Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase zeta /beta by the yeast substrate-trapping system.
We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases zeta (PTPzeta/RPTPbeta). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPzeta (PTPzeta-D1902A) as bait. By using this system, several substrate candidates for PTPzeta were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor 1/Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1/Cat-1 bound to PTPzeta-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTPzeta in vitro. Immunoprecipitation experiments indicated that PTPzeta-D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTPzeta and GIT1/Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTPzeta, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTPzeta.