Figure 1
Common NSCLC-associated EGFR mutations lead to increased autophosphorylation of an YFP-tagged EGFR intracellular domain. A. Schematic representation of full-length EGFR and YFP-EGFR-ICD. The amino acid numbering includes the 24 residues of the signal peptide. YFP-EGFR-ICD contains the tyrosine kinase (TK) domain and part of the regulatory region (Reg), but lacks the extracellular and juxtamembrane (JM) domains. B. Expression of YFP-EGFR-ICD Del746 induces morphological changes in MCF-7 cells. Unlike cells transfected with wild type (WT) YFP-EGFR-ICD, MCF-7 cells expressing YFP-EGFR-ICD Del746 frequently show long lamellipodial protrusions (arrowheads). C Using immunofluorescence, increased autophosphorylation of YFP-EGFR-ICD Del746 at tyrosine residues Y869 (left set of panels) and Y1092 (right set of panels) can be detected. Phosphorylation is virtually undetectable in cells expressing YFP-EGFR-ICD WT. Images were taken using 160 × magnification and the exposure time indicated inside the panels. The fluorescent signal was consistently brighter using the anti-pY1092 antibody (note the shorter exposure time used). D. Semi-quantitative comparison of YFP-EGFR-ICD autophosphorylation level using computer-assisted image analysis. Images of several transfected cells (400 × magnification) were taken using 40 ms (YFP) or 160 ms (AF-594) exposure times. The fluorescence intensity in the green and the red channels was measured within a cytoplasmic area (YFP signal and AF-594 signal), and within an area outside the cells (background). In the graph, the intensity of the YFP and AF-594 fluorophores for each cell was plotted against each other using Excel, and the best-fitting trend lines (highest R2) were added. At similar expression levels (YFP intensity), the level of pY1092 is higher for YFP-EGFR-ICD bearing the Del746 mutation (white squares) than for the wild type protein (circles). The V948R mutation (open triangles) virtually abrogated autophosphorylation. The experiment was repeated twice with similar results. Graph shows the data from one experiment. au: arbitrary units.