Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu - PubMed (original) (raw)

Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu

Wanping Xu et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression in vivo is associated with poor prognosis. We now show that the chaperone-binding ubiquitin ligase CHIP efficiently ubiquitinates and down-regulates ErbB2. CHIP expression shortens the half-life of both nascent and mature ErbB2 protein. In vitro ubiquitination assay shows that CHIP serves as a ubiquitin ligase for ErbB2, and both exogenously expressed and endogenous CHIP coprecipitate with the kinase. Furthermore, CHIP association with ErbB2 requires a chaperone intermediate and is increased by the chaperone-binding drug geldanamycin, a potent stimulator of ErbB2 ubiquitination and degradation. These data describe a previously unrecognized pathway, amenable to pharmacologic manipulation, that mediates ErbB2 stability.

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Figures

Figure 1

Figure 1

The E3 ubiquitin ligase CHIP down-regulates ectopically expressed ErbB2 protein in COS7 cells. Cotransfected COS7 cells in six-well plates were lysed with TMNS buffer (50 mM Tris, pH 7.5/150 mM NaCl/20 mM Na2MoO4/0.09% Nonidet P-40). Cell lysate was separated by SDS/PAGE and probed with antibodies against ErbB2 (L87, Neomarker), hemagglutinin epitope (HA)-tagged c-Cbl (mouse anti-HA), tubulin (mouse monoclonal; Oncogene), or CHIP (rabbit polyclonal). (A) Cells were transfected with different ErbB2 constructs (Upper), with or without cotransfection of wild-type CHIP. One set of cells was treated with 1 μM GA for 6 h before lysis. (B) Cells were transfected with ErbB2 and c-Cbl constructs. GA treatment was as in A. (C) Cells were transfected with ErbB2, together with different constructs of CHIP or vector alone.

Figure 2

Figure 2

CHIP interacts with ErbB2 in vivo by a chaperone intermediate. (A) CHIP specifically associates with ErbB2 proteins. COS7 cells in 150 × 25 mm plates were transfected with CHIP, plus full-length or truncated ErbB2. Twenty-four hours after transfection, cells were lysed with TMNS buffer. For immunoprecipitation (IP), 1 mg of cell lysate was incubated with 2 μg of anti-ErbB2 mAb (Ab2/Ab5, Oncogene), and the immunocomplexes were precipitated with protein G-agarose beads. Precipitated proteins were dissolved in SDS sample buffer and separated by SDS/PAGE. Blot was probed with ErbB2 antibody to check immunoprecipitation efficiency. IB, immunoblotting. Association of CHIP with ErbB2 was detected by rabbit polyclonal anti-CHIP antibody. (B) Association of CHIP with ErbB2 requires intact TPR domain. COS7 cells were transfected with wild-type ErbB2 plus different CHIP constructs. Immunoprecipitation and Western blotting were performed as in A. (C) GA increases binding of CHIP to ErbB2 protein. Transfected COS7 cells were treated with 1 μM GA and 100 μM ALLnL for 1 h before being lysed with TMNS buffer. Immunoprecipitation and Western blotting were as in A. (D) Endogenous CHIP and ErbB2 associate with each other in the presence of GA. SKOV3 cells were treated with ALLnL and GA, alone or in combination, and processed as in C. Immunoprecipitation and Western blotting were performed as in A. (E) Effects of GA on association of CHIP with chaperone and ErbB2 proteins. COS7 cells were cotransfected with ErbB2 and Myc-His-tagged CHIPs. Twenty hours after transfection, one set of transfected cells was treated with 1 μM GA for 30 min. Cells were lysed with PBS containing 1% Nonidet P-40, and CHIP proteins were precipitated from the supernatant by using Talon beads (CLONTECH). Precipitated proteins were separated on SDS/4–20% polyacrylamide gels, transferred to a poly(vinylidene difluoride) membrane, and probed with different antibodies. (F) CHIP alters composition of the chaperone complexes that bind ErbB2. COS7 cells were transfected with full-length ErbB2, with or without wild-type CHIP. Twenty hours after transfection, cells were treated with 1 μM GA and 100 μM ALLnL for 6 h. Immunoprecipitation was as in A. Association of CHIP, Hsc/Hsp70, and Hsp90 was detected with appropriate antibodies.

Figure 3

Figure 3

CHIP induces ErbB2 ubiquitination. (A) CHIP enhances ubiquitination of ErbB2 protein in vivo. COS7 cells in 150 × 25 mm plates were transfected with 5 μg of ErbB2 and 15 μg of CHIP plasmid DNA. Twenty hours after transfection, cells were treated with 100 μM ALLnL, with or without 1 μM GA, for 6 h. Cells were then lysed with 2.5 ml of TMNS buffer. ErbB2 proteins were immunoprecipitated (IP) as described for Fig. 2_A_. Precipitated proteins were separated on an SDS/4–20% polyacrylamide gel, transferred to nitrocellulose membrane, and autoclaved to maximize the detection of polyubiquitinated proteins. The membrane was probed with rabbit anti-ubiquitin antibody, followed by mouse anti-ErbB2 mAb. IB, immunoblotting. (B) CHIP serves as an E3 ubiquitin ligase for ErbB2 in an in vitro ubiquitination assay. The entire intracellular domain of ErbB2 was translated in rabbit reticulocyte lysate, immunoprecipitated, and subjected to in vitro ubiquitination. The ubiquitination of ErbB2 (seen as slower migrating species) was detected by Western blotting with an ErbB2-specific antibody. a, Glutathione _S_-transferase (GST)-CHIP(1–197), lacking the U box domain; b, GST-CHIP(198–303), lacking the TPR domain.

Figure 4

Figure 4

CHIP is not the only E3 capable of mediating GA-induced down-regulation of ErbB2 protein. (A) CHIP−/− cells were infected with an adenovirus containing the CHIP gene (Ad-CHIP). Uninfected (control) or CHIP-infected cells were treated with increasing concentrations of GA (0.0625–0.5 μM) for 4 h before lysis. Endogenous ErbB2 protein in cell lysate was detected by Western blotting. (B) CHIP reduces the half-life of mature ErbB2 protein. CHIP−/− cells were infected as in A. Four hours later, they, and uninfected CHIP −/− and +/+ cells, were treated with 100 μg/ml cycloheximide (Chx), and lysed at various times. Levels of ErbB2, CHIP, and β-tubulin were detected by Western blotting of total cell lysate. (C) CHIP reduces the half-life of nascent ErbB2 protein. COS7 cells, transfected with ErbB2 alone (Upper) or together with CHIP (Lower), were pulse-labeled with [35S]methionine for 45 min, and then chased in nonradioactive complete medium for various times. ErbB2 protein was immunoprecipitated, and separated by SDS/PAGE. (Upper) The dried gel was exposed to Kodak X-Omat AR film. (Lower) Quantitation of the results.

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