Transforming growth factor beta (TGFb) is a potent growth inhibitor for a wide variety of cells including those of epithelial lineage. The fact that the majority of the transformed counterparts of these normal cells have lost their growth inhibitory response to TGFb has led to the hypothesis that this loss of sensitivity to TGFb may be an initial step in the carcinogenic process. Recent work by the applicant has generated a TGFb-regulated cell line which can either be induced to proliferate or die in a TGFb-dependent fashion in the appropriate media. In this application it is proposed to generate recessive stable mutants defective in their response to TGFb which can subsequently be employed for functional complementation cloning of the affected genes. In addition to isolating cDNAs capable of complementing TGFb mutants, genes which encode negative regulators or suppressors of TGFb signaling will be isolated and characterized. cDNAs encoding proteins that either complement TGFb mutants or suppress TGFb signaling will be isolated, sequenced, and characterized for conserved functional domains (i.e., kinases, phosphatases, transcription factors, SH2, SH3 domains, etc.) for some clues to their function. These will then be manipulated by deletional and site directed mutagenesis and introduced into the mutant cells for structure-function analyses. Antibodies will be generated to assay for post-translational modification(s) in response to growth factors and cytokines and to determine protein-protein interactions by co-precipitation analyses.
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