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Advances in Brief

Tumor-inhibitory Antibodies to HER-2/ErbB-2 May Act by Recruiting c-Cbl and Enhancing Ubiquitination of HER-2

Leah N. Klapper, Hadassa Waterman, Michael Sela and Yosef Yarden
Leah N. Klapper
Departments of Immunology [L. N. K., M. S.] and Biological Regulation [H. W., Y. Y.], The Weizmann Institute of Science, Rehovot 76100, Israel
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Hadassa Waterman
Departments of Immunology [L. N. K., M. S.] and Biological Regulation [H. W., Y. Y.], The Weizmann Institute of Science, Rehovot 76100, Israel
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Michael Sela
Departments of Immunology [L. N. K., M. S.] and Biological Regulation [H. W., Y. Y.], The Weizmann Institute of Science, Rehovot 76100, Israel
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Yosef Yarden
Departments of Immunology [L. N. K., M. S.] and Biological Regulation [H. W., Y. Y.], The Weizmann Institute of Science, Rehovot 76100, Israel
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DOI:  Published July 2000
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    Fig. 1.

    Tumor inhibition and down-regulation of cell surface expressed ErbB-2 by the L26 mAb. A, athymic mice received a s.c. injection of 5 × 106 N87 human gastric cancer cells that overexpress ErbB-2. Three, 7, and 10 days later, mAb L26 was injected i.p. at a total dose of 0.1 mg (▪), 0.25 mg (▴), or 2 mg (○). Tumor volumes were measured at the end of the indicated time periods. Saline-injected mice were used for control (□). Bars represent SDs for groups of six mice. B, N87 cells were grown to confluence in 24-well plates, washed with binding buffer, and incubated at 37°C for the indicated time intervals in the presence (•) or absence (○) of mAb L26 (20μ g/ml). Monolayers were then rinsed twice and stripped of surface-bound antibody by using a 7-min long incubation in a low pH buffer. The level of ErbB-2 molecules that remained at the cell surface was determined by binding of a radiolabeled mAb L26. Each point represents the average ± SD of duplicates.

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    Fig. 2.

    Antibody- and ligand-induced degradation of ErbB-2 involves increased receptor ubiquitination. A, N87 cells were incubated at 37°C for the indicated time intervals in the presence of mAbs L26 or N28 (each at 20 μg/ml). ErbB-2 was immunoprecipitated (IP) by using a mixture of mAbs specific to its extracellular domain and immunoblotted (IB) with a rabbit polyclonal serum. Untreated cells served as controls (lanes labeled —). B, CHO cells were cotransfected with an ErbB-2 expression vector, along with plasmids encoding a HA-tagged ubiquitin. Forty-eight h later, cell monolayers were treated for 30 min at 37°C with or without mAb L26 (20 μg/ml). ErbB-2 ubiquitination was detected by IB receptor precipitates (top) or whole cell lysates (bottom) with an anti-AH antibody. C, CHO cells were transfected, as described above, in the presence of a plasmid encoding ErbB-1. Following stimulation by EGF (100 ng/ml, 10 min), cells were lysed and ErbB-2 was specifically precipitated under conditions that break ErbB-1•ErbB-2 heterodimers. The precipitated receptor was examined for ubiquitination, as described above. Whole cell lysates were subjected to anti-ErbB-2 IB to compare receptor expression (bottom).

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    Fig. 3.

    c-Cbl recruitment is involved in ubiquitination of ErbB-2. A, an expression vector encoding the transforming mutant of human ErbB-2 (V659E) was transfected into CHO cells. Alongside, plasmids encoding a sequence-tagged ubiquitin, c-Cbl (+), or a corresponding empty vector (−) were also used. Twenty-four h later, receptor molecules were immunoprecipitated (IP) and their ubiquitination was detected by using an anti-HA antibody (top). Whole cell lysates were examined for the effect on receptor level (bottom). B, CHO cells were cotransfected with plasmids encoding the WT form of ErbB-2 or the transforming mutant (V659E), along with a c-Cbl vector (+) or the respective empty vector (−). Total cell proteins were examined for receptor expression 48 h later by immunoblotting (IB) with an anti-ErbB-2 antibody. C, amino acid sequence alignment of the predicted Cbl-binding sites of ErbB-1 (10) , ErbB-2, and ZAP-70 (18) is shown. The consensus tyrosine and proline residues are shown in bold. The indicated amino acid numbering refers to the corresponding human proteins. D, HEK-293T cells were transfected with plasmids encoding ErbB-1 and ErbB-2 (either WT or Y1112F), and 24 h later cells were exposed for 10 min to EGF (100 ng/ml). Control cultures (Cont.) were mock-treated prior to cell lysis. Cell extracts were directly analyzed by IB with an anti-ErbB-2 antibody (bottom). Alternatively, extracts were first incubated with an immobilized v-Cbl-GST protein and association with ErbB-2 was analyzed by IB (top). Note the up-smearing of ErbB-2, which is attributed to poly-ubiquitination of the WT form. E, ubiquitination effects of EGF (left) and a bivalent Fab fragment of the L26 mAb (right) were examined in CHO cells. For stimulation by EGF, the WT form of ErbB-2 and a Y1112F mutant were introduced along with a plasmid encoding ErbB-1. Antibody stimulation was tested in cells expressing the V659E mutant of ErbB-2, and a double mutant (V659E and Y112F) denoted EF. Ubiquitination was detected in receptor immunoprecipitates (top), and the ErbB-2 level was determined in whole cell lysates (bottom).

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    Fig. 4.

    The Cbl docking site at tyrosine 1112 is essential for antibody-, ligand-, and mutation-induced enhanced turnover of ErbB-2. A, receptor degradation was followed in HEK-293T cells transfected with plasmids encoding ErbB-2 (WT) or its mutant (Y1112F). Cells were incubated with or without mAb L26 (20μ g/ml) at 37°C for the indicated time intervals. Whole cell lysates were immunoblotted (IB) with an anti-ErbB-2 antiserum. B, ErbB-2 degradation was examined as above, except cells were cotransfected with an ErbB-1 plasmid. Forty-eight h later, cells were treated with EGF (15 min at 37°C) and the level of ErbB-2 was determined in whole cell lysates. C, ErbB-2 turnover was determined in cells transfected with the indicated constructs. Following biosynthetic labeling with radioactive methionine, cells were chased with fresh medium for the indicated time intervals, and ErbB-2 was immunoprecipitated (IP). Subsequent to gel electrophoresis, labeled proteins were detected by directly exposing the dried gel to an X-ray film. D, V659E and its corresponding Y1112F double mutant (EF) were compared for their ability to undergo antibody-induced turnover. Receptor turnover was followed as described in C, following incubation at 37°C with or without the L26 mAb (20 μg/ml) for the indicated time intervals.

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July 2000
Volume 60, Issue 13
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Tumor-inhibitory Antibodies to HER-2/ErbB-2 May Act by Recruiting c-Cbl and Enhancing Ubiquitination of HER-2
Leah N. Klapper, Hadassa Waterman, Michael Sela and Yosef Yarden
Cancer Res July 1 2000 (60) (13) 3384-3388;

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Tumor-inhibitory Antibodies to HER-2/ErbB-2 May Act by Recruiting c-Cbl and Enhancing Ubiquitination of HER-2
Leah N. Klapper, Hadassa Waterman, Michael Sela and Yosef Yarden
Cancer Res July 1 2000 (60) (13) 3384-3388;
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