Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase ζ/β by the yeast substrate-trapping system

We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases zeta (PTP zeta/ RPTP beta). 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 PTP zeta (PTP zeta -D1902A) as bait. By using this system, several substrate candidates for PTP zeta were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor l:Cool-associated, tyrosine-phosphorylated l)was examined further. GIT1/Cat-1 bound to PTP zeta -D1902A dependent on the substrate tyrosine phosphorylation, Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTP zeta in vitro. Immunoprecipitation experiments indicated that PTP zeta -D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTP zeta 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 messy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTP zeta, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTP zeta.