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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↗
PubMed2004

Control of APC-Cdc20 by the tumor suppressor RASSF1A.

The tumor suppressor gene RASSF1A is frequently hypermethylated in various tumors. However, how RASSF1A functions in tumor suppression was unknown. Our study shows that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression by inhibiting APC-Cdc20. RASSF1A appears to work in early pro-metaphase, after Emi1 destruction and before activation of the Mad2-dependent spindle checkpoint. Loss of RASSF1A expression induces premature APC activation, thereby resulting in acceleration of mitotic cyclin degradation and mitotic progression as well as induction of mitotic abnormalities. Thus, RASSF1A plays a critical role in mitotic progression and tumor suppression by controlling APC-Cdc20 activity.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

Don't skip the G1 phase: how APC/CCdh1 keeps SCFSKP2 in check.

By keeping the levels of Skp2 and Cks1 low during G(1) progression, APC/C(Cdh1) prevents unscheduled degradation of SCF(Skp2) substrates and premature entry into S phase. Thus, APC/C(Cdh1), a ubiquitin ligase involved in mitotic exit and maintenance of G(0)/G(1) phase, directly controls SCF(Skp2), a ubiquitin ligase involved in the regulation of S phase entry.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Bub1 multitasking in mitosis.

Accurate partition of duplicated genetic material to the daughter cells during mitosis relies on the maintenance of the physical linkage (cohesion) between sister chromatids until their bipolar attachment to the mitotic spindle. In response to a single straying chromatid within a cell, a surveillance mechanism called the spindle checkpoint blocks the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), stabilizes securin (an APC/C substrate and an inhibitor of separase), and delays the activation of separase. This in turn prevents cleavage of cohesin by separase, preserves sister chromatid cohesion, and delays the onset of anaphase. The protein kinase, Bub1, is a key component of the spindle checkpoint. Bub1 has an upstream function in regulating the kinetochore localization of Mad2 and other downstream checkpoint components. In addition, recent biochemical studies have shown that Bub1 directly phosphorylates the APC/C activator, Cdc20, and inhibits APC/C. Finally, Bub1 has a noncheckpoint function at the kinetochores and preserves centromeric cohesion through the MEI-S332/shugoshin family of proteins. Therefore, Bub1 performs multiple tasks in mitosis that ensure the proper inheritance of chromosomes.

Anaphase↗
PubMed2005

The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs.

The study of animal viruses has provided extraordinary insights into cell cycle dynamics and tumor biology. The significance of the p53 and Rb tumor suppressor proteins, for example, was discovered due to their interactions with viral oncogenes. In the past several years, investigations with four viral proteins, human immunodeficiency virus type 1 (HIV-1) vpr, adenovirus E4orf4, chicken anemia virus (CAV) apoptin and human T lymphotropic virus type I (HTLV-I) Tax, have indicated that there are also critical viral targets involved in G2/M control. In particular, recent studies with E4orf4 and apoptin have shown that they induce G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Notably, these two viral proteins induce apoptosis selectively in transformed cells in a p53-independent manner; thus pathways affected by these proteins are of significant therapeutic interest. Further investigation of the underlying mechanism of G2/M arrest and subsequent apoptosis induced by viral APC/C inhibitors may shed light on the mechanisms of current cancer therapies and provide the foundation for developing novel therapeutic targets.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Rewiring the exit from mitosis.

The mitotic cell cycle can be described as an alternation between two states. During mitosis, MPF (mitosis promoting factor) is high and keeps inactive its numerous molecular antagonists. In interphase, MPF is inactivated, and the antagonists prevail. The transition between the two states is ensured by 'helper' molecules that favor one state over the other. It has long been assumed that active MPF (a dimer of cyclin B and cyclin-dependent kinase 1) induces exit from mitosis by activating APC:Cdc20, a ubiquitin ligase responsible for cyclin B degradation. The molecular details have not been fully worked out yet, but recent results show that MPF and the ubiquitin ligase are not involved in a simple negative feedback loop. While it is proven that MPF activates APC, new data suggest that MPF inhibits Cdc20, i.e., that MPF and Cdc20 are antagonists. We introduce this new idea into a published model for cell cycle regulation in Xenopus laevis, and study its dynamical behavior. We show that the new wiring permits oscillations with a simpler and smaller network than previously envisaged and that the antagonism between MPF and Cdc20 suggests a new interpretation of the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Arabidopsis anaphase-promoting complexes: multiple activators and wide range of substrates might keep APC perpetually busy.

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an essential regulator of the cell cycle from metaphase until S phase in yeast and metazoans. APC mediates degradation of numerous cell cycle-related proteins, including mitotic cyclins and its activation and substrate-specificity are determined by two adaptor proteins, Cdc20 and Cdh1. Plants have multiple APC activators and the Cdh1-type proteins, in addition, are represented by two subclasses, known as Ccs52A and Ccs52B. The Arabidopsis genome contains five cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. In Schizosaccharomyces pombe, expression of the three Atccs52 genes elicited distinct phenotypes supporting nonredundant function of the AtCcs52 proteins. Consistent with these activities, the AtCcs52 proteins were able to bind both to the yeast and the Arabidopsis APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts were present from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1 and ccs52A2 were from late M until early G2, suggesting consecutive action of these APC activators in the plant cell cycle. The AtCcs52 proteins interacted with different subsets of mitotic cyclins, in accordance with their expression profiles, either in free- or CDK-bound forms. Expression of most APC subunits was constitutive, whereas cdc27a and cdc27b, corresponding to two forms of apc3, and ubc19 and ubc20 encoding E2-C type ubiquitin-conjugating enzymes displayed differences in their cell cycle regulation. These data indicate the existence of numerous APC(Cdc20/Ccs52/Cdc27) forms in Arabidopsis, which in conjunction with different E2 enzymes might have distinct or complementary functions at distinct stages of the cell cycle.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1991

A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Alleles↗
PubMed1995

Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16.

In an effort to identify genes that are differentially regulated during mast cell development, subtracted cDNA prepared from wild-type murine P815 mastocytoma cells and a P815 subline that exhibits properties of mast cell differentiation was used to screen mast cell cDNA libraries. Several known mast cell-specific cDNAs were isolated including mast cell carboxypeptidase A (MC-CPA), murine mast cell protease-5 (MMCP-5), and gp49. A novel cDNA, designated Tbc1, was identified that showed differential expression in the two mast cell lines. The amino acid sequence predicted from the cDNA contains a 200 amino acid domain that is homologous to regions in the tre-2 oncogene and the yeast regulators of mitosis, BUB2 and cdc16. The N-terminal region contains a number of cysteine and histidine residues, potentially encoding a zinc finger domain. Tbc1 is a nuclear protein and is expressed in highest levels in hematopoietic cells, testis and kidney. Within these tissues, expression of Tbc1 is cell- and stage-specific. Based on sequence similarity, pattern of expression and subcellular localization, Tbc1 may play a role in the cell cycle and differentiation of various tissues.

3T3 Cells↗
PubMed1994

Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association.

Like several other ubiquitin-conjugating enzymes, the yeast cell cycle enzyme CDC34 (UBC3) has a carboxyl-terminal extension or tail. These tails appear to carry out unique functions that can vary from one ubiquitin-conjugating enzyme to the next. Using biophysical techniques we have determined that the tail of CDC34 constitutes a highly structured and extended domain. Although the tail of CDC34 is the largest tail identified to date (125 residues), we have found that only 39 residues lying adjacent to the catalytic domain are necessary and sufficient for full cell cycle function and that this region fulfills a novel function that may be common to the tails of other ubiquitin-conjugating enzymes. Cross-linking studies demonstrate that this region facilitates a physical interaction between CDC34 monomers in vitro. Furthermore, phenotypic analysis of various CDC34 derivatives expressed in different cdc34 mutant strains indicates that this region facilitates the same interaction in vivo. Based on these findings, it appears that the cell cycle function of CDC34 is dependent upon the ability of CDC34 monomers to interact with one another and that this interaction is mediated by a small region of the CDC34 tail. The similarity of this region with sequences contained within the tails of the UBC1 and UBC6 enzymes suggests that these tails may function in a similar manner.

Anaphase-Promoting Complex-Cyclosome↗