Regulation Of TGF-? Signaling By Protein Tyrosine Phosphatase PTPN2

Transforming Growth Factor-?(TGF-?)plays important roles in cells,regulating multiple cellular activities such as proliferation,differentiation and apoptosis.The canonical TGF-? pathway includes Type ? and Type ? receptors,R-Smads and Co-Smad.Among the Smad proteins,Co-Smad complexes with phosphorylated R-Smads,which are then translocated to the nucleus to regulate the expression of downstream genes.Therefore,Co-Smad,i.e.Smad4 in mammals,represents an important checkpoint in TGF-? signaling.Phosphatase refers to a group of enzymes that can remove phosphate groups from substrates by hydrolysis.The reversible phosphorylation and dephosphorylation are of great importance in regulating cellular signaling.The human genome encodes at least 125 protein tyrosine phosphatases.These phosphatases maintain the homeostasis of the cell through dephosphorylating specific substrates.Our lab previously found that Smad4 is tyrosine phosphorylated by NPM-ALK and this inhibits TGF-? signaling.In this project we screened a library of human protein tyrosine phosphatases(PTP)to identify phosphatases that can specifically dephosphorylate Smad4.We found 21 tyrosine phosphatases for Smad4.In further studies we performed various experiments to test the function of these PTPs in TGF-P signaling.Here we focused on a significant candidate named PTPN2.We found that PTPN2,which belongs to the classic non-receptor typed PTP family,can dephosphorylate Smad4 and NPM-ALK both in vivo and in vitro.Results from reporter assays revealed that PTPN2 can reverse the suppression of response to TGF-P by constitutively active tyrosine kinase such as NPM-ALK,and this effect is dependent on its phosphatese activity.Results of Co-immunoprecipitation and GST pull-down assays determined that PTPN2 directly binds to NPM-ALK.High expression of PTPN2 coincided with high NPM-ALK level in lymphoma cells.Although PTPN2 exhibits auto-inhibition,a small molecule,Spermidine(SPD),can activate PTPN2.Treatment of SPD significantly up-regulated the activity of PTPN2 and decreased the phosphorylation level of Smad4 and NPM-ALK.Treatment of SPD also up-regulated TGF-? triggered responses.In summary,we screened a human PTP library and identified a few PTPs that can dephosphorylate Smad4,including PTPN2.PTPN2 promotes TGF-? signaling through dephosphorylating Smad4 and NPM-ALK in lymphoma cells.Overexpressing or activating PTPN2 reversed the inhibition of TGF-? signaling by NPM-ALK.Because tyrosine phosphorylation of Smad4 by NPM-ALK promotes tumorigenesis partly through the suppression of TGF-? signaling,PTPN2 is a potential therapeutic target for restoring TGF-?-dependent tumor suppression.Therefore,our study is of both scientific and clinical implication.