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The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor
H Waterman1, G Levkowitz, I Alroy
1Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.
Abstract:
Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.
Insights
The RING finger of c-Cbl is crucial for epidermal growth factor receptor (EGFR) ubiquitination and desensitization. This zinc finger domain directly mediates EGFR down-regulation and signal transduction termination.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Surface receptor activation, like epidermal growth factor receptor (EGFR), leads to desensitization via endocytosis and degradation.
- c-Cbl, a signaling pathway substrate, promotes EGFR desensitization by enhancing receptor polyubiquitination.
Purpose of the Study:
- To investigate the role of the RING type zinc finger of c-Cbl in epidermal growth factor receptor (EGFR) ubiquitination and desensitization.
- To determine the functional significance of the c-Cbl RING finger in EGFR signal transduction.
Main Methods:
- Site-directed mutagenesis of the c-Cbl RING finger domain.
- Assessment of EGFR ubiquitination and down-regulation in living cells.
- In vitro reconstitution assays for EGFR ubiquitination.
- Analysis of downstream signaling inhibition.
Main Results:
- Mutagenesis of a single cysteine residue in the c-Cbl RING finger abolished its ability to enhance EGFR ubiquitination and down-regulation.
- The mutant c-Cbl retained binding to activated EGFR but exhibited dominant inhibitory effects.
- In vitro assays confirmed the direct role of the RING finger in ubiquitin ligation.
- EGFR signaling downstream of the receptor was not inhibited by the mutant c-Cbl.
Conclusions:
- The RING finger of c-Cbl is essential for the ubiquitination and subsequent desensitization of EGFR.
- This domain plays a direct, causative role in the endocytic sorting and signal transduction desensitization of EGFR.