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At least 163 records · Page 9Linked to original sources
PubMed2002

The anaphase-promoting complex: proteolysis in mitosis and beyond.

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.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex.

Cell-cycle transitions are driven by waves of ubiquitin-dependent degradation of key cell-cycle regulators. SCF (Skp1/Cullin/F-box protein) complexes and anaphase-promoting complexes (APC) represent two major classes of ubiquitin ligases whose activities are thought to regulate primarily the G1/S and metaphase/anaphase cell-cycle transitions, respectively. The major target of the Skp1/Cul1/Skp2 (SCF(SKP2)) complex is thought to be the Cdk inhibitor p27 during S phase, whereas the principal targets for the APC are thought to be involved in chromatid separation (securin) and exit from mitosis (cyclin B). Although the role of the APC in mitosis is relatively clear, there is mounting evidence that APCs containing Cdh1 (APC(CDH1)) also have a function in the G1 phase of the cell cycle. Here, we show that the F-box protein Skp2 is polyubiquitinated, and hence earmarked for destruction, by APC(CDH1). As a result, accumulation of SCF(SKP2) requires prior inactivation of APC(CDH1). These findings provide an insight into the orchestration of SCF and APC activities during cell-cycle progression, and into the involvement of the APC in G1.

Amino Acid Motifs↗
PubMed2005

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation.

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

The Doc1 subunit is a processivity factor for the anaphase-promoting complex.

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity.

Anaphase↗
PubMed2005

Anaphase-promoting complex-dependent proteolysis of cell cycle regulators and genomic instability of cancer cells.

Genomic instability can be found in most cancer cells. Cell proliferation is under tight control to ensure accurate DNA replication and chromosome segregation. Cyclin-dependent kinases (Cdks) and their activating subunits, the cyclins, are the driving forces of the cell division cycle. Regulation of cyclin oscillation by ubiquitin-dependent proteolysis thereby has a central role in cell cycle regulation. The anaphase-promoting complex (APC) is a specific ubiquitin ligase and is essential for chromosome segregation, exit from mitosis and a stable subsequent G1 phase allowing cell differentiation or accurate DNA replication in the following S phase. The APC is activated by the regulatory subunits Cdc20 (APC(Cdc20)) and Cdh1 (APC(Cdh1)) to target securin, mitotic cyclins and other cell cycle regulatory proteins for proteasomal degradation. This review is focused on the role of APC-dependent proteolysis in cell cycle regulation and how its deregulation may lead to genomic instability of cancer cells.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair.

BACKGROUND: In mammals, proteins containing BIR domains (IAPs and survivin) are implicated in inhibiting apoptosis and sister chromatid separation. In the nematode, Bir1 is required for a proper localization of aurora kinase, which moves from the mitotic chromosome in metaphase to the spindle midzone in anaphase as a passenger. Fission yeast Bir1/Pbh1 is essential for normal mitosis. RESULTS: A temperature sensitive mutant cut17-275 exhibits the defect in condensation and spindle elongation at 36 degrees C, while securin is degraded. Gene cloning shows that the cut17+ gene is identical to bir1+/pbh1+. At 26 degrees C, cut17-275 is UV sensitive as the repair of DNA damage is severely compromised. Bir1/Cut17 is a nuclear protein in interphase, which is then required for recruiting condensin to the mitotic nucleus, and concentrates to form a discrete number of dots from prometaphase to metaphase. Once the chromatids are separated, Bir1/Cut17 no longer binds to kinetochores and instead moves to the middle of spindle. Chromatin immunoprecipitation suggested that Bir1/Cut17 associates with the outer repetitious centromere region in metaphase. Following the initiation of anaphase the protein switches from being a chromosomal protein to a spindle protein. This transit is stringently regulated by the state of sister chromatid cohesion proteins Mis4 and Rad21. Ark1, is an aurora kinase homologue whose mitotic distribution is identical to, and under the control of Bir1/Cut17. CONCLUSIONS: Bir1/Cut17 and Ark1 act as "passengers" but they may play a main role as a recruitment factor, essential for condensation, spindle elongation and DNA repair. Bir1/Cut17 should have roles both in mitotic and in interphase chromosome. The proper location of Ark1 requires Bir1/Cut17, and the mitotic localization of Bir1/Cut17 requires sister cohesion.

Amino Acid Sequence↗
PubMed2001

Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a.

Two forms of the anaphase-promoting complex (APC) mediate the degradation of critical cell cycle regulators. APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis. Here we show that phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC. A human homologue of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1). In contrast, hCdc14a does not affect the activity of APC(Cdc20). hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells. Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

Structural and functional analysis of the human mitotic-specific ubiquitin-conjugating enzyme, UbcH10.

Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-A crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH2-terminal extension in UbcH10 is disordered and that a conserved 3(10)-helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH2-terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.

Amino Acid Motifs↗
PubMed2005

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase.

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.

Anaphase↗
PubMed2002

Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells.

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20-GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance.

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.

Calcium-Binding Proteins↗
PubMed2001

Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint.

The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.

Animals↗
PubMed2004

Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation.

The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome. APC-Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins. APC-Cdh1 promotes degradation of cyclins and other proteins during G(1). We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5). To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice. Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure. Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced. We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of APC/C activity.

Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. It was previously shown that the Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation remained unclear, the existence of a putative APC/C inhibitor was postulated whose activity would be neutralized by Plx1 upon CSF release. Here we identify XErp1, a novel Plx1-regulated inhibitor of APC/C activity, and we demonstrate that XErp1 is required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, our data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation.

Amino Acid Sequence↗
PubMed2001

Inhibition of Cdh1-APC by the MAD2-related protein MAD2L2: a novel mechanism for regulating Cdh1.

Exit from mitosis requires the degradation of regulatory proteins including the mitotic cyclins and securin through ubiquitination by the anaphase promoting complex (APC) bound to Cdc20 or Cdh1. Cdc20-APC is regulated through inhibition by the spindle assembly checkpoint protein MAD2. Knowledge of Cdh1-APC regulation is limited to the phosphorylation-dependent dissociation of Cdh1 from APC. We report a novel means of regulating Cdh1 by the MAD2-related gene, MAD2L2. MAD2L2 specifically binds and inhibits Cdh1-APC, paralleling the effect of MAD2 on Cdc20. We suggest that MAD2L2 and MAD2 inhibit the release of substrates from APC and propose a mechanism of inhibition.

Adaptor Proteins, Signal Transducing↗