TGFβ enforces activation of eukaryotic elongation factor-2 (eEF2) via inactivation of eEF2 kinase by p90 ribosomal S6 kinase (p90Rsk) to induce mesangial cell hypertrophy

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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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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Eukaryotic elongation factor 2 (eEF2) kinase/eEF2 plays protective roles against glucose deprivation-induced cell death in H9c2 cardiomyoblasts

APOPTOSIS ◽

10.1007/s10495-019-01525-z ◽

2019 ◽

Vol 24 (3-4) ◽

pp. 359-368 ◽

Cited By ~ 4

Author(s):

Satoshi Kameshima ◽

Muneyoshi Okada ◽

Hideyuki Yamawaki

Keyword(s):

Cell Death ◽

Elongation Factor ◽

Glucose Deprivation ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

H9c2 Cardiomyoblasts ◽

Eef2 Kinase

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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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Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and −insensitive pathways

Biochemical Journal ◽

10.1042/bj20020916 ◽

2002 ◽

Vol 367 (2) ◽

pp. 525-532 ◽

Cited By ~ 63

Author(s):

Axel KNEBEL ◽

Claire E. HAYDON ◽

Nick MORRICE ◽

Philip COHEN

Keyword(s):

Protein Kinase ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Tnf Α ◽

High Concentrations ◽

Eef2 Kinase ◽

Sb 203580

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38Δ) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360—4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370—4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-α (TNF-α) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-α and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their ‘upstream’ activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38Δ in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-α or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-α inhibit eEF2 kinase via the phosphorylation of Ser-359.

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