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Critical amino acid substitutions in the Src SH3 domain that convert c-Src to be oncogenic
K Miyazaki1, T Senga, S Matsuda
1First Department of Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Abstract:
The Src homology 3 (SH3) domain, originally identified in v-Crk, plays an important role in signal transduction. The comparative study with c-src has revealed that v-src oncogene of Schmidt-Ruppin 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 introduced 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 further activated Src kinase. By use of GST-fusion forms of v-Src SH3 and c-Src 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 those of c-SrcSH3. Taken together, our results suggest that point mutations accumulated in SH3 region can activate, in concert, Src kinase by relaxing the interaction between SH3 and the linker region and subsequently convert Src to be oncogenic.
Insights
Point mutations in the Src homology 3 (SH3) domain activate Src kinase, converting it into an oncogene. These mutations relax interactions within the SH3 region, leading to enhanced kinase activity and cell transformation.
Area of Science:
- Molecular Biology
- Signal Transduction
- Oncogenesis
Background:
- The Src homology 3 (SH3) domain is crucial for signal transduction pathways.
- The v-src oncogene contains mutations in its SH3 domain compared to c-Src.
- Understanding these mutations' impact on Src activity is key to oncogenesis research.
Purpose of the Study:
- To investigate the role of specific point mutations in the v-src SH3 domain.
- To determine how these mutations affect c-Src kinase activity and cell transformation.
- To elucidate the mechanism by which SH3 domain mutations activate Src.
Main Methods:
- Oligonucleotide-directed mutagenesis to introduce single and multiple mutations into c-Src.
- Cell transformation assays to assess oncogenic potential.
- GST-fusion protein binding assays to analyze SH3 domain interactions.
Main Results:
- Single mutations in the SH3 domain did not confer transforming ability to c-Src.
- Double mutations at positions 95 and 117 rendered c-Src oncogenic and kinase-active.
- Further mutation at position 124 enhanced Src kinase activity.
- Mutations suppressed SH3 binding to the c-Src linker region but retained binding to other cellular proteins.
Conclusions:
- Accumulated point mutations in the SH3 domain cooperatively activate Src kinase.
- Activation occurs by relieving the interaction between the SH3 domain and its linker region.
- These mutations convert Src into an oncogenic protein, driving cell transformation.