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Signal transduction by the epidermal growth factor receptor after functional desensitization of the receptor tyrosine
1Howard Hughes Medical Institute, Program in Molecular Medicine, Worcester, MA.
Abstract:
Previous work identified a protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669. An assay for this protein kinase activity present in homogenates prepared from A431 human epidermoid carcinoma cells was developed using a synthetic peptide substrate corresponding to residues 663-681 of the EGF receptor (peptide T669). Here we report that a greater initial rate of T669 phosphorylation was observed in experiments using homogenates prepared from EGF- or phorbol ester-treated cells compared with control cells. EGF and 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a 6-fold and a 2-fold increase in protein kinase activity, respectively. A kinetic analysis of T669 phosphorylation demonstrated that the increase in protein kinase activity observed was accounted for by an increase in Vmax. To examine the interaction between protein kinase C and signal transduction by the EGF receptor, the effect of pretreatment of cells with PMA on the subsequent response to EGF was investigated. Treatment of cells with PMA caused greater than 90% inhibition of the EGF-stimulated tyrosine phosphorylation of the EGF receptor and abolished the EGF-stimulated formation of soluble inositol phosphates. In contrast, PMA was not observed to inhibit the stimulation of T669 protein kinase activity caused by EGF. Thus, the apparent functional desensitization of the EGF receptor caused by PMA does not inhibit signal transduction mediated by the T669 protein kinase. Our results demonstrate that EGF receptor transmodulation alters the pattern of signal-transduction pathways that are utilized by the EGF receptor.
Insights
Epidermal growth factor (EGF) receptor signaling is altered by phorbol ester (PMA) treatment. PMA treatment desensitizes EGF receptor tyrosine phosphorylation but not T669 protein kinase activity, indicating altered signal transduction pathways.
Area of Science:
- Cellular signaling
- Molecular biology
- Oncology
Background:
- A protein kinase phosphorylates the epidermal growth factor (EGF) receptor at Thr669.
- An assay was developed to measure this protein kinase activity using a synthetic peptide substrate (peptide T669).
Purpose of the Study:
- To investigate the effect of EGF and phorbol ester (PMA) on T669 protein kinase activity.
- To examine the interaction between protein kinase C and EGF receptor signal transduction.
- To determine if PMA-induced EGF receptor desensitization affects T669 protein kinase activity.
Main Methods:
- Utilized homogenates from A431 human epidermoid carcinoma cells.
- Measured T669 phosphorylation rates using a synthetic peptide substrate.
- Performed kinetic analysis to determine changes in Vmax.
- Investigated the effect of PMA pretreatment on EGF-stimulated responses.
Main Results:
- EGF and PMA treatment increased T669 protein kinase activity by 6-fold and 2-fold, respectively, due to increased Vmax.
- PMA pretreatment inhibited EGF-stimulated EGF receptor tyrosine phosphorylation and inositol phosphate formation by over 90%.
- PMA did not inhibit EGF-stimulated T669 protein kinase activity.
Conclusions:
- PMA-induced functional desensitization of the EGF receptor does not inhibit signal transduction mediated by the T669 protein kinase.
- EGF receptor transmodulation alters the pattern of utilized signal-transduction pathways.