All together these findings unveil the ability of c-Cbl to regulate inducible Hrs monoubiquitination. Hrs is found in the cytosol and on limiting membranes of early endosomes [22, 23]. Urbè and coauthors [23] have proposed that Hrs tyrosine phosphorylation may relocate Hrs from endosomal limiting membranes to the cytosol. Moreover, a more recent study [28] supported a role for Hrs poly-ubiquitination downstream of EGF stimulation in regulating MDV3100 cell line Hrs cytosolic relocation and degradation. Thus, we initially examined the distribution

of phosphorylated and ubiquitinated Hrs species between particulate-membrane and cytosolic constituents of an RBL-2H3 cell postnuclear supernatant. After having established the validity of our fractionation procedure by anti-β and anti-tubulin immunoblot

performed on total proteins from membranes and cytosol fractions (Fig. 5A), equal protein amounts of both fractions obtained from unstimulated (-) and stimulated cells were immunoprecipitated with an anti-Hrs Ab and sequentially immunoblotted as indicated (Fig. Abiraterone in vitro 5B). Five minutes after receptor cross-linking, phosphorylated Hrs was highly enriched in membrane fraction (Fig. 5B, upper panel), and this enrichment correlated with accumulation of Syk on the same location (Fig. 5A, bottom panel), suggesting that Syk-dependent Hrs phosphorylation occurs at membrane level. Interestingly, a persistence of phosphorylated Hrs in membrane compartment was observed after longer time of stimulation (data not shown). Conversely, the anti-Ub and anti-Hrs immunoblots showed that the majority of monoubiquitinated Hrs was enriched in the cytosol upon Ag stimulation (Fig. 5B, middle and bottom panels). Based on these results, we conclude that tyrosine phosphorylated Hrs preferentially localizes in the membrane fraction whereas monoubiquitinated forms of Hrs Demeclocycline are predominantly cytosolic. To investigate whether Hrs modifications could affect protein stability, RBL-2H3

cells were pretreated with cycloheximide to block protein synthesis before antigen stimulation, and the levels of Hrs were analyzed by western blot of whole-cell lysates (Supporting Information Fig. 5). While a time-dependent decrease of Syk expression (∼50%) was observed in line with previous reported susceptibility of Syk to proteolysis [17, 31, 32], Hrs expression remained substantially stable until 8 h after FcεRI engagement, supporting the notion that Hrs modifications do not act as a degradation signal on mast cells. All together, our findings indicate that Syk/Cbl-induced Hrs modifications, without affecting protein stability, might impact on the ability of Hrs to function as endocytic adapter. In addition to its key role in IR-mediated signaling transduction events, Syk is also required for endocytosis of engaged BCR and FcεRI in B and mast cells, respectively [8, 10, 11].