The mechanisms whereby integrins signal to the interior of the cell are largely unknown. Evidence indicates that tyrosine phosphorylation of intracellular proteins may be a key event p130Cas (Cas) is one of the prominent proteins that are tyrosine-phosphorylated upon integrin- mediated cell attachment. Cas is an SH3-domain containing protein that has a cluster of putative SH2-binding sites; Cas also has several proline-rich regions that are candidates for SH3-binding sites. Preliminary results suggest that Cas is important in integrin signaling; Cas bound to multiple SH2-domain containing signaling proteins upon integrin ligand binding, cell adhesion resulted in changes in the subcellular localization of Cas, and Cas antisense mRNA expression reduced integrin-dependent MAP kinase activation. The studies proposed here will explore the molecular mechanisms and significance of Cas in integrin signaling. Because Cas has no known enzymatic activity, Cas most likely exerts its signaling effects through protein-protein interactions. It is thus important to identify the proteins that interact with Cas. The first part of the study will characterize the protein-protein interactions between Cas and known SH2-domain containing proteins.
This aim also includes an analysis of the signaling molecules known to interact with the SH2- proteins present in the Cas signaling complex. Next, novel signaling proteins interacting with Cas in an adhesion- dependent manner will be identified and characterized. These experiments use two powerful genetic systems suitable for direct cloning of proteins that interact with tyrosine-phosphorylated Cas. In another approach, proteins interacting with Cas in an adhesion-dependent manner regardless of the phosphorylation status of Cas will be identified. The functional effects of Cas on integrin signaling will be studied. These experiments will be designed based on what is currently known about the Cas-associated molecules, and on the outcome of experiments described above. Cell lines expressing reduced levels of Cas have been generated by using antisense technology, and will be used in these studies. These studies will provide insights into the role of Cas in intracellular signaling of integrins. This work should also lead to a better understanding of mechanisms whereby extracellular matrix influences cell behavior, such as normal growth regulation and adhesion dependency.
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