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1.
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.  相似文献   

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By keeping the levels of Skp2 and Cks1 low during G1 progression, APC/CCdh1 prevents unscheduled degradation of SCFSkp2 substrates and premature entry into S phase. Thus, APC/CCdh1, a ubiquitin ligase involved in mitotic exit and maintenance of G0/G1 phase, directly controls SCFSKP2, a ubiquitin ligase involved in the regulation of S phase entry.  相似文献   

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Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).  相似文献   

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The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint   总被引:2,自引:0,他引:2  
In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint.  相似文献   

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Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G1/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/CCdh1) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs in two steps in vertebrate cells (1-3). In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and removed from chromosome arms (1, 4). Then, cleavage of centromeric cohesin by separase takes place at the metaphase-to-anaphase transition to allow sister-chromatid separation (5). The shugoshin (Sgo) family of proteins plays an important role in the protection of centromeric cohesion (6, 7). Human cells depleted of Sgo1 by RNAi undergo massive chromosome missegregation (8-11). In cells with compromised Sgo1 function, centromeric cohesin is improperly phosphorylated and removed (4, 11), resulting in premature sister-chromatid separation. It has been shown recently that Sgo1 collaborates with PP2A to counteract the action of Plk1 and other mitotic kinases and to protect centromeric cohesin from premature removal (12-14). In addition, Sgo1 has also been shown to promote stable kinetochore-microtubule attachment and sense tension across sister kinetochores (8, 15). Thus, Sgo1 is crucial for mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C),2 a large multiprotein ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome (16). APC/C selects substrates for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize specific sequences called APC/C degrons within target proteins (17). Several APC/C degrons have been characterized, including the destruction box (D box) and the Lys-Glu-Asn box (KEN box) (18, 19). The D box, with the consensus amino acid sequence of RXXLXXXN(X indicates any amino acid), are found in many APC/C substrates, including mitotic cyclins and are essential for their ubiquitin-mediated destruction. The KEN box, which contains a consensus KEN motif, is also found in several APC/C substrates and is preferentially but not exclusively recognized by APC/CCdh1. When APC/C is active, it directs progression through and exit from mitosis by catalyzing the ubiquitination and timely destruction of mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor securin (16). The APC/C activity needs to be tightly controlled to prevent unscheduled substrate degradation. An important mechanism for APC/C regulation is the spindle checkpoint, which prevents the activation of APC/C and destruction of its substrates in response to kinetochores that have not properly attached to the mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein levels oscillate during the cell cycle (8, 9). In this article we study the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during mitotic exit, and this degradation depends on APC/CCdh1. We further show that both KEN and D boxes are required for Sgo1 degradation in vivo and ubiquitination in vitro. Removal of these motifs stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1 protein in human cells does not change the kinetics of chromosome segregation and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes place but is not required for mitotic exit. Finally, we show that Bub1 regulates Sgo1 protein levels through a mechanism that does not involve APC/C-mediated degradation.  相似文献   

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Mammalian Cdh1/Fzr mediates its own degradation   总被引:4,自引:0,他引:4  
The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.  相似文献   

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We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/CCdh1, the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/CCdh1 is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/CCdh1 activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/CCdh1 activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.  相似文献   

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Receptor-associated protein 80 (RAP80) is a component of the BRCA1-A complex that recruits BRCA1 to DNA damage sites in the DNA damage-induced ubiquitin signaling pathway. RAP80-depleted cells showed defective G(2)-M phase checkpoint control. In this study, we show that RAP80 protein levels fluctuate during the cell cycle. Its expression level peaked in the G(2) phase and declined during mitosis and progression into the G(1) phase. Also, RAP80 is polyubiquitinated and degraded by the anaphase-promoting complex (APC/C)(Cdc20) or (APC/C)(Cdh1). Consistent with this, knockdown of Cdc20 or Cdh1 expression by transfecting with small interfering RNAs blocked RAP80 degradation during mitosis or the G(1) phase, respectively. A conserved destruction box (D box) in RAP80 affected its stability and ubiquitination, which was dependent on APC/cyclosome(Cdc20) (C(Cdc20)) or APC/cyclosome(Cdh1) (C(Cdh1)). In addition, overexpression of RAP80 destruction box1 deletion mutant attenuated mitotic progression. Thus, APC/C(Cdc20) or APC/C(Cdh1) complexes regulate RAP80 stability during mitosis to the G(1) phase, and these events are critical for a novel function of RAP80 in mitotic progression.  相似文献   

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The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).  相似文献   

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The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G(1) phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G(1) phase of the cell cycle in mammalian cells.  相似文献   

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