Mutational analysis of the nucleotide binding site of the epidermal growth factor receptor and v-Src protein-tyrosine kinases

Abstract

Tyrosine kinases differ from serine/threonine kinases in sequences located at the active site where ATP and substrate bind. In the structure of cyclic AMP-dependent protein kinase, the catalytic loop contains the sequence Lys-Pro-Glu where the Lys residue contacts the gamma-phosphate of ATP and the Glu residue contacts a basic residue located in the peptide substrate. In tyrosine kinases, the analogous sequence is Ala-Ala-Arg in the receptor tyrosine kinase subfamily and Arg-Ala-Ala in the Src tyrosine kinase subfamily. To deduce the role of these residues in tyrosine kinase function, site-directed mutations were prepared in the epidermal growth factor receptor (EGFR) and in v-Src and effects on ATP binding and kinase activity were determined. Changing Arg to either Lys or Ala dramatically reduced activity of both tyrosine kinases and this correlated with loss of ATP binding. Changing the orientation of this sequence impaired activity of EGFR to a greater extent than that of v-Src but did not change substrate specificity of the two enzymes. These results support the hypothesis that Arg functions to coordinate the gamma-phosphate of ATP. Analysis of sequence inversions in the catalytic loop indicate that the active site of v-Src exhibits greater flexibility than that of EGFR.