Figure 2.

Ligand-independent EGFR activation is not sufficient for induction of cyclin D1 and proliferation. (A) Schematic representation of C-terminal Flag-tagged EGFR and interleukin-2 receptor (ILR)-EGFR-chimera constructs. The ILR-wt contains ILR ectodomain fused to the EGFR cytoplasmic region; ILR-ΔJD contains ILR ectodomain and EGFR cytoplasmic region lacking 33 amino acids at the juxtadomain and ILR-ΔKD contains ILR ectodomain and the EGFR cytoplasmic region lacking juxtadomain and a segment of the kinase domain. TM, transmembrane; KD, kinase domain; YAPS, tyrosine autophosphorylation sites. (B) Protein lysates from HEK293 cells transfected with the various constructs were immunoblotted as indicated. (C) HEK293 cells transiently expressing the indicated constructs were cultured in the absence of serum for 18 h and with or without EGF (10 ng/ml). Protein lysates were then prepared and analyzed by immunoblotting as indicated. The phospho-EGFR and p-AKT membranes were stripped and reprobed with anti-EGFR and AKT antibody, respectively. Note that the p-AKT level in presence of serum and EGF are not prominent compared to the others. This is likely due to global endogenous AKT activity emancipating from the adherent cells. The results are representative of 2 independent experiments.