Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.
Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.
Garcia
P
P
Department of Physiology and Biophysics, School of Medicine State University of New York, Stony Brook 11794.
Shoelson
S E
SE
Drew
J S
JS
Miller
W T
WT
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D001665:Binding Sites; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D010748:Phosphopeptides; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)