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Insights into anaphase promoting complex TPR subdomain assembly from a CDC26-APC6 structure.

ABSTRACT: The multisubunit anaphase promoting complex (APC) is an essential cell-cycle regulator. Although CDC26 is known to have a role in APC assembly, its molecular function has remained unclear. Biophysical, structural and genetic studies presented here reveal that CDC26 stabilizes the structure of APC6, a core TPR protein required for APC integrity. Notably, CDC26-APC6 association involves an intermolecular TPR mimic composed of one helix from each protein.

SUBMITTER: Wang J

PROVIDER: S-EPMC2759704 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Insights into anaphase promoting complex TPR subdomain assembly from a CDC26-APC6 structure.

Wang Jing J Dye Billy T BT Rajashankar Kanagalaghatta R KR Kurinov Igor I Schulman Brenda A BA

Nature structural & molecular biology 20090809 9

The multisubunit anaphase promoting complex (APC) is an essential cell-cycle regulator. Although CDC26 is known to have a role in APC assembly, its molecular function has remained unclear. Biophysical, structural and genetic studies presented here reveal that CDC26 stabilizes the structure of APC6, a core TPR protein required for APC integrity. Notably, CDC26-APC6 association involves an intermolecular TPR mimic composed of one helix from each protein. ...[more]

PMID: 19668213

Similar Datasets

Structure of an APC3-APC16 complex: insights into assembly of the anaphase-promoting complex/cyclosome.

Project description:The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the "Platform" centers around a cullin-RING-like E3 ligase catalytic core; the "Arc Lamp" is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.

| S-EPMC4444369 | biostudies-literature

Regulated degradation of spindle assembly factors by the anaphase-promoting complex.

Project description: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 beta 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.

| S-EPMC2883244 | biostudies-literature

Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex.

Project description: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/C(Cdc20)). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C(Cdc20). 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.

| S-EPMC2989460 | biostudies-literature

Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly.

Project description:Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

| S-EPMC4272865 | biostudies-literature

Crystal structure of the N-terminal domain of anaphase-promoting complex subunit 7.

Project description:Anaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 A. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C.

| S-EPMC2685695 | biostudies-literature

Anaphase-promoting complex/cyclosome controls HEC1 stability.

Project description:ObjectiveChromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit.Materials and methods and resultsTo investigate how HEC1 protein is controlled post-translation, we analysed the role of anaphase-promoting complex/cyclosome (APC/C)-Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin-proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C-hCdh1 E3 ligase. The evolutionally conserved D-box at the C-terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C-Cdh1. Overexpression of non-degradable HEC1 (D-box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest.ConclusionAPC/C-Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.

| S-EPMC6496518 | biostudies-literature

Anaphase-promoting complex-dependent control of cell identity

Project description:Tissue development and homeostasis rely on the proliferation of progenitor cells, whose identities are established by tightly controlled gene expression networks. However, mRNA synthesis is inhibited during mitosis, and the transcriptional programs that define a cell type must be restarted upon entry into each new cell cycle; how this is accomplished is poorly understood. Here, we identified a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active promoters during interphase, recruit the E3 ligase anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows the APC/C to locally decorate histones with K11/K48-linked ubiquitin chains, which destabilizes nucleosomes and ensures continued gene expression in the next cell cycle. Mitotic exit and transcription re-initiation are therefore controlled by the same essential regulator, the APC/C, which provides a robust mechanism to maintain cell identity through cell division.

2020-02-03 | GSE122298 | GEO

Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound.

Project description:BackgroundAnaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20.Materials and methodsThe structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated.ResultsIt was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown.ConclusionThe findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds.

| S-EPMC5359995 | biostudies-literature

Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1.

Project description:Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.

| S-EPMC549351 | biostudies-literature

The anaphase-promoting complex coordinates initiation of lens differentiation.

Project description:Lens development requires the precise coordination of cell division and differentiation. The mechanisms by which the differentiation program is initiated after cell cycle arrest remains not well understood. Cyclin-dependent kinase inhibitors (CKIs), such as p15 and p21, have been suggested to be critical components that inhibit G1 progression and therefore, their activation is necessary for quiescence and important for the onset of differentiation. Regulation of p15 and p21 is principally governed by transforming growth factor (TGF)-beta-signaling pathway. We have identified that Cdh1/APC, a critical ubiquitin protein ligase, plays an important role in regulating lens differentiation by facilitating TGF-beta-induced degradation of SnoN, a transcriptional corepressor that needs to be removed for transcriptional activation of p15 and p21. The depletion of Cdh1 by RNA interference attenuates the TGF-beta-mediated induction of p15 and p21 and significantly blocks lens differentiation. Expression of nondegradable SnoN also noticeably attenuates lens induction. Furthermore, we have shown that Cdh1 and SnoN form a complex at the onset of lens differentiation. In vivo histological analysis confirms our biochemical and genetic results. Thus, Cdh1/APC is crucial to the coordination of cell cycle progression and the initiation of lens differentiation through mediating TGF-beta-signaling-induced destruction of SnoN.

| S-EPMC1805114 | biostudies-literature

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