Critical amino acid substitutions in the Src SH3 domain that convert c-Srcto be oncogenic

The Src homology 3 (SH3) domain, originally identified in v-Crk, plays an i mportant role in signal transduction. The comparative study with c-src has revealed that v-src oncogene of Schmidt-Buppin strain of Rous sarcoma virus has three point mutations in its SH3 domain and one in the upstream of SH3 . To assess the role of these mutations, each of the single mutations was i ntroduced into c-Src by oligonucleotide-directed mutagenesis and its effect on cell transformation was examined. While variant Src proteins that carry each one of single mutations could not transform cells, double mutation at positions; 95 and 117 converted c-Src to be oncogenic and active in kinase . An additional mutation at position 124 together with one at 95 and 117 fu rther activated Src kinase. By use of GST-fusion forms of v-Src SH3 and c-S rc SH3, we found that these mutations in SH3 suppressed the binding of SH3 with c-Src protein, possibly with a linker region, while v-SrcSH3 retained the ability to bind a subset of cellular protein to the level similar to th ose of c-SrcSH3. Taken together, our results suggest that point mutations a ccumulated in SH3 region can activate, in concert, Src kinase by relaxing t he interaction between SH3 and the linker region and subsequently convert S rc to be oncogenic. (C) 1999 Academic Press.