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PubMed2004

APC activators caught by their tails?

The complexity of the Anaphase-Promoting Complex (APC), the major ubiquitin ligase in mitotic control, has been puzzling investigators ever since its discovery. Recent biochemical and genetic studies have now provided insights not only into the architecture of the complex, but also into how activators are recruited to the APC. In this article, we discuss the implications of these findings on our current understanding of APC activation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1999

Genomic organization and expression of the ubiquitin-proteasome complex-associated protein Rbx1/ROC1/Hrt1.

Rbx1/ROC1/Hrt1 (Rbx1) has been recently shown to be involved in the regulation of protein turn-over. Here, we report the organization of the human Rbx1 gene, established by both a cloning and a functional genomics approach. The human gene, composed of five exons, encompasses 22.3 kb on chromosome 22q 13. The expression of Rbx1 transcripts (0.5 kb) appears developmentally regulated during mouse embryonic development and is prevalent in the adult mouse genital tract. A Genbank database search for Rbx1 related sequences in various species, from plants to mammals, is indicative of a high degree of evolutionary conservation in mouse rat and zebra fish and also, for the main functional part of the molecule, in other living species, although their gene structures can be significantly altered.

Amino Acid Sequence↗
PubMed2000

The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.

Amino Acid Sequence↗
PubMed2001

Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F1 by multiple ROC-cullin ligases.

Ubiquitin-dependent proteolysis plays a critical role in the control of many cellular processes and is mediated by a cascade of enzymes involving ubiquitin activating (El), conjugating (E2), and ligating (E3) activities. Cullin 1/CDC53 functions as an E3 ligase by interacting with RING finger protein ROC1 and recruiting phosphorylated substrate. We report here that E2F1 transcription factor can be ubiquitinated in vitro and in vivo by multiple ROC-cullin ligases. In vitro, E2F1 can be ubiquitinated by E2/Ubc5 but not by E2/CDC34, is dependent on catalytically active ROC1, and is protected by the Rb protein. In contrast to substrates of the SKP1-Cullin 1-F box (SCF) complexes, in vitro ubiquitination of E2F1 by CUL1-ROC1 ligase does not require E2F1 phosphorylation, is not stimulated by overexpression of F box protein SKP2, and is not affected by immunodepletion of SKP1 or mutations in CUL1 disrupting SKPI binding. These results suggest a novel, SKP1-independent mechanism for targeting E2F1 ubiquitination.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia.

Ubiquitin-dependent protein degradation impacts many cellular processes.However, the regulation of ubiquitin-conjugating enzymes (UBCs) in cancer is unknown. We find that the human CDC34 UBC protein is expressed at a 3-4 fold higher level (P < 0.001) in pediatric T cell than in pre-B-cell acute lymphoblastic leukemia (ALL) before treatment in two independent patient sets. The level of CDC34 mRNA was similar in both types of leukemia. CDC34 expression levels in normal resting T cells, B cells and activated T lymphocytes was comparable with pre-B-cell ALL. CDC34 protein (but not mRNA) was also increased in T-cell ALL compared with pre-B-cell ALL cell lines. The difference in expression was not attributable to mutation or associated with altered CDC34 stability. Immunohistochemistry and cellular fractionation reveals a heterogeneous CDC34 expression pattern including cells containing primarily cytoplasmic or nuclear protein. Thus, a feature of pediatric T-cell ALL is posttranscriptional up-regulation and heterogeneous localization of the human CDC34 UBC.

Anaphase-Promoting Complex-Cyclosome↗
PubMedDate not supplied

The dephosphorylated form of the anaphase-promoting complex protein Cdc27/Apc3 concentrates on kinetochores and chromosome arms in mitosis.

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.

Animals↗
PubMed2003

Induction of C-anaphase and diplochromosome through dysregulation of spindle assembly checkpoint by sodium arsenite in human fibroblasts.

Cytogenetic alterations induced by arsenite are associated with its carcinogenic activity. Cytogenetic analysis revealed first that arsenite induced c-anaphases in a time- and dose-dependent manner in human fibroblasts (HFW). With additional incubation of arsenite-arrested mitotic cells in drug-free medium for 0-48 h, approximately 35% exited from mitosis without cell division. This was confirmed by the appearance of tetraploid metaphase, mainly diplochromosomes, in the subsequent cell division. Treatment of HFW cells with both nocodazole, a known agent of microtubular depolymerization, and Taxol, which induces tubulin polymerization and inhibits disassembly of microtubules, resulted in remarkable mitotic arrest but induced only negligible c-anaphase, tetraploidy, and diplochromosomes. Staurosporine, a kinase inhibitor that could effectively reduce arsenite-induced c-anaphase, could also decrease the development of diplochromosomes in the subsequent cell division cycle. These results imply that arsenite-induced c-anaphases mainly exited from mitosis without cell division and became tetraploid in the subsequent cell cycle. Antitubulin immunofluorescent staining confirmed no formation of bipolar spindles in nocodazole-arrested mitotic HFW cells, whereas in arsenite-arrested mitotic cells bipolar spindles were present but distorted in appearance and apparently dysfunctional. Mitotic arrest deficient 2 (Mad2) signal was, as expected, clearly visible at centromeres of nocodazole-arrested mitotic cells. However, the Mad2 signal at centrosomes became insignificant in either arsenite-arrested or nocodazole/arsenite-arrested mitotic cells. In addition, the association of Mad2 with the APC/C(cdc20) complex and the accumulation of Pds1, an anaphase inhibitor, were remarkably reduced in arsenite-arrested mitotic cells as compared with nocodazole-arrested mitotic cells. These results support the observation that nocodazole can inhibit spindle formation and, hence, activate spindle assembly checkpoint to arrest cells at metaphase. In contrast, the dysfunctional bipolar spindles in arsenite-arrested mitotic cells could not effectively activate spindle assembly checkpoint and, hence, resulted in formation of c-anaphase and diplochromosomes in the subsequent cell division.

Anaphase↗
PubMed2003

The mitotic checkpoint: a signaling pathway that allows a single unattached kinetochore to inhibit mitotic exit.

The mitotic checkpoint is a failsafe mechanism for the cell to ensure accurate chromosome segregation during mitosis. Mutations in genes encoding essential checkpoint proteins lead to chromosome instability and promote carcinogenesis. The BUB and MAD genes are essential components of the mitotic checkpoint pathway. BUB and MAD inhibit the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C) during mitosis to ensure cells with unaligned chromosomes do not prematurely enter anaphase. Two models explain how the APC/C is inhibited by the checkpoint. The Sequestration Model postulates that Mad2 and BubR1 bind and sequester Cdc20, an APC/C activator, away from APC/C so substrates whose destruction drives mitotic exit are no longer ubiquitinated. In this model, the unattached kinetochore is postulated to catalytically convert Mad2 to a form that binds Cdc20. In the Direct Inhibition Model, the Mitotic Checkpoint Complex (MCC) consisting of BubR1, Bub3, Mad2 and Cdc20 binds and inhibits the APC/C independently of the kinetochore. However, the "wait anaphase" signal generated by unattached kinetochores sensitizes the APC/C to prolonged inhibition by the MCC. A single unattached kinetochore is proposed to amplify the "wait anaphase" signal through a kinase cascade involving checkpoint kinases such as hBubR1, hBub1 and Mps1.

Anaphase↗
PubMed2004

Cdc20 in S-phase: the Banquo at replication's banquet.

The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that covalently attaches ubiquitins onto proteins to target them for proteolysis by the 26S proteasome. During mitosis, the APC/C is instrumental in allowing the cell to enter and exit from mitosis. The APC/C accomplishes this by using different specificity factors to recognize, interact with, and ubiquitylate key proteins that block cell cycle progression. The specificity factors, Cdc20p and Cdh1p, are not always associated with the APC/C and indeed they have the ability to interact with substrates in isolation. The molecular events that take place in order for Cdc20p and Cdh1p to couple substrates and APC/C are currently being resolved. Meanwhile, evidence has emerged suggesting that at least one of the specificity factors, Cdc20p, might be capable of functioning independently of the APC/C.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

Cycling without the cyclosome: modeling a yeast strain lacking the APC.

The construction of viable Saccharomyces cerevisiae strains that lack the anaphase promoting complex (APC) was recently reported. The normally lethal deletions of APC genes were suppressed by the double deletion of the PDS1 and CLB5 genes in conjunction with the insertion of multiple copies of the SIC1 gene controlled by its endogenous promoter. It was proposed that cyclic expression and degradation of Sic1 results in oscillations of Clb/CDK activity necessary for the cell cycle. We have used an updated version of a mathematical model of the yeast cell cycle to model strains that lack the APC. With a few modifications, the model accurately simulates the viability of Apc- strains, as well as the phenotypes of 27 other previously characterized strains. We discuss a few minor inconsistencies between the model and experiment, and how these may inform future revisions to the model.

Anaphase-Promoting Complex-Cyclosome↗