EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.
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.
Department of Dermatology, Northwestern University, Chicago, IL 60611, USA.
Bethany E
BE
Perez White
Department of Dermatology, Northwestern University, Chicago, IL 60611, USA.
Rosa
R
Ventrella
Department of Dermatology, Northwestern University, Chicago, IL 60611, USA.
Nihal
N
Kaplan
Department of Dermatology, Northwestern University, Chicago, IL 60611, USA.
Calvin J
CJ
Cable
Proteomics Center of Excellence, Northwestern University, Chicago, IL 60611, USA.|Department of Molecular Biosciences, Northwestern University, Chicago, IL 60611, USA.
Paul M
PM
Thomas
Department of Dermatology, Northwestern University, Chicago, IL 60611, USA spigetsi@gmail.com.|Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.
Spiro
0000-0002-1141-4562
S
Getsios
D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002478:Cells, Cultured; D004817:Epidermis / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000378:metabolism*; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics; D036104:Receptor, EphA2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D019108:Tight Junctions / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C108835:afadin; D036104:Receptor, EphA2; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
3D culture; Afadin; EphA2; Keratinocytes; Proteomics; Tight junction