Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

Abstract

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.

Figure 1.

Degradation and pH dependent phosphorylation of the EGFR upon incubation with EGF and TGFα. (A) Hep2 cells were incubated with CHX (25 μg/ml) and EGF (10 nM) (top) or TGFα (10 nM) (bottom) at 37°C for the indicated time periods. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR (Stang et al., 2000). c, control cells not incubated with ligand nor CHX. (B) Hep2 cells were incubated with EGF (10 nM) or TGFα (10 nM) on ice for 15 min. The medium was removed, and the cells were incubated on ice for 20 min in MEM without bicarbonate with BSA and MES (10 mM) adjusted to pH 5.0, 6.0, 7.0 and 7.4, containing EGF (10 nM) or TGFα (10 nM). The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to activated EGFR (pY1173). (C) Hep2 cells were incubated with 10 nM EGF (▴) or 10 nM TGFα (▵) at 37°C for indicated times, before the cells were biotinylated on ice. Biotinylated EGFR was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting. Biotinylated EGFR was normalized for precipitation efficiency using total EGFR immunoprecipitated. The data represent the mean of four independent experiments ± SEM.

Figure 2.

The EGFR shows different localization within MVBs upon incubation with EGF compared with TGFα. Hep2 cells incubated with EGF (10 nM) (A–C) or TGFα (10 nM) (D-E) on ice for 15 min and chased at 37°C for 30 min were processed for immuno-EM, sectioned and labeled using antibody to EGF (A) or EGFR (B–E). Upon activation with EGF, labeling for EGF (A), as well as for EGFR (B–C), is found on MVBs at all stages of formation. The labeling on MVBs is found on the limiting membrane (small arrowheads), as well as on internal vesicles. In MVBs with a high number of internal vesicles (B and C), labeling is concentrated on the inner membranes. Some labeling for the EGFR is also found on small vesicles within the cytoplasm (large arrowheads). In cells activated with TGFα (D–E), labeling intensity is generally reduced (D), and labeling is mainly localized to the outer limiting membrane of MVBs. Note that the labeling of small vesicles within the cytoplasm appears to be most frequent in TGFα-treated cells. Bar, 100 nm.

Figure 3.

Ubiquitination and phosphorylation of the EGFR and tyrosine phosphorylation of c-Cbl induced by EGF and TGFα. (A) Hep2 cells were incubated with EGF (10 nM) or TGFα (10 nM) on ice for 15 min and chased at 37°C for indicated times. The cells were lysed, and the lysates were subjected to immunoprecipitation with antibody to conjugated ubiquitin, followed by immunoblotting with antibody to EGFR (top). Lysates were also subjected to SDS-PAGE and immunoblotting with antibody to EGFR (middle) or activated EGFR (pY1173) (bottom). c, control cells not incubated with ligand. (B) Hep2 cells were incubated as in (A), and c-Cbl was immunoprecipitated. The precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to phosphotyrosine (pTyr). c, control cells not incubated with ligand. (C) Hep2 cells were preincubated with PD153035 (100 nM) for 2 h at 37°C, before incubation with EGF (10 nM) on ice for 15 min. The cells were subsequently chased in MEM without bicarbonate containing PD153035 (100 nM) at 37°C for the indicated time periods. Conjugated ubiquitin was immunoprecipitated, and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to EGFR (left). c-Cbl was immunoprecipitated, and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to phosphotyrosine (pTyr) (right).

Figure. 4.

Localization of EGFR and c-Cbl upon incubation with EGF and TGFα. Hep2 cells were incubated without ligand (A–C) or with EGF (10 nM) (D–F and J–L) or TGFα (10 nM) (G–I and M–O) on ice for 15 min. In J–O, the cells were further chased at 37°C for 15 min. The cells were processed and immunostained, as described in Materials and methods, using sheep anti-EGFR (1:500) and rabbit anti–c-Cbl (1:500), followed by Cy™2-conjugated donkey anti-sheep (1:250) (green) and Alexa Fluor 594–conjugated goat anti–rabbit (1:1,000) (red). In nonstimulated cells the EGFR is concentrated at the plasma membrane (A, arrowhead), whereas c-Cbl is localized to the cytoplasm. Upon incubation with EGF or TGFα on ice, c-Cbl is recruited to the plasma membrane and colocalizes with EGFR (D–I, arrowheads). The EGFR colocalizes with c-Cbl in vesicular compartments upon incubation with EGF and chase at 37°C for 15 min, as indicated by arrowheads in J–L. After incubation with TGFα and chase at 37°C for 15 min the EGFR is localized at the plasma membrane, and c-Cbl is localized to the cytoplasm, giving no colocalization. Bar, 50 μm.

Figure 5.

Overexpression of N-Cbl does not affect the endocytosis of EGF, but inhibits degradation of EGF and EGFR. (A) Rh-EGF (10 nM) was added to COS-1 cells transfected with a plasmid encoding HA-tagged N-Cbl. The cells were incubated for 15 min at 37°C, before being fixed and immunostained with anti-HA antibody. The HA-N-Cbl–positive cells demonstrate green fluorescence. Both transfected and nontransfected cells clearly endocytosed Rh-EGF (red fluorescence). Bar, 25 μm. (B) Mock-transfected COS-1 cells (transfected with empty pCDNA3) or COS-1 cells transfected with a plasmid encoding HA-N-Cbl were incubated with ¹²⁵I-EGF (0.2 nM) on ice for 15 min and chased at 37°C for the indicated time periods. Analysis of internalized EGF (•, ○) was performed as described (Skarpen et al., 1998). The data represent the mean of three independent experiments ± SEM. (C) Mock-transfected COS-1 cells (▪, ▾) or COS-1 cells transfected with a plasmid encoding HA-N-Cbl (□, ▿) were loaded with 8.5 nM ¹²⁵I-EGF for 20 min at 37°C. The cells were washed and stripped of surface-associated ¹²⁵I-EGF before being chased for indicated times. Analysis of recycled EGF (▪, □), intact intracellular EGF and degraded EGF (▾, ▿) was performed as described in Materials and methods. The fraction representing intact intracellular EGF was not affected by N-Cbl and is not demonstrated. The data represent the mean of three independent experiments ± SEM. (D) The effect of overexpression of N-Cbl on degradation of EGFR was investigated by Western blotting. Mock-transfected COS-1 cells (control) and COS-1 cells transfected with a plasmid encoding HA-N-Cbl were incubated without or with EGF (10 nM) in the presence of CHX for 3 or 5 h. The cell lysates were subjected to SDS-PAGE and immunoblotting with anti-EGFR antibody. (E) The effect of overexpression of N-Cbl on ubiquitination of EGFR was investigated by immunoprecipitation and Western blotting. EGF (10 nM) was added to mock-transfected COS-1 cells (control) and to N-Cbl–transfected COS-1 cells, and EGF was bound for 15 min on ice. Then the cells were washed, and subsequently incubated at 37°C for 10 min. The cells were lysed and the lysate immunoprecipitated with antibody to conjugated ubiquitin. Immunoprecipitated material was subsequently analyzed by SDS-PAGE and immunoblotting with anti-EGFR antibody.

Figure 6.

Degradation of the EGFR and ¹²⁵I-EGF in the presence of NH₄Cl, MG132, and lactacystin. (A–C) Hep2 cells were preincubated with either NH₄Cl, MG132 or lactacystin, as described in Materials and methods. (A) EGF (10 nM) and CHX (25 μg/ml) was added, and the cells were further incubated at 37°C for the indicated time periods. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR. c, control cells not incubated with ligand or CHX. Both NH₄Cl, MG132 and lactacystin inhibited the degradation of EGFR. The presented data is representative of seven independent experiments. (B) EGF (10 nM) was added to cells on ice for 15 min before chase at 37°C for the indicated times in medium containing the same inhibitors as during the preincubation. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to activated EGFR (pY1173). c, control cells not incubated with ligand. (C) Cells preincubated with or without inhibitors were incubated with ¹²⁵I-EGF (0.2 nM) on ice for 15 min and chased in medium containing the inhibitors at 37°C for the indicated time periods. Analysis of EGF in the medium (▿), EGF at the cell surface (○), internalized EGF (•) and degraded EGF (▾) was performed as described (Skarpen et al., 1998). Both NH₄Cl and MG132, but not lactacystin, inhibited the degradation of ¹²⁵I-EGF. (D) Hep2 cells were preincubated for 2 h with and without lactacystin. The cells were then loaded with 8.5 nM ¹²⁵I-EGF for 20 min at 37°C, and recycling and degradation was investigated as described in Fig. 5 C. Lactacystin was found to enhance recycling and to inhibit degradation of ¹²⁵I-EGF. The fraction representing intact intracellular EGF was not affected by lactacystin and is not demonstrated. The data in C and D represent the mean of 3 independent experiments ± SEM.

Figure 7.

Translocation of EGF-EGFR to internal membranes of MVBs depends on proteasomal activity. Control and lactacystin-treated Hep2 cells were incubated with EGF for 15 min on ice followed by chase in EGF free medium at 37°C for 1 h. To localize endocytosed EGF, ultra-thin frozen sections were labeled with anti-EGF antibodies followed by 15 nm protein A gold. In control cells (A) EGF is localized within MVBs with a high number of internal vesicles, and small amounts of labeling is also found within electron dense, compact MVBs (arrowhead). In lactacystin-treated cells (B) EGF is localized to MVBs with very few internal vesicles. (C) Lactacystin-treated Hep2 cells (preincubated for 2 h) were incubated with 10 nm BSA-gold for 60 min at 37°C. Endocytosed gold was found to localize within MVBs with high numbers of internal vesicles. Bars, 100 nm.

Figure 8.

Preincubation of Hep2 cells with lactacystin for 3 and 5 h does not inhibit subsequent EGF-induced ubiquitination of the EGFR. Hep2 cells were preincubated with or without lactacystin and CHX for 3 or 5 h. Then control cells and cells pretreated with lactacystin/CHX were incubated without or with EGF for 15 min on ice before being washed and incubated at 37°C for 10 min. The cell lysates were subjected to SDS-PAGE and immunoblotting with antibody to EGFR. The upsmearing illustrates ubiquitination.

Figure 9.

The COOH-terminal part of EGFR–GFP is intact upon translocation of EGFR to inner membranes of MVBs . Hep2 cells transfected with EGFR-GFP, as described in Materials and methods, were incubated with 10 nM EGF for 30 min at 37°C before processing for immuno-EM. Thawed cryosections were double labeled using anti-GFP antibodies followed by 15 nm protein A gold and anti-EGFR antibodies followed by 10 nm protein A gold (arrowheads). The results show that both GFP- and EGFR-labeling localize to the limiting membrane as well as to inner membranes of MVBs. Bar, 100 nm.