3385_Ch12 - Chapter 12 Functional Analysis of FAK and...

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12 © 1999 by CRC Press LLC Chapter Functional Analysis of FAK and Associated Molecules in Cell Migration Leslie Cary and Jun-Lin Guan Contents I. Introduction II. CHO Cell Stable Transfection A. General Considerations B. Protocol C. Material and Equipment III. Cell Migration A. General Considerations B. Protocol C. Material and Equipment IV. Biochemical Analysis of FAK and Associated Proteins A. General Considerations B. Protocol C. Material and Equipment Acknowledgments References I. Introduction Cell migration plays a critical role in many biological processes, including embry- onic development, wound healing, and tumor metastasis. 1 Cell migration through extracellular matrix (ECM) proteins is generally mediated by cell surface integrin
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© 1999 by CRC Press LLC receptors. 2-5 In addition to serving as ECM adhesion receptors, integrins transduce a variety of biochemical signals across the plasma membrane. 6-8 Although many components of integrin signaling pathways have been identified, the molecular mechanisms by which integrins regulate cell migration processes are not well understood. Focal adhesion kinase (FAK), 9,10 a non-receptor protein tyrosine kinase which is localized to focal contacts in cultured fibroblasts, has been identified as an important mediator of integrin signal transduction pathways. 11 FAK is activated and tyrosine phosphorylated in response to integrin activation, such as by plating cells on the appropriate ECM. 9,12-14 FAK associates with a number of signaling molecules including Src family kinases, 15,16 Grb2, 17 phos- phatidylinositol 3-kinase (PI 3-kinase), 18,19 and p130 Cas . 20 It also associates with the cytoskeletal proteins paxillin 21,22 and talin, 23 and is believed to associate with tensin. 24 FAK is clearly an important mediator of integrin signaling, but its roles in specific cellular functions have only recently begun to be identified. 25-29 We have recently shown that stable overexpression of FAK in Chinese hamster ovary (CHO) cells increased cell migration on fibronectin (FN). 30 These results are consistent with other reports suggesting a role for FAK in cell migration using other approaches. 25,27 Our CHO cell transfection system has allowed us to dissect the molecular mechanisms of FAK-dependent cell motility by analyzing various FAK mutants. We have shown that FAK association with Src family members is crucial for its promotion of cell migration. 30 In addition, FAK association with p130 Cas plays a critical role for cell motility, while FAK binding to Grb2 is not important. We have also correlated p130 Cas tyrosine phosphorylation by the FAK/Src complex with increased cell motility. 31 Using this system, we are exam- ining the roles of signaling molecules downstream of the FAK/Src/p130 Cas com- plex in integrin-mediated cell migration. Our CHO cell transfection system for functional analysis of FAK provides
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This note was uploaded on 05/06/2010 for the course MECH. 28197 taught by Professor Dr.shafii during the Spring '10 term at Sharif University of Technology.

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3385_Ch12 - Chapter 12 Functional Analysis of FAK and...

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