Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

Abstract

b; Université catholique de Louvain, Brussels, Belgium c Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal ␣-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.

Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

“…This position corresponds to an autophosphorylation site in the related enzyme myosin heavy chain kinase (MHCK), which, when phosphorylated, docks into a binding pocket to allow the enzyme to adopt an active conformation [11] . A similar mechanism appears to apply to eEF2K [12,13] . This site can even be phosphorylated in E coli, which lacks CaM, suggesting that the activity of eEF2K in autophosphorylation is not strictly Ca/CaM dependent [13] .…”

“…The first input from mTORC1 to be identified involves the 70 kDa ribosomal protein S6 kinase, p70S6K, which is activated by mTORC1 and phosphorylates eEF2K at Ser366 (human sequence numbering), inactivating it [17] (Figure 1, 2). Since then, additional links between mTORC1 and eEF2K have been delineated, including direct phosphorylation of eEF2K by mTORC1 in an in vitro kinase assay, a nutrient-regulated protein kinase [12,18] (Figure 1, 2). Ser78 in eEF2K is also phosphorylated in an mTORC1-dependent manner [19] ( Figure 1); this residue is adjacent to the CaM-binding motif, and its phosphorylation impairs CaM binding, and thus, the activation of eEF2K.…”

Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression

“…Equal amount of lysis buffer (hypotonic buffer 2% DOC, 2% Triton X-100, 2.5 mM DTT) was added, and subsequent cell lysates were vigorously vortexed, incubated 20 min on ice, and centrifuged at 15,000 g for 10 min at 4°C. Post-nuclear lysates were layered on 10 ml 10-50% (w/v) sucrose gradients (50, 40,30,20, and 10% sucrose in 80 mM NaCl, 5 mM MgCl 2 , 20 mM Tris pH 7.4, 1 mM DTT, and 100 lg/ ml CHX in DEPC H 2 O). Gradients were centrifuged at 30,000 rpm for 4 h at 4°C and separated through a live OD254 nm ultraviolet spectrometer.…”

Regulation and roles of elongation factor 2 kinase

“…Conversely, eEF2K is inactivated by the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway. This involves the phosphorylation of eEF2K at several sites [1,6], some of which may be direct substrates for mTORC1 while others are targets for the ribosomal protein S6 kinases which are themselves activated by mTORC1 or for other kinases [6,7]. The inactivation of eEF2K by mTORC1 signalling probably serves to couple this anabolic pathway to the activation of translation elongation and the accelerated synthesis of new polypeptides.…”