AIP4/Itch Regulates Notch Receptor Degradation in the Absence of Ligand

International audienceBACKGROUND: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

The von Hippel-Lindau Tumor Suppressor Protein Promotes c-Cbl-Independent Poly-Ubiquitylation and Degradation of the Activated EGFR

Somatic mutations or reduced expression of the von Hippel-Lindau (VHL) tumor suppressor occurs in the majority of the clear cell renal cell carcinoma (ccRCC) and is a causal factor for the pathogenesis of ccRCC. pVHL was reported to suppress the oncogenic activity of Epidermal Growth Factor Receptor (EGFR) by reducing the expression of the EGFR agonist TGF-α and by reducing the translation efficiency of EGFR itself. Furthermore, it was reported that pVHL down-regulates activated EGFR by promoting efficient lysosomal degradation of the receptor. These modes of negative regulation of EGFR by pVHL were dependent on Hypoxia Inducible Factor (HIF). In this study, we report that HIF was not the only factor stabilizing the activated EGFR in VHL-deficient ccRCC cells. Down-regulation of endogenous HIF in these cells had little effect on the turnover rates of the activated EGFR. Furthermore, neither pretreatment with lysomomal inhibitors pretreatment nor down-regulation of c-Cbl, a major E3 ubiquitin ligase that targets the activated EGFR for lysosomal degradation, significantly increased the stabilities of EGFR in VHL-expressing ccRCC cells. In contrast, pretreatment with proteasomal inhibitors extended EGFR lifetime and led to similar EGFR half-lives in VHL-expressing and VHL-deficient ccRCC cells. Down-regulation of c-Cbl in VHL-deficient ccRCC cells revealed that the c-Cbl and pVHL collaborated to down-regulate the activated EGFR. Finally, we found that pVHL promoted the poly-ubiquitylation of the activated EGFR, and this function was c-Cbl-independent. Thus these results indicate that pVHL limits EGFR signaling by promoting c-Cbl-independent poly-ubiquitylation of the activated receptor, which likely results in its degradation by proteasome

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment

Employment protection regulations in local public transport: how do they affect production efficiency and competition?

Employment protection in local public transport raises crucial questions in a period of changing regimes of how transport services are purchased by the government. Traditionally the government either produced passenger services itself or negotiated with private businesses. However, competitive tendering is increasingly introduced in European countries. This occasions much debate, as many – especially labour unions – argue that competitive tendering worsens working conditions. They are of the opinion that employees pay for the increased production efficiency gained through competitive tendering, as “slack” is reduced and employment relations become unstable. On this background there is a debate going on in Norway on how to regulate employment protection in the event of competitive tendering processes in local public transport. One alternative is to give the workers the same rights when competitive tendering is carried out, as they would have had in the events of transfer of an undertaking. This is practised in the capital areas of Denmark and Sweden and introduced in one Norwegian region. In a report by the Institute of Transport Economics (Osland/Leiren 2006), we map the expected results of franchising on employment relations which refer to employment protection, wages and pensions, working time, qualification requirements, and workers’ participation. We conclude that franchising is most crucial to employment protection. We suggest alternative ways of limiting this effect. One of the alternatives is to adopt employees’ rights in the events of transfer of an undertaking. However, the effects of such a regulation may vary according to different factors such as whether the employees have already been through a process of competitive tendering. We propose to map such factors and discuss the extent of how the regulation may affect employment relations. Moreover, we ask the question: How will the regulation affect other areas such as competition, production costs and administrative costs? Does the regulation cause unwanted risks for the Public Transport Administration and bus companies, thereby rising costs and distortion of competition

Mapping of Unstable and Potentially Unstable Slopes in Sogne og Fjordane

For the past three years NGU has worked on 25 unstable and potentially unstable rock slopes in Sogn og Fjordane. In addition Fjærlandsfjord, Hyenfjord and Årdalsvatnet were systematically mapped for deposits of prehistoric and historic rock slope failures onshore and with help of a bathymetry. Mapping on land included structural mapping of ten sites by on-site field mapping and nine sites by remote structural mapping using terrestrial laser scanning technology (TLS). Field work also included periodic monitoring of 14 sites using differential Global Positioning Systems (dGPS) and TLS at 4 sites. Synthetic Aperture Radar was app lied for the entire county but slide velocities could only be mapped out at one locality at Osmundneset (Gloppen municipality). A large amount of work was carried out on the slope east of Flåm in the Aurland valley, and results have been reported separately (NGU 20 11 .025). Three large instabilities have been discovered or taken into the monitoring program. These are Osmundneset in Gloppen municipality, Skrednipa in Sogndal municipality, and the Ovris valley in Vik municipality. The largest movements with 1.5 cm horizontal and 1.5 cm vertical displacement were measured on the instability with a volume of approx. 1 Mm3 in Ovris valley. Opening of cracks has been measured at that site also in the 1960’s, 1970’s and 1980’s. Our data suggest a slight acceleration of this instability. Acceleration was also documented for a 100.000 m3 large instability called Lifjellet, although velocity of that site is less than half of the velocity of the block in Ovris valley. However at that site a collapse of a rockslope with a volume of 25.000 to 30.000 m3 occurred only 19 years ago. Installation of continuous monitoring and early-warning systems should be considered at both of those sites as well as at similar sites where relatively small instabilities that might fail without a long acceleration phase are positioned above settlements (Gråberget in Høyanger municipality). Similarly all other monitored instabilities in Sogn og Fjordane are in the order of mm/yr and not considered to be critical on a short term. However, periodic monitoring has to be continued. Cosmogenic nuclide dating (CN) has been applied to determine ages of rockslide deposits in Fjærlandfjord (Sogne municipality) and at the slope E of Flåm (Aurland community), resulting in Late Pleistocene and Holocene ages. CN dating has also applied to the sliding planes at Skjeringahaugane (Luste municipality). The results indicate that the movement initiated at the beginning of the Holocene and is progressive. Long term slip rates are in the same order of slip rates measured by dGPS