Functional role of GTPase-activating protein in cell transformation by pp60v-src.
Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.
DeClue
J E
JE
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892.
Vass
W C
WC
Johnson
M R
MR
Stacey
D W
DW
Lowy
D R
DR
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002471:Cell Transformation, Neoplastic; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D011905:Genes, ras; D016391:Genes, src; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism; D019000:Phosphotyrosine; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D009227:Myristic Acids; D010750:Phosphoproteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019814:Myristic Acid; D019000:Phosphotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases