Prolyl Hydroxylase-dependent Modulation of Eukaryotic Elongation Factor 2 Activity and Protein Translation under Acute Hypoxia

Romero-Ruíz, Antonio; Bautista, Lucía; Navarro, Virginia; Heras‐Garvin, Antonio; March-Díaz, Rosana; Castellano, Antonio; Gómez-Díaz, Raquel; Castro, María José; Berra, Edurne; López‐Barneo, José; Pascual, Alberto

doi:10.1074/jbc.m111.299180

Cited by 32 publications

(27 citation statements)

References 42 publications

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“…1A). Our findings are distinct from those of an earlier study, which reported a rapid increase in eEF2K phosphorylation during hypoxia (17); our data also revealed the existence of a second, slower, and much more marked rise in eEF2 phosphorylation. The rapid changes in eEF2 phosphorylation observed in the previous study likely reflect different regulatory inputs into eEF2 (17).…”

Section: Hypoxia Elicits a Delayed Increased In Eef2 Phosphorylationcontrasting

confidence: 57%

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

Moore

Mikolajek

Mota

et al. 2015

Molecular and Cellular Biology

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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 ␣-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. Many cells require aerobic metabolism to generate energy, necessitating an adequate supply of oxygen. Protein synthesis, especially translation elongation, is a major energy-consuming process, and translation elongation uses both ATP (for aminoacyl-tRNA charging) and GTP (at least two GTP equivalents are used during each round of the elongation process). Overall, at least four ATP equivalents are used for each amino acid added to the growing chain during elongation. Elongation rates can be regulated through the phosphorylation of eukaryotic elongation factor 2 (eEF2) (1). Phosphorylation of eEF2 on Thr56 by eEF2 kinase (eEF2K) inhibits its ability to interact with ribosomes (2), thereby impairing translation elongation. Indeed, a range of studies has shown that increased phosphorylation of eEF2 is associated with slower ribosomal movement along the mRNA (e.g., see references 3 to 5).eEF2K interacts with calmodulin (CaM) through a binding site which lies almost immediately N terminal to its catalytic domain (6, 7). The catalytic domain belongs to the small group of (six) mammalian ␣-kinases, rather than the main protein kinase superfamily; ␣-kinases show no sequence homology and only limited three-dimensional structural homology to other protein kinases (8, 9). eEF2K activity is regulated through several signaling pathways linked, e.g., to nutrient availability; these include signaling through the mammalian target of rapamycin complex 1 (mTORC1), which represses eEF2K activity, and the AMPactivated protein kinase (AMPK), a key cellular energy sensor (10) which causes activation of eEF2K (11, 12), probably in part by inhibiting mTORC1 signaling. Both inputs operate such th...

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Section: Hypoxia Elicits a Delayed Increased In Eef2 Phosphorylationcontrasting

confidence: 57%

“…The activation of AMPK or inhibition of mTORC1 signaling, each of which can acutely activate eEF2K, likely serves to provide a short-term modulation of eEF2K activity. The long-term adaptive response revealed in this study is distinct from the rapid responses to PHD inhibition reported recently (17).…”

Section: Discussionmentioning

confidence: 40%

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

Moore

Mikolajek

Mota

et al. 2015

Molecular and Cellular Biology

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“…Their role in HIF-α hydroxylation evolved in animals, possibly via fusion of a basic helix-loop-helix domain transcription factor with an EF-Tu, collagen, or Skp1-related or Skp1-derived substrate. The observation that PHD2 interacts with, but apparently does not hydroxylate, eEF2, the eukaryotic counterpart to prokaryotic EF-G, an EF-Tu homolog (15), is interesting because it may reflect an ancestral relationship between PPHD and EF-Tu. Competition between HIF-α and eEF2 [and potentially other PHD substrates; e.g., pyruvate kinase M2 (38) or centrosomal protein 192 (39)], may help regulate the hypoxic response in higher animals.…”

Section: Discussionmentioning

confidence: 99%

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

Scotti

Leung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolylhydroxylation in human hypoxia sensing has ancient origins.T he hypoxia-inducible transcription factor (HIF) is a major regulator of the response to limited oxygen availability in humans and other animals (1-3). A hypoxia-sensing component of the HIF system is provided by 2-oxoglutarate (2OG)-dependent and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydroxylation of HIF-α subunits, a posttranslational modification that enhances binding of HIF-α to the von Hippel-Lindau protein (pVHL), so targeting HIF-α for proteasomal degradation. The HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG oxygenases that catalyze prolyl-hydroxylation, which also includes the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydroxylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hydroxylation of Skp1, a ubiquitin ligase subunit (5-9). The HIF-PHD-VHL triad is likely present in all animals, but probably not in other organisms (3). However, structurally informed bioinformatic analyses imply the presence of PHD homologs in bacteria (10, 11), including in Pseudomonas spp, suggesting PHDs may have ancient origins. ResultsPseudomonas spp. Cont...

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“…Hypoxia (0.5-1.5% O 2 ) inhibits protein synthesis through suppression of multiple key regulators of eIF2␣, eEF2, p70 S6 , mTOR, and rpS6 (4,(13)(14)(15). Moderate hypoxia (1.5% O 2 ) in combination with serum deprivation effectively inhibits mTOR activity and causes hypophosphorylation of the mTOR substrates 4E-BP1and S6K in normal cells (16).…”

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confidence: 99%

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Papadopoulos

Hagner

et al. 2013

Journal of Biological Chemistry

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Background: Hypoxia promotes tumor growth, but connections to translation mechanisms are obscure. Results: Hypoxia-enhanced tumorsphere growth of breast cancer cells is HIF-1␣-dependent, and HIF-1␣ up-regulates eIF4E1 in hypoxic cancer cells. Conclusion: HIF-1␣ promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1 in cancer cells at hypoxia. Significance: Our study provides new insights into the translation mechanisms in cancer cells under low O 2 concentrations.

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Prolyl Hydroxylase-dependent Modulation of Eukaryotic Elongation Factor 2 Activity and Protein Translation under Acute Hypoxia

Cited by 32 publications

References 42 publications

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

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

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

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