Abstract 2071: Regulation of EF-2 kinase stability by the mTOR/S6 kinase- and AMP kinase-mediated phosphorylation in human glioma cells

Elongation factor-2 (EF-2) kinase is a Ca 2+ /calmodulin-dependent enzyme involved in regulating protein synthesis. EF-2 kinase inhibits protein synthesis by phosphorylating EF-2, resulting in termination of peptide elongation. Our laboratory has reported that the activity of EF-2 kinase is up-regulated in several types of cancer including glioma, and that this kinase promotes survival of cancer cells under stresses such as nutrient deprivation and hypoxia. Although it is appreciated that EF-2 kinase plays an important role in tumor progression and sensitivity to therapies, how the activity of EF-2 kinase is regulated remains largely unclear. As EF-2 kinase has sites that are phosphorylated by both mTOR/S6 kinase (at Ser78 and Ser366) and AMP kinase (at Ser398), here we determined how phosphorylation at these sites affected the activity or stability of the kinase under stresses. In human glioma cell lines (T98G and LN229) stressed with nutrient deprivation or hypoxia, we found a 2-5 fold increase in expression and activity of EF-2 kinase. EF-2 kinase mRNA was transiently increased and shortly returned to basal, protein levels continued to increase in stressed cells. To determine whether stresses inhibited the turnover of EF-2 kinase protein, which has a half-life (t 1/2 ) of ∼ 6-8 hours, we carried out pulse-chase experiments. A seemingly paradoxical increase in EF-2 kinase turnover (t 1/2 = 2-4h) was observed in stressed cells. We next examined the roles of the phosphorylation of EF-2 kinase in regulating its turnover using the phosphorylation-defective mutants of the kinase. We found that in the cells expressing the S398A mutant, the t 1/2 of the EF-2 kinase protein increased to greater than 24 hours under both normal and stress conditions, suggesting that the phosphorylation of Ser398 by AMP kinase plays an important role in the degradation of the kinase. In the cells expressing the S78A/S366A mutant, the t 1/2 of the kinase also increased to greater than 24 hours under normal culture condition; however, when these cells were subjected to metabolic stress the turnover rate of the mutant EF-2 kinase was similar to that of the wild-type kinase (t 1/2 = 8h). Inhibition of the mTOR pathway by rapamycin caused a ∼ 5 fold increase in EF-2 kinase expression, but further decreased EF-2 kinase stability of the mutated kinase under normal and stress conditions (t 1/2 = 2-6h). These experiments suggest that phosphorylation of Ser78 and Ser366 by the mTOR/S6 kinase is essential for turnover of EF-2 kinase under normal, but not under stress conditions. Our results indicate that phosphorylations at Ser398, Ser78 and Ser366 have differential effects on stability of EF-2 kinase in response to stress, and the differential roles of these phosphorylation sites in the regulation of the kinase stability may be exploited to inhibit tumor progression or modulate response of cancer cells to therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2071. doi:10.1158/1538-7445.AM2011-2071