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Activation of Akt increases Cell Surface Expression of System x c −
Author(s) -
Versluis Philip,
Gibson Amanda,
Schmidt Mackenzie,
Smith Daniel,
Chase Leah
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.950.8
Subject(s) - protein kinase b , phosphorylation , chemistry , intracellular , western blot , microbiology and biotechnology , glioma , cell membrane , cell , biochemistry , biology , cancer research , gene
System x c − 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 H 2 O 2 in confluent U138MG human glioma cells. The study described herein sought to begin to characterize the mechanism by which H 2 O 2 regulates the trafficking of xCT. We hypothesized that Akt signaling is necessary for H 2 O 2 ‐mediated trafficking of of xCT. A significant increase in Akt phosphorylation was observed in U138MG cells following ten‐minute exposure to 3 mM H 2 O 2 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 H 2 O 2 exposure resulted in a decrease in H 2 O 2 ‐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 H 2 O 2 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 H 2 O 2 ‐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 Information This work was supported by NSF‐RUI 0843564.