Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor

Abstract

The ϳ230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ϳ80 residues of the tail are inhibitory, as demonstrated previously. The entire ϳ80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

“…Because EGFR is the only active kinase in this complex, the resulting receptor phosphorylation is a result of its activity. Although phosphorylation of EGFR in cis in the active dimer has been shown to be less pronounced than in trans, it does occur (36). Why then cannot EGFR autophosphorylate itself when stimulated with NRG in the presence of HER3?…”

Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases

“…The intrinsically disordered properties of these CTTs also explain why these regions are mostly absent from crystallographic studies on EGFR (7,55). Portions of the EGFR tail located close to the kinase domain have been shown to be responsible for EGFR autoinhibition (56).…”

“…The deletion of residues 982-1054 in the EGFR vIVb mutant is of particular importance in downstream signaling and kinase activation and has been shown to promote tumor formation. This region has been characterized via crystallography as containing an AP2-helix and other secondary structure elements, which appear to interact with the kinase domain in a manner that inhibits activation (4,55,56). In a study by Kovacs et al (55), serial deletions of tail regions revealed specific regions important for autoinhibition as they relate to the oncogenic vIVb mutation.…”

Combination of a STAT3 Inhibitor and an mTOR Inhibitor Against a Temozolomide-resistant Glioblastoma Cell Line

“…Another important issue regarding mTOR signal activation in TMZ-R U87 cells is genetic alteration of the mTOR gene (29)(30)(31). Briefly, three studies regarding mTOR mutation research have been cited.…”

“…Interestingly, a novel mutation (E1004K) located in the C-terminal tail domain of EGFR was found in Table I. Considering that the N-terminal half of the EGFR C-terminal tail domain has an inhibitory function in EGFR signal activation (31) and that a neighboring mutation (ER1005-1006KD) was reported to induce spontaneous activation of EGFR phosphorylation (17), this mutation (E1004K) might be involved in EGFR signaling. Alternatively, a novel somatic mutation of the YKL-40 gene (G292R) was also found, but it likely dose not function in signal transduction.…”