scholarly journals Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

2004 ◽  
Vol 18 (5) ◽  
pp. 1301-1312 ◽  
Author(s):  
Kathryn A. Nguyen ◽  
Sharon J. Santos ◽  
Marit K. Kreidel ◽  
Alejandro L. Diaz ◽  
Rodolfo Rey ◽  
...  
Keyword(s):  
Cell Line ◽  
Translation Initiation ◽  
Translational Control ◽  
Promoter Activity ◽  
Gnrh Receptor ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Eukaryotic Initiation Factor ◽  
Western Blots ◽  
Stimulation Of

Abstract The hypothalamic neuropeptide hormone GnRH is the central regulator of reproductive function. GnRH stimulates the synthesis and release of the gonadotropins LH and FSH by the gonadotropes of the anterior pituitary through activation of the G-protein-coupled GnRH receptor. In this study, we investigated the role of translational control of hormone synthesis by the GnRH receptor in the novel gonadotrope cell line LβT2. Using immunohistochemical and RIA studies with this model, we show that acute GnRH-induced synthesis and secretion of LH are dependent upon new protein synthesis but not new mRNA synthesis. We examined the response to GnRH and found that activation of cap-dependent translation occurs within 4 h. LHβ promoter activity was also examined, and we found no increases in LHβ promoter activity after 6 h of GnRH stimulation. Additionally, we show that increased phosphorylation of translation initiation proteins, 4E-binding protein 1, eukaryotic initiation factor 4E, and eukaryotic initiation factor 4G, occur in a dose- and time-dependent manner in response to GnRH stimulation. Quantitative luminescent image analysis of Western blots shows that 10 nm GnRH is sufficient to cause a maximal increase in factor phosphorylation, and maximal responses occur within 30 min of stimulation. Further, we demonstrate that the MAPK kinase inhibitor, PD 98059, abolishes the GnRH-mediated stimulation of a cap-dependent translation reporter. More specifically, we demonstrate that PD 98059 abolishes the GnRH-mediated stimulation of a downstream target of the ERK pathway, MAPK-interacting kinase. Based on these findings, we conclude that acute GnRH stimulation of LβT2 cells increases translation initiation through ERK signaling. This may contribute to the acute increases in LHβ subunit production.

scholarly journals Effect of Brain Ischemia and Reperfusion on the Localization of Phosphorylated Eukaryotic Initiation Factor 2α

1997 ◽  
Vol 17 (12) ◽  
pp. 1291-1302 ◽  
Author(s):  
Donald J. DeGracia ◽  
Jonathon M. Sullivan ◽  
Robert W. Neumar ◽  
Sarah S. Alousi ◽  
Katie R. Hikade ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Initiation Rate ◽  
Western Blots ◽  
Α Subunit ◽  
Neuronal Nuclei ◽  
Initiation Factor 2 ◽  
Brain Reperfusion

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the α-subunit of eukaryotic initiation factor 2 (eIF-2α), we used an antibody specific to phosphorylated eIF-2α [eIF-2(αP)] to study the regional and cellular distribution of eIF-2(αP) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that ~1% of all eIF-2α is phosphorylated in controls, eIF-2(αP) is not reduced by up to 30 minutes of ischemia, and eIF-2(αP) is increased ~20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(αP) to astrocytes in normal brains, a massive increase in eIF-2(αP) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(αP) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(αP) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(αP) inhibits translation initiation, make a compelling case that eIF-2(αP) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.

scholarly journals Interactome Mapping of eIF3A in a Colon Cancer and an Immortalized Embryonic Cell Line Using Proximity-Dependent Biotin Identification

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1293
Author(s):  
Diep-Khanh Vo ◽  
Alexander Engler ◽  
Darko Stoimenovski ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Line ◽  
Translation Initiation ◽  
Rna Binding ◽  
Embryonic Cell ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Interacting Proteins ◽  
Eukaryotic Initiation Factor ◽  
Embryonic Cell Line

Translation initiation comprises complex interactions of eukaryotic initiation factor (eIF) subunits and the structural elements of the mRNAs. Translation initiation is a key process for building the cell’s proteome. It not only determines the total amount of protein synthesized but also controls the translation efficiency for individual transcripts, which is important for cancer or ageing. Thus, understanding protein interactions during translation initiation is one key that contributes to understanding how the eIF subunit composition influences translation or other pathways not yet attributed to eIFs. We applied the BioID technique to two rapidly dividing cell lines (the immortalized embryonic cell line HEK-293T and the colon carcinoma cell line HCT-166) in order to identify interacting proteins of eIF3A, a core subunit of the eukaryotic initiation factor 3 complex. We identified a total of 84 interacting proteins, with very few proteins being specific to one cell line. When protein biosynthesis was blocked by thapsigargin-induced endoplasmic reticulum (ER) stress, the interacting proteins were considerably smaller in number. In terms of gene ontology, although eIF3A interactors are mainly part of the translation machinery, protein folding and RNA binding were also found. Cells suffering from ER-stress show a few remaining interactors which are mainly ribosomal proteins or involved in RNA-binding.

scholarly journals Translational Control by Neuroguidin, a Eukaryotic Initiation Factor 4E and CPEB Binding Protein

2006 ◽  
Vol 26 (11) ◽  
pp. 4277-4287 ◽  
Author(s):  
Mi-Young Jung ◽  
Lori Lorenz ◽  
Joel D. Richter
Keyword(s):  
Neural Crest ◽  
Neural Tube ◽  
Neural Development ◽  
Translational Control ◽  
Binding Protein ◽  
Neural Tube Closure ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E

ABSTRACT CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.

scholarly journals Insulin and IGF-I stimulate the formation of the eukaryotic initiation factor 4F complex and protein synthesis in C2C12 myotubes independent of availability of external amino acids

2005 ◽  
Vol 185 (2) ◽  
pp. 275-289 ◽  
Author(s):  
Wei-Hua Shen ◽  
David W Boyle ◽  
Paul Wisniowski ◽  
Aashia Bade ◽  
Edward A Liechty
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Glycogen Synthase ◽  
Initiation Factor ◽  
C2c12 Myotubes ◽  
Dependent Manner ◽  
Eukaryotic Initiation Factor ◽  
Akt Phosphorylation ◽  
Igf I

The objective of this study was to investigate the effect of insulin and IGF-I on protein synthesis and translation initiation in C2C12 myotubes in nutrient-deprived Dulbecco’s phosphate buffered saline (DPBS). The results showed that insulin and IGF-I increased protein synthesis by 62% and 35% respectively in DPBS, and the effect was not affected by rapamycin, but was blocked by LY294002. Insulin and IGF-I stimulated eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP1) phosphorylation in a dose-dependent manner, and the stimulation was independent of availability of external amino acids. Both LY294002 and rapamycin blocked the insulin and IGF-I-induced increases in 4EBP1 phosphorylation. The results also showed that insulin and IGF-I were able to stimulate PKB/Akt phosphorylation, glycogen synthase kinase (GSK) 3β phosphorylation and mTOR phosphorylation in DPBS. Insulin and IGF-I increased the amount of eIF4G associated with eIF4E in nutrient-deprived C2C12 myotubes. The amount of 4EBP1 associated with eIF4E was decreased after insulin or IGF-I stimulation. We conclude that in C2C12 myotubes, insulin and IGF-I may regulate protein synthesis and translation initiation independent of external amino acid supply via the phosphatidylinositol-3 kinase-PKB/Akt-mTOR pathway.

scholarly journals Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

1997 ◽  
Vol 17 (12) ◽  
pp. 6876-6886 ◽  
Author(s):  
S Z Tarun ◽  
A B Sachs
Keyword(s):  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

scholarly journals Focus on Translation Initiation of the HIV-1 mRNAs

2018 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
Sylvain de Breyne ◽  
Théophile Ohlmann
Keyword(s):  
Translation Initiation ◽  
Translational Control ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Cellular Environment ◽  
Key Steps ◽  
Hiv 1

To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

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