Morales Shawn A, Mareninov Sergey, Coulam Paige, Wadehra Madhuri, Goodglick Lee, Braun Jonathan, Gordon Lynn K
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA.
Invest Ophthalmol Vis Sci. 2009 Oct;50(10):4949-56. doi: 10.1167/iovs.08-3315. Epub 2009 Jun 3.
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.
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.
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.
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).
ARPE - 19细胞对胶原凝胶的收缩受上皮膜蛋白2（EMP2）通过粘着斑激酶（FAK）激活的调控。本研究旨在测试EMP2在FAK激活的细胞环境中的作用。
对ARPE - 19细胞系进行重组修饰以增加EMP2的表达，并用于本研究。使用针对不同磷酸化靶位点的特异性抗体，通过全细胞裂解物的蛋白质印迹分析来测定FAK和Src磷酸化的定量。用抗EMP2抗体对全细胞裂解物进行免疫共沉淀，随后进行蛋白质印迹分析并鉴定FAK。通过免疫荧光和共聚焦显微镜鉴定粘着斑及其与EMP2的关系。使用荧光显微镜鉴定F - 肌动蛋白分布，并用蛋白质印迹分析和特异性抗体对α - 平滑肌肌动蛋白（α - SMA）表达进行定量。通过结合试验测定对I型胶原的粘附。
EMP2过表达导致在所有测量的磷酸化位点FAK磷酸化增加。免疫共沉淀和共聚焦显微镜为EMP2与FAK之间的物理关联提供了证据。EMP2增加还与粘着斑分布改变、肌动蛋白组织变化、α - SMA表达增加以及对胶原包被表面的粘附增加有关。
EMP2 - FAK关联代表一种新的蛋白质 - 蛋白质相互作用，此前未见报道，在增殖性玻璃体视网膜病变（PVR）的体外模型中显示出显著的细胞功能反应。
