scholarly journals Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

2000 ◽  
Vol 20 (10) ◽  
pp. 3558-3567 ◽  
Author(s):  
Isabelle Mothe-Satney ◽  
Daqing Yang ◽  
Patrick Fadden ◽  
Timothy A. J. Haystead ◽  
John C. Lawrence
Keyword(s):  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Translational Repression ◽  
Phosphorylation Sites ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Dependent Processes ◽  
Constitutive Phosphorylation

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

2000 ◽  
Vol 279 (4) ◽  
pp. E715-E729 ◽  
Author(s):  
O. Jameel Shah ◽  
Joshua C. Anthony ◽  
Scot R. Kimball ◽  
Leonard S. Jefferson
Keyword(s):  
Translational Control ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Translational Repressor ◽  
Initiation Factors ◽  
Initiation Phase ◽  
Eukaryotic Initiation Factor 4E ◽  
Integration Sites ◽  
A Site

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.

scholarly journals Caspase Cleavage of Initiation Factor 4E-Binding Protein 1 Yields a Dominant Inhibitor of Cap-Dependent Translation and Reveals a Novel Regulatory Motif

2002 ◽  
Vol 22 (6) ◽  
pp. 1674-1683 ◽  
Author(s):  
Andrew R. Tee ◽  
Christopher G. Proud
Keyword(s):  
Potent Inhibitor ◽  
Chimeric Protein ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Insulin Stimulation ◽  
Terminal Sequence ◽  
Regulatory Motif ◽  
Caspase Cleavage ◽  
Eukaryotic Initiation Factor 4E

ABSTRACT Eukaryotic initiation factor 4E (eIF4E) binding proteins (4E-BPs) regulate the assembly of initiation complexes required for cap-dependent mRNA translation. 4E-BP1 undergoes insulin-stimulated phosphorylation, resulting in its release from eIF4E, allowing initiation complex assembly. 4E-BP1 undergoes caspase-dependent cleavage in cells undergoing apoptosis. Here we show that cleavage occurs after Asp24, giving rise to the N-terminally truncated polypeptide Δ4E-BP1, which possesses the eIF4E-binding site and all the known phosphorylation sites. Δ4E-BP1 binds to eIF4E and fails to become sufficiently phosphorylated upon insulin stimulation to bring about its release from eIF4E. Therefore, Δ4E-BP1 acts as a potent inhibitor of cap-dependent translation. Using a mutagenesis approach, we identify a novel regulatory motif of four amino acids (RAIP) which lies within the first 24 residues of 4E-BP1 and which is necessary for efficient phosphorylation of 4E-BP1. This motif is conserved among sequences of 4E-BP1 and 4E-BP2 but is absent from 4E-BP3. Insulin increased the phosphorylation of 4E-BP3 but not sufficiently to cause its release from eIF4E. However, a chimeric protein that was generated by replacing the N terminus of 4E-BP3 with the N-terminal sequence of 4E-BP1 (containing this RAIP motif) underwent a higher degree of phosphorylation and was released from eIF4E. This suggests that the N-terminal sequence of 4E-BP1 is required for optimal regulation of 4E-BPs by insulin.

scholarly journals Distinct Signaling Events Downstream of mTOR Cooperate To Mediate the Effects of Amino Acids and Insulin on Initiation Factor 4E-Binding Proteins

2005 ◽  
Vol 25 (7) ◽  
pp. 2558-2572 ◽  
Author(s):  
Xuemin Wang ◽  
Anne Beugnet ◽  
Mirei Murakami ◽  
Shinya Yamanaka ◽  
Christopher G. Proud
Keyword(s):  
Amino Acids ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Tissue Growth ◽  
Mtor Signaling ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor 4E ◽  
Terminal Site ◽  
New Strategies

ABSTRACT Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.

scholarly journals Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

2003 ◽  
Vol 163 (6) ◽  
pp. 1197-1204 ◽  
Author(s):  
James E. Wilhelm ◽  
Meredith Hilton ◽  
Quinlan Amos ◽  
William J. Henzel
Keyword(s):  
Translational Control ◽  
Mrna Localization ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Microtubule Transport ◽  
Rnp Complex ◽  
Made In

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end–directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.

scholarly journals Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo

2001 ◽  
Vol 268 (20) ◽  
pp. 5375-5385 ◽  
Author(s):  
Linda McKendrick ◽  
Simon J. Morley ◽  
Virginia M. Pain ◽  
Rosemary Jagus ◽  
Bhavesh Joshi
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E

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.

Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1129-1138 ◽  
Author(s):  
Y.S. Lie ◽  
P.M. Macdonald
Keyword(s):  
Translational Control ◽  
Untranslated Region ◽  
Rna Binding ◽  
Mrna Translation ◽  
Posterior Pole ◽  
Translational Repression ◽  
Yeast Two Hybrid ◽  
Functional Interaction ◽  
Translational Repressor ◽  
Two Hybrid

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3′ untranslated region of the oskar mRNA. To begin to understand how Bruno acts in translational repression, we performed a yeast two-hybrid screen to identify Bruno-interacting proteins. One interactor, described here, is the product of the apontic gene. Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. Given this interaction, Apontic is likely to act together with Bruno in translational repression of oskar mRNA. Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the oskar mRNA 3′ untranslated region.

scholarly journals Cytoplasmic Expression of the ALS/FTD-Related Protein TDP-43 Decreases Global Translation Both in vitro and in vivo

2020 ◽  
Vol 14 ◽  
Author(s):  
Santiago E. Charif ◽  
Luciana Luchelli ◽  
Antonella Vila ◽  
Matías Blaustein ◽  
Lionel M. Igaz
Keyword(s):  
De Novo ◽  
Brain Slices ◽  
Mrna Translation ◽  
Hek293 Cells ◽  
Cytoplasmic Inclusions ◽  
Cytoplasmic Expression ◽  
Global Translation

TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.

scholarly journals 5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

2020 ◽  
Vol 295 (33) ◽  
pp. 11693-11706 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Specific Subset ◽  
Independent Translation

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5′ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5′ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5′ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

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