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Peter K Jackson - One of the best experts on this subject based on the ideXlab platform.

  • a specific form of phospho protein phosphatase 2 regulates anaphase promoting Complex cyclosome association with spindle poles
    Molecular Biology of the Cell, 2010
    Co-Authors: Jorge Z Torres, Kenneth Hon Kim Ban, Peter K Jackson
    Abstract:

    In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the Anaphase-Promoting Complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

  • the end network couples spindle pole assembly to inhibition of the anaphase promoting Complex cyclosome in early mitosis
    Developmental Cell, 2007
    Co-Authors: Kenneth H K Ban, Conly L Rieder, Julie J Miller, Maxence V Nachury, Jeffrey J Tung, Jorge Z Torres, Alexei Mikhailov, Peter K Jackson
    Abstract:

    Summary Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the Anaphase-Promoting Complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin Complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.

  • oncogenic regulators and substrates of the anaphase promoting Complex cyclosome are frequently overexpressed in malignant tumors
    American Journal of Pathology, 2007
    Co-Authors: Norman L Lehman, Robert Tibshirani, Yasodha Natkunam, Brent T Harris, Robert B West, Marilyn Masek, Kelli Montgomery, Matt Van De Rijn, Peter K Jackson
    Abstract:

    The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting Complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a “mitotic profile” in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.

  • emi1 stably binds and inhibits the anaphase promoting Complex cyclosome as a pseudosubstrate inhibitor
    Genes & Development, 2006
    Co-Authors: Julie J Miller, Matthew K Summers, David V Hansen, Maxence V Nachury, Norman L Lehman, Alex V Loktev, Peter K Jackson
    Abstract:

    The periodic destruction of mitotic cyclins is triggered by the activation of the Anaphase-Promoting Complex/cyclosome (APC/C) in mitosis. Although the ability of the APC/C to recognize destruction box (D-box) substrates oscillates throughout the cell cycle, the mechanism regulating APC/C binding to D-box substrates remains unclear. Here, we show that the APC/C inhibitor Emi1 tightly binds both the APC/C and its Cdh1 activator, binds to the D-box receptor site on the APC/CCdh1, and competes with APC/C substrates for D-box binding. Emi1 itself contains a conserved C-terminal D-box, which provides APC/C-binding affinity, and a conserved zinc-binding region (ZBR), which antagonizes APC/C E3 ligase activity independent of tight APC binding. Mutation of the ZBR converts Emi1 into a D-box-dependent APC/C substrate. The identification of a direct Emi1–APC/C Complex further explains how Emi1 functions as a stabilizing factor for cyclin accumulation and the need to destroy Emi1 for APC/C activation in mitosis. The combination of a degron/E3 recognition site and an anti-ligase function in Emi1 suggests a general model for how E3 substrates evolve to become pseudosubstrate inhibitors.

  • a role for the anaphase promoting Complex inhibitor emi2 xerp1 a homolog of early mitotic inhibitor 1 in cytostatic factor arrest of xenopus eggs
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Jeffrey J Tung, Matthew K Summers, David V Hansen, Alexander V Loktev, John R Adler, Peter K Jackson
    Abstract:

    Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the Anaphase-Promoting Complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCFβTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.

Janmichael Peters - One of the best experts on this subject based on the ideXlab platform.

  • structure of the anaphase promoting Complex cyclosome interacting with a mitotic checkpoint Complex
    Science, 2009
    Co-Authors: Franz Herzog, Ivana Primorac, B. Sander, Peter Lenart, Karl Mechtler, Prakash Dube, Janmichael Peters
    Abstract:

    Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the Anaphase-Promoting Complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/CCdc20). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint Complex (MCC) that inhibits APC/CCdc20. We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a “closed” state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.

  • the anaphase promoting Complex cyclosome a machine designed to destroy
    Nature Reviews Molecular Cell Biology, 2006
    Co-Authors: Janmichael Peters
    Abstract:

    The anaphase promoting Complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most Complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa Complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.

  • mitotic regulation of the human anaphase promoting Complex by phosphorylation
    The EMBO Journal, 2003
    Co-Authors: Claudine Kraft, Franz Herzog, Karl Mechtler, Christian Gieffers, Anja Hagting, Jonathon Pines, Janmichael Peters
    Abstract:

    The anaphase‐promoting Complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. We have identified 43 phospho‐sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro , at least 15 of the mitotic phospho‐sites can be generated by cyclin‐dependent kinase 1 (Cdk1), and 3 by Polo‐like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho‐antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho‐APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo . These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.

  • tpr subunits of the anaphase promoting Complex mediate binding to the activator protein cdh1
    Current Biology, 2003
    Co-Authors: Hartmut C Vodermaier, Christian Gieffers, Sebastian Maurerstroh, Frank Eisenhaber, Janmichael Peters
    Abstract:

    Abstract Background: Chromosome segregation and mitotic exit depend on activation of the Anaphase-Promoting Complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. Results: We have biochemically characterized subComplexes of the human APC. One subComplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subComplex contains all known APC subunits except APC2/11. This subComplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. Conclusions: APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. In analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10.

  • the anaphase promoting Complex proteolysis in mitosis and beyond
    Molecular Cell, 2002
    Co-Authors: Janmichael Peters
    Abstract:

    Key events in mitosis such as sister chromatid separation and subsequent inactivation of cyclin-dependent kinase 1 are regulated by ubiquitin-dependent proteolysis. These events are mediated by the Anaphase-Promoting Complex (APC), a cell cycle-regulated ubiquitin ligase that assembles multiubiquitin chains on regulatory proteins such as securin and cyclins and thereby targets them for destruction by the 26S proteasome.

Michael Rape - One of the best experts on this subject based on the ideXlab platform.

  • gene expression and cell identity controlled by anaphase promoting Complex
    Nature, 2020
    Co-Authors: Eugene Oh, Edmond R Watson, Kevin G Mark, Annamaria Mocciaro, Rajan J Prabu, Martin Kampmann, Nathan Gamarra, Coral Y Zhou, Michael Rape
    Abstract:

    Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2–5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the Anaphase-Promoting Complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division. WDR5 and TBP recruit Anaphase-Promoting Complex to specific transcription start sites in mitosis, initiating a ubiquitin-dependent mechanism that preserves cell identity by linking gene expression and cell division.

  • regulated degradation of spindle assembly factors by the anaphase promoting Complex
    Molecular Cell, 2010
    Co-Authors: Ling Song, Michael Rape
    Abstract:

    Summary The ubiquitin ligase Anaphase-Promoting Complex (APC/C) is essential for cell division in all eukaryotes. Loss of APC/C activity arrests cells at metaphase and results in severe aberrations of the mitotic spindle, but how the APC/C regulates spindle formation is not understood. Here, we report that the APC/C promotes the ubiquitination and degradation of four proteins required for Ran-dependent spindle assembly: Bard1, Hmmr, HURP, and NuSAP. Among these substrates, HURP and NuSAP can be degraded during spindle formation when the spindle checkpoint is active. Their degradation requires additional layers of regulation, and both SAFs are only degraded after being released from their inhibitor importin β by Ran GTP . Our findings reveal a tightly regulated mechanism by which the APC/C and the GTPase Ran control the abundance of active spindle assembly factors to achieve the accurate formation of the mitotic spindle.

  • identification of a physiological e2 module for the human anaphase promoting Complex
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Adam Williamson, Katherine E Wickliffe, Barbara G Mellone, Ling Song, Gary H Karpen, Michael Rape
    Abstract:

    Ubiquitination by the Anaphase-Promoting Complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.

  • mechanism of ubiquitin chain formation by the human anaphase promoting Complex
    Cell, 2008
    Co-Authors: Adam Williamson, Sudeep Banerjee, Isabelle Philipp, Michael Rape
    Abstract:

    Summary The Anaphase-Promoting Complex (APC/C) orchestrates progression through mitosis by decorating cell-cycle regulators with ubiquitin chains. To nucleate chains, the APC/C links ubiquitin to a lysine in substrates, but to elongate chains it modifies lysine residues in attached ubiquitin moieties. The mechanism enabling the APC/C, and ubiquitin ligases in general, to switch from lysine residues in substrates to specific ones in ubiquitin remains poorly understood. Here, we determine the topology and the mechanism of assembly for the ubiquitin chains mediating functions of the human APC/C. We find that the APC/C triggers substrate degradation by assembling K11-linked ubiquitin chains, the efficient formation of which depends on a surface of ubiquitin, the TEK-box. Strikingly, homologous TEK-boxes are found in APC/C substrates, where they facilitate chain nucleation. We propose that recognition of similar motifs in substrates and ubiquitin enables the APC/C to assemble ubiquitin chains with the specificity and efficiency required for tight cell-cycle control.

  • ubiquitination by the anaphase promoting Complex drives spindle checkpoint inactivation
    Nature, 2007
    Co-Authors: Michael Rape, Sashank Reddy, W A Margansky, Marc W Kirschner
    Abstract:

    During cell division the spindle checkpoint ensures that chromosome segregation is delayed until all chromosomes are properly attached to the mitotic spindle. Two papers now identify a new regulatory mechanism that controls the spindle checkpoint. This involves the fine-tuned ubiquitination and de-ubiquitination of a coactivator of the anaphase promoting Complex APC/C to regulate the timing of APC/C activation and thereby the onset of anaphase. One of two papers that identify a new regulatory mechanism that controls the spindle checkpoint. This involves the fine-tuned ubiquitination and de-ubiquitination of a co-activator of the anaphase promoting Complex APC to regulate the timing of APC activation and thereby the onset of anaphase. Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle1,2,3. Checkpoint proteins tightly inhibit the Anaphase-Promoting Complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes4. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme5. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes6.

Marc W Kirschner - One of the best experts on this subject based on the ideXlab platform.

  • specificity of the anaphase promoting Complex a single molecule study
    Science, 2015
    Co-Authors: Ying Lu, Weiping Wang, Marc W Kirschner
    Abstract:

    Biological processes require specific enzymatic reactions, paradoxically involving short recognition sequences. As an example, cell-cycle timing depends on a sequence of ubiquitylation events mediated by the Anaphase-Promoting Complex (APC) based on short redundant motifs. To understand the origin of specificity, we designed single-molecule fluorescence assays that capture transient ubiquitylation reactions. We find that the APC-mediated ubiquitylation involves a highly processive initial reaction on the substrate, followed by multiple encounters and reactions at a slower rate. The initial ubiquitylation greatly enhances the substrate's binding affinity in subsequent reactions, by both increasing the on-rate and decreasing the off-rate. We postulate that these cycles of positive feedback enable high specificity for substrates with short recognition motifs in a Complex cellular environment.

  • emi1 preferentially inhibits ubiquitin chain elongation by the anaphase promoting Complex
    Nature Cell Biology, 2013
    Co-Authors: Weiping Wang, Marc W Kirschner
    Abstract:

    The Emi1 protein inhibits the anaphase promoting Complex (APC), an E3 ubiquitin ligase and critical cell cycle regulator, but it has remained unclear how it does this. Wang and Kirschner demonstrate that Emi1 suppresses the elongation of ubiquitin chains on APC substrates by interfering in different ways with the activities of the E2 enzymes UBCH10 and Ube2S.

  • different phosphorylation states of the anaphase promoting Complex in response to antimitotic drugs a quantitative proteomic analysis
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Judith A Steen, Hanno Steen, Ann Georgi, Kenneth C Parker, Michael Springer, Marc Kirchner, Fred A Hamprecht, Marc W Kirschner
    Abstract:

    The anaphase promoting Complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the Complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy.

  • ubiquitination by the anaphase promoting Complex drives spindle checkpoint inactivation
    Nature, 2007
    Co-Authors: Michael Rape, Sashank Reddy, W A Margansky, Marc W Kirschner
    Abstract:

    During cell division the spindle checkpoint ensures that chromosome segregation is delayed until all chromosomes are properly attached to the mitotic spindle. Two papers now identify a new regulatory mechanism that controls the spindle checkpoint. This involves the fine-tuned ubiquitination and de-ubiquitination of a coactivator of the anaphase promoting Complex APC/C to regulate the timing of APC/C activation and thereby the onset of anaphase. One of two papers that identify a new regulatory mechanism that controls the spindle checkpoint. This involves the fine-tuned ubiquitination and de-ubiquitination of a co-activator of the anaphase promoting Complex APC to regulate the timing of APC activation and thereby the onset of anaphase. Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle1,2,3. Checkpoint proteins tightly inhibit the Anaphase-Promoting Complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes4. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme5. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes6.

  • sororin a substrate of the anaphase promoting Complex is required for sister chromatid cohesion in vertebrates
    Molecular Cell, 2005
    Co-Authors: Susannah Rankin, Nagi G Ayad, Marc W Kirschner
    Abstract:

    We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through Anaphase-Promoting Complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin Complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin Complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.

Guowei Fang - One of the best experts on this subject based on the ideXlab platform.

  • anaphase promoting Complex cyclosome controls the stability of tpx2 during mitotic exit
    Molecular and Cellular Biology, 2005
    Co-Authors: Scott Stewart, Guowei Fang
    Abstract:

    TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the Anaphase-Promoting Complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.

  • anillin is a substrate of anaphase promoting Complex cyclosome apc c that controls spatial contractility of myosin during late cytokinesis
    Journal of Biological Chemistry, 2005
    Co-Authors: Weimeng Zhao, Guowei Fang
    Abstract:

    Abstract Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the Anaphase-Promoting Complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/CCdh1 increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.

  • checkpoint protein bubr1 acts synergistically with mad2 to inhibit anaphase promoting Complex
    Molecular Biology of the Cell, 2002
    Co-Authors: Guowei Fang
    Abstract:

    Abstract The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the Anaphase-Promoting Complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive Complexes, a lower affinity Complex with Mad2 and a higher affinity Complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.

  • direct binding of cdc20 protein family members activates the anaphase promoting Complex in mitosis and g1
    Molecular Cell, 1998
    Co-Authors: Guowei Fang, Hongtao Yu, Marc W Kirschner
    Abstract:

    Abstract Activation of the Anaphase-Promoting Complex (APC) is required for anaphase initiation and for exit from mitosis. We show that APC is activated during mitosis and G1 by two regulatory factors, hCDC20 and hCDH1. These proteins directly bind to APC and activate its cyclin ubiquitination activity. hCDC20 confers a strict destruction-box (D-box) dependence on APC, while hCDH1 shows a much more relaxed specificity for the D-box. In HeLa cells, the protein levels of hCDC20 as well as its binding to APC peak in mitosis and decrease drastically at early G1. Thus, hCDC20 is the mitotic activator of APC and directs the degradation of substrates containing the D-box. The hCDH1 protein level remains constant during the cell cycle and may target specific substrates lacking the D-box in G1, such as polo-like kinase, for ubiquitination.