A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain-containing protein, and Src substrate

We describe a method for identifying tyrosine kinase substrates using anti‐phosphotyrosine antibodies to screen tyrosine‐phosphorylated cDNA expression libraries. Several potential Src substrates were identified including Fish, which has five SH3 domains and a recently discovered phox homology (PX) domain. Fish is tyrosine‐phosphorylated in Src‐transformed fibroblasts (suggesting that it is a target of Src in vivo ) and in normal cells following treatment with several growth factors. Treatment of cells with cytochalasin D also resulted in rapid tyrosine phosphorylation of Fish, concomitant with activation of Src. These data suggest that Fish is involved in signalling by tyrosine kinases, and imply a specialized role in the actin cytoskeleton.