scholarly journals Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

1998 ◽  
Vol 18 (4) ◽  
pp. 2282-2297 ◽  
Author(s):  
Patrick R. Romano ◽  
Minerva T. Garcia-Barrio ◽  
Xiaolong Zhang ◽  
Qizhi Wang ◽  
Deborah R. Taylor ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Regulatory Region ◽  
Yeast Cells ◽  
Mass Spectrometry Analysis ◽  
Initiation Factor ◽  
Interferon Response ◽  
Activation Loop ◽  
Eukaryotic Initiation Factor

ABSTRACT The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

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 A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

1996 ◽  
Vol 16 (10) ◽  
pp. 5328-5334 ◽  
Author(s):  
N Méthot ◽  
M S Song ◽  
N Sonenberg
Keyword(s):  
Aspartic Acid ◽  
Ribosomal Subunit ◽  
Direct Interaction ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Ribosome Binding ◽  
Initiation Factors ◽  
Self Association

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

scholarly journals GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2.

1993 ◽  
Vol 13 (6) ◽  
pp. 3541-3556 ◽  
Author(s):  
M J Marton ◽  
D Crouch ◽  
A G Hinnebusch
Keyword(s):  
Kinase Activity ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 2

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

scholarly journals Initiation of Protein Synthesis by Hepatitis C Virus Is Refractory to Reduced eIF2 · GTP · Met-tRNAiMetTernary Complex Availability

2006 ◽  
Vol 17 (11) ◽  
pp. 4632-4644 ◽  
Author(s):  
Francis Robert ◽  
Lee D. Kapp ◽  
Shakila N. Khan ◽  
Michael G. Acker ◽  
Sarah Kolitz ◽  
...  
Keyword(s):  
Cellular Response ◽  
Mrna Translation ◽  
Ternary Complexes ◽  
Initiation Factor ◽  
Interferon Response ◽  
Ternary Complex Formation ◽  
In Vivo Analysis ◽  
Hcv Ires

A cornerstone of the antiviral interferon response is phosphorylation of eukaryotic initiation factor (eIF)2α. This limits the availability of eIF2·GTP·Met-tRNAiMetternary complexes, reduces formation of 43S preinitiation complexes, and blocks viral (and most cellular) mRNA translation. However, many viruses have developed counterstrategies that circumvent this cellular response. Herein, we characterize a novel class of translation initiation inhibitors that block ternary complex formation and prevent the assembly of 43S preinitiation complexes. We find that translation driven by the HCV IRES is refractory to inhibition by these compounds at concentrations that effectively block cap-dependent translation in vitro and in vivo. Analysis of initiation complexes formed on the HCV IRES in the presence of inhibitor indicates that eIF2α and Met-tRNAiMetare present, defining a tactic used by HCV to evade part of the antiviral interferon response.

scholarly journals Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast

2006 ◽  
Vol 26 (8) ◽  
pp. 2984-2998 ◽  
Author(s):  
Klaus H. Nielsen ◽  
Leos Valášek ◽  
Caroah Sykes ◽  
Antonina Jivotovskaya ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Eukaryotic Initiation Factor ◽  
Wild Type ◽  
Leaky Scanning ◽  
Rrm Domain ◽  
Subunit B

ABSTRACT We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts− phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts− phenotype is exacerbated by tif32-Δ6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Δ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

scholarly journals The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation

2018 ◽  
Vol 38 (16) ◽  
Author(s):  
Ryo Murakami ◽  
Chingakham Ranjit Singh ◽  
Jacob Morris ◽  
Leiming Tang ◽  
Ian Harmon ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Hydrophobic Pocket ◽  
Reading Frame ◽  
Ribosomal Stalk

ABSTRACTRibosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activityin vitro. In yeast cells, the eIF5B mutation affected growth and impairedGCN4expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame inGCN4mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-inducedGCN4expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiationin vivo.

scholarly journals Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent

2001 ◽  
Vol 75 (24) ◽  
pp. 12141-12152 ◽  
Author(s):  
Daniel R. Gallie
Keyword(s):  
Large Subunit ◽  
Tobacco Etch Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4G ◽  
Independent Translation

ABSTRACT The 5′ leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

scholarly journals Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

2015 ◽  
Vol 89 (9) ◽  
pp. 4966-4979 ◽  
Author(s):  
Yeu-Yang Tseng ◽  
Fong-Yuan Lin ◽  
Sun-Fang Cheng ◽  
David Tscharke ◽  
Songkhla Chulakasian ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Sindbis Virus ◽  
Full Length ◽  
Translation Initiation Factor ◽  
Orf Virus ◽  
Initiation Factor ◽  
Interferon Response ◽  
Virus Protein

ABSTRACTOrf virus (ORFV)OV20.0Lis an ortholog of vaccinia virus (VACV) geneE3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACVE3L,OV20.0Lencodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of functionin vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model.IMPORTANCEThe OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon responsein vitro.In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.

Association and Phosphorylation of Deoxyhypusine Synthase with CK2

2005 ◽  
Vol 277-279 ◽  
pp. 102-106
Author(s):  
Kee Ryeon Kang
Keyword(s):  
Protein Kinase Ck2 ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Deoxyhypusine Synthase ◽  
Cellular Event ◽  
Phosphoamino Acid Analysis ◽  
Kinase Ck2

Deoxyhypusine synthase (DHS) catalyzes the first step in the posttranslational synthesis of hypusine in the eukaryotic initiation factor 5A (eIF5A) precursor protein. As such, the phosphorylation of DHS by the protein kinase CK2 was investigated to define the role of DHS in the regulation of eIF5A in cells. The results showed that DHS was phosphorylated by CK2 in vivo as well as in vitro. The endogenous CK2 in HeLa cells and cell lysates was able to phosphorylate DHS and this modification was enhanced or decreased by the addition of CK2 effectors, such as polylysine, heparin, or poly (Glu, Tyr). A phosphoamino acid analysis of the enzyme revealed that the DHS was mainly phosphorylated into the Thr residue, with the remainder into the Ser residue. Therefore, it would appear that the phosphorylation of DHS was a CK2-dependent cellular event, thereby opening the path for possible regulation of the interaction with the eIF5A precursor for hypusine synthesis.

scholarly journals Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

1997 ◽  
Vol 17 (5) ◽  
pp. 2497-2501 ◽  
Author(s):  
J Feng ◽  
B A Witthuhn ◽  
T Matsuda ◽  
F Kohlhuber ◽  
I M Kerr ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Kinase Domain ◽  
Detectable Effect ◽  
Activation Loop ◽  
Receptor Complexes ◽  
Induced Aggregation

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

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