{"gene":"PLK1","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1997,"finding":"PLK1 (Plk) is a functional homolog of S. cerevisiae Cdc5; Glu206 and Thr210 in the kinase domain are critical for catalytic activity, and replacement of Thr210 with a negatively charged residue (T210D) elevates Plk specific activity. Ectopic expression of Plk complements the cdc5-1 cell division defect in yeast in a kinase-activity-dependent manner.","method":"In vitro kinase assay with immunoprecipitates from Sf9 cells; site-directed mutagenesis (K82M, D194N, D194R, T210D); yeast complementation of cdc5-1 mutation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis plus genetic epistasis (yeast complementation)","pmids":["9154840"],"is_preprint":false},{"year":2002,"finding":"PLK1 phosphorylates the microtubule-stabilizing protein TCTP on two serine residues in vitro and in vivo; this phosphorylation decreases TCTP's microtubule-stabilizing activity, and overexpression of a phosphorylation-deficient TCTP mutant causes multinucleate cells and cell death similar to anti-Plk antibody microinjection phenotypes.","method":"Two-hybrid screen to identify Plk-interacting proteins; in vitro kinase assay; co-fractionation; in vivo phosphorylation analysis; overexpression of phosphomutant TCTP","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis and in vivo validation using phospho-deficient mutant","pmids":["12167714"],"is_preprint":false},{"year":2003,"finding":"Plk1 depletion in cancer cells causes mitotic arrest with 4N DNA content, formation of dumbbell-like chromatin structures (failure of sister chromatid separation), and apoptosis involving caspase-3 activation and p53 stabilization; DNA damage also occurs in p53-null cells. ATM inhibition potentiates lethality of Plk1 depletion.","method":"Vector-based siRNA depletion; FACS; caspase-3 activation assay; rescue with non-degradable Plk1 constructs; ATM inhibitor treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — KO/KD with defined cellular phenotype replicated with rescue experiment; multiple orthogonal readouts","pmids":["12732729"],"is_preprint":false},{"year":2004,"finding":"PLK1 physically binds to the DNA-binding region of p53 (identified by systematic immunoprecipitation with deletion mutants) and phosphorylates p53 in a kinase-dependent manner, thereby inhibiting p53 transcriptional activity and its pro-apoptotic function; a kinase-dead Plk1 mutant fails to suppress p53 activity.","method":"Co-immunoprecipitation with deletion mutants; confocal co-localization; luciferase reporter assay; kinase-dead mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — Co-IP with mapping, kinase-dead mutant, and functional transcription assay","pmids":["15024021"],"is_preprint":false},{"year":2005,"finding":"PLK1's polo-box domain (PBD) interacts with MCM2 and MCM7 (and the whole MCM2-7 complex) in a chromatin-associated fraction, suggesting a role for PLK1 in coordinating DNA replication and mitotic events.","method":"PBD-based protein interaction screen; co-immunoprecipitation of endogenous proteins; immunoblot; chromatin fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP with biochemical fractionation, single lab, no functional rescue","pmids":["15654075"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the PLK1 kinase domain in complex with the inhibitor BI 2536 reveals that Leu132 in the hinge region creates a small selectivity pocket exploited by BI 2536's methoxy group, providing structural basis for PLK1 selectivity over other kinases.","method":"X-ray crystallography (co-crystal structure of PLK1 kinase domain with BI 2536); selectivity profiling","journal":"Chemical biology & drug design","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with selectivity data","pmids":["18005335"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of wild-type PLK1 kinase domain in complex with DARPin 3H10 solved at 2.3 Å, revealing the active conformation of PLK1 and enabling structure-based drug design.","method":"X-ray crystallography using DARPin as crystallization chaperone","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 — crystal structure at 2.3 Å resolution","pmids":["18391401"],"is_preprint":false},{"year":2008,"finding":"PLK1 is recruited to centromeres through a self-priming mechanism: PLK1 phosphorylates the centromeric component PBIP1, generating its own PBD-binding site (phospho-epitope) for initial kinetochore recruitment; PLK1 subsequently induces PBIP1 delocalization and degradation to allow binding to other kinetochore components.","method":"Cell-based localization studies; biochemical analysis of PLK1-PBIP1 interaction; site-directed mutagenesis","journal":"Cell division","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic model supported by localization and phosphorylation studies; single lab review with primary data","pmids":["18215321"],"is_preprint":false},{"year":2008,"finding":"In C. elegans embryos, PLK-1 is asymmetrically distributed between AB and P1 blastomeres under control of anterior-posterior PAR polarity cues, and this asymmetry promotes mitotic entry preferentially in AB; mild plk-1 RNAi delays mitosis specifically in P1 but not AB, independently of ATL-1/CHK-1.","method":"RNAi depletion in C. elegans; live imaging; genetic analysis with PAR mutants","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with polarity pathway combined with live imaging and quantitative cell biology","pmids":["18305005"],"is_preprint":false},{"year":2009,"finding":"PLK1 interacts with the microtubule depolymerase Kif2a during mitosis in a kinase-activity-dependent manner and phosphorylates Kif2a to enhance its depolymerase activity in vitro; PLK1 inhibition decreases microtubule-associated Kif2a signals and increases spindle microtubule intensity in vivo. Aurora A antagonizes this by phosphorylating Kif2a to suppress its depolymerase activity.","method":"Proteomic identification; co-immunoprecipitation; in vitro kinase/depolymerase assay; PLK1 inhibition/depletion in vivo","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay combined with in vivo functional readout; antagonistic regulation by Aurora A provides mechanistic context","pmids":["19351716"],"is_preprint":false},{"year":2009,"finding":"PLK1 phosphorylates TAp63 at Ser-52 of the TA domain (demonstrated by in vitro kinase assay), decreasing TAp63 protein stability and suppressing TAp63-induced apoptotic cell death in liver tumor cells; PLK1 binds p63 through its kinase domain via p63's DNA-binding region.","method":"Immunoprecipitation; in vitro pull-down; in vitro kinase assay; siRNA knockdown; double knockdown epistasis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with substrate mapping plus epistasis (double knockdown)","pmids":["19668228"],"is_preprint":false},{"year":2010,"finding":"PICH (a PLK1-binding protein) and PLK1 kinase coordinately maintain chromosome arm architecture during prometaphase; PICH knockdown causes loss of PLK1 from chromosome arms, resulting in 'wavy' chromosome disorganization that can be prevented by topoisomerase II inhibition, suggesting the PICH-PLK1 complex normally acts via topoisomerase II activity.","method":"siRNA knockdown; live and fixed-cell imaging; chromosome architecture analysis; topoisomerase II inhibitor treatment","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — KD with defined phenotype and chemical epistasis with topo II inhibitor","pmids":["20130082"],"is_preprint":false},{"year":2010,"finding":"p53 represses PLK1 transcription directly: chromatin immunoprecipitation shows p53 binds two sites (p53RE1 and p53RE2) on the PLK1 promoter; DNA damage stimulates p53 recruitment to p53RE2 coincident with repression-associated chromatin changes including HDAC recruitment; this repression is independent of p21 and the CDE/CHR element.","method":"Chromatin immunoprecipitation (ChIP); reporter gene assay; siRNA; HDAC inhibitor treatment","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 2 — ChIP with multiple mutant promoter constructs and siRNA epistasis; multiple orthogonal methods","pmids":["20962589"],"is_preprint":false},{"year":2012,"finding":"PLK1 directly phosphorylates the kinesin-13 Kif2b at Thr125 and Ser204; phosphorylation of S204 is required for kinetochore localization of Kif2b in prometaphase, while T125 phosphorylation is required for Kif2b activity in correcting k-MT attachment errors.","method":"Mass spectrometry phospho-mapping; in vitro kinase assay; phosphomutant overexpression; kinetochore localization assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with site-specific phosphomutants and functional readout","pmids":["22535524"],"is_preprint":false},{"year":2013,"finding":"The CUL3-KLHL22 E3 ubiquitin ligase ubiquitylates PLK1 at Lys492 within the PBD, causing PLK1 dissociation from kinetochore phosphoreceptors without proteolytic degradation; loss of KLHL22 causes PLK1 accumulation at kinetochores and sustained SAC activation; expression of non-ubiquitylatable PLK1-K492R phenocopies KLHL22 inactivation.","method":"Co-immunoprecipitation; mass spectrometry; siRNA; non-ubiquitylatable mutant expression; kinetochore localization; SAC analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — ubiquitylation site mapped to K492, phenocopied by K492R mutant, multiple orthogonal methods","pmids":["23455478"],"is_preprint":false},{"year":2013,"finding":"Bora-Aurora-A remains the major activating complex for PLK1 in mitosis (not only G2); a small amount of Aurora-A activity with residual Bora is sufficient to sustain PLK1 T210 phosphorylation in mitosis, creating a bistable switch where PLK1 is sensitive to Aurora-A inhibition during initial activation but refractory once fully activated.","method":"Kinase inhibitor treatment; immunoblotting of T210 phosphorylation; RNAi depletion of Bora","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative phosphorylation analysis with inhibitors and knockdowns; 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 phospho-epitope) followed by PLK1 at S305 suppresses extrinsic apoptosis during mitosis; non-phosphorylatable caspase-8 S305A increases Fas-stimulated cell death, and BI 2536 (PLK1 inhibitor) lowers the threshold for Fas-induced apoptosis.","method":"In vitro kinase assay; PBD-binding assay; RNAi replacement with phosphomutants; Fas-stimulation assay; BI 2536 treatment","journal":"Molecular oncology","confidence":"High","confidence_rationale":"Tier 1 — sequential kinase assay with phosphomutant rescue; mechanistic PBD-docking model validated","pmids":["24484936"],"is_preprint":false},{"year":2014,"finding":"CLIP-170, phosphorylated by CDK1 at T287, acts as a PLK1 recruiter at kinetochores during early mitosis; depletion of CLIP-170 or expression of non-phosphorylatable CLIP-170-T287A reduces PLK1 kinetochore localization, destabilizes kinetochore fibers, and impairs chromosome alignment.","method":"Co-localization; siRNA depletion; phosphomutant expression; kinetochore fiber stability assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — KD and phosphomutant rescue with defined localization and functional phenotype","pmids":["24777477"],"is_preprint":false},{"year":2014,"finding":"PLK1 regulates NEK2 activity, which in turn phosphorylates and stabilizes β-catenin at mitotic centrosomes; Plk1 inhibition or depletion disrupts this pathway. In vitro and cell-based assays show Nek2 phosphorylates β-catenin at N-terminal regulatory sites, blocking β-TrCP binding and preventing ubiquitination and degradation.","method":"In vitro kinase assay; co-immunoprecipitation; ubiquitination assay; PLK1 inhibitor treatment; siRNA","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay with mechanistic pathway (Plk1→Nek2→β-catenin) validated by multiple methods","pmids":["24501426"],"is_preprint":false},{"year":2015,"finding":"CDK-1 phosphorylates Bora/SPAT-1 at conserved Sp/Tp residues, promoting Bora interaction with PLK1 and stimulating Aurora A-dependent T-loop phosphorylation of PLK1 to trigger mitotic entry; phospho-SPAT-1 activates PLK1 by enabling Aurora A phosphorylation in vitro; this mechanism is conserved between C. elegans and humans.","method":"In vitro kinase assay (CDK1 phosphorylation of Bora; Aurora A phosphorylation of PLK1); non-phosphorylatable SPAT-1 mutant analysis in C. elegans embryos; human Bora/PLK1 reconstitution","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution across two organisms; phosphomutant analysis; multiple orthogonal methods","pmids":["25753036"],"is_preprint":false},{"year":2015,"finding":"PLK1 is required for efficient meiotic resumption (nuclear envelope breakdown), recruitment of centrosomal proteins to acentriolar MTOCs, spindle formation, stable kinetochore-microtubule attachment, APC/C activation (by promoting EMI1 degradation), and chromosome condensation maintenance during meiosis I-II transition in mouse oocytes.","method":"Specific small-molecule PLK1 inhibition combined with live mouse oocyte imaging; time-lapse microscopy; APC/C activity assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — specific inhibitor combined with live imaging; multiple defined phenotypic readouts","pmids":["25658810"],"is_preprint":false},{"year":2016,"finding":"Drosophila Polo/PLK1's polo-box domain (PBD) binds phospho-Pon (phosphorylated by Cdk1 at Thr63), and crystal structure of Plk1 PBD/phospho-Pon complex reveals that two phospho-Pon-bound PBDs dimerize; this PBD dimerization relieves Plk1 autoinhibition and promotes sequential Plk1 phosphorylation of Pon.","method":"Crystal structure of PBD/phospho-Pon complex; in vitro binding assay; mutagenesis; in vitro kinase assay","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and in vitro kinase assay; structural mechanism for autoinhibition relief","pmids":["27238966"],"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; a phospho-mimetic PLK1 mutant causes polyploidy, and expression of non-phosphorylatable PLK1 or PLK1 inhibition reduces polyploid cell populations.","method":"Co-immunoprecipitation; phosphatase assay; phosphomimetic and non-phosphorylatable PLK1 mutant expression; ploidy analysis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with phosphatase activity and mutant analysis; single lab","pmids":["27398835"],"is_preprint":false},{"year":2017,"finding":"PLK1 phosphorylates Mre11 at S649, priming subsequent CK2-mediated phosphorylation at S688; dual phosphorylation inhibits MRN complex loading onto damaged DNA, leading to premature DNA damage checkpoint termination and impaired DNA repair.","method":"In vitro kinase assay; phosphomimetic/non-phosphorylatable Mre11 mutants; DNA damage focus assay; checkpoint termination assay; xenograft tumor sensitivity assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with site-specific mutants and multiple functional readouts","pmids":["28512243"],"is_preprint":false},{"year":2017,"finding":"PLK1 coordinates nuclear pore complex (NPC) disassembly at mitotic entry (but not lamin A/C disassembly) and negatively regulates NPC reassembly on lagging chromatin; this reveals independent regulatory pathways for lamin A/C (CDK1-dependent) and NPCs (PLK1-dependent) during mitosis.","method":"PLK1 inhibition; live cell imaging; immunofluorescence; functional analysis of NPC/lamin assembly on lagging chromatin","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — PLK1 inhibitor with defined localization phenotype distinguishing NPC from lamin pathway","pmids":["29487689"],"is_preprint":false},{"year":2017,"finding":"Plk1 phosphorylates Numb, leading to enhanced proteasomal degradation of Numb and impairment of the Numb/p53 pathway, thereby antagonizing p53 during DNA damage response; cancer cells expressing non-phosphorylatable Numb are more sensitive to doxorubicin.","method":"In vitro kinase assay; co-immunoprecipitation; proteasomal degradation assay; phosphomutant expression; xenograft model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay with functional rescue and in vivo xenograft validation","pmids":["29059161"],"is_preprint":false},{"year":2017,"finding":"HMMR acts at centrosomes in a PLK1-dependent pathway that localizes active Ran and modulates cortical NuMA-dynein complexes for spindle orientation; HMMR loss causes spindle misorientation that phenocopies disruption of this PLK1-dependent pathway.","method":"Hmmr-knockout mouse model; live cell imaging; Ran-GTP localization; spindle orientation assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined spindle orientation phenotype; pathway placement via genetic analysis","pmids":["28994651"],"is_preprint":false},{"year":2018,"finding":"DNA replication restricts CDK1 and PLK1 activation; preventing DNA replication licensing or firing causes premature activation of CDK1 and PLK1 in S phase; in the presence of DNA replication, CHK1 and p38 inhibition also leads to premature mitotic kinase activation causing replication stress.","method":"Double-degron system for protein depletion; kinase inhibitors; cell cycle synchronization; kinase activity assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — engineered degron system with multiple kinase inhibitors; mechanistic epistasis showing replication as upstream brake","pmids":["30008317"],"is_preprint":false},{"year":2018,"finding":"PLK1 overexpression causes cytokinesis defects 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; live cell imaging; Cep55/ESCRT localization assay; kinase-dead mutant","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vivo knock-in model with kinase-dependent mechanistic analysis of Cep55/ESCRT loading","pmids":["30069007"],"is_preprint":false},{"year":2019,"finding":"SETD6 binds and methylates PLK1 at K209 and K413 during mitosis; lack of PLK1 methylation results in increased PLK1 kinase activity, accelerated mitosis, and faster cellular proliferation, revealing SETD6-mediated methylation as a negative regulator of PLK1 kinase activity.","method":"In vitro methyltransferase assay; SETD6-deficient cells; non-methylatable PLK1 mutants; kinase activity assay; time-lapse microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro methylation assay with non-methylatable mutants and functional kinase activity readout","pmids":["30622182"],"is_preprint":false},{"year":2019,"finding":"PLK1 inhibition reveals a centromere disintegration mechanism: PLK1 inactivation allows BLM helicase to unwind DNA at a specific centromere domain, and under bipolar spindle pulling, the distorted centromeres are decompacted into threadlike DNA molecules leading to centromere rupture and chromosome arm splitting.","method":"PLK1 small-molecule inhibition; live cell imaging; BLM depletion epistasis; centromere structure analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — PLK1 inhibition with genetic epistasis (BLM depletion); novel mechanism supported by live imaging and structural analysis","pmids":["31253795"],"is_preprint":false},{"year":2020,"finding":"BUB1 (outer kinetochore) and CENP-U (inner kinetochore) are the main PLK1 receptors at kinetochores; both share a constellation of sequence motifs (PP2A-docking motif and two PLK1-docking sites); PLK1 recruitment requires priming phosphorylation by CDK1 and PLK1 itself, and promotes PLK1 dimerization.","method":"Ectopic localization assay; in vitro reconstitution; kinetochore localization studies; biochemical binding assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution combined with ectopic localization and kinetochore assays; multiple orthogonal methods","pmids":["33248027"],"is_preprint":false},{"year":2020,"finding":"BRCA2 is phosphorylated by PLK1 at T207 (a bona fide PLK1 docking site); BRCA2 bound to PLK1 forms a complex with PP2A and phospho-BUBR1; disruption of BRCA2-PLK1 binding (BRCA2 variants S206C and T207A) destabilizes this complex, causing unstable kinetochore-microtubule interactions, chromosome misalignment, and aneuploidy.","method":"Phosphorylation site mapping; co-immunoprecipitation; BRCA2 variant analysis; kinetochore-microtubule stability assay; chromosome alignment analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — phospho-site identification with disease variants and reconstitution of tetrameric complex; multiple functional readouts","pmids":["32286328"],"is_preprint":false},{"year":2020,"finding":"SET7/9 dimethylates PLK1 at Lys191 at kinetochores during early mitosis, tuning down PLK1 kinase activity by limiting ATP utilization; overexpression of non-methylatable PLK1 or SET7/9 inhibition causes mitotic arrest due to destabilized kinetochore-microtubule attachments.","method":"In vitro methyltransferase assay; non-methylatable PLK1 mutant expression; PLK1 kinase activity assay; kinetochore-microtubule stability analysis; SET7/9 inhibitor treatment","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro methylation assay with non-methylatable mutant and activity readout","pmids":["31863092"],"is_preprint":false},{"year":2021,"finding":"PLK1 dimerization during early G2 phase (supported by Bora) fine-tunes PLK1 activation timing and nuclear entry; T210 phosphorylation by Aurora-A triggers dimer dissociation generating active PLK1 monomers; interfering with this dimer/monomer switch prevents importin association and nuclear PLK1 localization during G2-M transition.","method":"Biochemical dimerization assay; T210 phosphorylation analysis; importin co-immunoprecipitation; nuclear localization assay; dimer interface mutants","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical demonstration of dimer/monomer switch linked to T210 phosphorylation and nuclear entry","pmids":["34759346"],"is_preprint":false},{"year":2021,"finding":"Apolo1 localizes to kinetochores during early mitosis, sustains PLK1 kinase activity at kinetochores for accurate kinetochore-microtubule attachment, and is itself a PLK1 substrate; PLK1-phosphorylated Apolo1 recruits PP1γ to dephosphorylate and inactivate PLK1, constituting a feedback loop that fine-tunes PLK1 activity.","method":"FRET-based PLK1 activity biosensor; co-immunoprecipitation; siRNA; kinase assay; phosphatase recruitment assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — FRET biosensor combined with biochemical kinase/phosphatase assays and genetic depletion","pmids":["34260926"],"is_preprint":false},{"year":2021,"finding":"PLK1 activity is essential for time-dependent release of 53BP1 from kinetochores (through CENP-F as docking partner); PLK1 inhibition causes 53BP1 persistence at kinetochores, preventing cytosolic 53BP1 association with p53 and blunting the mitotic surveillance pathway (mitotic stopwatch).","method":"PLK1 inhibitor treatment; 53BP1 localization imaging; CENP-F-53BP1 binding disruption; mitotic surveillance pathway activation assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — PLK1 inhibition with mechanistic dissection of CENP-F-53BP1 and cytosolic p53 interaction; multiple readouts","pmids":["37888778"],"is_preprint":false},{"year":2021,"finding":"PLK1 regulates PrimPol by phosphorylating it at a conserved residue between its RPA binding motifs, preventing aberrant PrimPol chromatin recruitment; this phosphorylation is cell cycle regulated and reversed during replication stress; loss of PLK1-dependent PrimPol regulation causes chromosome breaks and micronuclei.","method":"In vitro kinase assay; chromatin recruitment assay; cell cycle phosphorylation analysis; chromosome instability readouts (breaks, micronuclei); drug sensitivity assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 — in vitro phosphorylation with chromatin recruitment and functional consequence readouts","pmids":["34860556"],"is_preprint":false},{"year":2021,"finding":"PLK1 promotes NLRP3 inflammasome activation at cell interphase; a proximity-dependent BioID screen identifies enhanced PLK1-NLRP3 proximity upon inflammasome activation; PLK1 orchestrates MTOC structure and NLRP3 subcellular positioning upon inflammasome activation; PLK1 inhibition suppresses IL-1β production in vivo.","method":"BioID proximity screen; co-immunoprecipitation (interaction domain mapping); PLK1 inhibitor in vivo; NLRP3 subcellular localization; MTOC organization assay","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — BioID screen with Co-IP validation and in vivo pharmacological confirmation; single lab","pmids":["37698938"],"is_preprint":false},{"year":2023,"finding":"PLK1 phosphorylates DNA polymerase theta (Polθ) to activate it specifically during mitosis; phosphorylated Polθ is recruited via direct interaction with BRCA1 C-terminal domains of TOPBP1 to mitotic DNA double-strand breaks, where it mediates DNA end joining; loss of Polθ leads to defective mitotic DSB repair and genome instability.","method":"In vitro kinase assay; direct protein interaction assay (Polθ-TOPBP1); KO phenotype analysis; DNA repair assay in mitosis; synthetic lethality assay with HR-deficient cells","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — in vitro phosphorylation with direct interaction mapping and KO functional rescue; published in Nature","pmids":["37674080"],"is_preprint":false},{"year":2023,"finding":"PLK1 maintains intralysosomal pH by regulating ATP6V1A phosphorylation, and PLK1 inhibition impairs lysosomal function leading to blockade of autophagic flux and reduced fibroblast activation in renal fibrosis.","method":"PLK1 pharmacological inhibition/genetic KO; lysosomal pH measurement; autophagic flux assay; ATP6V1A phosphorylation analysis; UUO mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO and inhibitor with biochemical phosphorylation analysis; in vivo model validation","pmids":["37640723"],"is_preprint":false},{"year":2024,"finding":"PLK1 promotes CENP-A deposition in early G1 by interacting with the Mis18 complex through recognition of self-primed phosphorylations on Mis18α (Ser54) and Mis18BP1 (Thr78 and Ser93) via its polo-box domain; PLK1 binding activates a conformational switch in Mis18α-Mis18β and promotes Mis18 complex-HJURP interaction required for CENP-A loading.","method":"Biochemical reconstitution; PLK1 PBD binding assay; phosphorylation site mapping; structural analysis; functional CENP-A deposition assay","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 — biochemical reconstitution with phospho-site mapping and conformational/functional analysis; published in Science","pmids":["39236163"],"is_preprint":false},{"year":2024,"finding":"PLK1 interacts with Mis18α and Mis18BP1 through their self-primed phosphorylations (Mis18α Ser54, Mis18BP1 Thr78 and Ser93) recognized by PLK1's polo-box domain; disrupting these phosphorylations perturbs HJURP centromere recruitment and new CENP-A loading; PLK1 binding activates Mis18α-Mis18β complex.","method":"Biochemical and functional analysis of Mis18 phosphorylation; PLK1 PBD-binding assay; HJURP recruitment assay; CENP-A loading assay","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 — phospho-site mapping with reconstitution and functional CENP-A deposition readout; independent replication of PMID 39236163 findings","pmids":["39236175"],"is_preprint":false}],"current_model":"PLK1 is a master mitotic Ser/Thr kinase activated by Aurora-A-mediated T210 phosphorylation (promoted by Bora/SPAT-1 primed by CDK1) that dynamically localizes to centrosomes, kinetochores (via BUB1, CENP-U, CLIP-170, and PBIP1 phospho-docking), and the midbody through its polo-box domain (PBD), where it phosphorylates a broad array of substrates — including TCTP, Kif2a, Kif2b, caspase-8, Mre11, Numb, PrimPol, Polθ, and TAp63 — to regulate mitotic entry, spindle assembly, kinetochore-microtubule attachment, chromosome integrity, cytokinesis (via Cep55/ESCRT loading), and CENP-A deposition; its activity is counterbalanced by non-proteolytic CUL3-KLHL22 ubiquitylation at K492 that removes PLK1 from kinetochores, by SETD6-mediated methylation at K209/K413 and SET7/9-mediated dimethylation at K191 that reduce its kinase activity, by a dimerization mechanism that controls its nuclear entry, and by feedback involving Apolo1-recruited PP1γ, while p53 transcriptionally represses PLK1 and PLK1 reciprocally inhibits p53 through direct phosphorylation."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing PLK1 as a conserved cell-division kinase: mutagenesis of T210 and the catalytic domain demonstrated that PLK1 kinase activity is essential for viability and that T210 is a key activating residue, while yeast complementation proved functional conservation with Cdc5.","evidence":"Site-directed mutagenesis (K82M, D194N, T210D) with in vitro kinase assay and S. cerevisiae cdc5-1 complementation","pmids":["9154840"],"confidence":"High","gaps":["Upstream activating kinase for T210 not yet identified","No structural information on the kinase domain available"]},{"year":2002,"claim":"Identifying the first mitotic substrate: PLK1 phosphorylation of TCTP attenuates its microtubule-stabilizing activity, providing direct evidence that PLK1 controls spindle microtubule dynamics through substrate phosphorylation.","evidence":"Two-hybrid screen; in vitro kinase assay; phospho-deficient TCTP mutant causes multinucleation phenocopying PLK1 loss","pmids":["12167714"],"confidence":"High","gaps":["Specific TCTP phosphorylation sites not fully mapped","Relative contribution of TCTP versus other substrates to spindle assembly unclear"]},{"year":2003,"claim":"Demonstrating that PLK1 is essential for chromosome segregation and cell survival: PLK1 depletion causes mitotic arrest, failure of sister chromatid separation, DNA damage, and caspase-3-dependent apoptosis, establishing PLK1 as indispensable for mitotic fidelity.","evidence":"Vector-based siRNA with FACS, caspase-3 activation, and rescue by non-degradable PLK1","pmids":["12732729"],"confidence":"High","gaps":["Direct substrates mediating sister chromatid separation not identified","Mechanism linking PLK1 loss to DNA damage not resolved"]},{"year":2004,"claim":"Revealing a reciprocal PLK1–p53 regulatory axis: PLK1 directly binds and phosphorylates p53 to inhibit its transcriptional and pro-apoptotic functions, while p53 reciprocally represses PLK1 transcription via direct promoter binding, establishing a feedback circuit that integrates mitotic signaling with tumor suppression.","evidence":"Co-IP with deletion mapping, kinase-dead mutant, luciferase reporter (2004); ChIP on PLK1 promoter with HDAC recruitment (2010)","pmids":["15024021","20962589"],"confidence":"High","gaps":["Specific p53 phosphorylation sites by PLK1 not mapped","In vivo physiological significance of this feedback in normal tissues not established"]},{"year":2007,"claim":"Solving the structural basis for PLK1 kinase selectivity: crystal structures of the kinase domain with BI 2536 and with a DARPin chaperone revealed the active conformation and a Leu132-dependent selectivity pocket, enabling rational drug design.","evidence":"X-ray crystallography of PLK1 kinase domain with BI 2536 (2007) and DARPin 3H10 at 2.3 Å (2008)","pmids":["18005335","18391401"],"confidence":"High","gaps":["Full-length PLK1 structure including PBD intramolecular contacts not determined","Structural basis of substrate recognition beyond PBD not resolved"]},{"year":2008,"claim":"Defining the self-priming mechanism for kinetochore recruitment: PLK1 phosphorylates PBIP1 to create its own PBD-docking site, then induces PBIP1 degradation to transition to other kinetochore receptors, establishing the principle that PLK1 generates and then consumes its own docking platforms.","evidence":"Cell-based localization with site-directed mutagenesis of PBIP1 phospho-epitope","pmids":["18215321"],"confidence":"Medium","gaps":["Temporal order of receptor switching at kinetochores not fully resolved","Quantitative contribution of PBIP1 versus other docking sites unclear"]},{"year":2009,"claim":"Expanding PLK1's substrate repertoire to microtubule depolymerases: PLK1 phosphorylates Kif2a to enhance its depolymerase activity while Aurora-A antagonizes this, revealing a kinase-counterkinase mechanism controlling spindle microtubule length.","evidence":"Co-IP and in vitro kinase/depolymerase assay; PLK1 inhibition increases spindle microtubule intensity in vivo","pmids":["19351716"],"confidence":"High","gaps":["Spatial coordination of PLK1 and Aurora-A on Kif2a at spindle poles not resolved"]},{"year":2012,"claim":"Demonstrating phospho-site-specific control of a kinesin: PLK1 phosphorylation of Kif2b at S204 governs kinetochore targeting while T125 phosphorylation activates error-correction function, showing that distinct phospho-events on a single substrate can control localization versus activity.","evidence":"Mass spectrometry phospho-mapping; in vitro kinase assay; phosphomutant localization and functional rescue","pmids":["22535524"],"confidence":"High","gaps":["Whether PLK1-Kif2b axis acts independently of or redundantly with Aurora-B error correction not established"]},{"year":2013,"claim":"Discovering non-proteolytic ubiquitylation as a kinetochore removal mechanism: CUL3–KLHL22 ubiquitylates PLK1 at K492 within the PBD to dissociate it from kinetochores without degradation, revealing ubiquitin as a spatial regulator of PLK1 rather than a destruction signal.","evidence":"Mass spectrometry site identification; K492R mutant phenocopies KLHL22 depletion (sustained SAC activation)","pmids":["23455478"],"confidence":"High","gaps":["Identity of deubiquitinase that reverses K492 modification unknown","Structural basis for how K492 ubiquitylation disrupts PBD binding not determined"]},{"year":2014,"claim":"Establishing a Cdk1→PLK1 sequential phosphorylation relay on procaspase-8 that suppresses extrinsic apoptosis during mitosis, integrating cell-cycle kinase signaling with death receptor signaling.","evidence":"In vitro kinase assay; PBD-docking site generated by Cdk1; S305A mutant sensitizes cells to Fas-induced apoptosis","pmids":["24484936"],"confidence":"High","gaps":["Whether this mechanism operates in non-cancer cell contexts not tested"]},{"year":2015,"claim":"Resolving the activation cascade: CDK1 phosphorylates Bora/SPAT-1, which then stimulates Aurora-A–dependent T210 phosphorylation of PLK1, establishing the CDK1→Bora→Aurora-A→PLK1 mitotic entry relay as a conserved mechanism from C. elegans to humans.","evidence":"In vitro reconstitution of CDK1-Bora-Aurora A-PLK1 phosphorylation cascade; non-phosphorylatable SPAT-1 mutants in C. elegans embryos","pmids":["25753036"],"confidence":"High","gaps":["Quantitative parameters of the bistable switch in vivo not measured","Spatial regulation of Bora–Aurora-A interaction at centrosomes not resolved"]},{"year":2016,"claim":"Revealing PBD dimerization as an autoinhibition-relief mechanism: crystal structure of Drosophila Polo PBD bound to phospho-Pon showed that two PBD molecules dimerize upon phosphopeptide binding, relieving intramolecular autoinhibition.","evidence":"Crystal structure of PBD/phospho-Pon complex; mutagenesis of dimer interface; in vitro kinase assay","pmids":["27238966"],"confidence":"High","gaps":["Whether human PLK1 PBD dimerizes through the same interface not confirmed structurally","In vivo relevance of PBD dimerization for PLK1 regulation in mammalian cells not tested"]},{"year":2017,"claim":"Linking PLK1 to DNA-damage checkpoint termination: PLK1 phosphorylates Mre11 at S649, priming CK2 phosphorylation at S688 to inhibit MRN complex loading, providing a mechanism by which mitotic entry overrides DNA repair checkpoints.","evidence":"In vitro kinase assay; phosphomimetic/non-phosphorylatable Mre11 mutants; DNA damage focus assay; xenograft sensitivity","pmids":["28512243"],"confidence":"High","gaps":["Whether PLK1-Mre11 regulation operates at all DSB types or only specific lesion classes unknown"]},{"year":2019,"claim":"Identifying lysine methylation as a negative regulatory mechanism: SETD6 methylates PLK1 at K209/K413 and SET7/9 dimethylates K191, each independently reducing PLK1 kinase activity; loss of these marks accelerates mitosis, establishing methylation as a kinase tuning mechanism complementary to phosphorylation and ubiquitylation.","evidence":"In vitro methyltransferase assays; non-methylatable PLK1 mutants with elevated activity; kinetochore-microtubule stability readout","pmids":["30622182","31863092"],"confidence":"High","gaps":["Demethylases that reverse PLK1 methylation not identified","Structural mechanism by which methylation reduces ATP utilization unknown"]},{"year":2019,"claim":"Demonstrating PLK1's role in centromere structural integrity: PLK1 inactivation permits BLM helicase-dependent unwinding of centromeric DNA, which under bipolar tension leads to centromere rupture and chromosome arm splitting.","evidence":"PLK1 inhibitor with BLM depletion epistasis; live cell imaging of centromere decompaction","pmids":["31253795"],"confidence":"High","gaps":["Direct PLK1 substrate at centromeres that restrains BLM not identified","Whether this mechanism operates in non-cancer cells unknown"]},{"year":2020,"claim":"Mapping the principal kinetochore docking receptors: BUB1 (outer) and CENP-U (inner) were identified as the main PLK1 receptors at kinetochores, each bearing paired PLK1-docking and PP2A-docking motifs created by CDK1/PLK1 priming phosphorylation, and promoting PLK1 dimerization at kinetochores.","evidence":"Ectopic localization assay; in vitro reconstitution; kinetochore PLK1 localization studies","pmids":["33248027"],"confidence":"High","gaps":["Functional consequence of PLK1 dimerization at kinetochores versus monomeric activity not resolved"]},{"year":2021,"claim":"Revealing the dimer-to-monomer activation switch: PLK1 dimerizes in early G2 supported by Bora; Aurora-A–mediated T210 phosphorylation triggers dimer dissociation, generating monomers competent for importin binding and nuclear entry, thereby coupling activation to nuclear translocation.","evidence":"Biochemical dimerization assay; importin co-IP; nuclear localization assay with dimer-interface mutants","pmids":["34759346"],"confidence":"High","gaps":["Structural basis of the PLK1 dimer interface in human PLK1 not determined","Whether dimer/monomer ratio is regulated by additional signals unknown"]},{"year":2021,"claim":"Discovering a self-limiting feedback loop at kinetochores: Apolo1 sustains PLK1 activity at kinetochores but, once phosphorylated by PLK1, recruits PP1γ to dephosphorylate and inactivate PLK1, constituting a substrate-mediated negative feedback timer.","evidence":"FRET-based PLK1 activity biosensor; co-IP; siRNA; phosphatase recruitment assay","pmids":["34260926"],"confidence":"High","gaps":["Temporal resolution of Apolo1 phosphorylation relative to kinetochore–microtubule stabilization not established"]},{"year":2021,"claim":"Connecting PLK1 to genome protection during replication stress: PLK1 phosphorylates PrimPol between its RPA-binding motifs to prevent aberrant chromatin loading; reversal during replication stress licenses PrimPol access, and loss of this regulation causes chromosome breaks and micronuclei.","evidence":"In vitro kinase assay; chromatin recruitment assay; chromosome instability readouts","pmids":["34860556"],"confidence":"High","gaps":["Phosphatase responsible for reversing PrimPol phosphorylation during stress not identified"]},{"year":2023,"claim":"Establishing PLK1 as the activating kinase for mitotic DNA repair via Polθ: PLK1 phosphorylates Polθ to activate it during mitosis, enabling TOPBP1-dependent recruitment to mitotic DSBs for end-joining; this pathway is essential for genome stability and is synthetically lethal with HR deficiency.","evidence":"In vitro kinase assay; Polθ–TOPBP1 direct interaction; KO phenotype; mitotic DNA repair assay; synthetic lethality with HR-deficient cells","pmids":["37674080"],"confidence":"High","gaps":["Full spectrum of Polθ phosphorylation sites by PLK1 not mapped","Whether PLK1-Polθ axis operates outside mitosis unknown"]},{"year":2024,"claim":"Revealing PLK1's role in epigenetic centromere maintenance: PLK1 binds self-primed phosphorylations on Mis18α (S54) and Mis18BP1 (T78/S93) via its PBD, triggering a conformational change in the Mis18α–Mis18β complex that promotes HJURP recruitment and new CENP-A deposition in early G1, extending PLK1 function beyond mitosis into chromatin inheritance.","evidence":"Biochemical reconstitution; PBD binding assay; phospho-site mapping; CENP-A deposition assay (two independent groups)","pmids":["39236163","39236175"],"confidence":"High","gaps":["How PLK1 activity persists into G1 after mitotic exit-associated degradation not explained","Whether PLK1-Mis18 regulation is perturbed in cancer aneuploidies unknown"]},{"year":null,"claim":"Full-length PLK1 structure capturing the intramolecular PBD–kinase domain interface and its modulation by methylation, ubiquitylation, and dimerization remains undetermined; a unified quantitative model integrating all post-translational inputs (phosphorylation, methylation, ubiquitylation, dephosphorylation) into a spatiotemporal activity profile is lacking.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length human PLK1 structure","No integrated quantitative model of combinatorial PTM regulation","Demethylases and deubiquitinases acting on PLK1 not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,9,10,13,16,23,25,29,33,37,39,41]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,9,10,13,16,23,25,37,39,41]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[7,14,17,30,31,33,35,36,41,42]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[20,26,28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[34]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,8,15,19,20,27,31,34]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[23,37,39]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,10,16,25]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,12,22,25,36]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[41,42]}],"complexes":[],"partners":["AURKA","BORA","BUB1","CENPU","KLHL22","SETD6","MIS18A","APOLO1"],"other_free_text":[]},"mechanistic_narrative":"PLK1 is a master mitotic serine/threonine kinase that orchestrates centrosome maturation, spindle assembly, kinetochore–microtubule attachment, chromosome integrity, cytokinesis, and centromeric CENP-A deposition through phosphorylation of a broad substrate repertoire. PLK1 activation requires Aurora-A–mediated phosphorylation of T210 in a process primed by CDK1-phosphorylated Bora/SPAT-1, and this activation operates as a bistable switch controlled by a dimerization-to-monomer transition that gates nuclear entry [PMID:25753036, PMID:34759346, PMID:24338364]. At kinetochores, PLK1 is recruited via its polo-box domain (PBD) to CDK1/self-primed phosphodocking sites on BUB1, CENP-U, CLIP-170, and PBIP1, and is removed by non-proteolytic CUL3–KLHL22 ubiquitylation at K492; its kinase output is further tuned by SETD6 methylation (K209/K413), SET7/9 dimethylation (K191), PTEN-dependent dephosphorylation, and an Apolo1–PP1γ feedback loop [PMID:33248027, PMID:23455478, PMID:30622182, PMID:31863092, PMID:34260926]. Beyond mitotic progression, PLK1 phosphorylates p53 and TAp63 to suppress apoptosis, phosphorylates Mre11 to terminate DNA-damage checkpoints, activates Polθ for mitotic DNA repair, restricts PrimPol chromatin loading to prevent replication-associated breaks, promotes CENP-A deposition by activating the Mis18 complex, and coordinates nuclear pore complex disassembly at mitotic entry [PMID:15024021, PMID:19668228, PMID:28512243, PMID:37674080, PMID:34860556, PMID:39236163, PMID:29487689]."},"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). Phosphorylates ATXN10 which may play a role in the regulation of cytokinesis and may stimulate the proteasome-mediated degradation of ATXN10 (PubMed:21857149)","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, spindle; Midbody","url":"https://www.uniprot.org/uniprotkb/P53350/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PLK1","classification":"Common Essential","n_dependent_lines":1207,"n_total_lines":1208,"dependency_fraction":0.9991721854304636},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000166851","cell_line_id":"CID001242","localizations":[{"compartment":"centrosome","grade":3},{"compartment":"cytoplasmic","grade":2},{"compartment":"nuclear_punctae","grade":2},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"RBM25","stoichiometry":4.0},{"gene":"C13ORF34;BORA","stoichiometry":0.2},{"gene":"FAM53C","stoichiometry":0.2},{"gene":"ANGPT1","stoichiometry":0.2},{"gene":"ERCC6L","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001242","total_profiled":1310},"omim":[{"mim_id":"621430","title":"FIGNL1-INTERACTING REGULATOR OF RECOMBINATION AND MITOSIS; FIRRM","url":"https://www.omim.org/entry/621430"},{"mim_id":"621412","title":"CENTROSOMAL PROTEIN 57-LIKE 1; CEP57L1","url":"https://www.omim.org/entry/621412"},{"mim_id":"620791","title":"CENTROSOMAL PROTEIN, 76-KD; 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PLK1.","date":"2021","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/34759346","citation_count":20,"is_preprint":false},{"pmid":"16223707","id":"PMC_16223707","title":"Polo-like kinase (Plk) 1 as a target for prostate cancer management.","date":"2005","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/16223707","citation_count":19,"is_preprint":false},{"pmid":"37640723","id":"PMC_37640723","title":"Plk1 promotes renal tubulointerstitial fibrosis by targeting autophagy/lysosome axis.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/37640723","citation_count":19,"is_preprint":false},{"pmid":"24067371","id":"PMC_24067371","title":"CUL3 and protein kinases: insights from PLK1/KLHL22 interaction.","date":"2013","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/24067371","citation_count":19,"is_preprint":false},{"pmid":"37979172","id":"PMC_37979172","title":"Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/37979172","citation_count":19,"is_preprint":false},{"pmid":"24722282","id":"PMC_24722282","title":"Liver kinase B1 regulates the centrosome via PLK1.","date":"2014","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/24722282","citation_count":19,"is_preprint":false},{"pmid":"31597699","id":"PMC_31597699","title":"PLK1 targets NOTCH1 during DNA damage and mitotic progression.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31597699","citation_count":18,"is_preprint":false},{"pmid":"34860556","id":"PMC_34860556","title":"PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle.","date":"2021","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/34860556","citation_count":18,"is_preprint":false},{"pmid":"29059161","id":"PMC_29059161","title":"Plk1 phosphorylation of Numb leads to impaired DNA damage response.","date":"2017","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/29059161","citation_count":18,"is_preprint":false},{"pmid":"30760542","id":"PMC_30760542","title":"LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway.","date":"2019","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/30760542","citation_count":18,"is_preprint":false},{"pmid":"37888778","id":"PMC_37888778","title":"PLK1 promotes the mitotic surveillance pathway by controlling cytosolic 53BP1 availability.","date":"2023","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/37888778","citation_count":17,"is_preprint":false},{"pmid":"37698938","id":"PMC_37698938","title":"PLK1 inhibition dampens NLRP3 inflammasome-elicited response in inflammatory disease models.","date":"2023","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/37698938","citation_count":17,"is_preprint":false},{"pmid":"32799774","id":"PMC_32799774","title":"SKA3 Promotes Cell Growth in Breast Cancer by Inhibiting PLK-1 Protein Degradation.","date":"2020","source":"Technology in cancer research & treatment","url":"https://pubmed.ncbi.nlm.nih.gov/32799774","citation_count":16,"is_preprint":false},{"pmid":"29282278","id":"PMC_29282278","title":"Gravin regulates centrosome function through PLK1.","date":"2017","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/29282278","citation_count":16,"is_preprint":false},{"pmid":"29749476","id":"PMC_29749476","title":"miR‑23a suppresses pancreatic cancer cell 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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43520,"output_tokens":10334,"usd":0.142785},"stage2":{"model":"claude-opus-4-6","input_tokens":14333,"output_tokens":5325,"usd":0.307185},"total_usd":0.44997,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"PLK1 (Plk) is a functional homolog of S. cerevisiae Cdc5; Glu206 and Thr210 in the kinase domain are critical for catalytic activity, and replacement of Thr210 with a negatively charged residue (T210D) elevates Plk specific activity. Ectopic expression of Plk complements the cdc5-1 cell division defect in yeast in a kinase-activity-dependent manner.\",\n      \"method\": \"In vitro kinase assay with immunoprecipitates from Sf9 cells; site-directed mutagenesis (K82M, D194N, D194R, T210D); yeast complementation of cdc5-1 mutation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis plus genetic epistasis (yeast complementation)\",\n      \"pmids\": [\"9154840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PLK1 phosphorylates the microtubule-stabilizing protein TCTP on two serine residues in vitro and in vivo; this phosphorylation decreases TCTP's microtubule-stabilizing activity, and overexpression of a phosphorylation-deficient TCTP mutant causes multinucleate cells and cell death similar to anti-Plk antibody microinjection phenotypes.\",\n      \"method\": \"Two-hybrid screen to identify Plk-interacting proteins; in vitro kinase assay; co-fractionation; in vivo phosphorylation analysis; overexpression of phosphomutant TCTP\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis and in vivo validation using phospho-deficient mutant\",\n      \"pmids\": [\"12167714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Plk1 depletion in cancer cells causes mitotic arrest with 4N DNA content, formation of dumbbell-like chromatin structures (failure of sister chromatid separation), and apoptosis involving caspase-3 activation and p53 stabilization; DNA damage also occurs in p53-null cells. ATM inhibition potentiates lethality of Plk1 depletion.\",\n      \"method\": \"Vector-based siRNA depletion; FACS; caspase-3 activation assay; rescue with non-degradable Plk1 constructs; ATM inhibitor treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO/KD with defined cellular phenotype replicated with rescue experiment; multiple orthogonal readouts\",\n      \"pmids\": [\"12732729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PLK1 physically binds to the DNA-binding region of p53 (identified by systematic immunoprecipitation with deletion mutants) and phosphorylates p53 in a kinase-dependent manner, thereby inhibiting p53 transcriptional activity and its pro-apoptotic function; a kinase-dead Plk1 mutant fails to suppress p53 activity.\",\n      \"method\": \"Co-immunoprecipitation with deletion mutants; confocal co-localization; luciferase reporter assay; kinase-dead mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — Co-IP with mapping, kinase-dead mutant, and functional transcription assay\",\n      \"pmids\": [\"15024021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLK1's polo-box domain (PBD) interacts with MCM2 and MCM7 (and the whole MCM2-7 complex) in a chromatin-associated fraction, suggesting a role for PLK1 in coordinating DNA replication and mitotic events.\",\n      \"method\": \"PBD-based protein interaction screen; co-immunoprecipitation of endogenous proteins; immunoblot; chromatin fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP with biochemical fractionation, single lab, no functional rescue\",\n      \"pmids\": [\"15654075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the PLK1 kinase domain in complex with the inhibitor BI 2536 reveals that Leu132 in the hinge region creates a small selectivity pocket exploited by BI 2536's methoxy group, providing structural basis for PLK1 selectivity over other kinases.\",\n      \"method\": \"X-ray crystallography (co-crystal structure of PLK1 kinase domain with BI 2536); selectivity profiling\",\n      \"journal\": \"Chemical biology & drug design\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with selectivity data\",\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 solved at 2.3 Å, revealing the active conformation of PLK1 and enabling structure-based drug design.\",\n      \"method\": \"X-ray crystallography using DARPin as crystallization chaperone\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure at 2.3 Å resolution\",\n      \"pmids\": [\"18391401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PLK1 is recruited to centromeres through a self-priming mechanism: PLK1 phosphorylates the centromeric component PBIP1, generating its own PBD-binding site (phospho-epitope) for initial kinetochore recruitment; PLK1 subsequently induces PBIP1 delocalization and degradation to allow binding to other kinetochore components.\",\n      \"method\": \"Cell-based localization studies; biochemical analysis of PLK1-PBIP1 interaction; site-directed mutagenesis\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic model supported by localization and phosphorylation studies; single lab review with primary data\",\n      \"pmids\": [\"18215321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In C. elegans embryos, PLK-1 is asymmetrically distributed between AB and P1 blastomeres under control of anterior-posterior PAR polarity cues, and this asymmetry promotes mitotic entry preferentially in AB; mild plk-1 RNAi delays mitosis specifically in P1 but not AB, independently of ATL-1/CHK-1.\",\n      \"method\": \"RNAi depletion in C. elegans; live imaging; genetic analysis with PAR mutants\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with polarity pathway combined with live imaging and quantitative cell biology\",\n      \"pmids\": [\"18305005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PLK1 interacts with the microtubule depolymerase Kif2a during mitosis in a kinase-activity-dependent manner and phosphorylates Kif2a to enhance its depolymerase activity in vitro; PLK1 inhibition decreases microtubule-associated Kif2a signals and increases spindle microtubule intensity in vivo. Aurora A antagonizes this by phosphorylating Kif2a to suppress its depolymerase activity.\",\n      \"method\": \"Proteomic identification; co-immunoprecipitation; in vitro kinase/depolymerase assay; PLK1 inhibition/depletion in vivo\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay combined with in vivo functional readout; antagonistic regulation by Aurora A provides mechanistic context\",\n      \"pmids\": [\"19351716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PLK1 phosphorylates TAp63 at Ser-52 of the TA domain (demonstrated by in vitro kinase assay), decreasing TAp63 protein stability and suppressing TAp63-induced apoptotic cell death in liver tumor cells; PLK1 binds p63 through its kinase domain via p63's DNA-binding region.\",\n      \"method\": \"Immunoprecipitation; in vitro pull-down; in vitro kinase assay; siRNA knockdown; double knockdown epistasis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with substrate mapping plus epistasis (double knockdown)\",\n      \"pmids\": [\"19668228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PICH (a PLK1-binding protein) and PLK1 kinase coordinately maintain chromosome arm architecture during prometaphase; PICH knockdown causes loss of PLK1 from chromosome arms, resulting in 'wavy' chromosome disorganization that can be prevented by topoisomerase II inhibition, suggesting the PICH-PLK1 complex normally acts via topoisomerase II activity.\",\n      \"method\": \"siRNA knockdown; live and fixed-cell imaging; chromosome architecture analysis; topoisomerase II inhibitor treatment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined phenotype and chemical epistasis with topo II inhibitor\",\n      \"pmids\": [\"20130082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"p53 represses PLK1 transcription directly: chromatin immunoprecipitation shows p53 binds two sites (p53RE1 and p53RE2) on the PLK1 promoter; DNA damage stimulates p53 recruitment to p53RE2 coincident with repression-associated chromatin changes including HDAC recruitment; this repression is independent of p21 and the CDE/CHR element.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP); reporter gene assay; siRNA; HDAC inhibitor treatment\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP with multiple mutant promoter constructs and siRNA epistasis; multiple orthogonal methods\",\n      \"pmids\": [\"20962589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PLK1 directly phosphorylates the kinesin-13 Kif2b at Thr125 and Ser204; phosphorylation of S204 is required for kinetochore localization of Kif2b in prometaphase, while T125 phosphorylation is required for Kif2b activity in correcting k-MT attachment errors.\",\n      \"method\": \"Mass spectrometry phospho-mapping; in vitro kinase assay; phosphomutant overexpression; kinetochore localization assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with site-specific phosphomutants and functional readout\",\n      \"pmids\": [\"22535524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CUL3-KLHL22 E3 ubiquitin ligase ubiquitylates PLK1 at Lys492 within the PBD, causing PLK1 dissociation from kinetochore phosphoreceptors without proteolytic degradation; loss of KLHL22 causes PLK1 accumulation at kinetochores and sustained SAC activation; expression of non-ubiquitylatable PLK1-K492R phenocopies KLHL22 inactivation.\",\n      \"method\": \"Co-immunoprecipitation; mass spectrometry; siRNA; non-ubiquitylatable mutant expression; kinetochore localization; SAC analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ubiquitylation site mapped to K492, phenocopied by K492R mutant, multiple orthogonal methods\",\n      \"pmids\": [\"23455478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Bora-Aurora-A remains the major activating complex for PLK1 in mitosis (not only G2); a small amount of Aurora-A activity with residual Bora is sufficient to sustain PLK1 T210 phosphorylation in mitosis, creating a bistable switch where PLK1 is sensitive to Aurora-A inhibition during initial activation but refractory once fully activated.\",\n      \"method\": \"Kinase inhibitor treatment; immunoblotting of T210 phosphorylation; RNAi depletion of Bora\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative phosphorylation analysis with inhibitors and knockdowns; 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 phospho-epitope) followed by PLK1 at S305 suppresses extrinsic apoptosis during mitosis; non-phosphorylatable caspase-8 S305A increases Fas-stimulated cell death, and BI 2536 (PLK1 inhibitor) lowers the threshold for Fas-induced apoptosis.\",\n      \"method\": \"In vitro kinase assay; PBD-binding assay; RNAi replacement with phosphomutants; Fas-stimulation assay; BI 2536 treatment\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — sequential kinase assay with phosphomutant rescue; mechanistic PBD-docking model validated\",\n      \"pmids\": [\"24484936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLIP-170, phosphorylated by CDK1 at T287, acts as a PLK1 recruiter at kinetochores during early mitosis; depletion of CLIP-170 or expression of non-phosphorylatable CLIP-170-T287A reduces PLK1 kinetochore localization, destabilizes kinetochore fibers, and impairs chromosome alignment.\",\n      \"method\": \"Co-localization; siRNA depletion; phosphomutant expression; kinetochore fiber stability assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KD and phosphomutant rescue with defined localization and functional phenotype\",\n      \"pmids\": [\"24777477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PLK1 regulates NEK2 activity, which in turn phosphorylates and stabilizes β-catenin at mitotic centrosomes; Plk1 inhibition or depletion disrupts this pathway. In vitro and cell-based assays show Nek2 phosphorylates β-catenin at N-terminal regulatory sites, blocking β-TrCP binding and preventing ubiquitination and degradation.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; ubiquitination assay; PLK1 inhibitor treatment; siRNA\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay with mechanistic pathway (Plk1→Nek2→β-catenin) validated by multiple methods\",\n      \"pmids\": [\"24501426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CDK-1 phosphorylates Bora/SPAT-1 at conserved Sp/Tp residues, promoting Bora interaction with PLK1 and stimulating Aurora A-dependent T-loop phosphorylation of PLK1 to trigger mitotic entry; phospho-SPAT-1 activates PLK1 by enabling Aurora A phosphorylation in vitro; this mechanism is conserved between C. elegans and humans.\",\n      \"method\": \"In vitro kinase assay (CDK1 phosphorylation of Bora; Aurora A phosphorylation of PLK1); non-phosphorylatable SPAT-1 mutant analysis in C. elegans embryos; human Bora/PLK1 reconstitution\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution across two organisms; phosphomutant analysis; multiple orthogonal methods\",\n      \"pmids\": [\"25753036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PLK1 is required for efficient meiotic resumption (nuclear envelope breakdown), recruitment of centrosomal proteins to acentriolar MTOCs, spindle formation, stable kinetochore-microtubule attachment, APC/C activation (by promoting EMI1 degradation), and chromosome condensation maintenance during meiosis I-II transition in mouse oocytes.\",\n      \"method\": \"Specific small-molecule PLK1 inhibition combined with live mouse oocyte imaging; time-lapse microscopy; APC/C activity assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — specific inhibitor combined with live imaging; multiple defined phenotypic readouts\",\n      \"pmids\": [\"25658810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Drosophila Polo/PLK1's polo-box domain (PBD) binds phospho-Pon (phosphorylated by Cdk1 at Thr63), and crystal structure of Plk1 PBD/phospho-Pon complex reveals that two phospho-Pon-bound PBDs dimerize; this PBD dimerization relieves Plk1 autoinhibition and promotes sequential Plk1 phosphorylation of Pon.\",\n      \"method\": \"Crystal structure of PBD/phospho-Pon complex; in vitro binding assay; mutagenesis; in vitro kinase assay\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and in vitro kinase assay; structural mechanism for autoinhibition relief\",\n      \"pmids\": [\"27238966\"],\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; a phospho-mimetic PLK1 mutant causes polyploidy, and expression of non-phosphorylatable PLK1 or PLK1 inhibition reduces polyploid cell populations.\",\n      \"method\": \"Co-immunoprecipitation; phosphatase assay; phosphomimetic and non-phosphorylatable PLK1 mutant expression; ploidy analysis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with phosphatase activity and mutant analysis; single lab\",\n      \"pmids\": [\"27398835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLK1 phosphorylates Mre11 at S649, priming subsequent CK2-mediated phosphorylation at S688; dual phosphorylation inhibits MRN complex loading onto damaged DNA, leading to premature DNA damage checkpoint termination and impaired DNA repair.\",\n      \"method\": \"In vitro kinase assay; phosphomimetic/non-phosphorylatable Mre11 mutants; DNA damage focus assay; checkpoint termination assay; xenograft tumor sensitivity assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with site-specific mutants and multiple functional readouts\",\n      \"pmids\": [\"28512243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLK1 coordinates nuclear pore complex (NPC) disassembly at mitotic entry (but not lamin A/C disassembly) and negatively regulates NPC reassembly on lagging chromatin; this reveals independent regulatory pathways for lamin A/C (CDK1-dependent) and NPCs (PLK1-dependent) during mitosis.\",\n      \"method\": \"PLK1 inhibition; live cell imaging; immunofluorescence; functional analysis of NPC/lamin assembly on lagging chromatin\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — PLK1 inhibitor with defined localization phenotype distinguishing NPC from lamin pathway\",\n      \"pmids\": [\"29487689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plk1 phosphorylates Numb, leading to enhanced proteasomal degradation of Numb and impairment of the Numb/p53 pathway, thereby antagonizing p53 during DNA damage response; cancer cells expressing non-phosphorylatable Numb are more sensitive to doxorubicin.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; proteasomal degradation assay; phosphomutant expression; xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay with functional rescue and in vivo xenograft validation\",\n      \"pmids\": [\"29059161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HMMR acts at centrosomes in a PLK1-dependent pathway that localizes active Ran and modulates cortical NuMA-dynein complexes for spindle orientation; HMMR loss causes spindle misorientation that phenocopies disruption of this PLK1-dependent pathway.\",\n      \"method\": \"Hmmr-knockout mouse model; live cell imaging; Ran-GTP localization; spindle orientation assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined spindle orientation phenotype; pathway placement via genetic analysis\",\n      \"pmids\": [\"28994651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DNA replication restricts CDK1 and PLK1 activation; preventing DNA replication licensing or firing causes premature activation of CDK1 and PLK1 in S phase; in the presence of DNA replication, CHK1 and p38 inhibition also leads to premature mitotic kinase activation causing replication stress.\",\n      \"method\": \"Double-degron system for protein depletion; kinase inhibitors; cell cycle synchronization; kinase activity assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — engineered degron system with multiple kinase inhibitors; mechanistic epistasis showing replication as upstream brake\",\n      \"pmids\": [\"30008317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PLK1 overexpression causes cytokinesis defects 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; live cell imaging; Cep55/ESCRT localization assay; kinase-dead mutant\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo knock-in model with kinase-dependent mechanistic analysis of Cep55/ESCRT loading\",\n      \"pmids\": [\"30069007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SETD6 binds and methylates PLK1 at K209 and K413 during mitosis; lack of PLK1 methylation results in increased PLK1 kinase activity, accelerated mitosis, and faster cellular proliferation, revealing SETD6-mediated methylation as a negative regulator of PLK1 kinase activity.\",\n      \"method\": \"In vitro methyltransferase assay; SETD6-deficient cells; non-methylatable PLK1 mutants; kinase activity assay; time-lapse microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro methylation assay with non-methylatable mutants and functional kinase activity readout\",\n      \"pmids\": [\"30622182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLK1 inhibition reveals a centromere disintegration mechanism: PLK1 inactivation allows BLM helicase to unwind DNA at a specific centromere domain, and under bipolar spindle pulling, the distorted centromeres are decompacted into threadlike DNA molecules leading to centromere rupture and chromosome arm splitting.\",\n      \"method\": \"PLK1 small-molecule inhibition; live cell imaging; BLM depletion epistasis; centromere structure analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — PLK1 inhibition with genetic epistasis (BLM depletion); novel mechanism supported by live imaging and structural analysis\",\n      \"pmids\": [\"31253795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BUB1 (outer kinetochore) and CENP-U (inner kinetochore) are the main PLK1 receptors at kinetochores; both share a constellation of sequence motifs (PP2A-docking motif and two PLK1-docking sites); PLK1 recruitment requires priming phosphorylation by CDK1 and PLK1 itself, and promotes PLK1 dimerization.\",\n      \"method\": \"Ectopic localization assay; in vitro reconstitution; kinetochore localization studies; biochemical binding assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution combined with ectopic localization and kinetochore assays; multiple orthogonal methods\",\n      \"pmids\": [\"33248027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BRCA2 is phosphorylated by PLK1 at T207 (a bona fide PLK1 docking site); BRCA2 bound to PLK1 forms a complex with PP2A and phospho-BUBR1; disruption of BRCA2-PLK1 binding (BRCA2 variants S206C and T207A) destabilizes this complex, causing unstable kinetochore-microtubule interactions, chromosome misalignment, and aneuploidy.\",\n      \"method\": \"Phosphorylation site mapping; co-immunoprecipitation; BRCA2 variant analysis; kinetochore-microtubule stability assay; chromosome alignment analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phospho-site identification with disease variants and reconstitution of tetrameric complex; multiple functional readouts\",\n      \"pmids\": [\"32286328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SET7/9 dimethylates PLK1 at Lys191 at kinetochores during early mitosis, tuning down PLK1 kinase activity by limiting ATP utilization; overexpression of non-methylatable PLK1 or SET7/9 inhibition causes mitotic arrest due to destabilized kinetochore-microtubule attachments.\",\n      \"method\": \"In vitro methyltransferase assay; non-methylatable PLK1 mutant expression; PLK1 kinase activity assay; kinetochore-microtubule stability analysis; SET7/9 inhibitor treatment\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro methylation assay with non-methylatable mutant and activity readout\",\n      \"pmids\": [\"31863092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 dimerization during early G2 phase (supported by Bora) fine-tunes PLK1 activation timing and nuclear entry; T210 phosphorylation by Aurora-A triggers dimer dissociation generating active PLK1 monomers; interfering with this dimer/monomer switch prevents importin association and nuclear PLK1 localization during G2-M transition.\",\n      \"method\": \"Biochemical dimerization assay; T210 phosphorylation analysis; importin co-immunoprecipitation; nuclear localization assay; dimer interface mutants\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical demonstration of dimer/monomer switch linked to T210 phosphorylation and nuclear entry\",\n      \"pmids\": [\"34759346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Apolo1 localizes to kinetochores during early mitosis, sustains PLK1 kinase activity at kinetochores for accurate kinetochore-microtubule attachment, and is itself a PLK1 substrate; PLK1-phosphorylated Apolo1 recruits PP1γ to dephosphorylate and inactivate PLK1, constituting a feedback loop that fine-tunes PLK1 activity.\",\n      \"method\": \"FRET-based PLK1 activity biosensor; co-immunoprecipitation; siRNA; kinase assay; phosphatase recruitment assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — FRET biosensor combined with biochemical kinase/phosphatase assays and genetic depletion\",\n      \"pmids\": [\"34260926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 activity is essential for time-dependent release of 53BP1 from kinetochores (through CENP-F as docking partner); PLK1 inhibition causes 53BP1 persistence at kinetochores, preventing cytosolic 53BP1 association with p53 and blunting the mitotic surveillance pathway (mitotic stopwatch).\",\n      \"method\": \"PLK1 inhibitor treatment; 53BP1 localization imaging; CENP-F-53BP1 binding disruption; mitotic surveillance pathway activation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — PLK1 inhibition with mechanistic dissection of CENP-F-53BP1 and cytosolic p53 interaction; multiple readouts\",\n      \"pmids\": [\"37888778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 regulates PrimPol by phosphorylating it at a conserved residue between its RPA binding motifs, preventing aberrant PrimPol chromatin recruitment; this phosphorylation is cell cycle regulated and reversed during replication stress; loss of PLK1-dependent PrimPol regulation causes chromosome breaks and micronuclei.\",\n      \"method\": \"In vitro kinase assay; chromatin recruitment assay; cell cycle phosphorylation analysis; chromosome instability readouts (breaks, micronuclei); drug sensitivity assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro phosphorylation with chromatin recruitment and functional consequence readouts\",\n      \"pmids\": [\"34860556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLK1 promotes NLRP3 inflammasome activation at cell interphase; a proximity-dependent BioID screen identifies enhanced PLK1-NLRP3 proximity upon inflammasome activation; PLK1 orchestrates MTOC structure and NLRP3 subcellular positioning upon inflammasome activation; PLK1 inhibition suppresses IL-1β production in vivo.\",\n      \"method\": \"BioID proximity screen; co-immunoprecipitation (interaction domain mapping); PLK1 inhibitor in vivo; NLRP3 subcellular localization; MTOC organization assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BioID screen with Co-IP validation and in vivo pharmacological confirmation; single lab\",\n      \"pmids\": [\"37698938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PLK1 phosphorylates DNA polymerase theta (Polθ) to activate it specifically during mitosis; phosphorylated Polθ is recruited via direct interaction with BRCA1 C-terminal domains of TOPBP1 to mitotic DNA double-strand breaks, where it mediates DNA end joining; loss of Polθ leads to defective mitotic DSB repair and genome instability.\",\n      \"method\": \"In vitro kinase assay; direct protein interaction assay (Polθ-TOPBP1); KO phenotype analysis; DNA repair assay in mitosis; synthetic lethality assay with HR-deficient cells\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro phosphorylation with direct interaction mapping and KO functional rescue; published in Nature\",\n      \"pmids\": [\"37674080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PLK1 maintains intralysosomal pH by regulating ATP6V1A phosphorylation, and PLK1 inhibition impairs lysosomal function leading to blockade of autophagic flux and reduced fibroblast activation in renal fibrosis.\",\n      \"method\": \"PLK1 pharmacological inhibition/genetic KO; lysosomal pH measurement; autophagic flux assay; ATP6V1A phosphorylation analysis; UUO mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO and inhibitor with biochemical phosphorylation analysis; in vivo model validation\",\n      \"pmids\": [\"37640723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLK1 promotes CENP-A deposition in early G1 by interacting with the Mis18 complex through recognition of self-primed phosphorylations on Mis18α (Ser54) and Mis18BP1 (Thr78 and Ser93) via its polo-box domain; PLK1 binding activates a conformational switch in Mis18α-Mis18β and promotes Mis18 complex-HJURP interaction required for CENP-A loading.\",\n      \"method\": \"Biochemical reconstitution; PLK1 PBD binding assay; phosphorylation site mapping; structural analysis; functional CENP-A deposition assay\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical reconstitution with phospho-site mapping and conformational/functional analysis; published in Science\",\n      \"pmids\": [\"39236163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLK1 interacts with Mis18α and Mis18BP1 through their self-primed phosphorylations (Mis18α Ser54, Mis18BP1 Thr78 and Ser93) recognized by PLK1's polo-box domain; disrupting these phosphorylations perturbs HJURP centromere recruitment and new CENP-A loading; PLK1 binding activates Mis18α-Mis18β complex.\",\n      \"method\": \"Biochemical and functional analysis of Mis18 phosphorylation; PLK1 PBD-binding assay; HJURP recruitment assay; CENP-A loading assay\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — phospho-site mapping with reconstitution and functional CENP-A deposition readout; independent replication of PMID 39236163 findings\",\n      \"pmids\": [\"39236175\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLK1 is a master mitotic Ser/Thr kinase activated by Aurora-A-mediated T210 phosphorylation (promoted by Bora/SPAT-1 primed by CDK1) that dynamically localizes to centrosomes, kinetochores (via BUB1, CENP-U, CLIP-170, and PBIP1 phospho-docking), and the midbody through its polo-box domain (PBD), where it phosphorylates a broad array of substrates — including TCTP, Kif2a, Kif2b, caspase-8, Mre11, Numb, PrimPol, Polθ, and TAp63 — to regulate mitotic entry, spindle assembly, kinetochore-microtubule attachment, chromosome integrity, cytokinesis (via Cep55/ESCRT loading), and CENP-A deposition; its activity is counterbalanced by non-proteolytic CUL3-KLHL22 ubiquitylation at K492 that removes PLK1 from kinetochores, by SETD6-mediated methylation at K209/K413 and SET7/9-mediated dimethylation at K191 that reduce its kinase activity, by a dimerization mechanism that controls its nuclear entry, and by feedback involving Apolo1-recruited PP1γ, while p53 transcriptionally represses PLK1 and PLK1 reciprocally inhibits p53 through direct phosphorylation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PLK1 is a master mitotic serine/threonine kinase that orchestrates centrosome maturation, spindle assembly, kinetochore–microtubule attachment, chromosome integrity, cytokinesis, and centromeric CENP-A deposition through phosphorylation of a broad substrate repertoire. PLK1 activation requires Aurora-A–mediated phosphorylation of T210 in a process primed by CDK1-phosphorylated Bora/SPAT-1, and this activation operates as a bistable switch controlled by a dimerization-to-monomer transition that gates nuclear entry [PMID:25753036, PMID:34759346, PMID:24338364]. At kinetochores, PLK1 is recruited via its polo-box domain (PBD) to CDK1/self-primed phosphodocking sites on BUB1, CENP-U, CLIP-170, and PBIP1, and is removed by non-proteolytic CUL3–KLHL22 ubiquitylation at K492; its kinase output is further tuned by SETD6 methylation (K209/K413), SET7/9 dimethylation (K191), PTEN-dependent dephosphorylation, and an Apolo1–PP1γ feedback loop [PMID:33248027, PMID:23455478, PMID:30622182, PMID:31863092, PMID:34260926]. Beyond mitotic progression, PLK1 phosphorylates p53 and TAp63 to suppress apoptosis, phosphorylates Mre11 to terminate DNA-damage checkpoints, activates Polθ for mitotic DNA repair, restricts PrimPol chromatin loading to prevent replication-associated breaks, promotes CENP-A deposition by activating the Mis18 complex, and coordinates nuclear pore complex disassembly at mitotic entry [PMID:15024021, PMID:19668228, PMID:28512243, PMID:37674080, PMID:34860556, PMID:39236163, PMID:29487689].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing PLK1 as a conserved cell-division kinase: mutagenesis of T210 and the catalytic domain demonstrated that PLK1 kinase activity is essential for viability and that T210 is a key activating residue, while yeast complementation proved functional conservation with Cdc5.\",\n      \"evidence\": \"Site-directed mutagenesis (K82M, D194N, T210D) with in vitro kinase assay and S. cerevisiae cdc5-1 complementation\",\n      \"pmids\": [\"9154840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream activating kinase for T210 not yet identified\", \"No structural information on the kinase domain available\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying the first mitotic substrate: PLK1 phosphorylation of TCTP attenuates its microtubule-stabilizing activity, providing direct evidence that PLK1 controls spindle microtubule dynamics through substrate phosphorylation.\",\n      \"evidence\": \"Two-hybrid screen; in vitro kinase assay; phospho-deficient TCTP mutant causes multinucleation phenocopying PLK1 loss\",\n      \"pmids\": [\"12167714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific TCTP phosphorylation sites not fully mapped\", \"Relative contribution of TCTP versus other substrates to spindle assembly unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating that PLK1 is essential for chromosome segregation and cell survival: PLK1 depletion causes mitotic arrest, failure of sister chromatid separation, DNA damage, and caspase-3-dependent apoptosis, establishing PLK1 as indispensable for mitotic fidelity.\",\n      \"evidence\": \"Vector-based siRNA with FACS, caspase-3 activation, and rescue by non-degradable PLK1\",\n      \"pmids\": [\"12732729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates mediating sister chromatid separation not identified\", \"Mechanism linking PLK1 loss to DNA damage not resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Revealing a reciprocal PLK1–p53 regulatory axis: PLK1 directly binds and phosphorylates p53 to inhibit its transcriptional and pro-apoptotic functions, while p53 reciprocally represses PLK1 transcription via direct promoter binding, establishing a feedback circuit that integrates mitotic signaling with tumor suppression.\",\n      \"evidence\": \"Co-IP with deletion mapping, kinase-dead mutant, luciferase reporter (2004); ChIP on PLK1 promoter with HDAC recruitment (2010)\",\n      \"pmids\": [\"15024021\", \"20962589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific p53 phosphorylation sites by PLK1 not mapped\", \"In vivo physiological significance of this feedback in normal tissues not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Solving the structural basis for PLK1 kinase selectivity: crystal structures of the kinase domain with BI 2536 and with a DARPin chaperone revealed the active conformation and a Leu132-dependent selectivity pocket, enabling rational drug design.\",\n      \"evidence\": \"X-ray crystallography of PLK1 kinase domain with BI 2536 (2007) and DARPin 3H10 at 2.3 Å (2008)\",\n      \"pmids\": [\"18005335\", \"18391401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length PLK1 structure including PBD intramolecular contacts not determined\", \"Structural basis of substrate recognition beyond PBD not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defining the self-priming mechanism for kinetochore recruitment: PLK1 phosphorylates PBIP1 to create its own PBD-docking site, then induces PBIP1 degradation to transition to other kinetochore receptors, establishing the principle that PLK1 generates and then consumes its own docking platforms.\",\n      \"evidence\": \"Cell-based localization with site-directed mutagenesis of PBIP1 phospho-epitope\",\n      \"pmids\": [\"18215321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Temporal order of receptor switching at kinetochores not fully resolved\", \"Quantitative contribution of PBIP1 versus other docking sites unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Expanding PLK1's substrate repertoire to microtubule depolymerases: PLK1 phosphorylates Kif2a to enhance its depolymerase activity while Aurora-A antagonizes this, revealing a kinase-counterkinase mechanism controlling spindle microtubule length.\",\n      \"evidence\": \"Co-IP and in vitro kinase/depolymerase assay; PLK1 inhibition increases spindle microtubule intensity in vivo\",\n      \"pmids\": [\"19351716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial coordination of PLK1 and Aurora-A on Kif2a at spindle poles not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating phospho-site-specific control of a kinesin: PLK1 phosphorylation of Kif2b at S204 governs kinetochore targeting while T125 phosphorylation activates error-correction function, showing that distinct phospho-events on a single substrate can control localization versus activity.\",\n      \"evidence\": \"Mass spectrometry phospho-mapping; in vitro kinase assay; phosphomutant localization and functional rescue\",\n      \"pmids\": [\"22535524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLK1-Kif2b axis acts independently of or redundantly with Aurora-B error correction not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovering non-proteolytic ubiquitylation as a kinetochore removal mechanism: CUL3–KLHL22 ubiquitylates PLK1 at K492 within the PBD to dissociate it from kinetochores without degradation, revealing ubiquitin as a spatial regulator of PLK1 rather than a destruction signal.\",\n      \"evidence\": \"Mass spectrometry site identification; K492R mutant phenocopies KLHL22 depletion (sustained SAC activation)\",\n      \"pmids\": [\"23455478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of deubiquitinase that reverses K492 modification unknown\", \"Structural basis for how K492 ubiquitylation disrupts PBD binding not determined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing a Cdk1→PLK1 sequential phosphorylation relay on procaspase-8 that suppresses extrinsic apoptosis during mitosis, integrating cell-cycle kinase signaling with death receptor signaling.\",\n      \"evidence\": \"In vitro kinase assay; PBD-docking site generated by Cdk1; S305A mutant sensitizes cells to Fas-induced apoptosis\",\n      \"pmids\": [\"24484936\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism operates in non-cancer cell contexts not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolving the activation cascade: CDK1 phosphorylates Bora/SPAT-1, which then stimulates Aurora-A–dependent T210 phosphorylation of PLK1, establishing the CDK1→Bora→Aurora-A→PLK1 mitotic entry relay as a conserved mechanism from C. elegans to humans.\",\n      \"evidence\": \"In vitro reconstitution of CDK1-Bora-Aurora A-PLK1 phosphorylation cascade; non-phosphorylatable SPAT-1 mutants in C. elegans embryos\",\n      \"pmids\": [\"25753036\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative parameters of the bistable switch in vivo not measured\", \"Spatial regulation of Bora–Aurora-A interaction at centrosomes not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealing PBD dimerization as an autoinhibition-relief mechanism: crystal structure of Drosophila Polo PBD bound to phospho-Pon showed that two PBD molecules dimerize upon phosphopeptide binding, relieving intramolecular autoinhibition.\",\n      \"evidence\": \"Crystal structure of PBD/phospho-Pon complex; mutagenesis of dimer interface; in vitro kinase assay\",\n      \"pmids\": [\"27238966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human PLK1 PBD dimerizes through the same interface not confirmed structurally\", \"In vivo relevance of PBD dimerization for PLK1 regulation in mammalian cells not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linking PLK1 to DNA-damage checkpoint termination: PLK1 phosphorylates Mre11 at S649, priming CK2 phosphorylation at S688 to inhibit MRN complex loading, providing a mechanism by which mitotic entry overrides DNA repair checkpoints.\",\n      \"evidence\": \"In vitro kinase assay; phosphomimetic/non-phosphorylatable Mre11 mutants; DNA damage focus assay; xenograft sensitivity\",\n      \"pmids\": [\"28512243\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLK1-Mre11 regulation operates at all DSB types or only specific lesion classes unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying lysine methylation as a negative regulatory mechanism: SETD6 methylates PLK1 at K209/K413 and SET7/9 dimethylates K191, each independently reducing PLK1 kinase activity; loss of these marks accelerates mitosis, establishing methylation as a kinase tuning mechanism complementary to phosphorylation and ubiquitylation.\",\n      \"evidence\": \"In vitro methyltransferase assays; non-methylatable PLK1 mutants with elevated activity; kinetochore-microtubule stability readout\",\n      \"pmids\": [\"30622182\", \"31863092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Demethylases that reverse PLK1 methylation not identified\", \"Structural mechanism by which methylation reduces ATP utilization unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating PLK1's role in centromere structural integrity: PLK1 inactivation permits BLM helicase-dependent unwinding of centromeric DNA, which under bipolar tension leads to centromere rupture and chromosome arm splitting.\",\n      \"evidence\": \"PLK1 inhibitor with BLM depletion epistasis; live cell imaging of centromere decompaction\",\n      \"pmids\": [\"31253795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PLK1 substrate at centromeres that restrains BLM not identified\", \"Whether this mechanism operates in non-cancer cells unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapping the principal kinetochore docking receptors: BUB1 (outer) and CENP-U (inner) were identified as the main PLK1 receptors at kinetochores, each bearing paired PLK1-docking and PP2A-docking motifs created by CDK1/PLK1 priming phosphorylation, and promoting PLK1 dimerization at kinetochores.\",\n      \"evidence\": \"Ectopic localization assay; in vitro reconstitution; kinetochore PLK1 localization studies\",\n      \"pmids\": [\"33248027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of PLK1 dimerization at kinetochores versus monomeric activity not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealing the dimer-to-monomer activation switch: PLK1 dimerizes in early G2 supported by Bora; Aurora-A–mediated T210 phosphorylation triggers dimer dissociation, generating monomers competent for importin binding and nuclear entry, thereby coupling activation to nuclear translocation.\",\n      \"evidence\": \"Biochemical dimerization assay; importin co-IP; nuclear localization assay with dimer-interface mutants\",\n      \"pmids\": [\"34759346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the PLK1 dimer interface in human PLK1 not determined\", \"Whether dimer/monomer ratio is regulated by additional signals unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovering a self-limiting feedback loop at kinetochores: Apolo1 sustains PLK1 activity at kinetochores but, once phosphorylated by PLK1, recruits PP1γ to dephosphorylate and inactivate PLK1, constituting a substrate-mediated negative feedback timer.\",\n      \"evidence\": \"FRET-based PLK1 activity biosensor; co-IP; siRNA; phosphatase recruitment assay\",\n      \"pmids\": [\"34260926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Temporal resolution of Apolo1 phosphorylation relative to kinetochore–microtubule stabilization not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connecting PLK1 to genome protection during replication stress: PLK1 phosphorylates PrimPol between its RPA-binding motifs to prevent aberrant chromatin loading; reversal during replication stress licenses PrimPol access, and loss of this regulation causes chromosome breaks and micronuclei.\",\n      \"evidence\": \"In vitro kinase assay; chromatin recruitment assay; chromosome instability readouts\",\n      \"pmids\": [\"34860556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase responsible for reversing PrimPol phosphorylation during stress not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Establishing PLK1 as the activating kinase for mitotic DNA repair via Polθ: PLK1 phosphorylates Polθ to activate it during mitosis, enabling TOPBP1-dependent recruitment to mitotic DSBs for end-joining; this pathway is essential for genome stability and is synthetically lethal with HR deficiency.\",\n      \"evidence\": \"In vitro kinase assay; Polθ–TOPBP1 direct interaction; KO phenotype; mitotic DNA repair assay; synthetic lethality with HR-deficient cells\",\n      \"pmids\": [\"37674080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of Polθ phosphorylation sites by PLK1 not mapped\", \"Whether PLK1-Polθ axis operates outside mitosis unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealing PLK1's role in epigenetic centromere maintenance: PLK1 binds self-primed phosphorylations on Mis18α (S54) and Mis18BP1 (T78/S93) via its PBD, triggering a conformational change in the Mis18α–Mis18β complex that promotes HJURP recruitment and new CENP-A deposition in early G1, extending PLK1 function beyond mitosis into chromatin inheritance.\",\n      \"evidence\": \"Biochemical reconstitution; PBD binding assay; phospho-site mapping; CENP-A deposition assay (two independent groups)\",\n      \"pmids\": [\"39236163\", \"39236175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PLK1 activity persists into G1 after mitotic exit-associated degradation not explained\", \"Whether PLK1-Mis18 regulation is perturbed in cancer aneuploidies unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Full-length PLK1 structure capturing the intramolecular PBD–kinase domain interface and its modulation by methylation, ubiquitylation, and dimerization remains undetermined; a unified quantitative model integrating all post-translational inputs (phosphorylation, methylation, ubiquitylation, dephosphorylation) into a spatiotemporal activity profile is lacking.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length human PLK1 structure\", \"No integrated quantitative model of combinatorial PTM regulation\", \"Demethylases and deubiquitinases acting on PLK1 not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 9, 10, 13, 16, 23, 25, 29, 33, 37, 39, 41]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 9, 10, 13, 16, 23, 25, 37, 39, 41]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [7, 14, 17, 30, 31, 33, 35, 36, 41, 42]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [20, 26, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 8, 15, 19, 20, 27, 31, 34]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [23, 37, 39]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 10, 16, 25]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 12, 22, 25, 36]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [41, 42]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"AURKA\",\n      \"BORA\",\n      \"BUB1\",\n      \"CENPU\",\n      \"KLHL22\",\n      \"SETD6\",\n      \"MIS18A\",\n      \"APOLO1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}