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Unformatted text preview: 6 © 1999 by CRC Press LLC Chapter p130 Cas in Integrin Signaling Fabrizio Dolfi, Miguel Garcia-Guzman, and Kristiina Vuori Contents I. Introduction II. Tyrosine Phosphorylation of Cas A. Overview B. Protocols C. Materials D. Buffers III. Protein-Protein Interactions Mediated by Tyrosine-Phosphorylated Cas A. Overview B. Protocols C. Materials D. Buffers IV. Functional Studies on the Cas-Crk Signaling Complex A. Overview B. Protocols C. Materials D. Buffers Acknowledgments References © 1999 by CRC Press LLC I. Introduction p130 Cas (Cas—Crk-Associated Substrate) was originally identified as a major tyrosine- phosphorylated protein in cells transformed by the p47 v-crk ( v-crk ) 1,2 and p60 v-src ( v-src ) oncoproteins. 3,4 The tyrosine phosphorylation levels of Cas correlate well with the transforming phenotypes of cells and are therefore thought to play a role in the process of cellular transformation. Molecular cloning of Cas revealed that Cas lacks any enzymatic activity but contains multiple domains suitable for protein-protein interactions ( Figure 1 ). 5 Cas has a SH3-domain in its amino-terminus; recent studies have demonstrated that the SH3-domain interacts directly with a proline-rich region in the focal adhesion kinase FAK, and also binds to two protein tyrosine phos- phatases, PTP1B and PTP-PEST. 6-8 The Src family tyrosine kinases, in turn, bind to a region near the carboxy-terminus of Cas via their SH2- and SH3-domains. 9 Interactions mediated by these two domains of Cas may therefore be responsible for the regulation of the tyrosine phosphorylation status of Cas and also target Cas to focal adhesions, which are sites of close cell-extracellular matrix (ECM) interac- tions. 10-12 Additionally, Cas has a cluster of multiple putative SH2-binding motifs (substrate domain; Tyr 377-Tyr 414 ); 5 nine of these are YDV/TP sequences that conform to the binding motif for the adaptor protein Crk SH2-domain. These structural characteristics indicate that Cas is a docking molecule, which can assemble and transmit cellular signals via interactions through the SH2- and SH3-domains of a wide variety of signaling proteins. Two recently cloned molecules, Efs (Embryonal Fyn-associated substrate)/Sin (Src-interacting or signal-integrating protein) 13,14 and HEF1 (Human Enhancer of Filamentation 1)/Cas-L, 15,16 have similar primary struc- ture to Cas and are assumed to comprise a new family of docking proteins. Consistent with a role as a signaling molecule, Cas has been reported to become tyrosine-phosphorylated in response to a number of different cellular stimuli, many of which affect the assembly of focal adhesions and stress fibers. These stimuli include integrin-mediated cell adhesion 17-19 and ligand engagement of a variety of different receptors, such as G-protein-coupled receptors and receptor tyrosine kinases....
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