Expression And Relationship Study Of LKB1,14-3-3γand AMPK In Liver Tumorigenesis Of Rat

LKB1, also known as STK11, is a serine/threonine kinase. LKB1has recently been shown to be frequently mutated in cancer, such as liver cancer, lung cancer and cervical cancer, thus defining LKB1as a tumor suppressor protein. The human LKB1tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, including the control of cell-cycle arrest, cell apoptosis, Wnt signaling. Ras-induced cell transformation, AMPK pathway and energy metabolism,and cell polarity. Thr336is a major autophosphorylation site on LKB1. It has been previously shown that the mutation of Thr336to Glu prevented LKB1from inhibiting G361cell growth. This implies that there may be some proteins involved in the negative regulation of LKB1function initiated by the phosphorylation of Thr336. LKB1protein is widely expressed in both embryonic and adult tissues, although at different levels. In murine embryos, LKB1expression is highly ubiquitous, but during development, expression becomes more prominent in the heart, lung, kidney. In adults, glial cells and epithelial tissues show the highest levels of LKB1protein expression. AMP-activated protein kinase (AMPK) is the primary regulator of the cellular response to lowered ATP levels in eukaryotic cells. AMPK is activated by stimuli that include pathological stresses, such as oxidative damage, osmotic shock and glucose deprivation.LKBl directly phosphorylates Thr-172of AMPK in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172phosphorylation. The LKB1-AMPK pathway plays critical roles in regulating important life-and-death processes including apoptosis and autophagy.14-3-3proteins are a family of highly conserved cellular proteins and play an important role in a wide variety of cellular-processes. These include signal transduction, cell cycle regulation, apoptosis, cellular metabolism, cytoskeleton organization and malignant transformation. There are seven distinct14-3-3isoforms (β,ε,ζ,η,θ,γ and σ), which are encoded by separate genes in mammals.14-3-3 isoforms have distinct tissue and subcellular localizations. For example, the σ isoform was mainly expressed in epithelial cells. The14-3-3γ protein is an important regulator of various cellular and physiologic functions. Overexpression promotes cell proliferation and induces cancer cell polyploidization. Many studies show that the expression of14-3-3γ in hepatocellular carcinoma was higher than control tissue. More than200proteins have been reported to associate with14-3-3proteins in vivo. Isoforms of14-3-3also have distinct binding partners.In present study, LKB1could interact with14-3-3, and only directly bind to three isoforms of14-3-3, such as14-3-3γ,τ and ξ. And indicated that phosphorylation of LKB1on Thr336is required for14-3-3binding. At the same time, the downstream of LKB1, AMPK, could interact with LKB1competively.In this study, we want to detect the expression levels of LKB,14-3-3γ, p-AMPK in the process of primary carcinoma of liver and different cells, and to find the interaction of these proteins, even to find the conditions of phosphorylation of LKB1T336.The results indicated that the expression of14-3-3γ in control was lower than in tumor cells, and expression in HepG2was higher than in A549and Hela. The expression of LKB1was higher in HL7702than HepG2and A549. But the expression levels of LKB1T336in HepG2and A549was higher than HL7702. Hela was LKB1deficient cell lines, so the expression of LKB1was not detected in Hela. In HCC models, our results showed that in the process of HCC the activation of14-3-3γ was significantly increased, but LKB1and p-AMPK were decreased. Notebaly, the p-AMPK was not activated in tumor. These results indicated there is a certain correlation among LKB1, p-AMPK and14-3-3γ.We investigated whether autophosphorylation at Thr336is inducible. Insulin, H2O2, and serum starvation were chosen to stimulate HEK-293cells as well as COS7cells. And we only found after serum starvation for6h, phosphorylation of LKB1at Thr336in HEK-293cells was detectable. Notably, the level of the phosphorylation increased significantly over time. The results from these experiments indicate that expression of WT LKB1and LKB1T336A both significantly induce G1cell cycle arrest and apoptosis. Importantly, we found that Gl cell cycle arrest and apoptosis induced by WT LKB1were attenuated by overexpression of14-3-3, while these processes were not affected by overexpression of14-3-3in LKB1T336A-expressing cellsConclusion:endogenous protein detect results demonstrated that there is an correlation among LKB1,14-3-3y and AMPK, and the interaction between LKB1and14-3-3y may produce an effect on the expression of LKB1,AMPK and14-3-3y. In the HCC models, LKB1and14-3-3y could be detection of indexes of tumor. After serum starvation for6h, the phosphorylation of LKB1Thr336and AMPKT172in HEK-293cells was detectable, but the expression of14-3-3was reduce. Overexpression of14-3-3could combine to LKB1compete into AMPK, so LKB1was not easy bind to AMPK. Gl cell cycle arrest was induced by WT LKB1were attenuated by overexpression of14-3-3.The results suggest that the interaction between14-3-3and LKB1had an effection on the cell cycle.