Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.
GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.
Park
S
S
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109.
Liu
X
X
Pawson
T
T
Jove
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002872:Chromosome Deletion; D020690:GTPase-Activating Proteins; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014357:Trypsin; D014443:Tyrosine; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010748:Phosphopeptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D014357:Trypsin