Phosphorylation Of eEF2K Research Articles

Age-dependent increase in activity of eukaryotic elongation factor 2 kinase in mesenteric arteries from spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) negatively regulates protein translation through the phosphorylation of its specific substrate, eEF2. We previously found that expression of eEF2K was increased in arteries from 13–15-week-old spontaneously hypertensive rats (SHR) as well as in left ventricles of cardiac hypertrophy models. Furthermore, we demonstrated that eEF2K mediates the development of essential hypertension and pulmonary arterial hypertension in animal models. Protein expression changes with age during development of hypertension in SHR. In the present study, we examined whether activity and expression of eEF2K change in isolated mesenteric arteries dependent on the age. After superior mesenteric arteries were isolated from 4–10-week-old Wistar Kyoto rats (WKY) and SHR, Western blotting was performed. The phosphorylation of eEF2K at Ser500, an activating phosphorylation site, was increased in the arteries from 10-week-old SHR, whereas the phosphorylation of eEF2K at Ser366, an inactivating phosphorylation site, was increased in the arteries from 4–5-week-old SHR compared with WKY. The expression of eEF2K was increased in the arteries from 10-week-old SHR compared with WKY. The phosphorylation of eEF2 at Thr56 was decreased in the arteries from 4–5-week-old SHR, whereas it was increased in the arteries from 10-week-old SHR compared with WKY. We for the first time revealed that eEF2K activity is lower in prehypertensive stage but higher in hypertensive stage in SHR, suggesting that an inhibition of eEF2K activity may be a potential therapeutic strategy for the treatment of essential hypertension.

Dynamic equilibrium of eEF‐2K and CaM as a regulatory logic circuit: investigations in MCF10A cells

Eukaryotic elongation factor 2 kinase (eEF‐2K) is a Ca2+/Calmodulin (CaM)‐dependent kinase that phosphorylates eEF‐2 and inactivates it, thereby blocking translation. Studies have correlated eEF‐2K expression with poorer outcomes in cancer, and it was recently shown to contribute to the Warburg effect. eEF‐2K is activated by the PKA and AMPK pathways, and inhibited by the ERK, mTORC1, and p38∂ pathways, among others. How eEF‐2K integrates these signals into a coherent output is not understood. We recently showed that phosphorylation of eEF‐2K on Ser500 by PKA, like Ca2+, drives the association of eEF‐2K and CaM to activate the kinase. We hypothesized that other regulatory signals converge on the equilibrium of eEF‐2K and CaM, either driving it toward the bound (fully active) or unbound (fully inactive) state. Here, we establish that at Ca2+ levels basal to MCF10A cells, removal of either ERK or mTORC1 activity is sufficient to fully activate eEF‐2K; increasing intracellular Ca2+ with ionomycin is sufficient to overcome these inhibitory signals. Once mTORC1 activity is already removed, additionally increasing intracellular Ca2+ produces no increase in eEF‐2K activity. Thus, inhibitory signals maintain eEF‐2K in the unbound (inactive) state, but sufficiently high Ca2+ can overcome this repression. Blocking glycolysis with 2‐deoxyglucose (2‐DG), which induces full eEF‐2K activation, results in the shutdown of ERK and mTORC1, increases intracellular Ca2+, and activates AMPK. Although AMPK phosphorylates eEF‐2K directly at Ser398, this has no effect on its association with CaM and is not required for activation by 2‐DG in cells. Similarly, directly activating AMPK (~7‐fold) with A769662 only weakly activates eEF‐2K (<2‐fold), and has no effect on ERK, mTORC1, or Ca2+. We show that, to a first approximation, the activation status of eEF‐2K is described by a simple Boolean “logic circuit”, where inhibitory inputs act to maintain the circuit in the OFF (unbound) state, and activating ones switch the circuit to the ON (CaM‐bound) state. AMPK appears to act on the circuit's inputs rather than forming a critical component itself. Further, we discuss how this “logic circuit” can be generalized to other cell types and ultimately reveal the mechanisms underpinning the regulation of eEF‐2K in cancer.Support or Funding InformationR01 GM123252, F‐1390 Welch Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 204: Protective Roles of Eukaryotic Elongation Factor 2 Kinase on Rat Cardiomyoblast Death Under Glucose-deprived Condition

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K; also known as calmodulin-dependent protein kinase III) has both active and inactive phosphorylation sites. An intracellular energy sensor, AMP-activated protein kinase (AMPK) was reported to activate eEF2K via increasing dephosphorylation at Ser366 (inactive site). Activated eEF2K phosphorylates and inactivates a specific substrate, eEF2, which results in the inhibition of protein translation consuming high energy. Glucose depletion (GD) is one of the primary causes for cardiomyocyte death in the developed cardiac hypertrophy. We have recently found that the expression and dephosphorylation of eEF2K (Ser366) and eEF2 phosphorylation were significantly increased in left ventricle of several cardiac hypertrophy models. However, it is almost unknown whether eEF2K/eEF2 signals affect GD-induced cardiomyocyte death. The aim of this study was to explore it. GD was induced by incubating H9c2 cells in a glucose-free medium. H9c2 cell viability, apoptotic-like nuclear condensation or protein expression was examined using a cell counting assay, DAPI staining or Western blotting, respectively. GD induced H9c2 cell death (p<0.01, n=6) and caspase-3 fragmentation (p<0.05, n=10-12). In addition, GD significantly increased phosphorylation of AMPK (p<0.05, n=6-8) and eEF2 (p<0.01, n=4-8) as well as eEF2K dephosphorylation at Ser366 (p<0.01, n=4-8). eEF2K gene knockdown (eEF2K KD) by siRNA transfection significantly increased GD-induced H9c2 cell death (p<0.05, n=7) and caspase-3 fragmentation (p<0.01, n=9). Moreover, eEF2K KD significantly facilitated GD-induced increase of nuclear condensation (44.0±3.3%, eEF2K siRNA vs. 30.9±2.4%, control siRNA p<0.01, n=5). AMPK KD did not affect GD-induced H9c2 cell death and eEF2K dephosphorylation. In conclusion, we for the first time revealed in H9c2 cells that activated eEF2K might play protective roles in GD-induced apoptosis via the inhibition of caspase-3 fragmentation, whereas AMPK activation is not directly related to the regulation of eEF2K/eEF2 signals in GD condition. The present results suggest eEF2K as a novel pharmacotherapeutic target for cardiac dysfunction.

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

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs.

Decreased Whole-Body Fat Mass Produced by Chronic Alcohol Consumption is Associated with Activation of S6K1-Mediated Protein Synthesis and Increased Autophagy in Epididymal White Adipose Tissue.

Chronic alcohol consumption leads to a loss of white adipose tissue (WAT) but the underlying mechanisms for this lipodystrophy are not fully elucidated. This study tested the hypothesis that the reduction in WAT mass in chronic alcohol-fed mice is associated with a decreased protein synthesis specifically related to impaired function of mammalian target of rapamycin (mTOR). Adult male mice were provided an alcohol-containing liquid diet for 24weeks or an isonitrogenous isocaloric control diet. In vivo protein synthesis was determined at this time and thereafter epididymal WAT (eWAT) was excised for analysis of signal transduction pathways central to controling protein synthesis and degradation. While chronic alcohol feeding decreased whole-body and eWAT mass, this was associated with a discordant increase in protein synthesis in eWAT. This increase was not associated with a change in mTOR, 4E-BP1, Akt, or PRAS40 phosphorylation. Instead, a selective increase in phosphorylation of S6K1 and its downstream substrates, S6 and eIF4B was detected in alcohol-fed mice. Alcohol also increased eEF2K phosphorylation and decreased eEF2 phosphorylation consistent with increased translation elongation. Alcohol increased Atg12-5, LC3B-I and -II, and ULK1 S555 phosphorylation, suggesting increased autophagy, while markers of apoptosis (cleaved caspase-3 and -9, and PARP) were unchanged. Lipolytic enzymes (ATGL and HSL phosphorylation) were increased and lipogenic regulators (PPARγ and C/EBPα) were decreased in eWAT by alcohol. Although alcohol increased TNF-α, IL-6, and IL-1β mRNA, no change in key components of the NLRP3 inflammasome (NLRP3, ACS, and cleaved caspase-1) was detected suggesting alcohol did not increase pyroptosis. Plasma insulin did not differ between groups. These results demonstrate that the alcohol-induced decrease in whole-body fat mass resulted in part from activation of autophagy in eWAT as protein synthesis was increased and mediated by the specific increase in the activity of S6K1.

Abstract 274: Expression and Phosphorylation States of Eukaryotic Elongation Factor 2 (EEF2) and EEF2 Kinase in Cardiomyocytes From Hypertrophy Models

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K, also known as calmodulin (CaM)-dependent protein kinase III) is regulated by both CaM-dependent and -independent mechanisms. Activated eEF2K phosphorylates and inactivates a specific substrate, eEF2. eEF2 activation facilitates protein translation. It is recognized that increased protein synthesis is one of the primary factors for cardiomyocyte hypertrophy. In fact, angiotensin II, which induces cardiomyocyte hypertrophy, was reported to facilitate eEF2 dephosphorylation (activation) and protein synthesis in rat isolated cardiomyocytes. We have previously demonstrated that protein expression of eEF2K was increased specifically in left ventricles (LV) of spontaneously hypertensive rats (SHR). However, expression and phosphorylation states of eEF2K and eEF2 in LV of other cardiac hypertrophy models are unknown. The aim of this study was to explore it. Male C57BL/6NJcl mice and Wistar rats received transverse aortic constriction (TAC) and isoproterenol (5 mg/kg; ISO) injection, respectively, which induced cardiac hypertrophy. After 3 and 28 days from TAC operation and 7 days from ISO injection, LV were isolated and used for Western blotting (WB) and immunohistochemistry (IHC). Echocardiography was done in TAC mice before LV isolation. In TAC-induced hypertrophied LV (3 days), eEF2K expression was significantly increased (p<0.01 vs. SHAM) and its phosphorylation at Ser366 was significantly decreased (p<0.05 vs. SHAM). Consistently, eEF2 phosphorylation was significantly increased (p<0.01 vs. SHAM). In LV from ISO rats, eEF2K phosphorylation at Ser366 was significantly decreased as determined by WB (p<0.01 vs. control). In addition, eEF2K- and phosphorylated eEF2-positive cardiomyocytes were increased as determined by IHC. These changes were also confirmed in LV from SHR. At 28 days after TAC, fractional shortening was significantly decreased (from 56.6±1.6% to 44.4±2.3%, p<0.01). Interestingly, eEF2 phosphorylation in LV was significantly decreased (p<0.05 vs. SHAM). The present results suggest the potential role of eEF2K/eEF2 signals in the pathogenesis of cardiac hypertrophy/failure.

Abstract 193: Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K)

Abstract 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.

Coordination of changes in expression and phosphorylation of eukaryotic elongation factor 2 (eEF2) and eEF2 kinase in hypertrophied cardiomyocytes

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is one of the Ca2+/calmodulin-dependent protein kinases. Activated eEF2K phosphorylates its specific substrate, eEF2, which results in inhibition of protein translation. We have recently shown that protein expression of eEF2K was specifically increased in hypertrophied left ventricles (LV) from spontaneously hypertensive rats (SHR). However, phosphorylation state of eEF2K and eEF2 in hypertrophied LV is not determined. In the present study, we examined expression and phosphorylation of eEF2K and eEF2 in LV from SHR as well as the pressure overload (transverse aortic constriction: TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy model. In LV from TAC mice, eEF2K expression was increased as determined by Western blotting. In LV from TAC mice and SHR, eEF2K phosphorylation at Ser366 (inactive site) was decreased. Consistently, eEF2 phosphorylation at Thr56 was increased. In LV from ISO rats, while eEF2K phosphorylation was decreased, eEF2K expression and eEF2 phosphorylation were not different as determined by Western blotting. In the results obtained from immunohistochemistry, however, total eEF2K and phosphorylated eEF2 (at Thr56) localized to cardiomyocytes were increased in LV cardiomyocytes from ISO rats. Accordingly, the increased expression and the decreased phosphorylation of eEF2K and the increased phosphorylation of eEF2 in hypertrophied LV were common to all models in this study. The present results thus suggest that cardiac hypertrophy may be regulated at least partly via eEF2K-eEF2 signaling pathway.

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells.

Eukaryotic elongation factor 2 kinase (eEF2K), also known as calmodulin (CaM)-dependent protein kinase (CaMK) III, is a unique member of CaMK family protein. We have recently found that expression of eEF2K protein increased in mesenteric artery from spontaneously hypertensive rats. As pathogenesis of hypertension is in part regulated by vascular structural remodelling via proliferation and migration of vascular smooth muscle cells (SMCs), we tested the hypothesis that eEF2K controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, an eEF2K inhibitor, A-484954 (10 μm), significantly inhibited platelet-derived growth factor (PDGF)-BB (10 ng mL(-1) )-induced SMCs proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay. PDGF-BB (10 ng mL(-1) )-induced SMCs migration was significantly inhibited by A-484954 (10 μm) as determined by a Boyden chamber assay. A-484954 (10 μm) significantly inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K, extracellular signal-regulated kinase (ERK), Akt, p38 and heat-shock protein (HSP) 27 as determined by Western blotting. It was confirmed that a CaM inhibitor, W-7 (50 μm), inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K. In an ex vivo mesenteric arterial ring assay, 10% foetal bovine serum-induced SMCs outgrowth was significantly inhibited by A-484954 (10 μm). We for the first time revealed that eEF2K mediates PDGF-BB-induced SMCs proliferation and migration through activating ERK, Akt, p38 and HSP27 signals in a CaM-dependent manner. Our results suggest eEF2K as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Modulation of eukaryotic elongation factor 2 kinase activity via phosphorylation on Ser‐500 (773.1)

Eukaryotic elongation factor 2 kinase (eEF‐2K) is an atypical calcium/calmodulin (Ca2+/CaM)‐dependent kinase known to modulate protein synthesis through the inhibitory phosphorylation of elongation factor 2. Despite its pivotal role in translation regulation and potential role in tumorigenesis, the mechanism of regulation of eEF‐2K activity by phosphorylation is still unclear. Our objective was to analyze the activation of eEF‐2K by phosphorylation at Ser‐500 (phosphorylated by protein kinase A or Ca2+/CaM‐induced autophosphorylation). In vitro analysis indicates that autophosphorylation of eEF‐2K induces Ca2+‐independent activity. Western blotting analysis as well as mutagenesis of Ser‐500 to Ala or Asp suggests that Ser‐500 phosphorylation is responsible for this Ca2+‐independent activity. Interestingly, CaM appears to be essential for this Ca2+‐independent activity of eEF‐2K. Mechanistic studies indicate that Ser‐500 phosphorylation increases eEF‐2K affinity for CaM in the presence or absence of Ca2+. Ser‐500 phosphorylation also increases the rate of Thr‐348 phosphorylation (a key site for the allosteric regulation of eEF‐2K activity). We are currently studying the dynamic nature of Ser‐500 phosphorylation in cells. Phosphorylation of eEF‐2K on Ser‐500 could be a significant mechanism for its regulation, especially upon the influx of Ca2+ during cellular signaling events.Grant Funding Source: NIH (GM059802 and CA167505) to KND

Abstract 2222: Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer.

Abstract Breast cancer is the most common malignancy in women in the Western world, with over 40,000 deaths attributed to this disease. Studies have demonstrated that breast cancer mitogens regulate the activity of eukaryotic elongation factor 2 kinase (eEF-2K). The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. We have recently shown for the first time that the targeting of eEF-2K in an in vivo orthotopic model inhibits growth of established breast cancer tumors and sensitizes the tumors to doxorubicin, an important agent in a number of chemotherapy regimens. Our data suggest that eEF-2K may enhance tumorigenesis through the up-regulation of pro-tumorigenic proteins and pathways including cyclin D1, c-Myc, c-Src/FAK and Akt, whose dysregulation are associated with a poor prognosis in breast cancer. Thus, the targeting of eEF-2K in breast cancer cells has potential as a therapeutic approach for the treatment of breast cancer. In order to develop inhibitors of eEF-2K, an understanding of the regulation of enzyme activity is essential. eEF-2K is an atypical calcium/calmodulin-dependent protein kinase (CaMK-III), that through its phosphorylation of elongation factor 2 (eEF-2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The purpose of our study was to analyze the role of calcium/calmodulin-stimulated autophosphorylation in the generation of a calcium-independent form of eEF-2K. Analysis of the kinase activity using [γ-32P]ATP indicates that autophosphorylation of eEF-2K induces calcium-independent activity. Site-directed mutagenesis of the autophosphorylation sites to alanine and aspartate suggests that the site responsible for this calcium-independent activity is Ser-500. Moreover, using a phospho-specific antibody we have determined that the time course of incorporation of phosphate at Ser-500 correlates with the induction of calcium-independent activity. Surprisingly, calmodulin appears to be essential for this calcium-independent activity of eEF-2K. Mechanistic studies indicate that phosphorylation of eEF-2K on Ser-500 increases its affinity for calmodulin in the absence of calcium, potentially by decreasing its rate of dissociation. We have also found that eEF-2K is indeed phosphorylated on Ser-500 in cells, and we are currently studying the dynamic nature of phosphorylation at this site, and its effect on stability of the enzyme and its affinity for calmodulin. Citation Format: Clint D. J. Tavares, Scarlett B. Ferguson, Rebecca M. Wellmann, Tamer S. Kaoud, John P. O'Brien, Bulent Ozpolat, Jennifer S. Brodbelt, Kevin N. Dalby. Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2222. doi:10.1158/1538-7445.AM2013-2222

Abstract 171: Eukaryotic Elongation Factor 2 Kinase (eEF2K) Mediates Vascular Inflammation and Development of Hypertension in Spontaneously Hypertensive Rats

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca 2+ /calmodulin-dependent protein kinase that mainly regulates protein translation. We have recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated in part by vascular inflammation. We examined whether eEF2K mediates vascular inflammatory responses and development of hypertension. In human umbilical vein endothelial cells (HUVECs) and rat mesenteric arterial smooth muscle cells (SMCs), eEF2K phosphorylation was increased by TNF-α (48% increase in HUVECs and 46% increase in SMCs, n=5, P<0.01). In HUVECs, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 (62% inhibition, n=4) and e-selectin (29% inhibition, n=6) as well as monocyte adhesion (43% inhibition, n=4) by TNF-α (P<0.01). eEF2K siRNA inhibited phosphorylation of JNK (76% inhibition) and NF-κB (54% inhibition) as well as reactive oxygen species (ROS) production (69% inhibition) by TNF-α (n=4, P<0.01). In SMCs, eEF2K siRNA inhibited VCAM-1 induction (41% inhibition, n=5, P<0.05) and phosphorylation of JNK (65% inhibition, n=6, P<0.01) and NF-κB (83% inhibition, n=4, P<0.05) by TNF-α. In vivo, increased blood pressure (systolic blood pressure, SHR; 199.5 mmHg vs. SHR+NH125; 159.4 mmHg, n=4, P<0.01) and increased eEF2K phosphorylation (68% inhibition, n=4), induction of VCAM-1 (62% inhibition, n=4, P<0.05) and hypertrophy (70% inhibition, n=4) in SHR mesenteric artery was normalized by long-term treatment with NH125 (500 μg/kg/day for 6 weeks). In SHR mesenteric artery, impairment of acetylcholine (ACh)-induced endothelium-dependent relaxation was normalized by NH125 (relaxation induced by 30 nM ACh, SHR; 60.4% vs. SHR+NH125; 90.0%, n=4, P<0.01). The present results for the first time demonstrated in cultured ECs and SMCs that eEF2K mediates TNF-induced inflammatory responses via ROS-dependent mechanism. It is also suggested that eEF2K may mediate development of hypertension in SHR likely via inflammation, hypertrophy and endothelial dysfunction.

Phosphorylation of elongation factor-2 kinase differentially regulates the enzyme’s stability under stress conditions

Eukaryotic elongation factor-2 kinase (eEF-2K) is a Ca2+/calmodulin-dependent enzyme that negatively regulates protein synthesis. eEF-2K has been shown to be up-regulated in cancer, and to play an important role in cell survival through inhibition of protein synthesis. Post-translational modification of protein synthesis machinery is important for its regulation and could be critical for survival of cancer cells encountering stress. The purpose of our study was to examine the regulation of eEF-2K during stress with a focus on the roles of phosphorylation in determining the stability of eEF-2K. We found that stress conditions (nutrient deprivation and hypoxia) increase eEF-2K protein. mRNA levels are only transiently increased and shortly return to normal, while eEF-2K protein levels continue to increase after further exposure to stress. A seemingly paradoxical decrease in eEF-2K stability was found when glioma cells were subjected to stress despite increased protein expression. We further demonstrated that phosphorylation of eEF-2K differentially affects the enzyme’s turnover under both normal and stress conditions, as evidenced by the different half-lives of phosphorylation-defective mutants of eEF-2K. We further found that the eEF-2K site (Ser398) phosphorylated by AMPK is pivotal to the protein’s stability, as the half-life of S398A mutant increases to greater than 24h under both normal and stress conditions. These data indicate that eEF-2K is regulated at multiple levels with phosphorylation playing a critical role in the enzyme’s turnover under stressful conditions. The complexity of eEF-2K phosphorylation highlights the intricacies of protein synthesis control during cellular stress.

Abstract 4985: The essential role of eEF-2 kinase in determining fate of glioma cells under ER stress

Abstract Endoplasmic reticulum (ER), an organelle that plays an essential role in protein folding and post-translational modification, can be stressed a variety of stimuli and pathological conditions. To recover normal ER function, an evolutionarily conserved response, termed unfolded protein response, is triggered. When ER homeostasis fails to be re-established, persistent ER stress can induce apoptosis. Recently, it has been demonstrated that ER stress can also induce autophagy, another mechanism for removing unfolded proteins that cannot be eliminated via the ubiquitin/proteasome system, thereby reducing ER stress. However, the precise mechanisms by which the ER stress-induced autophagy is regulated and the functional association between apoptosis and autophagy in tumor cells under ER stress remains unclear. We report here that eukaryotic elongation factor-2 kinase (eEF-2K) is a critical mediator of the ER stress-induced autophagy. We found in the human glioma cells (T98G and LN229) that eEF-2K and autophagy were simultaneously activated following treatment with the ER stress inducers, thapsigargin and tunicamycin; silencing of eEF-2K expression blunted the autophagic response to ER stress. To explore the pathway linking eEF-2K activation and ER stress, we tested the role of REDD1, a stress-induced protein involved in repression of the mTOR signaling. We demonstrated that under ER stress, silencing of REDD1 blocked the phosphorylation of eEF-2, the only known substrate of eEF-2K, indicating that REDD1 is required for transducing the signal for eEF-2K activation. As phosphorylation of eEF-2K at different sites can either activate or inhibit the activity of the enzyme, we next determined the roles of phosphorylation of the kinase in regulating autophagy. We showed that phosphorylation of eEF-2K at Ser398 was essential for the induction of autophagy, while phosphorylation at Ser 366 and Ser78 exerted an inhibitory effect on the kinase activity and autophagy. We further observed that suppression of the ER stress-activated autophagy via silencing of eEF-2K expression aggravated ER stress and promoted apoptotic cell death. Moreover, inhibiting eEF-2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered the glioma cells 2∼3-fold more sensitive to curcumin and velcade, two cytotoxic agents that trigger anti-tumor activities by inducing ER stress. Our study indicates that REDD1-eEF-2K pathway is essential for inducing autophagy and for determining fate of tumor cells under ER stress, and suggests that inhibiting the eEF-2K-mediated autophagy can enhance ER stress and lead to greater apoptotic response, thereby potentiating the efficacy of anticancer agents. These results underscore the potential of eEF-2K as a novel target for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4985. doi:1538-7445.AM2012-4985

Compartment-specific, differential regulation of eukaryotic elongation factor 2 and its kinase within Aplysia sensory neurons

Long-term facilitation (LTF) in Aplysia is a leading model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. LTF requires translational control downstream of target of rapamycin complex 1. Our lab has previously shown that treatment with the facilitating neurotransmitter, 5-hydroxytryptamine (5-HT), causes a target of rapamycin complex 1-mediated decrease in phosphorylation of eukaryotic elongation factor 2 (eEF2) within the neurites of sensory neurons involved in LTF. Here, we characterize the Aplysia orthologue of eEF2 kinase (eEF2K). We show that the Aplysia eEF2K orthologue contains an S6 kinase phosphorylation site and that a serine-to-alanine mutation at this site blocks the ability of 5-HT to decrease eEF2 phosphorylation in neurites. We also show that within the soma, 5-HT has the opposite effect, decreasing eEF2K phosphorylation at the S6 kinase site and, concomitantly, increasing eEF2 phosphorylation. Surprisingly, while eEF2K over-expression inhibits translation of a marker for internal ribosome entry site-dependent translation, it stimulates the translation of a marker for cap-dependent translation. This study demonstrates that eEF2 is differentially regulated in separate compartments and contributes to a growing body of evidence that inhibition of elongation can stimulate the translation of certain transcripts.

Nonlethal aluminum maltolate can reduce brain-derived neurotrophic factor-induced Arc expression through interrupting the ERK signaling in SH-SY5Y neuroblastoma cells

Although many studies have demonstrated that aluminum (Al) exposure impairs learning and memory, its underlying mechanism is still uncertain. Long-lasting forms of synaptic plasticity that underlie memory are dependent on new protein synthesis. In particular, activity-regulated cytoskeleton-associated protein (Arc) has a versatile role in synaptic plasticity, and its synthesis can be induced by brain-derived neurotrophic factor (BDNF). BDNF-induced Arc expression has been suggested to play a fundamental role in the stabilization of synaptic plasticity. In the present study, the pretreatment of Al(malt) 3 at nonlethal level (200 μM, 24 h) significantly reduced BDNF (10 ng/ml, 1 h)-induced Arc expression in SH-SY5Y human neuroblastoma cells. BDNF-induced activation of ERK but not PI3K signaling pathway was interfered with the Al(malt) 3 pretreatment, resulting in the subsequent reduction of BDNF-induced phosphorylation of 4EBP1, p70S6K, and eIF4E. Reduced phospho-4EBP1 and phospho-eIF4E hindered the initiation step of translation, which may lead to a reduction in BDNF-induced Arc expression. However, reduced phospho-p70S6K did not influence the phosphorylation of eEF2K and eEF2, indicating no significant effect on BDNF-enhanced translation elongation. Therefore, even at nonlethal level, Al(malt) 3 pretreatment reduced BDNF-induced Arc expression, which was caused by interrupting the ERK signaling pathway as well as the subsequent translation initiation.

It's no go for protein when it's all go

It's no go for protein when it's all go

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes

1 Bisindolylmaleimide inhibitors of protein kinase C (PKC), such as GF109203X and Ro31-8220, have been used to investigate the roles of PKC isoforms in many cellular processes in cardiac myocytes, but these agents may also inhibit p90RSK activity. 2 In in vitro kinase assays utilising 50 microM [ATP], GF109203X and Ro31-8220 inhibited p90RSK isoforms (IC50 values for inhibition of RSK1, RSK2 and RSK3, respectively, were 610, 310 and 120 nM for GF109203X, and 200, 36 and 5 nM for Ro31-8220) as well as classical and novel PKC isoforms (IC50 values for inhibition of PKCalpha and PKCepsilon, respectively, were 8 and 12 nM for GF109203X, and 4 and 8 nM for Ro31-8220). 3 At physiological [ATP] (5 mM), both GF109203X and Ro31-8220 exhibited reduced potency as inhibitors of RSK2, PKCalpha and PKCepsilon (IC50 values of 7400, 310 and 170 nM, respectively, for GF109203X, and 930, 150 and 140 nM, respectively, for Ro31-8220), with the latter agent retaining its relatively greater potency. 4 To determine the effects of GF109203X and Ro31-8220 on p90RSK activity in cultured adult rat ventricular myocytes (ARVM), phosphorylation of the eukaryotic elongation factor 2 kinase (eEF2K) at Ser366, a known p90RSK target, was used as the index of such activity. Adenoviral expression of a constitutively active form of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) was used to induce PKC-independent p90RSK activation and downstream phosphorylation of eEF2K. 5 eEF2K phosphorylation was abolished by U0126 (1 microM), a selective inhibitor of MEK1, and was significantly reduced by GF109203X at > or =3 microM and by Ro31-8220 at > or =1 microM. At 1 microM, both agents inhibited PMA-induced PKC activity in ARVM. 6 These data show that GF109203X and Ro31-8220 inhibit various isoforms of PKC and p90RSK in vitro and in intact ARVM, with the former agent exhibiting relatively greater selectivity for PKC.

A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta.

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.