-E is autoinhibited by its tail, relieved by MPS1 or CDK1-cyclin B phosphorylation [#21]; an Aurora/PP1 switch controls congression versus stable attachment, with distinct Aurora A and Aurora B phosphorylation events governing corona dynamics [#26, #41]; and CDK1/MPF phosphorylation of a C-terminal domain suppresses microtubule cross-linking until anaphase [#3]. Kinetochore targeting requires SUMO-2/3 chain binding via a SIM motif, dependent on poly-SUMOylation of Nuf2 and SUMOylated NKAP [#20, #39, #33]. CENP-E additionally recruits CLASP1/2 to regulate microtubule flux, scaffolds dynein/RZZS recruitment to the corona, and forms a complex with PRC1 for central spindle assembly [#25, #42, #44]. Genetic deletion in mouse cells destabilizes kinetochore microtubule capture and causes chromosome missegregation and chromosomal instability [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Establishing CENP-E as a kinesin-family motor that cycles through mitosis explained how a centromere protein could physically move chromosomes, framing it as a mechanochemical effector rather than a passive scaffold.\",\n      \"evidence\": \"Molecular cloning, sequence analysis, and immunofluorescence cell-cycle localization\",\n      \"pmids\": [\"1406971\", \"2022189\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Directionality and in vitro motility not yet demonstrated\", \"Kinetochore-binding domain unmapped\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Defining timed accumulation and abrupt degradation, plus MPF phosphorylation of a microtubule cross-linking domain, showed CENP-E activity is temporally gated within the mitotic proteolytic and kinase cascade.\",\n      \"evidence\": \"Elutriation synchronization, pulse-chase immunoblotting, and in vitro MPF kinase/microtubule cross-linking assays\",\n      \"pmids\": [\"8207059\", \"8023161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin ligase responsible for degradation not identified here\", \"Physiological cross-linking targets at anaphase undefined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Immunodepletion, antibody injection, and immuno-EM established CENP-E as a plus-end-directed motor in the kinetochore corona essential for aligning chromosomes at the metaphase plate.\",\n      \"evidence\": \"Xenopus egg extract depletion, in vitro polarity/motility assays, antibody microinjection, and immunoelectron microscopy\",\n      \"pmids\": [\"9363944\", \"9396744\", \"9334346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How motor activity is coupled to kinetochore tethering not resolved\", \"Lateral vs end-on attachment modes not yet distinguished\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Mapping a mitosis-specific kinetochore-binding domain and identifying BubR1 and CENP-F partners revealed CENP-E as part of a motor-kinase complex assembled in defined order.\",\n      \"evidence\": \"Yeast two-hybrid screen, co-immunoprecipitation from HeLa cells, and sequential-assembly immunofluorescence\",\n      \"pmids\": [\"9763420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of BubR1 binding not yet tested\", \"Direct vs indirect nature of CENP-F association unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Loss-of-function in cells and extracts coupled to stoichiometric BubR1 association established CENP-E as the link transducing microtubule-attachment status into checkpoint signaling.\",\n      \"evidence\": \"Antisense suppression, immunodepletion with MAD2 rescue epistasis, and immunoprecipitation\",\n      \"pmids\": [\"10934468\", \"11030625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CENP-E activates BubR1 kinase not yet shown\", \"Molecular switch coupling capture to silencing undefined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Sequential kinase-dependence experiments placed Aurora B, Bub1, and MPS1 upstream of CENP-E kinetochore recruitment, defining the assembly hierarchy for the motor.\",\n      \"evidence\": \"Pharmacological inhibition (ZM447439), RNAi, and immunofluorescence localization\",\n      \"pmids\": [\"12719470\", \"12686615\", \"15020684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular receptors for CENP-E at the kinetochore not identified\", \"Whether kinase effects are direct or via intermediate proteins unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The motor-domain crystal structure with MgADP and a docked linker confirmed at atomic resolution the plus-end directionality inferred from motility assays.\",\n      \"evidence\": \"X-ray crystallography at 2.5 Angstrom of the human motor domain and linker\",\n      \"pmids\": [\"15236970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length architecture and tail regulation not captured\", \"Microtubule-bound state not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Reconstitution of a BubR1-CENP-E-microtubule ternary complex showed CENP-E binding activates BubR1 kinase and that motor-mediated microtubule capture silences this signaling, defining the mechanistic core of attachment-to-checkpoint coupling.\",\n      \"evidence\": \"In vitro kinase assays with purified components plus motorless-fragment expression in cells\",\n      \"pmids\": [\"16144904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reciprocal phosphorylation of CENP-E by BubR1 not yet demonstrated\", \"In vivo relevance of silencing step partly inferred\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mouse gene deletion provided in vivo proof that CENP-E is required for stable kinetochore microtubule capture and chromosome stability, linking its molecular activity to chromosomal instability.\",\n      \"evidence\": \"Conditional knockout with EM quantification of kinetochore microtubules and chromosome counting\",\n      \"pmids\": [\"12361599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of microtubule-number reduction not dissected\", \"Tissue-specific requirements not fully mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Single-molecule and structural work defined CENP-E as a slow, highly processive motor with a long flexible stalk that is autoinhibited by its tail and relieved by MPS1/CDK1 phosphorylation, explaining how its activity is spatially and temporally licensed.\",\n      \"evidence\": \"Single-molecule motility, EM of full-length Xenopus protein, tail-deletion constructs, and kinase assays\",\n      \"pmids\": [\"18443223\", \"18342609\", \"11382767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo phosphorylation sites mediating relief not fully mapped\", \"Coordination of autoinhibition with kinetochore loading unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying SUMO-2/3 chain binding via a SIM motif as essential for kinetochore targeting revealed a non-phosphorylation PTM-reader mechanism for CENP-E localization.\",\n      \"evidence\": \"SUMOylation and SUMO-chain binding assays, SIM mutant analysis, and global SUMOylation inhibition\",\n      \"pmids\": [\"18374647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMOylated kinetochore receptor not yet identified\", \"Whether CENP-E itself or its receptor carries the chain unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Enzymological kinetics and CLASP recruitment work explained how CENP-E processivity favors stable fibers and linked the motor to kinetochore microtubule flux regulation.\",\n      \"evidence\": \"Pre-steady-state ATPase kinetics, MS-based interactor identification, co-IP, and FRAP flux measurements\",\n      \"pmids\": [\"22637578\", \"19733075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the slow association step is structurally encoded unclear\", \"Mechanism by which CENP-E delivers CLASPs not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"An Aurora/PP1 phosphorylation switch and demonstration of ATP-dependent plus-end elongation showed how CENP-E affinity for microtubules is tuned to first tow polar chromosomes then form stable attachments.\",\n      \"evidence\": \"In vitro kinase and single-molecule assays, PP1-binding mutants, phospho-antibodies, and microtubule polymerization assays\",\n      \"pmids\": [\"20691903\", \"20797864\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The plus-end elongation activity (Medium) is from a single lab and not extensively corroborated\", \"Coupling of PP1 rebinding to end-on conversion in vivo partly inferred\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Single-molecule trapping, attachment-intermediate imaging, and stalk-truncation studies defined how CENP-E converts from a lateral transporter and wall-tether into a bidirectional tip-tracker dependent on motor, tail, and stalk domains.\",\n      \"evidence\": \"Laser trapping, computational modeling, live-cell attachment-intermediate imaging, and Bonsai stalk mutant assays\",\n      \"pmids\": [\"23955301\", \"23891108\", \"23892111\", \"24920822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger of the lateral-to-end-on conversion not fully resolved\", \"Quantitative contribution of the non-kinesin microtubule-binding domain in vivo unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Biochemical reconstitution established that CENP-E binds BubR1 directly through a dimeric coiled-coil engaging the BubR1 kinase domain, and that CENP-E loads onto kinetochores independently of the RZZ complex.\",\n      \"evidence\": \"Reconstitution with purified proteins, co-IP, RNAi epistasis, and domain mapping\",\n      \"pmids\": [\"29748388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy relative to SUMO and ubiquitin receptors not integrated\", \"Whether BubR1 binding contributes to CENP-E retention unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating BubR1 is a genuine kinase that phosphorylates CENP-E to switch it from transporter to tip-tracker closed the reciprocal regulatory loop with the checkpoint kinase and tied it to central spindle assembly.\",\n      \"evidence\": \"BubR1 kinase domain crystal structure, in vitro kinase and single-molecule assays, live imaging, and inhibitor design\",\n      \"pmids\": [\"31201382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phospho-site identity on CENP-E not fully defined here\", \"In vivo timing relative to attachment status partly modeled\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of TRAMM/TrappC12 and CTCF as CENP-E recruiters expanded the set of localization determinants beyond canonical kinetochore proteins to include phospho-regulated and chromatin-anchored pathways.\",\n      \"evidence\": \"Co-IP, ChIP, phosphomimetic/non-phosphorylatable and dominant-negative constructs, and immunofluorescence\",\n      \"pmids\": [\"25918224\", \"26321640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRAMM and CTCF links rest on single-lab co-IP plus mutant assays without reconstitution\", \"How these pathways integrate with SUMO/ubiquitin recruitment unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing SUMOylated NKAP, recruited by Bub3, is required to stabilize CENP-E-BubR1 binding connected the SUMO-dependent localization mechanism to a specific kinetochore adaptor.\",\n      \"evidence\": \"RNAi, SUMOylation assays, co-IP, and SUMOylation-deficient mutant rescue\",\n      \"pmids\": [\"27694884\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab evidence; relationship to the CENP-E SIM not fully reconciled\", \"Whether NKAP SUMOylation is the chain read by the CENP-E SIM untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"LUBAC-catalyzed linear ubiquitination read by KNL1 was shown to anchor CENP-E specifically at attached kinetochores, adding a ubiquitin-dependent retention mechanism coupled to the KMN network.\",\n      \"evidence\": \"Linear ubiquitination assays, KNL1 interaction assays, RNAi/knockout, and attachment-status immunofluorescence\",\n      \"pmids\": [\"30655516\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab demonstration; the ubiquitinated residues on CENP-E not mapped\", \"How this coexists with SUMO-dependent targeting unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying a CENP-E-PRC1 complex established a role for CENP-E in organizing kinetochore microtubules into stable midzone arrays at the metaphase-anaphase transition.\",\n      \"evidence\": \"Biotinylated-syntelin affinity purification, co-IP, chemical inhibition, and light-sheet live imaging\",\n      \"pmids\": [\"31174204\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab affinity-capture interaction; directness of CENP-E-PRC1 binding unclear\", \"Whether motor activity is needed for midzone assembly untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Distinct Aurora A (pole) and Aurora B (kinetochore) phosphorylation events, and a CENP-E/RZZS platform for dynein recruitment, revealed how CENP-E spatially controls fibrous corona dynamics and dynein loading.\",\n      \"evidence\": \"Phospho-specific antibodies, Aurora inhibitors, phospho-mutant constructs, MPS1 inhibition, RZZS phosphomimetics, and live imaging\",\n      \"pmids\": [\"37658044\", \"37984321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The EMBO dynein-platform study (Medium) is single-lab\", \"How corona disassembly timing integrates with checkpoint silencing unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating HPV16 E6/E6AP-driven proteasomal degradation of CENP-E producing chromosomal instability connected CENP-E turnover to viral oncogenesis independently of p53.\",\n      \"evidence\": \"HPV16 E6 expression, proteasome inhibitor rescue, E6AP knockdown in p53-independent cells, and congression imaging\",\n      \"pmids\": [\"36989302\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab; direct CENP-E ubiquitination by E6AP not shown\", \"Relevance to spontaneous tumor CIN beyond HPV context unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple parallel recruitment inputs (SUMO-2/3 chains, linear ubiquitin/KNL1, BubR1, NKAP, TRAMM, CTCF, RZZS) are integrated and ordered to set CENP-E levels, residence time, and attachment-mode at a single kinetochore remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling SUMO, ubiquitin, and protein-protein receptor pathways\", \"Quantitative stoichiometry of CENP-E loading mechanisms unknown\", \"Direct in vivo phospho-site map across regulatory kinases incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003774\", \"supporting_discovery_ids\": [5, 22, 24, 29]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [24, 27]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 31, 32]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 19, 25, 42]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [26, 41]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7, 27]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 5, 11, 14]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [42, 44]}\n    ],\n    \"complexes\": [\"kinetochore fibrous corona\", \"BubR1-CENP-E complex\"],\n    \"partners\": [\"BUBR1\", \"CENP-F\", \"CLASP1\", \"SKAP\", \"PRC1\", \"KIF18A\", \"Skp1\", \"KNL1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"CENPE","tier":"IDENTITY","verdict":"Identity concern","subtype":"corpus_ungrounded","uniprot_band":"rich","rules_fired":"R1","issue":"R1: gene named in 11/100 (11%) of its own corpus abstracts (< 25%) — corpus likely a paralog/alias collision"},"evaluation":{"pairwise":"tie","faith_supported":10,"faith_total":10,"faith_pct":100.0}}