Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling.
The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P-Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony-stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.
Yu
J C
JC
Laboratory of Cellular and Molecular Biology, National Cancer Institute (37-1E24), Bethesda, Maryland 20892.
Gutkind
J S
JS
Mahadevan
D
D
Li
W
W
Meyers
K A
KA
Pierce
J H
JH
Heidaran
M A
MA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002633:Chemotaxis; D004789:Enzyme Activation; D004790:Enzyme Induction; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D019950:Mitogen-Activated Protein Kinase 1; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D010982:Platelet-Derived Growth Factor; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D017479:Receptors, Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases