Analysis of substrate recognition determinants in a synthetic peptide containing the Tyr 1173 autophosphorylation site of the epidermal growth factor receptor.
The epidermal growth factor receptor contains five autophosphorylation sites in its C-terminal region. Synthetic peptides based on the major autophosphorylation site at Tyr 1173 were tested as substrates of the intracellular domain of the epidermal growth factor receptor. A peptide containing acidic residues N-terminal to the substrate Tyr as well as the Tyr-Met-Xaa-Met motif of the insulin receptor substrate 1 had a Km value of 15 microM, the lowest value for a synthetic peptide reported to date. Another important residue contributing to substrate binding is the Tyr itself, or more specifically, the hydroxyl group of the Tyr. Substituting Phe for Tyr results in a peptide that is ineffective as an inhibitor of kinase phosphorylation. However, substitution of a Ser residue does not restore a functional substrate, indicating specificity for the Tyr hydroxyl. Secondary structure algorithms predicted that the peptide substrate based on the native sequence at Tyr 1173 would have a propensity to adopt a helical conformation in solution. Circular dichroism spectroscopy confirmed this prediction. The secondary structure of the peptide substrate is significant in its consistency with the idea that secondary structure is an important determinant in substrate recognition by protein tyrosine kinases.
Klingbeil
C K
CK
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla 92093-0650.
Gill
G N
GN
Cadena
D L
DL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D066246:ErbB Receptors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D010446:Peptide Fragments; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases