spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 19 November 2003
doi: 10.1242/dev.00883

Development 130, 6411-6421 (2003)
Published by The Company of Biologists 2003
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lei, L.
Right arrow Articles by Irvine, K. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lei, L.
Right arrow Articles by Irvine, K. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

An O-fucose site in the ligand binding domain inhibits Notch activation

Liang Lei, Aiguo Xu, Vladislav M. Panin* and Kenneth D. Irvine{dagger}

Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA



View larger version (44K):

[in a new window]
  		Fig. 1. O-fucosylation of Notch. (A) Schematic of EGF repeats in the extracellular domain of Notch. Top: Drosophila Notch, with EGF repeats that conform to a narrow consensus sequence for O-fucosylation [C2XXGG(S/T)C3] shaded pink, and those that conform only to a broader consensus sequence [C2XXX(A/G/S)(S/T)C3] shaded orange. Green bar marks EGF11-12, asterisks mark repeats to which NAx mutations map, X marks repeats mutated in this study. Bottom: Consensus O-fucosylation pattern, with EGF repeats shaded according to the fraction of the 15 Notch receptors analyzed that have a broad consensus O-fucose site in that repeat (15/15=black, 0/15=white, intermediate levels of conservation are shaded proportionately gray). (B) Schematic of an EGF domain, illustrating the first two steps in the O-fucosylation pathway. In CHO cells, further elongation occurs; the extent of elongation in Drosophila is unknown. Adapted with permission from Moloney et al. (Moloney et al., 2000aGo). (C) Amino acid sequences of O-fucose sites mutated in this study. The O-fucose attachment site is underlined; the mutation created is in parenthesis. (D) To characterize O-fucosylation of EGF12, a polypeptide including EGF10-12 and a V5 tag was expressed in S2 cells, partially purified, and assayed as an acceptor substrate for Fringe as described previously (Panin et al., 2002Go). Fluorography reveals that N-EGF10-12 is a substrate for Fringe, but that the N-EGF10-12f mutant is not. (E) Schematic of part of the wing disc, illustrating Notch signaling at the DV boundary. Serrate (SER) is expressed by dorsal cells and activates Notch (N) in ventral cells; it is blocked from activating Notch in dorsal cells by Fringe. Delta (DL) is expressed by dorsal and ventral cells, but activates Notch principally in dorsal cells, potentiated by Fringe. Notch activation results in the expression of downstream genes, including wg (magenta). (F) Portion of a wing disc, stained for WG protein.

 


View larger version (75K):

[in a new window]
  		Fig. 2. An ectopic expression assay for Notch activity in the wing. Wing imaginal discs, with ventral bottom and anterior left, stained for expression of WG (magenta), GFP (green) and ß-galactosidase (red). Arrows point to ectopic dorsal expression; arrowheads point to absence of expression. Panels marked with a prime show a single channel of the double stain to the left. (A,B) GFP marks the ptc-expression stripe. Higher or lower Notch expression can affect the length of the ectopic Notch activation stripe (i.e. how far it extends towards the proximal wing), but not the width (Notch activation is always confined to the AP boundary). (A) ptc-Gal4 UAS-N.865 UASGFP. (B) ptc-Gal4 UAS-N.865 UAS-fng[22a]UAS-GFP. Notch activation in ventral cells is due to potentiation of Delta signaling by Fringe (Panin et al., 1997Go). (C-F) GFP marks wild-type cells; insets show a portion of the ptc expression stripe (red). (C) ptc-Gal4 UASN.865 UAS-lacZ with anterior Dlrev10 clone. (D) ptc-Gal4 UAS-N.865 UAS-lacZ with posterior Dlrev10 clone. (E) ptc-Gal4 UAS-N.865 UAS-lacZ with anterior Serrev2-11 clone. (F) ptc-Gal4 UAS-N[865]UAS-lacZ with posterior Serrev2-11 clone. This clone also disrupts normal Notch activation at the DV border.

 


View larger version (126K):

[in a new window]
  		Fig. 3. Influence of O-fucose site mutants on Notch activity. WG expression (magenta) in wing discs expressing a Notch receptor with O-fucose attachment sites mutated. Arrows point to ectopic expression. (A) ptc-Gal4 UAS-N-EGF24f[c]. (B) ptc-Gal4 UAS-NEGF26f[b]. (C) ptc-Gal4 UAS-N-EGF24+26f[a]. (D) ptc-Gal4 UASN-EGF31f[b]. (E) ptc-Gal4 UAS-N-EGF12f[M4a]. (F) ptc-Gal4 UAS-N-EGF12f[M4a]UAS-fng[22a]. Fringe also results in Notch activation in ventral cells.

 


View larger version (140K):

[in a new window]
  		Fig. 4. Notch ligands are required for N EGF12f induced Notch activation. Wing discs stained for WG (magenta) or SER (red), and GFP or MYC (green). (A,C,E) Arrows point to ectopic expression inside clones; arrowheads point to its absence. (B,D,F,G) Arrows point to ectopic expression at the edge of clones. (A-G) MARCM clones, in which cells expressing Gal4 under tub-Gal4 control are identified by GFP expression. In C-G the clones are also mutant for Notch ligands. (A) tub-Gal4 UAS-GFP UAS-N-EGF12f[M4a]. (B) tub-Gal4 UAS-GFP UAS-N.865. (C) tub-Gal4 UAS-GFP UAS-N-EGF12f[M4a] Serrev2-11. (D) tub-Gal4 UAS-GFP UAS-N.865 Serrev2-11. (E) tub-Gal4 UAS-GFP UAS-N-EGF12f[M4a] Dlrev10. (F) tub-Gal4 UAS-GFP UAS-N.865 Dlrev10. (G) tub-Gal4 UAS-GFP Serrev2-11 Dlrev10. (H) SER expression in Dl mutant clones (outlined by white dashes). Faint staining in some regions is from the peripodial epithelium. In a large clone that spans the DV boundary (1), SER expression is lost non-autonomously. In a narrow spanning clone (2), SER is not significantly affected. In a dorsal clone (3), SER expression is reduced but not absent. (I) SER expression is lost autonomously from Notch clones.

 


View larger version (126K):

[in a new window]
  		Fig. 5. N-EGF12f is a functional receptor, but cannot rescue DV boundary formation. Wing discs stained for expression of WG (magenta), with MARCM clones expressing a Notch receptor under tub-Gal4 control and mutant for the endogenous Notch gene identified by GFP (green). (A) N55e11; tub-Gal4 UAS-GFP UASN.865. Notch activation is continuous through a clone that spans the DV border (arrow). (B) N55e11; tub-Gal4 UAS-GFP UAS-NEGF12f[M4a]. Notch is activated ectopically within a dorsal clone (arrow). (C) N55e11; tub-Gal4 UAS-GFP UAS-N-EGF12f[M4a]. Notch activation is disrupted at the DV boundary (arrowhead).

 


View larger version (87K):

[in a new window]
  		Fig. 6. N-EGF12f can rescue neurogenesis in Notch embryos. Embryos from a cross of N55e11/FM7; UAS-NEGF12f[M4a]/+ to da-Gal4, stained for expression of ELAV (green), Notch (red) and SXL (cyan). Absence of SXL identifies embryos as male. Anti-Notch staining allowed effective identification of embryos with Notch overexpression, but did not allow effective discrimination between zygotically wild-type and Notch mutant embryos. Staining with the markers thus allowed division of male embryos into four classes, as indicated in Table 1. (A) Class II embryo. (B) Class III embryo. A portion of a female embryo is also visible (asterisk).

 


View larger version (16K):

[in a new window]
  		Fig. 7. Mutation of the O-fucose site in EGF12 enhances Notch-ligand binding. Histograms show the results of binding assays conducted with conditioned media, including 4800 mOD/minute AP activity of the indicated fusion proteins (Media), mixed with (A) Stably transfected DL-expressing cells, (B) transiently transfected SER-expressing cells, or, as a control, transfected S2 cells. Fc:AP is a control protein, consisting of the Fc domain of human IgG fused to AP. Values represent an average of three experiments, error bars indicate one s.d.

 


View larger version (25K):

[in a new window]
  		Fig. 8. Influence of O-fucosylation of EGF12 on Notch-Serrate interactions. Schematic of the extracellular domains of Notch (N) and Serrate (SER), with EGF domains that include consensus O-fucose sites (orange), LN repeats (blue) and the DSL motif (yellow). (A) In the absence of Fringe, SER binds to and activates Notch. The structure of the SER-N complex is unknown, but the DSL motif of ligands and EGF11-12 of Notch are crucial, and might interact (Fleming, 1998Go). (B) When EGF12 can not be O-fucosylated, SER-N binding is enhanced. (C) When O-fucose is extended by Fringe, SER can not bind or activate Notch. The O-fucose glycan is shown extended to the trisaccharide, which appears to be crucial for inhibition of Jagged1-Notch1 signaling in CHO cells (Chen et al., 2001Go). (D) When the O-fucose site in EGF12 is mutant, SER can still bind despite the presence of elongated O-fucose glycans at other sites, and consequently can still activate Notch. Green triangles, fucose; red squares, N-acetylglucosamine; blue circles, galactose.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003