Analyzing FAK and Pyk2 in early integrin signaling events.
Integrins are a family of heterodimeric alpha/beta transmembrane cell adhesion receptors that play important roles in the regulation of cell migration, proliferation, and survival. Integrins do not possess intrinsic catalytic activity, and signaling events are mediated by their lateral association with other cell surface receptors or clustering of their cytoplasmic domains with signaling proteins. Rapid activation of protein-tyrosine kinases is one of the first signaling events associated with integrin binding to the extracellular matrix protein fibronectin. The intracellular focal adhesion kinase (FAK) is recruited to sites of integrin clustering, and this unit describes the methods with which to analyze FAK phosphorylation, activity, and localization within fibroblasts. Additional methods on how to grow primary FAK+/+ and FAK-/- fibroblasts and measure integrin-stimulated cell motility are described as well as methods for evaluating the activity of the FAK-related kinase, Pyk2, which is expressed in FAK-/- cells.
Bernard-Trifilo
Joie A
JA
The Scripps Research Institute, La Jolla, California, USA.
Lim
Ssang-Taek
ST
Hou
Shihe
S
Schlaepfer
David D
DD
Ilic
Dusko
D
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D054443:Cell Migration Assays / Q000379:methods; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D005347:Fibroblasts / Q000201:enzymology; D051418:Focal Adhesion Kinase 2 / Q000032:analysis*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis*; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D015398:Signal Transduction / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology
D016023:Integrins; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases