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 activates the protein tyrosine kinase, Src. To determine whether this interaction is critical for middle T transformation, we examined the ability 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 formation on cell monolayers and colony formation in soft agar in Src-negative 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 with 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 kinases in these cells. In Src-positive cell lysates, there was a similar level of middle T phosphorylation in Src and Yes immunoprecipitates, suggesting that middle T can bind to Src and Yes to a similar extent in this cell type. Fyn immunoprecipitates displayed fourfold lower levels of middle T phosphorylation than that detected in the Src and Yes immunoprecipitates. In Src-negative cells, the level of middle T phosphorylation in Yes and Fyn immunoprecipitates was not significantly different from that detected in the Src-positive cells, suggesting that the absence of Src does not lead to a compensating increase in the proportion 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. Phosphatidylinositol 3'-kinase activity associated with middle T was reduced 30-60% in Src-negative cells, suggesting that Src contributes at least one-third of the total middle T associated in wild-type cells. Taken together, these results indicate that Src is not required for middle T-induced hemangiomas in mice or for focus induction in immortalized fibroblasts, and that the residual level of Yes, Fyn and phosphatidylinositol kinase activity associated with middle T in Src-negative cells may compensate for the absence of Src.
Thomas
J E
JE
Howard Hughes Medical Institute, Department of Microbiology, University of Pennsylvania, Philadelphia.
Aguzzi
A
A
Soriano
P
P
Wagner
E F
EF
Brugge
J S
JS
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D019061:src-Family Kinases
D000952:Antigens, Polyomavirus Transforming; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src); C496639:Yes1 protein, mouse; D019061:src-Family Kinases