EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

Abstract

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions.

AFDN (afadin, adherens junction formation factor)

“…ZO-1 is a member of F-actin-binding ZO proteins, which bind CAMs of tight junctions (TJs) such as claudins, occludin ( OCLN), and junctional adhesion molecules (JAMs) and link them to the actin cytoskeleton (Sakisaka et al, 2007) (see Figure 4). Occludin, EPHA2, a transmembrane tyrosine kinase receptor, and afadin also cooperate in tight junction organization (Perez White et al, 2017). Afadin is therefore a peripheral component of tight junctions in epithelial cells.…”

Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial Proximity

“…BirA converts biotin into reactive biotineadenosine monophosphate, whereas APEX and HRP create free radicals of biotin as intermediates. BirA can be used in living tissue, as has been successfully applied within organotypic skin models (Perez-White et al, 2017). Unlike the simple biotin used for BirA, the biotin-phenol reagent used for APEX and the hydrogen peroxide reagent used for HRP are not suitable for organoid or in vivo studies because of the tissue toxicity of SUMMARY Proximity protein labeling allows detection of spatially proximal proteins.…”

Scaffolding proteins in the development and maintenance of the epidermal permeability barrier

“…90 Recently, an additional, unexpected role for afadin in tight junction assembly that involves its interactions with the Ephrin receptor EphA2 has been described. 91 EphA2 is a transmembrane receptor tyrosine kinase expressed in a polarized fashion in the upper suprabasal layers of the human epidermis. 91 This receptor fulfills several functions in the epidermis, including protection against chemically-induced carcinogenesis, 92 modulation of keratinocyte adhesion and tight junction formation.…”

“…91 EphA2 is a transmembrane receptor tyrosine kinase expressed in a polarized fashion in the upper suprabasal layers of the human epidermis. 91 This receptor fulfills several functions in the epidermis, including protection against chemically-induced carcinogenesis, 92 modulation of keratinocyte adhesion and tight junction formation. 93 Afadin binds to EphA2, and in the absence of the latter the subcellular distribution of afadin and occludin changes from mainly junctional to cytoplasmic.…”

“…93 Afadin binds to EphA2, and in the absence of the latter the subcellular distribution of afadin and occludin changes from mainly junctional to cytoplasmic. 91 These changes are also associated with defects in the tight junction permeability barrier to ions and a dextran tracer. Thus, the interactions of afadin with EphA2 are essential for tight junction assembly.…”