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Kyung S Lee - One of the best experts on this subject based on the ideXlab platform.
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identification of a new heterocyclic scaffold for inhibitors of the Polo box domain of Polo Like Kinase 1
Journal of Medicinal Chemistry, 2020Co-Authors: Celeste N Alverez, Jungeun Park, Jeong K Bang, Kiran S Toti, Yangliu Xia, Kristopher W Krausz, Ganesha Rai, Frank J Gonzalez, Kenneth A Jacobson, Kyung S LeeAbstract:As a mitotic-specific target widely deregulated in various human cancers, Polo-Like Kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic Polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.
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phase separation of Polo Like Kinase 4 by autoactivation and clustering drives centriole biogenesis
Nature Communications, 2019Co-Authors: Jungeun Park, Liang Zhang, Jeong Kyu Bang, Thorkell Andresson, Frank Dimaio, Kyung S LeeAbstract:Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-Like Kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique Kinase that utilizes its autophosphorylated noncatalytic cryptic Polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4's dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.
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effects on Polo Like Kinase 1 Polo box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Wenjian Qian, Jungeun Park, Fa Liu, Kyung S Lee, Terrence R BurkeAbstract:Abstract Protein–protein interactions (PPIs) mediated by the Polo-box domain (PBD) of Polo-Like Kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr β-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.
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identification of high affinity Polo Like Kinase 1 plk1 Polo box domain binding peptides using oxime based diversification
ACS Chemical Biology, 2012Co-Authors: Fa Liu, Michael B Yaffe, Wenjian Qian, Jungeun Park, Kyung S Lee, Daniel Lim, Thorsten Berg, Andrej Scharow, Terrence R BurkeAbstract:In an effort to develop improved binding antagonists of the Polo-Like Kinase 1 (Plk1) Polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein–protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.
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regulation of microtubule based microtubule nucleation by mammalian Polo Like Kinase 1
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Yoshikazu Johmura, Raymond L Erikson, Jungeun Park, Nak Kyun Soung, Timothy D Veenstra, Jeong K Bang, Ming Zhou, Bo Yeon Kim, Kyung S LeeAbstract:Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian Polo-Like Kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.
Terrence R Burke - One of the best experts on this subject based on the ideXlab platform.
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a new genre of fluorescence recovery assay to evaluate Polo Like Kinase 1 atp competitive inhibitors
Analytical Methods, 2020Co-Authors: Kohei Tsuji, David Hymel, Terrence R BurkeAbstract:Using a probe consisting of a fluorescein-labeled variant of the potent Polo-Like Kinase 1 (Plk1) inhibitor BI2536 [FITC-PEG-Lys(BI2536) 4], we were able to determine half maximal inhibitory concentration (IC50) of ATP-competitive Type 1 inhibitors of Plk1 by means of a fluorescence recovery assay. This methodology represents a cost-effective and simple alternative to traditional Kinase assays for initial screening of potential Plk1 inhibitors.
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enhancing Polo Like Kinase 1 selectivity of Polo box domain binding peptides
Bioorganic & Medicinal Chemistry, 2017Co-Authors: Xue Zhi Zhao, David Hymel, Terrence R BurkeAbstract:Abstract An important goal in the development of Polo-Like Kinase 1 (Plk1) Polo-box domain (PBD) binding inhibitors is selectivity for Plk1 relative to Plk2 and Plk3. In our current work we show that Plk1 PBD selectivity can be significantly enhanced by modulating interactions within a previously discovered “cryptic pocket” and a more recently identified proximal “auxiliary pocket.”
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phosphatase stable phosphoamino acid mimetics that enhance binding affinities with the Polo box domain of Polo Like Kinase 1
ChemMedChem, 2017Co-Authors: David Hymel, Terrence R BurkeAbstract:(2S,3R)-2-Amino-3-methyl-4-phosphonobutanoic acid (Pmab) is a phosphatase-stable analogue of phosphothreonine (pThr), which has been used in a variety of biological contexts. Among these applications are peptidomimetic ligands that bind to the Polo-box domain (PBD) of Polo-Like Kinase 1 (Plk1) with affinities approaching that of the corresponding pThr-containing peptides. However, Pmab is not widely used, because there are no direct, high-yield preparations of suitably protected reagent. We have now achieved an efficient synthesis of protected Pmab, as well as variants with different substituents at the 3R center. When incorporated into our peptidomimetic scaffold, these new Pmab analogues exhibit Plk1 PBD-binding affinities that are several-fold higher than Pmab, yet retain good selectivity for Plk1 relative to the PBDs of Plk2 and Plk3. These findings will significantly impact the future development of PBD-binding inhibitors, as well as ligands directed against a broad spectrum of pThr-dependent processes.
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effects on Polo Like Kinase 1 Polo box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Wenjian Qian, Jungeun Park, Fa Liu, Kyung S Lee, Terrence R BurkeAbstract:Abstract Protein–protein interactions (PPIs) mediated by the Polo-box domain (PBD) of Polo-Like Kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr β-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.
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identification of high affinity Polo Like Kinase 1 plk1 Polo box domain binding peptides using oxime based diversification
ACS Chemical Biology, 2012Co-Authors: Fa Liu, Michael B Yaffe, Wenjian Qian, Jungeun Park, Kyung S Lee, Daniel Lim, Thorsten Berg, Andrej Scharow, Terrence R BurkeAbstract:In an effort to develop improved binding antagonists of the Polo-Like Kinase 1 (Plk1) Polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein–protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.
Jungeun Park - One of the best experts on this subject based on the ideXlab platform.
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identification of a new heterocyclic scaffold for inhibitors of the Polo box domain of Polo Like Kinase 1
Journal of Medicinal Chemistry, 2020Co-Authors: Celeste N Alverez, Jungeun Park, Jeong K Bang, Kiran S Toti, Yangliu Xia, Kristopher W Krausz, Ganesha Rai, Frank J Gonzalez, Kenneth A Jacobson, Kyung S LeeAbstract:As a mitotic-specific target widely deregulated in various human cancers, Polo-Like Kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic Polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.
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phase separation of Polo Like Kinase 4 by autoactivation and clustering drives centriole biogenesis
Nature Communications, 2019Co-Authors: Jungeun Park, Liang Zhang, Jeong Kyu Bang, Thorkell Andresson, Frank Dimaio, Kyung S LeeAbstract:Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-Like Kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique Kinase that utilizes its autophosphorylated noncatalytic cryptic Polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4's dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.
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effects on Polo Like Kinase 1 Polo box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Wenjian Qian, Jungeun Park, Fa Liu, Kyung S Lee, Terrence R BurkeAbstract:Abstract Protein–protein interactions (PPIs) mediated by the Polo-box domain (PBD) of Polo-Like Kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr β-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.
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identification of high affinity Polo Like Kinase 1 plk1 Polo box domain binding peptides using oxime based diversification
ACS Chemical Biology, 2012Co-Authors: Fa Liu, Michael B Yaffe, Wenjian Qian, Jungeun Park, Kyung S Lee, Daniel Lim, Thorsten Berg, Andrej Scharow, Terrence R BurkeAbstract:In an effort to develop improved binding antagonists of the Polo-Like Kinase 1 (Plk1) Polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein–protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.
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regulation of microtubule based microtubule nucleation by mammalian Polo Like Kinase 1
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Yoshikazu Johmura, Raymond L Erikson, Jungeun Park, Nak Kyun Soung, Timothy D Veenstra, Jeong K Bang, Ming Zhou, Bo Yeon Kim, Kyung S LeeAbstract:Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian Polo-Like Kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.
Kelsie L Thu - One of the best experts on this subject based on the ideXlab platform.
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reply to oegema et al cfi 400945 and Polo Like Kinase 4 inhibition
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguezcanales, Barbara Mino, Jonathan M Kurie, Jason Roszik, Pamela Villalobos, Kelsie L ThuAbstract:In “CFI-400945 is not a selective cellular PLK4 inhibitor,” Oegema et al. (1) raise thoughtful comments about our article (2). We appreciate their interest and critique. They propose that CFI-400945 activity was not from Polo-Like Kinase (PLK4) inhibition and argue that antineoplastic activity was through Aurora B Kinase (1), rather than PLK4 inhibition (2). Kinases often share related catalytic pockets, and targeting one Kinase without affecting another is difficult to achieve. This is true for CFI-400945 (2) and centrinone, the PLK4 inhibitor (3) used by Oegema et al. (1). CFI-400945 is (by IC50) 38-fold more potent in causing PLK4 inhibition than in antagonizing Aurora B Kinase (4, 5). Despite their claim … [↵][1]1Email: tak.mak{at}uhnresearch.ca. [1]: #xref-corresp-1-1
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abstract 1948 Polo Like Kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers
Cancer Research, 2018Co-Authors: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguezcanales, Barbara Mino, Jonathan M Kurie, Jason Roszik, Pamela Villalobos, Kelsie L ThuAbstract:Despite current treatments, lung cancers remain a major public health problem. Innovative ways are needed to treat or prevent these cancers. Centrosomes are critical for fidelity of mitosis. Abnormal centrosome numbers can cause aberrant mitosis and cell death. Polo-Like Kinase 4 (PLK4) is a serine/threonine Kinase regulating centriole duplication and its deregulation alters centrosome number and mitosis. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Here, we explored CFI-400945 activity against lung cancer using in vitro and in vivo models. CFI-400945 caused polyploidy, growth inhibition and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with > 4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored by individually staining treated and control cells with γ-tubulin, α-tubulin, and DAPI. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in the examined lung cancer cell lines. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was statistically significantly reduced at dosages that were well tolerated. Phospho-histone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguez-Canales, Barbara Mino, Jonathan M. Kurie, Jason Roszik, Pamela Andrea Villalobos, Kelsie L. Thu, David W. Cescon, Jennifer Silvester, Ignacio Wistuba, Tak W. Mak, Xi Liu, Ethan Dmitrovsky. Polo-Like Kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1948.
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Polo Like Kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguezcanales, Barbara Mino, Jonathan M Kurie, Jason Roszik, Pamela Villalobos, Kelsie L ThuAbstract:Polo-Like Kinase 4 (PLK4) is a serine/threonine Kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent Kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.
Rene H Medema - One of the best experts on this subject based on the ideXlab platform.
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switching Polo Like Kinase 1 on and off in time and space
Trends in Biochemical Sciences, 2012Co-Authors: Wytse Bruinsma, Jonne A Raaijmakers, Rene H MedemaAbstract:Polo-Like Kinase (Plk)1 executes several essential functions to promote cell division. These functions range from centrosome maturation in late G2 phase to the regulation of cytokinesis, which necessitates precise separation of Plk1-dependent substrate phosphorylation over time. Multiple levels of control are in place to ensure that Plk1-dependent phosphorylation of its various substrates is properly coordinated in time and space. Here, we review the current knowledge on the mechanisms that enforce the temporal and spatial control of Plk1 activity, and how this results in coordinated phosphorylation of its many different substrates. We also review a number of newly discovered functions of Plk1 that provide more insights into the spatiotemporal control of Plk1-dependent substrate phosphorylation.
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Polo Like Kinase 1 is activated by aurora a to promote checkpoint recovery
Nature, 2008Co-Authors: Libor Macůrek, Michael A Lampson, Rob Klompmaker, Michael B Yaffe, Arne Lindqvist, Daniel Lim, Raimundo Freire, Christophe Clouin, Stephen S Taylor, Rene H MedemaAbstract:Polo-Like Kinase-1 (PLK1) is an essential mitotic Kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 Kinase domain, but the Kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.
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Polo Like Kinase 1 controls proteasome dependent degradation of claspin during checkpoint recovery
Current Biology, 2006Co-Authors: Ivan Mamely, Veronique A J Smits, Rene H Medema, Marcel A T M Van Vugt, Jennifer I Semple, Bennie Lemmens, Anastassis Perrakis, Raimundo FreireAbstract:DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint Kinase ATR phosphorylates and activates its effector Kinase Chk1 in a process that critically depends on Claspin [1]. However, it is not known how exactly this Kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 Kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the β-TrCP-SCF ubiquitin ligase and Polo-Like Kinase-1 (Plk1). We demonstrate that Claspin interacts with both β-TrCP and Plk1 and that inactivation of these components or the β-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.
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getting in and out of mitosis with Polo Like Kinase 1
Oncogene, 2005Co-Authors: Marcel A T M Van Vugt, Rene H MedemaAbstract:Research in different species has shown that Polo-Like Kinases are essential for successful cell division. In human cells, Polo-Like Kinase-1 (Plk1) has been implicated in the regulation of different processes, including mitotic entry, spindle formation and cytokinesis. Recently, a range of new downstream targets of Plk1 has been identified, as well as a molecular mechanism that explains recruitment of Plk1 to potential substrate proteins through its Polo-box domain. On the basis of these reports, we discuss possible mechanisms by which Polo-Like Kinases can exert their multiple functions during mitosis. Polo-Like Kinases also function in DNA damage checkpoints. Plk1 has been shown to be a target of the G2 DNA damage checkpoint, while Cdc5, the Polo-Like Kinase in Saccharomyces cerevisiae, has long been known to be required for adaptation to persistent DNA damage. Just recently, a similar requirement for Polo-Like Kinases during checkpoint adaptation was demonstrated in multicellular organisms. Moreover, Plk1 was also shown to be required for checkpoint recovery following checkpoint inactivation, that is, in cells where the damage is completely repaired. Thus, Plk1 appears to play a role at multiple points during a restart of the cell cycle following DNA damage. Based on these novel observations, we discuss possible consequences of using Plk1 as a target in anticancer strategies.
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Polo Like Kinase 1 is a target of the dna damage checkpoint
Nature Cell Biology, 2000Co-Authors: Veronique A J Smits, Erich A Nigg, Rob Klompmaker, Lionel Arnaud, Gert Rijksen, Rene H MedemaAbstract:Polo-Like Kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-Like Kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the Kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.