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PubMed1997

The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase.

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

Anaphase↗
PubMed1998

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition.

The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+. Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.

Anaphase↗
PubMed1996

Clinical implications of cytogenetic abnormalities in melanoma.

Unlike leukemia, in which specific reciprocal translocations are frequently observed, melanomas involve complex recurring chromosome anomalies. Analysis of the constituted genome of melanoma patients should identify cancer susceptibility genes and at-risk individuals in families with a history of melanoma. The first of these genes to be cloned is the cell cycle regulatory protein inhibitor--the p16 gene-- and a second gene locus for melanoma predisposition has been linked to the chromosome 1p36 band region. Detection of the most common somatic genetic alterations in melanoma enhances our understanding of molecular mechanisms of melanoma development and may lead to genetic markers in melanoma. Some alterations may be used to identify interesting subpopulations. Others may be of prognostic value when they are considered in tandem with clinical data.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

CDC16 controls initiation at chromosome replication origins.

The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. (1997), that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

TFDP1, CUL4A, and CDC16 identified as targets for amplification at 13q34 in hepatocellular carcinomas.

We carried out molecular cytogenetic characterization of 11 cell lines derived from hepatocellular carcinomas (HCCs) and 51 primary HCCs. Comparative genomic hybridization (CGH) revealed frequent amplification at 13q34, where we had detected amplification in several other types of tumor, including esophageal squamous cell carcinomas (ESC). Previously, we suggested possible involvement of TFDP1, encoding a transcription factor DP-1, in the 13q34 amplification observed in a primary ESC. Therefore, we investigated amplifications and expression levels of 5 genes mapped on the amplified region, including TFDP1, for exploring amplification targets at 13q34 in HCCs. 3 of those genes, TFDP1, CUL4A (cullin 4A), and CDC16 (cell division cycle 16), showed distinct amplification and consequent over-expression in some cell lines. Moreover, each was amplified in 3 or 4 of the 51 primary HCCs, and all 3 were amplified in 2 tumors, in which their expression patterns correlated with amplification patterns. To elucidate the functional role of TFDP1 in HCC, we examined expression levels of genes downstream of TFDP1 with real-time quantitative polymerase chain reaction (PCR). Expression of cyclin E gene (CCNE1) correlated closely with that of TFDP1 in not only cell lines, but also primary tumors. Treatment of HCC cells with the antisense oligonucleotide targeting TFDP1 resulted in down-regulation of CCNE1, suggesting that TFDP1 overexpression led to up-regulation of CCNE1 that encoded a positive regulator for cell cycle G1/S transition. In conclusion, our findings suggest that TFDP1, CUL4A, and CDC16 are probable targets of an amplification mechanism and therefore may be involved, together or separately, in development and/or progression of some HCCs.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed1997

The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex.

The evolutionarily conserved multisubunit complex known as the cyclosome or anaphase-promoting complex is involved in catalyzing the ubiquitination of diverse substrates in M phase, allowing their destruction by the 26 S proteasome and the completion of mitosis. Three of the eight subunits of the anaphase-promoting complex (CDC16, CDC23, and CDC27) have been shown to be phosphorylated in M phase, and their phosphorylation is required for the anaphase-promoting complex to be active as a ubiquitin ligase. Several subunits of the anaphase-promoting complex contain tetratricopeptide repeats, a protein motif involved in protein/protein interactions. PP5 is a serine/threonine phosphatase that also contains four copies of the tetratricopeptide repeats motif. Here we show by a combination of two-hybrid analysis and in vitro binding that PP5 interacts with CDC16 and CDC27, two subunits of the anaphase-promoting complex. Only the NH2-terminal domain of PP5, containing all four tetratricopeptide repeats, is required for this physical interaction. Deletion analysis suggests that the site of binding to PP5 is localized to the COOH-terminal block of tetratricopeptide repeats in CDC16 and CDC27. In addition, indirect immunofluorescence showed that PP5 localizes to the mitotic spindle apparatus. The direct interaction of PP5 with CDC16 and CDC27, as well as its overlapping spindle localization in mitosis, suggests that PP5 may be involved in the regulation of the activity of the anaphase-promoting complex.

Amino Acid Sequence↗
PubMed2001

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP.

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.

Amino Acid Sequence↗
PubMed1997

A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

Amino Acid Sequence↗
PubMed1999

Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability.

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

A screen for Schizosaccharomyces pombe mutants defective in rereplication identifies new alleles of rad4+, cut9+ and psf2+.

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit cut9(+), the initiation/checkpoint factor rad4(+)/cut5(+), and the first mutant allele of psf2(+), a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.

Alleles↗
PubMed2001

Down-regulation of the human CDC16 gene after exposure to ionizing radiation: a possible role in the radioadaptive response.

We have used the method of differential display of mRNAs to search for changes in gene expression associated with radioadaptation triggered by low doses of ionizing radiation in human lymphoblasts. We isolated a cDNA designated PB13 that was down-regulated as early as 1 h after irradiation with 4 Gy in cells adapted by a pre-exposure to a dose of 2 cGy, compared to 3 h in nonadapted cells (4 Gy alone). Northern analysis confirmed the differential expression of a 2.4-kb transcript that was repressed for at least 10 h after irradiation. The major part of the PB13 cDNA was identical to the human CDC16 mRNA. When using either PB13 or CDC16 cDNA as probes, similar radiation-induced alterations in gene expression were observed. Expression of the CDC16 gene was also repressed after oxidative stress with H(2)O(2). The CDC16 protein belongs to the anaphase-promoting complex (APC) that controls progression through mitosis. The repression of expression of the CDC16 gene by ionizing radiation could result in delayed progression of damaged cells through mitosis. This cycle delay would occur earlier in adapted cells and would allow a more rapid and efficient repair that could contribute to the tolerance to subsequent irradiation.

Adaptation, Biological↗
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↗
PubMed2001

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex.

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.

Amino Acid Sequence↗
PubMed1999

Control of metaphase-anaphase progression by proteolysis: cyclosome function regulated by the protein kinase A pathway, ubiquitination and localization.

Ubiquitin-mediated proteolysis is fundamental to cell cycle progression. In the fission yeast Schizosaccharomyces pombe, a mitotic cyclin (Cdc13), a key cell cycle regulator, is degraded for exiting mitosis, while Cut2 has to be destroyed for the onset of sister chromatid separation in anaphase. Ubiquitination of these proteins requires the special destruction box (DB) sequences locating in their N-termini and the large, 20S complex called the anaphase-promoting complex or cyclosome. Here we show that cyclosome function during metaphase-anaphase progression is regulated by the protein kinase A (PKA) inactivation pathway, ubiquitination of the cyclosome subunit, and cellular localization of the target substrates. Evidence is provided that the cyclosome plays pleiotropic roles in the cell cycle: mutations in the subunit genes show a common anaphase defect, but subunit-specific phenotypes such as in G1/S or G2/M transition, septation and cytokinesis, stress response and heavy metal sensitivity, are additionally produced, suggesting that different subunits take distinct parts of complex cyclosome functions. Inactivation of PKA is important for the activation of the cyclosome for promoting anaphase, perhaps through dephosphorylation of the subunits such as Cut9 (Apc6). Cut4 (Apc1), the largest subunit, plays an essential role in the assembly and functional regulation of the cyclosome in response to cell cycle arrest and stresses. Cut4 is highly modified, probably by ubiquitination, when it is not assembled into the 20S cyclosome. Sds23 is implicated in DB-mediated ubiquitination possibly through regulating de-ubiquitination, while Cut8 is necessary for efficient proteolysis of Cdc13 and Cut2 coupled with cytokinesis. Unexpectedly, the timing of proteolysis is dependent on cellular localization of the substrate. Cdc13 enriched along the spindle disappears first, followed by decay of the nuclear signal, whereas Cut2 in the nucleus disappears first, followed by decline in the spindle signal during metaphase-anaphase progression.

Anaphase↗
PubMed1998

Identification of a cullin homology region in a subunit of the anaphase-promoting complex.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Amino Acid Sequence↗