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Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and
A Sorkin1, C Waters, K A Overholser
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Abstract:
We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.
Insights
Multiple autophosphorylations of the epidermal growth factor (EGF) receptor are crucial for its kinase activity and the internalization and processing of the EGF-receptor complex. Disrupting these sites significantly impairs EGF receptor function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The epidermal growth factor (EGF) receptor plays a critical role in cellular signaling pathways.
- Autophosphorylation sites on the EGF receptor are known regulators of its activity.
- Understanding these regulatory mechanisms is key to comprehending cellular responses to EGF.
Purpose of the Study:
- To investigate the role of specific autophosphorylation sites on the EGF receptor.
- To determine the impact of these mutations on receptor kinase activity and ligand-induced endocytosis.
- To elucidate the contribution of EGF receptor autophosphorylation to cellular processing of the EGF-receptor complex.
Main Methods:
- Site-directed mutagenesis was used to create EGF receptor mutants with altered autophosphorylation sites.
- Kinase activity was assessed in vivo using PLC gamma 1 as a specific substrate.
- Internalization, recycling, and degradation of EGF were analyzed using experimental assays and mathematical modeling.
Main Results:
- Single and double mutations of autophosphorylation sites did not significantly inhibit EGF receptor kinase activity.
- A triple mutation of key autophosphorylation sites (Y1173, Y1148, Y1068) reduced EGF-induced PLC gamma 1 phosphorylation by over 50%.
- The triple mutation substantially inhibited EGF-receptor endocytosis, with a 2-fold lower internalization rate and slower degradation of the EGF-receptor complex.
Conclusions:
- Multiple autophosphorylations at the carboxyl terminus of the EGF receptor are essential for robust kinase activation.
- These autophosphorylations are critical for the efficient internalization and intracellular processing of the EGF-receptor complex.
- The findings highlight the complex regulatory role of EGF receptor autophosphorylation in signal transduction and cellular dynamics.