Related Experiment Video
Updated: Mar 17, 2026
In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and
Chaeuk Chung1, Geon Yoo2, Tackhoon Kim3
1Cancer Institute of Chungnam National University, Daejeon 35015, South Korea; Division of Pulmonology, Department of Internal Medicine, College of Medicine, Chungnam National University, Daejeon 35015, South Korea.
Abstract:
Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.
Insights
C-terminus of Hsc70-interacting protein (CHIP) selectively targets and degrades mutant epidermal growth factor receptor (EGFR) in lung adenocarcinoma. CHIP overexpression inhibits tumor growth, suggesting it as a therapeutic target for EGFR TKI resistance.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Somatic mutations in epidermal growth factor receptor (EGFR) predict therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma.
- Heat shock protein 90 (Hsp90) stabilizes mutant EGFR, and its co-chaperone, C-terminus of Hsc70-interacting protein (CHIP), possesses E3 ubiquitin ligase activity.
- CHIP, as an Hsp70/Hsp90 co-chaperone, regulates client proteins through the Hsp90-CHIP complex or independently via its ligase activity.
Purpose of the Study:
- To investigate the role of CHIP in regulating EGFR in lung adenocarcinoma.
- To evaluate the specificity of CHIP's effects on wild-type (WT) versus mutant EGFR.
- To explore CHIP as a potential therapeutic target for overcoming EGFR TKI resistance.
Main Methods:
- Overexpression of CHIP in HEK 293T cells transfected with WT or mutant EGFR.
- Pull-down assays to assess CHIP-EGFR interaction and ubiquitination.
- Western blot analysis to determine protein expression levels in various lung cancer cell lines (PC9, H1975, A549).
- Cell proliferation and xenograft tumor growth assays.
Main Results:
- CHIP overexpression selectively decreased the expression of specific EGFR mutants (G719S, L747_E749del A750P, L858R) but not WT EGFR.
- CHIP selectively interacted with EGFR mutants, inducing their ubiquitination and proteasomal degradation.
- CHIP diminished mutant EGFR expression in PC9 and H1975 cells, with minimal effect on WT EGFR in A549 cells.
- CHIP overexpression inhibited proliferation and tumor growth in EGFR mutant cell lines, but not in WT EGFR cell lines.
Conclusions:
- CHIP specifically targets and degrades mutant EGFR through ubiquitination and proteasomal pathways.
- CHIP-mediated regulation of mutant EGFR offers a crucial mechanism in lung adenocarcinoma.
- CHIP represents a novel therapeutic target for overcoming resistance to EGFR TKIs in lung adenocarcinoma.
Related Concept Videos
01:15Receptor Downregulation in MVBs
The EGFR can initiate signaling pathways that lead to cell proliferation, migration, and differentiation. Overexpression of EGFR stimulates cells to proliferate. Excessive EGFR...
02:58Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
02:38Mitogens and the Cell Cycle
01:37The Unfolded Protein Response
01:13The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
02:18The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...