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 ofafadinandoccludin indifferentiated 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.
Original language | English (US) |
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Pages (from-to) | 111-118 |
Number of pages | 8 |
Journal | Journal of cell science |
Volume | 130 |
Issue number | 1 |
DOIs | |
State | Published - 2017 |
Funding
This work was supported by the National Institutes of Health (NIH) [grant number R01-AR062110 to S.G.], a Skin Disease Research Center Grant [NIH grant number P30-AR057216-01], and NIH training fellowships to B.E.P.W. [grant number T32-AR060710] and R.V. [grant number T32-GM08061]. B.E.P.W. also received support from the Chicago Biomedical Consortium. C.J.C. was supported by the Northwestern Undergraduate Research Program. Proteomics were performed by the Northwestern Proteomics Core Facility [NIH grant numbers P30-CA060553 and P41-GM108569]. Deposited in PMC for release after 12 months.
Keywords
- 3D culture
- Afadin
- EphA2
- Keratinocytes
- Proteomics
- Tight junction
ASJC Scopus subject areas
- Cell Biology