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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↗
PubMed2006

Dual regulation of the anaphase promoting complex in human cells by cyclin A-Cdk2 and cyclin A-Cdk1 complexes.

In mammalian somatic cells, the Anaphase Promoting Complex (APC) is inactivated during S phase by active cyclin A-Cyclin dependent kinase (Cdk) 2 complexes promoting accumulation of mitotic regulators, such as cyclin B and Polo like kinase 1 (Plk1). However, mitotic entry does not appear to be perturbed in some human cancer cells or in normal mouse cells following Cdk2 RNA interference (i) or deletion of the Cdk2 gene. These results suggest functional complementation of APC regulation by a compensatory kinase. Using Plk1 protein level as readout of APC activity, we show that APC is inactivated during S phase in human cells by both cyclin A-Cdk2 and cyclin A-Cdk1 complexes. Expression of a dominant negative mutant of Cdk2 or Cdk2 RNAi in early S phase destabilizes Plk1 as it begins to accumulate. However, this effect wanes in late S phase, where destabilization of Plk1 also requires Cdk1 RNAi. Although Cdk2 is the dominant partner of cyclin A in these settings, cyclin A also binds Cdk1. Both complexes bind the APC targeting factor Cdh1, but Cdk1 complexes are inactive in early S phase, accounting for the stronger regulation of APC function by Cdk2. These results provide further evidence that cyclin A-Cdk2 and -Cdk1 complexes display overlapping and partially redundant roles in preparing cells for mitosis, through regulation of the APC.

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↗
PubMed1993

The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein.

The CDC34 gene of Saccharomyces cerevisiae encodes a 295-residue ubiquitin-conjugating enzyme (E2). The function of this ubiquitin-conjugating activity remains to be defined as its in vivo substrates are presently unknown. The bacterially expressed and purified Cdc34 protein is shown here to catalyze its own ubiquitination via an intramolecular transfer of its thiol ester-linked ubiquitin to a lysine. In this process, multiple ubiquitin groups are added to Cdc34, and these ubiquitin groups were shown to be arranged predominantly in the form of a single Lys48-specific multiubiquitin chain. Analysis of the hydroxylamine-dependent cleavage of ubiquitin-Cdc34 conjugates at the single Asn-Gly sequence in Cdc34 placed the major ubiquitin linkage site within the C-terminal 215-295 residues of Cdc34. The 4 Lys residues (Lys273, Lys277, Lys293, and Lys294) in this region of CDC34 were substituted by arginine either singly or in combination to produce a set of Cdc34 mutants. Analysis of these Cdc34 mutants for autoubiquitination revealed that the multiubiquitin chain can be formed on any one of these 4 lysines although most Cdc34 conjugates contain a single multiubiquitin chain. Since the presence of a Lys48-specific multiubiquitin chain in protein conjugates is known to target acceptor proteins for degradation in the ubiquitin-mediated proteolytic pathway, the present result raises the possibility that one function of the ubiquitin-conjugating activity in CDC34 may be used to target its own degradation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1996

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.

Alleles↗
PubMed1997

Kip1 degradation via the ubiquitin-proteasome pathway.

The cell cycle has been the object of extensive studies for the past years. A complex network of molecular interactions has been identified. In particular, a class of cell cycle inhibitory proteins has been identified but details of the molecular mechanism of their action have yet to be resolved. These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity. The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis. Kip1 is a potent inhibitor of Cdks. In quiescent cells Kip1 accumulates without an increase in mRNA or protein synthesis. We demonstrated that cell cycle regulation of Kip1 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway. In a crude in vitro system, Kip1 is ubiquitinated and degraded in an ATP dependent manner and inhibition or depletion of the proteasome blocks Kip1 degradation. Human Ubc2 and Ubc3, the homologs of yeast Rad6 and Cdc34 gene products respectively, are specifically involved in the ubiquitination of Kip1. Compared to proliferating cells, quiescent cells contain a far lower amount of Kip1 ubiquitinating activity. These results represent the first demonstration that the ubiquitin-proteasome pathway plays a role in the regulation of a cell cycle protein in human cells, namely the Cdk inhibitor Kip1. The specific proteolysis of Kip1 may be involved in the pathway of inactivation of Cdks.

Adenosine Triphosphate↗