Arsenite-induced Cdc25C degradation is through the KEN-box and ubiquitin-proteasome pathway.
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Arsenite is a known human carcinogen that induces tumorigenesis through either a genotoxic or an epigenetic mechanism. In this study, the effect of arsenite on cell cycle regulation and the mechanisms that contribute to this effect were investigated. Treatment of the cells with arsenite suppressed cell proliferation and reduced cell viability in a dose- or time-dependent manner. Analysis of cell cycle profile and cell cycle regulatory proteins indicated that arsenite arrested the cell cycle at G(2)/M phase, partially through induction of cell division cycle 25 (Cdc25) isoform C (Cdc25C) degradation via ubiquitin-proteasome pathways. Mutation of the putative KEN box within the region 151 to 157 of human Cdc25C or treatment of the cells with a peptide competitor encompassing the KEN box partially inhibited arsenite-induced ubiquitination of Cdc25C. Thus, these results indicate that the regulated ubiquitination of Cdc25C may be involved in the arsenite-induced proteolytic down-regulation of Cdc25C activity in the G(2)/M phase of the cell cycle and suggest a link between cell cycle and the carcinogenic effects of arsenite. (+info)
Elovl1 and p55Cdc genes are localized in a tail-to-tail array and are co-expressed in proliferating cells.
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Elovl1 is a ubiquitously expressed gene, the product of which belongs to a highly conserved family of microsomal enzymes, which are involved in the formation of very long chain fatty acids and sphingolipids in yeast to man. To elucidate the structure and regulation of the Elovl gene we have isolated a lambda phage genomic DNA clone containing the entire mouse gene and found that Elovl1 consists of eight exons that are dispersed over 5.4 kb of genomic sequence. Interestingly, sequencing of the lambda clone to completion revealed that the insert contained a segment of the cell cycle gene p55Cdc directed in the opposite orientation. The genes are very tightly linked so that the 3'-end of the long mRNA species are complementary over a short stretch of nucleotides. Although both Elovl1 and p55Cdc are highly conserved genes, a BLAST search implies that the tail-to-tail arrangement has evolved in vertebrates. Despite the non-similar expression pattern in different tissues, mRNA analysis of the two genes disclosed simultaneous transcription during a proliferation-differentiation transition state, which suggests that the two genes may be regulated through a common bi-directional transcription mechanism under specific conditions. (+info)
Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex.
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The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC. (+info)
Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma.
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BACKGROUND: Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC. METHODS: Aneuploidy and the function of the spindle assembly checkpoint were examined using DNA flow cytometry and morphologic analysis with microtubule disrupting drugs. To explore the molecular basis, the authors examined the expression and alterations of the mitotic checkpoint gene, BUB1, using Northern hybridization and direct sequencing in 8 HCC cell lines and 50 HCC specimens. Furthermore, the authors examined the alterations of other mitotic checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, using direct sequencing in HCC cell lines with aneuploidy. RESULTS: An impaired spindle assembly checkpoint was found in five (62.5%) of the eight aneuploid cell lines. Transcriptional expressions of the BUB1 gene appeared in all cell lines. While some polymorphic base changes were noted in BUB1, BUBR1, and CDC20, no mutations responsible for impairment of the mitotic checkpoint were found in either the HCC cell lines or HCC specimens, which suggests that these genes did not seem to be involved in tumor development in HCC. CONCLUSIONS: The loss of spindle assembly checkpoint occurred with a high frequency in HCC with CI. However, other mechanisms might also contribute to CI in HCC. (+info)
Degradation of human Aurora-A protein kinase is mediated by hCdh1.
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Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, which are key regulators of the structure and function of the mitotic spindle. We previously showed that human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity. (+info)
BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1.
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The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores. (+info)
Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing.
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Early Xenopus embryos are large, and during the egg to gastrula stages, when there is little extracellular matrix, the cytoskeletons of the individual blastomeres are thought to maintain their spherical architecture and provide scaffolding for the cellular movements of gastrulation. We showed previously that depletion of plakoglobin protein during the egg to gastrula stages caused collapse of embryonic architecture. Here, we show that this is due to loss of the cortical actin skeleton after depletion of plakoglobin, whereas the microtubule and cytokeratin skeletons are still present. As a functional assay for the actin skeleton, we show that wound healing, an actin-based behavior in embryos, is also abrogated by plakoglobin depletion. Both wound healing and the amount of cortical actin are enhanced by overexpression of plakoglobin. To begin to identify links between plakoglobin and the cortical actin polymerization machinery, we show here that the Rho family GTPase cdc42, is required for wound healing in the Xenopus blastula. Myc-tagged cdc42 colocalizes with actin in purse-strings surrounding wounds. Overexpression of cdc42 dramatically enhances wound healing, whereas depletion of maternal cdc42 mRNA blocks it. In combinatorial experiments we show that cdc42 cannot rescue the effects of plakoglobin depletion, showing that plakoglobin is required for cdc42-mediated cortical actin assembly during wound healing. However, plakoglobin does rescue the effect of cdc42 depletion, suggesting that cdc42 somehow mediates the distribution or function of plakoglobin. Depletion of alpha-catenin does not remove the cortical actin skeleton, showing that plakoglobin does not mediate its effect by its known linkage through alpha-catenin to the actin skeleton. We conclude that in Xenopus, the actin skeleton is a major determinant of cell shape and overall architecture in the early embryo, and that plakoglobin plays an essential role in the assembly, maintenance, or organization of this cortical actin. (+info)
Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells.
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Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20-GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle. (+info)