GAPex-5 Mediates Ubiquitination, Trafficking, and Degradation of Epidermal Growth Factor Receptor

Xiong Su,Philip D. Stahl,Chen Kong

doi:10.1074/jbc.m703725200

Abstract

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.

Highlights

Downstream signaling molecules either on the plasma membrane or in signaling endosomes, its degradation has been shown to be important in prevention of excessive signaling and cell proliferation [3]
Depletion of GAPex-5 Inhibits epidermal growth factor receptor (EGFR) Degradation—GAPex-5 is a newly identified Rab5 exchange factor, and we have recently demonstrated that its depletion inhibits insulin receptor internalization and signaling [25]
Because EGFR and insulin receptor follow similar endocytic itineraries upon stimulation, we examined the effect of GAPex-5 depletion on EGFR internalization

Summary

Introduction

Downstream signaling molecules either on the plasma membrane or in signaling endosomes, its degradation has been shown to be important in prevention of excessive signaling and cell proliferation [3]. Previous study demonstrated that depletion of all three Rab5 isoforms in HeLa cells decreased 125I-EGF internalization rate by ϳ50% [32]. To examine whether GAPex-5 mediates EGFR ubiquitination, HeLa cells transfected with scrambled negative control siRNA or a siRNA against GAPex-5 were starved and stimulated with EGF.

Results

Conclusion