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First published online 21 September 2004
doi: 10.1242/jcs.01354

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c-Cbl directs EGF receptors into an endocytic pathway that involves the ubiquitin-interacting motif of Eps15

Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (20K): [in a new window]   Fig. 1. Ubiquitin ligase activity of c-Cbl contributes to clathrin-mediated EGFR internalization. (A) Wild-type c-Cbl and 70Z-Cbl proteins used in this study. 70Z-Cbl lacks 17 amino acids including part of the Ring finger domain. (B) CHO cells were transfected with empty vector, c-Cbl or 70Z-Cbl. Western blots of total cell lysates were probed with antibodies against c-Cbl. (C) CHO cells were co-transfected with EGFR, HA-ubiquitin and empty vector, c-Cbl or 70Z-Cbl. Anti-EGFR immunoprecipitates from EGF-stimulated cells were immunoblotted with anti-HA mAb to detect ubiquitinated EGFR and pAb to the EGFR to detect total EGFR protein amounts. (D) CHO cells co-expressing the EGFR and empty vector, c-Cbl or 70Z-Cbl were incubated at 37°C with 1 ng/ml 125I-labelled EGF. Internalization was monitored as outlined in Materials and Methods. Presented is the ratio of internalized to total-cell-associated radioactivity. Expression controls of the Cbl proteins are shown in B. (E) CHO cells expressing wild-type EGFR (EGFR-WT) or EGFR-Y1045 in the presence or absence of exogenous c-Cbl were used in an internalization assay as described in D. (F) CHO cells were co-transfected with full length c-Cbl and wild-type EGFR and treated as described in D, using 20 ng/ml radiolabelled EGF (control), or subjected to hypotonic shock followed by incubation with ligand in the absence () or presence () of K+ ions. Experiments in D and E contained triplicate samples, error bars represent standard deviations. In F, both values of duplicate samples are shown and means are connected by a line.

View larger version (74K): [in a new window]   Fig. 2. EGF-induced Eps15 phosphorylation depends on the ubiquitination function of c-Cbl and Y1045 of the EGFR. (A) CHO cells co-transfected with wild-type EGFR, FLAG-tagged Eps15 and empty vector, wild-type c-Cbl, or 70Z-Cbl were stimulated with EGF. Anti-FLAG immunoprecipitates (IP) were first probed with mAb to PY, stripped and reprobed with mAb to the FLAG tag. Anti-EGFR IPs were similarly probed in succession with anti-PY and anti-EGFR mAb and total cell lysates were immunoblotted (IB) with mAb to the HA-tag, as indicated. (B) CHO cells co-transfected with full-length c-Cbl, FLAG-tagged Eps15 and EGFR-WT or EGFRY1045F were stimulated with EGF. Anti-FLAG and anti-EGFR immunoprecipitates were immunoblotted with antibodies to PY (PY), FLAG-tag or EGFR.

View larger version (73K): [in a new window]   Fig. 3. Ubiquitin ligase activity of c-Cbl determines EGF-induced recruitment of Eps15 towards plasma membrane and endosomes. CHO cells co-expressing FLAG-Eps15, wild-type EGFR and HA-c-Cbl (A-L) or HA-70Z-Cbl (M-X) were incubated with EGF-TxR (red) (A,E,I,M,Q,U) on ice for 1 hour. After stimulation at 37°C for 0 minutes (A-D,M-P), 1 minute (E-H,Q-T) or 5 minutes (I-L,U-X), cells were fixed and stained with anti-FLAG mAb and FITC-conjugated secondary antibody to detect Eps15 proteins (green) (B,F,J,N,R,V) and with anti-HA mAb and Cy5-conjugated secondary antibody to detect Cbl proteins (blue) (C,G,K,O,S,W). Cells were analyzed by confocal microscopy. The displayed confocal planes are from the basal half of the cells.

View larger version (58K): [in a new window]   Fig. 4. EGF-induced re-distribution of Eps15 depends on Y1045 of the EGFR. CHO cells co-expressing FLAG-tagged Eps15, c-Cbl and wild-type EGFR (A-F) or EGFR-Y1045F (G-L) were incubated with EGF-TxR (red) on ice for 1 hour. After stimulation at 37°C for 0 minutes (A,B,G,H), 1 minute (C,D,I,J) or 5 minutes (E,F,K,L), cells were fixed and stained with anti-FLAG, followed by FITC-conjugated secondary antibody (green). Cells were analyzed by confocal microscopy. The displayed confocal planes are from the basal half of the cells. XZ sections are shown below each panel.

View larger version (68K): [in a new window]   Fig. 5. Mutation of the second UIM of Eps15 abrogates EGF-induced Eps15 phosphorylation and re-distribution. (A) Wild-type and mutant Eps15 constructs used in this study. Eps15-UIM- contains leucine to alanine substitutions at positions 883 and 885, indicated by an asterisk. UIM-WT consists of amino acids 741-897 of murine Eps15. UIM-mut in addition contains L883A/L885A substitutions. (B) CHO cells were co-transfected with EGFR-WT, c-Cbl and Eps15-WT or Eps15-UIM-. FLAG-tagged Eps15 and EGFR were immunoprecipitated from stimulated or control cells. Immunoprecipitates (IP) and total cell lysates were separated on gel and immunoblotted (IB) using mAbs to PY, FLAG-tag or HA-tag. (C-N) For immunofluorescence, CHO cells expressing EGFR-WT, c-Cbl and Eps15-WT (C-H) or Eps15-UIM- (I-N) were incubated with EGF-TxR (red) on ice for 1 hour. After stimulation at 37°C for 0 minutes (C,D,I,J), 1 minute (E,F,K,L) or 5 minutes (G,H,M,N), cells were stained with anti-FLAG mAb, followed by FITC-conjugated secondary antibody (green). The displayed confocal planes are from the basal half of the cells. XZ sections are shown below each panel.

View larger version (17K): [in a new window]   Fig. 6. Dominant negative effect of UIM-WT on c-Cbl-mediated EGFR internalization depends on intact UIM. CHO cells expressing the EGFR alone () or co-expressing the EGFR and FLAG-tagged UIM-WT (A) or UIM-mut (B) constructs (see Fig. 5A) in the presence () or absence of c-Cbl () were incubated at 37°C with 1 ng/ml 125I-labelled EGF. Internalization was determined as outlined in Materials and Methods. Presented is the ratio of internalized to total-cell-associated radioactivity. The experiments contained triplicate samples. Error bars represent standard deviations.

View larger version (73K): [in a new window]   Fig. 7. UIM-WT, but not UIM-mut, inhibits EGF-induced re-distribution of endogenous Eps15. CHO cells expressing EGFR-WT together with an empty vector (A-D), FLAG-UIM WT (E-H) or FLAG-UIM-mut (I-L) were incubated with EGF-TxR (red) on ice for 1 hour. After stimulation at 37°C for 1 minute (A-C, E-G, I-K) or 5 minutes (D,H,L), cells were stained with anti-Eps15 pAb, followed by FITC-conjugated secondary antibodies (green). Cells were analyzed by confocal microscopy. The displayed confocal planes are from the basal half of the cells, except for panel (L), which is from the upper part of the cell. XZ sections are shown below each panel. (M) For each transfectant used in the confocal experiment described above, 3x50 cells were analyzed at t=1 minute and scored for plasma membrane recruitment of Eps15. Black bars, control cells; grey bars, UIM-WT-expressing cells; white bars, UIM-mut-expressing cells. Error bars represent standard deviations between the three independent analyses.

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