Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development

Webb, Tom R.; Cross, Sally H.; McKie, Lisa; Edgar, Ruth; Vizor, Lucie; Harrison, Jackie; Peters, Jo; Jackson, Ian J.

doi:10.1242/jcs.035550

Cited by 63 publications

(67 citation statements)

References 35 publications

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“…Diphthamide deficiency can be induced by deletion of the genes involved in the diphthamide biosynthesis (20)(21)(22). However, these genes may have pleiotropic functions, as seen with Dph3 involvement in tRNA anticodon modifications (23).…”

Section: Resultsmentioning

confidence: 99%

“…OVCA1 −/− and Dph4 −/− mice phenocopied each other with a delay in embryo development starting as early as E8.5, and having 100% prenatal lethality (20)(21)(22) (Table S2). Interestingly, the eEF2 G717R/G717R mice displayed a milder phenotype, with a small fraction surviving to adulthood.…”

Section: Resultsmentioning

confidence: 99%

“…Surprisingly, none of the diphthamide-deficient mutants identified in either yeast or CHO cells exhibit strong phenotypes (5, 6, 8-10, 18, 19), demonstrating that the diphthamide modification is not essential for cell survival. However, recent studies of OVCA1 (Dph1), Dph3, and Dph4 knockout mice have shed light on the physiological role of diphthamide in multicellular organisms (20)(21)(22). Remarkably, OVCA1-, Dph3-, and Dph4-null mice die during embryonic development, suggesting an important role of diphthamide in embryonic development.…”

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confidence: 99%

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Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Liu

Bachran

Gupta

et al. 2012

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

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To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly 717 Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly 717 -to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1 −/− mice, eEF2 G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2 G717R/G717R mice showed milder phenotypes than OVCA1 −/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2 G717R/G717R /OVCA1 −/− double mutant mice displayed the milder phenotypes of the eEF2 G717R/G717R mice, suggesting that the embryonic lethality of OVCA1 −/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly 717 -to-Arg mutation cannot rescue this defect. E ukaryotic elongation factor 2 (eEF2) is a member of the GTPbinding translation elongation factor family, and an essential factor for protein synthesis and cell survival. eEF2 drives the GTP-dependent translocation of the nascent polypeptide chain from the A site to the P site of the ribosome and advances mRNA by three bases during the elongation cycle of protein synthesis (1). eEF2 is highly homologous in all eukaryotes. In fact, eEF2 of humans, rats, mice, hamsters, and other mammals have exactly the same amino acid sequence. Intriguingly, all eukaryotic eEF2 proteins contain a unique posttranslationally modified histidine residue termed diphthamide (2, 3). Diphthamide modification occurs after eEF2 is translated and is irreversible, marking the completion of the biosynthesis of eEF2.Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig. S1), thus inactivating eEF2, halting cellular protein synthesis, and causing cell death.Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective a...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Liu

Bachran

Gupta

et al. 2012

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

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“…Homozygous Wt1 mutant embryos die between E13 and E15 (Kreidberg et al 1993). Dph4 homozygotes die prior to E14.5 when carried on a C3H/ HeH genetic background (Webb et al 2008). Homozygous Pax6 intragenic null mutations are lethal shortly after birth.…”

Section: Discussionmentioning

confidence: 99%

“…Sey-H (Hogan et al 1986;Kent et al 1997;Kleinjan et al 2002;Webb et al 2008) and Pax6 Sey-Dey (Theiler et al 1978;Hogan et al 1987;Glaser et al 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations.…”

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confidence: 99%

Analysis of Pax6 Contiguous Gene Deletions in the Mouse, Mus musculus, Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

et al. 2009

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In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca 12 -binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

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Proteomics analysis of in vitro protein methylation during Src‐induced transformation

Chiou¹,

Fu²,

Lin³

et al. 2012

Electrophoresis

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Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and β-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of β-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.

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Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development

Cited by 63 publications

References 35 publications

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Analysis of Pax6 Contiguous Gene Deletions in the Mouse, Mus musculus, Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

Proteomics analysis of in vitro protein methylation during Src‐induced transformation

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