Functional consequences of interactions between FAK and epithelial membrane protein 2 (EMP2).
OBJECTIVE
Collagen gel contraction by ARPE-19 is controlled by epithelial membrane protein 2 (EMP2) through focal adhesion kinase (FAK) activation. The purpose of this study was to test the role of EMP2 in the cellular context of FAK activation.


METHODS
The ARPE-19 cell line was recombinantly modified to increase the expression of EMP2 and was used in this study. Quantification of FAK and Src phosphorylation was determined with Western blot analysis of whole cell lysates with the use of specific antibodies for different target sites of phosphorylation. Coimmunoprecipitation of whole cell lysates with an antibody against EMP2, followed by Western blot analysis and identification of FAK, was performed. Focal adhesions and their relationship to EMP2 were identified with immunofluorescence and confocal microscopy. F-actin distribution was identified using fluorescence microscopy, and alpha- smooth muscle actin (alpha-SMA) expression was quantified with Western blot analysis and specific antibodies. Adhesion to collagen type I was determined with a binding assay.


RESULTS
EMP2 overexpression led to increased FAK phosphorylation at all measured phosphorylation sites. Coimmunoprecipitation and confocal microscopy provided evidence for a physical association between EMP2 and FAK. Increased EMP2 was also associated with altered distribution of focal adhesions, changes in actin organization, increased alpha-SMA expression, and increased adherence to a collagen-coated surface.


CONCLUSIONS
The EMP2-FAK association represents a novel protein-protein interaction, not previously reported, that demonstrates significant functional cellular responses in the context of in vitro models of proliferative vitreoretinopathy (PVR).
Morales
Shawn A
SA
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA.
Mareninov
Sergey
S
Coulam
Paige
P
Wadehra
Madhuri
M
Goodglick
Lee
L
Braun
Jonathan
J
Gordon
Lynn K
LK
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000502:physiology*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism
C541116:ACTA2 protein, human; D000199:Actins; D024042:Collagen Type I; C486403:EMP2 protein, human; D008562:Membrane Glycoproteins; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases