The overall goal of this research program is to elucidate mechanisms of intracellular signalling by pp60V-src, with an emphasis on the interaction of pp60v-src with signalling pathways utilized during normal mitogenesis. For the first Specific Aim, we propose a molecular characterization of pp42, a protein whose tyrosine phosphorylation accompanies a wide variety of signalling events, including transformation by src, and mitogenic stimulation. The second and third Specific Aims propose feasibility studies for, respectively, the development of improved biochemical or genetic tools to identify and isolate additional signalling intermediates. 1. Purification and characterization of pp42. Protein tyrosyl phosphorylation is undoubtedly a key first step in the mechanism of action of pp60v-src and of the tyrosine kinase mitogen receptors. We have identified and are purifying a 42,000 Mr protein which is the best candidate so far identified for a signalling intermediary for pp60v-src and other tyrosine kinases. We propose to finish the purification of this protein, and to characterize it by obtaining amino acid sequence information, raising antisera against it and cloning and sequencing the gene. 2. Identification and isolation of pp60v-src substrates. We propose to use partially transforming src mutants which we have isolated, cloned and sequenced, to identify and purify additional cellular proteins which might interact with pp60v-src in functionally significant ways. We will analyze the biochemical basis for the substrate selectivity of these mutant src proteins, an over-express them with a Baculovirus vector to produce sufficient quantities of wild-type and mutant pp60v-src to use as affinity reagents for the isolation of cellular target proteins. 3. Cellular mutants refractory to oncogenesis by src. We wish to generate biological reagents which will allow us to study signal transduction pathways utilizing the same protocols which have been used successfully in identifying oncogenes (e.g. DNA transfection and molecular genetics). Thus, we propose to construct or identify cell lines refractory to transformation by src, and will use DNA transfection to search for genes which confer transformability on these cells. Such genes are candidate """"""""signal transduction"""""""" genes. We already have identified one such cell line, TNR9 cells, a 3T3 derivative isolated on the basis of its resistance to TPA-induced mitogenesis. We also describe protocols to isolate additional src refractory cell lines.
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