The activities of protein-tyrosine kinases are normally regulated in the cell. A mutation in a gene for a protein-tyrosine kinase that overrides normal regulation can engender an oncogene. Such mutations can be gene rearrangements (as occur in the c-abl proto- oncogene in human chronic myelogenous leukemia) or point mutations (as with the neu or HER2 proto-oncogene in rat neuroblastomas). To understand how mutations activate oncogenesis by protein- tyrosine kinases genes, it is necessary to investigate the enzymatic regulation of the kinase products. The sequences of seven of the kinase proto-oncogenes are closely related to each other. The paradigm of this family is c-src. The kinase activity of the c-src product, p60c-src, is regulated by sequences in its amino- and carboxy-terminal regions. The carboxy- terminus is phosphorylated at a tryosine residue. Mutations that remove this tyrosine or prevent its phosphorylation activate oncogenesis by c-src. The mechanism of enzymatic regulation of p60c-src and the closely related gene product, p56lck, will be studied and regulatory molecules with which the kinases interact in fibroblasts wil be identified. Specific mutations that affect regulation will be generated by making point mutations in c-src and chimeras between c-src and lck. The altered proteins will be expressed in fibroblasts and in yeast cells and their sites of phosphorylation and enzymatic activities determined. Sub-cellular localizations of the chimeric molecules and their interactions with the medium T antigen (a regulatory molecule encoded by polymavirus) will be investigated. In these ways the sequences involved in phosphorylation of the carboxy terminus and in regulation, sub-cellular localization and activation by medium T antigen will be characterized. Protein- tyrosine kinases present in fibroblast extracts that phosphorylate p60src will be indentified by in vitro assays and their significance explored using mutant substrates. A search will be made for physiological stimuli that alter the phosphorylation state of the carboxy-terminal tyrosine. The interactions between amino- and carabosy-terminal regulatory domains will be studied.
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