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PubMed2006

The emerging role of pituitary tumor transforming gene in tumorigenesis.

Pituitary tumor transforming gene (PTTG) is a newly discovered oncogene, and serves as a marker of malignancy grades in several forms of cancer, particularly endocrine malignancies such as pituitary adenomas. PTTG appears also to have a role in the genesis of some types of cancer. Also known as a human form of securin, PTTG is an anaphase inhibitor that prevents premature chromosome separation through inhibition of separase activity; hence, its degradation is required to start anaphase. Through this important function, PTTG participates in several key cellular events such as mitosis, cell cycle progression, DNA repair and apoptosis. The physiological importance of PTTG is indicated by the study of PTTG-null mice that have cell growth abnormalities in testis and pancreatic beta cells. Overexpression of PTTG has been observed in thyroid and colon cancers. In addition, 90% of pituitary adenomas overexpress PTTG, qualifying it as the best available marker for this disease. Although the exact mechanism is unknown, PTTG participates in the pathogenesis of various tumors, including pituitary tumors, by inducing aneuploidy and upregulating FGF-2, a potent mitogenic and angiogenic factor. Various growth factors, nuclear factors and hormones regulate PTTG expression in different tumor cells, which could be important to understand in order to obtain insight into the tumorigenic and tumor progression process. Here, we review the current knowledge of the biological and pathophysiological roles of PTTG.

Adenoma↗
PubMed2005

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Regulation of Separase in meiosis: Separase is activated at the metaphase I-II transition in Xenopus oocytes during meiosis.

Separase is a cysteine protease conserved in all eukaryotes and functions to remove sister chromatid cohesion in anaphase by cleaving the SCC1 subunit of the cohesin complex. Separase activity is regulated by its inhibitor securin and by an inhibitory phosphorylation in vertebrates. However, these regulations have never been directly investigated in the meiotic cell cycle of vertebrates. In this study, we cloned the full-length gene encoding Xenopus separase from an oocyte cDNA library. Purified xSeparase can cleave the human alpha-kleisin subunit of cohesin in vitro but cannot bind to hSecurin when these two proteins are coexpressed in 293T cells. Similar to its human counterpart, xSeparase cleaves itself upon activation but at a single site. The cleavage site is conserved with one of the three self-cleavage sites in hSeparase. Using self-cleavage as a reporter for its activation, we demonstrated that xSeparase is transiently activated between the two meioses and may be involved in homolog separation, as is observed in other organisms. Taking advantage of the inability of xSecurin to interact with hSeparase, we demonstrated that CSF extract can reinhibit both full-length and auto-cleaved hSeparase, indicating that inhibition of separase by phosphorylation does occur under physiological conditions. In addition, we found that endogenous xSecurin accumulated in response to progesterone-induced oocyte maturation and was degraded at both anaphase I and II in an APC/C-dependent manner.

Amino Acid Sequence↗
PubMed2004

[Activity of separase and its regulation].

Sister chromatids separation is under precise regulation during cell cycle. Any turbulence happened in the separation process can cause instability in the transmission of inherited material, and may cause: death or disease of cell or even individual. In eukaryotic cells, one conserved mechanism governs the separation of sister chromatids. Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. At the metaphase to anaphase transition, separase is activated by proteolysis of securin. Separase can cleave one of cohesin's subunits, and then promote cohesin dissociation and sister chromatids separation.

Anaphase↗
PubMed2006

Increased expression of pituitary tumor-transforming gene (PTTG)-1 is correlated with poor prognosis in glioma patients.

Pituitary tumor-transforming gene (PTTG), which is homologous to a mammalian securin, plays a pivotal role in cell transformation and is overexpressed in numerous cancer cell lines and tissues. PTTG functions in the control of mitosis, cell transformation, DNA repair and gene regulation. In the present study, we investigated whether the expression of PTTG1 is correlated with tumorigenicity and prognosis in glioma patients. Expression of PTTG1 was confirmed in three glioma cell lines at the mRNA and protein levels using RT-PCR analysis and Western blotting, respectively. Furthermore, PTTG1 protein was detected in 44 glioma tissue samples using immunohistochemical techniques, markedly increased in high-grade gliomas compared to low-grade gliomas and associated with an unfavorable patient outcome. Moreover, siRNA against the PTTG1 gene inhibited cell proliferation and invasion in glioma cell lines. These data suggest that increased expression of PTTG1 contributes to the tumorigenicity of glioma cells and may be useful as a prognostic marker for glioma patients.

Adult↗
PubMed2006

Cdc48 is required for the stability of Cut1/separase in mitotic anaphase.

Separase, a large protease essential for sister chromatid separation, cleaves the cohesin subunit Scc1/Rad21 during anaphase and leads to dissociation of the link between sister chromatids. Securin, a chaperone and inhibitor of separase, is ubiquitinated by APC/cyclosome, and degraded by 26S proteasome in anaphase. Cdc48/VCP/p97, an AAA ATPase, is involved in a variety of cellular activities, many of which are implicated in the proteasome-mediated degradation. We previously reported that temperature-sensitive (ts) fission yeast Schizosaccharomyces pombe cdc48 mutants were suppressed by multicopy plasmid carrying the cut1(+)/separase gene and that the defective mitotic phenotypes of cut1 and cdc48 were similar. We here describe characterizations of Cdc48 mutant protein and the role of Cdc48 in sister chromatid separation. Mutant residue resides in the conserved D1 domain within the central hole of hexamer, while Cdc48 mutant protein possesses the ATPase activity. Consistent with the phenotypic similarity and the rescue of cdc48 mutant by overproduced Cut1/separase, the levels of Cut1 and also Cut2 are diminished in cdc48 mutant. We show that the stability of Cut1 during anaphase requires Cdc48. Cells lose viability during the traverse of anaphase in cdc48 mutant cells. Cdc48 may protect Cut1/separase and Cut2/securin against the instability during polyubiquitination and degradation in the metaphase-anaphase transition.

Adenosine Triphosphatases↗
PubMed2006

Novel functions of thyroid hormone receptor mutants: beyond nucleus-initiated transcription.

Study of molecular actions of thyroid hormone receptor beta (TRbeta) mutants in vivo has been facilitated by creation of a mouse model (TRbetaPV mouse) that harbors a knockin mutant of TRbeta (denoted PV). PV, which was identified in a patient with resistance to thyroid hormone, has lost T3 binding activity and transcription capacity. The striking phenotype of thyroid cancer exhibited by TRbeta(PV/PV) mice has allowed the elucidation of novel oncogenic activity of a TRbeta mutant (PV) [PAS1] beyond nucleus-initiated transcription. PV was found to physically interact with the regulatory p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) in both the nuclear and cytoplasmic compartments. This protein-protein interaction activates the PI3K signaling by increasing phosphorylation of AKT, mammalian target of rapamycin (mTOR), and p70(S6K). PV, via interaction with p85alpha, also activates the PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathway in the extra-nuclear compartment. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. In addition to affecting these membrane-initiated signaling events, PV affects the stability of the pituitary tumor-transforming gene (PTTG) product. PTTG (also known as securin), a critical mitotic checkpoint protein, is physically associated with TRbeta or PV in vivo. Concomitant with T3-induced degradation of TRbeta, PTTG is degraded by the proteasome machinery, but no such degradation occurs when PTTG is associated with PV. The degradation of PTTG/TRbeta is activated by the direct interaction of the T3-bound TRbeta with the steroid receptor coactivator-3 (SRC-3) that recruits a proteasome activator (PA28gamma). PV that does not bind T3 cannot interact directly with SRC-3/PA28gamma to activate proteasome degradation, and the absence of degradation results in an aberrant accumulation of PTTG. The PV-induced failure of timely degradation of PTTG results in mitotic abnormalities. PV, via novel protein-protein interaction and transcription regulation, acts to antagonize the functions of wild-type TRs and contributes to the oncogenic functions of this mutation.

Animals↗
PubMed2005

Mitosis: a matter of getting rid of the right protein at the right time.

There are two major problems for the cell to solve in mitosis: how to ensure that each daughter cell receives an equal and identical complement of the genome, and how to prevent cell separation before chromosome segregation. Both these problems are solved by controlling when two specific proteins are destroyed: securin, an inhibitor of chromosome segregation, and cyclin B, which inhibits cell separation (cytokinesis). It has recently become clear that several other proteins are degraded at specific points in mitosis. This review (which is part of the Chromosome Segregation and Aneuploidy series) focuses on how specific proteins are selected for proteolysis at defined points in mitosis and how this contributes to the proper coordination of chromosome segregation and cytokinesis.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex.

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

Anaphase specific auto-cleavage of separase.

Sister-chromatid separation is triggered by a specific proteolytic cleavage of chromosomal cohesins catalyzed by the endopeptidase separase. Prior to anaphase, separase is inhibited independently by affinity binding to securin and by specific inhibitory phosphorylation. Here we show that separase itself is also subjected to proteolytic cleavages at three adjacent sites. The cleavages are auto-catalyzed and occur specifically at anaphase coincident with separase activation. The cleaved fragments remain associated with each other and are catalytically active. Mapping of the cleavage sites reveals that all three sites are conserved in vertebrates underlining a significant function for this regulation.

Amino Acid Sequence↗
PubMed2003

Western blot screening for monoclonal antibodies against human separase.

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.

Animals↗
PubMed2005

Enzymology of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is an ubiquitin-protein ligase that promotes mitotic progression by catalyzing the ubiquitination of numerous proteins, including securin and cyclin. Its complex subunit composition and extensive regulation make the APC an active subject of investigation for both cell biologists and enzymologists. This chapter describes a system for the reconstitution and quantitative analysis of APC activity from budding yeast in vitro. We focus in particular on the measurement of processive ubiquitination, which complements traditional analysis of the reaction rate as a means to elucidate the molecular details of substrate recognition and ubiquitination by the APC.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

High-throughput screening for inhibitors of the e3 ubiquitin ligase APC.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.

Drug Evaluation, Preclinical↗
PubMed2000

Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase.

In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase. We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase-anaphase transition.

Anaphase↗
PubMed2004

Recycling the cell cycle: cyclins revisited.

I discuss advances in the cell cycle in the 21 years since cyclin was discovered. The surprising redundancy amongst the classical cyclins (A, B, and E) and cyclin-dependent kinases (Cdk1 and Cdk2) show that the important differences between these proteins are when and where they are expressed rather than the proteins they phosphorylate. Although the broad principles of the cell cycle oscillator are widely accepted, we are surprisingly ignorant of its detailed mechanism. This is especially true of the anaphase promoting complex (APC), the machine that triggers chromosome segregation and the exit of mitosis by targeting securin and mitotic cyclins for destruction. I discuss how a cyclin/Cdk-based engine could have evolved to assume control of the cell cycle from other, older protein kinases.

Animals↗
PubMed2001

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

Acetyltransferases↗
PubMed2001

The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition.

Clb2 mitotic cyclin inhibits cell cycle progression by preventing mitotic exit and DNA synthesis. To allow cell cycle progression, Clb2 proteolysis is triggered by Cdc20 during the metaphase-to-anaphase (M-A) transition and by Hct1 during mitotic exit and G1 [1-6]. A cis element called the destruction box is required for this proteolysis [7-11]. Recently, an additional cis element called the "KEN box" was also shown to be required for proteolysis of human CDC20 and Securin [3,12]. Using a novel color assay, we show that a Clb2 KEN box is required to target a fusion protein containing the first 124 amino acids of Clb2 for proteolysis. We further show that full-length Clb2 bearing mutations in the KEN box is degraded efficiently during the M-A transition, but poorly during G1. If the destruction box of Clb2 is mutated in combination with mutation of the KEN box, then this form of Clb2 is more stable than Clb2 bearing either mutation by itself during both M-A and G1. Our results show that the KEN box and the destruction box act together during both M-A and G1 to regulate Clb2 proteolysis.

Anaphase↗
PubMed2002

Completion of mitosis requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog.

Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(FZY)/Cdc20 and Fizzy-related(FZR)/Hct1/Cdh1 proteins. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous FZR is not involved during exit from mitosis. However, the potential involvement of minor amounts of maternally derived FZR was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1(-/-) cells might be explained by the recent identification of multiple Cdh1 genes [10]. Here, we have carefully analyzed the FZR requirement during exit from mitosis in Drosophila, which, apart from fzr, has only one additional homolog. We find that this fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, we demonstrate that completion of mitosis including Cyclin B degradation does not require FZR. However, fzr is an essential gene corresponding to the rap locus, and FZR, which accumulates predominantly in the cytoplasm, is clearly required during G1.

Amino Acid Sequence↗