A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition

Cordle, Jemima; Johnson, Steven; Tay, Joyce Zi Yan; Roversi, Pietro; Wilkin, Marian B.; Madrid, Beatriz Hernandez de; Shimizu, Hideyuki; Jensen, Sacha A.; Whiteman, Pat; Jin, Boquan; Redfield, Christina; Baron, Martín; Lea, Susan M.; Handford, Penny A.

doi:10.1038/nsmb.1457

Cited by 231 publications

(265 citation statements)

References 54 publications

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“…These results add to the current view of the effect of these ligands on angiogenesis (31,59). Jagged has been suggested to counteract Dll4 signaling through cis-inhibition by direct binding to the Notch receptors and inhibition of Dll4-mediated activation in the signal receiving cell (35,60,61). By contrast, our data show that the phenotype can be rescued by externally presented recombinant Jagged ligands, which support the existence of transsignaling mechanisms in the proangiogenic functions of Jagged.…”

Section: Discussioncontrasting

confidence: 56%

“…Dll4 activation of Notch in neighboring cells reduces expression of VEGFR2 and inhibits tip cell selection. In contrast to Dll4, Jagged-Notch signaling promotes tip cell selection and sprouting by antagonizing Dll4-Notch signaling (31,(33)(34)(35)(36). How the competition between the ligands is balanced and how ligand-receptor signaling specificity is achieved is under intense investigation (31,33,34,37,38).…”

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confidence: 99%

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Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

Antfolk

Sjöqvist

Cheng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Notch signaling is a key regulator of angiogenesis, in which sprouting is regulated by an equilibrium between inhibitory Dll4-Notch signaling and promoting Jagged-Notch signaling. Whereas Fringe proteins modify Notch receptors and strengthen their activation by Dll4 ligands, other mechanisms balancing Jagged and Dll4 signaling are yet to be described. The intermediate filament protein vimentin, which has been previously shown to affect vascular integrity and regenerative signaling, is here shown to regulate ligand-specific Notch signaling. Vimentin interacts with Jagged, impedes basal recycling endocytosis of ligands, but is required for efficient receptor ligand transendocytosis and Notch activation upon receptor binding. Analyses of Notch signal activation by using chimeric ligands with swapped intracellular domains (ICDs), demonstrated that the Jagged ICD binds to vimentin and contributes to signaling strength. Vimentin also suppresses expression of Fringe proteins, whereas depletion of vimentin enhances Fringe levels to promote Dll4 signaling. In line with these data, the vasculature in vimentin knockout (VimKO) embryos and placental tissue is underdeveloped with reduced branching. Disrupted angiogenesis in aortic rings from VimKO mice and in endothelial 3D sprouting assays can be rescued by reactivating Notch signaling by recombinant Jagged ligands. Taken together, we reveal a function of vimentin and demonstrate that vimentin regulates Notch ligand signaling activities during angiogenesis.

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Section: Discussioncontrasting

confidence: 56%

mentioning

confidence: 99%

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

Antfolk

Sjöqvist

Cheng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…It is somewhat surprising that elimination of the O-glucose site in EGF 12 has no effect on Notch activity in cell-based assays. EGF 12 is known to be part of the ligand-binding domain (59,60), and recent modeling studies suggest that the O-glucose modification site sits in the interface between human Notch1 and Jagged1 (61). The site is also present in most but not all known Notch receptors (Fig.…”

Section: Discussionmentioning

confidence: 99%

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Rana¹,

Nita‐Lazar²,

Takeuchi

et al. 2011

Journal of Biological Chemistry

117

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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 ...

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“…X-ray crystallographic structures of proteins modified with the O-fucose monosaccharide and GlcNAc-fucose disaccharide on T466 were obtained (SI Appendix, Table S2). Structures were solved to <2 Å and were compared with the previously described structure of the unmodified protein (35) (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Taylor

Takeuchi

Sheppard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

121

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The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.glycosylation | mass spectrometry | X-ray

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A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition

Cited by 231 publications

References 54 publications

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

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