We are interested in studying the mechanism of viral transformation by retroviruses, an avian Fujinami sarcoma virus (FSV), as the model system. Our long term goals are to understand how the transforming gene of FSV, v-fps, evolves and how its product, the transforming protein, p140, transforms cells and induces tumors. In order to achieve this goal, we propose to elucidate the structure and function relationship of the FSV transforming protein, p140, and to explore how it interacts with other cellular proteins. This approach is based on our previous studies in which we have defined the genetic structure of FSV, characterized the transforming protein, p140, and analyzed the structure of proto-fps (cellular homologue of v-fps). We plan to take advantage of gene-splicing techniques and site-directed mutagenesis to systematically construct different mutants of p140 in vitro and to correlate structure and function. The putative domain and function relationship will be explored by constructing various deletion mutants and by assessing the alterations in the expression of transformation parameter and in host cell response as a consequence of modification of p140. We shall further characterize the ts lesion in the pFL-5 FSV mutant, which is temperature sensitive for transformation. We have to establish changes in which amino acid residue(s) affect the phosphorylation of p140 and alter its transforming activity. We will be able to assign the site(s) of phosphorylation and to define the possible function(s) of each phosphorylation site(s) of p140. Having assigned the phosphylation site(s) precisely, we plan to use the site directed mutagenesis to construct mutants with altered specific phosphorylation site(s) and to examine the functional consequence of such alterations. The combination of the studies of deletion mutants, ts mutants and phosphorylation mutants, should clarify how the oncogene v-fps product, p140, transforms cells. Finally, we shall attempt to express p140 or its fragments in E. coli for purification of the p140 protein and its further characterization. We also plan to make antisera against synthetic peptides deduced from p140 amino acid sequence and to use such antisera in immunoaffinity chromatography to purify the p140 for further studies. These antisera will be also useful in examining the expression of deletion mutants described above. The results from these studies should shed lights on the biochemical properties of p140.
| Cheng, J; Scully, P; Shew, J Y et al. (1990) Homozygous deletion of the retinoblastoma gene in an acute lymphoblastic leukemia (T) cell line. Blood 75:730-5 |
| Lee, E Y; To, H; Shew, J Y et al. (1988) Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 241:218-21 |
| Lee, E Y; Bookstein, R; Young, L J et al. (1988) Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79. Proc Natl Acad Sci U S A 85:6017-21 |
| Chen, L H; Hatada, E; Wheatley, W et al. (1986) Single amino acid substitution, from Glu1025 to Asp, of the fps oncogenic protein causes temperature sensitivity in transformation and kinase activity. Virology 155:106-19 |
