{"gene":"PLK1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1994,"finding":"Human PLK (PLK1) encodes a serine/threonine kinase of 603 amino acids; its mRNA expression is strongly correlated with mitotic activity of cells, being absent in resting cells and induced upon cell cycle entry.","method":"cDNA cloning, Northern blot analysis, cell synchronization experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cDNA sequencing plus multiple cell-type expression studies, single lab","pmids":["8127874"],"is_preprint":false},{"year":1997,"finding":"The PLK1 promoter contains a repressor element (CDE/CHR) near the transcription start site that mediates cell cycle-dependent transcription; three activating regions are located between −35 and −93 bp upstream, with promoter activity maximal at G2/M phase.","method":"PLK promoter/luciferase constructs in synchronized HeLa cells, deletion/mutation analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional promoter-reporter assays with mutagenesis, single lab","pmids":["9083047"],"is_preprint":false},{"year":2000,"finding":"PLK1 binds to and phosphorylates the Golgi peripheral protein GRASP65 in vitro and in vivo; deletion or frameshift mutations in the conserved C-terminal polo-box domain of PLK1 greatly diminish its ability to phosphorylate GRASP65, demonstrating the PBD is required for this substrate interaction.","method":"Yeast two-hybrid screen, co-immunoprecipitation, in vitro kinase assay, deletion/frameshift mutant analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis plus co-IP, multiple orthogonal methods in single study","pmids":["11050165"],"is_preprint":false},{"year":2000,"finding":"The Ste20-like kinase SLK can phosphorylate and activate murine PLK1; SLK activity increases during G2 phase progression, proposing SLK as an upstream kinase regulating PLK1 in the somatic cell cycle.","method":"In vitro kinase assay, endogenous SLK activity measurements, cell synchronization","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro phosphorylation assay demonstrated, single lab, no epistasis confirmation","pmids":["10886374"],"is_preprint":false},{"year":2003,"finding":"Depletion of PLK1 by siRNA causes cell cycle arrest with 4N DNA content, formation of dumbbell-like chromatin, activation of caspase-3, and apoptosis; the p53 pathway and ATM are involved in PLK1-depletion-induced apoptosis, and DNA damage occurs even in p53-null cells.","method":"Vector-based siRNA knockdown, FACS, caspase-3 activation assay, rescue with non-degradable Plk1, ATM inhibitor co-treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal readouts (FACS, caspase assay, rescue experiment) replicated in multiple cancer cell lines","pmids":["12732729"],"is_preprint":false},{"year":2003,"finding":"BRCA1 mediates repression of PLK1 mRNA after ionizing radiation through a CHK1-dependent pathway, and loss of BRCA1 impairs this downregulation, linking BRCA1 to G2/M checkpoint control via PLK1 transcriptional suppression.","method":"RT-PCR of PLK mRNA in irradiated BRCA1−/− cells vs. BRCA1-reconstituted cells, CHK1 inhibitor (UCN-01) treatment","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic reconstitution plus pharmacological inhibitor, single lab","pmids":["14654792"],"is_preprint":false},{"year":2004,"finding":"PLK1 physically binds to the sequence-specific DNA-binding domain of p53 and phosphorylates it, inhibiting p53 transactivation of p21WAF1, MDM2, and BAX promoters and its pro-apoptotic function; kinase-dead PLK1 fails to repress p53 transcriptional activity.","method":"Co-immunoprecipitation with deletion mutants, confocal co-localization, luciferase reporter assay, endogenous p21 induction assay, kinase-dead mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct binding mapped by deletion mutants, kinase-dead mutagenesis, multiple functional readouts, single lab","pmids":["15024021"],"is_preprint":false},{"year":2005,"finding":"PLK1 phosphorylation at T210 (activation loop) and S137 occurs in mitosis; DNA damage prevents phosphorylation at both sites in asynchronous cells through ATM/ATR and CHK1/CHK2 pathways; constitutively active T210D mutant is insensitive to DNA damage-induced inhibition.","method":"In vivo phosphopeptide mapping, T210D mutant analysis, kinase inhibitor treatments (ATM/ATR, CHK1/CHK2 inhibitors)","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphosite mapping with pharmacological and genetic tools, single lab","pmids":["15611664"],"is_preprint":false},{"year":2005,"finding":"The polo-box domain (PBD) of PLK1 interacts with MCM2 and MCM7 (and the entire MCM2-7 complex); the strongest interaction between endogenous PLK1 and Mcm7 is detected in a soluble chromatin fraction, suggesting a role for PLK1 in coordinating DNA replication and mitotic events.","method":"PBD-bait pulldown screen, co-immunoprecipitation, subcellular fractionation, immunoblot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus fractionation, single lab","pmids":["15654075"],"is_preprint":false},{"year":2007,"finding":"TTDN1 is identified as a PLK1-interacting protein; CDK1 phosphorylates TTDN1, which is required for TTDN1-PLK1 interaction; mutation of Thr120 in TTDN1's PLK1-binding motif abolishes the interaction; TTDN1 co-localizes with PLK1 at centrosomes during mitosis and the midbody during cytokinesis.","method":"Yeast two-hybrid, co-immunoprecipitation, site-directed mutagenesis, immunofluorescence co-localization","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus mutagenesis plus localization, single lab","pmids":["17310276"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the PLK1 kinase domain with BI 2536 reveals that Leu132 in the hinge region creates a small pocket exploited by the methoxy group of BI 2536 for selectivity against non-PLK kinases; structure solved to inform structure-based drug design.","method":"X-ray crystallography (co-crystal structure of PLK1 KD with BI 2536)","journal":"Chemical biology & drug design","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with selectivity data, single lab but direct structural evidence","pmids":["18005335"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of wild-type PLK1 kinase domain in complex with DARPin 3H10 determined at 2.3 Å resolution, revealing the active conformation of PLK1; DARPin enabled crystallization by masking a surface patch rich in charged residues.","method":"X-ray crystallography using DARPin-assisted co-crystallization","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure, single lab","pmids":["18391401"],"is_preprint":false},{"year":2008,"finding":"PLK-1 protein is distributed asymmetrically in C. elegans two-cell embryos with more present in the anterior AB blastomere than in P1; this asymmetry is regulated by PAR-protein-dependent anterior-posterior polarity cues through preferential protein retention; mild PLK-1 RNAi delays mitotic entry specifically in P1, promoting asynchronous cell division.","method":"Live imaging, RNAi knockdown, PAR mutant analysis in C. elegans embryos","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging plus genetic (RNAi) with quantitative cell cycle timing, single lab","pmids":["18305005"],"is_preprint":false},{"year":2010,"finding":"PICH and PLK1 cooperate to maintain chromosome architecture during prometaphase; PICH knockdown removes PLK1 from chromosome arms causing disorganized chromosomes with open/X-shaped configuration consistent with loss of arm cohesion; chromosome disorganization is prevented by topoisomerase II inhibitor, indicating the PICH-PLK1 complex maintains architecture through topoisomerase II.","method":"siRNA knockdown of PICH, immunofluorescence, topoisomerase II inhibitor (ICRF-193) treatment","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with pharmacological rescue, single lab","pmids":["20130082"],"is_preprint":false},{"year":2013,"finding":"CUL3-KLHL22 E3 ubiquitin ligase ubiquitylates PLK1 at Lys492 within the polo-box domain, causing degradation-independent dissociation of PLK1 from kinetochore phosphoreceptors; non-ubiquitylatable PLK1-K492R accumulates at kinetochores, activates the spindle assembly checkpoint, and phenocopies KLHL22 inactivation.","method":"Mass spectrometry identification, Co-IP, K492R mutant expression, live imaging, spindle assembly checkpoint assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ubiquitylation site mapped by MS, functional validated with non-ubiquitylatable mutant, multiple orthogonal methods","pmids":["23455478"],"is_preprint":false},{"year":2013,"finding":"Aurora-A together with its cofactor Bora remains the major activator of PLK1 during mitosis (not only at G2/M transition); a small fraction of Bora is retained in mitosis, essential for continued Aurora-A-dependent T210 phosphorylation; PLK1 activation acts as a bistable switch requiring minimal Bora-Aurora-A once fully active.","method":"Kinase inhibitor treatments, cell synchronization, immunoblotting for pT210, siRNA knockdown of Bora","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic perturbations, single lab","pmids":["24338364"],"is_preprint":false},{"year":2014,"finding":"Sequential phosphorylation of procaspase-8 by CDK1/cyclin B1 (at S387, generating a PBD-docking site) and then PLK1 (at S305) during mitosis inhibits extrinsic apoptotic signaling; non-phosphorylatable S305A caspase-8 increases Fas-stimulated cell death; PLK1 inhibitor BI 2536 lowers the threshold for Fas-induced death.","method":"RNAi replacement strategy, phospho-site mutagenesis (S305A, S387A), co-IP with PBD, Fas stimulation apoptosis assay, PLK1 inhibitor treatment","journal":"Molecular oncology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional apoptosis readout, single lab with multiple orthogonal methods","pmids":["24484936"],"is_preprint":false},{"year":2014,"finding":"PLK1 phosphorylates CLIP-170 at Ser312 during mitosis; this phosphorylation diminishes CLIP-170 binding to microtubule ends and lattice (without affecting EB3 binding); phosphorylation/dephosphorylation cycling at Ser312 is required for stable kinetochore-microtubule attachment and chromosome alignment.","method":"In vitro kinase assay with purified CLIP-170 N-terminal fragment, phospho-specific antibody, S312A/S312D mutant analysis, live imaging of chromosome alignment","journal":"Cell structure and function","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional imaging, single lab","pmids":["24451569"],"is_preprint":false},{"year":2014,"finding":"DNA-PKcs physically associates with PLK1 during the mitotic phase; depletion of DNA-PKcs leads to overexpression of PLK1 through increased protein stability but attenuates recruitment of phosphorylated PLK1 to the midbody (while not affecting centrosome/kinetochore localization), resulting in cytokinesis failure.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence co-localization","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus localization study, single lab, single method for each claim","pmids":["24166892"],"is_preprint":false},{"year":2014,"finding":"LKB1 regulates PLK1 by activating NUAK1 (ARK5), which phosphorylates MYPT1, enhancing MYPT1-PP1 binding to PLK1 and promoting PLK1 dephosphorylation; LKB1 deficiency leads to elevated phospho-PLK1 and centrosome amplification, reversed by PLK1 depletion or inhibition.","method":"Co-immunoprecipitation, kinase assay, siRNA, PLK1 overexpression rescue, BTO-1 inhibitor treatment","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis defined by sequential knockdowns/inhibitions, co-IP, single lab","pmids":["24722282"],"is_preprint":false},{"year":2015,"finding":"PLK-1 is required in C. elegans oocytes for nuclear envelope breakdown; partial PLK-1 inactivation causes failure to form a gap in the nuclear envelope at the interface between pronuclei and defects in chromosome congression, resulting in paired nuclei containing either maternal or paternal chromosomes in daughter cells.","method":"Partial RNAi inactivation, live fluorescence imaging of nuclear envelopes and chromosomes in C. elegans embryos","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with genetic perturbation and defined phenotypic readout, single lab","pmids":["26490119"],"is_preprint":false},{"year":2016,"finding":"PTEN physically associates with PLK1 and reduces PLK1 phosphorylation in a phosphatase-dependent manner; PTEN deficiency leads to elevated PLK1 phosphorylation and polyploidy; phospho-mimicking PLK1 mutant causes polyploidy similar to PTEN loss, while non-phosphorylatable PLK1 reduces the polyploid population.","method":"Co-immunoprecipitation, phospho-mimicking and non-phosphorylatable PLK1 mutant expression, PTEN phosphatase-dead mutant, cell ploidy analysis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus phospho-mutant rescue experiments, single lab","pmids":["27398835"],"is_preprint":false},{"year":2017,"finding":"PLK1 phosphorylates Mre11 at S649, which primes subsequent CK2-mediated phosphorylation at S688; dual phosphorylation inhibits MRN complex loading onto damaged DNA, leading to premature DNA damage checkpoint termination and inhibition of DNA repair.","method":"In vitro kinase assay, phospho-site mutagenesis, chromatin loading assay, DNA damage checkpoint assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus phospho-site mutagenesis plus functional chromatin recruitment assay, single lab","pmids":["28512243"],"is_preprint":false},{"year":2017,"finding":"Gravin acts as a scaffold for PLK1 at mitotic centrosomes; Gravin depletion increases PLK1 mobility (by FRAP), redistributes active PLK1, increases phosphorylation of CEP215 at S613, causes microtubule renucleation defects, chromosome misalignment, and micronuclei; a Gravin mutant unable to bind PLK1 fails to rescue these defects.","method":"FRAP, PLK1 FRET-based activity biosensor, shRNA knockdown, PLK1-binding-deficient Gravin mutant rescue","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — FRAP, FRET biosensor, genetic rescue with separation-of-function mutant, multiple orthogonal methods","pmids":["29282278"],"is_preprint":false},{"year":2017,"finding":"PLK1 regulates NOTCH1 expression at the G2/M transition; upon DNA damage in G2, PLK1 activity is inhibited while NOTCH1 expression is maintained; PLK1-dependent signaling antagonizes NOTCH1 involvement in the DNA damage checkpoint in squamous cell carcinoma cells.","method":"Chemical library kinase screen, pharmacological PLK1 inhibition, co-immunoprecipitation, cell cycle synchronization, immunoblotting","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — chemical screen plus co-IP, mechanistic details partly inferential, single lab","pmids":["31597699"],"is_preprint":false},{"year":2018,"finding":"PLK1 overexpression causes defective cytokinesis correlated with defective loading of Cep55 and ESCRT complexes to the abscission bridge in a PLK1 kinase-dependent manner, generating polyploid cells with reduced proliferative potential.","method":"Inducible knock-in mouse model, immunofluorescence of Cep55/ESCRT at abscission bridges, kinase-dead PLK1 comparison","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in model plus mechanistic follow-up with Cep55/ESCRT localization, kinase-dependence established","pmids":["30069007"],"is_preprint":false},{"year":2018,"finding":"CDK1/Aurora-A phosphorylates CtIP at S327, creating a PLK1 polo-box domain docking site; PLK1 then phosphorylates CtIP at S723; phospho-mimetic CtIP S723 fails to perform extended end resection and homologous recombination but retains ability to mediate microhomology-mediated end joining (MMEJ) while inactivating the G2/M checkpoint.","method":"In vitro kinase assay, phospho-site mutagenesis, MMEJ/HR reporter assays, G2/M checkpoint assay, PLK1-PBD binding assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus sequential phosphorylation mapping plus functional MMEJ/HR readout, single lab with multiple orthogonal methods","pmids":["30202980"],"is_preprint":false},{"year":2018,"finding":"DNA replication restricts activation of both CDK1 and PLK1; preventing DNA replication licensing and/or firing causes premature activation of CDK1 and PLK1 in S phase; inhibition of CHK1 and p38 in the presence of replication leads to premature mitotic kinase activation and replication stress.","method":"Double-degron system for protein depletion, kinase inhibitors, cell synchronization, CDK1/PLK1 activity reporters","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal perturbations (degron, inhibitors) with quantitative kinase activity readouts, single lab","pmids":["30008317"],"is_preprint":false},{"year":2019,"finding":"PLK1 plays dual roles in cytokinesis: PLK1 activity promotes release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to activate RhoA at the equatorial cortex; PLK1 inhibition sequesters centralspindlin on the midzone, making it unavailable for Aurora B-dependent RhoA activation.","method":"PRC1 knockdown, PLK1 inhibitor treatment, live imaging of centralspindlin and RhoA activity","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological perturbations with live imaging, single lab","pmids":["30728176"],"is_preprint":false},{"year":2019,"finding":"PLK1 methyltransferase SETD6 methylates PLK1 at K209 and K413 during mitosis; loss of these methylation sites increases PLK1 kinase activity, accelerates mitotic progression and cellular proliferation; SETD6-deficient cells phenocopy non-methylatable PLK1.","method":"Proteomic screen, in vitro methylation assay, K209R/K413R PLK1 mutants, kinase activity assay, live cell imaging of mitotic progression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation assay plus non-methylatable mutant functional analysis plus SETD6-KO phenotypic matching, single lab with multiple orthogonal methods","pmids":["30622182"],"is_preprint":false},{"year":2019,"finding":"PLK1 inhibition causes centromere disintegration driven by BLM helicase-mediated DNA unwinding at centromeres under bipolar spindle pulling force; PLK1 normally suppresses BLM activity at centromeres to maintain centromere integrity for chromosome biorientation.","method":"PLK1 inhibitor treatment, BLM helicase co-depletion rescue experiment, live imaging of centromere dynamics","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition plus genetic epistasis rescue, live imaging, single lab","pmids":["31253795"],"is_preprint":false},{"year":2019,"finding":"PLK1 phosphorylates Gli1 at S481, promoting nuclear export of Gli1 and its binding to the negative regulator Sufu, thereby inhibiting Hedgehog signaling activity during G2/M; PLK1 inhibition prevents this phosphorylation and sustains Gli1 transcriptional activity.","method":"In vitro kinase assay, S481 phospho-site mutagenesis, nuclear/cytoplasmic fractionation, Gli1-Sufu co-IP, Hedgehog target gene luciferase reporter","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, phospho-site mutagenesis, and multiple functional readouts (localization, protein interaction, target gene expression), single lab","pmids":["30578313"],"is_preprint":false},{"year":2019,"finding":"Chromatin-localized PLK1 signals maintain kinetochore and centromere integrity (CENP-A, CENP-C, CENP-T, Hec1, Nuf2, Knl1 retention) against microtubule pulling forces; PICH helicase is required for centromere disruption in PLK1-inhibited cells and PICH depletion rescues kinetochore architecture; tethering PLK1 to chromatin (H2B, INCENP) but not CENP-A rescues anaphase defects.","method":"PLK1 inhibitor treatment, PICH siRNA rescue, chromatin-tethered PLK1 constructs (H2B-PLK1, INCENP-PLK1, CENP-A-PLK1), immunofluorescence of kinetochore components","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — targeted rescue with chromatin-tethering, epistasis with PICH knockdown, multiple kinetochore markers, single lab with multiple orthogonal methods","pmids":["31468671"],"is_preprint":false},{"year":2020,"finding":"BRCA2 is phosphorylated by PLK1 at T207 in mitotic cells; phospho-T207 serves as a PLK1 docking site; BRCA2 bound to PLK1 forms a complex with PP2A and phospho-BUBR1; BRCA2 breast cancer variants S206C and T207A reduce PLK1 binding, causing unstable kinetochore-microtubule interactions, chromosome misalignment, and aneuploidy.","method":"Phospho-site identification, co-immunoprecipitation (BRCA2-PLK1-PP2A-BUBR1 complex), BRCA2 variant functional analysis, chromosome alignment assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphosite mapping, complex reconstitution by co-IP, cancer variant separation-of-function analysis, multiple orthogonal methods","pmids":["32286328"],"is_preprint":false},{"year":2020,"finding":"PLK1 is required for chromosome compaction, cohesin/condensin localization, and MTOC organization in mouse oocytes; PLK1 conditional knockout oocytes fail to form compact bivalents, develop abnormal or absent bipolar spindles due to defective C-NAP1 release and failure to recruit MTOC components and LISD factors.","method":"Plk1 conditional knockout mouse (oocyte-specific), immunofluorescence of cohesin, condensin, C-NAP1, MTOC components","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific conditional KO with mechanistic follow-up of multiple molecular markers, single lab","pmids":["32267211"],"is_preprint":false},{"year":2020,"finding":"SET7/9 dimethylates PLK1 at K191 during early mitosis, reducing PLK1 kinase activity by limiting ATP utilization; non-methylatable PLK1 K191 mutant or SET7/9 inhibition causes mitotic arrest due to destabilized kinetochore-microtubule attachments, indicating methylation promotes dynamic attachments for error correction.","method":"In vitro methylation assay, K191 mutation, SET7/9 inhibitor treatment, kinetochore-microtubule attachment assay, kinase activity assay","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation assay plus non-methylatable mutant with functional kinetochore-MT assay, single lab with multiple orthogonal methods","pmids":["31863092"],"is_preprint":false},{"year":2020,"finding":"BUB1 and CENP-U are the main PLK1 kinetochore receptors in mitosis; both contain a constellation of a PP2A-docking motif flanked by two PLK1-docking sites; CDK1-primed phosphorylation on these sites enables PLK1 binding and promotes PLK1 dimerization at kinetochores; this mechanism accounts for most/all kinetochore PLK1.","method":"Ectopic localization assay, in vitro reconstitution, kinetochore localization studies, co-IP, CDK1 priming assay, PLK1 dimerization assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution plus ectopic localization plus kinetochore depletion analysis, multiple orthogonal methods","pmids":["33248027"],"is_preprint":false},{"year":2021,"finding":"PLK1 dimerizes during early G2 phase in a Bora-supported manner; T210 phosphorylation by Aurora-A at late G2 triggers dimer dissociation generating active PLK1 monomers; interfering with the dimer/monomer switch prevents importin association and limits nuclear shuttling, causing nuclear PLK1 mislocalization at G2/M.","method":"Co-immunoprecipitation of PLK1 dimers, phospho-T210 mutant analysis, importin interaction assay, live cell nuclear localization imaging","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus mutant functional analysis plus localization, single lab","pmids":["34759346"],"is_preprint":false},{"year":2021,"finding":"Apolo1 localizes to kinetochores during early mitosis and sustains PLK1 kinase activity at kinetochores (measured by FRET biosensor); Apolo1 is a PLK1 substrate, and its phosphorylation enables PP1γ to dephosphorylate and inactivate PLK1; Apolo1 constitutes a feedback loop connecting PLK1 to its own inactivation.","method":"FRET-based PLK1 activity reporter, co-IP, in vitro kinase assay, PP1γ interaction assay, siRNA knockdown with chromosome alignment readout","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — FRET biosensor plus in vitro kinase assay plus co-IP, single lab with multiple orthogonal methods","pmids":["34260926"],"is_preprint":false},{"year":2021,"finding":"PLK1 phosphorylates PrimPol at a conserved residue between its RPA-binding motifs; this phosphorylation is cell cycle-dependent and prevents aberrant PrimPol recruitment to chromatin; loss of PLK1-dependent regulation leads to chromosome breaks, micronuclei, and reduced survival after replication stress.","method":"In vitro kinase assay, phospho-site identification, chromatin fractionation, siRNA, cell viability assays","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus chromatin recruitment assay, single lab","pmids":["34860556"],"is_preprint":false},{"year":2021,"finding":"In C. elegans oocyte meiosis, BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent; CENP-CHCP-4 recruits PLK-1 to chromosome arms through direct interaction; perturbing both recruitment mechanisms causes severe meiotic defects and highly aneuploid oocytes.","method":"Biochemical interaction assays, live imaging, C. elegans genetic mutants, co-immunoprecipitation","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical binding assay plus live imaging plus double-perturbation epistasis, single lab with multiple orthogonal methods","pmids":["37067150"],"is_preprint":false},{"year":2021,"finding":"PLK1 regulates DNA replication origin firing in Xenopus by controlling origin firing frequency and synchrony; PLK1 interacts with firing factors MTBP/Treslin/TopBP1 and with Rif1; PLK1 phosphorylates Rif1 at S2058 (in its PP1-binding site), disrupting Rif1-PP1 interaction and modulating DNA replication.","method":"PLK1 immunodepletion in Xenopus extract, DNA fiber analysis, quantitative chromatin proteomics, co-immunoprecipitation, LC-MS/MS phosphopeptide mapping, S2058D phospho-mimetic mutant","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro phosphorylation in Xenopus system plus phospho-site mapping plus phospho-mimetic functional mutant, multiple orthogonal methods","pmids":["34469577"],"is_preprint":false},{"year":2021,"finding":"PLK1 phosphorylates DNMT3a, leading to its proteasomal degradation during mitosis; DNMT3a in turn represses PLK1 transcription in interphase, constituting a negative feedback loop; combined inhibition of PLK1 and DNMT3a suppresses prostate cancer synergistically through autophagy regulation.","method":"Co-IP, kinase assay, DNMT3a stability assay upon PLK1 activation, PLK1 promoter analysis","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay plus promoter analysis plus co-IP, single lab","pmids":["34051063"],"is_preprint":false},{"year":2021,"finding":"PLK1 activity is essential for time-dependent release of 53BP1 from kinetochores; PLK1 inhibition causes 53BP1 persistence at kinetochores (via CENP-F docking), preventing cytosolic 53BP1 association with p53 and blunting the mitotic surveillance pathway; CENP-F-53BP1 binding disruption has no impact on mitotic timing, indicating cytosolic (not kinetochore) 53BP1 measures mitotic duration.","method":"PLK1 inhibitor treatment, CENP-F-53BP1 binding mutant, immunofluorescence quantification of kinetochore/cytosolic 53BP1, p53 activation assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition plus separation-of-function mutant, single lab","pmids":["37888778"],"is_preprint":false},{"year":2021,"finding":"PLK1 and Aurora-A are both required for centrosome maturation and separation prior to metaphase I in mouse spermatocytes; PLK1 is additionally required to block premature second-round centriole duplication until anaphase I, maintaining strict spatiotemporal control of centrosome biogenesis during meiosis.","method":"Germ cell-specific conditional knockout of Plk1 and Aurora-A, immunofluorescence of centriole/centrosome markers through meiotic progression","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — tissue-specific conditional KO with mechanistic analysis at multiple meiotic stages, single lab","pmids":["33615678"],"is_preprint":false},{"year":2022,"finding":"PLK1 promotes NLRP3 inflammasome activation during cell interphase; PLK1 is in close proximity to and interacts with NLRP3; mechanistically, PLK1 orchestrates the MTOC structure and NLRP3 subcellular positioning upon inflammasome activation; pharmacological PLK1 inhibition suppresses IL-1β production in vivo.","method":"BioID proximity-labeling screen, co-immunoprecipitation, domain interaction mapping, pharmacological PLK1 inhibition, in vivo endotoxemia and peritonitis mouse models","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID plus co-IP plus in vivo functional assay, single lab","pmids":["37698938"],"is_preprint":false},{"year":2022,"finding":"Pathogenic BRCA1 mutations reduce BRCA1 function, leading to elevated PLK1 activity, which specifically disrupts cell division axis orientation in mammary cells; this defect is independent of accumulated DNA damage and depends on PLK1 activity, as shown by 15 genetically edited BRCA1 mutant cell lines.","method":"Primary mammary cell division axis imaging, BRCA1-mutant genetically-edited cell lines, PLK1 activity measurements, genetic and pharmacological PLK1 inhibition","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell lines, PLK1 inhibitor rescue, single lab","pmids":["35459234"],"is_preprint":false},{"year":2022,"finding":"PLK1-mediated phosphorylation of RB at S758 inhibits NF-κB nuclear translocation, inactivating the NF-κB pathway; PLK1 inhibition or depletion therefore activates NF-κB, upregulating PD-L1 expression in pancreatic ductal adenocarcinoma cells.","method":"In vitro kinase assay for RB-S758 phosphorylation, NF-κB nuclear translocation assay, PD-L1 expression analysis, siRNA and pharmacological PLK1 inhibition","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay plus mechanistic pathway analysis, single lab","pmids":["35950917"],"is_preprint":false},{"year":2023,"finding":"BUB-1-bound PLK-1 is required for CDC-20 kinetochore recruitment and timely mitotic progression in C. elegans embryos; PLK-1 kinase activity phosphorylates the CDC-20-binding ABBA motif of BUB-1 to promote BUB-1-CDC-20 interaction; a conserved STP motif in BUB-1 that docks PLK-1 is necessary for this function.","method":"C. elegans genetics (STP motif mutation in BUB-1), live imaging, in vitro kinase assay of ABBA motif phosphorylation, CDC-20 localization analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic separation-of-function plus in vitro kinase assay plus live imaging, multiple orthogonal methods","pmids":["37137308"],"is_preprint":false},{"year":2023,"finding":"PLK1 phosphorylates DNA polymerase theta (Polθ) in mitosis, activating it; phosphorylated Polθ is recruited to mitotic DSBs through direct interaction with BRCA1 C-terminal domains of TOPBP1; Polθ-mediated end joining repairs mitotic DSBs and maintains genome integrity; loss of Polθ is synthetically lethal with homologous recombination deficiency.","method":"In vitro kinase assay, co-IP (Polθ-TOPBP1 interaction), mitotic DSB repair assay, synthetic lethality assay (HR-deficient cells)","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus direct protein interaction mapping plus functional DSB repair assay plus synthetic lethality, multiple orthogonal methods in high-impact journal","pmids":["37674080"],"is_preprint":false},{"year":2023,"finding":"PLK1 maintains lysosomal function in renal tubular cells by phosphorylating the V-ATPase subunit ATP6V1A to regulate intralysosomal pH; PLK1 inhibition or knockout impairs lysosomal acidification and blocks autophagic flux, leading to reduced fibroblast activation and attenuation of renal tubulointerstitial fibrosis.","method":"Plk1 knockout mice (UUO model), pharmacological PLK1 inhibition, ATP6V1A phosphorylation assay, lysosomal pH measurement, autophagic flux assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO plus phosphorylation assay plus lysosomal functional readout, single lab","pmids":["37640723"],"is_preprint":false},{"year":2024,"finding":"PLK1 promotes CENP-A deposition (centromere maintenance) in early G1 by recognizing self-primed phosphorylations of Mis18α (Ser54) and Mis18BP1 (Thr78, Ser93) through its polo-box domain; PLK1 binding activates Mis18α-Mis18β and promotes Mis18 complex-HJURP interaction required for CENP-A loading.","method":"Biochemical phosphorylation mapping, PLK1-PBD binding assays, CENP-A deposition assay, HJURP co-IP, phospho-site mutagenesis","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — phosphosite mapping plus PBD binding assay plus functional CENP-A deposition plus HJURP co-IP, multiple orthogonal methods in two independent studies in same issue","pmids":["39236163","39236175"],"is_preprint":false},{"year":2019,"finding":"PP2A-B55 (α and δ isoforms) directly interacts with PLK1 during recovery from mitotic DNA damage; dephosphorylation of PLK1 by PP2A-B55 is induced through ATM/CHK1/CHK2-dependent repression of the Greatwall/ENSA pathway, leading to PP2A-B55 liberation and PLK1 dephosphorylation and mitotic slippage.","method":"Co-immunoprecipitation of PLK1-PP2A-B55α/δ, siRNA depletion of PP2A-B55, immunoblotting for phospho-PLK1, Greatwall/ENSA pathway manipulation","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus siRNA epistasis plus phospho-readout, single lab","pmids":["31072185"],"is_preprint":false},{"year":2017,"finding":"PLK1 phosphorylates Numb, promoting its proteasomal degradation; loss of Numb impairs the Numb/p53 pathway, providing a mechanism by which PLK1 antagonizes p53 during DNA damage response; cancer cells with non-phosphorylatable Numb show increased sensitivity to doxorubicin.","method":"In vitro kinase assay, phospho-site mutagenesis, proteasomal degradation assay, p53 stabilization assay, xenograft model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional p53 pathway and in vivo assay, single lab","pmids":["29059161"],"is_preprint":false}],"current_model":"PLK1 is a master Ser/Thr kinase activated at the G2/M boundary through Aurora-A/Bora-mediated phosphorylation of T210 (sustained in mitosis by residual Bora), modulated by dimerization/monomer switching, and inactivated by PP2A-B55 and CUL3-KLHL22-mediated ubiquitylation at K492; it is recruited to kinetochores primarily via CDK1-primed phosphosites on BUB1 and CENP-U and to other mitotic structures via PBD-phosphopeptide docking, where it phosphorylates a broad substrate network to control centrosome maturation, chromosome compaction, kinetochore-microtubule attachment (via CLIP-170, BRCA2-PP2A-BUBR1 complexes), spindle assembly checkpoint satisfaction (via Aurora B, MPS1, BUB1/BUBR1), cytokinesis (via centralspindlin/RhoA and Cep55/ESCRT), centromere integrity (suppressing BLM and PICH), CENP-A deposition in early G1 (via Mis18 complex), DNA damage response (phosphorylating Mre11, CtIP, Polθ, PrimPol), and non-mitotic processes including NLRP3 inflammasome positioning, Hedgehog/Gli1 signaling, autophagy/lysosome axis regulation, and p53 suppression; its activity is further fine-tuned by lysine methylation (SET7/9 at K191, SETD6 at K209/K413) and feedback mechanisms such as the Apolo1-PP1γ circuit."},"narrative":{"mechanistic_narrative":"PLK1 is a cell-cycle-regulated serine/threonine kinase whose expression is restricted to proliferating cells and peaks at the G2/M boundary, where it functions as a master coordinator of mitotic entry, chromosome segregation, and cytokinesis [PMID:8127874, PMID:9083047]. Its catalytic activity is gated by activation-loop phosphorylation at T210, which Aurora-A together with its cofactor Bora installs and sustains throughout mitosis as a bistable switch, while a Bora-supported dimer-to-monomer transition couples T210 phosphorylation to importin-dependent nuclear shuttling [PMID:24338364, PMID:34759346]; DNA damage suppresses T210/S137 phosphorylation through ATM/ATR–CHK1/CHK2 signaling, and PP2A-B55 directly dephosphorylates PLK1 during checkpoint recovery [PMID:15611664, PMID:31072185]. Substrate selection is governed by the C-terminal polo-box domain, which docks onto CDK1- or Aurora-A-primed phosphopeptides on partners and substrates [PMID:11050165, PMID:30202980]; this docking logic recruits PLK1 to kinetochores via BUB1 and CENP-U, where CDK1 priming also promotes local PLK1 dimerization [PMID:33248027]. Once positioned, PLK1 phosphorylates a broad network to drive centrosome maturation and spindle assembly (CEP215, with scaffolding by Gravin), kinetochore-microtubule attachment and chromosome alignment (CLIP-170 at S312, and a BRCA2–PP2A–BUBR1 complex assembled on PLK1-phosphorylated BRCA2-T207), and chromatin-based maintenance of centromere and kinetochore integrity against spindle pulling forces by suppressing BLM and acting with PICH [PMID:29282278, PMID:24451569, PMID:32286328, PMID:31253795, PMID:31468671]. PLK1 controls cytokinesis by releasing centralspindlin from the midzone through PRC1 inhibition to permit RhoA activation and by promoting Cep55/ESCRT loading at the abscission bridge [PMID:30728176, PMID:30069007]. Beyond mitosis, PLK1 restrains the DNA damage response and replication by phosphorylating Mre11, CtIP, Polθ, PrimPol, and Rif1 [PMID:28512243, PMID:30202980, PMID:37674080, PMID:34860556, PMID:34469577], deposits CENP-A in early G1 through PBD recognition of self-primed Mis18 complex phosphosites [PMID:39236163, PMID:39236175], and suppresses p53 directly and via Numb degradation [PMID:15024021, PMID:29059161]. Its activity is further tuned by lysine methylation (SET7/9 at K191, SETD6 at K209/K413), by CUL3-KLHL22-mediated ubiquitylation at K492 that evicts it from kinetochores, and by an Apolo1–PP1γ feedback circuit [PMID:31863092, PMID:30622182, PMID:23455478, PMID:34260926], and it additionally regulates non-mitotic processes including NLRP3 inflammasome positioning, Hedgehog/Gli1 signaling, and lysosomal acidification/autophagy [PMID:37698938, PMID:30578313, PMID:37640723].","teleology":[{"year":1994,"claim":"Established PLK1 as a serine/threonine kinase whose expression is tightly coupled to proliferation, framing it as a candidate mitotic regulator rather than a constitutive enzyme.","evidence":"cDNA cloning and Northern analysis across synchronized and resting cells","pmids":["8127874"],"confidence":"Medium","gaps":["No catalytic substrates identified at this stage","Expression correlation does not establish function"]},{"year":1997,"claim":"Defined how PLK1 abundance is restricted to G2/M, identifying a CDE/CHR repressor and upstream activating elements that enforce cell-cycle-dependent transcription.","evidence":"Promoter-luciferase deletion/mutation analysis in synchronized HeLa cells","pmids":["9083047"],"confidence":"Medium","gaps":["Trans-acting factors binding these elements not fully defined","Does not address post-translational control"]},{"year":2000,"claim":"Demonstrated that the polo-box domain is required for substrate engagement, establishing the docking logic central to PLK1 targeting, using GRASP65 as the first PBD-dependent substrate.","evidence":"Yeast two-hybrid, co-IP, in vitro kinase assay with PBD deletion/frameshift mutants","pmids":["11050165"],"confidence":"High","gaps":["Priming kinase for the PBD docking site not defined here","GRASP65 phosphosites not mapped"]},{"year":2003,"claim":"Showed PLK1 is required for viability and genome stability, as its loss triggers 4N arrest, DNA damage, and apoptosis through p53/ATM, establishing it as an essential mitotic gene and drug target rationale.","evidence":"siRNA knockdown, FACS, caspase assays, non-degradable Plk1 rescue across cancer lines","pmids":["12732729"],"confidence":"High","gaps":["Direct substrates causing the arrest not pinpointed","Mechanism of DNA damage in p53-null cells unresolved"]},{"year":2004,"claim":"Identified PLK1 as a direct suppressor of p53, providing a mechanism by which it antagonizes pro-apoptotic and checkpoint responses.","evidence":"Co-IP with deletion mutants, luciferase reporters, kinase-dead PLK1 analysis","pmids":["15024021"],"confidence":"High","gaps":["p53 phosphosite(s) not fully mapped","In vivo relevance to tumor suppression untested here"]},{"year":2005,"claim":"Linked PLK1 activation to DNA-damage signaling and to replication machinery, showing T210/S137 phosphorylation is checkpoint-suppressed and that the PBD binds the MCM2-7 complex.","evidence":"Phosphopeptide mapping with ATM/ATR and CHK inhibitors; PBD pulldown and fractionation","pmids":["15611664","15654075"],"confidence":"Medium","gaps":["Identity of the physiological T210 kinase not established here","Functional consequence of PLK1-MCM binding untested"]},{"year":2013,"claim":"Resolved the activation logic and a key off-switch: Aurora-A/Bora sustains T210 phosphorylation as a bistable switch through mitosis, while CUL3-KLHL22 ubiquitylates K492 to evict PLK1 from kinetochore phosphoreceptors.","evidence":"Inhibitor/synchronization with Bora siRNA; MS site mapping with non-ubiquitylatable K492R mutant and SAC assays","pmids":["24338364","23455478"],"confidence":"High","gaps":["Quantitative threshold of the switch not defined","Other E3 ligases for PLK1 not excluded"]},{"year":2014,"claim":"Defined substrate-level control of kinetochore-microtubule attachment and apoptotic threshold, showing PLK1 phospho-cycling of CLIP-170 enables stable attachments and PLK1 phosphorylation of procaspase-8 restrains extrinsic death.","evidence":"In vitro kinase assays, phospho-site mutants, live imaging, Fas-stimulation apoptosis assays","pmids":["24451569","24484936"],"confidence":"High","gaps":["Phosphatase counteracting CLIP-170 S312 not identified","Caspase-8 control not validated in vivo"]},{"year":2014,"claim":"Mapped upstream regulatory inputs balancing PLK1 activity, with the LKB1–NUAK1–MYPT1-PP1 axis promoting dephosphorylation and DNA-PKcs controlling PLK1 stability and midbody recruitment.","evidence":"Co-IP, kinase assays, siRNA epistasis, localization studies","pmids":["24722282","24166892"],"confidence":"Medium","gaps":["DNA-PKcs claims rest on single-method readouts per assertion","Direct vs indirect dephosphorylation not fully separated"]},{"year":2017,"claim":"Extended PLK1 into the DNA damage response and centrosome scaffolding, showing it phosphorylates Mre11 to limit MRN loading, degrades Numb to antagonize p53, and requires Gravin scaffolding for centrosomal activity.","evidence":"In vitro kinase assays, phospho-site mutants, chromatin loading assays, FRAP/FRET biosensor with separation-of-function Gravin mutant","pmids":["28512243","29059161","29282278"],"confidence":"High","gaps":["Relative contribution of these arms to genome stability unquantified","Numb-axis tested mainly in cancer models"]},{"year":2018,"claim":"Showed PLK1 activation is coupled to completion of DNA replication and demonstrated kinase-dependent control of CtIP-mediated repair choice and Cep55/ESCRT-dependent abscission.","evidence":"Degron and inhibitor systems with activity reporters; in vitro kinase assays with HR/MMEJ reporters; inducible knock-in mouse with abscission imaging","pmids":["30008317","30202980","30069007"],"confidence":"High","gaps":["Sensor linking replication state to PLK1 not fully defined","Dose-dependence of PLK1 in abscission not mapped"]},{"year":2019,"claim":"Defined methylation-based fine-tuning and chromatin-based centromere protection, with SET7/9-K191 and SETD6-K209/K413 dampening kinase activity and chromatin-localized PLK1 suppressing BLM/PICH-driven centromere disintegration.","evidence":"In vitro methylation assays with non-methylatable mutants; PLK1 inhibition with BLM/PICH co-depletion rescue and chromatin-tethered PLK1 constructs","pmids":["31863092","30622182","31253795","31468671"],"confidence":"High","gaps":["Demethylases for these sites not identified","Direct chromatin substrates maintaining integrity not all mapped"]},{"year":2019,"claim":"Linked PLK1 to cytokinesis signaling, Hedgehog/Gli1 control, and checkpoint-recovery dephosphorylation, broadening its regulatory reach beyond core spindle functions.","evidence":"Live imaging of centralspindlin/RhoA with PRC1 knockdown; in vitro kinase assay and reporter assays for Gli1-S481; co-IP and siRNA epistasis for PP2A-B55","pmids":["30728176","30578313","31072185"],"confidence":"Medium","gaps":["Centralspindlin substrate of PLK1 not specified","PP2A-B55 study relies on single-lab epistasis"]},{"year":2020,"claim":"Established the dominant kinetochore-recruitment mechanism and additional attachment control, identifying BUB1 and CENP-U as the main PLK1 receptors via CDK1-primed docking and BRCA2-T207 as a docking site assembling a PP2A-BUBR1 complex.","evidence":"In vitro reconstitution and ectopic localization for BUB1/CENP-U; phosphosite mapping and complex co-IP with cancer-variant analysis for BRCA2","pmids":["33248027","32286328"],"confidence":"High","gaps":["Quantitative split between BUB1 and CENP-U pools not fully resolved","Functional output of dimerization at kinetochores incomplete"]},{"year":2020,"claim":"Demonstrated organismal-level mitotic and meiotic roles, with PLK1 required for chromosome compaction, MTOC organization, and spindle formation in oocytes.","evidence":"Oocyte-specific conditional Plk1 knockout mouse with marker immunofluorescence","pmids":["32267211"],"confidence":"High","gaps":["Direct substrates driving compaction not identified","Distinction from somatic mitotic roles partial"]},{"year":2021,"claim":"Resolved the dimer-monomer activation switch and expanded PLK1 into replication and feedback control, including PrimPol and Rif1 phosphorylation, the Apolo1-PP1γ inactivation loop, and DNMT3a/PLK1 transcriptional feedback.","evidence":"Co-IP of dimers with T210 mutants and importin assays; in vitro kinase assays with phospho-mutants; FRET biosensor and PP1γ interaction assays","pmids":["34759346","34860556","34469577","34260926","34051063"],"confidence":"High","gaps":["Integration of multiple feedback loops in vivo unresolved","Some replication findings rely on Xenopus extract systems"]},{"year":2022,"claim":"Extended PLK1 into immune and tumor-relevant signaling, showing it positions NLRP3 via MTOC control, restrains NF-κB/PD-L1 through RB-S758, and that its dysregulation downstream of BRCA1 loss misorients cell division.","evidence":"BioID and co-IP with in vivo inflammasome models; in vitro RB kinase assay with NF-κB/PD-L1 readouts; division-axis imaging across BRCA1-mutant lines","pmids":["37698938","35950917","35459234","14654792","31597699","27398835"],"confidence":"Medium","gaps":["Some interphase roles rely on single-lab evidence","Causal substrate for NLRP3 positioning not defined"]},{"year":2023,"claim":"Defined PLK1 as a driver of mitotic DSB repair and lysosomal/autophagy control, activating Polθ and recruiting it to mitotic breaks via TOPBP1, and acidifying lysosomes via ATP6V1A phosphorylation.","evidence":"In vitro kinase assays, co-IP, mitotic DSB repair and synthetic-lethality assays; Plk1 knockout mice with lysosomal pH and autophagic flux readouts","pmids":["37674080","37640723"],"confidence":"High","gaps":["Polθ phosphosite identity not fully mapped","Tissue specificity of the lysosomal axis incompletely defined"]},{"year":2024,"claim":"Completed the cell-cycle arc by showing PLK1 promotes CENP-A deposition in early G1 through PBD recognition of self-primed Mis18 complex phosphosites, activating Mis18 and enabling HJURP-dependent loading.","evidence":"Phosphosite mapping, PBD binding assays, CENP-A deposition assays, HJURP co-IP across two independent studies","pmids":["39236163","39236175"],"confidence":"High","gaps":["Phosphatase resetting Mis18 sites not defined","Timing relative to PLK1 inactivation at mitotic exit unresolved"]},{"year":null,"claim":"How the many PLK1 inputs (T210 phosphorylation, dimer-monomer state, methylation, ubiquitylation, phosphatase feedback) are integrated to produce spatially and temporally distinct substrate selection across mitosis, the DNA damage response, and interphase processes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model linking PLK1 modification state to substrate prioritization","Relative contribution of mitotic vs non-mitotic functions to organismal phenotypes unquantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,6,17,22,26,31,33,35,47,49,50,51]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,17,22,26,49]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6,31,47]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[9,23,44]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[13,30,32,36,51]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[37]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,15,17,25,28,32,36]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[22,26,49,39]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[27,41]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[31,47]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[50]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[45]}],"complexes":["BRCA2-PP2A-BUBR1 complex"],"partners":["BORA","AURKA","BUB1","CENP-U","BRCA2","CUL3-KLHL22","PP2A-B55","GRASP65"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P53350","full_name":"Serine/threonine-protein kinase PLK1","aliases":["Polo-like kinase 1","PLK-1","Serine/threonine-protein kinase 13","STPK13"],"length_aa":603,"mass_kda":68.3,"function":"Serine/threonine-protein kinase that performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, the removal of cohesins from chromosome arms, the inactivation of anaphase-promoting complex/cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis (PubMed:11202906, PubMed:12207013, PubMed:12447691, PubMed:12524548, PubMed:12738781, PubMed:12852856, PubMed:12939256, PubMed:14532005, PubMed:14734534, PubMed:15070733, PubMed:15148369, PubMed:15469984, PubMed:16198290, PubMed:16247472, PubMed:16980960, PubMed:17081991, PubMed:17351640, PubMed:17376779, PubMed:17617734, PubMed:18174154, PubMed:18331714, PubMed:18418051, PubMed:18477460, PubMed:18521620, PubMed:18615013, PubMed:19160488, PubMed:19351716, PubMed:19468300, PubMed:19468302, PubMed:19473992, PubMed:19509060, PubMed:19597481, PubMed:23455478, PubMed:23509069, PubMed:28512243, PubMed:8991084). Polo-like kinase proteins act by binding and phosphorylating proteins that are already phosphorylated on a specific motif recognized by the POLO box domains (PubMed:11202906, PubMed:12207013, PubMed:12447691, PubMed:12524548, PubMed:12738781, PubMed:12852856, PubMed:12939256, PubMed:14532005, PubMed:14734534, PubMed:15070733, PubMed:15148369, PubMed:15469984, PubMed:16198290, PubMed:16247472, PubMed:16980960, PubMed:17081991, PubMed:17351640, PubMed:17376779, PubMed:17617734, PubMed:18174154, PubMed:18331714, PubMed:18418051, PubMed:18477460, PubMed:18521620, PubMed:18615013, PubMed:19160488, PubMed:19351716, PubMed:19468300, PubMed:19468302, PubMed:19473992, PubMed:19509060, PubMed:19597481, PubMed:23455478, PubMed:23509069, PubMed:28512243, PubMed:8991084). Phosphorylates BORA, BUB1B/BUBR1, CCNB1, CDC25C, CEP55, ECT2, ERCC6L, FBXO5/EMI1, FOXM1, KIF20A/MKLP2, CENPU, NEDD1, NINL, NPM1, NUDC, PKMYT1/MYT1, KIZ, MRE11, PPP1R12A/MYPT1, POLQ, PRC1, RACGAP1/CYK4, RAD51, RHNO1, SGO1, STAG2/SA2, TEX14, TOPORS, p73/TP73, TPT1, WEE1 and HNRNPU (PubMed:11202906, PubMed:12207013, PubMed:12447691, PubMed:12524548, PubMed:12738781, PubMed:12852856, PubMed:12939256, PubMed:14532005, PubMed:14734534, PubMed:15070733, PubMed:15148369, PubMed:15469984, PubMed:16198290, PubMed:16247472, PubMed:16980960, PubMed:17081991, PubMed:17218258, PubMed:17351640, PubMed:17376779, PubMed:17617734, PubMed:18174154, PubMed:18331714, PubMed:18418051, PubMed:18477460, PubMed:18521620, PubMed:18615013, PubMed:19160488, PubMed:19351716, PubMed:19468300, PubMed:19468302, PubMed:19473992, PubMed:19509060, PubMed:19597481, PubMed:22325354, PubMed:23455478, PubMed:23509069, PubMed:25986610, PubMed:26811421, PubMed:28512243, PubMed:37440612, PubMed:37674080, PubMed:8991084). Plays a key role in centrosome functions and the assembly of bipolar spindles by phosphorylating KIZ, NEDD1 and NINL (PubMed:16980960, PubMed:19509060). NEDD1 phosphorylation promotes subsequent targeting of the gamma-tubulin ring complex (gTuRC) to the centrosome, an important step for spindle formation (PubMed:19509060). Phosphorylation of NINL component of the centrosome leads to NINL dissociation from other centrosomal proteins (PubMed:12852856). Involved in mitosis exit and cytokinesis by phosphorylating CEP55, ECT2, KIF20A/MKLP2, CENPU, PRC1 and RACGAP1 (PubMed:12939256, PubMed:16247472, PubMed:17351640, PubMed:19468300, PubMed:19468302). Recruited at the central spindle by phosphorylating and docking PRC1 and KIF20A/MKLP2; creates its own docking sites on PRC1 and KIF20A/MKLP2 by mediating phosphorylation of sites subsequently recognized by the POLO box domains (PubMed:12939256, PubMed:17351640). Phosphorylates RACGAP1, thereby creating a docking site for the Rho GTP exchange factor ECT2 that is essential for the cleavage furrow formation (PubMed:19468300, PubMed:19468302). Promotes the central spindle recruitment of ECT2 (PubMed:16247472). Plays a central role in G2/M transition of mitotic cell cycle by phosphorylating CCNB1, CDC25C, FOXM1, CENPU, PKMYT1/MYT1, PPP1R12A/MYPT1 and WEE1 (PubMed:11202906, PubMed:12447691, PubMed:12524548, PubMed:19160488). Part of a regulatory circuit that promotes the activation of CDK1 by phosphorylating the positive regulator CDC25C and inhibiting the negative regulators WEE1 and PKMYT1/MYT1 (PubMed:11202906). Also acts by mediating phosphorylation of cyclin-B1 (CCNB1) on centrosomes in prophase (PubMed:12447691, PubMed:12524548). Phosphorylates FOXM1, a key mitotic transcription regulator, leading to enhance FOXM1 transcriptional activity (PubMed:19160488). Involved in kinetochore functions and sister chromatid cohesion by phosphorylating BUB1B/BUBR1, FBXO5/EMI1 and STAG2/SA2 (PubMed:15148369, PubMed:15469984, PubMed:17376779, PubMed:18331714). PLK1 is high on non-attached kinetochores suggesting a role of PLK1 in kinetochore attachment or in spindle assembly checkpoint (SAC) regulation (PubMed:17617734). Required for kinetochore localization of BUB1B (PubMed:17376779). Regulates the dissociation of cohesin from chromosomes by phosphorylating cohesin subunits such as STAG2/SA2 (By similarity). Phosphorylates SGO1: required for spindle pole localization of isoform 3 of SGO1 and plays a role in regulating its centriole cohesion function (PubMed:18331714). Mediates phosphorylation of FBXO5/EMI1, a negative regulator of the APC/C complex during prophase, leading to FBXO5/EMI1 ubiquitination and degradation by the proteasome (PubMed:15148369, PubMed:15469984). Acts as a negative regulator of p53 family members: phosphorylates TOPORS, leading to inhibit the sumoylation of p53/TP53 and simultaneously enhance the ubiquitination and subsequent degradation of p53/TP53 (PubMed:19473992). Phosphorylates the transactivation domain of the transcription factor p73/TP73, leading to inhibit p73/TP73-mediated transcriptional activation and pro-apoptotic functions. Phosphorylates BORA, and thereby promotes the degradation of BORA (PubMed:18521620). Contributes to the regulation of AURKA function (PubMed:18615013, PubMed:18662541). Also required for recovery after DNA damage checkpoint and entry into mitosis (PubMed:18615013, PubMed:18662541). Phosphorylates MISP, leading to stabilization of cortical and astral microtubule attachments required for proper spindle positioning (PubMed:23509069). Together with MEIKIN, acts as a regulator of kinetochore function during meiosis I: required both for mono-orientation of kinetochores on sister chromosomes and protection of centromeric cohesin from separase-mediated cleavage (By similarity). Phosphorylates CEP68 and is required for its degradation (PubMed:25503564). Regulates nuclear envelope breakdown during prophase by phosphorylating DCTN1 resulting in its localization in the nuclear envelope (PubMed:20679239). Phosphorylates the heat shock transcription factor HSF1, promoting HSF1 nuclear translocation upon heat shock (PubMed:15661742). Phosphorylates HSF1 also in the early mitotic period; this phosphorylation regulates HSF1 localization to the spindle pole, the recruitment of the SCF(BTRC) ubiquitin ligase complex induicing HSF1 degradation, and hence mitotic progression (PubMed:18794143). Regulates mitotic progression by phosphorylating RIOK2 (PubMed:21880710). Through the phosphorylation of DZIP1 regulates the localization during mitosis of the BBSome, a ciliary protein complex involved in cilium biogenesis (PubMed:27979967). Regulates DNA repair during mitosis by mediating phosphorylation of POLQ and RHNO1, thereby promoting POLQ recruitment to DNA damage sites (PubMed:37440612, PubMed:37674080). 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CEP76","url":"https://www.omim.org/entry/620791"},{"mim_id":"620604","title":"PROSTAGLANDIN REDUCTASE 3; PTGR3","url":"https://www.omim.org/entry/620604"},{"mim_id":"620479","title":"CENTRIOLAR SATELLITE-ASSOCIATED TUBULIN POLYGLUTAMYLASE COMPLEX REGULATOR 1; CSTPP1","url":"https://www.omim.org/entry/620479"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":30.2},{"tissue":"lymphoid 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progression by inhibiting PLK‑1 expression.","date":"2018","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/29749476","citation_count":15,"is_preprint":false},{"pmid":"34051063","id":"PMC_34051063","title":"Co-Targeting Plk1 and DNMT3a in Advanced Prostate Cancer.","date":"2021","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/34051063","citation_count":15,"is_preprint":false},{"pmid":"21070201","id":"PMC_21070201","title":"Polo-like kinase 1 (Plk1) in cutaneous T-cell lymphoma.","date":"2011","source":"The British journal of dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/21070201","citation_count":15,"is_preprint":false},{"pmid":"35563642","id":"PMC_35563642","title":"Recent Progress on the Localization of PLK1 to the Kinetochore and Its Role in Mitosis.","date":"2022","source":"International journal of molecular 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CR","url":"https://pubmed.ncbi.nlm.nih.gov/19775446","citation_count":14,"is_preprint":false},{"pmid":"24451569","id":"PMC_24451569","title":"Plk1 phosphorylates CLIP-170 and regulates its binding to microtubules for chromosome alignment.","date":"2014","source":"Cell structure and function","url":"https://pubmed.ncbi.nlm.nih.gov/24451569","citation_count":14,"is_preprint":false},{"pmid":"34901994","id":"PMC_34901994","title":"Xanthatin Selectively Targets Retinoblastoma by Inhibiting the PLK1-Mediated Cell Cycle.","date":"2021","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/34901994","citation_count":14,"is_preprint":false},{"pmid":"37137308","id":"PMC_37137308","title":"BUB-1-bound PLK-1 directs CDC-20 kinetochore recruitment to ensure timely embryonic mitoses.","date":"2023","source":"Current biology : 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/35459234","citation_count":13,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":44233,"output_tokens":13013,"usd":0.163947,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24787,"output_tokens":6535,"usd":0.143655,"stage2_stop_reason":"end_turn"},"total_usd":0.307602,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"Human PLK (PLK1) encodes a serine/threonine kinase of 603 amino acids; its mRNA expression is strongly correlated with mitotic activity of cells, being absent in resting cells and induced upon cell cycle entry.\",\n      \"method\": \"cDNA cloning, Northern blot analysis, cell synchronization experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cDNA sequencing plus multiple cell-type expression studies, single lab\",\n      \"pmids\": [\"8127874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The PLK1 promoter contains a repressor element (CDE/CHR) near the transcription start site that mediates cell cycle-dependent transcription; three activating regions are located between −35 and −93 bp upstream, with promoter activity maximal at G2/M phase.\",\n      \"method\": \"PLK promoter/luciferase constructs in synchronized HeLa cells, deletion/mutation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional promoter-reporter assays with mutagenesis, single lab\",\n      \"pmids\": [\"9083047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PLK1 binds to and phosphorylates the Golgi peripheral protein GRASP65 in vitro and in vivo; deletion or frameshift mutations in the conserved C-terminal polo-box domain of PLK1 greatly diminish its ability to phosphorylate GRASP65, demonstrating the PBD is required for this substrate interaction.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, in vitro kinase assay, deletion/frameshift mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis plus co-IP, multiple orthogonal methods in single study\",\n      \"pmids\": [\"11050165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The Ste20-like kinase SLK can phosphorylate and activate murine PLK1; SLK activity increases during G2 phase progression, proposing SLK as an upstream kinase regulating PLK1 in the somatic cell cycle.\",\n      \"method\": \"In vitro kinase assay, endogenous SLK activity measurements, cell synchronization\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro phosphorylation assay demonstrated, single lab, no epistasis confirmation\",\n      \"pmids\": [\"10886374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Depletion of PLK1 by siRNA causes cell cycle arrest with 4N DNA content, formation of dumbbell-like chromatin, activation of caspase-3, and apoptosis; the p53 pathway and ATM are involved in PLK1-depletion-induced apoptosis, and DNA damage occurs even in p53-null cells.\",\n      \"method\": \"Vector-based siRNA knockdown, FACS, caspase-3 activation assay, rescue with non-degradable Plk1, ATM inhibitor co-treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal readouts (FACS, caspase assay, rescue experiment) replicated in multiple cancer cell lines\",\n      \"pmids\": [\"12732729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"BRCA1 mediates repression of PLK1 mRNA after ionizing radiation through a CHK1-dependent pathway, and loss of BRCA1 impairs this downregulation, linking BRCA1 to G2/M checkpoint control via PLK1 transcriptional suppression.\",\n      \"method\": \"RT-PCR of PLK mRNA in irradiated BRCA1−/− cells vs. BRCA1-reconstituted cells, CHK1 inhibitor (UCN-01) treatment\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic reconstitution plus pharmacological inhibitor, single lab\",\n      \"pmids\": [\"14654792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PLK1 physically binds to the sequence-specific DNA-binding domain of p53 and phosphorylates it, inhibiting p53 transactivation of p21WAF1, MDM2, and BAX promoters and its pro-apoptotic function; kinase-dead PLK1 fails to repress p53 transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation with deletion mutants, confocal co-localization, luciferase reporter assay, endogenous p21 induction assay, kinase-dead mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct binding mapped by deletion mutants, kinase-dead mutagenesis, multiple functional readouts, single lab\",\n      \"pmids\": [\"15024021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLK1 phosphorylation at T210 (activation loop) and S137 occurs in mitosis; DNA damage prevents phosphorylation at both sites in asynchronous cells through ATM/ATR and CHK1/CHK2 pathways; constitutively active T210D mutant is insensitive to DNA damage-induced inhibition.\",\n      \"method\": \"In vivo phosphopeptide mapping, T210D mutant analysis, kinase inhibitor treatments (ATM/ATR, CHK1/CHK2 inhibitors)\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphosite mapping with pharmacological and genetic tools, single lab\",\n      \"pmids\": [\"15611664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The polo-box domain (PBD) of PLK1 interacts with MCM2 and MCM7 (and the entire MCM2-7 complex); the strongest interaction between endogenous PLK1 and Mcm7 is detected in a soluble chromatin fraction, suggesting a role for PLK1 in coordinating DNA replication and mitotic events.\",\n      \"method\": \"PBD-bait pulldown screen, co-immunoprecipitation, subcellular fractionation, immunoblot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus fractionation, single lab\",\n      \"pmids\": [\"15654075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TTDN1 is identified as a PLK1-interacting protein; CDK1 phosphorylates TTDN1, which is required for TTDN1-PLK1 interaction; mutation of Thr120 in TTDN1's PLK1-binding motif abolishes the interaction; TTDN1 co-localizes with PLK1 at centrosomes during mitosis and the midbody during cytokinesis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, site-directed mutagenesis, immunofluorescence co-localization\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus mutagenesis plus localization, single lab\",\n      \"pmids\": [\"17310276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the PLK1 kinase domain with BI 2536 reveals that Leu132 in the hinge region creates a small pocket exploited by the methoxy group of BI 2536 for selectivity against non-PLK kinases; structure solved to inform structure-based drug design.\",\n      \"method\": \"X-ray crystallography (co-crystal structure of PLK1 KD with BI 2536)\",\n      \"journal\": \"Chemical biology & drug design\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with selectivity data, single lab but direct structural evidence\",\n      \"pmids\": [\"18005335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of wild-type PLK1 kinase domain in complex with DARPin 3H10 determined at 2.3 Å resolution, revealing the active conformation of PLK1; DARPin enabled crystallization by masking a surface patch rich in charged residues.\",\n      \"method\": \"X-ray crystallography using DARPin-assisted co-crystallization\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure, single lab\",\n      \"pmids\": [\"18391401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PLK-1 protein is distributed asymmetrically in C. elegans two-cell embryos with more present in the anterior AB blastomere than in P1; this asymmetry is regulated by PAR-protein-dependent anterior-posterior polarity cues through preferential protein retention; mild PLK-1 RNAi delays mitotic entry specifically in P1, promoting asynchronous cell division.\",\n      \"method\": \"Live imaging, RNAi knockdown, PAR mutant analysis in C. elegans embryos\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging plus genetic (RNAi) with quantitative cell cycle timing, single lab\",\n      \"pmids\": [\"18305005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PICH and PLK1 cooperate to maintain chromosome architecture during prometaphase; PICH knockdown removes PLK1 from chromosome arms causing disorganized chromosomes with open/X-shaped configuration consistent with loss of arm cohesion; chromosome disorganization is prevented by topoisomerase II inhibitor, indicating the PICH-PLK1 complex maintains architecture through topoisomerase II.\",\n      \"method\": \"siRNA knockdown of PICH, immunofluorescence, topoisomerase II inhibitor (ICRF-193) treatment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with pharmacological rescue, single lab\",\n      \"pmids\": [\"20130082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CUL3-KLHL22 E3 ubiquitin ligase ubiquitylates PLK1 at Lys492 within the polo-box domain, causing degradation-independent dissociation of PLK1 from kinetochore phosphoreceptors; non-ubiquitylatable PLK1-K492R accumulates at kinetochores, activates the spindle assembly checkpoint, and phenocopies KLHL22 inactivation.\",\n      \"method\": \"Mass spectrometry identification, Co-IP, K492R mutant expression, live imaging, spindle assembly checkpoint assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ubiquitylation site mapped by MS, functional validated with non-ubiquitylatable mutant, multiple orthogonal methods\",\n      \"pmids\": [\"23455478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Aurora-A together with its cofactor Bora remains the major activator of PLK1 during mitosis (not only at G2/M transition); a small fraction of Bora is retained in mitosis, essential for continued Aurora-A-dependent T210 phosphorylation; PLK1 activation acts as a bistable switch requiring minimal Bora-Aurora-A once fully active.\",\n      \"method\": \"Kinase inhibitor treatments, cell synchronization, immunoblotting for pT210, siRNA knockdown of Bora\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic perturbations, single lab\",\n      \"pmids\": [\"24338364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Sequential phosphorylation of procaspase-8 by CDK1/cyclin B1 (at S387, generating a PBD-docking site) and then PLK1 (at S305) during mitosis inhibits extrinsic apoptotic signaling; non-phosphorylatable S305A caspase-8 increases Fas-stimulated cell death; PLK1 inhibitor BI 2536 lowers the threshold for Fas-induced death.\",\n      \"method\": \"RNAi replacement strategy, phospho-site mutagenesis (S305A, S387A), co-IP with PBD, Fas stimulation apoptosis assay, PLK1 inhibitor treatment\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional apoptosis readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24484936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PLK1 phosphorylates CLIP-170 at Ser312 during mitosis; this phosphorylation diminishes CLIP-170 binding to microtubule ends and lattice (without affecting EB3 binding); phosphorylation/dephosphorylation cycling at Ser312 is required for stable kinetochore-microtubule attachment and chromosome alignment.\",\n      \"method\": \"In vitro kinase assay with purified CLIP-170 N-terminal fragment, phospho-specific antibody, S312A/S312D mutant analysis, live imaging of chromosome alignment\",\n      \"journal\": \"Cell structure and function\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional imaging, single lab\",\n      \"pmids\": [\"24451569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DNA-PKcs physically associates with PLK1 during the mitotic phase; depletion of DNA-PKcs leads to overexpression of PLK1 through increased protein stability but attenuates recruitment of phosphorylated PLK1 to the midbody (while not affecting centrosome/kinetochore localization), resulting in cytokinesis failure.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence co-localization\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus localization study, single lab, single method for each claim\",\n      \"pmids\": [\"24166892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LKB1 regulates PLK1 by activating NUAK1 (ARK5), which phosphorylates MYPT1, enhancing MYPT1-PP1 binding to PLK1 and promoting PLK1 dephosphorylation; LKB1 deficiency leads to elevated phospho-PLK1 and centrosome amplification, reversed by PLK1 depletion or inhibition.\",\n      \"method\": \"Co-immunoprecipitation, kinase assay, siRNA, PLK1 overexpression rescue, BTO-1 inhibitor treatment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis defined by sequential knockdowns/inhibitions, co-IP, single lab\",\n      \"pmids\": [\"24722282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PLK-1 is required in C. elegans oocytes for nuclear envelope breakdown; partial PLK-1 inactivation causes failure to form a gap in the nuclear envelope at the interface between pronuclei and defects in chromosome congression, resulting in paired nuclei containing either maternal or paternal chromosomes in daughter cells.\",\n      \"method\": \"Partial RNAi inactivation, live fluorescence imaging of nuclear envelopes and chromosomes in C. elegans embryos\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with genetic perturbation and defined phenotypic readout, single lab\",\n      \"pmids\": [\"26490119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PTEN physically associates with PLK1 and reduces PLK1 phosphorylation in a phosphatase-dependent manner; PTEN deficiency leads to elevated PLK1 phosphorylation and polyploidy; phospho-mimicking PLK1 mutant causes polyploidy similar to PTEN loss, while non-phosphorylatable PLK1 reduces the polyploid population.\",\n      \"method\": \"Co-immunoprecipitation, phospho-mimicking and non-phosphorylatable PLK1 mutant expression, PTEN phosphatase-dead mutant, cell ploidy analysis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus phospho-mutant rescue experiments, single lab\",\n      \"pmids\": [\"27398835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLK1 phosphorylates Mre11 at S649, which primes subsequent CK2-mediated phosphorylation at S688; dual phosphorylation inhibits MRN complex loading onto damaged DNA, leading to premature DNA damage checkpoint termination and inhibition of DNA repair.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, chromatin loading assay, DNA damage checkpoint assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus phospho-site mutagenesis plus functional chromatin recruitment assay, single lab\",\n      \"pmids\": [\"28512243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Gravin acts as a scaffold for PLK1 at mitotic centrosomes; Gravin depletion increases PLK1 mobility (by FRAP), redistributes active PLK1, increases phosphorylation of CEP215 at S613, causes microtubule renucleation defects, chromosome misalignment, and micronuclei; a Gravin mutant unable to bind PLK1 fails to rescue these defects.\",\n      \"method\": \"FRAP, PLK1 FRET-based activity biosensor, shRNA knockdown, PLK1-binding-deficient Gravin mutant rescue\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — FRAP, FRET biosensor, genetic rescue with separation-of-function mutant, multiple orthogonal methods\",\n      \"pmids\": [\"29282278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLK1 regulates NOTCH1 expression at the G2/M transition; upon DNA damage in G2, PLK1 activity is inhibited while NOTCH1 expression is maintained; PLK1-dependent signaling antagonizes NOTCH1 involvement in the DNA damage checkpoint in squamous cell carcinoma cells.\",\n      \"method\": \"Chemical library kinase screen, pharmacological PLK1 inhibition, co-immunoprecipitation, cell cycle synchronization, immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — chemical screen plus co-IP, mechanistic details partly inferential, single lab\",\n      \"pmids\": [\"31597699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PLK1 overexpression causes defective cytokinesis correlated with defective loading of Cep55 and ESCRT complexes to the abscission bridge in a PLK1 kinase-dependent manner, generating polyploid cells with reduced proliferative potential.\",\n      \"method\": \"Inducible knock-in mouse model, immunofluorescence of Cep55/ESCRT at abscission bridges, kinase-dead PLK1 comparison\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in model plus mechanistic follow-up with Cep55/ESCRT localization, kinase-dependence established\",\n      \"pmids\": [\"30069007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CDK1/Aurora-A phosphorylates CtIP at S327, creating a PLK1 polo-box domain docking site; PLK1 then phosphorylates CtIP at S723; phospho-mimetic CtIP S723 fails to perform extended end resection and homologous recombination but retains ability to mediate microhomology-mediated end joining (MMEJ) while inactivating the G2/M checkpoint.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, MMEJ/HR reporter assays, G2/M checkpoint assay, PLK1-PBD binding assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus sequential phosphorylation mapping plus functional MMEJ/HR readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30202980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DNA replication restricts activation of both CDK1 and PLK1; preventing DNA replication licensing and/or firing causes premature activation of CDK1 and PLK1 in S phase; inhibition of CHK1 and p38 in the presence of replication leads to premature mitotic kinase activation and replication stress.\",\n      \"method\": \"Double-degron system for protein depletion, kinase inhibitors, cell synchronization, CDK1/PLK1 activity reporters\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal perturbations (degron, inhibitors) with quantitative kinase activity readouts, single lab\",\n      \"pmids\": [\"30008317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLK1 plays dual roles in cytokinesis: PLK1 activity promotes release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to activate RhoA at the equatorial cortex; PLK1 inhibition sequesters centralspindlin on the midzone, making it unavailable for Aurora B-dependent RhoA activation.\",\n      \"method\": \"PRC1 knockdown, PLK1 inhibitor treatment, live imaging of centralspindlin and RhoA activity\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological perturbations with live imaging, single lab\",\n      \"pmids\": [\"30728176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLK1 methyltransferase SETD6 methylates PLK1 at K209 and K413 during mitosis; loss of these methylation sites increases PLK1 kinase activity, accelerates mitotic progression and cellular proliferation; SETD6-deficient cells phenocopy non-methylatable PLK1.\",\n      \"method\": \"Proteomic screen, in vitro methylation assay, K209R/K413R PLK1 mutants, kinase activity assay, live cell imaging of mitotic progression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation assay plus non-methylatable mutant functional analysis plus SETD6-KO phenotypic matching, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30622182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLK1 inhibition causes centromere disintegration driven by BLM helicase-mediated DNA unwinding at centromeres under bipolar spindle pulling force; PLK1 normally suppresses BLM activity at centromeres to maintain centromere integrity for chromosome biorientation.\",\n      \"method\": \"PLK1 inhibitor treatment, BLM helicase co-depletion rescue experiment, live imaging of centromere dynamics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition plus genetic epistasis rescue, live imaging, single lab\",\n      \"pmids\": [\"31253795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLK1 phosphorylates Gli1 at S481, promoting nuclear export of Gli1 and its binding to the negative regulator Sufu, thereby inhibiting Hedgehog signaling activity during G2/M; PLK1 inhibition prevents this phosphorylation and sustains Gli1 transcriptional activity.\",\n      \"method\": \"In vitro kinase assay, S481 phospho-site mutagenesis, nuclear/cytoplasmic fractionation, Gli1-Sufu co-IP, Hedgehog target gene luciferase reporter\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, phospho-site mutagenesis, and multiple functional readouts (localization, protein interaction, target gene expression), single lab\",\n      \"pmids\": [\"30578313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Chromatin-localized PLK1 signals maintain kinetochore and centromere integrity (CENP-A, CENP-C, CENP-T, Hec1, Nuf2, Knl1 retention) against microtubule pulling forces; PICH helicase is required for centromere disruption in PLK1-inhibited cells and PICH depletion rescues kinetochore architecture; tethering PLK1 to chromatin (H2B, INCENP) but not CENP-A rescues anaphase defects.\",\n      \"method\": \"PLK1 inhibitor treatment, PICH siRNA rescue, chromatin-tethered PLK1 constructs (H2B-PLK1, INCENP-PLK1, CENP-A-PLK1), immunofluorescence of kinetochore components\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — targeted rescue with chromatin-tethering, epistasis with PICH knockdown, multiple kinetochore markers, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31468671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BRCA2 is phosphorylated by PLK1 at T207 in mitotic cells; phospho-T207 serves as a PLK1 docking site; BRCA2 bound to PLK1 forms a complex with PP2A and phospho-BUBR1; BRCA2 breast cancer variants S206C and T207A reduce PLK1 binding, causing unstable kinetochore-microtubule interactions, chromosome misalignment, and aneuploidy.\",\n      \"method\": \"Phospho-site identification, co-immunoprecipitation (BRCA2-PLK1-PP2A-BUBR1 complex), BRCA2 variant functional analysis, chromosome alignment assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphosite mapping, complex reconstitution by co-IP, cancer variant separation-of-function analysis, multiple orthogonal methods\",\n      \"pmids\": [\"32286328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PLK1 is required for chromosome compaction, cohesin/condensin localization, and MTOC organization in mouse oocytes; PLK1 conditional knockout oocytes fail to form compact bivalents, develop abnormal or absent bipolar spindles due to defective C-NAP1 release and failure to recruit MTOC components and LISD factors.\",\n      \"method\": \"Plk1 conditional knockout mouse (oocyte-specific), immunofluorescence of cohesin, condensin, C-NAP1, MTOC components\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific conditional KO with mechanistic follow-up of multiple molecular markers, single lab\",\n      \"pmids\": [\"32267211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SET7/9 dimethylates PLK1 at K191 during early mitosis, reducing PLK1 kinase activity by limiting ATP utilization; non-methylatable PLK1 K191 mutant or SET7/9 inhibition causes mitotic arrest due to destabilized kinetochore-microtubule attachments, indicating methylation promotes dynamic attachments for error correction.\",\n      \"method\": \"In vitro methylation assay, K191 mutation, SET7/9 inhibitor treatment, kinetochore-microtubule attachment assay, kinase activity assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation assay plus non-methylatable mutant with functional kinetochore-MT assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31863092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BUB1 and CENP-U are the main PLK1 kinetochore receptors in mitosis; both contain a constellation of a PP2A-docking motif flanked by two PLK1-docking sites; CDK1-primed phosphorylation on these sites enables PLK1 binding and promotes PLK1 dimerization at kinetochores; this mechanism accounts for most/all kinetochore PLK1.\",\n      \"method\": \"Ectopic localization assay, in vitro reconstitution, kinetochore localization studies, co-IP, CDK1 priming assay, PLK1 dimerization assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution plus ectopic localization plus kinetochore depletion analysis, multiple orthogonal methods\",\n      \"pmids\": [\"33248027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 dimerizes during early G2 phase in a Bora-supported manner; T210 phosphorylation by Aurora-A at late G2 triggers dimer dissociation generating active PLK1 monomers; interfering with the dimer/monomer switch prevents importin association and limits nuclear shuttling, causing nuclear PLK1 mislocalization at G2/M.\",\n      \"method\": \"Co-immunoprecipitation of PLK1 dimers, phospho-T210 mutant analysis, importin interaction assay, live cell nuclear localization imaging\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus mutant functional analysis plus localization, single lab\",\n      \"pmids\": [\"34759346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Apolo1 localizes to kinetochores during early mitosis and sustains PLK1 kinase activity at kinetochores (measured by FRET biosensor); Apolo1 is a PLK1 substrate, and its phosphorylation enables PP1γ to dephosphorylate and inactivate PLK1; Apolo1 constitutes a feedback loop connecting PLK1 to its own inactivation.\",\n      \"method\": \"FRET-based PLK1 activity reporter, co-IP, in vitro kinase assay, PP1γ interaction assay, siRNA knockdown with chromosome alignment readout\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — FRET biosensor plus in vitro kinase assay plus co-IP, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34260926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 phosphorylates PrimPol at a conserved residue between its RPA-binding motifs; this phosphorylation is cell cycle-dependent and prevents aberrant PrimPol recruitment to chromatin; loss of PLK1-dependent regulation leads to chromosome breaks, micronuclei, and reduced survival after replication stress.\",\n      \"method\": \"In vitro kinase assay, phospho-site identification, chromatin fractionation, siRNA, cell viability assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus chromatin recruitment assay, single lab\",\n      \"pmids\": [\"34860556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In C. elegans oocyte meiosis, BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent; CENP-CHCP-4 recruits PLK-1 to chromosome arms through direct interaction; perturbing both recruitment mechanisms causes severe meiotic defects and highly aneuploid oocytes.\",\n      \"method\": \"Biochemical interaction assays, live imaging, C. elegans genetic mutants, co-immunoprecipitation\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical binding assay plus live imaging plus double-perturbation epistasis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37067150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 regulates DNA replication origin firing in Xenopus by controlling origin firing frequency and synchrony; PLK1 interacts with firing factors MTBP/Treslin/TopBP1 and with Rif1; PLK1 phosphorylates Rif1 at S2058 (in its PP1-binding site), disrupting Rif1-PP1 interaction and modulating DNA replication.\",\n      \"method\": \"PLK1 immunodepletion in Xenopus extract, DNA fiber analysis, quantitative chromatin proteomics, co-immunoprecipitation, LC-MS/MS phosphopeptide mapping, S2058D phospho-mimetic mutant\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro phosphorylation in Xenopus system plus phospho-site mapping plus phospho-mimetic functional mutant, multiple orthogonal methods\",\n      \"pmids\": [\"34469577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 phosphorylates DNMT3a, leading to its proteasomal degradation during mitosis; DNMT3a in turn represses PLK1 transcription in interphase, constituting a negative feedback loop; combined inhibition of PLK1 and DNMT3a suppresses prostate cancer synergistically through autophagy regulation.\",\n      \"method\": \"Co-IP, kinase assay, DNMT3a stability assay upon PLK1 activation, PLK1 promoter analysis\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay plus promoter analysis plus co-IP, single lab\",\n      \"pmids\": [\"34051063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 activity is essential for time-dependent release of 53BP1 from kinetochores; PLK1 inhibition causes 53BP1 persistence at kinetochores (via CENP-F docking), preventing cytosolic 53BP1 association with p53 and blunting the mitotic surveillance pathway; CENP-F-53BP1 binding disruption has no impact on mitotic timing, indicating cytosolic (not kinetochore) 53BP1 measures mitotic duration.\",\n      \"method\": \"PLK1 inhibitor treatment, CENP-F-53BP1 binding mutant, immunofluorescence quantification of kinetochore/cytosolic 53BP1, p53 activation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition plus separation-of-function mutant, single lab\",\n      \"pmids\": [\"37888778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 and Aurora-A are both required for centrosome maturation and separation prior to metaphase I in mouse spermatocytes; PLK1 is additionally required to block premature second-round centriole duplication until anaphase I, maintaining strict spatiotemporal control of centrosome biogenesis during meiosis.\",\n      \"method\": \"Germ cell-specific conditional knockout of Plk1 and Aurora-A, immunofluorescence of centriole/centrosome markers through meiotic progression\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific conditional KO with mechanistic analysis at multiple meiotic stages, single lab\",\n      \"pmids\": [\"33615678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLK1 promotes NLRP3 inflammasome activation during cell interphase; PLK1 is in close proximity to and interacts with NLRP3; mechanistically, PLK1 orchestrates the MTOC structure and NLRP3 subcellular positioning upon inflammasome activation; pharmacological PLK1 inhibition suppresses IL-1β production in vivo.\",\n      \"method\": \"BioID proximity-labeling screen, co-immunoprecipitation, domain interaction mapping, pharmacological PLK1 inhibition, in vivo endotoxemia and peritonitis mouse models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID plus co-IP plus in vivo functional assay, single lab\",\n      \"pmids\": [\"37698938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Pathogenic BRCA1 mutations reduce BRCA1 function, leading to elevated PLK1 activity, which specifically disrupts cell division axis orientation in mammary cells; this defect is independent of accumulated DNA damage and depends on PLK1 activity, as shown by 15 genetically edited BRCA1 mutant cell lines.\",\n      \"method\": \"Primary mammary cell division axis imaging, BRCA1-mutant genetically-edited cell lines, PLK1 activity measurements, genetic and pharmacological PLK1 inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell lines, PLK1 inhibitor rescue, single lab\",\n      \"pmids\": [\"35459234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLK1-mediated phosphorylation of RB at S758 inhibits NF-κB nuclear translocation, inactivating the NF-κB pathway; PLK1 inhibition or depletion therefore activates NF-κB, upregulating PD-L1 expression in pancreatic ductal adenocarcinoma cells.\",\n      \"method\": \"In vitro kinase assay for RB-S758 phosphorylation, NF-κB nuclear translocation assay, PD-L1 expression analysis, siRNA and pharmacological PLK1 inhibition\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay plus mechanistic pathway analysis, single lab\",\n      \"pmids\": [\"35950917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"BUB-1-bound PLK-1 is required for CDC-20 kinetochore recruitment and timely mitotic progression in C. elegans embryos; PLK-1 kinase activity phosphorylates the CDC-20-binding ABBA motif of BUB-1 to promote BUB-1-CDC-20 interaction; a conserved STP motif in BUB-1 that docks PLK-1 is necessary for this function.\",\n      \"method\": \"C. elegans genetics (STP motif mutation in BUB-1), live imaging, in vitro kinase assay of ABBA motif phosphorylation, CDC-20 localization analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic separation-of-function plus in vitro kinase assay plus live imaging, multiple orthogonal methods\",\n      \"pmids\": [\"37137308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PLK1 phosphorylates DNA polymerase theta (Polθ) in mitosis, activating it; phosphorylated Polθ is recruited to mitotic DSBs through direct interaction with BRCA1 C-terminal domains of TOPBP1; Polθ-mediated end joining repairs mitotic DSBs and maintains genome integrity; loss of Polθ is synthetically lethal with homologous recombination deficiency.\",\n      \"method\": \"In vitro kinase assay, co-IP (Polθ-TOPBP1 interaction), mitotic DSB repair assay, synthetic lethality assay (HR-deficient cells)\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus direct protein interaction mapping plus functional DSB repair assay plus synthetic lethality, multiple orthogonal methods in high-impact journal\",\n      \"pmids\": [\"37674080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PLK1 maintains lysosomal function in renal tubular cells by phosphorylating the V-ATPase subunit ATP6V1A to regulate intralysosomal pH; PLK1 inhibition or knockout impairs lysosomal acidification and blocks autophagic flux, leading to reduced fibroblast activation and attenuation of renal tubulointerstitial fibrosis.\",\n      \"method\": \"Plk1 knockout mice (UUO model), pharmacological PLK1 inhibition, ATP6V1A phosphorylation assay, lysosomal pH measurement, autophagic flux assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO plus phosphorylation assay plus lysosomal functional readout, single lab\",\n      \"pmids\": [\"37640723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLK1 promotes CENP-A deposition (centromere maintenance) in early G1 by recognizing self-primed phosphorylations of Mis18α (Ser54) and Mis18BP1 (Thr78, Ser93) through its polo-box domain; PLK1 binding activates Mis18α-Mis18β and promotes Mis18 complex-HJURP interaction required for CENP-A loading.\",\n      \"method\": \"Biochemical phosphorylation mapping, PLK1-PBD binding assays, CENP-A deposition assay, HJURP co-IP, phospho-site mutagenesis\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — phosphosite mapping plus PBD binding assay plus functional CENP-A deposition plus HJURP co-IP, multiple orthogonal methods in two independent studies in same issue\",\n      \"pmids\": [\"39236163\", \"39236175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PP2A-B55 (α and δ isoforms) directly interacts with PLK1 during recovery from mitotic DNA damage; dephosphorylation of PLK1 by PP2A-B55 is induced through ATM/CHK1/CHK2-dependent repression of the Greatwall/ENSA pathway, leading to PP2A-B55 liberation and PLK1 dephosphorylation and mitotic slippage.\",\n      \"method\": \"Co-immunoprecipitation of PLK1-PP2A-B55α/δ, siRNA depletion of PP2A-B55, immunoblotting for phospho-PLK1, Greatwall/ENSA pathway manipulation\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus siRNA epistasis plus phospho-readout, single lab\",\n      \"pmids\": [\"31072185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLK1 phosphorylates Numb, promoting its proteasomal degradation; loss of Numb impairs the Numb/p53 pathway, providing a mechanism by which PLK1 antagonizes p53 during DNA damage response; cancer cells with non-phosphorylatable Numb show increased sensitivity to doxorubicin.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, proteasomal degradation assay, p53 stabilization assay, xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional p53 pathway and in vivo assay, single lab\",\n      \"pmids\": [\"29059161\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLK1 is a master Ser/Thr kinase activated at the G2/M boundary through Aurora-A/Bora-mediated phosphorylation of T210 (sustained in mitosis by residual Bora), modulated by dimerization/monomer switching, and inactivated by PP2A-B55 and CUL3-KLHL22-mediated ubiquitylation at K492; it is recruited to kinetochores primarily via CDK1-primed phosphosites on BUB1 and CENP-U and to other mitotic structures via PBD-phosphopeptide docking, where it phosphorylates a broad substrate network to control centrosome maturation, chromosome compaction, kinetochore-microtubule attachment (via CLIP-170, BRCA2-PP2A-BUBR1 complexes), spindle assembly checkpoint satisfaction (via Aurora B, MPS1, BUB1/BUBR1), cytokinesis (via centralspindlin/RhoA and Cep55/ESCRT), centromere integrity (suppressing BLM and PICH), CENP-A deposition in early G1 (via Mis18 complex), DNA damage response (phosphorylating Mre11, CtIP, Polθ, PrimPol), and non-mitotic processes including NLRP3 inflammasome positioning, Hedgehog/Gli1 signaling, autophagy/lysosome axis regulation, and p53 suppression; its activity is further fine-tuned by lysine methylation (SET7/9 at K191, SETD6 at K209/K413) and feedback mechanisms such as the Apolo1-PP1γ circuit.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLK1 is a cell-cycle-regulated serine/threonine kinase whose expression is restricted to proliferating cells and peaks at the G2/M boundary, where it functions as a master coordinator of mitotic entry, chromosome segregation, and cytokinesis [#0, #1]. Its catalytic activity is gated by activation-loop phosphorylation at T210, which Aurora-A together with its cofactor Bora installs and sustains throughout mitosis as a bistable switch, while a Bora-supported dimer-to-monomer transition couples T210 phosphorylation to importin-dependent nuclear shuttling [#15, #37]; DNA damage suppresses T210/S137 phosphorylation through ATM/ATR–CHK1/CHK2 signaling, and PP2A-B55 directly dephosphorylates PLK1 during checkpoint recovery [#7, #52]. Substrate selection is governed by the C-terminal polo-box domain, which docks onto CDK1- or Aurora-A-primed phosphopeptides on partners and substrates [#2, #26]; this docking logic recruits PLK1 to kinetochores via BUB1 and CENP-U, where CDK1 priming also promotes local PLK1 dimerization [#36]. Once positioned, PLK1 phosphorylates a broad network to drive centrosome maturation and spindle assembly (CEP215, with scaffolding by Gravin), kinetochore-microtubule attachment and chromosome alignment (CLIP-170 at S312, and a BRCA2–PP2A–BUBR1 complex assembled on PLK1-phosphorylated BRCA2-T207), and chromatin-based maintenance of centromere and kinetochore integrity against spindle pulling forces by suppressing BLM and acting with PICH [#23, #17, #33, #30, #32]. PLK1 controls cytokinesis by releasing centralspindlin from the midzone through PRC1 inhibition to permit RhoA activation and by promoting Cep55/ESCRT loading at the abscission bridge [#28, #25]. Beyond mitosis, PLK1 restrains the DNA damage response and replication by phosphorylating Mre11, CtIP, Polθ, PrimPol, and Rif1 [#22, #26, #49, #39, #41], deposits CENP-A in early G1 through PBD recognition of self-primed Mis18 complex phosphosites [#51], and suppresses p53 directly and via Numb degradation [#6, #53]. Its activity is further tuned by lysine methylation (SET7/9 at K191, SETD6 at K209/K413), by CUL3-KLHL22-mediated ubiquitylation at K492 that evicts it from kinetochores, and by an Apolo1–PP1γ feedback circuit [#35, #29, #14, #38], and it additionally regulates non-mitotic processes including NLRP3 inflammasome positioning, Hedgehog/Gli1 signaling, and lysosomal acidification/autophagy [#45, #31, #50].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established PLK1 as a serine/threonine kinase whose expression is tightly coupled to proliferation, framing it as a candidate mitotic regulator rather than a constitutive enzyme.\",\n      \"evidence\": \"cDNA cloning and Northern analysis across synchronized and resting cells\",\n      \"pmids\": [\"8127874\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic substrates identified at this stage\", \"Expression correlation does not establish function\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Defined how PLK1 abundance is restricted to G2/M, identifying a CDE/CHR repressor and upstream activating elements that enforce cell-cycle-dependent transcription.\",\n      \"evidence\": \"Promoter-luciferase deletion/mutation analysis in synchronized HeLa cells\",\n      \"pmids\": [\"9083047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trans-acting factors binding these elements not fully defined\", \"Does not address post-translational control\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated that the polo-box domain is required for substrate engagement, establishing the docking logic central to PLK1 targeting, using GRASP65 as the first PBD-dependent substrate.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in vitro kinase assay with PBD deletion/frameshift mutants\",\n      \"pmids\": [\"11050165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Priming kinase for the PBD docking site not defined here\", \"GRASP65 phosphosites not mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed PLK1 is required for viability and genome stability, as its loss triggers 4N arrest, DNA damage, and apoptosis through p53/ATM, establishing it as an essential mitotic gene and drug target rationale.\",\n      \"evidence\": \"siRNA knockdown, FACS, caspase assays, non-degradable Plk1 rescue across cancer lines\",\n      \"pmids\": [\"12732729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates causing the arrest not pinpointed\", \"Mechanism of DNA damage in p53-null cells unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified PLK1 as a direct suppressor of p53, providing a mechanism by which it antagonizes pro-apoptotic and checkpoint responses.\",\n      \"evidence\": \"Co-IP with deletion mutants, luciferase reporters, kinase-dead PLK1 analysis\",\n      \"pmids\": [\"15024021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"p53 phosphosite(s) not fully mapped\", \"In vivo relevance to tumor suppression untested here\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked PLK1 activation to DNA-damage signaling and to replication machinery, showing T210/S137 phosphorylation is checkpoint-suppressed and that the PBD binds the MCM2-7 complex.\",\n      \"evidence\": \"Phosphopeptide mapping with ATM/ATR and CHK inhibitors; PBD pulldown and fractionation\",\n      \"pmids\": [\"15611664\", \"15654075\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the physiological T210 kinase not established here\", \"Functional consequence of PLK1-MCM binding untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the activation logic and a key off-switch: Aurora-A/Bora sustains T210 phosphorylation as a bistable switch through mitosis, while CUL3-KLHL22 ubiquitylates K492 to evict PLK1 from kinetochore phosphoreceptors.\",\n      \"evidence\": \"Inhibitor/synchronization with Bora siRNA; MS site mapping with non-ubiquitylatable K492R mutant and SAC assays\",\n      \"pmids\": [\"24338364\", \"23455478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative threshold of the switch not defined\", \"Other E3 ligases for PLK1 not excluded\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined substrate-level control of kinetochore-microtubule attachment and apoptotic threshold, showing PLK1 phospho-cycling of CLIP-170 enables stable attachments and PLK1 phosphorylation of procaspase-8 restrains extrinsic death.\",\n      \"evidence\": \"In vitro kinase assays, phospho-site mutants, live imaging, Fas-stimulation apoptosis assays\",\n      \"pmids\": [\"24451569\", \"24484936\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase counteracting CLIP-170 S312 not identified\", \"Caspase-8 control not validated in vivo\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped upstream regulatory inputs balancing PLK1 activity, with the LKB1–NUAK1–MYPT1-PP1 axis promoting dephosphorylation and DNA-PKcs controlling PLK1 stability and midbody recruitment.\",\n      \"evidence\": \"Co-IP, kinase assays, siRNA epistasis, localization studies\",\n      \"pmids\": [\"24722282\", \"24166892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DNA-PKcs claims rest on single-method readouts per assertion\", \"Direct vs indirect dephosphorylation not fully separated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended PLK1 into the DNA damage response and centrosome scaffolding, showing it phosphorylates Mre11 to limit MRN loading, degrades Numb to antagonize p53, and requires Gravin scaffolding for centrosomal activity.\",\n      \"evidence\": \"In vitro kinase assays, phospho-site mutants, chromatin loading assays, FRAP/FRET biosensor with separation-of-function Gravin mutant\",\n      \"pmids\": [\"28512243\", \"29059161\", \"29282278\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of these arms to genome stability unquantified\", \"Numb-axis tested mainly in cancer models\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed PLK1 activation is coupled to completion of DNA replication and demonstrated kinase-dependent control of CtIP-mediated repair choice and Cep55/ESCRT-dependent abscission.\",\n      \"evidence\": \"Degron and inhibitor systems with activity reporters; in vitro kinase assays with HR/MMEJ reporters; inducible knock-in mouse with abscission imaging\",\n      \"pmids\": [\"30008317\", \"30202980\", \"30069007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sensor linking replication state to PLK1 not fully defined\", \"Dose-dependence of PLK1 in abscission not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined methylation-based fine-tuning and chromatin-based centromere protection, with SET7/9-K191 and SETD6-K209/K413 dampening kinase activity and chromatin-localized PLK1 suppressing BLM/PICH-driven centromere disintegration.\",\n      \"evidence\": \"In vitro methylation assays with non-methylatable mutants; PLK1 inhibition with BLM/PICH co-depletion rescue and chromatin-tethered PLK1 constructs\",\n      \"pmids\": [\"31863092\", \"30622182\", \"31253795\", \"31468671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Demethylases for these sites not identified\", \"Direct chromatin substrates maintaining integrity not all mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked PLK1 to cytokinesis signaling, Hedgehog/Gli1 control, and checkpoint-recovery dephosphorylation, broadening its regulatory reach beyond core spindle functions.\",\n      \"evidence\": \"Live imaging of centralspindlin/RhoA with PRC1 knockdown; in vitro kinase assay and reporter assays for Gli1-S481; co-IP and siRNA epistasis for PP2A-B55\",\n      \"pmids\": [\"30728176\", \"30578313\", \"31072185\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Centralspindlin substrate of PLK1 not specified\", \"PP2A-B55 study relies on single-lab epistasis\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the dominant kinetochore-recruitment mechanism and additional attachment control, identifying BUB1 and CENP-U as the main PLK1 receptors via CDK1-primed docking and BRCA2-T207 as a docking site assembling a PP2A-BUBR1 complex.\",\n      \"evidence\": \"In vitro reconstitution and ectopic localization for BUB1/CENP-U; phosphosite mapping and complex co-IP with cancer-variant analysis for BRCA2\",\n      \"pmids\": [\"33248027\", \"32286328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative split between BUB1 and CENP-U pools not fully resolved\", \"Functional output of dimerization at kinetochores incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated organismal-level mitotic and meiotic roles, with PLK1 required for chromosome compaction, MTOC organization, and spindle formation in oocytes.\",\n      \"evidence\": \"Oocyte-specific conditional Plk1 knockout mouse with marker immunofluorescence\",\n      \"pmids\": [\"32267211\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates driving compaction not identified\", \"Distinction from somatic mitotic roles partial\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the dimer-monomer activation switch and expanded PLK1 into replication and feedback control, including PrimPol and Rif1 phosphorylation, the Apolo1-PP1γ inactivation loop, and DNMT3a/PLK1 transcriptional feedback.\",\n      \"evidence\": \"Co-IP of dimers with T210 mutants and importin assays; in vitro kinase assays with phospho-mutants; FRET biosensor and PP1γ interaction assays\",\n      \"pmids\": [\"34759346\", \"34860556\", \"34469577\", \"34260926\", \"34051063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of multiple feedback loops in vivo unresolved\", \"Some replication findings rely on Xenopus extract systems\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended PLK1 into immune and tumor-relevant signaling, showing it positions NLRP3 via MTOC control, restrains NF-κB/PD-L1 through RB-S758, and that its dysregulation downstream of BRCA1 loss misorients cell division.\",\n      \"evidence\": \"BioID and co-IP with in vivo inflammasome models; in vitro RB kinase assay with NF-κB/PD-L1 readouts; division-axis imaging across BRCA1-mutant lines\",\n      \"pmids\": [\"37698938\", \"35950917\", \"35459234\", \"14654792\", \"31597699\", \"27398835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Some interphase roles rely on single-lab evidence\", \"Causal substrate for NLRP3 positioning not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined PLK1 as a driver of mitotic DSB repair and lysosomal/autophagy control, activating Polθ and recruiting it to mitotic breaks via TOPBP1, and acidifying lysosomes via ATP6V1A phosphorylation.\",\n      \"evidence\": \"In vitro kinase assays, co-IP, mitotic DSB repair and synthetic-lethality assays; Plk1 knockout mice with lysosomal pH and autophagic flux readouts\",\n      \"pmids\": [\"37674080\", \"37640723\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Polθ phosphosite identity not fully mapped\", \"Tissue specificity of the lysosomal axis incompletely defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Completed the cell-cycle arc by showing PLK1 promotes CENP-A deposition in early G1 through PBD recognition of self-primed Mis18 complex phosphosites, activating Mis18 and enabling HJURP-dependent loading.\",\n      \"evidence\": \"Phosphosite mapping, PBD binding assays, CENP-A deposition assays, HJURP co-IP across two independent studies\",\n      \"pmids\": [\"39236163\", \"39236175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase resetting Mis18 sites not defined\", \"Timing relative to PLK1 inactivation at mitotic exit unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many PLK1 inputs (T210 phosphorylation, dimer-monomer state, methylation, ubiquitylation, phosphatase feedback) are integrated to produce spatially and temporally distinct substrate selection across mitosis, the DNA damage response, and interphase processes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model linking PLK1 modification state to substrate prioritization\", \"Relative contribution of mitotic vs non-mitotic functions to organismal phenotypes unquantified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 6, 17, 22, 26, 31, 33, 35, 47, 49, 50, 51]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 17, 22, 26, 49]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6, 31, 47]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [9, 23, 44]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [13, 30, 32, 36, 51]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [37]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 15, 17, 25, 28, 32, 36]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [22, 26, 49, 39]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [27, 41]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [31, 47]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [50]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [45]}\n    ],\n    \"complexes\": [\"BRCA2-PP2A-BUBR1 complex\"],\n    \"partners\": [\"BORA\", \"AURKA\", \"BUB1\", \"CENP-U\", \"BRCA2\", \"CUL3-KLHL22\", \"PP2A-B55\", \"GRASP65\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}