AIP4/Itch regulates Notch receptor degradation in the absence of ligand - PubMed (original) (raw)
AIP4/Itch regulates Notch receptor degradation in the absence of ligand
Patricia Chastagner et al. PLoS One. 2008.
Abstract
Background: The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the post-endocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet.
Methodology/principal findings: Using Itch-/- fibroblasts expressing the Notch1 receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified.
Conclusions/significance: Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Notch activation by EDTA in WT (MD-FL) and Itch-/- (ID-FL) cells.
Cells were activated by a 15 minutes EDTA treatment, in the presence of DAPT when indicated. Extracts were then directly prepared and tested by immunoblotting with antibodies recognizing the activated form of Notch (V1744 antibody, lanes 1 to 6), or the whole intracellular domain (Notch IC antibody, lanes 7 to 12). The open arrowhead indicates the S2 cleavage product appearing after Notch activation. p300 and p120 represent respectively the Notch precursor and its furin-generated transmembrane product.
Figure 2. Notch endocytosis and degradation.
Pulse-chase antibody uptake assay on MD-FL or ID-FL cells. Alexa 488-coupled anti-HA antibody (green) was taken into the cells by endocytosis during various periods of time at 37°C. When indicated, cells were incubated 1 h before adding antibody with leupeptin or lactacystin, and maintained during the entire experiment in the presence of these inhibitors. The data are representative of 4 independent experiments. Bottom, quantification of Notch degradation: the percentage of cells showing HA staining after 60 or 90 minutes of incubation was calculated as an average from at least 40 cells providing from 4 independent experiments.
Figure 3. Notch degradation occurs in the lysosomes.
The anti-HA antibody uptake assay was performed as in figure 2. After 90 minutes of chase, cells were fixed, permeabilized and incubated with an anti-LAMP-1 antibody, followed by a mouse-adsorbed rabbit anti-rat and then Al555-coupled anti-rabbit antibodies. Insets represent enlargements (fourfold) of the boxed regions. The photographs are representative of a large number of observed fields. The relative amount of Notch-containing vesicles also positive for LAMP-1 was calculated from 10 cells in each condition. In average less than 25% of Notch vesicles were LAMP-1 negative in MD-FL+Leupeptin, whereas less than 25% of Notch vesicles were positive for LAMP-1 in ID-FL cells (+/− Leupeptin).
Figure 4. Subcellular fractionation of Notch-expressing cells.
MD-FL or ID-FL cells (non-treated or after leupeptin treatment as indicated) were mechanically lysed and a post-nuclear supernatant (P) was prepared. Early (E) or late (L) endosomes were prepared by floatation in a sucrose gradient, and blotted for the presence of Notch in A. p300 is the full-length Notch molecule retained in the Golgi apparatus before its maturation by furin protease, p120 is the mature membrane-associated C-terminal part of the molecule. In B, the fractions were analyzed with rabbit anti-EGFR, anti-GM130 (as a Golgi marker) and monoclonal anti-Itch, as indicated on the left.
Figure 5. AIP4 complementation in ID-FL cells.
The anti-HA antibody uptake assay (see figure 2) was performed on ID-FL cells prealably transfected with Flag-AIP4 expression vector. After 0 or 60 minutes of chase, cells were fixed, permeabilized and incubated with a CY3-coupled anti-Flag antibody.
Figure 6. AIP4 stimulates polyubiquitination of Notch through K29-linked chains after early endocytosis.
A. HEK293T were transfected with Notch FL-CT, AIP4 WT or DN, and 6×His-ubiquitin expression vectors when indicated. His-Ub-conjugated forms of Notch were purified in denaturing conditions on Ni+-sepharose (lanes 1–4) and tested by immunoblotting with anti-Notch IC. A Notch-containing cell lysate was tested in parallel (lane 5) to visualize the non-ubiquitinated Notch. The * indicates the starting point of the ubiquitinated products derived from Notch. Bottom, the corresponding cell extracts were directly analyzed with the same antibody. B. HEK293T were transfected with Notch FL(HA), AIP4 and VSV-tagged ubiquitin (Ub) expression vectors. The number indicates the only lysine residue remaining in the ubiquitin molecule. Cell extracts were directly tested for Notch (lanes 5–8) or Notch ubiquitinated products were purified via denaturing immunoprecipitation (lanes 1–4) and revealed by anti-VSV western blotting. Bottom, the cell extracts were directly analyzed by SDS-PAGE on a 4–20% gradient gel followed by immunoblotting with anti-VSV antibody, which revealed the free ubiquitin molecules (arrow) and a smear corresponding to the ubiquitinated proteins in the whole extracts. C. HEK293T were transfected with expression vectors encoding for Notch FL(−CT), Ub K29, AIP4 (WT) and a dominant-negative form of rab5 linked to GFP (rab5DN) when indicated. Notch products were purified by immunoprecipitation and the ubiquitinated molecules were revealed by anti-VSV (lanes 1–8) followed by anti-Notch (bottom) western blotting. As controls, cell extracts were directly tested for Notch, rab5 (anti-GFP) and Itch (lanes 9–16).
Figure 7. Notch does not interact directly with Itch/AIP4.
The Notch IC+CT, IC-CT, numb and DTX proteins were in vitro-translated in the presence of 35S met, and their ability to be retained onto a GST-AIP4 fusion protein or control GST adsorbed to glutathione-agarose beads was analyzed by SDS-PAGE analysis. The input lanes (7, 8, 15, 16, 23, 24, 25) show the different in vitro-translated products prior to incubation with the beads. Two concentrations of in vitro translated DTX (0.5 or 3 µl, indicated by + and + respectively) were tested. White lines indicate that intervening lanes have been spliced out.
Figure 8. AIP4/Itch associates with Notch in non-activated cells.
Digitonin solubilized extracts from MD-FL cells were immunoprecipitated with antibodies for Notch EC (anti-HA antibody, lane 1), Notch IC (lane 2), a rabbit preimmune serum (PI, lane 3) or rabbit anti Itch (lane 4). The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with monoclonal anti-Itch and anti-Notch as indicated. Lane 5 contains an aliquot of the starting material. Bottom panel shows a longer exposure of the anti-Notch blot allowing detection of p120 in lane 4.
References
- Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7:678–689. - PubMed
- Perry WL, Hustad CM, Swing DA, O'Sullivan TN, Jenkins NA, et al. The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a18H mice. Nat Genet. 1998;18:143–146. - PubMed
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