Biochemical Basis For Protein Tyrosine Phosphatase SHP2in Regulation Of Pulmonary Epithelial Mesenchymal Transition

Aims:The epithelial-to-mesenchymal transition (EMT) is an essential process during embryogenesis. It also plays a critical role in the initiation of tumor metastasis. EMT is an important form of cell differentiation. SHP2is a ubiquitously expressed tyrosine phosphatase and regulates extensive cellular functions, including cell differentiation. Somatic activated SHP2mutations have also been detected in various human tumors, suggesting that SHP2participates in the formation and progression of tumors. The function role of SHP2in EMT remains largely unknown. Our aims are to explore the roles of SHP2in EMT during the initiation of tumor metastasis.Methods:To investigate the role of SHP2in EMT, we used TGFβ1-induced EMT in non-small cell lung cancer cell line-A549as a model. To explore the effects of the expression levels of SHP2on EMT, we overexpressed WT SHP2or transfected SHP2siRNA in A549. Further, A549cells were treated with PHPS1or transfected dominant-negative mutant (SHP2C459S), activated mutant (SHP2E76V) to find the effects of SHP2activity on EMT. To explore the molecular mechanisms of the regulatory role of SHP2on EMT, Yeast two-hybrid system was used to screen the binding proteins of SHP2.Results:Downregulation of SHP2inhibited TGFβ1induced EMT in A549cells. Dominant negative SHP2mutant (SHP2C459S) and SHP2inhibitor (PHPS1) downregulated mesenchymal markers. Activated mutant SHP2(SHP2E76V) promoted TGFβ1-induced EMT. Using yeast two-hybrid screening, we found Hookl was a new binding protein of SHP2-PTP. Co-IP assay was used to verify the interaction of SHP2and Hookl. Hookl was downregulated in TGFβ1-treated A549. Hookl knockdown promoted EMT, and overexprssion of Hookl inhibited EMT. Further, we found that Hookl not only binded to SHP2PTP domain, but also to SHP2NSH2domain. Moreover, Hookl knockdown up-regulated SHP2activity. Co-transfection of Hookl and SHP2E76V mostly offset the promotional role of SHP2E76V on EMT. These results suggested that Hookl participated in the negative regulation of SHP2activation.Conclusion:1. SHP2positively regulated TGFβ1-induced EMT.2. Hookl was a new binding protein of SHP2, not only binded to SHP2-PTP domain, but also to SHP2-NSH2domain.3. Hookl was downregulated by TGFβ1and negatively regulated EMT.4. Hookl potentially acts as an endogenous negative regulator of SHP2activation.5. SHP2-Hookl interaction potentially participates in regulation of EMT.