Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor.
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 kinetic analysis comparing peptide phosphorylation using unphosphorylated and prephosphorylated enzyme indicated that the self-phosphorylation site can act as a competitive inhibitor (alternate substrate) versus peptide substrates. These results suggest that self-phosphorylation of the epidermal growth factor receptor removes a competitive constraint so that exogenous substrates can be more readily phosphorylated.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013379:Substrate Specificity
D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases