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Published on: December 13, 2013
c-Cbl-mediated ubiquitinylation is required for epidermal growth factor receptor exit from the early endosomes
Tommer Ravid1, Jill M Heidinger, Peter Gee
1Signal Transduction, Department of Internal Medicine, University of California, School of Medicine, Davis, CA 95616, USA.
Abstract:
Epidermal growth factor receptor (EGFR) controls cell growth and has a key role in tumorigenic processes. The extent of EGFR signaling is tightly regulated by post-transcriptional modifications leading to down-regulation of the levels of the receptor. Previous studies from our laboratory demonstrated that the reactive oxidant hydrogen peroxide activates the EGFR, yet, without down-regulation of the receptor levels, which results in prolonged receptor signaling. In the present study we examined the role of the E3 ligase c-Cbl, as a possible link between oxidative stress, EGFR signaling, and tumorigenic responses. First, we ectopically expressed a mutant EGFR (Tyr-1045 --> Phe) in cells lacking endogenous receptor, to determine whether the lack of phosphorylation at this site is the cause for EGFR retention at the membrane under oxidative stress, as we have previously suggested. Our findings suggest that abrogation of tyrosine 1045 phosphorylation alone is not enough to retain the EGFR at the plasma membrane under oxidative stress. Second, through the use of the Src inhibitor PP1, our findings establish EGFR movement out of the early endosomes as the exact location where c-Cbl-mediated ubiquitinylation is essential for EGFR trafficking. Finally, our studies substantiate the findings that c-Cbl-mediated ubiquitinylation is needed for degradation, but not for internalization of the EGFR in both transfection-dependent Chinese hamster ovary cells and transfection-independent A549 lung epithelial cells. These findings only begin to explain the features seen under oxidative stress, but they yield a greater understanding of the role of c-Cbl in EGFR trafficking.
Insights
Oxidative stress prolongs epidermal growth factor receptor (EGFR) signaling. The E3 ligase c-Cbl is crucial for EGFR trafficking and degradation, but not internalization, impacting tumorigenic responses.
Area of Science:
- Cell Biology
- Molecular Oncology
- Signal Transduction
Background:
- Epidermal growth factor receptor (EGFR) signaling regulates cell growth and is implicated in tumorigenesis.
- EGFR levels are controlled by post-transcriptional modifications, typically leading to receptor down-regulation.
- Oxidative stress, like hydrogen peroxide, activates EGFR but prevents its down-regulation, causing prolonged signaling.
Purpose of the Study:
- To investigate the role of the E3 ligase c-Cbl in linking oxidative stress, EGFR signaling, and tumorigenesis.
- To determine if abrogating EGFR phosphorylation at Tyr-1045 causes receptor retention at the plasma membrane under oxidative stress.
- To pinpoint the cellular location where c-Cbl-mediated ubiquitination is essential for EGFR trafficking.
Main Methods:
- Ectopic expression of a mutant EGFR (Tyr-1045 --> Phe) in EGFR-null cells.
- Utilizing the Src inhibitor PP1 to study EGFR trafficking.
- Assessing EGFR internalization and degradation in Chinese hamster ovary and A549 lung epithelial cells.
Main Results:
- Abrogation of EGFR phosphorylation at Tyr-1045 alone does not retain EGFR at the plasma membrane under oxidative stress.
- EGFR movement out of early endosomes is the critical site for c-Cbl-mediated ubiquitination essential for EGFR trafficking.
- c-Cbl-mediated ubiquitination is required for EGFR degradation, but not for its internalization.
Conclusions:
- EGFR retention under oxidative stress is not solely due to the lack of Tyr-1045 phosphorylation.
- c-Cbl-mediated ubiquitination is essential for EGFR degradation following its exit from early endosomes.
- These findings enhance the understanding of c-Cbl's role in EGFR trafficking dynamics, particularly under oxidative stress conditions.
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