Elongation factor eEF2 kinase and autophagy jointly promote survival of cancer cells

Cells within solid tumours can become deprived of nutrients; in order to survive, they need to invoke mechanisms to conserve these resources. Using cancer cells in culture in the absence of key nutrients, we have explored the roles of two potential survival mechanisms, autophagy and elongation factor 2 kinase (eEF2K), which, when activated, inhibits the resource-intensive elongation stage of protein synthesis. Both processes are regulated through the nutrient-sensitive AMP-activated protein kinase and mechanistic target of rapamycin complex 1 signalling pathways. We find that disabling both autophagy and eEF2K strongly compromises the survival of nutrient-deprived lung and breast cancer cells, whereas, for example, knocking out eEF2K alone has little effect. Contrary to some earlier reports, we find no evidence that eEF2K regulates autophagy. Unexpectedly, eEF2K does not facilitate survival of prostate cancer PC3 cells. Thus, eEF2K and autophagy enable survival of certain cell-types in a mutually complementary manner. To explore this further, we generated, by selection, cells which were able to survive nutrient starvation even when autophagy and eEF2K were disabled. Proteome profiling using mass spectrometry revealed that these ‘resistant’ cells showed lower levels of diverse proteins which are required for energy-consuming processes such as protein and fatty acid synthesis, although different clones of ‘resistant cells’ appear to adapt in dissimilar ways. Our data provide further information of the ways that human cells cope with nutrient limitation and to understanding of the utility of eEF2K as a potential target in oncology.

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Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

Molecular and Cellular Biology ◽

10.1128/mcb.01457-14 ◽

2015 ◽

Vol 35 (10) ◽

pp. 1788-1804 ◽

Cited By ~ 38

Author(s):

Claire E. J. Moore ◽

Halina Mikolajek ◽

Sergio Regufe da Mota ◽

Xuemin Wang ◽

Justin W. Kenney ◽

...

Keyword(s):

Elongation Factor ◽

Translation Elongation ◽

Calmodulin Binding ◽

Calcium Calmodulin Dependent ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Energy Consuming ◽

Proline Hydroxylation ◽

Eef2 Kinase

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

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Faculty Opinions recommendation of beta-Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium-independent eEF2 kinase activity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1159737.621143 ◽

2009 ◽

Author(s):

Marc Jeschke

Keyword(s):

Kinase Activity ◽

Elongation Factor ◽

Adrenergic Agonists ◽

Beta Adrenergic ◽

Beta Adrenergic Agonists ◽

Elongation Factor 2 ◽

Eef2 Kinase

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A Novel mTOR-Regulated Phosphorylation Site in Elongation Factor 2 Kinase Modulates the Activity of the Kinase and Its Binding to Calmodulin

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2986-2997.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2986-2997 ◽

Cited By ~ 178

Author(s):

Gareth J. Browne ◽

Christopher G. Proud

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Phosphorylation Site ◽

Elongation Factor ◽

Mtor Pathway ◽

Mtor Signaling ◽

Chain Elongation ◽

Binding Motif ◽

Elongation Factor 2 ◽

Eef2 Kinase

ABSTRACT Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.

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Abstract 4054: Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells

10.1158/1538-7445.am2014-4054 ◽

2014 ◽

Author(s):

Ahmed A. Ashour ◽

Sultan N. Alpay ◽

Nilgun Gurbuz ◽

Abdel-Aziz H. Abdel-Aziz ◽

Ahmed M. Mansour ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cell Invasion ◽

Epithelial Mesenchymal Transition ◽

Elongation Factor ◽

Human Pancreatic Cancer ◽

Mesenchymal Transition ◽

Pancreatic Cancer Cells ◽

Elongation Factor 2

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FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells

Oncotarget ◽

10.18632/oncotarget.7672 ◽

2016 ◽

Vol 7 (13) ◽

pp. 16619-16635 ◽

Cited By ~ 41

Author(s):

Zuhal Hamurcu ◽

Ahmed Ashour ◽

Nermin Kahraman ◽

Bulent Ozpolat

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cells ◽

Triple Negative ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor

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

Circulation Research ◽

10.1161/res.119.suppl_1.274 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Satoshi Kameshima ◽

Muneyoshi Okada ◽

Shiro Ikeda ◽

Yuki Watanabe ◽

Hideyuki Yamawaki

Keyword(s):

Protein Synthesis ◽

Cardiac Hypertrophy ◽

Elongation Factor ◽

Protein Translation ◽

Cardiomyocyte Hypertrophy ◽

Specific Substrate ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Eef2 Kinase

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.

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A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase α1 in liver and lung

Biochemical Journal ◽

10.1042/bj20050269 ◽

2005 ◽

Vol 392 (1) ◽

pp. 201-209 ◽

Cited By ~ 20

Author(s):

Russell M. Crawford ◽

Kate J. Treharne ◽

O. Giles Best ◽

Richmond Muimo ◽

Claudia E. Riemen ◽

...

Keyword(s):

Protein Kinase ◽

Mammalian Cells ◽

Nucleoside Diphosphate Kinase ◽

Cell Types ◽

Acid Synthesis ◽

Cellular Fatty Acid ◽

Energy Status ◽

Amp Activated Protein Kinase

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (∼16–20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK α1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the α1 (but not α2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK α1-mediated phosphorylation is present in mammalian cells.

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Activation of AMP-Activated Protein Kinase Leads to the Phosphorylation of Elongation Factor 2 and an Inhibition of Protein Synthesis

Current Biology ◽

10.1016/s0960-9822(02)01077-1 ◽

2002 ◽

Vol 12 (16) ◽

pp. 1419-1423 ◽

Cited By ~ 318

Author(s):

Sandrine Horman ◽

Gareth J. Browne ◽

Ulrike Krause ◽

Jigna V. Patel ◽

Didier Vertommen ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Inhibition Of Protein Synthesis ◽

Amp Activated Protein Kinase

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Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A–Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.01270-12 ◽

2012 ◽

Vol 33 (3) ◽

pp. 596-604 ◽

Cited By ~ 44

Author(s):

Asli A. Hizli ◽

Yong Chi ◽

Jherek Swanger ◽

John H. Carter ◽

Yi Liao ◽

...

Keyword(s):

Cell Cycle ◽

Elongation Factor ◽

Cyclin A ◽

Cyclin Dependent Kinase ◽

Regulatory Pathways ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Eef2 Kinase ◽

Cell Cycle Machinery

ABSTRACTProtein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A–cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylationin vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2Kin vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.

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