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PDGF induction of tyrosine phosphorylation of GTPase activating protein
C J Molloy1, D P Bottaro, T P Fleming
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system.
Insights
Platelet-derived growth factor (PDGF) rapidly triggers tyrosine phosphorylation of GTPase-activating protein (GAP) in fibroblasts. This links PDGF signaling to the Ras protein pathway, crucial for cell growth and cancer.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Signaling
Background:
- Growth factor signaling pathways regulate normal cell growth and are often dysregulated in cancer.
- Ras proteins (p21ras) are key mediators in signal transduction pathways activated by growth factors like PDGF.
- GTPase-activating protein (GAP) interacts with Ras proteins to regulate their activity.
Purpose of the Study:
- To investigate the direct biochemical link between Platelet-Derived Growth Factor (PDGF) receptor activation and the Ras-GTPase-activating protein (Ras-GAP) signaling system.
- To determine if PDGF induces modifications in Ras-GAP relevant to mitogenic signaling.
Main Methods:
- Utilized quiescent fibroblasts to study cellular responses to growth factors.
- Administered specific growth factors: Platelet-Derived Growth Factor (PDGF) AA and BB homodimers, insulin, and basic fibroblast growth factor (bFGF).
- Assessed tyrosine phosphorylation of GAP and its cellular localization (cytosol vs. membrane-associated).
Main Results:
- PDGF (both AA and BB homodimers) rapidly induced tyrosine phosphorylation of GAP in intact quiescent fibroblasts.
- Insulin and basic fibroblast growth factor (bFGF) were ineffective in inducing GAP tyrosine phosphorylation under the same conditions.
- While GAP is primarily cytosolic, the tyrosine-phosphorylated form was found associated with the cell membrane, the site of p21ras activity.
Conclusions:
- Established a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signaling pathway.
- Demonstrated that PDGF specifically modulates Ras-GAP activity through tyrosine phosphorylation, positioning it at the cell membrane.
- Provided evidence for PDGF's role in activating the Ras signaling cascade, relevant to cell proliferation and cancer biology.