| Transforming Growth Factor beta(TGF-β)is a multifunctional cytokine that regulates cell proliferation,differentiation,and apoptosis in several cells and tissues.During the early stage of tumorigenesis,TGF-βfunctions as a tumor suppressor by inhibiting cell proliferation and inducing apoptosis of cancer cells.In the contrast,dysregulation of the TGF-βsignaling pathway is an important pathological mechanism underlying tumor progression.Researches in recent years reveal that deletion and variation of SMAD4 gene,are relatively rare in most cancer types,compared to TGF-βreceptors and TGF-βinduced transcription factors.In 2019,Zhang et al.reported a new mechanism that anaplastic lymphoma kinase(ALK)phosphorylates Smad4 at Tyr 95,which compromises the DNA-binding ability of Smad4 and blocks the TGF-βsignaling pathway in cancer cells~1.The novel mechanism,that ALK regulates the TGF-βsignaling pathway by post-translational modification of Smad4 provides a new direction for exploring tumor pathogenesis.Protein phosphorylation and dephosphorylation are two widespread physiological processes.The reversible dynamic processes are the biological basis of many cascade reactions and regulatory functions.Therefore,it is of great clinical significance to screen and identify tyrosine phosphatase in cells that can dephosphorylate Smad4,thereby reconstructing the TGF-βsignaling pathway in cells.In our lab’s previous work,we discovered PTPN2,a classical non-receptor protein phosphatase that dephosphorylates Smad4.In this research,we investigated the regulation of the TGF-βsignaling pathway by PTPN2 in ALK-positive cells in depth.We demonstrated that Smad4 is a novel substrate for the tyrosine phosphatase PTPN2.In vivo and in vitro phosphatase assays showed that tyrosine phosphatase PTPN2 dephosphorylates Smad4on tyr95,but not its inactive mutant PTPN2-D182A.Co-immunoprecipitation assays were performed in HEK293T and SUDHL-1 cells.To characterize the direct interaction between Smad4 and PTPN2,in vitro GST-pulldown was also conducted.This series of experiments have shown that Smad4 interacts with PTPN2 and that both Smad4phosphorylation and mimic phosphorylation enhance the interaction.Meanwhile,DNA-pulldown experiments showed that PTPN2 rescues the DNA binding ability of Smad4 by dephosphorylating Smad4.We further explored the function of PTPN2 in ALK-positive cancer cells.Data showed that both activation and overexpression of PTPN2 restored ALK-suppressed TGF-βsignaling pathway responses in different cell lines.By promoting TGF-β-mediated apoptosis and restoring TGF-β-induced cell proliferation arrest,PTPN2 reestablishes the tumor suppressor function of the TGF-βsignaling pathway in ALK-positive cells.In conclusion,our study found that PTPN2 regulates the TGF-βsignaling pathway through dephosphorylation of Smad4,and restores the TGF-βsignaling pathway in cancer cell lines.This finding also opens up a new view for the clinical application of the TGF-βsignaling pathway. |
