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Abstract. The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pls ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of ~85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60 src in Rous sarcoma virus-transformed ceils (Glenney, J. R., and L.

Abstract. We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virustransformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215-and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and >90% of the 22-kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.M ANY oncogenes are known to encode tyrosinespecific protein kinases and presumably exert their effect by phosphorylating proteins present in normal cells (6,32,44). The target substrates of tyrosine kinases have proven difficult to identify and characterize since they are generally minor proteins present in cultured cells. A variety of tyrosine kinase substrates are known. These include the cytoskeletal proteins vinculin (34, 50) and talin (15, 42) together with integrin (31) that are thought to participate in a cytoskeleton to membrane linkage (for reviews, See references 8, 33). Proteins termed calpactins (also referred to as p35 and p36 or lipocortins) are also known to be phosphorylated by both the transforming tyrosine protein kinases (10,16,45) and the epidermal growth factor receptor kinase (24, 49). Calpactins have been shown to associate with membrane lipids (22, 24) and the cytoskeletal proteins actin and spectrin (20, 24) in a Ca÷÷-dependent manner. They have been localized just under the membrane in a number of cell types (2,13,28,35,40). Other tyrosine kinase substrates include calmodulin (18), microtubule associated proteins (1), glycolytic enzymes (12), and a 50-kD protein (21).Thus far there is little evidence Suggesting a functional connection between the phosphorylation of the known substrates and any of the properties of transformed cells. Indeed, recent studies have shown that cells expressing the nonmyristylated mutants of the transforming protein of Rous sarcoma virus, pp60 ~, are not transformed (30,37). In this system, nonmyristylated p60src is no longer targeted to the membrane (14, 37), but it still phosphorylates th...

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