3385_Ch05 - Chapter 5 Analysis of Paxillin as a...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
5 © 1999 by CRC Press LLC Chapter Analysis of Paxillin as a Multi-Domain Scaffolding Protein Mary C. Riedy, Michael C. Brown, and Christopher E. Turner Contents I. Introduction II. Transfection A. Overview B. Protocols C. Materials III. Rate of Paxillin Localization to Focal Adhesions and Role in Cell Adhesion A. Overview B. Protocols C. Materials D. Buffers IV. GST-Paxillin Precipitation Kinase Assays A. Overview B. Protocols C. Materials D. Buffers V. Cell Migration A. Overview B. Protocols C. Materials
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
© 1999 by CRC Press LLC VI. Immunoprecipitation and Western Blotting A. Overview B. Protocols C. Materials D. Buffers References I. Introduction Cells communicate with their extracellular environment via a variety of cellular receptors. In one example, integrins associate with the extracellular matrix (ECM), initiating a cascade of intracellular signaling events including association of these receptors with the actin cytoskeleton, cytoskeletal reorganization, and protein phos- phorylation. 1 Cytoskeletal proteins that become phosphorylated include the focal adhesion kinase (FAK), p130 cas , and paxillin. These proteins are also phosphorylated during stimulation by growth factors that include angiotensin II, platelet derived growth factor (PDGF), activin A, bombesin, nerve growth factor, and TGF- β . 2-6 Paxillin co-localizes with FAK and vinculin, along with several other structural and regulatory proteins, to points of contact of the cell membrane with the ECM, termed focal adhesions. 7-9 The formation of these structures is stimulated by both cell adhesion and growth factors, and they likely act as important nucleation centers for efficient signal transduction. Paxillin is composed of multiple protein binding sites such as SH3- and SH2-binding domains, five LD motifs, and four LIM domains. 8-12 Additionally, sites which are phosphorylated by FAK and/or Src func- tion as SH2-binding sites for the adaptor protein Crk and the tyrosine kinases Csk and Lyn. 12,13 A series of paxillin-homologous proteins including Hic-5, 14 PaxB, 15 and leupaxin 16 is emerging. The methods described in this chapter have been used to define the role paxillin and related family members play as molecular adaptors, as well as to evaluate their participation in growth factor and adhesion-dependent signaling events. II. Transfection A. Overview In an attempt to understand the role(s) of the focal adhesion-related protein paxillin in cellular function in vivo , it is necessary to be able to identify important domains of this protein that specify the role(s). This approach has been instrumental in the determination that the LIM domains of paxillin direct the protein to focal adhesions. 10
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

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.

Page1 / 18

3385_Ch05 - Chapter 5 Analysis of Paxillin as a...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online