Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.
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) beta receptor mutants (beta-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 beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR 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 beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), 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-gamma 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-gamma are key initiators of such signal relay cascades.
DeMali
K A
KA
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Whiteford
C C
CC
Ulug
E T
ET
Kazlauskas
A
A
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma