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 there is an increase in cell surface expression within ten minutes of exposure to H2O2 in confluent U138MG human glioma cells. The study described herein sought to begin to characterize the mechanism by which H2O2 regulates the trafficking of xCT. 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. Our preliminary data suggests that Akt activation is necessary for H2O2‐induced trafficking xCT to the plasma membrane in cultured glioma cells. To determine if the regulation of xCT cell surface expression is not limited to human glioma cells where xCT is oven overexpressed, we also studied the role Akt plays in the trafficking of recombinant, transiently‐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 form of Akt. Collectively, these data suggest that Akt is an important regulator of xCT cell surface expression.Support or Funding InformationThis work was supported by NSF‐RUI 0843564.

System xc− exchanges intracellular glutamate for extracellular cystine across the membrane of many cell types, including astrocytes. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in some areas of the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc‐. Using biotinylation assays and immunocytochemical analysis, we have demonstrated that mutation of the N‐terminal lysine residues increases the cell surface expression of the transporter. We are currently assessing the ubiquitination status of these mutant transporters to determine if the changes in ubiquitination of the transporter are associated with changes in the cell surface expression and activity of the transporter. In addition, we have identified a putative ubiquitin ligase binding site in the C‐terminus of the transporter. Disruption of this binding site also leads to an increase in cell surface expression of the transporter. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.Support or Funding InformationThis work was supported by RUI 0843564.

No abstract

System xc− is a Na+‐independent, Cl−‐dependent transporter which exchanges intracellular glutamate for extracellular cystine across the plasma membrane. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. System xc− belongs to the heteromeric amino acid (HAT) transport family, consisting of the transport‐specific light chain subunit xCT and the heavy chain 4f2HC, which associates with numerous light chains in the HAT family. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc−. Using biotinylation assays and immunocytochemistry analysis, we have demonstrated that mutation of the N‐terminal lysine residues, but not the C‐terminal residues, increases the cell surface expression of the transporter. In addition, we have identified a GVPAYYLFI domain near the C‐terminus that appears to regulate cell surface expression of xCT. This domain may serve as a u ubiquitin ligase binding or as a tyrosine‐based AP‐2 binding site. We are currently assessing the ubiquitination status of these mutant transporters to determine if ubiquitination regulates cell surface expression of xCT. In addition, we are developing a N‐terminal fluorescence activated peptide (FAP)‐xCT fusion construct that will allow us to rapidly assess xCT cell surface expression. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.Support or Funding InformationThis work was supported by NSF‐RUI #0848564 and the Biology, Chemistry and Neuroscience Programs at Hope CollegeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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