Synthetic peptide substrates for the membrane tyrosine protein kinase stimulated by epidermal growth factor

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Abstract
The substrate specificity of the epidermal-growth-factor-stimulated tyrosine protein kinase of A431 cell [human epidermoid carcinoma] membranes was studied using a series of synthetic peptide analogs of the sequence around the phosphorylated tyrosine-419 of pp60src. The 9-residue peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Thr-Ala was phosphorylated on tyrosine with an apparent Km of 0.4 mM and a V of 5.7 nmol .cntdot. min-1 .times. mg-1. Synthetic peptide tyrosine phosphorylation was stimulated by epidermal growth factor but not by insulin or relaxin. Extension of the 9-residue peptide to include the basic residues, arginine-412, arginine-422 and lysine-424 led to an increased apparent Km. Subsitution of glutamic-418 by leucine also increased the apparent Km. In the model peptide Ile-Xaa-Xaa-Ala-Ala-Tyr-Thr-Ala a lower apparent Km was obtained when Xaa was glutamic rather than aspartic acid. Poly(aspartic acid) and poly(glutamic acid) had only weak effects on peptide tyrosine phosphorylation. Acidic residues and not basic residues evidently are important specificity determinants for the epidermal-growth-factor-stimulated tyrosine protein kinase. [Tyrosine phosphorylation is now recognized as an important regulatory mechanism in the transformation of host cells by a group of RNA tumor viruses.].