The long term goal of this project is to understand the regulation of the assembly of actin filaments and the attachment of the filaments to other cellular structures for force generation and transmission. In the past several years, the focus of our research has been on the structure and function of tensin, a multifunctional protein with properties suggesting that it plays a key role in filament-membrane association and in signal transduction: (1) Tensin has been immunolocalized to muscle Z-lines and cell-cell and cell-substrate adherens junctions, locations where actin filament ends are associated with other structures. (2) Tensin is the only protein at these locations with discrete functional domains for specific binding to the ends of actin filaments, vinculin, and integrin, proteins also found at adherens junctions. The latter plays a key role in cell-substrate attachment and signal transduction. (3) Tensin also has an SH2 domain that binds phosphotyrosine-containing proteins, and is phosphorylated at threonine, serine, and tyrosine residues. Tyrosine phosphorylation increases with viral transformation, cell-substrate adhesion, and stimulation by growth factors. The proposed research employs an integrative approach using methods of cell biology, biochemistry- molecular biology, and biophysics to study the structure and function of the different tensin domains. The project is composed of the following components; (a) The use of biochemical and biophysical assays to characterize in detail the in vitro interaction of tensin with other proteins present at cell-substrate and cell-cell junctions, including actin, vinculin, and talin. (b) Analysis of the activity of recombinant proteins and mutants to define the submolecular domains and critical amino acid residues involved in the binding of these proteins to each other. (c) Investigation of the effects of modifications of the domains by phosphorylation, proteolysis, etc., and the possible interplay among the different domains. (d) Determination of the structural requirements and functional roles of the tensin domains by studying their localization in cultured cells following microinjection, transfection, and the use of neutralizing monoclonal antibodies. (e) Determination of the 3- dimensional atomic structures of the tensin domains by NMR spectroscopy and X-ray crystallography, and to study structure-function relationships with the help of molecular modelling programs. / The results of the proposed work will increase our understanding of the molecular basis of actin-based cytoskeletal and contractile functions in normal and diseased cells and tissues.
