SUBSTRATE SPECIFICITY OF NONRECEPTOR TYROSINE KINASES

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Cellular transformation and tumor formation by Rous sarcoma virus is dependent on the expression of the viral v-src gene. The product of this gene is a 60-kilodalton tyrosine protein kinase designated as v-Src. Likewise, the transforming gene of Abelson murine leukemia virus (which causes B-cell lymphomas in vivo) encodes a tyrosine protein kinase designated as v-Abl. This proposal focuses on the role of protein-protein interactions in the function of the v-Src and v-Abl tyrosine kinases. It has been suggested that the Src homology (SH) regions 2 and 3 of nonreceptor tyrosine kinases are important both in the regulation of kinase activity and in the recognition of cellular substrates. Regions of v-Src and v-Abl which are involved in intra- or intermolecular recognition will be identified by peptide-based photoaffinity labelling experiments. Peptide substrate analogs for these enzymes containing the photoactive amino acid p-benzoyl-Phe will be used as the affinity labels. Modified regions inside and outside the SH2 and SH3 domains will be studied further by site-directed mutagenesis, and the mutant enzymes will be tested by in vitro phosphorylation with tyrosine-containing synthetic peptides. These results will be correlated with a model of the three-dimensional structure of v-Src based on the recent crystal structure of the catalytic domain of the cAMP-dependent protein kinase. To identify determinants in protein substrates for v-Src which confer recognition by the wild-type and mutant enzymes, synthetic peptide "libraries" will be used to select those peptides which give maximal phosphorylation by v-Src. Mutant v-Src kinases will be used to study in vivo substrate recognition. The model system for these studies will be Rat-1 cells transfected with v-Src constructs encoding kinases with altered specificity. Substrate recognition by the mutant enzymes will be assessed by three criteria: effects on total tyrosine phosphorylation, effects on phosphorylation of phospholipase C-gamma and other specific substrates, and effects on cellular transformation. The goal of these studies is to describe at a molecular level the steps leading to the formation of the "signalling complexes" which play a central role in signal transduction and oncogenic transformation.