Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor

Abstract

Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR.

Figure 1

Epidermal growth factor receptor (EGFR) constructs and their expression. (a) Structures of the various EGFR mutants. EGFR‐Wt, wild‐type human EGFR; EGFR‐Wt‐Tr, wild‐type kinase domain of EGFR with C‐terminal truncation at amino acid 980; EGFR‐D, EGFR with a 15‐bp deletion from the tyrosine kinase domain (delE746_A750); EGFR‐D7F, 15‐bp deletion of EGFR (delE746_A750) and substitution of seven tyrosine residues to phenylalanine (Y992F, Y1068F, Y1045F, Y1068F, Y1086F, Y1148F, Y1173F); and EGFR‐D‐Tr, 15‐bp deletion of EGFR (delE746_A750) with C‐terminal truncation at amino acid 980. EGFR‐Wt, EGFR‐D, EGFR‐D7F, and EGFR‐D‐Tr contained a myc‐tag. EGFR‐Wt‐Tr contained a flag‐tag. ECD, extracellular domain; TK, tyrosine kinase; TM, transmembrane. (b) Stable transfectants were lysed and cell lysates containing equal amounts of protein were immunoblotted with anti‐EGFR antibody recognizing the extracellular domain of EGFR. A band with a molecular weight of ~170 kDa was detected in the HEK293/Wt, HEK293/D, and HEK293/D7F cells, and a band of lower molecular weight was detected in the HEK293/D‐Tr and HEK293/Wt‐Tr cells. Mock, HEK293/Mock; Wt, HEK293/Wt; Wt‐Tr, HEK293/Wt‐Tr; D, HEK293/D; D7F, HEK293/D7F; and D‐Tr, HEK293/D‐Tr.

Figure 2

Epidermal growth factor receptor (EGFR) lacking C‐terminal autophosphorylation sites retains EGFR signal transduction. (a) The HEK293/Mock, HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells were lysed, and the cell lysates were immunoblotted with anti‐phospho‐EGFR (p‐EGFR Y845, Y1068, Y1173), anti‐EGFR (recognizing the extracellular domain), anti‐phospho‐ERK, anti‐ERK, anti‐phospho‐Akt, and anti‐Akt antibodies. (b) The HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells were incubated in 1% serum starve medium for 12 h, followed by treatment with 10 ng/mL epidermal growth factor for 10 min at 37°C. The cell lysates were immunoblotted. Mock, HEK293/Mock; Wt, HEK293/Wt; D, HEK293/D; D7F, HEK293/D7F; and D‐Tr, HEK293/D‐Tr.

Figure 3

Sensitivity of cell growth and downstream epidermal growth factor receptor (EGFR) signaling to AG1478 in the mutant EGFR transfectants. (a) The growth‐inhibitory effect of AG1478 in HEK293/Wt, HEK293/Wt‐Tr, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells. The seeded cells were exposed to AG1478 for 72 h and the cellular proliferative activity was determined by MTT assay. (b) The HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells were incubated in 1% serum starve medium for 12 h, followed by exposure to 20 or 200 nM AG1478 for 3 h at 37°C. The cell lysates were immunoblotted with anti‐phospho‐EGFR (p‐EGFR Y1068), anti‐EGFR (recognizing the extracellular domain), anti‐phospho‐ERK, or anti‐ERK antibodies. Mock, HEK293/Mock; Wt, HEK293/Wt; Wt‐Tr, HEK293/Wt‐Tr; D, HEK293/D; D7F, HEK293/D7F; D‐Tr, HEK293/D‐Tr.

Figure 4

Heterodimerization of mutant epidermal growth factor receptor (EGFR) with endogenous receptors of the HER family in mutant EGFR transfectants. Expression of endogenous EGFR and response to epidermal growth factor stimulation. HEK293/Mock, HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells were incubated in 1% serum starve medium for 12 h followed by the addition of 10 ng/mL epidermal growth factor for 10 min at 37°C. (a) The whole‐cell lysates of HEK293/Mock and HEK293/W+ cells containing equal amounts of protein were immunoblotted with anti‐phospho‐EGFR (p‐EGFR Y1068) and anti‐EGFR (recognized extra‐cellular domain). (b,c) The lysates were immunoprecipitated with anti‐EGFR, anti‐HER2, or anti‐HER3 antibodies, and immunoblotted with anti‐EGFR, anti‐HER2, anti‐HER3, or anti‐phosphotyrosine antibodies to detect the dimerization and phosphorylation of EGFR and endogenous HER2 or HER3. HER2, HER2‐introduced HEK293 cells as a positive control; HER3, HER3‐introduced HEK293 cells as a positive control.

Figure 5

The cells under 1% serum starved medium were allowed to react with 2 mM of the chemical crosslinking regent BS₃ before the crosslinking reaction was quenched. The cell lysates were immunoblotted with (a) anti‐epidermal growth factor receptor (EGFR) (recognizing the extracellular domain) and (b) anti‐phosphoEGFR (p‐EGFR Y1173) antibodies to detect the dimerization and phosphorylation of wild‐type and mutant EGFR. Black arrow, EGFR dimer; open arrow, EGFR monomer. Mock, HEK293/Mock; Wt, HEK293/Wt; D, HEK 293/D; D7F, HEK293/D7F; D‐Tr, HEK293/D‐Tr. EGF, epidermal growth factor.

Figure 6

Interaction between mutant epidermal growth factor receptor (EGFR) and adaptor proteins. The cells were cultured under normal conditions. (a) The lysates of HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D‐Tr cells were immunoprecipitated with anti‐myc tag antibody; the precipitates were immunoblotted with anti‐son of sevenless homolog (Sos) and anti‐growth factor receptor‐bound protein (Grb) 2 antibodies. (b) Whole‐cell lysates containing equal amounts of protein were immunoblotted with anti‐phospho‐Src homology and collagen homology (Shc) and anti‐Shc antibodies. (c) The cells incubated in 1% serum starve medium for 12 h were treated with 20 or 200 nM AG1478 for 3 h and then the lysates were immunoblotted with anti‐phospho‐Shc or anti‐Shc antibodies. Mock, HEK293/Mock; Wt, HEK293/Wt; D, HEK293/D; D7F, HEK293/D7F; D‐Tr, HEK293/D‐Tr. PY, anti‐phospho‐tyrosine.