Multiple SH2-Mediated Interactions In v-src-Transformed Cells

Abstract

The Src homology 2 (SH2) domain is a noncatalytic region which is conserved among a number of signaling and transforming proteins, including cytoplasmic protein-tyrosine kinases and Ras GTPase-activating protein (GAP). Genetic and biochemical data indicate that the SH2 domain of the p60^v-src (v-Src) protein-tyrosine kinase is required for full v-src transforming activity and may direct the association of v-Src with specific tyrosine-phosphorylated proteins. To test the ability of the v-Src SH2 domain to mediate protein-protein interactions, v-Src polypeptides were expressed as fusion proteins in Escherichia coli. The bacterial v-Src SH2 domain bound a series of tyrosine-phosphorylated proteins in a lysate of v-src-transformed Rat-2 cells, including prominent species of 130 and 62 kDa (pl30 and p62). The pl30 and p62 tyrosine-phosphorylated proteins that complexed v-Src SH2 in vitro also associated with v-Src in v-src-transformed Rat-2 cells; this in vivo binding was dependent on the v-Src SH2 domain. In addition to binding soluble p62 and pl30, the SH2 domains of v-Src, GAP, and v-Crk directly recognized these phosphotyrosine-containing proteins which had been previously denatured and immobilized on a filter. In addition, the SH2 domains of GAP and v-Crk bound to the GAP-associated protein pl90 immobilized on a nitrocellulose membrane. These results show that SH2 domains bind directly to tyrosine-phosphorylated proteins and that the Src SH2 domain can bind phosphor-ylated targets of the v-Src kinase domain. In v-srotransformed cells, p62 is a prominent tyrosine-phosphorylated SH2-binding protein that is found in independent complexes with v-Src and GAP. The transformation-related assembly of these complexes suggests a network of SH2-mediated interactions involving the SH2 domains of signaling proteins and their tyrosine-phosphorylated ligands, which are important for v-Src transforming activity and regulation of GAP function.