Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Abstract

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser991 and Tyr998 accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser1039 and Thr1041. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser1039 and Thr1041 were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr998, Ser991, Ser1039, and Thr1041 governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

The Ubiquitin Ligase MYCBP2 Regulates Transient Receptor Potential Vanilloid Receptor 1 (TRPV1) Internalization through Inhibition of p38 MAPK Signaling

“…This notion that p38 MAPK fulfills different roles in receptor trafficking is further supported by the finding that regulation of receptor trafficking by p38 MAPK can occur at different stages. In this regard, it has been reported that phosphorylation of epidermal growth factor (EGF)-receptor between amino acids 1002-1022 mediates stress induced internalization (19) while EGFR phosphorylation at serine 1046 and 1047 by p38 MAPK mediates ubiquitylation and degradation of already internalized EGF receptor but not internalization itself (55,56).…”

Phosphorylation and ubiquitylation are opposing players in regulating endocytosis of the water channel Aquaporin-2

“…Both modifications have the potential to mediate inducible proteinprotein interactions and thereby regulate protein function. For membrane proteins, induction of phosphorylation, including polyphosphorylation, is a mechanism for regulating the activity and intracellular trafficking of channels and transporters (Moeller et al, 2010;Rosenbaek et al, 2012;Tong et al, 2009;Yang et al, 2006). Ubiquitylation of membrane proteins through specific linkage is a common way of mediating internalization and promotion of protein degradation by the lysosomal pathway (Kazazic et al, 2009;Piper and Luzio, 2007;Vina-Vilaseca et al, 2011).…”

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress

“…Consistent with this, EGFR phosphorylation sites linked with receptor trafficking are present within or proximal to this part of the receptor. For example, EGFR phosphorylation at S 991 and Y 998 accumulate with relatively slow kinetics following stimulation of cells with EGF (29). Phosphorylation-defective variants Y998F and S991A are impaired for ligand-stimulated down-regulation relative to wild type (WT) EGFR, but remain proficient for rapid EGFR-to-ERK signaling (29).…”

“…For example, EGFR phosphorylation at S 991 and Y 998 accumulate with relatively slow kinetics following stimulation of cells with EGF (29). Phosphorylation-defective variants Y998F and S991A are impaired for ligand-stimulated down-regulation relative to wild type (WT) EGFR, but remain proficient for rapid EGFR-to-ERK signaling (29). Non-phosphorylated Y 998 was cited as part of an AP-2 binding motif (Y 998 RAL) (22), while a nearby di-leucine motif (LL 1034/35 ) also serves as an AP-2 binding site (22,30).…”