Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.
The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.
Gorga
F R
FR
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
Riney
C E
CE
Benjamin
T L
TL
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004789:Enzyme Activation; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D007294:Inositol; D015544:Inositol 1,4,5-Trisphosphate; D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases