Epidermal growth factor (EGF) signaling is critical in normal and aberrant cellular behavior. Extracellular signal-regulated kinase (ERK) mediates important downstream aspects of EGF signaling. Additionally, EGFR undergoes MEK1-dependent ERK consensus site phosphorylation in response to EGF or cytokines such as growth hormone (GH) and prolactin (PRL). GH- or PRL-induced EGFR phosphorylation alters subsequent EGF-induced EGFR downregulation and signal characteristics in an ERK-dependent fashion. We now use reconstitution to study mutation of the sole EGFR ERK phosphorylation consensus residue, 669T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A (669T changed to alanine) EGFRs at similar abundance. Treatment of cells with GH or EGF caused phosphorylation of WT, but not T669A EGFR, in an ERK activity-dependent fashion that was detected with an antibody that recognizes phosphorylation of ERK consensus sites, indicating that 669T is required for this phosphorylation. Notably, EGF-induced downregulation of EGFR abundance was much more rapid in cells expressing EGFR T669A vs. WT EGFR. Further, pretreatment with the MEK1/ERK inhibitor PD98059 enhanced EGF-induced EGFR loss in cells expressing WT EGFR, but not EGFR T669A, suggesting that the ERK-dependent effects on EGFR downregulation required phosphorylation of 669T. In signaling experiments, EGFR T669A displayed enhanced acute (15 min) EGFR tyrosine phosphorylation (reflecting EGFR kinase activity) compared to WT EGFR. Further, acute EGF-induced ubiquitination of WT EGFR was markedly enhanced by PD98059 pretreatment and was increased in EGFR T669A-expressing cells independent of PD98059. These signaling data suggest that ERK-mediated 669T phosphorylation negatively modulates EGF-induced EGFR kinase activity. We furthered these investigations using a human fibrosarcoma cell line that endogenously expresses EGFR and ErbB-2 and also harbors an activating Ras mutation. In these cells, EGFR was constitutively detected with the ERK consensus site phosphorylation-specific antibody and EGF-induced EGFR downregulation was modest, but was substantially enhanced by pretreatment with MEK1/ERK inhibitor. Collectively, these data indicate that ERK activity, by phosphorylation of a threonine residue in the EGFR juxtamembrane cytoplasmic domain, modulates EGFR trafficking and signaling. © 2008 Elsevier Inc. All rights reserved.