3385_Ch03 - Chapter 3 Use of the Yeast Two-Hybrid System...

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3 © 1999 by CRC Press LLC Chapter Use of the Yeast Two-Hybrid System for the Characterization of Integrin Signal Transduction Pathways: Identification of the Integrin-Linked Kinase, p59 ILK Gregory E. Hannigan and Shoukat Dedhar Contents I. Introduction II. Preparation of Bait Plasmids for Interaction Screening A. Overview B. Protocols C. Technical Comments III. Testing of Baits for Autoactivation and Fusion Protein Expression Protocols IV. Conducting cDNA Library Screens with Confirmed Baits Protocols
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© 1999 by CRC Press LLC V. Rescue of Interaction Plasmids and Confirmation of the Interaction A. Overview B. Protocols C. Technical Comments VI. Materials and Reagents Yeast Growth Media (Ref. 16) Other Reagents VII. Concluding Remarks and Future Directions Acknowledgments References I. Introduction Specific protein-protein interactions underlie many fundamental cellular processes, such as gene transcription, mRNA splicing, and signal transduction. In the latter case, intracellular protein complexes are commonly formed in response to ligand activation of cell surface receptors, such as the receptor tyrosine kinases, and discrete protein domains mediate interactions which stabilize the complex and/or propagate the signal. 1 Similarly, occupation and clustering of integrin receptors induces the assembly of focal adhesion complexes, formation of which is dependent on protein interactions of integrin cytoplasmic domains. 2-4 Many biochemical, genetic, and molecular biological techniques have provided the opportunity for investigators to identify specific protein interactions. As a result, great strides have been made over the past decade toward understanding the signal transduction mechanisms regulating cellular responses to environmental stimuli. The focus of this chapter is the application of the yeast two-hybrid genetic screen in the isolation of a novel protein serine/threonine kinase, ILK, mediating integrin signal transduction. We selected the LexA-based interaction trap, developed in the laboratory of Roger Brent, 5 as the two-hybrid system of choice for analysis of integrin signal transduction pathways. This system and its use have recently been described in detail by its developers, 5 and LexA-based two-hybrid systems are now commercially available, e.g., LexA MatchMaker ® (Clontech, Inc., Palo Alto, CA), Hybrid Hunter ® (Invitrogen Corp., Carlsbad, CA), and DupLEX-A ® (OriGene Technologies, Inc., Rockville, MD). Excellent manuals are available for downloading as .pdf files from the company web sites ( www.Clontech.com ; www.invitrogen.com ; www.ori gene.com ). These manuals detail use of the two-hybrid system compo- nents, and provide general protocols for yeast growth, transformation, and selec- tion. These companies are rapidly developing interaction libraries from a variety of human tissues from various organisms, greatly increasing the versatility of the system for use in tissue-specific and developmental stage-specific screens. We will limit ourselves to a practical discussion of the interaction trap as it is used
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3385_Ch03 - Chapter 3 Use of the Yeast Two-Hybrid System...

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