Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

Abstract

The high affinity receptor for IgE, FcepsilonRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcepsilonRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcepsilonRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcepsilonRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcepsilonRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcepsilonRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcepsilonRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Figure 1

Ubiquitinated FcɛRI and Nedd4 are present within DRMs in RBL cells. RBL cells were transfected with an HA-tagged ubiquitin chimera. (A) Cellular extracts from sensitized RBL cells activated with DNP-BSA for less than 5 min at room temperature and treated (lane 1) or not (lane 2) with MeCD were analyzed by Western blotting. Anti-HA antibody was used to detect HA-tagged ubiquitinated proteins. Arrows indicate where β and γ subunits of the FcɛRI migrate. (B) Sensitized RBL cells were activated (+Ag) or not (−Ag) with DNP-BSA for less than 5 min at room temperature before lysis and analysis by SDS/PAGE and Western blotting. After floatation of Triton X-100 cell extracts, proteins were precipitated by using MeOH/CHCl₃ followed by an acetone wash. The analysis was carried out by SDS/PAGE and Western blotting using an anti-HA antibody. Arrows indicate where β and γ subunits of the FcɛRI migrate. (C) Filters presented in B were reprobed with anti-Lyn (C), anti-Cbl (D), and anti-Nedd4 (E) antibodies.

Figure 2

Distribution of ubiquitinated proteins and Lyn upon activation with multivalent antigen. (A) RBL cells were transfected with a HA-tagged ubiquitin chimera. Sensitized RBL cells were activated (+Ag) or not (−Ag) with DNP-BSA for less than 5 min at room temperature and then fixed and processed for immunolabeling with FITC-conjugated anti-DNP and polyclonal anti-HA antibodies plus tetramethyl isothiocyanate (TRITC)-conjugated anti-rabbit antibodies. Two magnifications are presented for activated cells. The red channel (Left), the green channel (Center), and the merged channels (Right) are shown. (B) RBL cells were activated, fixed, and immunolabeled with FITC-conjugated anti-DNP and polyclonal anti-Lyn antibodies plus TRITC-conjugated anti-rabbit antibodies (with or without MeCD treatment) or with polyclonal anti-HA antibodies plus FITC-conjugated anti-rabbit antibodies and monoclonal anti-Lyn antibodies plus TRITC-conjugated anti-rat antibodies. Only merged pictures are shown. (Bars = 10 μm.)

Figure 3

Distribution of ubiquitinated proteins and Cbl upon activation with multivalent antigen with or without MeCD treatment. Activated RBL cells were immunolabeled with polyclonal anti-Cbl antibodies plus TRITC-conjugated anti-rabbit antibodies. Costaining was performed by using either FITC-conjugated anti-DNP antibodies or monoclonal anti-HA primary plus FITC-conjugated secondary antibodies. Merged pictures are shown. (Bars = 10 μm.)

Figure 4

Distribution of ubiquitinated proteins and Nedd4 upon activation with multivalent antigen with or without MeCD treatment. Activated RBL cells were immunolabeled with FITC-conjugated anti-DNP and polyclonal anti-Nedd4 plus TRITC-conjugated anti-rabbit antibodies. The red channel (Left), the green channel (Center), and the merged channels (Right) are shown. (Bars = 10 μm.)