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3385_Ch04 - Chapter 4 Focal Adhesion Kinase and its...

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4 © 1999 by CRC Press LLC Chapter Focal Adhesion Kinase and its Associated Proteins Jill K. Slack, Judith Lacoste, and J. Thomas Parsons Contents I. Introduction II. Protein-Protein Interactions A. Preparation of Cell Lysates B. Co-Immunoprecipitation of FAK and Associated Proteins C. In vitro Protein Association Using GST Fusion Proteins D. Far-Western Analysis III. Subcellular Localization A. Immunofluorescence in Fixed Cells B. Use of GFP Fusion Protein in Living Cells IV. Epilogue Acknowledgments References I. Introduction Cellular adhesion to the extracellular matrix (ECM) is a critical regulator of fundamental processes including growth, differentiation, death, and migration. Loss of adhesive influences stimulates apoptosis in many non-transformed cells. 1 Indeed, a hallmark of transformation is the loss of adhesion-dependent growth control, which is often accom- panied by increased cellular migration. Cells recognize and respond to the ECM through specialized adhesive structures called focal adhesions. In addition to linking structural components of the cytoskeleton to the underlying matrix, focal adhesions likely organize
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© 1999 by CRC Press LLC a competent signal transduction cascade initiated by receptor-mediated recognition of ECM components (e.g., fibronectin, laminin, collagen). The ability of focal adhesions to organize a competent signal transducing com- plex is suggested by the observed interaction of heterodimeric transmembrane inte- grin receptors with focal adhesion associated tyrosine phosphorylated proteins. 2 Clustering integrin molecules without necessarily occupying the ligand binding domain results in increased tyrosine phosphorylation. 3,4 Among the proteins phos- phorylated as a result of integrin clustering is a protein tyrosine kinase found within the focal adhesion (focal adhesion kinase or FAK). 5-10 Because integrins themselves lack catalytic activity, the stimulation of FAK kinase activity upon integrin occu- pancy suggests a role for FAK in the signaling that underlies integrin-mediated cell adhesion and migration. Indeed, the results from several experimental systems pro- vide evidence for such a role for FAK in cell adhesion and migration. Mouse embryos deficient for FAK expression die early in development and cells derived from such early embryos exhibit reduced mobility in vitro . 11 The stable overexpression of FAK in Chinese hamster ovary cells stimulates cell migration on fibronectin. 12 Recent studies have shown that overexpression of FRNK (FAK-related non-kinase), the autonomously expressed C-terminal domain of FAK, 13 in chick embryo fibroblasts reduces FAK tyrosine phosphorylation concomitant with a delay in cell spreading on fibronectin. 14 Similar findings were obtained by microinjecting BALB/c 3T3 cells and human umbilical vein endothelial cells with a glutathione- S -transferase (GST) fusion protein containing the FAK C-terminal domain.
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