Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis - PubMed (original) (raw)
Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis
Marcus Glittenberg et al. EMBO J. 2006.
Abstract
Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.
Figures
Figure 1
Wing disc assay for Serrate functions and details of mutant Serrate proteins tested. Cut (A–C, green) expression in wild type (A, B) and in discs ectopically expressing SerWT (C); ventral (v) and dorsal (d) are indicated. (A) Endogenous Serrate (red); (B) Src-GFP (red) expressed in the ptc∷Gal4 domain (ptc∷GAL4/UAS∷Src-GFP); (C, C′) Ectopic Cut expression (trans-activation, arrows) is induced in cells flanking the domain with highest levels of SerWT (_ptc∷GAL4/UAS∷Ser_WT; red), most evident at the posterior side (right) where there is a sharp boundary between SerWT expressing/non-expressing cells. Endogenous Notch signalling at the d/v boundary is inhibited due to cis-inhibition (arrowhead). Note: At the levels of detection used, endogenous Serrate expression is not visible in (B, C), or in most subsequent images. (D) Diagram depicting domains of Serrate as labelled: N-terminal (NT), green; DSL, yellow; EGF-repeats, grey; cysteine-rich region (Cys) black oval; transmembrane (TM), red. Sites of mutations are indicated. *DSL, RPRDD (residues 251–55) substituted with AAAAA; ΔC20, terminal 20 residues deleted; *LL, LL (1352–3) substituted with AA; Δint, residues 1269–1285 deleted; ΔT, truncation after residue 1290 fused to Serrate C-terminal tripeptide VMV; *KK, both K(1269) and K(1287) substituted with A; KKYL, addition of KKYL tetrapetide to C-terminus. Table summarises results from expression in wing discs: + to +++ indicates increasing strength of effects; no, indicates no effect; * indicates there may also be inhibition of Notch on adjacent cells; ^ indicates possible weak inhibition of Cut at larval stages, this does not affect the adult wing. (E) ClustalW alignment of juxtamembrane region from the intracellular domains of the Serrate/Jagged proteins indicated: black shading, identities; grey shading, similarities; yellow shading indicates region with conservation between invertebrates and vertebrates; blue box, 17amino-acids deleted in Δint; thicker blue lines highlight the NNL (aa1271–3) and NPL (aa1284–6) triplets; red boxes outline lysine residues mutated (K1269 and K1287); green triangle indicates ΔT truncation (T1290).
Figure 2
Differential effects of Serrate mutants on Notch activation. (A–D) Effects on Cut (green) when the indicated ligands are expressed using ptc∷Gal4: arrows, ventral stripes of ectopic Cut (trans-activation); arrowheads, differing levels of inhibition. (E–H) Distribution of Serrate proteins (x/z sections), yellow line indicates ptc∷GAL4 domain: SerWT (E) and Ser*DSL (F) are detected in intracellular puncta (arrowheads) and apically at the membrane (arrows); Ser*LL (G) and SerΔint (H) are restricted apically at the membrane (arrows). (I–K) Similar activation characteristics (α-Cut, green) are observed when wild-type (I) and mutant (J, K) Serrate proteins are expressed in clones (red). In (K), SerΔint clones that touch the d/v boundary eliminate activity in both compartments, as occurs with Notch mutant clones (de Celis et al, 1996).
Figure 3
Role of the Serrate intracellular motif in Notch activation. (A, B, D, E) Cut expression (green) in wing discs expressing mutant Serrate proteins (red) as indicated. Ser*NNL (A) and SerΔT (D) activate to varying extents (arrows) and retain cis-inhibition (arrowheads); Ser*NNL/NPL (B) and Ser*KK (E) no longer trans-activate but still inhibit (arrowhead). (C, F) x/z section showing that Ser*NNL/NPL (C) and Ser*KK (F) are predominantly localised apically at the cell membrane, no prominent intracellular staining is detected (although some Ser*NNL/NPL is visible basally). In (F), Src-GFP (green) expressed in the ptc∷Gal4 domain (ptc∷Gal4/UAS∷Src-GFP) labels cell membranes. Yellow line indicates ptc∷Gal4 domain in all panels.
Figure 4
The conserved intracellular motif confers an interaction with ubiquitin ligases. (A–K) S2 cells were transfected with the constructs indicated and the subcellular distribution of Serrate monitored. (A–C) All Serrate variants are predominantly localised at the cell surface when expressed alone. (D–I) Co-expression of Mib1 or Neur causes significant relocalisation of SerWT (D, G) and Ser*LL (E, H), but not of SerΔint (F, I); cells were double labelled to confirm the presence of Mib1 or Neur (not shown). (J, K) Results from analysis of >50 cells for each combination, scored for high (black), low (green) or no (orange) detectable surface Serrate staining in the absence (−, dark shading) or presence (+, light shading) of Mib1 (J) or Neur (K). (L) SerWT and Ser*LL but not SerΔint co-immunoprecipitate with NeurΔRING. Input: Serrate levels (detected as two bands) in extracts from larvae expressing Serrate and Neur constructs as indicated. SerWT and Ser*LL are at reduced levels (asterisks) in the presence of full-length Neur, SerΔint is not affected. None show reduced levels in the presence of NeurΔRING. Extracts probed with antitubulin (α-Tub) as loading control. α-Neur IP: proteins complexed with Neur were immunoprecipitated from larval extracts expressing the constructs shown and probed with α-Neur (upper panel) or α-Ser (lower panel). Lower panel: levels of SerWT, Ser*LL or SerΔint co-immunoprecipitated with Neur (arrowhead) or NeurΔRING (arrows). SerWT and Ser*LL can be co-precipitated with NeurΔRING (black arrows) which does not promote ligand degradation, SerΔint does not co-precipitate even under these conditions (grey arrows).
Figure 5
Differential effects of Serrate mutants on Notch distribution. (A–K) Notch protein (A–H, anti-NotchICD white/green; I–K, anti-NotchECD) distribution in discs expressing SerWT (A, B, I), Ser*DSL (C, D), Ser*LL (E, F, J) and SerΔint (G, H, K). Yellow line marks ptc∷Gal4 domain, yellow arrows indicate d/v boundary. (A, C, E, G, I–K) Apical x/y sections: Notch is depleted from the membrane in the domain of ectopic SerWT but is enriched at the membrane in cells expressing Ser*LL or SerΔint. In (I–K), discs were not permeablised so that only surface protein is detected. (B, D, F, H) x/z sections, arrowheads indicate Notch puncta which colocalise with SerWT (B, B′) and Ser*DSL (D, D′).
Figure 6
Cis-inhibition is compromised by an ER retention signal, enhanced by NeurΔRING and independent of Fringe. (A–C) Expression of Cut (green) in discs expressing SerWT (red, A) and SerKKYL (red B, C). Addition of KKYL motif results in reduced/no activation (arrows) and reduced cis-inhibition (arrowheads). (D, E, E′) SerKKYL has different intracellular distribution from SerWT. In x/z sections, SerWT (D) is detected at the membrane (arrow) and in vesicles (arrowhead); SerKKYL (E, E′) accumulates apically and basally within the cells (white lines). (F, G) SerWT (red, F) induces E(spl)m_β_∷CD2 (green F, white F′) in flanking ventral cells (arrows) and inhibits expression throughout the ptc∷GAL4/UAS∷Ser stripe (yellow line). Wild-type E(spl)m_β_∷CD2 (G, white) is shown for comparison. To aid comparison, yellow asterisks indicate area of high expression around L3 sensilla, yellow line indicates position of ptc∷GAL4 domain and yellow arrows indicate d/v boundary expression. (H–K) Ectopic Notch activity as indicated by Cut expression (green) is detected in few Dl (H) and in most Dl Ser (I) mutant clones (red) in the wing disc (e.g. arrows). Expression of NeurΔRING (J, K) within the mutant cells prevents ectopic Cut expression in Dl (J) but not in Dl Ser (K) mutant clones (arrow). Data from multiple samples are presented in Table I.
References
- Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284: 770–776 - PubMed
- Bonifacino JS, Traub LM (2003) Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 72: 395–447 - PubMed
- Bruckner K, Perez L, Clausen H, Cohen S (2000) Glycosyltransferase activity of fringe modulates Notch-Delta interactions. Nature 406: 411–415 - PubMed
- Cooper MT, Tyler DM, Furriols M, Chalkiadaki A, Delidakis C, Bray S (2000) Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes. Dev Biol 221: 390–403 - PubMed
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