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

Bethany E. Perez White; Rosa Ventrella; Nihal Kaplan; Calvin J. Cable; Paul M. Thomas; Spiro Getsios

doi:10.1242/jcs.188169

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

Bethany E. Perez White, Rosa Ventrella, Nihal Kaplan, Calvin J. Cable, Paul M. Thomas, Spiro Getsios^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

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)
Pages (from-to)	111-118
Number of pages	8
Journal	Journal of cell science
Volume	130
Issue number	1
DOIs	https://doi.org/10.1242/jcs.188169
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

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10.1242/jcs.188169

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@article{410e55a9418140a0abcbe9505efed952,

title = "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 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.",

keywords = "3D culture, Afadin, EphA2, Keratinocytes, Proteomics, Tight junction",

author = "{Perez White}, {Bethany E.} and Rosa Ventrella and Nihal Kaplan and Cable, {Calvin J.} and Thomas, {Paul M.} and Spiro Getsios",

note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2017. Published by The Company of Biologists Ltd.",

year = "2017",

doi = "10.1242/jcs.188169",

language = "English (US)",

volume = "130",

pages = "111--118",

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T1 - EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

AU - Perez White, Bethany E.

AU - Ventrella, Rosa

AU - Kaplan, Nihal

AU - Cable, Calvin J.

AU - Thomas, Paul M.

AU - Getsios, Spiro

N1 - Funding Information: 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. Publisher Copyright: © 2017. Published by The Company of Biologists Ltd.

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - 3D culture

KW - Afadin

KW - EphA2

KW - Keratinocytes

KW - Proteomics

KW - Tight junction

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SN - 0021-9533

VL - 130

SP - 111

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JO - Journal of cell science

JF - Journal of cell science

IS - 1

ER -

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

Abstract

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Keywords

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