Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.
Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
L E
LE
Ling
B J
BJ
Druker
L C
LC
Cantley
T M
TM
Roberts
D019870:1-Phosphatidylinositol 4-Kinase; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D066298:In Vitro Techniques; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011120:Polyomavirus / Q000235:genetics; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D010716:Phosphatidylinositols; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D016392:Proto-Oncogene Proteins pp60(c-src)