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Kyung S Lee - One of the best experts on this subject based on the ideXlab platform.
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mammalian polo like kinase 1 PLK1 promotes proper chromosome segregation by phosphorylating and delocalizing the pbip1 cenp q complex from kinetochores
Journal of Biological Chemistry, 2015Co-Authors: Chi Hoon Park, Ming Zhou, Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Tae Sung Kim, Nam Kim, Kyung S LeeAbstract:Mammalian PLK1 is critically required for proper M phase progression. PLK1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating PLK1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. PLK1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine PLK1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although PLK1 did not alter the level of PBIP1 and CENP-Q ubiquitination, PLK1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that PLK1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting PLK1-dependent mitotic progression, but also for resetting the timing of PLK1 recruitment to prekinetochores in the next cell cycle.
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mammalian polo like kinase 1 dependent regulation of the pbip1 cenp q complex at kinetochores
Journal of Biological Chemistry, 2011Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Chi Hoon Park, Tae Sung Kim, Bo Y Kim, Kyung S LeeAbstract:Mammalian polo-like kinase 1 (PLK1) plays a pivotal role during M-phase progression. PLK1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that PLK1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of PLK1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. PLK1 did not appear to interact with CENP-Q directly. However, PLK1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for PLK1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates PLK1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how PLK1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.
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direct quantification of polo like kinase 1 activity in cells and tissues using a highly sensitive and specific elisa assay
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Jungeun Park, Joobae Park, Rainald Knecht, Klaus Strebhardt, Stuart H Yuspa, Kyung S LeeAbstract:Polo-like kinase 1 (PLK1) plays a pivotal role in the regulation of cellular proliferation. PLK1 is overexpressed in ≈80% of human tumors of diverse origins, and overexpression of PLK1 promotes neoplastic transformation of human cells. A growing body of evidence suggests that deregulation of PLK1 closely correlates with prognosis of various cancers in humans. Thus, accurate assessment of PLK1 deregulation would provide clear clinical advantages. However, because of the limited amount of cancer tissues available, quantification of the PLK1 activity has not been feasible. Here, we report the development of a rapid, highly sensitive, and specific ELISA-based PLK1 assay that can quantify the level of PLK1 activity with a small amount (2–20 μg) of total cellular proteins. Unlike the conventional immunocomplex kinase assay, this assay directly utilizes total cellular lysates and does not require a PLK1 enrichment step such as immunoprecipitation or affinity purification. Using this assay, we demonstrated that PLK1 activity is elevated in tumors but not in the surrounding normal tissues and that the level of PLK1 activity significantly diminishes after an antiproliferative chemotherapy. The method described here may provide an innovative tool for assessing the predisposition for cancer development, monitoring early tumor response after therapy, and estimating the prognosis of patients with cancers from multiple organ sites.
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self regulated PLK1 recruitment to kinetochores by the PLK1 pbip1 interaction is critical for proper chromosome segregation
Molecular Cell, 2006Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Sang Moon Yun, Jeong K Bang, Yeon Sun Seong, Susan H Garfield, Timothy D Veenstra, Kyung S LeeAbstract:The polo-box domain (PBD) of mammalian polo-like kinase 1 (PLK1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying PLK1 recruitment to the kinetochores and the role of PLK1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting PLK1 to the interphase and mitotic kinetochores. Unprecedentedly, PLK1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of PLK1, but not Plk2 or Plk3. Later in mitosis, PLK1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent PLK1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, PLK1 self-regulates the PLK1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.
Jungeun Park - One of the best experts on this subject based on the ideXlab platform.
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bimodal interaction of mammalian polo like kinase 1 and a centrosomal scaffold cep192 in the regulation of bipolar spindle formation
Molecular and Cellular Biology, 2015Co-Authors: Lingjun Meng, Ming Zhou, Jungeun Park, Suk Youl Park, Jeong K BangAbstract:: Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study with Xenopus laevis egg extracts suggested that the PLK1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif of Xenopus Cep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and PLK1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and PLK1. Strikingly, PLK1 interacted with Cep192 through either the p-T44 (analogous to Xenopus p-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between PLK1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of PLK1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-PLK1 interactions induced an additive defect in recruiting PLK1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that PLK1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.
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mammalian polo like kinase 1 PLK1 promotes proper chromosome segregation by phosphorylating and delocalizing the pbip1 cenp q complex from kinetochores
Journal of Biological Chemistry, 2015Co-Authors: Chi Hoon Park, Ming Zhou, Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Tae Sung Kim, Nam Kim, Kyung S LeeAbstract:Mammalian PLK1 is critically required for proper M phase progression. PLK1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating PLK1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. PLK1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine PLK1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although PLK1 did not alter the level of PBIP1 and CENP-Q ubiquitination, PLK1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that PLK1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting PLK1-dependent mitotic progression, but also for resetting the timing of PLK1 recruitment to prekinetochores in the next cell cycle.
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mammalian polo like kinase 1 dependent regulation of the pbip1 cenp q complex at kinetochores
Journal of Biological Chemistry, 2011Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Chi Hoon Park, Tae Sung Kim, Bo Y Kim, Kyung S LeeAbstract:Mammalian polo-like kinase 1 (PLK1) plays a pivotal role during M-phase progression. PLK1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that PLK1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of PLK1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. PLK1 did not appear to interact with CENP-Q directly. However, PLK1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for PLK1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates PLK1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how PLK1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.
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structural and functional analyses of minimal phosphopeptides targeting the polo box domain of polo like kinase 1
Nature Structural & Molecular Biology, 2009Co-Authors: Sang Moon Yun, Young H Kang, Jungeun Park, Daniel Lim, Tinoush Moulaei, Jeong K Bang, Shilpa R Shenoy, Fa Liu, Chenzhong Liao, Nak Kyun SoungAbstract:Polo-like kinase-1 (PLK1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of PLK1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.
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direct quantification of polo like kinase 1 activity in cells and tissues using a highly sensitive and specific elisa assay
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Jungeun Park, Joobae Park, Rainald Knecht, Klaus Strebhardt, Stuart H Yuspa, Kyung S LeeAbstract:Polo-like kinase 1 (PLK1) plays a pivotal role in the regulation of cellular proliferation. PLK1 is overexpressed in ≈80% of human tumors of diverse origins, and overexpression of PLK1 promotes neoplastic transformation of human cells. A growing body of evidence suggests that deregulation of PLK1 closely correlates with prognosis of various cancers in humans. Thus, accurate assessment of PLK1 deregulation would provide clear clinical advantages. However, because of the limited amount of cancer tissues available, quantification of the PLK1 activity has not been feasible. Here, we report the development of a rapid, highly sensitive, and specific ELISA-based PLK1 assay that can quantify the level of PLK1 activity with a small amount (2–20 μg) of total cellular proteins. Unlike the conventional immunocomplex kinase assay, this assay directly utilizes total cellular lysates and does not require a PLK1 enrichment step such as immunoprecipitation or affinity purification. Using this assay, we demonstrated that PLK1 activity is elevated in tumors but not in the surrounding normal tissues and that the level of PLK1 activity significantly diminishes after an antiproliferative chemotherapy. The method described here may provide an innovative tool for assessing the predisposition for cancer development, monitoring early tumor response after therapy, and estimating the prognosis of patients with cancers from multiple organ sites.
Jeong K Bang - One of the best experts on this subject based on the ideXlab platform.
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bimodal interaction of mammalian polo like kinase 1 and a centrosomal scaffold cep192 in the regulation of bipolar spindle formation
Molecular and Cellular Biology, 2015Co-Authors: Lingjun Meng, Ming Zhou, Jungeun Park, Suk Youl Park, Jeong K BangAbstract:: Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study with Xenopus laevis egg extracts suggested that the PLK1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif of Xenopus Cep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and PLK1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and PLK1. Strikingly, PLK1 interacted with Cep192 through either the p-T44 (analogous to Xenopus p-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between PLK1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of PLK1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-PLK1 interactions induced an additive defect in recruiting PLK1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that PLK1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.
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mammalian polo like kinase 1 PLK1 promotes proper chromosome segregation by phosphorylating and delocalizing the pbip1 cenp q complex from kinetochores
Journal of Biological Chemistry, 2015Co-Authors: Chi Hoon Park, Ming Zhou, Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Tae Sung Kim, Nam Kim, Kyung S LeeAbstract:Mammalian PLK1 is critically required for proper M phase progression. PLK1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating PLK1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. PLK1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine PLK1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although PLK1 did not alter the level of PBIP1 and CENP-Q ubiquitination, PLK1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that PLK1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting PLK1-dependent mitotic progression, but also for resetting the timing of PLK1 recruitment to prekinetochores in the next cell cycle.
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mammalian polo like kinase 1 dependent regulation of the pbip1 cenp q complex at kinetochores
Journal of Biological Chemistry, 2011Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Chi Hoon Park, Tae Sung Kim, Bo Y Kim, Kyung S LeeAbstract:Mammalian polo-like kinase 1 (PLK1) plays a pivotal role during M-phase progression. PLK1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that PLK1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of PLK1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. PLK1 did not appear to interact with CENP-Q directly. However, PLK1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for PLK1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates PLK1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how PLK1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.
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self regulated PLK1 recruitment to kinetochores by the PLK1 pbip1 interaction is critical for proper chromosome segregation
Molecular Cell, 2006Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Sang Moon Yun, Jeong K Bang, Yeon Sun Seong, Susan H Garfield, Timothy D Veenstra, Kyung S LeeAbstract:The polo-box domain (PBD) of mammalian polo-like kinase 1 (PLK1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying PLK1 recruitment to the kinetochores and the role of PLK1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting PLK1 to the interphase and mitotic kinetochores. Unprecedentedly, PLK1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of PLK1, but not Plk2 or Plk3. Later in mitosis, PLK1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent PLK1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, PLK1 self-regulates the PLK1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.
Nak Kyun Soung - One of the best experts on this subject based on the ideXlab platform.
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mammalian polo like kinase 1 PLK1 promotes proper chromosome segregation by phosphorylating and delocalizing the pbip1 cenp q complex from kinetochores
Journal of Biological Chemistry, 2015Co-Authors: Chi Hoon Park, Ming Zhou, Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Tae Sung Kim, Nam Kim, Kyung S LeeAbstract:Mammalian PLK1 is critically required for proper M phase progression. PLK1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating PLK1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. PLK1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine PLK1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although PLK1 did not alter the level of PBIP1 and CENP-Q ubiquitination, PLK1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that PLK1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting PLK1-dependent mitotic progression, but also for resetting the timing of PLK1 recruitment to prekinetochores in the next cell cycle.
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mammalian polo like kinase 1 dependent regulation of the pbip1 cenp q complex at kinetochores
Journal of Biological Chemistry, 2011Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Jeong K Bang, Chi Hoon Park, Tae Sung Kim, Bo Y Kim, Kyung S LeeAbstract:Mammalian polo-like kinase 1 (PLK1) plays a pivotal role during M-phase progression. PLK1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that PLK1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of PLK1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. PLK1 did not appear to interact with CENP-Q directly. However, PLK1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for PLK1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates PLK1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how PLK1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.
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structural and functional analyses of minimal phosphopeptides targeting the polo box domain of polo like kinase 1
Nature Structural & Molecular Biology, 2009Co-Authors: Sang Moon Yun, Young H Kang, Jungeun Park, Daniel Lim, Tinoush Moulaei, Jeong K Bang, Shilpa R Shenoy, Fa Liu, Chenzhong Liao, Nak Kyun SoungAbstract:Polo-like kinase-1 (PLK1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of PLK1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.
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self regulated PLK1 recruitment to kinetochores by the PLK1 pbip1 interaction is critical for proper chromosome segregation
Molecular Cell, 2006Co-Authors: Young H Kang, Jungeun Park, Nak Kyun Soung, Sang Moon Yun, Jeong K Bang, Yeon Sun Seong, Susan H Garfield, Timothy D Veenstra, Kyung S LeeAbstract:The polo-box domain (PBD) of mammalian polo-like kinase 1 (PLK1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying PLK1 recruitment to the kinetochores and the role of PLK1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting PLK1 to the interphase and mitotic kinetochores. Unprecedentedly, PLK1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of PLK1, but not Plk2 or Plk3. Later in mitosis, PLK1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent PLK1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, PLK1 self-regulates the PLK1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.
Erich A Nigg - One of the best experts on this subject based on the ideXlab platform.
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the PLK1 dependent phosphoproteome of the early mitotic spindle
Molecular & Cellular Proteomics, 2011Co-Authors: Anna Santamaria, Herman H W Sillje, Roman Korner, Bin Wang, Sabine Elowe, Rainer Malik, Feng Zhang, Manuel Bauer, Alexander Schmidt, Erich A NiggAbstract:Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (PLK1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of PLK1, the full range of PLK1 substrates remains to be explored. To investigate the PLK1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with PLK1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique PLK1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the PLK1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the PLK1 consensus motif. Collectively, our data provide a rich source of information on PLK1-dependent phosphorylation, PLK1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between PLK1 and Aurora A on the early mitotic spindle.
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phosphorylation of mitotic kinesin like protein 2 by polo like kinase 1 is required for cytokinesis
Journal of Cell Biology, 2003Co-Authors: Riidiger Neef, Erich A Nigg, Robe Kopajtich, Christian Preisinger, Josephine Sutcliffe, Thomas U Mayer, Francis A BarrAbstract:We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (PLK1). MKlp2 is a target for PLK1, and phosphorylated MKlp2 binds to the polo box domain of PLK1. PLK1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of PLK1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by PLK1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by PLK1 is necessary for the spatial restriction of PLK1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.
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antibody microinjection reveals an essential role for human polo like kinase 1 PLK1 in the functional maturation of mitotic centrosomes
Journal of Cell Biology, 1996Co-Authors: Heidi A Lane, Erich A NiggAbstract:Mammalian polo-like kinase 1 (PLK1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human PLK1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of PLK1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-PLK1 antibodies (PLK1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of PLK1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in PLK1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that PLK1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for PLK1 at later stages of mitosis are not excluded, although injection of PLK1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of PLK1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of PLK1 function.
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cell cycle analysis and chromosomal localization of human PLK1 a putative homologue of the mitotic kinases drosophila polo and saccharomyces cerevisiae cdc5
Journal of Cell Science, 1994Co-Authors: Roy M Golsteyn, Sharon J Schultz, Jiri Bartek, Andrew Ziemiecki, Thomas Ried, Erich A NiggAbstract:polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regulating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as PLK1 (for polo-like kinase 1). The largest open reading frame of the PLK1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of PLK1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single PLK1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, consistent with a possible function of PLK1 in cell proliferation. The PLK1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignancies are known. The PLK1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of PLK1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human PLK1 is likely to function in cell cycle progression.
