Ts transformation mutants of avian sarcoma virus PRCII: Lack of strict correlation between transforming ability and properties of the P105-associated kinase

Abstract

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive ( ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type ( wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.