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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↗
PubMed2001

The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23.

Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.

Animals↗
PubMed2004

APC activators caught by their tails?

The complexity of the Anaphase-Promoting Complex (APC), the major ubiquitin ligase in mitotic control, has been puzzling investigators ever since its discovery. Recent biochemical and genetic studies have now provided insights not only into the architecture of the complex, but also into how activators are recruited to the APC. In this article, we discuss the implications of these findings on our current understanding of APC activation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1999

Genomic organization and expression of the ubiquitin-proteasome complex-associated protein Rbx1/ROC1/Hrt1.

Rbx1/ROC1/Hrt1 (Rbx1) has been recently shown to be involved in the regulation of protein turn-over. Here, we report the organization of the human Rbx1 gene, established by both a cloning and a functional genomics approach. The human gene, composed of five exons, encompasses 22.3 kb on chromosome 22q 13. The expression of Rbx1 transcripts (0.5 kb) appears developmentally regulated during mouse embryonic development and is prevalent in the adult mouse genital tract. A Genbank database search for Rbx1 related sequences in various species, from plants to mammals, is indicative of a high degree of evolutionary conservation in mouse rat and zebra fish and also, for the main functional part of the molecule, in other living species, although their gene structures can be significantly altered.

Amino Acid Sequence↗
PubMed2000

The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.

Amino Acid Sequence↗
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↗