SEQUENCE SPECIFICITY IN THE RECOGNITION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR BY THE ABL SRC-HOMOLOGY-2 DOMAIN

The transforming activity of the abl gene product requires a functiona l src homology 2 (SH2) domain. An assay was developed to evaluate this function by examining binding of a bacterially-expressed abl SH2 doma in to the activated EGF receptor, used as a surrogate tyrosine phospho rylated protein. The sequence specificity of this interaction has been explored with a series of point mutants of EGF receptor. Analysis of equilibrium binding reveals that substitution of Tyr(1086) for Phe in the EGF receptor produced a 10-fold reduced affinity for abl SH2 domai n binding as compared to the wildtype receptor. Moreover, a phosphoryl ated peptide modeled on the sequences surrounding Tyr(1086) specifical ly inhibits abl SH2 binding, with an IC50 of approximately 10 mu M. Ev aluation of a series of additional peptides, modeled on the Tyr(1086) sequence, revealed that the carboxy terminal residues directly next to the phosphotyrosine were particularly critical to this binding. Molec ular modeling studies of the pTyr(1086) peptide revealed the potential hydrophobic, ionic and hydrogen bonding interactions involved in the functions of the abl SH2 domain.