Src-GFP mutants
| Src mutant . | Description . |
|---|---|
| Src-WT-GFP | WT chicken c-Src |
| Src-K295M-GFP | Kinase-dead mutant |
| Src-Y527F-GFP | Y527F mutation, open conformation, constitutively active |
| Src-Y527F/K295M-GFP | Double mutant, open conformation, kinase dead |
| Src-Y527F/R175A-GFP | Double mutant, open conformation, inactivated SH2 domain |
| Src-Y527F/W118A-GFP | Double mutant, open conformation, inactivated SH3 domain |
| Src-Y527F/K295M/ R175A-GFP | Triple mutant, open conformation, kinase dead, inactivated SH2 |
| Src-251-GFP | Truncated N-terminal half, containing SH3/SH2 domains, no kinase region |
| Src mutant . | Description . |
|---|---|
| Src-WT-GFP | WT chicken c-Src |
| Src-K295M-GFP | Kinase-dead mutant |
| Src-Y527F-GFP | Y527F mutation, open conformation, constitutively active |
| Src-Y527F/K295M-GFP | Double mutant, open conformation, kinase dead |
| Src-Y527F/R175A-GFP | Double mutant, open conformation, inactivated SH2 domain |
| Src-Y527F/W118A-GFP | Double mutant, open conformation, inactivated SH3 domain |
| Src-Y527F/K295M/ R175A-GFP | Triple mutant, open conformation, kinase dead, inactivated SH2 |
| Src-251-GFP | Truncated N-terminal half, containing SH3/SH2 domains, no kinase region |
Table shows Src-GFP constructs used in the studies. Chicken c-Src-WT-GFP was shown to undergo normal regulation in cells; Src-Y527F-GFP and Src-251-GFP were demonstrated to be active and possess correct cellular localization (Timpson et al., 2001; Sandilands et al., 2004). Additional mutants were constructed by site-directed mutagenesis using Src-WT-GFP or Src-Y527F-GFP as templates; the Y527F/K295M double mutant served as a template for the triple mutant. All constructs are in pEGFP-N1, except Src-251-GFP (pBabePuro). The K295M mutation inactivates Src kinase activity (Kamps et al., 1984; Kamps and Sefton, 1986), the R175A mutation inactivates pTyr binding by the SH2 domain (Waksman et al., 1993), and the W118A mutation inactivates SH3 domain binding (Musacchio et al., 1994; Erpel et al., 1995).