Activation of System xc- Trafficking via an Akt-dependent Signal Transduction Pathway

System xc- is a heterodimeric plasma membrane transporter involved in the exchange of intracellular glutamate for extracellular cystine. As such, this transporter plays a critical role in the production of the antioxidant glutathione. Previous studies in our lab have demonstrated that xCT cell surface expression increases within ten minutes of exposure to H2O2 in confluent U138MG human glioma cells. This study sought to begin to characterize the mechanism by which H2O2 regulates xCT trafficking. We hypothesized that Akt signaling is necessary for H2O2-mediated trafficking of of xCT. A significant increase in Akt phosphorylation was observed in U138MG cells following ten-minute exposure to 3 mM H2O2 compared to vehicle-treated cells using western blot analysis. Treatment with the Akt inhibitor 10-DEBC (2.5µM) for 30 minutes prior to and during H2O2 exposure resulted in a decrease in H2O2-induced phosphorylation of Akt at Ser473. Similar inhibition of Akt phosphorylation at Thr308 was observed following treatment of cells with 1.0µM API-2. Next, we used simultaneous treatment of cultured glioma cells with both inhibitors in the presence of H2O2 to determine if such treatment led to a reduction in the trafficking of endogenously expressed xCT to the plasma membrane. Preliminary data suggests that Akt activation is necessary for H2O2-induced trafficking xCT to the membrane in cultured glioma cells. To determine if the regulation of xCT cell surface expression is ubiquitous, not limited to human glioma cells where xCT is often overexpressed, we studied the role Akt plays in the trafficking of recombinantly expressed xCT in COS-7 cells. COS-7 cells transfected with myc-tagged xCT, 4F2HC and a constitutively active form of Akt showed higher levels of xCT localized to the membrane compared with cells transfected with a dominant negative Akt. These data suggest that Akt is an important regulator of xCT cell surface expression