Figure 1.

Schematic representations of the yeast substrate-trapping system and GIT1/Cat-1 protein isolated by this system. (A) In the presence of 1 mM methionine when v-src is not induced, only the standard two-hybrid bindings occur (not shown). In the absence of methionine, prey proteins (substrates for PTPζ) could be phosphorylated by the induced v-src, and trapped by the bait consisting of the whole intracellular domain with an Asp-1902 → Ala mutation of PTPζ (PTPζICR-D1902A). This complex formation leads to activation of transcription of the reporter genes, HIS3 and LacZ. We performed screenings in two steps and selected the colonies that showed an increase in blue color development on expression of v-src (see Materials and Methods). (B) GIT1/Cat-1 contains a zinc-finger region, three ankyrin-repeat regions, a potential Src-homology 3 (SH3) binding site and a G protein-coupled receptor kinase (GRK) binding site. The region encoded by clone 10–1 is shown by the solid line underneath. (C) GIT1/Cat-1 (amino acid residues 251–555; ●) and RCM lysozyme (○) were tyrosine phosphorylated by p60^c-src and then incubated with GST-PTPζICR for various periods. GST-PTPζICR efficiently dephosphorylated GIT1/Cat-1.