Receptor tyrosine phosphatase sigma (RPTPσ) regulates, p250GAP, a novel substrate that attenuates Rac signaling.

Cumulative evidence supports an important role for RPTPsigma in the development of the nervous system and nerve regeneration. However, the signaling mechanisms regulated by RPTPsigma remain largely unknown and the identification of RPTPsigma substrate(s) and binding partners is essential to understanding its mechanisms of action. We employed a modified yeast-two-hybrid approach, the yeast substrate-trapping system, to identify new substrates and interacting partners of RPTPsigma. The binding proteins RPTPdelta, Liprinalpha4, p130Cas and Trio were found to interact with RPTPsigma in the yeast system independently of tyrosine phosphorylation. Importantly, using the trapping mutant of RPTPsigma we identified p250GAP as a novel substrate and RPTPsigma displayed its phosphatase specificity toward p250GAP in vitro. In the mammalian expression system, the trapping mutant of RPTPsigma recognized p250GAP as its physiological substrate in the presence of active Fyn, suggesting that the interaction of the two proteins is primarily dependent on tyrosine phosphorylation. Furthermore, p250GAP activity increased in the presence of RPTPsigma leading to attenuated Rac activity. Overexpression of p250GAP and RPTPsigma inhibited axonal outgrowth in differentiated PC12 cells. Cumulative evidence implicates that RPTPsigma modulates the actin cytoskeleton by regulating Rac GTPase activity through p250GAP. Taken together, our results demonstrate for the first time that RPTPsigma modulates Rac dependent activity through regulating a novel substrate, p250GAP.