REGULATION OF THE HUMAN C-FES PROTEIN-TYROSINE KINASE (P93C-FES) BY ITS SRC HOMOLOGY-2 DOMAIN AND MAJOR AUTOPHOSPHORYLATION SITE (TYR-713)

The c-fes proto-oncogene product is expressed predominantly in hematop oietic cells of the myeloid lineage and has been implicated in the reg ulation of myeloid differentiation. The c-fes locus encodes a 93-kDa p rotein tyrosine kinase (p93c-fes) that possesses several structural fe atures characteristic of the cytoplasmic class of protein tyrosine kin ases, including a consensus sequence for autophosphorylation surroundi ng Tyr-713 and a src homology 2 (SH2) domain. To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using th e polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mu tants were expressed in Escherichia coli and assayed for changes in pr otein tyrosine kinase activity using an immune complex kinase assay. B oth mutations produced a marked decrease in the rate and extent of aut ophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosp horylated kinase domain, the SH2 domain was expressed as a fusion prot ein with glutathione S-transferase and immobilized on glutathione-agar ose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was c ompletely dependent upon autophosphorylation, as a kinase-defective mu tant of p93c-fes was not precipitated by the SH2 domain. High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions betwee n various members of the cytoplasmic PTK family. These results indicat e that the c-fes SH2 domain and consensus autophosphorylation site (Ty r-713) play major roles in the positive regulation of p93c-fes tyrosin e kinase activity, possibly through intramolecular interaction.