Study On The Relationship Between Tctp And Rheb By Yeast Two Hybrid And The Identification On The Fuction Of TCTP

Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein that is widely expressed in many eukaryotic. It is similar with Mss4 in structure, and plays important roles in a number of cell physiological events. Ras homolog enriched in brian (Rheb) is a small GTPase belongs to Ras superfamily and is a upstream positive regulator factor in mammalian target of rapamycin (mTOR) signal pathway. It has two states, GTP-bound state and GDP-bound state. It regulates the cell process such as cell growth, cell proliferation and cell differentiation. TCTP may function as a guanine nucleotide exchange factor of Rheb, which changes Rheb from GDP-bound to active GTP-bound, but such function is still in uncertainty at present. Taking cashmere goat fetal fibroblasts cells (GFb cells) as materials, this research discusses interactive relation between TCTP and Rheb and makes a preliminary study on the function of TCTP.Firstly, we made use of RT-PCR to clone the cDNA of TCTP, and analyzing its expression pattern. Secondly, Co-immunopricipitation (co-IP) and Western-Blot were used to detect the co-existed of Rheb and TCTP in immunoprecipitates. Then constructing yeast two hybrid bait vector and target vector of TCTP and Rheb. To defined the direct interaction relationship between Rheb and TCTP by using yeast two-hybrid. Finally, constructing overexpression vector and RNA interference vector of TCTP and transfecting GFb cells. We detected the expression and phosphorylation level of effector molecules of mTOR signaling pathway, mTOR and S6, to assure whether TCTP participates in the control of mTOR signaling pathway. We carried out flow cytometry to assure whether TCTP has the function of controlling cell cycle.The ORF of TCTP gene with total length 519 bp, encodes 172 amino acid residues. The expression quantity of such gene is comparatively higher in liver, but lower in brain. Co-immunoprecipitation showed that there is an interaction between TCTP and Rheb. The further yeast two hybrid indicated that they have direct interaction. Overexpression or silence of TCTP gene had significant effect on expression of mTOR and S6, but had no effect on their phosphorylation level. The flow cytometry results showed that overexpression TCTP has no effect on cell cycle; and TCTP posttranscriptional gene silencing induces cell cycle arrest, which indicates that TCTP may related with process of cell cycle.This study enriched genetic information of TCTP. TCTP interacts with Rheb directly, but it may not be a GEF to activate Rheb. It is needed to research that the machenism of TCTP participates in regulation of mTOR signaling pathway and process of GFb cell cycle.