Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638 -4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an ϳ38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an ϳ50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of ϳ10,000-fold purified Golgi GCS with the same reagents did not yield crosslinked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.Glucosylceramide is synthesized by UDP-glucose:ceramide glucosyltransferase (glucosylceramide synthase (GCS) 1 ) (1), a resident integral membrane protein of the cis/medial-Golgi membrane (2-4). Glucosylceramide is the common precursor of most higher order glycosphingolipids, which are important cell membrane constituents and have been implicated as important factors in development, differentiation, tumor progression, and pathogen/host interactions (5-11). Thus, GCS may play significant roles in several biological processes by regulating the overall synthesis of glucosylceramide-derived glycosphingolipids. However, surprisingly little is known about the polypeptide responsible for GCS activity.We recently solubilized and partially purified (ϳ10,000-fold) enzymatically active rat liver GCS (12). We found that detergent-solubilized GCS peaked in glycerol gradients at an apparent molecular mass of ϳ60 kDa, but were unable to conclusively identify the GCS polypeptide on SDS-polyacrylamide gels. Since then, GCS was cloned from a human cDNA library by rescue of a mutant mouse cell line deficient in GCS activity (13). The predicted amino acid sequence of the cloned enzyme encodes a protein with a calculated molecular mass of ϳ45 kDa, but the cloned enzyme was not visualized by SDS-polyacrylamide gel ele...

Arsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFB, suggesting that NFAT, rather than AP-1 or NFB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between ؊111 to ؉65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1-or NFB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells.Arsenic is an environmental toxin widely distributed in water, food, air, and soil (1). Arsenic, combined with oxygen, chlorine, and sulfur, is called inorganic arsenic, which represents the most common forms of either arsenite or arsenate in the environment (2). Humans are exposed to arsenic mainly by inhalation, ingestion, and skin contact (3, 4). The inhalation route is mainly associated with occupational exposure of ore smelters, insecticide manufacturers, and sheep dip workers. Previous studies have shown that environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin and lung cancers. Although arsenic itself is not a mutagen of DNA, it has some deleterious effects, such as the potential for DNA damage by other agents and inhibition of DNA repair (5-8). More importantly, arsenite resembles many other classic carcinogens in inducing cell tumorigenesis by activating certain genes, especially those involved in tumor promotion. Nonetheless, the mechanism by which arsenite causes human lung cancers remains to be intensively elucidated (9 -11).Cyclooxygenase (COX) 3 -2, also named prostaglandin endoperoxide synthase 2, is an essential enzyme involved in the inflammation processes and other pathogenesis (12). Previous studies have demonstrated that COX-2 is consti...

BRCA2 is a tumor suppressor gene that has been implicated in response to DNA damage, cell cycle control, and transcription. BRCA2 has been found to be overexpressed in many breast tumors, suggesting that altered expression of the BRCA2 gene may contribute to breast tumorigenesis. To determine how BRCA2 is overexpressed in tumors, we investigated the transcriptional regulation of the BRCA2 promoter. Deletion mapping of the BRCA2 promoter identified three regions associated with 3-fold activation or repression and one upstream stimulatory factor binding site associated with 20-fold activation. Gel shift and cotransfection studies verified the role of USF in regulation of BRCA2 transcription. Analysis of the ؊144 to ؊59 region associated with 3-fold activation identified a putative NFB binding site. Cotransfection of the p65 and p50 subunits of NFB upregulated the BRCA2 promoter 16-fold in a luciferase reporter assay, whereas mutations in the binding site ablated the effect. Gel shift and supershift assays with anti-p65 and -p50 antibodies demonstrated that NFB binds specifically to the NFB site. In addition, ectopic expression of NFB resulted in increased levels of endogeneous BRCA2 expression. Thus, NFB and USF regulate BRCA2 expression through the BRCA2 promoter.BRCA2 is a tumor suppressor gene associated with familial predisposition to breast and ovarian cancer (1, 2). Mutations in BRCA2 are thought to account for 20 -35% of all inherited breast cancers and are associated with a 37-85% lifetime risk of developing cancer (3, 4). The great majority of disease-associated mutations in BRCA2 result in truncation of the BRCA2 protein, suggesting that loss of function of BRCA2 results in tumor susceptibility. However, the mechanisms by which the BRCA2 protein suppresses tumor cell growth are largely unknown.The BRCA2 gene encodes a 3418-amino acid nuclear protein (2, 5), that has been implicated in the cellular response to DNA damage. BRCA2 interacts directly with RAD51, a protein involved in meiotic and mitotic recombination, DNA doublestranded break repair, and chromosome segregation (6, 7), through the BRC repeats and a C-terminal binding site. BRCA2Ϫ/Ϫ animals die as early embryos (8 -11), and viable BRCA2 Ϫ/Ϫ early mouse embryos are highly sensitive to ␥-irradiation-induced DNA damage (9). Moreover, cells expressing mutant BRCA2 are more sensitive to methyl methanesulfonate-induced DNA damage than cells expressing wild type BRCA2 (12), and BRCA2 appears to be required for ionizing radiation-induced assembly of a RAD51 protein complex in vivo (13).BRCA2 may be also involved in regulation of the cell cycle and genome instability. BRCA2 is expressed in a cell cycle-dependent manner with peak expression in the S and G 2 phases of the cell cycle. Low levels of expression are detected in G 0 , G 1 , and M phase (14). Cell cycle-dependent expression has recently been associated with binding of the upstream stimulatory factor (USF) 1 protein and Elf-1 transcription factor to the BRCA2 promoter (15). In addition, BRCA2 ex...

Adriamycin and other DNA-damaging agents have been shown to reduce BRCA2 mRNA levels in breast cancer cell lines, but the mechanism by which this occurs is unknown. In this study, we show that adriamycin and mitomycin C, but not other DNA-damaging agents, repress BRCA2 promoter activity in a dose-and time-dependent manner. We demonstrate that the effect is dependent on wild type p53 and that adriamycin and p53 mediate repression of the BRCA2 promoter by inhibiting binding of an upstream stimulatory factor protein complex to the promoter. In addition, we present evidence indicating that adriamycin and other DNA-damaging agents reduce BRCA2 mRNA and protein levels by altering both BRCA2 mRNA stability and protein stability. Thus, BRCA2 levels in the cell are regulated by three independent mechanisms in a p53-dependent manner.The BRCA2 gene was identified in 1996 as a breast and ovarian cancer susceptibility gene (1, 2). The BRCA2 gene encodes a 3,418-amino acid, cell cycle-regulated, nuclear phosphoprotein (3, 4) that has been implicated in the response to DNA damage. The evidence for a role in DNA repair came initially from the observation that BRCA2 binds directly with RAD51 through the exon 11-encoded BRC repeats (5, 6) and through an additional C-terminal binding site in the mouse (7). This association with a protein involved in meiotic and mitotic recombination and DNA double-stranded break repair suggests a similar role for BRCA2. Further support for a role in DNA repair comes from the observation that cells expressing a wild type BRCA2 BRC4 domain show hypersensitivity to ␥-irradiation, an inability to form RAD51 radiation-induced foci, and a failure of radiation-induced G 2 /M, but not G 1 /S, checkpoint control (8). Moreover, cells expressing mutant BRCA2 are more sensitive to methyl methanesulfonate-induced DNA damage than cells expressing wild type BRCA2 (9). Animal models have also been used to demonstrate an association between BRCA2 and the DNA damage response. Brca2-null mouse embryos that do not survive past day 8 of embryogenesis are highly sensitive to ␥-irradiation (7). Similarly, mouse embryo fibroblast cell lines derived from viable brca2 Ϫ/Ϫ animals are highly sensitive to DNA damage induced by a number of agents (10, 11). BRCA2 has also been directly implicated in homologous recombination and gene conversion using CAPAN-1 BRCA2 mutant cell lines and homozygous mutant brca2 embryonic stem cells (12, 13) and in transcription-coupled repair in response to 8-oxoguanine treatment (14). Most recently, the C terminus of BRCA2 has been shown to bind directly to singlestranded DNA and to promote strand transfer and RAD51 loading onto DNA during homologous recombination (15). The finding that BRCA2 was involved in the response to DNA damage led to the hypothesis that DNA damage might result in the induction of BRCA2 expression. However, the opposite has proven true. Specifically, BRCA2 mRNA levels were significantly down-regulated in breast and ovarian cancer cell lines after exposure to various DNA-d...

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89 -278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive ␤-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Sitedirected mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive ␤-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive ␤-glycosyltransferases possess a conserved substratebinding/catalytic domain.