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 (a lpha PDGFR) required for association with PI-3 kinase have been identi fied as tyrosines 731 and 742. Mutation of either tyrosine substantial ly reduced PDGF-induced PI-3 kinase activity but did not impair the re ceptor-mediated mitogenic response. We sought to determine whether PDG F-induced PI-3 kinase activity could be further ablated so as to exclu de a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y7 31F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF ind uced 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 wi ld-type alpha PDGFR levels of mitogenic and chemotactic responses to P DGF. To examine the effect of the double mutation in cells that normal ly respond to PDGF, we generated chimeras in which the cytoplasmic dom ains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to t he extracellular domain of colony-stimulating factor-1 (CSF-1) recepto r (fms). After introduction of the chimeric receptors into mouse NIH/3 T3 fibroblasts, the ability of CSF-1 to stimulate growth of these tran sfectants was examined. Our data show that all these chimeric receptor s exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase acti vity is not required for PDGF-stimulated mitogenic pathway in both NIH /3T3 fibroblasts and 32D hematopoietic cells.