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A role for activated p21 ras in inhibition/regulation of platelet-derived growth factor (PDGF) type-beta receptor
M Zubiaur1, L W Forman, L L Stice
1Cancer Research Center, Boston University School of Medicine, MA 02118, USA.
Abstract:
Ligand-stimulated Platelet-Derived Growth Factor (PDGF) type-beta receptor autophosphorylation, and tyrosine phosphorylation of receptor-associated signalling proteins, is blocked in cells expressing activated Ras genes. A factor present in membrane fractions of v-ras-expressing fibroblasts (Kbalb cells) dominantly inhibits the autophosphorylation of the PDGF type-beta receptor. Purification of this factor, via ion exchange, reveals that the inhibitor can be physically separated from the PDGF type-beta receptor, with reconstitution of PDGF type-beta receptor kinase activity in response to ligand binding. The inhibitor exhibited specificity for the PDGF type-beta receptor, and consistently co-purified with activated p21 ras, with Syp/PTP-2, and with Grb2. Neutralization of the p21 ras protein from the Kbalb cell membranes by p21 ras-specific monoclonal antibodies, however, completely removed the inhibition of PDGF type-beta receptor, rendering the PDGF type-beta receptor molecule capable of autophosphorylation in response to ligand. These results indicate that activated p21 ras either interacts directly with the PDGF type-beta receptor to inhibit autokinase activity, or complexes with different molecules such as Syp and/or Grb2 at the cell membrane to act on another effector which then inhibits PDGF type-beta receptor function.
Insights
Activated Ras genes block Platelet-Derived Growth Factor (PDGF) receptor signaling. A membrane factor co-purifying with Ras protein inhibits PDGF receptor autophosphorylation, suggesting Ras directly or indirectly controls receptor activity.
Area of Science:
- Cellular signaling pathways
- Receptor tyrosine kinases
- Ras GTPases
Background:
- Ligand-stimulated Platelet-Derived Growth Factor (PDGF) type-beta receptor autophosphorylation is crucial for cellular responses.
- Activated Ras genes are known to interfere with PDGF receptor signaling pathways.
Purpose of the Study:
- To investigate the mechanism by which activated Ras genes inhibit PDGF type-beta receptor autophosphorylation.
- To identify the inhibitory factor present in Ras-expressing cells.
Main Methods:
- Cell membrane fractionation and protein purification using ion exchange chromatography.
- Assays for PDGF type-beta receptor kinase activity.
- Western blotting and immunoprecipitation with specific antibodies, including p21 ras-specific monoclonal antibodies.
Main Results:
- A dominant inhibitor of PDGF type-beta receptor autophosphorylation was identified in membrane fractions of v-ras-expressing fibroblasts.
- The inhibitor co-purified with activated p21 ras, Syp/PTP-2, and Grb2.
- Neutralization of p21 ras protein abolished the inhibitory effect, restoring PDGF type-beta receptor autophosphorylation.
Conclusions:
- Activated p21 ras plays a critical role in inhibiting PDGF type-beta receptor autokinase activity.
- Activated Ras may directly interact with the PDGF type-beta receptor or form complexes with other signaling molecules (Syp, Grb2) to mediate inhibition.