2), indicating that Syk kinase this website activity is required for receptor degradation. Taken together our results demonstrate that Syk knockdown negatively affects ligand-induced FcεRI endocytosis, and partially prevents the targeting of activated receptors to a degradative compartment.
We have previously demonstrated the requirement of Syk kinase activity in Cbl-mediated receptor ubiquitination . Thus, it is possible that, Syk, by regulating receptor ubiquitination, may affect FcεRI trafficking and fate indirectly. Syk might also regulate receptor endocytic trafficking by directly targeting endocytic adapter(s) that become specific substrate(s) of the kinase upon receptor engagement. We decided to concentrate our attention on Hrs, since we have previously demonstrated that it is required for FcεRI entry into lysosomes . We initially evaluate whether Hrs undergoes antigen-dependent phosphorylation and ubiquitination in RBL-2H3 cells (Fig. 2 A and B) and in mouse bone marrow-derived mast cells (BMMCs) (Fig. 2 C and D). A strong increase of Hrs phosphorylation was observed upon FcεRI engagement (Fig. 2A and C): Hrs phosphorylation peaked within 5–10 min, and subsequently declined. Beside the main form migrating around 115 kDa, the anti-Hrs blot clearly revealed the presence of a specific activation-induced form of a Mr compatible with the
addition of a single Ub molecule, characteristic of monoubiquitination (Fig. learn more 2 B, C, and D, lower panels). This latter band (indicated as Ub∼Hrs) was, indeed, recognized by the FK2 anti-Ub mAb (Fig. 2 B and D, upper panels), that can reveal both mono- and polyubiquitinated proteins, but not by the FK1 mAb, that recognize only polyubiquitinated proteins (data not shown). Samples immunoprecipitated with an isotype-matched control Ab did not show any reactivity at the 115 kDa or higher Mr range (Fig. 2 A, B, and D). To investigate whether Hrs could interact with Syk, lysates obtained from RBL-2H3 cells unstimulated (-) and stimulated for the indicated
lengths of time were subjected to immunoprecipitation with an anti-Syk mAb, and the immunoprecipitates probed with anti-Hrs Ab, and stiripentol after stripping with the immunoprecipitating Ab (Supporting Information Fig. 3). The relative amount of Hrs associated with Syk changed with a time-course similar to Hrs coimmunoprecipitation with engaged FcεRI complexes : it was maximal at 5 min and decreased to near-baseline levels within 20 min of stimulation. Notably, the level of Syk/Hrs association also remarkably correlated with that of Hrs phosphorylation, consistent with the idea that upon receptor engagement Hrs may become a substrate for Syk-mediated phosphorylation. We therefore investigated whether active Syk is able to directly phosphorylate Hrs in vitro.