The Interaction Between LKB1and Pten In HeLa Cells

Objective:Tumor suppressors LKB1(Liver Kinase B1) and PTEN (phosphatase and tensin homolog deleted on chromosome ten) regulate mTOR (mammalian Target Of Rapamycin) signaling pathway. Germ-line mutations of LKB1and PTEN cause Peutz-Jeghers syndrome (PJS) and Cowden’s disease (CD), respectively, and they share similar clinical manifestations. A series of research results suggest that LKB1and PTEN may interact with each other. In this study, we constructed polycistronic expression vectors of LKB1and PTEN and explored their relationship in HeLa cells.Methods:Human liver total RNA was extracted and used as template in reverse transcription PCR to obtain Ikbl and Pten cDNA. IRES2fragment was amplified by PCR from pIRES2-EGFP vector. Expression vectors pIRES2-DsRed2-LKB1(abbreviated as pIRL)、pIRES2-DsRed2-PTEN (abbreviated as pIRP) and pIRES2-DsRed2-PTEN-IRES2-LKB1(abbreviated as pIRPIL) were constructed using pIRES2-DsRed2vector. To explore the interaction between LKB1and PTEN, polycistronic expression vectors were transfected into HeLa cells and the expression of reporter gene DsRed was detected by fluorescent microscope. Western blotting was used to detect the expression of LKB1and PTEN. Furthermore, we investigated the phosphorylation level and phosphatase activity of PTEN induced by LKB1。Results:We successfully obtained Ikbl and Pten cDNA as well as IRES2fragment through PCR. Vectors for expression of LKB1, PTEN, LKB1+PTEN were constructed using pIRES2-DsRed2vector and named as pIRL、pIRP and pIRPIL, respectively., Sequence analysis confirmed that the insert sequences were correct. We detected red fluorescence after pIRL and pIRP plasmids were transfected into HeLa cells. Western blot results showed that target proteins were expressed. The phosphorylation level and phosphatase activity of PTEN were up-regulated by LKB1. Conclusion:LKB1interacts with PTEN and LKB1can up-regulate the phosphorylation level and phosphatase activity of PTEN.