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Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor
Abstract:
4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.
Insights
4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) affects epidermal growth factor (EGF) receptor binding and phosphorylation. PMA decreases EGF receptor binding at low concentrations and increases its phosphorylation, suggesting a mechanism for PMA
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor (EGF) receptor plays a crucial role in cell growth and proliferation.
- Tumor promoters like 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) can modulate cellular responses.
- The interaction between PMA and the EGF receptor is not fully understood.
Purpose of the Study:
- To investigate the effect of PMA on EGF receptor binding and phosphorylation.
- To elucidate the mechanism by which PMA influences cellular responses to EGF.
Main Methods:
- A431 human epidermoid carcinoma cells were used.
- 125I-labeled EGF binding assays were performed.
- Affinity labeling with disuccinimidyl suberate was employed.
- Receptor phosphorylation was assessed using [32P]phosphate labeling and EGF-Sepharose purification.
- Phosphoamino acid analysis was conducted.
Main Results:
- PMA markedly inhibited 125I-EGF binding at low concentrations (<10(-9) M) but not at high concentrations (>10(-8) M).
- PMA decreased affinity labeling of the EGF receptor at low 125I-EGF concentrations.
- PMA treatment increased EGF receptor phosphorylation by 38%, primarily on serine residues.
- The observed effects were dose-dependent and specific to tumor-promoting phorbol diesters.
Conclusions:
- The EGF receptor is a direct target for PMA.
- PMA-induced phosphorylation of the EGF receptor contributes to its mechanism of action.
- This phosphorylation may mediate PMA's effects on cellular responses to EGF.