Nucleotide Binding by the Epidermal Growth Factor Receptor Protein-tyrosine Kinase

Abstract

The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2´(3´)-O-(2,4,6-trinitrophenyl)adenosine 5´-triphosphate.Binding affinities for ATP and Mn·ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn·ATP binding, although the binding affinity for the Mn·ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction.