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 hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation. The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding 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 the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay. Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant 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 between various members of the cytoplasmic PTK family. These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.
Hjermstad
S J
SJ
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198-6805.
Peters
K L
KL
Briggs
S D
SD
Glazer
R I
RI
Smithgall
T E
TE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D015681:Fusion Proteins, gag-onc / Q000737:chemistry; D017353:Gene Deletion; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051578:Proto-Oncogene Proteins c-fes; D014443:Tyrosine / Q000031:analogs & derivatives
D015681:Fusion Proteins, gag-onc; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D051578:Proto-Oncogene Proteins c-fes; C068415:v-fps oncogene protein, Fujinami sarcoma virus