Polo-box Domain Research Articles

Self-priming of Plk1 binding to BubR1 ensures accurate mitotic progression

Plk1 is a key mitotic kinase that localizes to distinct subcellular structures to promote accurate mitotic progression. Plk1 recruitment depends on direct interaction between polo-box domain (PBD) on Plk1 and PBD binding motif (PBD BM) on the interactors. However, recent study showed that PBD BM alone is not enough for stable binding between CENP-U and Plk1 highlighting the complexity of the interaction which warrants further investigation. An important interactor for Plk1 during mitosis is the checkpoint protein BubR1. Plk1 bound to BubR1 via PBD interaction with pT620 phosphorylates BubR1 S676/T680 to promote BubR1-PP2A/B56 interaction. The BubR1-PP2A/B56 complex counteracts the destablizing effect on kinetochore-microtubule attachments by mitotic kinases to promote mitotic progression. Here we show that Plk1 phosphorylates T600/T608 on BubR1 and the double phosphorylation is critical for BubR1-Plk1 interaction. A similar mechanism for Plk1-Bub1 interaction also exists indicating a general principle for Plk1 kinetochore recruitment through self-priming. Mechanistically preventing BubR1 T600/T608 phosphorylation impairs chromosome congression and checkpoint silencing by reducing Plk1 and PP2A/B56 binding to BubR1. Increasing the binding affinity towards Plk1 and PP2A/B56 in BubR1 through protein engineering bypasses the requirement of T600/T608 phosphorylation for mitotic progression. These results reveal a new layer of regulation for accurate mitotic progression.

PQSAR-guided Genetic Evolution of Peptide Ligands toward Improved Binding Capability to Rheumatic Immune-related Prk Polo-box Domain

Human Prk kinase plays an important role in the maintenance of inflammatory stability and has been established as an attractive target of diverse rheumatic immune diseases. The kinase possesses a highly conserved polo-box domain (PBD) in its C-terminal noncatalytic region, which exhibits a wide specificity across its various substrates to regulate the kinase-mediated substrate phosphorylation. In this study, peptide quantitative structure-activity relationship (pQSAR) was systematically modeled based on a gene ontology-enriched set of Prk PBD-binding peptide data, which was structurally characterized using global descriptors and statistically correlated with peptide affinities via both linear and nonlinear machine learning regressions. The optimal pQSAR predictor was then employed to guide the genetic evolution (GE) of a random peptide group toward improved potency to Prk PBD domain, from which a number of hits were measured to have moderate or high affinity. The resulting Pm7 peptide was determined as a good binder that can effectively interact with the domain at sub-micromolar level, which was then used as a parent template to further derive other promising candidates by combining systematic residue mutagenesis and structure-based rescoring. Two Pm7-derived counterparts Pm72 and Pm74 were rationally designed to exhibit a satisfactory binding profile to Prk PBD domain, with affinity improved by 3.1-fold and 4.6-fold relative to their parent Pm7. Structural and energetic analyses revealed that the potent Prk PBD-binding sequences can be divided into a N-terminal polar section, a middle positively charged anchor and a C-terminal hydrophobic section, thus conferring both stability and specificity to the domain-peptide recognition and association.

Plk4 regulates centriole duplication in the embryonic development of zebrafish

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish.

Structure-guided Development of Novel Benzothiophene Derivatives as PLK1-PBD Inhibitors

Background: Polo-like kinase 1 (PLK1), a validated target for tumor therapy, plays a key role in mitosis and is over-expressed in many tumors. In addition to its N-terminal kinase domain, PLk1 also harbors a C-terminal polo-box domain (PBD). Objective: A candidate based on PLK1-PBD was developed as a promising compound for future development. Methods: Seventeen small molecule PLK1-PBD inhibitors were designed, synthesized and evaluated for PLK1-PBD inhibitory activities by fluorescence polarization (FP) assay. The compounds with better inhibitory activities were further assessed for their anti-proliferative activities using a CCK-8 method. Results: The inhibitory rates of compounds 7a, 7d, 14a, 14d, 14e and 14f exceeded 98%. The IC50 values of compounds 7d, 14d, 14e, and 14f were 0.73 μM, 0.67 μM, 0.89 μM and 0.26 μM, proving better than MCC1019. Compound 14f showed the best inhibitory activity (IC50: 0.26 μM) and antiproliferative activities against three cancer cell lines (HeLa, HepG2 and MG63). Especially, compound 14f also exhibited acceptable safety profiles in the human ether-a-go-go related gene (hERG) and normal cell tests. The results of docking and prediction studies indicated that compound 14f had a high binding affinity to the target, with good drug-like absorption, distribution, metabolism, elimination, and toxicity (ADMET) properties. Conclusion: Compound 14f can be a promising compound for future development.

Targeting polo-like kinase 1 to treat kidney diseases.

Globally, ∼850 million individuals suffer from some form of kidney disease. This staggering figure underscores the importance of continued research and innovation in the field of nephrology to develop effective treatments and improve overall global kidney health. In current research, the polo-like kinase (Plk) family has emerged as a group of highly conserved enzyme kinases vital for proper cell cycle regulation. Plks are defined by their N-terminal kinase domain and C-terminal polo-box domain, which regulate their catalytic activity, subcellular localization, and substrate recognition. Among the Plk family members, Plk1 has garnered significant attention due to its pivotal role in regulating multiple mitotic processes, particularly in the kidneys. It is a crucial serine-threonine (Ser-Thr) kinase involved in cell division and genomic stability. In this review, we delve into the types and functions of Plks, focusing on Plk1's significance in processes such as cell proliferation, spindle assembly, and DNA damage repair. The review also underscores Plk1's vital contributions to maintaining kidney homeostasis, elucidating its involvement in nuclear envelope breakdown, anaphase-promoting complex/cyclosome activation, and the regulation of mRNA translation machinery. Furthermore, the review discusses how Plk1 contributes to the development and progression of kidney diseases, emphasizing its overexpression in conditions such as acute kidney injury, chronic kidney disease, and so forth. It also highlights the importance of exploring Plk1 modulators as targeted therapies for kidney diseases in future. This review will help in understanding the role of Plk1 in kidney disease development, paving the way for the discovery and development of novel therapeutic approaches to manage kidney diseases effectively.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 μM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

Abstract 5782: Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors

Abstract Polo Like Kinase 1 (PLK1), a sought-after target for drug discovery, is an essential mitotic kinase consisting of a catalytic domain (KD) and a polobox domain (PBD), the latter of which plays roles in substrate recognition, subcellular localization and further acting as an autoinhibitory regulatory domain. PLK1 can exist either in an inhibited closed conformation or an open active state allowing interaction with and phosphorylation of substrates. PLK1 has also been shown to be regulated by interchange between monomeric and dimeric forms through binding to the Bora protein and by activation loop phosphorylation. We have described PBD-binding molecules termed abbapolins that inhibit cellular phosphorylation of a PLK1 substrate and induce loss of intracellular PLK1. In addition, due to their engagement of the cryptic pocket (ligand induced) of the PBD, we proposed that abbapolins would interfere with formation of PLK1 dimers. Here we provide evidence further supporting the ability of abbapolins to block PLK1 cellular dimerization and that this is related to their mechanism of anti-proliferative activity. In a separate study, using a cellular thermal shift assay, KD inhibitors were shown to decrease soluble PLK1, indicating that catalytic-site binding results in less stable PLK1 suggestive of an open conformation. We have extended these observations using a sensitive readout of cellular target engagement employing a Microtag overexpressed PLK1. In this format, we observe heat-induced destabilization of PLK1 upon nanomolar engagement by two lead KD inhibitors, BI2536 and onvansertib, currently being clinically evaluated. Furthermore, cyclapolin 9, a low molecular weight kinase inhibitor that, unlike the other two KD inhibitors does not protrude from the active site, did not induce significant destabilization. We have also carried out experiments using a fluorescently labeled version of BI2536 to probe the conformational effects of PBD binding upon the KD. These data solidify the observations that relief of autoinhibited PLK1 is induced by certain KD binders and highlight the conformational perturbations from KD versus PBD binding. These observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 non-catalytic functions, which may explain their lack of clinical efficacy to date. Furthermore, the demonstration that the abbapolins are mechanistically unique in terms of blocking the dimerization of PLK through the PBD provides impetus for their development as next generation PLK1 inhibitors. Citation Format: Mourad Sanhaji, Monika Raab, George Merhej, Chintada Nageswara Rao, Ivan Babic, Elmar Nurmemmedov, Klaus Strebhardt, MIchael Wyatt, Campbell McInnes. Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5782.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Computational assessment of potential nonpeptidic inhibitors for PLK1 polo-box domain

Computational assessment of potential nonpeptidic inhibitors for PLK1 polo-box domain

Affinity enhancement of polo-like kinase 1 polo box domain-binding ligands by a bivalent approach using a covalent kinase-binding component.

The polo-like kinase 1 (Plk1) is an important cell cycle regulator that is recognized as a target molecule for development of anti-cancer agents. Plk1 consists of a catalytic kinase domain (KD) and a polo-box domain (PBD), which engages in protein-protein interactions (PPIs) essential to proper Plk1 function. Recently, we developed extremely high-affinity PBD-binding inhibitors based on a bivalent approach using the Plk1 KD-binding inhibitor, BI2536, and a PBD-binding peptide. Certain of the resulting bivalent constructs exhibited more than 100-fold Plk1 affinity enhancement relative to the best monovalent PBD-binding ligands. Herein, we report an extensive investigation of bivalent ligands that utilize the non-selective kinase inhibitor Wortmannin as a Plk1 KD-binding component. We found that bivalent ligands incorporating Wortmannin demonstrated affinity enhancements that could be similar to what we had obtained with BI2536 and that they could tightly bind to the protein. This suggests that these tight binding ligands might be useful for structural analysis of full-length Plk1.

Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote.

Plk1 (polo-like kinase 1) is an evolutionarily conserved serine/threonine kinase instrumental for mitotic entry and progression. Beyond these canonical functions, Plk1 also regulates cell polarization and cell fate during asymmetric cell divisions in C. elegans and D. melanogaster. Plk1 contains a specialized phosphoserine-threonine binding domain, the polo-box domain (PBD), which localizes and concentrates the kinase at its various sites of action within the cell in space and time. Here we present protocols to express and purify the C. elegans Plk1 kinase along with biochemical and phosphoproteomic approaches to interrogate the PBD interactome and to dissect Plk1 substrate interactions. These protocols are most suitable for the identification of Plk1 targets in C. elegans embryos but can be easily adapted to identify and study Plk1 substrates from any source."

Degradation of Polo-like Kinase 1 by the Novel Poly-Arginine N-Degron Pathway PROTAC Regulates Tumor Growth in Nonsmall Cell Lung Cancer.

Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.

METTL16-mediated N6-methyladenosine modification of Soga1 enables proper chromosome segregation and chromosomal stability in colorectal cancer.

N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian messenger RNAs and is associated with numerous biological processes. However, its role in chromosomal instability remains to be established. Here, we report that an RNA m6A methyltransferase, METTL16, plays an indispensable role in the progression of chromosome segregation and is required to preserve chromosome stability in colorectal cancer (CRC) cells. Depletion or inhibition of the methyltransferase activity of METTL16 results in abnormal kinetochore-microtubule attachment during mitosis, leading to delayed mitosis, lagging chromosomes, chromosome mis-segregation and chromosomal instability. Mechanistically, METTL16 exerts its oncogenic effects by enhancing the expression of suppressor of glucose by autophagy 1 (Soga1) in an m6A-dependent manner. CDK1 phosphorylates Soga1, thereby triggering its direct interaction with the polo box domain of PLK1. This interaction facilitates PLK1 activation and promotes mitotic progression. Therefore, targeting the METTL16-Soga1 pathway may provide a potential treatment strategy against CRC because of its essential role in maintaining chromosomal stability.

Synthesis and evaluation of small molecule-based derivatives as inhibitors of polo-box domain of polo-like kinase-1

ObjectivesPolo-like kinase 1 (Plk1) is an important mitotic protein. In particular, this protein is highly overexpressed in many types of tumors and has been identified as a potential biomarker for the treatment and diagnosis of tumors. Plk1 is composed of two domains, an N-terminal kinase domain and a C-terminal polo-box domain (PBD). Presently, inhibitors with improved selectivity and specificity for Plk1 are unavailable. Therefore, we aimed to develop an inhibitor targeting the C-terminal PBD present only in Plk1.Methods & resultsIn this study, three derivatives targeting PBD for Plk1 were designed by protein–protein interactions, which showed high levels of selectivity and specificity for Plk1 PBD, and were evaluated to inhibit tumor cell proliferation through an apoptotic process during tumor cell division. The investigation of the in vitro and in vivo antitumor effects of these inhibitors demonstrated that one of the new small molecules, 1, is a promising anticancer agent.ConclusionOur findings can provide new insights for the design of novel Plk1 peptide inhibitors in the future.

PLK1 inhibitors as a new targeted treatment for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options. Polo-like kinase 1 (PLK1) is a promising drug target; PLK1 inhibitors (PLK1i) have been investigated in solid cancers and are more effective in TP53-mutated cases. We evaluated PLK1 expression in ACC samples and the efficacy of two PLK1i in ACC cell lines with different genetic backgrounds. PLK1 protein expression was investigated by immunohistochemistry in tissue samples and correlated with clinical data. The efficacy of rigosertib (RGS), targeting RAS/PI3K, CDKs and PLKs, and poloxin (Pol), specifically targeting the PLK1 polo-box domain, was tested in TP53-mutated NCI-H295R, MUC-1, and CU-ACC2 cells and in TP53 wild-type CU-ACC1. Effects on proliferation, apoptosis, and viability were determined. PLK1 immunostaining was stronger in TP53-mutated ACC samples vs wild-type (P = 0.0017). High PLK1 expression together with TP53 mutations correlated with shorter progression-free survival (P= 0.041). NCI-H295R showed a time- and dose-dependent reduction in proliferation with both PLK1i (P< 0.05at 100 nM RGS and 30 µM Pol). In MUC-1, a less pronounced decrease was observed (P< 0.05at 1000 nM RGS and 100 µM Pol). 100 nM RGS increased apoptosis in NCI-H295R (P< 0.001), with no effect on MUC-1. CU-ACC2 apoptosis was induced only at high concentrations (P < 0.05 at 3000 nM RGS and 100 µM Pol), while proliferation decreased at 1000 nM RGS and 30 µM Pol. CU-ACC1 proliferation reduced, and apoptosis increased, only at 100 µM Pol. TP53-mutated ACC cell lines demonstrated better response to PLK1i than wild-type CU-ACC1. These data suggest PLK1i may be a promising targeted treatment of a subset of ACC patients, pre-selected according to tumour genetic signature.

Leveraging the Fragment Molecular Orbital Method to Explore the PLK1 Kinase Binding Site and Polo-Box Domain for Potent Small-Molecule Drug Design.

Polo-like kinase 1 (PLK1) plays a pivotal role in cell division regulation and emerges as a promising therapeutic target for cancer treatment. Consequently, the development of small-molecule inhibitors targeting PLK1 has become a focal point in contemporary research. The adenosine triphosphate (ATP)-binding site and the polo-box domain in PLK1 present crucial interaction sites for these inhibitors, aiming to disrupt the protein's function. However, designing potent and selective small-molecule inhibitors can be challenging, requiring a deep understanding of protein-ligand interaction mechanisms at these binding sites. In this context, our study leverages the fragment molecular orbital (FMO) method to explore these site-specific interactions in depth. Using the FMO approach, we used the FMO method to elucidate the molecular mechanisms of small-molecule drugs binding to these sites to design PLK1 inhibitors that are both potent and selective. Our investigation further entailed a comparative analysis of various PLK1 inhibitors, each characterized by distinct structural attributes, helping us gain a better understanding of the relationship between molecular structure and biological activity. The FMO method was particularly effective in identifying key binding features and predicting binding modes for small-molecule ligands. Our research also highlighted specific "hot spot" residues that played a critical role in the selective and robust binding of PLK1. These findings provide valuable insights that can be used to design new and effective PLK1 inhibitors, which can have significant implications for developing anticancer therapeutics.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

The centrosome assembles a bipolar spindle for faithful chromosome segregation during mitosis. To prevent the inheritance of DNA damage, the DNA damage response (DDR) triggers programmed spindle multipolarity and concomitant death in mitosis through a poorly understood mechanism. We identified hornerin, which forms a complex with checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1) to mediate phosphorylation at the polo-box domain (PBD) of Plk1, as the link between the DDR and death in mitosis. We demonstrate that hornerin mediates DDR-induced precocious centriole disengagement through a dichotomous mechanism that includes sequestration of Sgo1 and Plk1 in the cytoplasm through phosphorylation of the PBD in Plk1 by Chk1. Phosphorylation of the PBD in Plk1 abolishes the interaction with Sgo1 and phosphorylation-dependent Sgo1 translocation to the centrosome, leading to precocious centriole disengagement and spindle multipolarity. Mechanistically, hornerin traps phosphorylated Plk1 in the cytoplasm. Furthermore, PBD phosphorylation inactivates Plk1 and disrupts Cep192::Aurora A::Plk1 complex translocation to the centrosome and concurrent centrosome maturation. Remarkably, hornerin depletion leads to chemoresistance against DNA damaging agents by attenuating DDR-induced death in mitosis. These results reveal how the DDR eradicates mitotic cells harboring DNA damage to ensure genome integrity during cell division.

Crystal Structures of Plk1 Polo-Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2-Derived Peptide.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.