INDUCTION OF TUMOR-FORMATION AND CELL-TRANSFORMATION BY POLYOMA MIDDLE T-ANTIGEN IN THE ABSENCE OF SRC

In polyomavirus-transformed cells, middle T antigen binds to and activ ates the protein tyrosine kinase, Src. To determine whether this inter action is critical for middle T transformation, we examined the abilit y of middle T to transform cells that lack endogenous Src (because of a targeted disruption of both Src alleles). Infection of newborn or 2- week-old Src-negative mice with a retrovirus encoding middle T led to the induction of visceral hemangiomas that were indistinguishable from tumors in wild-type mice with respect to their morphology, frequency or latency period. In addition, middle T was able to induce foci forma tion on cell monolayers and colony formation in soft agar in Src-negat ive immortalized fibroblasts. These results indicated that Src is not essential for middle T-induced transformation of the cells targeted in these assays. To examine the protein tyrosine kinases that interact w ith middle T in the absence of Src, we compared the level of middle T phosphorylation in immune complex kinase assays from Src-negative and Src-positive cell lysates, and identified the middle T-associated kina ses in these cells. In Src-positive cell lysates, there was a similar level of middle T phosphorylation in Src and Yes immunoprecipitates, s uggesting that middle T can bind to Src and Yes to a similar extent in this cell type. Fyn immunoprecipitates displayed fourfold lower level s of middle T phosphorylation than that detected in the Src and Yes im munoprecipitates. In Src-negative cells, the level of middle T phospho rylation in Yes and Fyn immunoprecipitates was not significantly diffe rent from that detected in the Src-positive cells, suggesting that the absence of Src does not lead to a compensating increase in the propor tion of middle T associated with these kinases. The level of middle T- associated phosphatidylinositol 3'-kinase was also examined since this kinase is known to interact with middle T-kinase complexes. Phosphati dylinositol 3'-kinase activity associated with middle T was reduced 30 -60% in Src-negative cells, suggesting that Src contributes at least o ne-third of the total middle T associated in wild-type cells. Taken to gether, these results indicate that Src is not required for middle T-i nduced hemangiomas in mice or for focus induction in immortalized fibr oblasts, and that the residual level of Yes, Fyn and phosphatidylinosi tol kinase activity associated with middle T in Src-negative cells may compensate for the absence of Src.