The major focus of this project is to investigate the molecular biology and biochemistry of the ras oncogenes and the ras p21 proteins. The long-range goal is to elucidate molecular mechanisms of cell transformation induced by these genes and their protein products, with the purpose of contributing to our understanding of the roles of oncogenes in human tumorigenesis, and to develop strategies for tumor detection, monitoring and intervention. We have characterized the functional domains of p21 proteins. We have chemically mapped the palmitoylation site of p21 to cysteine-186, which appears to be the membrane binding domain. We have characterized the GTP binding domain by chemical, immunochemical and site-directed mutagenesis approaches. The structure of the GTP binding domain is very similar to that of the EF-Tu, the E. coli elongation factor, of which a three dimensional structure has recently been determined. The functional role of p21 in the adenylate cyclase system was studied using the S49 lymphoma cells lacking the regulatory G proteins. The results indicate that p21 does not function directly in the adenylate cyclase system. Chemical reactivity of the sulfhydryl group of p21 was also studied by a thiol-specific reagent, N-ethylmaleimide. A rapid method for detection of ras-transformed cells by flow cytometry was developed. Another immunocytochemical method was developed for assessing p21 in fixed tissue sections. Revertants of Ha-MuSV-transformed MDCK cells were isolated and characterized. Studies in progress involve the role of protein kinase C in p21 function, identification of p21 palmitoylation enzymes, and the roles of ras oncogenes in mammary carcinomas of human patients.
