Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

Abstract Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) β receptor mutants (β-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the β-PDGFR but no PDGF-α receptor (α-PDGFR). Hence, this cell system can be used to study signaling of mutant αPDGFRs or α/β chimeras. We constructed chimeric receptors containing the αPDGFR extracellular domain and the βPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type β-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cγ (PLC-γ), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-γ or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-γ are key initiators of such signal relay cascades.