The cytoskeleton is a complex set of proteins which provides stability and structure to all cells. There is increasing evidence that the cytoskeleton can be rapidly modified in response to signals from membrane bound receptors. Such signals induce changes in the cytoskeleton which are reflected in alterations in cell shape, rigidity, mobility, adhesiveness, and the ability of intracellular proteins to interact with the cytoskeleton. One form of signaling which is believed to be important in modifying functions is phosphorylation. One of the cytoskeleton proteins that may be an important link between membrane receptors and the cytoskeleton is paxillin. Paxillin is a focal contact (actin binding) protein with a MW of 68 kDa which can bind to vinculin. Paxillin was initially identified in Rous Sarcoma Virus transformed chick embryo fibroblasts in a blind screening of monoclonal antibodies to proteins containing phosphotyrosine. Up to 20-30% of paxillin is phosphorylated on tyrosine residues in RSV transformed cells, and paxillin has been shown to be transiently tyrosine phosphorylated in fibroblasts in response to epidermal growth factor. Thus, paxillin has been suggested to be a direct link between growth factor receptors and oncogene (v-SRC) which activate tyrosine kinases and the cytoskeleton. Since many growth factors and oncogenes alter the structure of the cytoskeleton in cells of many different lineages, paxillin may play an important role in signal transduction. Recent data shows paxillin is immediately phosphorylated after stimulation of hematopoietic cells with various cytokines and the oncogene p210BCR/ABL (Philadelphia chromosome in chronic myelogenous leukemia). Also, growth factor and oncogene stimulation causes paxillin to associate with vinculin and with several unidentified intracellular proteins. In an effort to understand the structure and function of paxillin, partial cDNA's encoding this protein have been cloned. It is proposed here in this project to determine the structure of paxillin and paxillin related proteins by cloning the cDNA's encoding these proteins. Also, to generate antibodies to various domains of paxillin. Finally, to determine the regions of paxillin necessary to bind vinculin and determine the effects of tyrosine phosphorylation of paxillin on vinculin binding. These studies will help elucidate the function of paxillin in the cytoskeleton and will provide a model system to study the mechanisms through which cell membrane receptors and oncogenes send signals to the cytoskeleton.
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