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PDGF-independent activation of PDGF-beta receptors in NIH-3T3 cells transformed by c-met protooncogene
K S Kochhar1, D Linnekin, A P Iyer
1Department of Pathology, Northwestern University, Chicago, Illinois 60611-3008.
Abstract:
We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-beta receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-gamma (PLC-gamma) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-gamma. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-beta receptor in NIH-3T3 cells transformed by c-met protooncogene.
Insights
Overexpression of the c-met protooncogene transforms NIH-3T3 cells, causing serum-free proliferation and constitutive protein phosphorylation. This includes platelet-derived growth factor (PDGF)-independent activation of PDGF-beta receptors, independent of PDGF signaling.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncogenesis
Background:
- The c-met protooncogene encodes a receptor tyrosine kinase involved in cell growth and differentiation.
- Overexpression of c-met can lead to cellular transformation and uncontrolled proliferation.
- Receptor tyrosine kinases play crucial roles in signal transduction pathways.
Purpose of the Study:
- To investigate the downstream signaling events in NIH-3T3 cells transformed by c-met protooncogene overexpression.
- To determine the mechanism of constitutive protein phosphorylation observed in these transformed cells.
- To explore the potential activation of platelet-derived growth factor (PDGF) receptors in a PDGF-independent manner.
Main Methods:
- Cell culture of NIH-3T3 cells overexpressing c-met protooncogene.
- Analysis of cellular proliferation in serum- and growth factor-free conditions.
- Western blotting and immunoprecipitation to detect protein phosphorylation and interactions.
- Phosphoamino acid analysis to identify phosphorylated residues.
- RNA and functional protein assays to assess PDGF activity.
Main Results:
- Transformed NIH-3T3 cells exhibited serum- and growth factor-independent proliferation.
- Constitutive tyrosine phosphorylation of cellular proteins, including p145 and p185 (met protooncogene products), was observed.
- Platelet-derived growth factor (PDGF)-beta receptors showed phosphorylation and activation independent of PDGF stimulation.
- Phosphorylation occurred at tyrosine and serine residues on PDGF receptors.
- No evidence of autocrine PDGF production was detected.
- Phospholipase C-gamma (PLC-gamma) was found to associate with PDGF receptors without PDGF stimulation.
Conclusions:
- c-met protooncogene transformation induces a signaling cascade leading to PDGF-independent PDGF-beta receptor phosphorylation and activation.
- This activation occurs independently of autocrine PDGF production.
- The findings suggest a cross-talk mechanism between c-met and PDGF signaling pathways.
- This aberrant signaling may contribute to the transformed phenotype.