CIN85 phosphorylation is essential for EGFR ubiquitination and sorting into multivesicular bodies

Abstract

Ubiquitination of the epidermal growth factor receptor (EGFR) by cbl and its cognate adaptor cbl-interacting protein of 85 kDa (CIN85) is known to play an essential role in directing this receptor to the lysosome for degradation. The mechanisms by which this ubiquitin modification is regulated are not fully defined, nor is it clear where this process occurs. In this study we show that EGFR activation leads to a pronounced src-mediated tyrosine phosphorylation of CIN85 that subsequently influences EGFR ubiquitination. Of importance, phospho-CIN85 interacts with the Rab5-positive endosome, where it mediates the sequestration of the ubiquitinated receptor into multivesicular bodies (MVBs) for subsequent degradation. These findings provide novel insights into how src- kinase-based regulation of a cbl adaptor regulates the fate of the EGFR.

FIGURE 1:

CIN85 is phosphorylated upon EGF stimulation in a src-dependent manner. (A) Representative blot showing EGF-induced CIN85 phosphorylation. CIN85 was immunoprecipitated from HuH7 cells stimulated with EGF for the indicated time points before Western blot analysis with an anti-phosphotyrosine antibody. (B) A substantial increase in tyrosine phosphorylation was observed by 30–60 min poststimulation and quantitated from three independent experiments as described in A. The pTyr signal was normalized to the amount of total precipitated CIN85 in each lane, and the data are represented as mean ± SE. (C) Representative blot showing induction of CIN85 phosphorylation by overexpression of active src (srcY530F) but not kinase-dead src (srcK297M) in HeLa and HuH7 cells under full serum conditions. (D) Representative blot showing rescue of CIN85 phosphorylation in SYF cells by reexpression of active src (srcY530F) but not kinase-dead src (srcK297M).

FIGURE 2:

A phospho-defective CIN85 mutant, CIN85-4F, does not interfere with EGFR endocytosis. (A) Mutation of four key tyrosines in CIN85 (CIN85-4F) prevents phosphorylation of CIN85. FLAG-tagged CIN85wt or CIN85-4F was precipitated from HeLa cells coexpressing active src (srcY530F), and the CIN85 phosphorylation status was assessed by Western blot analysis. (B) Overexpression of CIN85-4F does not affect EGFR endocytosis. Representative images of RhEGF internalization in HuH7 cells expressing either CIN85wt (asterisk, top) or -4F (asterisk, bottom). Internalization was allowed for 15 min, and cells were acid stripped to visualize only internalized ligand. (C) Quantitation of three independent experiments as described in B. The data are represented as mean ± SE. (D) Representative blot of a surface biotinylation assay used to examine the effects of CIN85wt and -4F on EGFR endocytosis. HuH7 cells expressing FLAG-CIN85wt or -4F were stimulated for the indicated time points; surface proteins were biotinylated and precipitated using streptavidin (SA)-coated beads. The amount of total and surface EGFR was assessed by Western blot analysis. (E) Quantitation of four independent experiments as described in D. The data are represented as mean ± SE.

FIGURE 3:

Inhibition of CIN85 phosphorylation causes a marked retention of EGFR at the early endosome. (A) RhEGF trafficking assay in HuH7 expressing GFP-Rab5 (green) together with either FLAG-CIN8wt or FLAG-CIN85-4F. CIN85wt and mutant proteins were expressed at comparable levels. Arrowheads point to the Rab5 endosomes positive for RhEGF, which are decreased substantially in wt-expressing cells as the ligand traffics through the endocytic pathway. In contrast, cells expressing the CIN85-4F mutant exhibit a marked retention of RhEGF in the Rab5 endosome. Insets 1–6 show magnifications of the boxed areas in the overview images. Bars, 10 μm. (B) Quantitation of the amount of RhEGF colocalizing with GFP-Rab5–positive endosomes at various time points in HuH7 cells expressing GFP-Rab5 together with either CIN85wt (blue line) or CIN85-4F (red line). Almost no transport of ligand from the Rab5 endosome is observed even after a 2-h chase. For each condition ≥30 cells were counted, and the data are represented as mean ± SE.

FIGURE 4:

Inhibition of CIN85 phosphorylation delays delivery of EGFR to late endosomes. (A) RhEGF trafficking assay in HuH7 expressing GFP-Rab7 (green) as a marker for the late endosome together with either FLAG-CIN85wt or FLAG-CIN85-4F. CIN85wt and mutant proteins were expressed at comparable levels. Arrowheads point to the Rab7 endosomes positive for RhEGF, which are markedly increased by 60 min after chase in wt-expressing cells but minimized in the CIN85 mutant–expressing cells. Insets 1–6 show magnifications of the boxed areas in the overview images. Bars, 10 μm. (B) Quantitation of the amount of RhEGF colocalizing with GFP-Rab7–positive endosomes at various time points in HuH7 cells expressing GFP-Rab7 together with either CIN85wt (blue line) or CIN85-4F (red line). Disruption of the CIN85–cbl complex caused a significant delay in EGFR trafficking to the late endosomes. For each condition ≥30 cells were counted, and the data are represented as mean ± SE.

FIGURE 5:

Phosphorylation of CIN85 is required for efficient degradation of EGFR. HuH7 cells expressing FLAG-CIN85wt or FLAG-CIN85-4F (A, B) or SYF cells with or without reexpression of src wt (C, D) were serum starved for 4 h in the presence of 50 μg/ml CHX and treated with 50 ng/ml EGF plus CHX for the indicated time points. Equal amounts of each sample were analyzed by Western blot (A and C) and show a strong persistence of EGFR levels in cells expressing the mutant CIN85 (A) and an acceleration of EGFR degradation in SYF cells reexpressing src wt (C). (B, D) Quantitation of ≥4 independent EGFR degradation assays comparing CIN85wt- and CIN85-4F–expressing cells demonstrating an 8- to 10-fold increase in the retention of intact EGFR in the mutant-expressing cells. SYF cells with src wt show three- to fourfold faster receptor degradation compared with control cells as assessed by quantitation of three independent experiments. The amount of EGFR in each sample was normalized to actin, and the data are represented as mean ± SE.

FIGURE 6:

Inhibition of CIN85 phosphorylation results in reduced EGFR ubiquitination and missorting of the receptor to the limiting endosomal membrane. (A) Representative blot of a coimmunoprecipitation assay from HeLa cells coexpressing HA-cbl and either FLAG-CIN85wt or FLAG-CIN85-4F that were treated with 50 ng/ml EGF for 30 min. The levels of coprecipitated CIN85 were normalized to that of precipitated cbl, and the average amount of cbl-bound CIN85-4F relative to the wt from three independent experiments is shown. (B) Representative blot of an EGFR ubiquitination assay in HeLa coexpressing HA-Ub and either FLAG-CIN85wt or FLAG-CIN85-4F. Cells were treated with 50 ng/ml EGF for 30 min. The amount of HA-Ub was normalized to the amount of precipitated EGFR, and the average amount of ubiquitinated EGFR in CIN85-4F–expressing cells relative to wt-expressing cells from three independent experiments is shown. (C) IF images of EGFR sorting into MVBs in HeLa cells expressing either GFP-Rab5Q79L alone (control) or together with FLAG-CIN85wt or FLAG-CIN85-4F. Cells were treated with 50 ng/ml EGF for 30 min. Insets 1–3 show higher-magnification images of the boxed areas in the overview pictures. Bars, 5 μm. (D) Quantitation of three independent ILV sorting assays as described in C. Localization of EGFR was assessed in >30 cells, and the data are represented as mean ± SE.