We have established and used a method to rapidly isolate tyrosine kinase substrates. The method entails inserting mammalian cDNA libraries into phage vectors. Protein production is induced, then plaque proteins are transferred to nitrocellulose and phosphorylated by the kinase of interest. Proof of principle for this technique was established by the isolation of a number of known Src substrates. We also implicated other known proteins as substrates for Src by this approach, and isolated a number of novel genes. Several of these are indeed Src substrates in mammalian cells. We have characterized further one of these novel substrates, Fish, which is a multi-domain adaptor protein.
- phosphatidylinositol 3-phosphate
- Phox homology (PX) domain
- Src homology 3 (SH3) domain
Signalling the Future, a Biochemical Society-sponsored meeting held at University of Liverpool, Liverpool, 4–6 September 2002
Abbreviations used: ADAM, a disintegrin and metalloprotease; EGF, extracellular growth factor; PI3K, phosphoinositide 3-kinase; PX, Phox homology; SH3, Src homology 3.
- Copyright 2003 Biochemical Society