scholarly journals Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm

2015 ◽  
Vol 212 (4) ◽  
pp. 581-596 ◽  
Author(s):  
Stephen P. Methot ◽  
Ludivine C. Litzler ◽  
Felipe Trajtenberg ◽  
Astrid Zahn ◽  
Francis Robert ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Elongation Factor ◽  
Chromosomal Translocation ◽  
Heat Shock Protein 90 ◽  
Free Form ◽  
Translation Elongation ◽  
Class Switch ◽  
Eukaryotic Elongation Factor ◽  
Activation Induced Deaminase ◽  
Functional Relevance

Activation-induced deaminase (AID) initiates mutagenic pathways to diversify the antibody genes during immune responses. The access of AID to the nucleus is limited by CRM1-mediated nuclear export and by an uncharacterized mechanism of cytoplasmic retention. Here, we define a conformational motif in AID that dictates its cytoplasmic retention and demonstrate that the translation elongation factor eukaryotic elongation factor 1 α (eEF1A) is necessary for AID cytoplasmic sequestering. The mechanism is independent of protein synthesis but dependent on a tRNA-free form of eEF1A. Inhibiting eEF1A prevents the interaction with AID, which accumulates in the nucleus and increases class switch recombination as well as chromosomal translocation byproducts. Most AID is associated to unspecified cytoplasmic complexes. We find that the interactions of AID with eEF1A and heat-shock protein 90 kD (HSP90) are inversely correlated. Despite both interactions stabilizing AID, the nature of the AID fractions associated with HSP90 or eEF1A are different, defining two complexes that sequentially produce and store functional AID in the cytoplasm. In addition, nuclear export and cytoplasmic retention cooperate to exclude AID from the nucleus but might not be functionally equivalent. Our results elucidate the molecular basis of AID cytoplasmic retention, define its functional relevance and distinguish it from other mechanisms regulating AID.

scholarly journals Insights Into the Pathologic Roles and Regulation of Eukaryotic Elongation Factor-2 Kinase

2021 ◽  
Vol 8 ◽  
Author(s):  
Darby J. Ballard ◽  
Hao-Yun Peng ◽  
Jugal Kishore Das ◽  
Anil Kumar ◽  
Liqing Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Elongation Factor ◽  
Protein Translation ◽  
Negative Regulator ◽  
Translation Elongation ◽  
Elongation Factor 2 ◽  
Global Rate ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor

Eukaryotic Elongation Factor-2 Kinase (eEF2K) acts as a negative regulator of protein synthesis, translation, and cell growth. As a structurally unique member of the alpha-kinase family, eEF2K is essential to cell survival under stressful conditions, as it contributes to both cell viability and proliferation. Known as the modulator of the global rate of protein translation, eEF2K inhibits eEF2 (eukaryotic Elongation Factor 2) and decreases translation elongation when active. eEF2K is regulated by various mechanisms, including phosphorylation through residues and autophosphorylation. Specifically, this protein kinase is downregulated through the phosphorylation of multiple sites via mTOR signaling and upregulated via the AMPK pathway. eEF2K plays important roles in numerous biological systems, including neurology, cardiology, myology, and immunology. This review provides further insights into the current roles of eEF2K and its potential to be explored as a therapeutic target for drug development.

scholarly journals Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

2012 ◽  
Vol 442 (3) ◽  
pp. 681-692 ◽  
Author(s):  
Sébastien Pyr Dit Ruys ◽  
Xuemin Wang ◽  
Ewan M. Smith ◽  
Gaëtan Herinckx ◽  
Nusrat Hussain ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Elongation Factor ◽  
Translation Elongation ◽  
Intact Cells ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
293 Cells ◽  
Eukaryotic Elongation Factor ◽  
A Site

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.

scholarly journals An aminoacylation-dependent nuclear tRNA export pathway in yeast

2000 ◽  
Vol 14 (7) ◽  
pp. 830-840 ◽  
Author(s):  
Helge Grosshans ◽  
Ed Hurt ◽  
George Simos
Keyword(s):  
Nuclear Export ◽  
Elongation Factor ◽  
Protein Translation ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Trna Processing ◽  
Trna Aminoacylation ◽  
Trna Species ◽  
Synthetically Lethal

Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

scholarly journals Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA

2002 ◽  
Vol 21 (22) ◽  
pp. 6216-6224 ◽  
Author(s):  
Angelo Calado ◽  
Nathalie Treichel ◽  
Eva-Christina Müller ◽  
Albrecht Otto ◽  
Ulrike Kutay
Keyword(s):  
Nuclear Export ◽  
Elongation Factor ◽  
Eukaryotic Elongation Factor

scholarly journals Regulation of tRNA Bidirectional Nuclear-Cytoplasmic Trafficking in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (4) ◽  
pp. 639-649 ◽  
Author(s):  
Athulaprabha Murthi ◽  
Hussam H. Shaheen ◽  
Hsiao-Yun Huang ◽  
Melanie A. Preston ◽  
Tsung-Po Lai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Subcellular Distribution ◽  
Nuclear Export ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Trna Aminoacylation ◽  
Data Support ◽  
Constitutive Process

tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the β-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the β-importin family. The β-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5.

scholarly journals Eukaryotic Elongation Factor 3 Protects Saccharomyces cerevisiae Yeast from Oxidative Stress

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1432
Author(s):  
Karolina Gościńska ◽  
Somayeh Shahmoradi Ghahe ◽  
Sara Domogała ◽  
Ulrike Topf
Keyword(s):  
Oxidative Stress ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Cellular Protein ◽  
Stress Conditions ◽  
Translation Elongation ◽  
Protein Levels ◽  
Eukaryotic Elongation Factor ◽  
Elongation Factor 3 ◽  
Elongation Process

Translation is a core process of cellular protein homeostasis and, thus, needs to be tightly regulated. The production of newly synthesized proteins adapts to the current needs of the cell, including the response to conditions of oxidative stress. Overall protein synthesis decreases upon oxidative stress. However, the selective production of proteins is initiated to help neutralize stress conditions. In contrast to higher eukaryotes, fungi require three translation elongation factors, eEF1, eEF2, and eEF3, for protein synthesis. eEF1 and eEF2 are evolutionarily conserved, but they alone are insufficient for the translation elongation process. eEF3 is encoded by two paralogous genes, YEF3 and HEF3. However, only YEF3 is essential in yeast, whereas the function of HEF3 remains unknown. To elucidate the cellular function of Hef3p, we used cells that were depleted of HEF3 and treated with H2O2 and analyzed the growth of yeast, global protein production, and protein levels. We found that HEF3 is necessary to withstand oxidative stress conditions, suggesting that Hef3p is involved in the selective production of proteins that are necessary for defense against reactive oxygen species.

The life and death of translation elongation factor 2

2006 ◽  
Vol 34 (1) ◽  
pp. 1-6 ◽  
Author(s):  
R. Jørgensen ◽  
A.R. Merrill ◽  
G.R. Andersen
Keyword(s):  
Functional Properties ◽  
Elongation Factor ◽  
Translation Elongation ◽  
Life And Death ◽  
Peptidyl Transfer ◽  
Structural Framework ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor ◽  
Structural And Functional Properties

eEF2 (eukaryotic elongation factor 2) occupies an essential role in protein synthesis where it catalyses the translocation of the two tRNAs and the mRNA after peptidyl transfer on the 80 S ribosome. Recent crystal structures of eEF2 and the cryo-electron microscopy reconstruction of its 80 S complex now provide a substantial structural framework for dissecting the functional properties of this factor. The factor can be modified by either phosphorylation or ADP-ribosylation, which results in cessation of translation. We review the structural and functional properties of eEF2 with particular emphasis on the unique diphthamide residue, which is ADP-ribosylated by diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa.

scholarly journals Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase

2004 ◽  
Vol 199 (9) ◽  
pp. 1235-1244 ◽  
Author(s):  
Kevin M. McBride ◽  
Vasco Barreto ◽  
Almudena R. Ramiro ◽  
Pete Stavropoulos ◽  
Michel C. Nussenzweig
Keyword(s):  
B Lymphocytes ◽  
Nuclear Export ◽  
Target Gene ◽  
Somatic Hypermutation ◽  
Nuclear Export Signal ◽  
Carboxyl Terminus ◽  
Class Switch ◽  
Activation Induced Deaminase ◽  
Export Signal ◽  
Dependent Pathway

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells.

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