Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the ubiquitination and degradation patterns of the human securin/PTTG protein. We show that, in contrast to budding yeast pds1, securin degradation is catalyzed by both fzy (fizzy/cdc20) and fzr (fizzy-related/cdh1/hct1). Both fzy and fzr also induce the APC/C to ubiquitinate securin in vitro. Securin degradation is mediated by an RXXL destruction box and a KEN box, and is inhibited only when both sequences are mutated. Interestingly, the non-degradable securin mutant is also partially ubiquitinated by fzy and fzr in vitro. Expressing the non-degradable securin mutant in cells frequently resulted in incomplete chromatid separation and gave rise to daughter cells connected by a thin chromatin fiber, presumably of chromosomes that failed to split completely. Strikingly, the mutant securin did not prevent the majority of sister chromatids from separating completely, nor did it prevent mitotic cyclin degradation and cytokinesis. This phenotype, reminiscent of the fission yeast cut (cells untimely torn) phenotype, is reported here for the first time in mammals.
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PMID:Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis. 1117 23

Nek2 is a NIMA-related kinase implicated in regulating centrosome structure at the G(2)/M transition. Two splice variants have been identified that exhibit distinct patterns of expression during cell cycle progression and development. Here we show that Nek2A, but not Nek2B, is destroyed upon entry into mitosis coincident with cyclin A destruction and in the presence of an active spindle assembly checkpoint. Destruction of Nek2A is mediated by the proteasome and is dependent upon the APC/C-Cdc20 ubiquitin ligase. Nek2 activity is not required for APC/C activation. Nek2A destruction in early mitosis is regulated by a motif in its extreme C-terminus which bears a striking resemblance to the extended destruction box (D-box) of cyclin A. Complete stabilization of Nek2A requires deletion of this motif and mutation of a KEN-box. Destruction of Nek2A is not inhibited by the cyclin B-type D-box, but the C-terminal domain of Nek2A inhibits destruction of both cyclins A and B. We propose that recognition of substrates by the APC/C-Cdc20 in early mitosis depends upon possession of an extended D-box motif.
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PMID:APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box. 1174 88

Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, which are key regulators of the structure and function of the mitotic spindle. We previously showed that human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity.
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PMID:Degradation of human Aurora-A protein kinase is mediated by hCdh1. 1202 18

The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G(1). We studied how d-boxes determine APC/C(fzy)/APC/C(fzr) specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/C(fzy) and APC/C(fzr); fzy has a KEN box and is degraded by APC/C(fzr) only. We characterized the degradation of substrates with swapped d-boxes. Cyclin B1 with KEN was degraded by APC/C(fzr) only. Fzy with RXXL could be degraded by APC/C(fzy) and APC/C(fzr). Interestingly, APC/C(fzy)- but not APC/C(fzr)-specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/C(fzr) is activated in early G(1). These observations demonstrate how d-box specificities of APC/C(fzy) and APC/C(fzr), and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/C(fzy), while others are restricted to APC/C(fzr).
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PMID:Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes. 1219 52

The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C(Cdh1). The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.
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PMID:Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit. 1220 50

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment.
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PMID:Dual mode of degradation of Cdc25 A phosphatase. 1223 27

Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, we show here that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, we also show that PIM contains a KEN-box, which is required for mitotic degradation in addition to the D-box, and that sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals.
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PMID:Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation. 1272 52

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.
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PMID:Mitotic degradation of human thymidine kinase 1 is dependent on the anaphase-promoting complex/cyclosome-CDH1-mediated pathway. 1470 26

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.
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PMID:5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. 2971 69

The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.
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PMID:Mechanism of Aurora-B degradation and its dependency on intact KEN and A-boxes: identification of an aneuploidy-promoting property. 1592 16


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