Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28 degrees C-30 degrees C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18 degrees C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.
SUMMARY In an approach aimed at defining interacting partners of ceramide synthases (CerS), we found that fatty acyl CoA synthase ACSL5 interacts with all CerS. We demonstrate that ACSL5 generated FA-CoA was utilized with de novo ceramide for the generation of acylceramides, poorly studied ceramide metabolites. Functionally, inhibition of ceramide channeling to acylceramide enhanced accumulation of de novo ceramide and resulted in augmentation of ceramide-mediated apoptosis. Mechanistically, we show that acylceramide generation is catalyzed by diacylglycerol acyltransferase 2 (DGAT2) on lipid droplets. In summary, this study identifies a metabolic pathway of acylceramide generation and its sequestration in LD in cells and in livers of mice on a high fat diet. The study also implicates this novel pathway in ceramide-mediated apoptosis, and has implications in co-regulation of triglyceride and sphingolipid metabolisms.
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.
Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C 1 XSXPC 2 . We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site-and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, nontraditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C 1 XSX(A/P)C 2 . We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.The Notch family of single-pass transmembrane receptors is essential for early metazoan development, activating the expression of many genes involved in cell differentiation and tissue morphogenesis (1-3). Defects in Notch signaling have been implicated in a number of human diseases, including several forms of cancer, vascular defects such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (4 -6), multiple sclerosis (6), and a number of developmental syndromes (4, 7-10). The canonical Notch signaling pathway is initiated by the interaction of Notch with its ligand on an apposed cell. Upon ligand binding, Notch undergoes presenilin-1-dependent proteolysis, releasing a soluble Notch intracellular domain, which enters the cell nucleus to interact directly with the transcription factors from the CSL family and regulate Notch target genes (2). There are four members of the Notch family in vertebrates (Notch1 to Notch4) interacting with several classes of Notch ligands: ligands of the DSL family (Delta-like 1, 3, and 4 and Jagged 1 and 2) (1) and newly characterized ligands without the DSL domain, such as DNER and MAGP-1 and -2 (11-13).The Notch proteins consist of a large extracellular domain (ECD), 5 a transmembrane region, and a large intracellular domain (1, 2, 14). The majority of the ECD consists of tandem epidermal growth factor-like (EGF) repeats (36 EGF repeats are ...
Notch signaling affects many developmental and cellular processes and has been implicated in congenital disorders, stroke, and numerous cancers. The Notch receptor binds its ligands Delta and Serrate and is able to discriminate between them in different contexts. However, the specific domains in Notch responsible for this selectivity are poorly defined. Through genetic screens in Drosophila, we isolated a mutation, Notchjigsaw, that affects Serrate- but not Delta-dependent signaling. Notchjigsaw carries a missense mutation in epidermal growth factor repeat-8 (EGFr-8) and is defective in Serrate binding. A homologous point mutation in mammalian Notch2 also exhibits defects in signaling of a mammalian Serrate homolog, Jagged1. Hence, an evolutionarily conserved valine in EGFr-8 is essential for ligand selectivity and provides a molecular handle to study numerous Notch-dependent signaling events.
The Notch family of receptors plays essential roles in many phases of development, and dysregulation of Notch activity is increasingly recognized as a player in many diseases. O-Glycosylation of the Notch extracellular domain is essential for Notch activity, and tissue-specific alterations in the glycan structures are known to regulate activity. Here we review recent advances in identification and characterization of the enzymes responsible for glycosylating Notch and molecular mechanisms for how these O-glycans affect Notch activity.
Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila . Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O -glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O -glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O -xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O -glucose consensus sequence ( ) of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence ( ), is also modified with either O -Xyl or O -glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O -Xyl but not O -glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used.
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