Abstract 193: Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K)
Authors: Giles David H. , Crittenden Chris M. , Brodbelt Jennifer S. , Dalby Kevin N. 
Published: July 2016

Eukaryotic elongation factor 2 kinase (eEF-2K) negatively regulates the rate of protein translation by phosphorylating and inactivating elongation factor 2 (eEF-2). Activation of eEF-2K by AMP-activated protein kinase (AMPK) contributes to the adaptive response of cancer cells to nutrient deprivation, promoting cancer cell survival and tumor progression. However, the mechanism by which AMPK activates eEF-2K is poorly described. Using LC-MS/MS, we confirmed previous findings that AMPK phosphorylates eEF-2K on three sites in vitro: Ser78, Ser366, and Ser398. Previous reports indicate that only Ser398 is phosphorylated by AMPK in cells, suggesting phosphorylation of Ser78 and Ser366 (which are known to inhibit eEF-2K via other kinases) are in vitro artifacts. To test the notion that phosphorylation of Ser398 activates eEF-2K, we purified recombinant eEF-2K S78/S366A, which cannot be phosphorylated at Ser78 or Ser366. Incubation of eEF-2K S78/S366A with AMPK led to significant phosphorylation of eEF-2K after four hours but did not alter eEF-2K activity against a peptide substrate. We then purified a form of eEF-2K S78/S366A in which Ser398 is ∼90% phosphorylated (eEF-2K SSA-pS398), as shown by LC-MS/MS and Western blot. The activity of eEF-2K SSA-pS398 against a peptide substrate was unchanged compared to a non-phosphorylated control, as was its activity against full-length eEF-2. Similarly, the EC50 and affinity of eEF-2K S78/S366A for Ca2+/calmodulin (CaM), which eEF-2K requires for activity, were unchanged by Ser398 phosphorylation. Phosphorylation of Ser398 did not alter the rate of autophosphorylation of eEF-2K S78/S366A at Thr348 or Ser500, which modulate its activity and dependence on Ca2+/CaM, respectively. Overall, our in vitro data suggests that AMPK may not directly regulate eEF-2K through phosphorylation of Ser398. In cells, mTOR activates several kinases, including p70S6K, that phosphorylate and inactivate eEF-2K. AMPK inhibits mTOR through phosphorylation of TSC2 and Raptor, suggesting AMPK could relieve eEF-2K from mTOR-mediated inhibition by suppressing mTOR activity. In line with this notion, we found that treatment of MDA-MB-231 breast cancer cells with Torin1 (a selective inhibitor of mTOR) or AICAR (an AMP-mimetic that activates AMPK) led to similar levels of eEF-2K activation, and activation of AMPK with AICAR reduced levels of mTOR activity. Combined application of Torin1 and AICAR resulted in levels of eEF-2K activity similar to those obtained with either compound separately. Taken together, our data suggests that the AMPK-dependent activation of eEF-2K likely involves the ability of AMPK to inhibit mTOR and relieve eEF-2K from mTOR-mediated inhibition, and questions the role of Ser398 phosphorylation in activating eEF-2K directly.

Citation Format: David H. Giles, Chris M. Crittenden, Jennifer S. Brodbelt, Kevin N. Dalby. Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 193.