Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor.

作者：Paul J. Bertics, Gordon N. Gill · 发表于：Journal of Biological Chemistry · 年份：1985 · DOI：10.1016/s0021-9258(17)38618-0 · 被引用次数：232 · 研究领域：Glycosylation and Glycoproteins Research、Monoclonal and Polyclonal Antibodies Research、Chemical Synthesis and Analysis

The effect of self-phosphorylation on the protein-tyrosine kinase activity of the epidermal growth factor receptor has been investigated using immunoaffinity-purified protein. Enzyme was first incubated for various times with excess ATP to phosphorylate it to differing extents; the ability of the enzyme to phosphorylate exogenous peptide substrates was then measured as a function of its self-phosphorylation state. Increasing self-phosphorylation to 1.3-1.8 mol of phosphate mol-1 of epidermal growth factor receptor enhanced protein-tyrosine kinase activity 2-3-fold. Comparison of the kinetics of protein-tyrosine kinase activity at different ATP concentrations revealed significant differences between unphosphorylated and phosphorylated enzyme. At low levels of ATP, a double reciprocal plot of the protein-tyrosine kinase activity of the unphosphorylated enzyme was hyperbolic, suggesting that ATP may act as an activator of the enzyme. At higher ATP concentrations, where greater levels of self-phosphorylation occurred during the reaction, the kinetics appeared linear and similar to those of the phosphorylated enzyme. Dose-response studies using three different peptide substrates (angiotensin II, gastrin, and a synthetic peptide corresponding to the self-phosphorylation site in p60v-src) showed that exogenous substrates inhibit receptor self-phosphorylation. In each case, half-maximal inhibition was observed at a peptide concentration approximately equal to the substrate's Km. A ki...