THE BOVINE PAPILLOMAVIRUS-E5 ONCOGENE CAN COOPERATE WITH RAS - IDENTIFICATION OF P21-AMINO ACIDS CRITICAL FOR TRANSFORMATION BY C-RASH BUT NOT V-RASH

We have previously used a series of insertion-deletion mutants of the mutationally activated v-ras(H) gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-ras(H), we have now tested many of the same insertion-deletion mutants in the c-ras(H) form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-ras(H) is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-ras(H) mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-ras(H) as well as for c-ras(H), which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-ras(H), of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-ras(H) transformation. Relative to the respective transforming activity of wild-type c-ras(H) and v-ras(H), mutants with lesions in codons 102 and 103 were significantly less active in their c-ras(H) forms than in their v-ras(H) forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-ras(H) than to v-ras(H). Guanine nucleotide exchange is one function that is compatible with such a phenotype.