We propose to investigate the localization of various viral tyrosyl protein kinases by both indirect immunofluorescence microscopy and by immunoelectron microscopy. These studies will be carried out on virally-infected cell cultures and on virally-induced tumors. Comparison of transformed cell cultures with tumors should be useful in helping to discern which subcellular residences are essential for onc product function and which cellular alterations are a sine qua non of transformation. Analysis of the mode of interaction of onc products with intracellular binding sites will be carried out using viral variants derived either from in vivo recombination or from site-directed mutagenesis. Such studies will allow us to determine which regions of the onc proteins are essential for specific intracellular localization and which domains are necessary for enzymatic activity. In particular, fatty acid acylation of onc products will be investigated to determine the nature of lipid binding sites on the onc proteins and the involvement of lipid in determining unique intracellular localizations. By means of double-label immunoelectron microscopy we plan to determine both the intracellular localizations and distances between onc gene prducts and their putative substrates. Proteins that are presumed to interact or have been shown to interact with viral tyrosyl protein kinases include the 34 k protein, pp90: pp50, 130 k-vinculin, and various cytoskeletal proteins. Subcellular fractions enriched for onc proteins will be examined for the presence of proteins containing phosphotyrosine. In addition, such fractions will be incubated with Gamma32P-ATP to test for proteins that are either autophosphorylated or become phosphorylated because of the presence of an onc protein kinase. We intend to investigate the metabolism of tyrosyl protein kinases from normal, untransformed tissues, and to determine the function of phosphorylated tyrosine residues in proteins. The influence of virally-encoded onc tyrosyl protein kinases on the metabolic pathway of normal tyrosyl protein kinases will be determined. Immunologic and nucleic acid probes will be used to determine the relationship between cellular and viral tyrosyl protein kinases.
| Radziejewski, C; Miller, W T; Mobashery, S et al. (1989) Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference. Biochemistry 28:9047-52 |
