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Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or
K A DeMali1, C C Whiteford, E T Ulug
1Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
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) 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.
Insights
Platelet-derived growth factor (PDGF) receptor activation triggers cell transformation through multiple pathways. Phosphatidylinositol 3 kinase (PI3K) or phospholipase Cgamma (PLC-gamma) are key initiators of these signaling cascades.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Signal Transduction
Background:
- Constitutive activation of receptor tyrosine kinases (RTKs) is a hallmark of cellular transformation.
- The specific signal relay pathways mediating RTK-driven transformation remain incompletely understood.
Purpose of the Study:
- To systematically investigate the signaling pathways required for platelet-derived growth factor (PDGF) beta receptor (beta-PDGFR)-dependent cellular transformation.
- To define the key initiating enzymes in PDGF-mediated signal relay cascades.
Main Methods:
- Utilized a panel of beta-PDGFR mutants and alpha/beta chimeras expressed in Ph cells.
- Assayed PDGF-dependent cellular transformation by measuring growth in soft agar.
- Systematically eliminated or restored specific signaling enzyme binding sites (e.g., PLC-gamma, PI3K, RasGAP, SHP-2).
Main Results:
- A wild-type beta-PDGFR chimera induced significant soft agar growth, indicating cellular transformation.
- A chimera lacking binding sites for PLC-gamma, PI3K, RasGAP, and SHP-2 failed to induce proliferation.
- Receptors associating with either PI3K or PLC-gamma rescued the PDGF-dependent growth response.
Conclusions:
- PDGF receptor activation engages multiple signaling pathways for cellular transformation.
- Either PI3K or PLC-gamma are critical initiators of the signal relay cascades leading to transformation.