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

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 tryrosine phosphosphorylation in the homo‐ and heterodimer asymmetric conformations of the EGFR.