Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation

Abstract

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.