In vivo tissue-specific, multi-omics analysis of Egf-stimulated Egfr signaling

Abstract

The epidermal growth factor receptor (EGFR) and its downstream effector pathways regulate essential cellular processes—including proliferation, growth, survival, and migration—in tissues throughout the body. Given the wide regulatory landscape of these pathways, the consequences of their dysfunction are diverse and known to be involved in many diseases, including cancer and disorders linked to chronic injury. Phenotypic consequences of specific aberrations in EGFR and its effector pathways are highly tissue-specific. Currently, it remains poorly understood how tissue type is linked to the in vivo regulation and output of EGFR-driven signaling at a molecular level. Although extensive efforts have been made to target both EGFR and its downstream effector pathways in disease, the heterogeneity of EGFR-driven signaling within different tissues makes these diseases difficult to treat. Here, we explore the association between tissue type and EGFR-driven signaling in the context of ligand stimulation in vivo. To that end, we deploy the Egfr ligand Egf to precisely evaluate how the response to Egfr stimulation is linked to tissue context at both the phosphoproteomic and transcriptomic levels. We show that tissue context influences the output of Egf stimulation at all levels. Moreover, we demonstrate that the homeostatic level of Egf-associated activity within each tissue is highly tissue-specific. Our results indicate that this baseline Egf-associated activity may influence both responsiveness to Egf stimulation and tissue-specific susceptibilities to EGFR-associated diseases. Despite the power of global phosphoproteomics analysis to illuminate general phosphorylation changes induced by Egfr stimulation, such analyses are limited in their ability to confirm direct kinase-substrate relationships. To more specifically characterize direct kinase-substrate relationships downstream of Egfr, we optimized the in vivo application of a previously developed thiophosphate ATP analog-sensitive mutant of Erk2, one of the major Egfr effectors, to label and identify direct Erk substrates in murine tissue lysates. This method can be combined with general phosphoproteomics approaches to verify direct kinase-substrate relationships. Altogether, the work presented here provides insight into the relationship between tissue type and Egfr-driven signaling. Additionally, it establishes generalizable approaches to studying ligand-stimulated signaling that can be broadly expanded to other ligands and signaling pathways.