Phosphorylation of the ras GTPase-activating protein (GAP) by the p93c-fes protein-tyrosine kinase in vitro and formation of GAP-fes complexes via an SH2 domain-dependent mechanism.
The protein-tyrosine kinase encoded by the human c-fes protooncogene (p93c-fes) plays a direct role in myeloid differentiation, but downstream substrates for this kinase have not been identified. Here we report that the human ras GTPase-activating protein (GAP) is a substrate for p93c-fes in vitro. Purified, recombinant GAP was readily phosphorylated on tyrosine residues by bacterially-expressed p93c-fes. Two-dimensional tryptic mapping revealed a single GAP phosphopeptide, consistent with specific phosphorylation of GAP by p93c-fes on one or several closely-spaced tyrosine residues. Autophosphorylated p93c-fes also formed a stable complex with GAP. Complex formation is likely to involve the src homology 2 (SH2) domains of GAP and autophosphorylated tyrosine residues of p93c-fes, as deletion of the fes SH2 domain did not abolish complex formation. Furthermore, immobilized recombinant fusion proteins containing either or both of the GAP SH2 domains were able to precipitate p93c-fes with an affinity equal to that observed with a monoclonal antibody against the recombinant fes protein. Fusion proteins containing the GAP N-terminal, C-terminal catalytic, or SH3 domains did not bind to p93c-fes. Interaction of the GAP SH2 domains with p93c-fes is phosphorylation-dependent, as the recombinant SH2 domain proteins were unable to bind to a kinase-defective c-fes mutant and showed reduced binding of a mutant in which one of the two tyrosine autophosphorylation sites was replaced with phenylalanine. Stimulation of c-fes autophosphorylation in vivo may induce interaction with GAP, resulting in altered p21ras function.
Hjermstad
S J
SJ
Eppley Institute for Research in Cancer, Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6805.
Briggs
S D
SD
Smithgall
T E
TE
D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051578:Proto-Oncogene Proteins c-fes; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051578:Proto-Oncogene Proteins c-fes; D014357:Trypsin