{"gene":"KIF11","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1995,"finding":"Phosphorylation of Eg5 at Thr937 (T937) by p34cdc2 (CDK1) is required for spindle localization: mutation of T937 to non-phosphorylatable alanine abolishes spindle localization, while T937S preserves it, establishing that cell-cycle-regulated phosphorylation at the conserved bimC C-terminal domain controls Eg5 targeting to the mitotic spindle.","method":"Transient transfection with myc-tagged Eg5 derivatives, site-directed mutagenesis, immunofluorescence localization in Xenopus A6 cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct mutagenesis of the phosphorylation site with clear localization readout; foundational study replicated in subsequent work","pmids":["7753799"],"is_preprint":false},{"year":2004,"finding":"Monomeric human Eg5 is a slow, plus-end-directed ATPase: mantATP binding is rapid (2–3 µM⁻¹s⁻¹), ATP hydrolysis is fast (~15 s⁻¹), but ATP-dependent microtubule dissociation is rate-limiting (~8 s⁻¹), making the ATPase mechanism more similar to conventional kinesin than to minus-end motors Ncd/Kar3.","method":"Sedimentation velocity, sedimentation equilibrium, analytical gel filtration, steady-state ATPase, stopped-flow kinetics with mantATP/mantADP","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — full pre-steady-state kinetic dissection with multiple orthogonal biochemical methods; rigorous in vitro reconstitution","pmids":["15247293"],"is_preprint":false},{"year":2006,"finding":"Eg5 is downstream of and regulated by Bcr-Abl tyrosine kinase in Philadelphia chromosome-positive cells: imatinib treatment downregulates Eg5 expression in imatinib-sensitive but not imatinib-resistant (T315I) cells, placing Eg5 in the Bcr-Abl signaling axis.","method":"Western blot and RT-PCR after imatinib treatment of sensitive vs. resistant cell lines; antisense oligonucleotide knockdown; xenograft mouse model","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic knockdown in matched sensitive/resistant lines with in vivo validation, single lab","pmids":["16969080"],"is_preprint":false},{"year":2006,"finding":"Inhibition of Eg5 upregulates Hsp70 transcriptionally via a cis-regulatory element in the Hsp70 promoter through the PI3K/Akt pathway; this Hsp70 induction is cytoprotective and reduces sensitivity to Eg5 inhibitors, while blocking Hsp70 or PI3K/Akt increases apoptosis.","method":"Small-molecule inhibition of Eg5, antisense/siRNA knockdown of Hsp70, PI3K/Akt pathway inhibitors, promoter reporter assays, apoptosis assays in multiple myeloma cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (promoter reporter, siRNA, pharmacological inhibition) in a single lab","pmids":["16627469"],"is_preprint":false},{"year":2008,"finding":"Parkin E3 ubiquitin ligase represses Eg5 gene transcription (without triggering proteasomal degradation of Eg5 protein) by blocking c-Jun binding to the AP-1 site in the Eg5 promoter; Parkin achieves this through multiple monoubiquitination of Hsp70, which inactivates JNK and reduces c-Jun phosphorylation.","method":"Co-transfection, promoter reporter assays, ubiquitination assays, Western blot for JNK/c-Jun phosphorylation, proteasome inhibitor controls","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, ubiquitination, kinase phosphorylation) in single lab establishing transcriptional mechanism","pmids":["18845538"],"is_preprint":false},{"year":2008,"finding":"Homozygous Eg5 (Knsl1) knockout mice die during early embryogenesis prior to implantation, while heterozygotes are viable and fertile, demonstrating that Eg5 is essential for early mammalian development and cannot be compensated by another motor.","method":"Gene targeting in mice, embryo analysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with definitive developmental phenotype; ortholog in mammalian model","pmids":["18474226"],"is_preprint":false},{"year":2010,"finding":"Tiam1-Rac signaling at centrosomes during prophase antagonizes Eg5-driven centrosome separation: Tiam1-depleted cells or Rac1-deficient cells escape monastrol-induced (Eg5-inhibited) mitotic arrest, and Eg5 suppression in Tiam1-depleted cells rescues increased centrosome separation and chromosome congression errors, placing Tiam1-Rac as the first identified force opposing Eg5 in prophase.","method":"siRNA depletion, conditional Rac1 knockout in vivo, monastrol treatment, live-cell imaging, centrosome separation measurements, epistasis analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vitro and in vivo with multiple orthogonal readouts and reciprocal rescue experiments","pmids":["20346677"],"is_preprint":false},{"year":2011,"finding":"Plk1 triggers centrosome separation in G2 phase through a two-step mechanism: CDK1 phosphorylates Eg5 first, then Plk1 activates Nek9, which through Nek6/7 phosphorylates Eg5 at Ser1033; both Ser1033 (Nek6/7 site) and Thr926 (CDK1 site) are required for Eg5 accumulation at centrosomes and subsequent centrosome separation.","method":"In vitro kinase assays, phospho-specific antibodies, siRNA knockdown, centrosome separation assays, epistasis analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assays combined with cell-based epistasis and phospho-specific antibody readouts; independently supported by companion paper","pmids":["21642957"],"is_preprint":false},{"year":2011,"finding":"Plk1 controls Eg5 localization to centrosomes and drives centrosome separation independently of CDK1; CDK2 can compensate for CDK1 by phosphorylating Eg5 at Thr927; actin-dependent opposing forces slow Plk1-dependent separation in G2, while CDK1 triggers faster centrosome movement; microtubule stability modulates the rate of centrosome separation.","method":"Plk1 and CDK1 inhibitors, CDK2 knockdown, phospho-Eg5 antibodies, centrosome tracking, actin depolymerization, MT stabilization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple kinase inhibitors and depletion with quantitative centrosome tracking; reciprocal epistasis; two EMBO papers from same period provide independent replication","pmids":["21522128"],"is_preprint":false},{"year":2013,"finding":"Eg5 restricts (acts as a brake on) anaphase B spindle elongation in mammalian cells: STLC (weakens Eg5-MT interaction) increases centrosome separation rate, while FCPT (rigor-like Eg5-MT interaction) decreases it; GFP-Eg5 accumulates in the spindle interzone during anaphase.","method":"Small-molecule perturbation (STLC, FCPT), live-cell centrosome tracking with GFP-γ-tubulin, fluorescence quantification of GFP-Eg5","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two mechanistically distinct inhibitors with opposite effects and quantitative live-cell readout, single lab","pmids":["24285623"],"is_preprint":false},{"year":2014,"finding":"RNF20/40 ubiquitin ligase complex physically interacts with Eg5 during mitosis, monoubiquitinates Eg5, and stabilizes it; loss of RNF20/40 reduces Eg5 protein levels, causes spindle assembly defects, cell cycle arrest and apoptosis.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, spindle assembly analysis, in vivo xenograft models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro ubiquitination assay, and rescue experiments; multiple orthogonal methods","pmids":["27557628"],"is_preprint":false},{"year":2015,"finding":"KIF11 directly binds ZBP1 (IGF2BP1) via defined regions on both proteins; this interaction is required for KIF11-mediated transport of β-actin mRNPs along microtubules; disruption of the KIF11-ZBP1 interaction in vivo delocalizes β-actin mRNA and impairs cell migration.","method":"Co-immunoprecipitation, pulldown, in vitro binding domain mapping, β-actin mRNA localization (FISH/imaging), cell migration assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro domain mapping, and functional rescue experiments with defined mutants; multiple orthogonal methods in single study","pmids":["25588836"],"is_preprint":false},{"year":2015,"finding":"TPX2 inhibits Eg5 velocity through two mechanisms: binding to microtubules (Kd ~200 nM, C-terminal tubulin tails not required) and direct interaction with the dimerization/neck region of Eg5; dimeric but not monomeric Eg5 is differentially inhibited by full-length vs. truncated TPX2.","method":"TIRF microscopy single-molecule assays, microtubule gliding assays, pulldown/binding assays with truncated TPX2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — single-molecule biophysical reconstitution with structure-function dissection using truncated constructs; multiple orthogonal in vitro assays","pmids":["26018074"],"is_preprint":false},{"year":2016,"finding":"PTEN associates and co-localizes with EG5 during mitosis; PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle, leading to shorter spindles and chromosome misalignment.","method":"Co-immunoprecipitation, immunofluorescence co-localization, PTEN knockdown/knockout, spindle geometry measurement, chromosome alignment analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional phenotype upon PTEN loss, single lab, without in vitro reconstitution of direct phosphorylation","pmids":["27492783"],"is_preprint":false},{"year":2017,"finding":"HDAC1 deacetylates Eg5 and co-localizes with Eg5 during mitosis; HDAC1 influences Eg5 ATPase activity; an HDAC1/2-selective inhibitor causes mitotic arrest and monopolar spindle formation consistent with Eg5 hyperacetylation impairing function, revealing a non-transcriptional mechanism of HDAC inhibitor-induced G2/M arrest.","method":"HDAC1 trapping mutant substrate identification, co-localization, ATPase activity assay, cell cycle analysis, spindle morphology","journal":"Cell chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — trapping-mutant strategy for substrate identification plus ATPase assay and cell-based phenotype, single lab","pmids":["28392145"],"is_preprint":false},{"year":2017,"finding":"PP2A/B55α complex physically associates with the Eg5 C-terminal tail domain and dephosphorylates Eg5 at Thr926, enabling mitotic exit; PP2A knockdown leads to elevated phospho-Eg5 in late metaphase and delayed mitotic exit, phenocopied by a phosphomimetic EG5/T926D mutant.","method":"Co-immunoprecipitation, phospho-specific antibodies, siRNA knockdown, phosphomimetic/non-phosphorylatable mutants, mitotic timing assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, phosphomimetic mutant rescue, and phospho-specific antibody readout; single lab","pmids":["28487562"],"is_preprint":false},{"year":2018,"finding":"NEK7 phosphorylates Eg5/KIF11, promoting its accumulation on microtubules in distal dendrites; there Eg5 limits retrograde microtubule polymerization through microtubule stabilization (independent of its motor activity), controlling dendrite growth and branching in postmitotic neurons.","method":"Targeted RNAi, kinase-dead NEK7 rescue, motor-dead Eg5 rescue, live-cell microtubule dynamics imaging, in vivo dendritic morphology analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinase-dead and motor-dead domain dissection with in vitro and in vivo rescue; multiple orthogonal approaches","pmids":["29899413"],"is_preprint":false},{"year":2018,"finding":"HMMR is required to dampen Eg5-mediated forces by localizing TPX2 and promoting formation of inhibitory TPX2-Eg5 complexes; HMMR silencing causes spindle disorganization, reduced K-fiber stability, and increased chromosome segregation errors that are rescued by chemical inhibition of Eg5 but not by KIF15 silencing.","method":"siRNA knockdown, immunofluorescence, Co-IP, Eg5 inhibitor rescue experiments, aneuploidy quantification","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for complex formation, chemical epistasis rescue, multiple phenotypic readouts; single lab","pmids":["29386294"],"is_preprint":false},{"year":2018,"finding":"KIF11 knockdown in hippocampal neurons increases excitatory post-synaptic current frequency and amplitude, enhances dendritic arborization and synapse number, and upregulates synaptic vesicle proteins; KIF11 requires pre-synaptic NMDAR activity for its constraining function, and Piccolo expression constrains KIF11 function in synaptic transmission.","method":"RNAi-mediated knockdown in primary hippocampal neurons, electrophysiology (EPSC recording), immunofluorescence for synaptic proteins","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct electrophysiological readout with RNAi knockdown; single lab, functional pathway placement via NMDAR dependence","pmids":["30479371"],"is_preprint":false},{"year":2018,"finding":"KIF11 interacts with death receptor 6 (DR6) and TRAF4 by co-immunoprecipitation and GST pulldown; overexpression of KIF11 or TRAF4 rescues the suppression of ovarian cancer cell migration caused by DR6 knockdown, placing KIF11 downstream of DR6/TRAF4 in migration signaling.","method":"Co-immunoprecipitation, GST pulldown, mass spectrometry, rescue overexpression experiments, migration assays","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and pulldown to establish interaction, epistasis rescue; single lab","pmids":["30186750"],"is_preprint":false},{"year":2020,"finding":"KIF11 localizes to basal bodies of primary cilia during interphase (in addition to its mitotic role); reduction of KIF11 expression increases the number of ciliated cells, increases cilium length, and attenuates cilia disassembly kinetics; exogenous KIF11 rescues these effects.","method":"Immunofluorescence localization, siRNA knockdown, cilia number/length quantification, rescue with exogenous KIF11","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional consequence, rescue validation; single lab","pmids":["32811879"],"is_preprint":false},{"year":2020,"finding":"TRIM8 E3 ubiquitin ligase interacts with KIF11/Eg5 (identified by proteomics in neural stem cells) and KIFC1; TRIM8 localizes to the mitotic spindle and plays a role in centrosome separation at mitosis onset, with consequent effects on mitotic progression and chromosomal stability.","method":"Co-immunoprecipitation, mass spectrometry interactome, immunofluorescence, TRIM8 knockdown, mitotic phenotype analysis","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus MS identification; functional knockdown phenotype; single lab without in vitro reconstitution","pmids":["31904480"],"is_preprint":false},{"year":2021,"finding":"EG5/kinesin-5 together with PRC1-dependent KIF4A/kinesin-4 constitute the force-generating mechanism for spindle elongation in human cells: combined depletion of EG5 and KIF4A causes total failure of chromosome segregation due to blocked midzone microtubule sliding without affecting microtubule stability, as shown by tubulin photoactivation, STED, and expansion microscopy.","method":"Combined siRNA depletion, CRISPR inactivation, tubulin photoactivation, STED microscopy, expansion microscopy, live-cell imaging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal imaging modalities, CRISPR/RNAi combined with photoactivation to directly measure microtubule sliding; rigorous epistasis design","pmids":["33910056"],"is_preprint":false},{"year":2022,"finding":"NAT10 acetyltransferase binds and co-localizes with Eg5 at the centrosome during mitosis, acetylates Eg5 at K771, and stabilizes Eg5; K771 acetylation is required for Eg5 motor function and centrosome loading; a K771Q (acetylation-mimic) but not K771R mutant rescues NAT10-depletion-induced spindle defects and mitotic catastrophe.","method":"Co-immunoprecipitation, acetylation-specific antibody generation, site-directed mutagenesis (K771Q/R), live-cell imaging, centrosome loading assay, spindle formation rescue","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-directed mutagenesis with acetylation-site-specific antibody and rescue experiments establishing both the modification and its functional requirement","pmids":["35210604"],"is_preprint":false},{"year":2022,"finding":"KIF11 interacts with SREBP2, the master regulator of the mevalonate pathway; KIF11 attenuates ubiquitination-mediated degradation of SREBP2, increasing its protein stability and accumulation, which in turn elevates mevalonate pathway gene expression and free cholesterol in PDAC cells.","method":"Co-immunoprecipitation, ubiquitination assays, Western blot for SREBP2 stability, GSEA pathway analysis, rescue experiments with statin treatment","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional rescue; single lab, non-canonical function","pmids":["35619540"],"is_preprint":false},{"year":2024,"finding":"UFMylation of Eg5 at Lys564 is required for proper spindle organization: Eg5 is a substrate of the UFM1 pathway; UFMylation does not alter Eg5 transcription, phosphorylation, or protein stability but affects its mono-ubiquitination and spindle localization; the K564R (UFMylation-defective) mutant shows shorter spindles, metaphase arrest, spindle checkpoint activation, and cell division failure.","method":"Identification of Eg5 as UFM1 substrate, site-directed mutagenesis (K564R), co-localization at centrosome/spindle, ubiquitination assays, spindle length and checkpoint assays in HeLa cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of modification site with functional phenotype readout; single lab; PTM site established but mechanism of UFMylation-mono-Ub crosstalk not fully resolved","pmids":["39085203"],"is_preprint":false},{"year":1998,"finding":"Eg5 is expressed in postmitotic rodent neurons, directly associates with the microtubule array, and is enriched in distal regions of developing axons and dendrites transiently during process outgrowth, re-appearing in newly forming dendritic sprouts, suggesting a role in regulating microtubule behavior at distal tips of developing neuronal processes.","method":"Western blot, immunofluorescence, microtubule co-pelleting from egg homogenates","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with biochemical co-sedimentation; functional inference is correlative but localization is directly demonstrated","pmids":["9742151"],"is_preprint":false},{"year":2014,"finding":"Inhibition of kinesin-5 (Eg5) by amyloid-beta (Aβ) or monastrol reduces transport of NGF/NTR(p75) and NMDA receptors to the cell surface, inhibits NGF-dependent neurite outgrowth from PC12 cells, blocks glutamate-dependent Ca²⁺ entry into primary neurons, and inhibits long-term potentiation; Eg5 activity is absent from APP/PS transgenic mouse brain.","method":"Cell-surface receptor quantification, neurite outgrowth assay, Ca²⁺ imaging, LTP electrophysiology, monastrol and Aβ treatment","journal":"Neurobiology of aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional readouts in neurons linking Eg5 inhibition to receptor trafficking; single lab","pmids":["24636920"],"is_preprint":false},{"year":2020,"finding":"In mice, postnatal EC-specific conditional knockout of Kif11 leads to severely stunted growth of the retinal vasculature and mildly stunted cerebellar vasculature, with little effect elsewhere in the CNS, demonstrating Kif11 function in endothelial cell mitosis is most critical in rapidly growing CNS vascular beds; this phenotype is not caused by reduced β-catenin signaling in ECs.","method":"Conditional knockout mouse (Kif11 CKO in endothelial cells), retinal flat-mount vasculature analysis, β-catenin signaling readouts","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific genetic knockout with defined vascular phenotype and mechanistic exclusion of β-catenin pathway","pmids":["31993640"],"is_preprint":false},{"year":2021,"finding":"ASPM physically interacts with KIF11 (by co-immunoprecipitation in HCC cells), and ASPM exerts its effects on HCC cell proliferation, invasion, and migration through KIF11; the ASPM-KIF11 complex promotes malignant progression by activating the Wnt/β-catenin signaling pathway.","method":"Co-immunoprecipitation, rescue overexpression of KIF11 after ASPM knockdown, Western blot for Wnt/β-catenin components","journal":"Experimental and therapeutic medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus rescue experiment; single lab, no in vitro reconstitution","pmids":["34504599"],"is_preprint":false},{"year":2020,"finding":"PAK6 specifically co-localizes with Eg5 at the centrosome; PAK6 depletion results in multipolar spindles and upregulation of Eg5; Eg5 knockdown reverses PAK6-depletion-induced multipolar spindles; PAK6 and Eg5 negatively inter-regulate each other's expression.","method":"Co-localization by immunofluorescence, siRNA knockdown epistasis, Western blot for protein levels","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-localization and genetic epistasis by knockdown, single lab, mechanism of inter-regulation not biochemically resolved","pmids":["33098954"],"is_preprint":false},{"year":2013,"finding":"Loss of Kif11 function in zebrafish radial glial cells (by mutation or STLC inhibition) causes monoastral spindle formation and mitotic arrest (~226× delay in mitotic exit), leading to apoptosis and specific reductions in oligodendroglia and secondary interneurons/motorneurons, demonstrating Kif11 is required for radial glia mitotic progression and subsequent neurogenesis.","method":"Forward genetic screen, pharmacological inhibition (STLC), anti-activated Caspase-3 staining, mathematical modeling, cell-type-specific marker analysis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mutant plus pharmacological validation with quantitative modeling; zebrafish ortholog; multiple readouts","pmids":["24370453"],"is_preprint":false}],"current_model":"KIF11/Eg5 is a homotetrameric, plus-end-directed kinesin-5 motor whose ATPase cycle (rate-limited by MT-dependent ADP release) powers antiparallel microtubule sliding to separate centrosomes and assemble/maintain the bipolar mitotic spindle; its spindle localization and activity are controlled by a multi-kinase phosphorylation cascade (CDK1/2 at Thr926/927, Plk1→Nek9→Nek6/7 at Ser1033) and balanced by dephosphorylation via PP2A/B55α, while protein stability is regulated by RNF20/40-mediated monoubiquitination, NAT10-mediated acetylation at K771, UFMylation at K564, and Parkin-dependent transcriptional repression through the Hsp70-JNK-c-Jun axis; beyond mitosis, KIF11 transports β-actin mRNPs via ZBP1, regulates dendrite morphogenesis through NEK7-mediated microtubule stabilization, controls primary cilia dynamics at basal bodies, and acts as an inhibitory constraint on excitatory synaptic transmission in neurons."},"narrative":{"mechanistic_narrative":"KIF11/Eg5 is a slow, plus-end-directed kinesin-5 ATPase whose mechanochemical cycle is rate-limited by ATP-dependent microtubule dissociation [PMID:15247293], and which generates the antiparallel microtubule-sliding forces that drive centrosome separation, bipolar spindle assembly, and spindle elongation; in human cells it cooperates with PRC1-dependent KIF4A to slide midzone microtubules, and combined loss of the two motors abolishes chromosome segregation [PMID:33910056]. Its essentiality for cell division is reflected in early embryonic lethality of homozygous knockout mice [PMID:18474226] and mitotic arrest with monoastral spindles upon loss of function [PMID:24370453]. Targeting of Eg5 to the spindle and centrosomes is governed by a phosphorylation cascade: CDK1 (with CDK2 compensation) phosphorylates the conserved C-terminal bimC domain (Thr926/Thr927/Thr937), and Plk1 acting through Nek9-Nek6/7 adds Ser1033, both required for centrosome accumulation and separation [PMID:7753799, PMID:21642957, PMID:21522128]; PP2A/B55α reverses Thr926 phosphorylation to permit mitotic exit [PMID:28487562]. Eg5 force output is constrained by inhibitory partners TPX2, which slows the motor through microtubule and neck-region binding [PMID:26018074], and HMMR, which localizes TPX2 to promote inhibitory TPX2-Eg5 complexes [PMID:29386294]. Eg5 abundance, motor activity, and spindle loading are further tuned by post-translational modifications: RNF20/40 monoubiquitination [PMID:27557628], NAT10 acetylation at K771 [PMID:35210604], HDAC1 deacetylation [PMID:28392145], and UFMylation at K564 [PMID:39085203], while Parkin represses Eg5 transcription via an Hsp70-JNK-c-Jun axis [PMID:18845538]. Beyond mitosis, Eg5 transports β-actin mRNPs through direct ZBP1 (IGF2BP1) binding to support cell migration [PMID:25588836], localizes to basal bodies to restrain primary cilium length and disassembly [PMID:32811879], and in postmitotic neurons is phosphorylated by NEK7 to stabilize distal dendritic microtubules independently of motor activity [PMID:29899413] and acts as a brake on excitatory synaptic transmission [PMID:30479371].","teleology":[{"year":1995,"claim":"Established that Eg5 spindle targeting is under cell-cycle control, defining the link between mitotic kinase activity and motor localization.","evidence":"Site-directed mutagenesis of the CDK1 phosphorylation site (T937) with immunofluorescence in Xenopus A6 cells","pmids":["7753799"],"confidence":"High","gaps":["Did not identify the full set of regulatory kinases","Did not connect localization to force generation"]},{"year":2004,"claim":"Defined the mechanochemical cycle of the motor, showing microtubule dissociation rather than hydrolysis is rate-limiting and the mechanism resembles conventional kinesin.","evidence":"Pre-steady-state and steady-state kinetics with mantATP/mantADP on monomeric human Eg5","pmids":["15247293"],"confidence":"High","gaps":["Used monomeric construct, not the native homotetramer","Did not address antiparallel sliding mechanics"]},{"year":2008,"claim":"Demonstrated absolute requirement of Eg5 for early mammalian development with no motor compensation, establishing biological essentiality.","evidence":"Knsl1 gene-targeted knockout mice with embryo analysis","pmids":["18474226"],"confidence":"High","gaps":["Pre-implantation lethality precludes analysis of later tissue-specific roles"]},{"year":2008,"claim":"Revealed transcriptional repression of Eg5 by Parkin through an Hsp70-JNK-c-Jun route, distinguishing transcriptional control from proteasomal degradation.","evidence":"Promoter reporter, ubiquitination, and JNK/c-Jun phosphorylation assays with proteasome controls","pmids":["18845538"],"confidence":"Medium","gaps":["Single lab","Physiological context of Parkin-Eg5 regulation not defined"]},{"year":2011,"claim":"Resolved the kinase hierarchy driving centrosome separation, placing CDK1/CDK2 and a Plk1-Nek9-Nek6/7 cascade upstream of distinct Eg5 phosphosites.","evidence":"In vitro kinase assays, phospho-specific antibodies, and centrosome separation epistasis (two companion EMBO studies)","pmids":["21642957","21522128"],"confidence":"High","gaps":["Quantitative contribution of each phosphosite to motor activity not isolated","Did not reconstitute phosphorylation-dependent sliding in vitro"]},{"year":2010,"claim":"Identified Tiam1-Rac as an opposing prophase force, framing Eg5-driven separation as a balance of antagonistic forces.","evidence":"siRNA depletion, conditional Rac1 knockout, monastrol, and reciprocal rescue with live-cell imaging","pmids":["20346677"],"confidence":"High","gaps":["Molecular target of the opposing force not pinpointed","Direct biochemical link to Eg5 not shown"]},{"year":2015,"claim":"Defined direct biophysical inhibition of the motor by TPX2 through dual microtubule and neck-region binding.","evidence":"TIRF single-molecule and gliding assays with truncated TPX2 constructs","pmids":["26018074"],"confidence":"High","gaps":["In vivo regulatory consequence addressed only later","Structural basis of neck-region binding not resolved"]},{"year":2018,"claim":"Showed HMMR positions TPX2 to assemble inhibitory TPX2-Eg5 complexes, controlling spindle force balance and segregation fidelity.","evidence":"siRNA, Co-IP, and chemical epistasis with Eg5 inhibitor rescue and aneuploidy quantification","pmids":["29386294"],"confidence":"Medium","gaps":["Single lab","Direct effect of HMMR on motor kinetics not measured"]},{"year":2021,"claim":"Directly measured Eg5's force-generating role in spindle elongation, showing it cooperates with KIF4A to slide midzone microtubules.","evidence":"Combined CRISPR/RNAi depletion with tubulin photoactivation, STED, and expansion microscopy","pmids":["33910056"],"confidence":"High","gaps":["Relative force contribution of each motor not quantified","Regulation of the EG5-KIF4A cooperation unknown"]},{"year":2024,"claim":"Expanded the PTM regulatory layer, establishing acetylation, monoubiquitination, deacetylation, and UFMylation as controllers of Eg5 stability, activity, and spindle loading.","evidence":"Site-directed mutagenesis (K771Q/R, K564R), modification-specific antibodies, Co-IP, ubiquitination assays, and rescue in HeLa cells","pmids":["27557628","28392145","35210604","39085203"],"confidence":"High","gaps":["Crosstalk between UFMylation and monoubiquitination not fully resolved","Temporal coordination of distinct PTMs during the cell cycle unclear"]},{"year":2015,"claim":"Uncovered a non-mitotic transport function, with Eg5 binding ZBP1 to deliver β-actin mRNPs and support cell migration.","evidence":"Reciprocal Co-IP, in vitro domain mapping, β-actin mRNA imaging, and migration assays","pmids":["25588836"],"confidence":"High","gaps":["In vivo physiological relevance of mRNP transport not established","Coupling to motor stepping not biophysically dissected"]},{"year":2020,"claim":"Extended Eg5 function to interphase and postmitotic cells, including basal body regulation of cilia and dendritic microtubule stabilization, the latter motor-independent.","evidence":"siRNA, NEK7 kinase-dead and motor-dead rescues, live-cell microtubule imaging, cilia quantification (multiple studies)","pmids":["32811879","29899413","30479371"],"confidence":"Medium","gaps":["Mechanism distinguishing motor-dependent vs motor-independent roles incompletely defined","Tissue-specific regulation of these functions unknown"]},{"year":2020,"claim":"Demonstrated tissue-selective dependence on Eg5-driven endothelial mitosis, most critical in rapidly growing CNS vascular beds and independent of β-catenin signaling.","evidence":"Endothelial-specific conditional Kif11 knockout with retinal flat-mount vasculature analysis","pmids":["31993640"],"confidence":"High","gaps":["Basis of vascular-bed selectivity not explained","Downstream pathway driving the phenotype not identified"]},{"year":null,"claim":"How the multiple PTMs, kinase cascades, and inhibitory partners are temporally integrated to tune Eg5 force output across mitotic stages and non-mitotic contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking PTM state to motor velocity in vivo","Mechanistic basis for switching between mitotic and post-mitotic functions unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003774","term_label":"cytoskeletal motor activity","supporting_discovery_ids":[1,22,23]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[1,14]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[16,26]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[7,23,30]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[16,26]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[20]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,7,22]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,28,31]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[16,18,27]}],"complexes":[],"partners":["TPX2","ZBP1","PTEN","RNF20","HMMR","NAT10","NEK7","PAK6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P52732","full_name":"Kinesin-like protein KIF11","aliases":["Kinesin-like protein 1","Kinesin-like spindle protein HKSP","Kinesin-related motor protein Eg5","Thyroid receptor-interacting protein 5","TR-interacting protein 5","TRIP-5"],"length_aa":1056,"mass_kda":119.2,"function":"Motor protein required for establishing a bipolar spindle and thus contributing to chromosome congression during mitosis (PubMed:19001501, PubMed:37728657). 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in benzo(a)pyrene-induced non-small cell lung cancer.","date":"2021","source":"Environmental toxicology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34800719","citation_count":14,"is_preprint":false},{"pmid":"32642733","id":"PMC_32642733","title":"KIF11 and KIF15 mitotic kinesins are potential therapeutic vulnerabilities for malignant peripheral nerve sheath tumors.","date":"2020","source":"Neuro-oncology advances","url":"https://pubmed.ncbi.nlm.nih.gov/32642733","citation_count":14,"is_preprint":false},{"pmid":"32165913","id":"PMC_32165913","title":"Kinesin-5 Eg5 is essential for spindle assembly and chromosome alignment of mouse spermatocytes.","date":"2020","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/32165913","citation_count":14,"is_preprint":false},{"pmid":"36929198","id":"PMC_36929198","title":"Cyclovirobuxine D inhibits growth and progression of non‑small cell lung cancer cells by suppressing the KIF11‑CDC25C‑CDK1‑CyclinB1 G2/M phase transition regulatory network and the NFκB/JNK signaling pathway.","date":"2023","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36929198","citation_count":14,"is_preprint":false},{"pmid":"36929571","id":"PMC_36929571","title":"Morphological Profiling Identifies the Motor Protein Eg5 as Cellular Target of Spirooxindoles.","date":"2023","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/36929571","citation_count":14,"is_preprint":false},{"pmid":"31428438","id":"PMC_31428438","title":"KIF11 microdeletion is associated with microcephaly, chorioretinopathy and intellectual disability.","date":"2018","source":"Human genome variation","url":"https://pubmed.ncbi.nlm.nih.gov/31428438","citation_count":14,"is_preprint":false},{"pmid":"31424977","id":"PMC_31424977","title":"KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.","date":"2019","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/31424977","citation_count":14,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47815,"output_tokens":7975,"usd":0.131535,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17650,"output_tokens":4153,"usd":0.096037,"stage2_stop_reason":"end_turn"},"total_usd":0.227572,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"Phosphorylation of Eg5 at Thr937 (T937) by p34cdc2 (CDK1) is required for spindle localization: mutation of T937 to non-phosphorylatable alanine abolishes spindle localization, while T937S preserves it, establishing that cell-cycle-regulated phosphorylation at the conserved bimC C-terminal domain controls Eg5 targeting to the mitotic spindle.\",\n      \"method\": \"Transient transfection with myc-tagged Eg5 derivatives, site-directed mutagenesis, immunofluorescence localization in Xenopus A6 cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct mutagenesis of the phosphorylation site with clear localization readout; foundational study replicated in subsequent work\",\n      \"pmids\": [\"7753799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Monomeric human Eg5 is a slow, plus-end-directed ATPase: mantATP binding is rapid (2–3 µM⁻¹s⁻¹), ATP hydrolysis is fast (~15 s⁻¹), but ATP-dependent microtubule dissociation is rate-limiting (~8 s⁻¹), making the ATPase mechanism more similar to conventional kinesin than to minus-end motors Ncd/Kar3.\",\n      \"method\": \"Sedimentation velocity, sedimentation equilibrium, analytical gel filtration, steady-state ATPase, stopped-flow kinetics with mantATP/mantADP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full pre-steady-state kinetic dissection with multiple orthogonal biochemical methods; rigorous in vitro reconstitution\",\n      \"pmids\": [\"15247293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Eg5 is downstream of and regulated by Bcr-Abl tyrosine kinase in Philadelphia chromosome-positive cells: imatinib treatment downregulates Eg5 expression in imatinib-sensitive but not imatinib-resistant (T315I) cells, placing Eg5 in the Bcr-Abl signaling axis.\",\n      \"method\": \"Western blot and RT-PCR after imatinib treatment of sensitive vs. resistant cell lines; antisense oligonucleotide knockdown; xenograft mouse model\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic knockdown in matched sensitive/resistant lines with in vivo validation, single lab\",\n      \"pmids\": [\"16969080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Inhibition of Eg5 upregulates Hsp70 transcriptionally via a cis-regulatory element in the Hsp70 promoter through the PI3K/Akt pathway; this Hsp70 induction is cytoprotective and reduces sensitivity to Eg5 inhibitors, while blocking Hsp70 or PI3K/Akt increases apoptosis.\",\n      \"method\": \"Small-molecule inhibition of Eg5, antisense/siRNA knockdown of Hsp70, PI3K/Akt pathway inhibitors, promoter reporter assays, apoptosis assays in multiple myeloma cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (promoter reporter, siRNA, pharmacological inhibition) in a single lab\",\n      \"pmids\": [\"16627469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Parkin E3 ubiquitin ligase represses Eg5 gene transcription (without triggering proteasomal degradation of Eg5 protein) by blocking c-Jun binding to the AP-1 site in the Eg5 promoter; Parkin achieves this through multiple monoubiquitination of Hsp70, which inactivates JNK and reduces c-Jun phosphorylation.\",\n      \"method\": \"Co-transfection, promoter reporter assays, ubiquitination assays, Western blot for JNK/c-Jun phosphorylation, proteasome inhibitor controls\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, ubiquitination, kinase phosphorylation) in single lab establishing transcriptional mechanism\",\n      \"pmids\": [\"18845538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Homozygous Eg5 (Knsl1) knockout mice die during early embryogenesis prior to implantation, while heterozygotes are viable and fertile, demonstrating that Eg5 is essential for early mammalian development and cannot be compensated by another motor.\",\n      \"method\": \"Gene targeting in mice, embryo analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with definitive developmental phenotype; ortholog in mammalian model\",\n      \"pmids\": [\"18474226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Tiam1-Rac signaling at centrosomes during prophase antagonizes Eg5-driven centrosome separation: Tiam1-depleted cells or Rac1-deficient cells escape monastrol-induced (Eg5-inhibited) mitotic arrest, and Eg5 suppression in Tiam1-depleted cells rescues increased centrosome separation and chromosome congression errors, placing Tiam1-Rac as the first identified force opposing Eg5 in prophase.\",\n      \"method\": \"siRNA depletion, conditional Rac1 knockout in vivo, monastrol treatment, live-cell imaging, centrosome separation measurements, epistasis analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vitro and in vivo with multiple orthogonal readouts and reciprocal rescue experiments\",\n      \"pmids\": [\"20346677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Plk1 triggers centrosome separation in G2 phase through a two-step mechanism: CDK1 phosphorylates Eg5 first, then Plk1 activates Nek9, which through Nek6/7 phosphorylates Eg5 at Ser1033; both Ser1033 (Nek6/7 site) and Thr926 (CDK1 site) are required for Eg5 accumulation at centrosomes and subsequent centrosome separation.\",\n      \"method\": \"In vitro kinase assays, phospho-specific antibodies, siRNA knockdown, centrosome separation assays, epistasis analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assays combined with cell-based epistasis and phospho-specific antibody readouts; independently supported by companion paper\",\n      \"pmids\": [\"21642957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Plk1 controls Eg5 localization to centrosomes and drives centrosome separation independently of CDK1; CDK2 can compensate for CDK1 by phosphorylating Eg5 at Thr927; actin-dependent opposing forces slow Plk1-dependent separation in G2, while CDK1 triggers faster centrosome movement; microtubule stability modulates the rate of centrosome separation.\",\n      \"method\": \"Plk1 and CDK1 inhibitors, CDK2 knockdown, phospho-Eg5 antibodies, centrosome tracking, actin depolymerization, MT stabilization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple kinase inhibitors and depletion with quantitative centrosome tracking; reciprocal epistasis; two EMBO papers from same period provide independent replication\",\n      \"pmids\": [\"21522128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Eg5 restricts (acts as a brake on) anaphase B spindle elongation in mammalian cells: STLC (weakens Eg5-MT interaction) increases centrosome separation rate, while FCPT (rigor-like Eg5-MT interaction) decreases it; GFP-Eg5 accumulates in the spindle interzone during anaphase.\",\n      \"method\": \"Small-molecule perturbation (STLC, FCPT), live-cell centrosome tracking with GFP-γ-tubulin, fluorescence quantification of GFP-Eg5\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two mechanistically distinct inhibitors with opposite effects and quantitative live-cell readout, single lab\",\n      \"pmids\": [\"24285623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF20/40 ubiquitin ligase complex physically interacts with Eg5 during mitosis, monoubiquitinates Eg5, and stabilizes it; loss of RNF20/40 reduces Eg5 protein levels, causes spindle assembly defects, cell cycle arrest and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, spindle assembly analysis, in vivo xenograft models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro ubiquitination assay, and rescue experiments; multiple orthogonal methods\",\n      \"pmids\": [\"27557628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"KIF11 directly binds ZBP1 (IGF2BP1) via defined regions on both proteins; this interaction is required for KIF11-mediated transport of β-actin mRNPs along microtubules; disruption of the KIF11-ZBP1 interaction in vivo delocalizes β-actin mRNA and impairs cell migration.\",\n      \"method\": \"Co-immunoprecipitation, pulldown, in vitro binding domain mapping, β-actin mRNA localization (FISH/imaging), cell migration assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro domain mapping, and functional rescue experiments with defined mutants; multiple orthogonal methods in single study\",\n      \"pmids\": [\"25588836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TPX2 inhibits Eg5 velocity through two mechanisms: binding to microtubules (Kd ~200 nM, C-terminal tubulin tails not required) and direct interaction with the dimerization/neck region of Eg5; dimeric but not monomeric Eg5 is differentially inhibited by full-length vs. truncated TPX2.\",\n      \"method\": \"TIRF microscopy single-molecule assays, microtubule gliding assays, pulldown/binding assays with truncated TPX2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule biophysical reconstitution with structure-function dissection using truncated constructs; multiple orthogonal in vitro assays\",\n      \"pmids\": [\"26018074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PTEN associates and co-localizes with EG5 during mitosis; PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle, leading to shorter spindles and chromosome misalignment.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, PTEN knockdown/knockout, spindle geometry measurement, chromosome alignment analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional phenotype upon PTEN loss, single lab, without in vitro reconstitution of direct phosphorylation\",\n      \"pmids\": [\"27492783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HDAC1 deacetylates Eg5 and co-localizes with Eg5 during mitosis; HDAC1 influences Eg5 ATPase activity; an HDAC1/2-selective inhibitor causes mitotic arrest and monopolar spindle formation consistent with Eg5 hyperacetylation impairing function, revealing a non-transcriptional mechanism of HDAC inhibitor-induced G2/M arrest.\",\n      \"method\": \"HDAC1 trapping mutant substrate identification, co-localization, ATPase activity assay, cell cycle analysis, spindle morphology\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — trapping-mutant strategy for substrate identification plus ATPase assay and cell-based phenotype, single lab\",\n      \"pmids\": [\"28392145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PP2A/B55α complex physically associates with the Eg5 C-terminal tail domain and dephosphorylates Eg5 at Thr926, enabling mitotic exit; PP2A knockdown leads to elevated phospho-Eg5 in late metaphase and delayed mitotic exit, phenocopied by a phosphomimetic EG5/T926D mutant.\",\n      \"method\": \"Co-immunoprecipitation, phospho-specific antibodies, siRNA knockdown, phosphomimetic/non-phosphorylatable mutants, mitotic timing assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, phosphomimetic mutant rescue, and phospho-specific antibody readout; single lab\",\n      \"pmids\": [\"28487562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NEK7 phosphorylates Eg5/KIF11, promoting its accumulation on microtubules in distal dendrites; there Eg5 limits retrograde microtubule polymerization through microtubule stabilization (independent of its motor activity), controlling dendrite growth and branching in postmitotic neurons.\",\n      \"method\": \"Targeted RNAi, kinase-dead NEK7 rescue, motor-dead Eg5 rescue, live-cell microtubule dynamics imaging, in vivo dendritic morphology analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinase-dead and motor-dead domain dissection with in vitro and in vivo rescue; multiple orthogonal approaches\",\n      \"pmids\": [\"29899413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HMMR is required to dampen Eg5-mediated forces by localizing TPX2 and promoting formation of inhibitory TPX2-Eg5 complexes; HMMR silencing causes spindle disorganization, reduced K-fiber stability, and increased chromosome segregation errors that are rescued by chemical inhibition of Eg5 but not by KIF15 silencing.\",\n      \"method\": \"siRNA knockdown, immunofluorescence, Co-IP, Eg5 inhibitor rescue experiments, aneuploidy quantification\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for complex formation, chemical epistasis rescue, multiple phenotypic readouts; single lab\",\n      \"pmids\": [\"29386294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KIF11 knockdown in hippocampal neurons increases excitatory post-synaptic current frequency and amplitude, enhances dendritic arborization and synapse number, and upregulates synaptic vesicle proteins; KIF11 requires pre-synaptic NMDAR activity for its constraining function, and Piccolo expression constrains KIF11 function in synaptic transmission.\",\n      \"method\": \"RNAi-mediated knockdown in primary hippocampal neurons, electrophysiology (EPSC recording), immunofluorescence for synaptic proteins\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct electrophysiological readout with RNAi knockdown; single lab, functional pathway placement via NMDAR dependence\",\n      \"pmids\": [\"30479371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KIF11 interacts with death receptor 6 (DR6) and TRAF4 by co-immunoprecipitation and GST pulldown; overexpression of KIF11 or TRAF4 rescues the suppression of ovarian cancer cell migration caused by DR6 knockdown, placing KIF11 downstream of DR6/TRAF4 in migration signaling.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, mass spectrometry, rescue overexpression experiments, migration assays\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and pulldown to establish interaction, epistasis rescue; single lab\",\n      \"pmids\": [\"30186750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KIF11 localizes to basal bodies of primary cilia during interphase (in addition to its mitotic role); reduction of KIF11 expression increases the number of ciliated cells, increases cilium length, and attenuates cilia disassembly kinetics; exogenous KIF11 rescues these effects.\",\n      \"method\": \"Immunofluorescence localization, siRNA knockdown, cilia number/length quantification, rescue with exogenous KIF11\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional consequence, rescue validation; single lab\",\n      \"pmids\": [\"32811879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRIM8 E3 ubiquitin ligase interacts with KIF11/Eg5 (identified by proteomics in neural stem cells) and KIFC1; TRIM8 localizes to the mitotic spindle and plays a role in centrosome separation at mitosis onset, with consequent effects on mitotic progression and chromosomal stability.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry interactome, immunofluorescence, TRIM8 knockdown, mitotic phenotype analysis\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus MS identification; functional knockdown phenotype; single lab without in vitro reconstitution\",\n      \"pmids\": [\"31904480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EG5/kinesin-5 together with PRC1-dependent KIF4A/kinesin-4 constitute the force-generating mechanism for spindle elongation in human cells: combined depletion of EG5 and KIF4A causes total failure of chromosome segregation due to blocked midzone microtubule sliding without affecting microtubule stability, as shown by tubulin photoactivation, STED, and expansion microscopy.\",\n      \"method\": \"Combined siRNA depletion, CRISPR inactivation, tubulin photoactivation, STED microscopy, expansion microscopy, live-cell imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal imaging modalities, CRISPR/RNAi combined with photoactivation to directly measure microtubule sliding; rigorous epistasis design\",\n      \"pmids\": [\"33910056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NAT10 acetyltransferase binds and co-localizes with Eg5 at the centrosome during mitosis, acetylates Eg5 at K771, and stabilizes Eg5; K771 acetylation is required for Eg5 motor function and centrosome loading; a K771Q (acetylation-mimic) but not K771R mutant rescues NAT10-depletion-induced spindle defects and mitotic catastrophe.\",\n      \"method\": \"Co-immunoprecipitation, acetylation-specific antibody generation, site-directed mutagenesis (K771Q/R), live-cell imaging, centrosome loading assay, spindle formation rescue\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-directed mutagenesis with acetylation-site-specific antibody and rescue experiments establishing both the modification and its functional requirement\",\n      \"pmids\": [\"35210604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KIF11 interacts with SREBP2, the master regulator of the mevalonate pathway; KIF11 attenuates ubiquitination-mediated degradation of SREBP2, increasing its protein stability and accumulation, which in turn elevates mevalonate pathway gene expression and free cholesterol in PDAC cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, Western blot for SREBP2 stability, GSEA pathway analysis, rescue experiments with statin treatment\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional rescue; single lab, non-canonical function\",\n      \"pmids\": [\"35619540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UFMylation of Eg5 at Lys564 is required for proper spindle organization: Eg5 is a substrate of the UFM1 pathway; UFMylation does not alter Eg5 transcription, phosphorylation, or protein stability but affects its mono-ubiquitination and spindle localization; the K564R (UFMylation-defective) mutant shows shorter spindles, metaphase arrest, spindle checkpoint activation, and cell division failure.\",\n      \"method\": \"Identification of Eg5 as UFM1 substrate, site-directed mutagenesis (K564R), co-localization at centrosome/spindle, ubiquitination assays, spindle length and checkpoint assays in HeLa cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of modification site with functional phenotype readout; single lab; PTM site established but mechanism of UFMylation-mono-Ub crosstalk not fully resolved\",\n      \"pmids\": [\"39085203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Eg5 is expressed in postmitotic rodent neurons, directly associates with the microtubule array, and is enriched in distal regions of developing axons and dendrites transiently during process outgrowth, re-appearing in newly forming dendritic sprouts, suggesting a role in regulating microtubule behavior at distal tips of developing neuronal processes.\",\n      \"method\": \"Western blot, immunofluorescence, microtubule co-pelleting from egg homogenates\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with biochemical co-sedimentation; functional inference is correlative but localization is directly demonstrated\",\n      \"pmids\": [\"9742151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Inhibition of kinesin-5 (Eg5) by amyloid-beta (Aβ) or monastrol reduces transport of NGF/NTR(p75) and NMDA receptors to the cell surface, inhibits NGF-dependent neurite outgrowth from PC12 cells, blocks glutamate-dependent Ca²⁺ entry into primary neurons, and inhibits long-term potentiation; Eg5 activity is absent from APP/PS transgenic mouse brain.\",\n      \"method\": \"Cell-surface receptor quantification, neurite outgrowth assay, Ca²⁺ imaging, LTP electrophysiology, monastrol and Aβ treatment\",\n      \"journal\": \"Neurobiology of aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional readouts in neurons linking Eg5 inhibition to receptor trafficking; single lab\",\n      \"pmids\": [\"24636920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mice, postnatal EC-specific conditional knockout of Kif11 leads to severely stunted growth of the retinal vasculature and mildly stunted cerebellar vasculature, with little effect elsewhere in the CNS, demonstrating Kif11 function in endothelial cell mitosis is most critical in rapidly growing CNS vascular beds; this phenotype is not caused by reduced β-catenin signaling in ECs.\",\n      \"method\": \"Conditional knockout mouse (Kif11 CKO in endothelial cells), retinal flat-mount vasculature analysis, β-catenin signaling readouts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific genetic knockout with defined vascular phenotype and mechanistic exclusion of β-catenin pathway\",\n      \"pmids\": [\"31993640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASPM physically interacts with KIF11 (by co-immunoprecipitation in HCC cells), and ASPM exerts its effects on HCC cell proliferation, invasion, and migration through KIF11; the ASPM-KIF11 complex promotes malignant progression by activating the Wnt/β-catenin signaling pathway.\",\n      \"method\": \"Co-immunoprecipitation, rescue overexpression of KIF11 after ASPM knockdown, Western blot for Wnt/β-catenin components\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus rescue experiment; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"34504599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PAK6 specifically co-localizes with Eg5 at the centrosome; PAK6 depletion results in multipolar spindles and upregulation of Eg5; Eg5 knockdown reverses PAK6-depletion-induced multipolar spindles; PAK6 and Eg5 negatively inter-regulate each other's expression.\",\n      \"method\": \"Co-localization by immunofluorescence, siRNA knockdown epistasis, Western blot for protein levels\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-localization and genetic epistasis by knockdown, single lab, mechanism of inter-regulation not biochemically resolved\",\n      \"pmids\": [\"33098954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of Kif11 function in zebrafish radial glial cells (by mutation or STLC inhibition) causes monoastral spindle formation and mitotic arrest (~226× delay in mitotic exit), leading to apoptosis and specific reductions in oligodendroglia and secondary interneurons/motorneurons, demonstrating Kif11 is required for radial glia mitotic progression and subsequent neurogenesis.\",\n      \"method\": \"Forward genetic screen, pharmacological inhibition (STLC), anti-activated Caspase-3 staining, mathematical modeling, cell-type-specific marker analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutant plus pharmacological validation with quantitative modeling; zebrafish ortholog; multiple readouts\",\n      \"pmids\": [\"24370453\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KIF11/Eg5 is a homotetrameric, plus-end-directed kinesin-5 motor whose ATPase cycle (rate-limited by MT-dependent ADP release) powers antiparallel microtubule sliding to separate centrosomes and assemble/maintain the bipolar mitotic spindle; its spindle localization and activity are controlled by a multi-kinase phosphorylation cascade (CDK1/2 at Thr926/927, Plk1→Nek9→Nek6/7 at Ser1033) and balanced by dephosphorylation via PP2A/B55α, while protein stability is regulated by RNF20/40-mediated monoubiquitination, NAT10-mediated acetylation at K771, UFMylation at K564, and Parkin-dependent transcriptional repression through the Hsp70-JNK-c-Jun axis; beyond mitosis, KIF11 transports β-actin mRNPs via ZBP1, regulates dendrite morphogenesis through NEK7-mediated microtubule stabilization, controls primary cilia dynamics at basal bodies, and acts as an inhibitory constraint on excitatory synaptic transmission in neurons.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KIF11/Eg5 is a slow, plus-end-directed kinesin-5 ATPase whose mechanochemical cycle is rate-limited by ATP-dependent microtubule dissociation [#1], and which generates the antiparallel microtubule-sliding forces that drive centrosome separation, bipolar spindle assembly, and spindle elongation; in human cells it cooperates with PRC1-dependent KIF4A to slide midzone microtubules, and combined loss of the two motors abolishes chromosome segregation [#22]. Its essentiality for cell division is reflected in early embryonic lethality of homozygous knockout mice [#5] and mitotic arrest with monoastral spindles upon loss of function [#31]. Targeting of Eg5 to the spindle and centrosomes is governed by a phosphorylation cascade: CDK1 (with CDK2 compensation) phosphorylates the conserved C-terminal bimC domain (Thr926/Thr927/Thr937), and Plk1 acting through Nek9-Nek6/7 adds Ser1033, both required for centrosome accumulation and separation [#0, #7, #8]; PP2A/B55\\u03b1 reverses Thr926 phosphorylation to permit mitotic exit [#15]. Eg5 force output is constrained by inhibitory partners TPX2, which slows the motor through microtubule and neck-region binding [#12], and HMMR, which localizes TPX2 to promote inhibitory TPX2-Eg5 complexes [#17]. Eg5 abundance, motor activity, and spindle loading are further tuned by post-translational modifications: RNF20/40 monoubiquitination [#10], NAT10 acetylation at K771 [#23], HDAC1 deacetylation [#14], and UFMylation at K564 [#25], while Parkin represses Eg5 transcription via an Hsp70-JNK-c-Jun axis [#4]. Beyond mitosis, Eg5 transports \\u03b2-actin mRNPs through direct ZBP1 (IGF2BP1) binding to support cell migration [#11], localizes to basal bodies to restrain primary cilium length and disassembly [#20], and in postmitotic neurons is phosphorylated by NEK7 to stabilize distal dendritic microtubules independently of motor activity [#16] and acts as a brake on excitatory synaptic transmission [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established that Eg5 spindle targeting is under cell-cycle control, defining the link between mitotic kinase activity and motor localization.\",\n      \"evidence\": \"Site-directed mutagenesis of the CDK1 phosphorylation site (T937) with immunofluorescence in Xenopus A6 cells\",\n      \"pmids\": [\"7753799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the full set of regulatory kinases\", \"Did not connect localization to force generation\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the mechanochemical cycle of the motor, showing microtubule dissociation rather than hydrolysis is rate-limiting and the mechanism resembles conventional kinesin.\",\n      \"evidence\": \"Pre-steady-state and steady-state kinetics with mantATP/mantADP on monomeric human Eg5\",\n      \"pmids\": [\"15247293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Used monomeric construct, not the native homotetramer\", \"Did not address antiparallel sliding mechanics\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated absolute requirement of Eg5 for early mammalian development with no motor compensation, establishing biological essentiality.\",\n      \"evidence\": \"Knsl1 gene-targeted knockout mice with embryo analysis\",\n      \"pmids\": [\"18474226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pre-implantation lethality precludes analysis of later tissue-specific roles\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed transcriptional repression of Eg5 by Parkin through an Hsp70-JNK-c-Jun route, distinguishing transcriptional control from proteasomal degradation.\",\n      \"evidence\": \"Promoter reporter, ubiquitination, and JNK/c-Jun phosphorylation assays with proteasome controls\",\n      \"pmids\": [\"18845538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Physiological context of Parkin-Eg5 regulation not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the kinase hierarchy driving centrosome separation, placing CDK1/CDK2 and a Plk1-Nek9-Nek6/7 cascade upstream of distinct Eg5 phosphosites.\",\n      \"evidence\": \"In vitro kinase assays, phospho-specific antibodies, and centrosome separation epistasis (two companion EMBO studies)\",\n      \"pmids\": [\"21642957\", \"21522128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each phosphosite to motor activity not isolated\", \"Did not reconstitute phosphorylation-dependent sliding in vitro\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified Tiam1-Rac as an opposing prophase force, framing Eg5-driven separation as a balance of antagonistic forces.\",\n      \"evidence\": \"siRNA depletion, conditional Rac1 knockout, monastrol, and reciprocal rescue with live-cell imaging\",\n      \"pmids\": [\"20346677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of the opposing force not pinpointed\", \"Direct biochemical link to Eg5 not shown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined direct biophysical inhibition of the motor by TPX2 through dual microtubule and neck-region binding.\",\n      \"evidence\": \"TIRF single-molecule and gliding assays with truncated TPX2 constructs\",\n      \"pmids\": [\"26018074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulatory consequence addressed only later\", \"Structural basis of neck-region binding not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed HMMR positions TPX2 to assemble inhibitory TPX2-Eg5 complexes, controlling spindle force balance and segregation fidelity.\",\n      \"evidence\": \"siRNA, Co-IP, and chemical epistasis with Eg5 inhibitor rescue and aneuploidy quantification\",\n      \"pmids\": [\"29386294\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct effect of HMMR on motor kinetics not measured\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Directly measured Eg5's force-generating role in spindle elongation, showing it cooperates with KIF4A to slide midzone microtubules.\",\n      \"evidence\": \"Combined CRISPR/RNAi depletion with tubulin photoactivation, STED, and expansion microscopy\",\n      \"pmids\": [\"33910056\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative force contribution of each motor not quantified\", \"Regulation of the EG5-KIF4A cooperation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded the PTM regulatory layer, establishing acetylation, monoubiquitination, deacetylation, and UFMylation as controllers of Eg5 stability, activity, and spindle loading.\",\n      \"evidence\": \"Site-directed mutagenesis (K771Q/R, K564R), modification-specific antibodies, Co-IP, ubiquitination assays, and rescue in HeLa cells\",\n      \"pmids\": [\"27557628\", \"28392145\", \"35210604\", \"39085203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between UFMylation and monoubiquitination not fully resolved\", \"Temporal coordination of distinct PTMs during the cell cycle unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Uncovered a non-mitotic transport function, with Eg5 binding ZBP1 to deliver \\u03b2-actin mRNPs and support cell migration.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro domain mapping, \\u03b2-actin mRNA imaging, and migration assays\",\n      \"pmids\": [\"25588836\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological relevance of mRNP transport not established\", \"Coupling to motor stepping not biophysically dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended Eg5 function to interphase and postmitotic cells, including basal body regulation of cilia and dendritic microtubule stabilization, the latter motor-independent.\",\n      \"evidence\": \"siRNA, NEK7 kinase-dead and motor-dead rescues, live-cell microtubule imaging, cilia quantification (multiple studies)\",\n      \"pmids\": [\"32811879\", \"29899413\", \"30479371\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism distinguishing motor-dependent vs motor-independent roles incompletely defined\", \"Tissue-specific regulation of these functions unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated tissue-selective dependence on Eg5-driven endothelial mitosis, most critical in rapidly growing CNS vascular beds and independent of \\u03b2-catenin signaling.\",\n      \"evidence\": \"Endothelial-specific conditional Kif11 knockout with retinal flat-mount vasculature analysis\",\n      \"pmids\": [\"31993640\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis of vascular-bed selectivity not explained\", \"Downstream pathway driving the phenotype not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple PTMs, kinase cascades, and inhibitory partners are temporally integrated to tune Eg5 force output across mitotic stages and non-mitotic contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking PTM state to motor velocity in vivo\", \"Mechanistic basis for switching between mitotic and post-mitotic functions unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003774\", \"supporting_discovery_ids\": [1, 22, 23]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [16, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [0, 13, 22]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [7, 23, 30]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [16, 26]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 7, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 28, 31]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [16, 18, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TPX2\", \"ZBP1\", \"PTEN\", \"RNF20\", \"HMMR\", \"NAT10\", \"NEK7\", \"PAK6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}