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Gary J Gorbsky - One of the best experts on this subject based on the ideXlab platform.
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Differences in spindle association of the mitotic checkpoint Protein Mad2 in mammalian spermatogenesis and oogenesis.
Developmental biology, 2000Co-Authors: Marko J Kallio, John E. Eriksson, Gary J GorbskyAbstract:We have investigated expression and subcellular localization of the spindle checkpoint Protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.
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mammalian p55cdc mediates association of the spindle checkpoint Protein Mad2 with the cyclosome anaphase promoting complex and is involved in regulating anaphase onset and late mitotic events
Journal of Cell Biology, 1998Co-Authors: Marko J Kallio, Jasminder Weinstein, Joseph R Daum, Daniel J. Burke, Gary J GorbskyAbstract:We have investigated the function of p55CDC, a mammalian Protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
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Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events
The Journal of cell biology, 1998Co-Authors: Marko J Kallio, Jasminder Weinstein, Joseph R Daum, Daniel J. Burke, Gary J GorbskyAbstract:We have investigated the function of p55CDC, a mammalian Protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
Xuelian Luo - One of the best experts on this subject based on the ideXlab platform.
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Mechanistic insight into TRIP13-catalyzed Mad2 structural transition and spindle checkpoint silencing
Nature Communications, 2017Co-Authors: Melissa L. Brulotte, Byung Cheon Jeong, Chad A. Brautigam, Xuelian LuoAbstract:The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint Protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2–Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding Protein p31comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31comet–Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13–p31comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region. The spindle checkpoint ensures the fidelity of chromosome segregation during mitosis and meiosis. Here the authors use a combination of biochemical and structural biology approaches to show how the TRIP13 ATPase and its adaptor, p31comet, catalyze the conversion of the checkpoint Protein Mad2 between latent and active forms
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Structure of an intermediate conformer of the spindle checkpoint Protein Mad2
Proceedings of the National Academy of Sciences, 2015Co-Authors: Mayuko Hara, Hongbin Sun, Engin Özkan, Xuelian LuoAbstract:The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint Protein mitotic arrest deficient 2 (Mad2) is an unusual Protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.
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Phosphorylation of the spindle checkpoint Protein Mad2 regulates its conformational transition
Proceedings of the National Academy of Sciences, 2010Co-Authors: Soonjoung Kim, Katja Wassmann, Hongbin Sun, Haydn L. Ball, Xuelian LuoAbstract:Regulated conformational changes of Proteins are critical for cellular signal transduction. The spindle checkpoint Protein Mad2 is an unusual Protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 Protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.
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Insights Into Mad2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric Mad2 Dimer.
PLoS Biology, 2008Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Chyong Jy Liu, Xuelian LuoAbstract:In response to misaligned sister chromatids during mitosis, the spindle checkpoint Protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding Proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/CCdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.
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Purification and assay of Mad2: a two-state inhibitor of anaphase-promoting complex/cyclosome.
Methods in Enzymology, 2005Co-Authors: Xuelian LuoAbstract:To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase‐promoting complex or cyclosome (APC/C). The spindle checkpoint Protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two‐state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.
Andrea Musacchio - One of the best experts on this subject based on the ideXlab platform.
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HORMA domains at the heart of meiotic chromosome dynamics.
Developmental cell, 2014Co-Authors: Gerben Vader, Andrea MusacchioAbstract:HORMA domain Proteins are required for the careful orchestration of chromosomal organization during meiosis. Kim et al. (2014) and Silva et al. (2014) now provide structural and functional insights into the roles of C. elegans HORMA Proteins, revealing parallels to the function of the HORMA Protein Mad2 in mitotic checkpoint signaling.
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Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae
Journal of Cell Biology, 2006Co-Authors: Luigi Nezi, Anna De Antoni, Simonetta Piatti, Giulia Rancati, Sebastiano Pasqualato, Andrea MusacchioAbstract:The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint Protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. Mad2 mutant alleles impaired in this interaction fail to restore the SAC in a Mad2 deletion strain. The corresponding mutant Proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.
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accumulation of Mad2 cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in saccharomyces cerevisiae
Journal of Cell Biology, 2006Co-Authors: Luigi Nezi, Anna De Antoni, Simonetta Piatti, Giulia Rancati, Sebastiano Pasqualato, Andrea MusacchioAbstract:The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint Protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. Mad2 mutant alleles impaired in this interaction fail to restore the SAC in a Mad2 deletion strain. The corresponding mutant Proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.
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Explaining the oligomerization properties of the spindle assembly checkpoint Protein Mad2
Philosophical Transactions of the Royal Society B: Biological Sciences, 2005Co-Authors: Anna Deantoni, Valeria Sala, Andrea MusacchioAbstract:Mad2 is an essential component of the spindle assembly checkpoint (SAC), a molecular device designed to coordinate anaphase onset with the completion of chromosome attachment to the spindle. Capture of chromosome by microtubules occur on Protein scaffolds known as kinetochores. The SAC Proteins are recruited to kinetochores in prometaphase where they generate a signal that halts anaphase until all sister chromatid pairs are bipolarly oriented. Mad2 is a subunit of the mitotic checkpoint complex, which is regarded as the effector of the spindle checkpoint. Its function is the sequestration of Cdc20, a Protein required for progression into anaphase. The function of Mad2 in the checkpoint correlates with a dramatic conformational rearrangement of the Mad2 Protein. Mad2 adopts a closed conformation (C-Mad2) when bound to Cdc20, and an open conformation (O-Mad2) when unbound to this ligand. Checkpoint activation promotes the conversion of O-Mad2 to Cdc20-bound C-Mad2. We show that this conversion requires a C-Mad2 template and we identify this in Mad1-bound Mad2. In our proposition, Mad1-bound C-Mad2 recruits O-Mad2 to kinetochores, stimulating Cdc20 capture, implying that O-Mad2 and C-Mad2 form dimers. We discuss Mad2 oligomerization and link our discoveries to previous observations related to Mad2 oligomerization.
Marko J Kallio - One of the best experts on this subject based on the ideXlab platform.
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Differences in spindle association of the mitotic checkpoint Protein Mad2 in mammalian spermatogenesis and oogenesis.
Developmental biology, 2000Co-Authors: Marko J Kallio, John E. Eriksson, Gary J GorbskyAbstract:We have investigated expression and subcellular localization of the spindle checkpoint Protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.
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mammalian p55cdc mediates association of the spindle checkpoint Protein Mad2 with the cyclosome anaphase promoting complex and is involved in regulating anaphase onset and late mitotic events
Journal of Cell Biology, 1998Co-Authors: Marko J Kallio, Jasminder Weinstein, Joseph R Daum, Daniel J. Burke, Gary J GorbskyAbstract:We have investigated the function of p55CDC, a mammalian Protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
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Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events
The Journal of cell biology, 1998Co-Authors: Marko J Kallio, Jasminder Weinstein, Joseph R Daum, Daniel J. Burke, Gary J GorbskyAbstract:We have investigated the function of p55CDC, a mammalian Protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
Guowei Fang - One of the best experts on this subject based on the ideXlab platform.
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Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope
The Journal of cell biology, 2007Co-Authors: Oi Kwan Wong, Guowei FangAbstract:Accurate chromosome segregation is controlled by the spindle checkpoint, which senses kinetochore– microtubule attachments and tension across sister kinetochores. An important step in the tension-signaling pathway involves the phosphorylation of an unknown Protein by polo-like kinase 1/Xenopus laevis polo-like kinase 1 (Plx1) on kinetochores lacking tension to generate the 3F3/2 phosphoepitope. We report here that the checkpoint Protein BubR1 interacts with Plx1 and that phosphorylation of BubR1 by Plx1 generates the 3F3/2 epitope. Formation of the BubR1 3F3/2 epitope by Plx1 requires a prior phosphorylation of BubR1 on Thr 605 by cyclin-dependant kinase 1 (Cdk1). This priming phosphorylation of BubR1 by Cdk1 is required for checkpoint-mediated mitotic arrest and for recruitment of Plx1 and the checkpoint Protein Mad2 to unattached kinetochores. Biochemically, formation of the 3F3/2 phosphoepitope by Cdk1 and Plx1 greatly enhances the kinase activity of BubR1. Thus, Cdk1-mediated phosphorylation of BubR1 controls checkpoint arrest and promotes the formation of the kinetochore 3F3/2 epitope.
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Mad2B is an inhibitor of the anaphase-promoting complex
Genes & development, 2001Co-Authors: James Chen, Guowei FangAbstract:Anaphase-promoting complex (APC), a ubiquitin ligase, controls both sister chromatid separation and mitotic exit. The APC is activated in mitosis and G1 by CDC20 and CDH1, and inhibited by the checkpoint Protein Mad2, a specific inhibitor of CDC20. We show here that a Mad2 homolog Mad2B also inhibits APC. In contrast to Mad2, Mad2B inhibits both CDH1-APC and CDC20-APC. This inhibition is targeted to CDH1 and CDC20, but not directly to APC. Unlike Mad2, whose interaction with MAD1 is required for mitotic checkpoint control, Mad2B does not interact with MAD1, suggesting that Mad2B may relay a different cellular signal to APC.
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Structure of the Mad2 spindle assembly checkpoint Protein and its interaction with Cdc20.
Nature Structural Biology, 2000Co-Authors: Xuelian Luo, Guowei Fang, Marc W Kirschner, Melissa Coldiron, Yingxi Lin, Gerhard WagnerAbstract:The checkpoint Protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering Cdc20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with Cdc20. Mad2 possesses a novel three-layered α/β fold with three α-helices packed between two β-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human Cdc20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to Cdc20. Mad2 and Cdc20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.
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Control of mitotic transitions by the anaphase-promoting complex.
Philosophical Transactions of the Royal Society B, 1999Co-Authors: Guowei Fang, Hongtao Yu, Marc W KirschnerAbstract:Proteolysis controls key transitions at several points in the cell cycle. In mitosis, the activation of a large ubiquitin–Protein ligase, the anaphase–promoting complex (APC), is required for anaphase initiation and for exit from mitosis. We show that APC is under complex control by a network of regulatory factors, CDC20, CDH1 and Mad2. CDC20 and CDH1 are activators of APC; they bind directly to APC and activate its cyclin ubiquitination activity. CDC20 activates APC at the onset of anaphase in a destruction box (DB)–dependent manner, while CDH1 activates APC from late anaphase through G1 with apparently a much relaxed specificity for the DB. Therefore, CDC20 and CDH1 control both the temporal order of activation and the substrate specificity of APC, and hence regulate different events during mitosis and G1. Counteracting the effect of CDC20, the checkpoint Protein Mad2 acts as an inhibitor of APC. When the spindle–assembly checkpoint is activated, Mad2 forms a ternary complex with CDC20 and APC to prevent activation of APC, and thereby arrests cells at prometaphase. Thus, a combination of positive and negative regulators establishes a regulatory circuit of APC, ensuring an ordered progression of events through cell division.
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the checkpoint Protein Mad2 and the mitotic regulator cdc20 form a ternary complex with the anaphase promoting complex to control anaphase initiation
Genes & Development, 1998Co-Authors: Guowei Fang, Hongtao Yu, Marc W KirschnerAbstract:Cell cycle progression is monitored by surveillance mechanisms that ensure the integrity of the genome and the fidelity of chromosome segregation (Elledge 1996). The spindle assembly checkpoint mechanism is thought to monitor the attachment of the kinetochores of all chromosomes to the mitotic spindle and the tension exerted on the kinetochores by microtubules (Murray 1994, 1995; Rudner and Murray 1996; Nicklas 1997). For example, the presence of a single unattached kinetochore will activate the spindle assembly checkpoint and prevent chromatid separation, anaphase initiation, and loss of Cdc2 kinase activity. The spindle assembly checkpoint can thus prevent aneuploidy from improper chromosome separation. This checkpoint mechanism may also function in every cell cycle to determine the normal timing of anaphase initiation (Taylor and McKeon 1997). Microtubule destabilizing drugs, such as nocodazole, activate the spindle assembly checkpoint. Using these drugs, genetic studies have identified several components of the checkpoint pathway in budding yeast (Hoyt et al. 1991; Li and Murray 1991). Mutations in any of these genes, MAD1, Mad2, MAD3 and BUB1, BUB2, BUB3, cause aberrant mitosis in the presence of nocodazole and eventually lead to cell death. MPS1, a gene required for spindle pole body duplication, has also been shown to be involved in checkpoint control (Hardwick et al. 1996). A combination of genetic and biochemical studies has shown that Bub1p, Bub3p, and Mps1p act upstream of Mad1p and Mad2p, whereas Bub2p and Mad3p act either downstream of Mad1p and Mad2p, or in a parallel pathway (Elledge 1996; Rudner and Murray 1996). Human and Xenopus homologs of the yeast Mad2p, hMad2 and xMad2, have been cloned and shown to be required for checkpoint control (Chen et al. 1996; Li and Benezra 1996). In addition to a general nuclear distribution, Mad2 Protein localizes to only those kinetochores that have not attached to the mitotic spindle. The murine homolog of yeast Bub1p also localizes to unattached kinetochores during mitosis (Taylor and McKeon 1997). Those observations suggest an important role of kinetochore structure in sensing and transducing the checkpoint signal. Little is known about how the checkpoint signal is transduced to the cell cycle machinery, leading to arrest of cell division. Activation of the anaphasepromoting complex (APC), a ubiquitin-cyclin ligase required for degradation of mitotic cyclins and other cell cycle regulators, has been shown to be required for metaphase → anaphase transition and for exit from mitosis during the normal cell cycle (Holloway et al. 1993; Irniger et al. 1995; King et al. 1995; Sudakin et al. 1995; Tugendreich et al. 1995). Although there could be many points of intervention, it seems logical that APC itself might be a target for the checkpoint control. Recent studies in fission yeast have identified a genetic interaction between Mad2 and APC (He et al. 1997). In addition, hMad2 has been reported to associate with APC in nocodazole-arrested HeLa cells, and it has been proposed that this association may mediate cell cycle arrest in the checkpoint-activated cells (Li et al. 1997). However, the activity of hMad2-associated APC has not been measured directly, and therefore, the effect of this association on the APC activity remains to be determined. The activity of APC is cell cycle regulated; it is active from anaphase up to late G1, but not in S-phase or G2 (Amon et al. 1994; King et al. 1995; Brandeis and Hunt 1996; Peters et al. 1996; G. Fang, H. Yu, and M.W. Kirschner, in prep.). Its activity is in part controlled by the regulatory factors CDC20 and CDH1/HCT1 (Schwab et al. 1997; Sigrist and Lehner 1997; Visintin et al. 1997; G. Fang, H. Yu, and M.W. Kirschner, in prep.), and in part by cell cycle-specific phosphorylation (King et al. 1995; Lahav-Baratz et al. 1995; Peters et al. 1996). CDC20 and CDH1 are WD-40 containing Proteins, conserved from yeast to human (Weinstein et al. 1994; Dawson et al. 1995; Matsumoto 1997; Yamaguchi et al. 1997). Recently we have shown that the human homologs, hCDC20 and hCDH1, bind directly to and activate APC (G. Fang, H. Yu, and M.W. Kirschner, in prep.). Formation of the hCDC20–APC complex during mitosis correlates roughly with the activation of the ubiquitination machinery during mitosis. However, there is a period in G2 and M where both CDC20 and APC are present, but where cyclin proteolysis does not occur, suggesting the existence of additional regulatory mechanism. Recent genetic studies in yeast show that Cdc20p interacts with Mad2p and is a target for the checkpoint control (Hwang et al. 1998; Kim et al. 1998). The role of hCDH1 in the cell cycle is less clear. Presumably it has different functions from hCDC20 and most likely is responsible for APC activity in G1. In summary, the discovery of APC, and its regulators CDC20 and CDH1 have complicated our picture of the regulatory events during mitosis. To better understand control of mitosis and G1, it is necessary to examine the progression of different activity states of APC, and to investigate how each is regulated. Because APC might also be the principal effector of the spindle checkpoint control system, it is expected that some of the known checkpoint genes could intervene in specific parts of this APC network. We report here that APC is a target for checkpoint intervention. The checkpoint Protein hMad2 forms a ternary complex with hCDC20 and APC in vivo, and prevents activation of APC when the checkpoint signal is present. Anaphase is initiated by dissociation of hMad2 from the complex; APC is then activated by bound hCDC20. Later in mitosis, hCDH1 binds to and activates APC, which allows cells to exit from mitosis. The checkpoint-mediated inhibition of APC has been reconstituted in vitro with purified hMad2, hCDC20, and APC. Interestingly, we found that the recombinant hMad2 Protein exists in two different forms, a tetramer and a monomer. Only the tetramer inhibits activation of APC by hCDC20 in vitro, suggesting a possible role of a hMad2 structure change in transducing the checkpoint signal.
