Inverse correlation between Thr-669 and constitutive tyrosine phosphorylation in the asymmetric epidermal growth factor receptor dimer conformation

Abstract

We have recently identified tumor necrosis factor (TNF)-α-induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr-669 and Ser-1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand-induced phosphorylation of serine and threonine residues in EGFR-overexpressing MDA-MB-468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr-669 and Ser-1046/1047. Inversely, constitutive tyrosine phosphorylation of the C-terminal domain, including Tyr-1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand-induced Thr-669 phosphorylation as well as Tyr-1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr-669 for Ala. In an asymmetric EGFR homodimer structure, one Thr-669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr-669 in an EGFR-ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK-mediated Thr-669 phosphorylation suppresses constitutive tyrosine phosphosphorylation in the homo- and heterodimer asymmetric conformations of the EGFR.

Figure 1

Regulation of serine and threonine phosphorylation of epidermal growth factor receptor (EGFR). (a) MDA‐MB‐468 cells were treated with 20 ng/mL tumor necrosis factor (TNF)‐α or 10 ng/mL EGF for the indicated periods. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047 and Tyr‐1068), EGFR, phospho‐mitogen‐activated protein kinase (MAPK), MAPK and Actin antibodies. (b,c) Cells were pretreated with gefitinib (GE; 10 μM), PD153035 (PD; 1 μM), SB203580 (SB; 10 μM) and U0126 (U; 5 μM) for 30 min, and then stimulated with TNF‐α (b), and EGF or heregulin (HRG) (c) for another 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047 and Tyr‐1068), EGFR, phospho‐ErbB3, ErbB3 and Actin antibodies.

Figure 2

Inverse phosphorylation at serine/threonine and tyrosine residues. (a–c) MDA‐MB‐468 cells were treated with EGF or heregulin (HRG) for the indicated periods. Whole cell lysates were immunoblotted with phospho‐epidermal growth factor receptor (EGFR) (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐992, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ErbB3, ErbB3, phospho‐mitogen‐activated protein kinase (MAPK) and Actin antibodies. (d) Cells were treated with EGF for 10 min and then cell surface EGFR expression was determined using a flow cytometer.

Figure 3

ERK‐mediated feedback control of constitutive tyrosine phosphorylation of epidermal growth factor receptor (EGFR). (a) MDA‐MB‐468 cells were pretreated with U0126 (5 μM) or vehicle (DMSO) for 30 min and then stimulated with EGF or heregulin (HRG) for another 10 min. (b) Cells were stimulated with TPA (100 ng/mL) for 10 min in the absence of presence of U0126. (c,d) PC‐9 cells were treated with gefitinib (1 μM) for 30 min and then stimulated with TPA for 10 min (c) or HGF (d) for 15–120 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK, phosphor‐MET, Tubulin and Actin antibodies.

Figure 4

Negative feedback regulation of epidermal growth factor receptor (EGFR) via Thr‐669 phosphorylation. (a) HEK293 cells stably transfected with wild‐type or S1046/1047A mutant (SS/AA) were stimulated with EGF for the indicated periods. (b,c) HEK293 cells stably transfected with wild‐type or T669A mutant were stimulated with EGF (b) or TPA (c) for 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK, phospho‐p38 and Actin antibodies.

Figure 5

Role of Thr‐669 in the asymmetric epidermal growth factor receptor (EGFR) homodimer. HEK293 cells were transiently transfected with (a) wild‐type or I682Q (Act‐M) and V924R (Rec‐M) mutants, (b) Act‐M and Rec‐M with or without additional T669A (TA) point mutation, (c) KK721/782AA (KK/AA) and Act‐M or Rec‐M with or without TA mutation, (d) ErbB3 and Act‐M or Rec‐M with or without TA mutation. At 24 h post‐transfection, whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR and ErbB3 antibodies.

Figure 6

Role of Thr‐669 in the asymmetric epidermal growth factor receptor (EGFR) homodimer. HEK293 cells were transiently transfected with I682Q (Act‐M) and V924R (Rec‐M) with or without TA mutation. At 24 h post‐transfection, cells were stimulated with TPA for 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK and Actin antibodies.

Figure 7

Schematic diagram of Thr‐669‐mediated inhibition of the asymmetric epidermal growth factor receptor (EGFR) dimer. Formation of an asymmetric dimer structure by docking the N‐lobe of the receiver to the C‐lobe of the activator induces EGFR autophosphorylation of tyrosine residues in the C‐terminal tail. ERK‐mediated Thr‐669 phosphorylation, especially in the receiver, induces conformational change to disrupt the active dimer structure, which leads to a decrease in C‐terminal autophosphorylation.