RAS GENES BY DOMINANT NEGATIVE MUTANTS

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It has been demonstrated that activated ras genes play vital roles in many types of human tumor. The immediate ras gene family consists of three members, H-ras, K-ras and N-ras. The ras genes belong to a group of proteins collectively called guanine-nucleotide binding proteins. Guanine-nucleotide binding proteins require guanine-nucleotides for their proper functions and share several conserved features among them. Many guanine-nucleotide binding proteins are involved in transducing the stimuli signals to their targets; therefore, it is conceivable that the ras gene products have similar biological roles. At present, not much is known about the signal transduction pathway of ras genes. In addition to their apparent role in cell proliferation, ras genes also have been shown to induce terminal differentiation in some cell types, such as rat pheochromocytoma cell line PC12, a in vitro neuronal cell model. Recently, we have described a class of H-ras mutants which can dominantly suppress the action of the c-H-ras gene as well as several other oncogenes, such as src, fms, sis and fes. The basis for this type of trans-dominant suppression might be attributed to sequestering of the essential element required for c-H-ras activation. These mutants will provide an extremely valuable tool for investigating the signal transduction pathway of ras genes and the interactions of ras genes with other oncogenes. We propose to study the underlying mechanism of trans-dominant suppression and will also utilize the trans-dominant properties of these mutants to study the biological role of the ras genes. 1. We will determine the physical requirements of the H-ras p2l protein for trans-dominant suppression; 2. we will determine the effects of K-ras (or N-ras) mutants on c-H-ras and other oncogenes as mentioned previously; 3. we will utilize the trans-dominant mutants as the affinity probe for purifying the element that interacts with ras p2l protein and 4. we will investigate the role of the ras p2l protein in differentiating PC12 cells by trans-dominant mutants.