TCTP as a Regulator of mTOR Signaling: Effects of Silencing and Over-expression

TCTP (Translationally Controlled Tumor Protein) is a highly conserved protein that has been suggested as a tumor associated antigen as shown by its deregulation in many different types of cancers. TCTP has been hypothesized to impact the mTOR pathway via its interaction with the small G-protein, Rheb. In this study a possible link between TCTP and mTOR signaling was investigated by gene silencing using RNA interference as well as over-expression of TCTP in mammalian cells. The effects were monitored via analysis of S6 phosphorylation and cell viability using immunoblotting and MTT proliferation assays respectively. Cells were treated with three different TCTP siRNAs in a dose and time dependent manner. Silencing of the TCTP gene was found to stimulate growth moderately and enhance S6 phosphorylation, implying that TCTP is inhibitory to mTOR signaling. Wild-type TCTP was over-expressed as a GFP fusion protein in the cell lines and phosphorylation status of S6 was analyzed under serum rich and starved conditions; preliminary results suggest that TCTP does not impact the starvation phenotype significantly. Recent reports have shown that TCTP itself is phosphorylated at two serine residues which may influence its function. We are currently synthesizing site-directed mutant forms of TCTP, targeting these serines. These mutant proteins will be over-expressed in cell lines and subsequent effects on mTOR signaling will be compared to the wild-type protein expression. Our present results indicate that TCTP may not be regulating the mTOR pathway by interacting directly with Rheb as has been previously thought.