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Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor
1Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.
Insights
Phorbol ester (TPA) affects epidermal growth factor (EGF) receptor binding and cell growth. Phosphorylation at Thr-654 by kinase C negatively controls EGF-induced cell division, a process not seen with a mutated EGF receptor.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal Growth Factor (EGF) receptor signaling is crucial for cell growth and proliferation.
- Tumor promoters like phorbol ester (TPA) can alter EGF receptor function.
- TPA-induced phosphorylation of EGF receptor at Thr-654 is implicated in regulating its activity.
Purpose of the Study:
- To investigate the role of Thr-654 phosphorylation in EGF receptor regulation by TPA.
- To determine if Thr-654 phosphorylation mediates the inhibitory effect of TPA on EGF-induced mitogenesis.
- To elucidate the mechanism by which TPA modulates EGF receptor binding affinity and signaling.
Main Methods:
- Site-directed mutagenesis was used to create a Tyr-654 EGF receptor mutant.
- The mutant and wild-type EGF receptors were expressed in NIH 3T3 cells.
- Binding affinity studies and EGF-stimulated DNA synthesis assays were performed in the presence and absence of TPA.
Main Results:
- The Tyr-654 mutant receptor exhibited normal high- and low-affinity binding sites.
- TPA modulated the binding affinity of both wild-type and mutant receptors.
- TPA inhibited EGF-induced mitogenesis in cells with wild-type EGF receptor but not in cells with the Tyr-654 mutant.
Conclusions:
- Phosphorylation of Thr-654 by kinase C acts as a negative control for EGF-induced mitogenesis in NIH 3T3 fibroblasts.
- While other phosphorylation sites may influence ligand binding, Thr-654 phosphorylation is key to TPA's inhibition of EGF-driven cell proliferation.
- This study highlights a specific regulatory mechanism of EGF receptor signaling by TPA.