Proteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress.

Jiefei Tong,Paul Taylor,Michael F Moran

doi:10.1074/mcp.m114.038596

Abstract

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S(1104). Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y(998) or in the S(1039) region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.

Highlights

Tion at Y998 or in the S1039 region abolished or greatly reduced epidermal growth factor receptor (EGFR) interactions with AP-2 and AP-1, and impaired receptor trafficking
Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR)1 are aberrantly activated by mutation and/or over-expression in numerous human cancers [1, 2]
The Western blot (WB) in Fig. 1A shows that the expression level of EGFR in stably transfected HEK293 cells gradually increased during a 48 h incubation period after cell treatment with 1 ␮g/ml tetracycline (Tet)

Summary

Introduction

Tion at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. Extracellular binding of ligand induces EGFR dimerization and trans-autophosphorylation at intracellular tyrosine residues, which serve as binding sites for various enzymes and adaptor proteins [11] These receptor-binding proteins are involved in signaling and/or receptor trafficking, and lead to further modulation of receptor PTMs. For example, binding of the E3 ubiquitin ligase CBL at EGFR pY1069 [13,14,15] or indirectly through the adaptor protein Grb, which binds primarily at pY1092 [16], are both involved in EGFR ubiquitinylation and down-regulation [17]. EGFR variants with amino acid substitutions at these positions were largely impaired for AP-1 and AP-2 interactions, showed altered patterns of ubiquitination, and resistance to EGF-stimulated receptor downregulation These results provide new insight into the dynamics and molecular events associated with EGFR function

Objectives

Results

Conclusion