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

Abstract

The substrate specificity of the epidermal-growth-factor-stimulated tyrosine protein kinase of A431 cell membranes has been studied using a series of synthetic peptide analogs of the sequence around the phosphorylated tyrosine-41 9 of pp60"'". The nine-residue peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Thr-Ala was phosphorylated on tyrosine with an apparent K, of 0.4mM and a V' of 5.7nmol xmin-' xmg-'. Synthetic peptide tyrosine phosphorylation was stimulated by epidermal growth factor but not by insulin or relaxin. Extension of the nineresidue peptide to include the basic residues, arginine-412, arginine-422 and lysine-423 led to an increased apparent K,. Substitution of glutamic-418 by leucine also increased the apparent K,. In the model peptide Ile-Xaa-Xaa-AlaAla-Tyr-Thr-Ala a lower apparent K, was obtained when Xaa was glutamic rather than aspartic acid. Poly(aspartic ' acid) and poly(g1utamic acid) had only weak effects on peptide tyrosine phosphorylation. The results support the concept that acidic residues and not basic residues are important specificity determinants for the epidermal-growthfactor-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 [I].Furthermore, tyrosine-specific protein kinase activity has been associated with the actions of several hormones and growth factors including insulin [2], epidermal growth factor (EGF) [3] and platelet-derived growth factor [4]. A number of cellular substrates for the viral tyrosine protein kinases have been identified (see [5] for review). Since the tyrosine protein kinases appear to be an important class of regulatory enzymes the molecular basis of their protein substrate specificity is of considerable interest. From the results of sequence studies [6 -91 it has been proposed that acidic residues on the NH2-terminal side of the phosphorylation site are important specificity determinants for the viral tyrosine protein kinase. The presence of more distant basic residues in this region has also been noted [7]. Recently the substrate specificity of tyrosine protein kinase has been examined using synthetic peptides in several laboratories [I0 -121. Hunter [lo] reported that the Y73 tyrosine protein kinase phosphorylated synthetic peptides corresponding to the region around tyrosine-419 in pp60"" [8]. In this peptide glutamic-415 had an influence on the kinetics of peptide phosphorylation. Similar observations have been made with the EGF-stimulated tyrosine protein kinase of A431 cell membranes where Pike et al. [ l l ] found that both aspartic-416 and glutamic-415 had modest effects on the kinetics of peptide phosphorylation. Further support for the concept that acidic residues may be important specificity determinants for tyrosine protein kinases comes from studies in which it was found that certain peptide hormones containing acidic residues and tyrosine were also substrates for these enzymes [I 2,131.The purpose of the present study wa...

Computational delineation of tyrosyl-substrate recognition and catalytic landscapes by the epidermal growth factor receptor tyrosine kinase domain

“…Moreover, it has been suggested that protein tyrosine kinase (including EGFR) select substrates with glutamic acid at the P−1 position and large hydrophobic amino acids at the P+1 position [22–25, 58, 59]. Our predicted peptide bound ternary complex suggests that a highly conserved region, corresponding to Val852-Pro853-Ile854-Lys855-Trp856 in EGFR TKD is essential for substrate binding: Lys855 forms a salt bridge with the highly preferred glutamic acid residue at the P−1 site, and the hydrophobic pocket formed by Val852, Pro853, Ile854 and Trp856 serves to anchor a large hydrophobic residue at P+1 position.…”

Structural Determinants of Substrate Selection by the Human Insulin‐Receptor Protein‐Tyrosine Kinase

“…Studies of peptides having amino acid sequences based on sites of PTK autophosphorylation and sites of phosphorylation in protein substrates of PTKs (e.g. House et al, 1984;Donella-Deana et al, 1993) have shown that an acidic residue immediately preceding the target tyrosine (position Y-1) may enhance the phosphorylation kinetics of the latter. It has also been suggested, in the case of potential tyrosine phosphorylation sites based on the IRPTK substrate IRS-1, that YMXM sequences form a recognition motif for IRPTK-catalysed phosphorylation (Myers and White, 1993).…”

Amino‐terminal sequence determinants for substrate recognition by platelet‐derived growth factor receptor tyrosine kinase

“…Synthetic peptides have proved to be powerful tools in studying the specificity of various protein kinases. Synthetic peptides based on known phosphorylation sites of natural substrates have been used to study the specificity of protein kinases such as cAMP-dependent and cGMP-dependent protein kinases [1][2][3], the Src family of tyrosine kinases [4,5] and receptor tyrosine kinases [6,7]. Kinetic analyses have been performed for various receptor protein tyrosine kinases using synthetic peptide substrates.…”

“…Kinetic analyses have been performed for various receptor protein tyrosine kinases using synthetic peptide substrates. Epidermal growth factor receptor (EGFR) tyrosine kinase has been shown to phosphorylate substrates such as Ile-Glu-Glu-Ala-Tyr-Leu-Gly, and peptides based on phosphorylation sites of insulin ]]-chain [6], p21 r~, and gastrin [8]. The platelet-derived growth factor receptor (PDGFR) is less extensively characterized.…”