This project will study the structure and function of the gene product, p37mos, encoded by the oncogene v-mos of Moloney murine sarcoma virus. The oncogene product p37mos is a member of the protein kinase family based on amino acid sequence homology, but has not yet been shown to possess intrinsic protein kinase activity. We also wish to probe the interchangeability of cellular localization signals as regards their role in the activation of oncogenic proteins. The oncogenic potential of normal cellular protein kinases which are related to the mos gene product will also be examined. Three areas of research are proposed. 1.) Structure-function studies: Site-directed mutations will be constructed in the ATP binding site of the mos gene product, and also in other regions defined by homology with the catalytic subunit of the cAMP dependent protein kinase. Linker insertion mutations will be constructed, to identify regions in p37mos which can tolerate insertion of additional amino acids. Potential enzymatic activities associated with the mos gene product will be examined using the site-directed mutants. Potential substrate specifications for p37mos will be studied by fusing the coding region for kinase substrate domains to the mos coding region. 2.) Retroviral vectors for redirecting cytological localization: By attaching a signal sequence for membrane translocation of the mos gene product, we have altered the cytological localization of this oncogene product as demonstrated by N-linked glycosylation and indirect immunofluorescence. When anchored to the membrane, this hybrid protein is still transforming. Additionally, we will examine the interchangeability of transmembrane anchor domains and myristoylation sites for membrane attachment. As part of this study, we will characterize mutant v-src proteins when localized to the membrane by means of a transmembrane anchor domain. We will also attempt the isolation of new transforming genes using a retroviral expression vector which donates a signal sequence for membrane translocation. 3.) Studies of cellular genes encoding proteins related to the mos gene product. We propose to randomly mutagenize normal cellular genes which encode gene products related to the mos gene product, after which we will attempt to isolate point mutants which are transforming. This will allow us to examine the oncogenic potential of these related genes.
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