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First published online 13 December 2005
doi: 10.1242/jcs.02707

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Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way

Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, Blegdamsvej 3C, University of Copenhagen, 2200 Copenhagen, Denmark

View larger version (84K): [in a new window]   Fig. 1. Measuring ErbB2 internalization in single cells and cell populations in a non-invasive manner. (A) The method used to study ErbB2 internalization and cleavage microscopically. The upper image shows fixed, non-permeabilized cells that are stained with an antibody (Sc08) recognizing an extracellular ErbB2 epitope followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (GM-488) (green, this staining is termed ErbB2surface). The lower image shows cells that subsequently are permeabilized and stained with Sc08 again plus Alexa Fluor 568-conjugated GM secondary antibody (GM-568) (red, this staining is termed ErbB2total) and an antibody recognizing a cytoplasmic C-terminal ErbB2 epitope (2242) followed by Alexa Fluor 633-conjugated goat-anti-rabbit secondary antibody (GR-633) (blue, this staining is termed ErbB2Cterminal). (B,C) Confocal images of SK-BR-3 cells stained for ErbB2 as described in A. ErbB2 is internalized after antibody crosslinking of ErbB2 (B). This was done by incubating the cells with Sc08 for 5 minutes followed by GM for 30 minutes at 37°C. Similarly, ErbB2 is internalized after 3 µM geldanamycin treatment as indicated (C). Arrows in the enlarged image indicate that surface exposed ErbB2 tends to aggregate before internalization. (D) Scatter diagrams of pixel intensities from an untreated cell and a cell treated with 3 µM geldanamycin for 2 hours and stained as in A. The x-axis depicts the fluorescence intensity of ErbB2surface (green), whereas the y-axis depicts the fluorescence intensity from ErbB2total (red). The analyzed cells are shown in the upper right corners of the scatter diagrams, and the region of interest is encircled in a green oval. The blue overlay represents pixel intensities that are below the threshold and therefore excluded from the analysis. The Pearson's correlation coefficients, r2i, are calculated from all pixels above the threshold in the selected area and shown in the top of the diagram. Thus, reduced r2i reflects internalization because of less correlation between ErbB2surface and ErbB2total. (E) Histograms of the colocalization values for ErbB2surface and ErbB2total (r2i) from SK-BR-3 cells treated with 3 µM geldanamycin as indicated and stained as described in A. Note that increased amounts of internalized ErbB2 are seen with time after geldanamycin treatment (decreasing r2i values). (F) Histogram of the r2i values from SK-BR-3 cells where ErbB2 is internalized after antibody crosslinking as in B. Cells were stained as described in A. Bars, 20 µm.

View larger version (113K): [in a new window]   Fig. 2. ErbB2 is internalized and degraded in lysosomes in response to geldanamycin treatment. (A,B) Confocal images of SK-BR-3 control cells and cells treated with 3 µM geldanamycin for 2 hours at 37°C. (A) Thirty minutes before fixation Alexa Fluor 633-conjugated transferrin was added to the cells. After fixation and permeabilization the cells were stained with primary antibody against ErbB2 (Sc08) and GM-488. Note that ErbB2 is internalized after geldanamycin (GA) treatment and colocalizes with transferrin (arrows). (B) After fixation and permeabilization the cells were stained with Sc08 and A561 against cathepsin D followed by GM-568 and GaR-633. Colocalization of internalized ErbB2 and cathepsin D is indicated with arrows. (C) Ultracryosections of SK-BR-3 cells treated with geldanamycin for 2 hours before fixation. Labeling with Sc08 followed by GM-10 shows ErbB2 in MVBs. (D)Ultracryosection of a geldanamycin-treated SK-BR-3 cell treated as in C. The section was first immunogold-labeled for cathepsin D (A561, PAG-5) and then for ErbB2 (Sc08, GM-10) as in C. Three MVBs are seen (M1-M3, M2 cut tangentially). In M1 and M3 double-labeling for cathepsin D and ErbB2 is seen. (E) Histogram of the r2i values from SK-BR-3 cells either pre-treated with 200 nM bafilomycin or left untreated for 1 hour before both samples were incubated with 3 µM geldanamycin added for 4 hours. Cells were stained as described in Fig. 1A, and the statistical analysis done using a two-sided Mann-Whitney U-test. Bars, 20 µm (A,B); 200 nm (C,D).

View larger version (38K): [in a new window]   Fig. 3. Geldanamycin affects ErbB2 internalization rather than recycling. (A) Confocal images of control and geldanamycin-treated SK-BR-3 cells where recycling was inhibited with monensin. The cells were treated with 3 µM geldanamycin (GA) and/or 10 µM monensin (Mon) and incubated for 2 hours at 37°C. (B) Histograms of the r2i values from SK-BR-3 cells. The upper graph shows cells that were treated with 3 µM geldanamycin for 2 hours or untreated. The lower graph shows cells that were treated for 2 hours with 3 µM geldanamycin and/or 10 µM monensin alone. Cells were stained as described in Fig. 1A, and the statistical analysis done using a two-sided Mann-Whitney U-test. Note that intracellular ErbB2 is seen only after geldanamycin treatment. Bar, 20 µm.

View larger version (128K): [in a new window]   Fig. 4. Geldanamycin treatment induces a redistribution of ErbB2 in the plasma membrane and increases receptor mobility. (A) Confocal images of live SK-BR-3 cells transfected with ErbB2-CFP before and after 1 hour of geldanamycin treatment. ErbB2 resides in protrusions of the plasma membrane before stimulation, but not after stimulation with geldanamycin. (B) Diagram showing the mobile fraction of ErbB2-CFP measured using FRAP before and after treatment with 3 µM geldanamycin for 15-60 minutes. The P-values are derived using a two-sided Student's t-test (n=42) and the error bars represent 95% confidence intervals. (C) Mobile fractions of ErbB2-CFP in SK-BR-3 cells treated with 3 µM geldanamycin for 1 hour. The cells were divided into two groups depending on their degree of internalized ErbB2 as described in the Materials and Methods. The P-value is derived using a two-sided Student's t-test (n=79) and the error bars represents 95% confidence intervals. Note that the cells with high internalization also exhibit a significantly higher mobile fraction. (D-G) Electron micrographs of SK-BR-3 cells immunogold labeled for ErbB2 on the cell surface (pre-embedding immunogold labeling). CP, clathrin-coated pits/profiles. (D) A control cell where ErbB2 is primarily associated with membrane protrusions. (E,F) The surface of two geldanamycin-treated cells where the ErbB2 distribution is more even on the surface of the cell. (G) The surface of a cell where ErbB2 has been crosslinked to antibody. ErbB2 is associated with the bulk membrane between protrusions including CPs. Bars, 20 µm (A); 200 nm (D-G).

View larger version (97K): [in a new window]   Fig. 5. Cleavage of the ErbB2 kinase domain is not needed for internalization. (A-D). Confocal images of SK-BR-3 cells stained for ErbB2 as described in Fig. 1A. (A) ErbB2Cterminal staining dislocates from ErbB2total and is diffusely localized in the cytoplasm after 3 µM geldanamycin treatment, suggesting cytoplasmic cleavage of the receptor. (B) In addition, detection of the C-terminus of ErbB2 using another antibody (Ab-1) also showed similar dislocation from ErbB2total after 3 µM geldanamycin treatment for 4 hours, supporting cleavage of ErbB2. (C) In SK-BR-3 cells transfected with ErbB2-CFP and exposed to 3 µM geldanamycin for 4 hours, the C-terminal CFP-tag (detected using an antibody against the fluorophore) is also dislocated from ErbB2total after geldanamycin treatment. (D) ErbB2 is both internalized and cleaved (seen as diffuse blue staining, indicated with *) after 3 µM geldanamycin treatment. The enlarged region and purple structures show that both the C- and N-termini of ErbB2 are internalized. (E) A biotinylation assay detecting internalized ErbB2. Cells were treated with 30 µM geldanamycin for 3 hours at 37°C, Sc08 1:100 for 1 hour followed by GM 1:100 for 1 hour at 37°C, or left untreated for 3 hours at 4 or 37°C. Both geldanamycin and crosslinking cause increased internalization of ErbB2 compared to the control situation but had no effect on the transferrin receptor (TfR). Note that in contrast to the other experiments we have used a tenfold increased concentration of geldanamycin. (F,G) Ultracryosections of SK-BR-3 cells treated with 3 µM geldanamycin for 2 hours before fixation. C-terminal ErbB2 was labeled with Ab-1 followed by PAG-5 (arrows), and then N-terminal ErbB2 with Sc08 followed by GM-10 (arrowheads) on the cell surface (F) as well as in a MVB (G). Bars, 20 µm (A-D); 200 nm (F,G).

View larger version (33K): [in a new window]   Fig. 6. Cleavage of the ErbB2 kinase domain is inversely correlated to internalization. (A) The upper left histogram shows r2c values from cells treated with 3 µM geldanamycin for 4 hours and untreated cells. The lower histogram shows similar r2c values for cells that in addition have been treated with the pancaspase inhibitor zVAD-fmk for 4 hours. The r2c values were calculated for individual cells after staining as described in Fig. 1A. P-values from two-sided Mann-Whitney U-tests are shown. Note that zVAD-fmk inhibited ErbB2 cleavage. (B) The r2i values from the same cells as in A, showing that zVAD-fmk has no effect on ErbB2 internalization. (C-E) Scatter diagrams, where each dot in the scatter diagram represents r2i and r2c values from a single SK-BR-3 cell treated as indicated and stained as described in Fig. 1A. (C) Untreated cells, cells treated with 3 µM geldanamycin for 1, 2 or 4 hours. (D) The cells were pre-treated with 50 µM zVAD-fmk and either added 3 µM geldanamycin or left in zVAD-fmk for 4 hours. The two images to the right show two different situations where either the internalization is dominant and no cleavage can be observed or where cleavage dominates and internalization is limited, which together with the scatter diagram suggest that these processes are competing events. (E) The cells were treated with 200 nM bafilomycin for 1 hour, then 3 µM geldanamycin was added for 4 hours. It is seen that cells with much intracellular ErbB2 had little cleavage and vice versa.

View larger version (75K): [in a new window]   Fig. 7. Geldanamycin-induced ErbB2 internalization requires proteasomal activity. (A) Western blot of ErbB2 from SK-BR-3 cells treated with 3 µM geldanamycin as indicated. In the lower panel the cells were pre-treated with the proteasome inhibitor lactacystin (10 µM) for 1 hour and added geldanamycin. Note that ErbB2 is degraded after geldanamycin treatment, and that this depends on proteasomal activity. (B) Confocal images of SK-BR-3 cells treated with the proteasomal inhibitor lactacystin (10 µM) for 1 hour and then 3 µM geldanamycin (upper right image) for 4 hours. Strikingly, the internalization was inhibited compared to cells that were only treated with geldanamycin (upper left image). Likewise, if cells were co-treated with both bafilomycin (200 nM) and lactacystin (lower right image) before addition of geldanamycin there was no internalization of ErbB2, confirming the importance of proteasome activity for internalization and as a consequence lysosomal degradation. (C) Upper histogram shows r2i values from cells treated with 3 µM geldanamycin for 4 hours and cells that were treated with lactacystin for 1 hour and then geldanamycin for 4 hours. Lower histogram shows similar r2i values for cells that furthermore have been pre-treated with bafilomycin. The r2i values were calculated for individual cells after staining as described in Fig. 1A, and P-values are from two-tailed Mann-Whitney U-tests. (D) Confocal images showing cells treated with monensin and geldanamycin for 2 hours (left image). When cells were pretreated with 10 µM lactacystin before 10 µM monensin and 3 µM geldanamycin were added, the geldanamycin-induced accumulation of intracellular ErbB2 in cells with inhibited recycling (left image) was efficiently inhibited (right image), suggesting that proteasomal activity is needed for ErbB2 internalization. Bars, 20 µm.

View larger version (43K): [in a new window]   Fig. 8. Tentative model of the mechanisms that influence ErbB2 downregulation in response to geldanamycin. Geldanamycin releases ErbB2 from restraints that inhibit its mobility, leading to internalization of the receptor. These processes require proteasomal activity. Cleavage of ErbB2 is not needed for internalization of the receptor, but is a competing event. Finally, ErbB2 is degraded in lysosomes rather than in proteasomes.