860 EGFR proteomics reveals novel EphA2-dependent trafficking and signaling pathways in epidermal keratinocytes

Abstract

Intricate signaling systems must be precisely controlled to maintain epidermal homeostasis. Receptor tyrosine kinases (RTKs) are integral in orchestrating these cell communication networks. In particular, EphA2 and EGFR transduce distinct signals to dictate keratinocyte fate. Our work shows that loss of EphA2 impairs keratinocyte differentiation and barrier formation. Underlying this defect are changes in EGFR signaling, localization, and degradation. We hypothesize that EphA2 promotes epidermal differentiation through upstream, negative regulation of EGFR signaling. To probe how EphA2 dampens EGFR, we performed biotin-identification proteomics (BioID) to identify disparities in EGFR-interacting proteins in keratinocytes with or without EphA2. As a result of these studies, we are the first to define a BioID proteome for EGFR in 3D epidermal cultures having identified 121 putative interactors. Of these, 87 are novel compared to reported EGFR associations. This protein subset was significantly enriched in vesicle- and junction-associated proteins (cellular component gene ontology; FDR: 1.44e-11 and 2.83e-8, respectively), highlighting EGFR trafficking and adhesion pathways exclusive to epidermal keratinocytes. In EphA2-deficient cells 73% of these vesicle proteins were lost, suggesting abnormal EGFR trafficking. We also uncovered novel EGFR interactions with two E3 ubiquitin ligases only in those cells expressing EphA2. These results align with the aberrant localization and ubiquitination status of EGFR seen with the loss of EphA2. KEGG analysis shows significant mapping of EGFR interactors to adherens and tight junctions (FDR: 3.34e-8 and 4.62e-6, respectively) only in the presence of EphA2. EGFR is emerging as a duplicitous regulator of cell-cell junctions, and our results indicate EphA2 may govern EGFR signaling at junctional complexes. Taken together, we suggest that RTK crosstalk between EphA2 and EGFR is critical for epidermal differentiation.