Analysis of Constitutive EGFR Signaling Regulating IRF3 Transcriptional Activity in Cancer Cells

Epidermal growth factor receptor (EGFR) plays an important role in various types of human cancers. Overexpression of EGFR leads to a constitutive tyrosine phosphorylation of multiple tyrosine residues in the EGFR. Recently, we have demonstrated that overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling depending on the presence or absence of ligand. EGFR constitutively activates transcription factor IRF3, which results in transcription of its target genes. Addition of EGF causes a loss of IRF3 transcriptional activity and activation of canonical signaling pathways such as ERK. The mechanistic basis of this bimodal signaling appears to be the association of a distinct set of signaling proteins with EGFR in the absence or presence of ligand. In this chapter, we describe a detailed protocol for analyses of constitutive EGFR signaling with a focus on IRF3 target genes.