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c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route

¹ Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
² Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

View larger version (136K): [in a new window]   Fig. 1. Colocalization of the EGFR and c-Cbl with clathrin in stimulated cells. Serum starved COS-7 cells transiently expressing human c-Cbl were incubated without (A,C) or with (B,D) 25 ng ml-1 EGF for 5 minutes at 37°C. Prior to fixation, cells were permeabilized with saponin. Cells were incubated with antibody to the EGFR (polyclonal antibody RK-2) (A,B) or c-Cbl (C,D), followed by Texas-Red-conjugated secondary antibody and double stained with an antibody to clathrin (mAb 23) followed by FITC-conjugated anti-mouse Ig. Merged confocal images of the EGFR or c-Cbl (red) and clathrin (green) are shown.

View larger version (43K): [in a new window]   Fig. 2. c-Cbl associates with and ubiquitinates the EGFR at 0°C. HA-tagged ubiquitin and HA-tagged c-Cbl were transiently overexpressed in COS-7 cells. Serum starved cells were stimulated with 25 ng ml-1 EGF for the indicated time periods on ice or at 37°C. Immunoprecipitates (IP) of the endogenous EGFR (mAb 528) and total cell lysates were separated on 6% SDS-polyacrylamide gels. Immunoblotting (IB) was used to detect EGFR (polyclonal antibody 282.7), c-Cbl and ubiquitin (both anti-HA).

View larger version (73K): [in a new window]   Fig. 3. c-Cbl associates with and ubiquitinates the EGFR in cells overexpressing a dominant negative form of dynamin. (A) HA-tagged c-Cbl or HA-tagged ubiquitin were transiently overexpressed in COS-7 cells in combination with wild-type dynamin, dynamin-K44A or empty vector. Serum starved cells were stimulated with 25 ng ml-1 EGF for the indicated times. Anti-EGFR immunoprecipitates (IP) were separated by SDS-PAGE and subjected to immunoblotting (IB) to detect the EGFR, or c-Cbl and ubiquitin (anti-HA). Expression levels of c-Cbl and dynamin were determined in total cell lysates. (B) The effect of the dynamin constructs on EGFR internalization was tested in COS cells transiently expressing c-Cbl in combination with wild-type dynamin (A,C) or dynamin-K44A (B,D). Serum starved cells were incubated without (A,B) or with 25 ng ml-1 EGF for 5 minutes (C,D), fixed with MeOH and stained with antibody to dynamin followed by FITC-conjugated secondary antibody (green) and with antibody to the EGFR (polyclonal antibody RK-2) followed by Texas-Red-conjugated secondary antibody (red). Analysis was performed by confocal microscopy.

View larger version (91K): [in a new window]   Fig. 4. c-Cbl and the activated EGFR colocalize at the plasma membrane of permeabilized COS cells. COS-7 cells transfected with c-cbl cDNA were plated on coverslips, serum starved and stimulated with 25 ng ml-1 EGF for the indicated time periods at 37°C (A-I) or on ice (J-L). Prior to fixation, cells were permeabilized with saponin. Cells were stained with mAb directed against the EGFR followed by FITC-conjugated secondary antibody (green; A,D,G,J) and with polyclonal antibody directed against c-Cbl followed by Texas-Red-conjugated secondary antibody (red; B,E,H,K) and analysed by confocal microscopy. Merged images are shown in C, F, I and L. The confocal image in panels A-C was taken at the level of the basal membrane, that in panels G-I at the middle of the cell. The insert in L shows a xz section.

View larger version (98K): [in a new window]   Fig. 5. Endogenous c-Cbl and the activated EGFR colocalize at the plasma membrane of non-permeabilized CHO cells. CHO cells stably expressing the human EGFR were plated on coverslips, serum starved and stimulated with 25 ng ml-1 EGF for the indicated time periods at 37°C (A-I) or on ice (J-L). Cells were fixed and stained as indicated for Fig. 4 to detect the EGFR (green; A,D,G,J), or c-Cbl (red; B,E,H,K) by confocal microscopy. Merged images are shown in C, F, I and L. The confocal image in panels G-I was taken at the middle of the cells.

View larger version (47K): [in a new window]   Fig. 6. c-Cbl and the EGFR colocalize at the plasma membrane in the presence of dominant negative dynamin. The human EGFR and c-Cbl were coexpressed in CHO cells, in combination with either the dynamin K44A mutant (A-F) or wild-type dynamin (G-I). Cells were serum starved and incubated without (A-C) or with 25 ng ml-1 EGF for 5 minutes (D-I). Cells were fixed and stained with antibodies directed against the EGFR (green; A,D,G) and c-Cbl (red; B,E,H) and analysed by confocal microscopy. Merge images are shown in C, F and I. The confocal image in panels G-I was taken at the middle of the cells. Inserts show xz sections.

View larger version (88K): [in a new window]   Fig. 7. Immuno-electron microscopy reveals localization of c-Cbl and EGFR outside, and at clathrin-coated pits. COS-7 cells transfected with c-cbl cDNA were stimulated with EGF for 2 minutes at 37°C (iA,iB), for 60 minutes at 0°C (iC,iD,ii) or for 5 minutes at 37°C, followed by treatment for 15 minutes with saponin on ice (iii). All preparations were fixed and ultrathin cryosections were prepared for immunogold labelling. Sections were single labelled with antibodies to EGFR (iA,iC) or c-Cbl (iB,iD,iii), or double labelled with antibodies to EGFR and c-Cbl (iiA) or antibodies to clathrin and c-Cbl (iiB). Abbreviations: c-Cbl10, c-Cbl 10 nm gold particle; Clt15, clathrin 15 nm gold particle; EGFR15, EGFR 15 nm gold particle; N, the nucleus; S, cell surface. Inset in iii is a higher magnification of the marked area (asterisk) showing c-Cbl on coated vesicles. Magnification: i and ii show the same magnification. Bar, 100 nm.

View larger version (112K): [in a new window]   Fig. 8. Immuno-electron microscopy reveals c-Cbl in multivesicular bodies. COS-7 cells transfected with c-cbl cDNA were stimulated with EGF for 15 minutes (A,B) or 60 minutes at 37°C (C). Ultrathin cryosections were single labelled to detect EGFR (A) or c-Cbl (B), or double labelled to detect both EGFR and c-Cbl (C). Abbreviations: c-Cbl10, c-Cbl 10 nm gold particle; EGFR15, EGFR 15 nm gold particle. Bar, 100 nm.

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