The long-term goal of these studies is to identify biochemical steps unique to the transformed cell which might be amenable to interference by specific pharmacological agents. Experiments are proposed which will help unravel the cell's genetic and biochemical contributions to the process of neoplastic transformation induced by the viral oncogene v-src. Cancer is an intrinsically complex phenomenon which is poorly understood at the molecular level. The current lack of understanding at the molecular level has prevented such a directed pharmacological approach; commonly used drugs attack all dividing cells, limiting the dose employed and inducing serious side effects. Three particular sets of experiments are proposed: (1) experiments involving further characterization of the unique mutant allele v-src-L, and the subsequent manipulation of v-src-L by further mutation; (2) experiments to test the role of mitotic specific phosphorylations in the process of transformation by v-src; and (3) (in collaboration with Dr. Harold Varmus, UCSF) analysis of a unique set of mutant of v-src. v-src-L transforms chicken but not rat cells; it is host-range dependent for transformation. The pp60v-src-L protein encoded by v-src-L will be characterized in detail for the Km and Vmax of the intrinsic tyrosine- specific protein kinase activity against several substrates; preliminary evidence suggests it exhibits a host-dependent substrate specificity (a unique property among all oncogenes reported thus far). Proposed biochemical and genetic experiments will address how this host-dependent regulation is accomplished. Further experiments will identify the relevant chicken and rat cellular genes involved in this regulation. Site-directed mutagenesis will be used to analyze the necessity of mitosis- specific phosphorylation of wt pp60v-src for (a) increased kinase activity associated with pp60v-src during mitosis and (b) transformation by v-src. Work by others in this field has focused on pp60c-src. Finally, a collection of naturally occurring, biologically selected v-src alleles (generated by H. Varmus) will be analyzed with regard to sequence in order to search for domains of pp60v-src which are critical for transformation. This collection of mutants is unique in that each member is (a) naturally occurring (implying no investigator bias), and (b) a point mutant (implying a relatively subtle lesion).
