scholarly journals An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

2015 ◽  
Vol 112 (27) ◽  
pp. E3466-E3475 ◽  
Author(s):  
Margarito Rojas ◽  
Gabriel Vasconcelos ◽  
Thomas E. Dever
Keyword(s):  
Protein Phosphatase ◽  
African Swine Fever Virus ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Protective Mechanism ◽  
Binding Motif ◽  
Α Subunit

Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x1–2Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

scholarly journals The MyD116 African Swine Fever Virus Homologue Interacts with the Catalytic Subunit of Protein Phosphatase 1 and Activates Its Phosphatase Activity

2007 ◽  
Vol 81 (6) ◽  
pp. 2923-2929 ◽  
Author(s):  
José Rivera ◽  
Charles Abrams ◽  
Bruno Hernáez ◽  
Alberto Alcázar ◽  
José M. Escribano ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Catalytic Subunit ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Wild Type ◽  
Α Subunit ◽  
Initiation Factor 2

ABSTRACT The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-α catalytic subunit expressed as a histidine fusion protein (6×His-PP1α) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α). ICP34.5 recruits PP1 to dephosphorylate the α subunit of eukaryotic translational initiation factor 2 (also known as eIF-2α); possibly the ASFV DP71L protein has a similar function.

scholarly journals The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence.

1994 ◽  
Vol 14 (4) ◽  
pp. 2307-2316 ◽  
Author(s):  
N Méthot ◽  
A Pause ◽  
J W Hershey ◽  
N Sonenberg
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Recognition Motif ◽  
Helicase Activity ◽  
Binding Region ◽  
Stimulation Of

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.

scholarly journals Gadd34 Requirement for Normal Hemoglobin Synthesis

2006 ◽  
Vol 26 (5) ◽  
pp. 1644-1653 ◽  
Author(s):  
Andrew D. Patterson ◽  
M. Christine Hollander ◽  
Georgina F. Miller ◽  
Albert J. Fornace
Keyword(s):  
Stress Responses ◽  
Protein Phosphatase ◽  
Biochemical Analysis ◽  
Growth Arrest ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Hemoglobin Content ◽  
Α Subunit ◽  
Hemoglobin Synthesis ◽  
Initiation Factor 2

ABSTRACT The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the α subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte.

scholarly journals The African Swine Fever Virus DP71L Protein Recruits the Protein Phosphatase 1 Catalytic Subunit To Dephosphorylate eIF2α and Inhibits CHOP Induction but Is Dispensable for These Activities during Virus Infection

2010 ◽  
Vol 84 (20) ◽  
pp. 10681-10689 ◽  
Author(s):  
Fuquan Zhang ◽  
Alice Moon ◽  
Kay Childs ◽  
Stephen Goodbourn ◽  
Linda K. Dixon
Keyword(s):  
Amino Acids ◽  
Protein Phosphatase ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Catalytic Subunit ◽  
Translation Initiation Factor ◽  
Protein Phosphatase 1 ◽  
Resting Cells ◽  
Eif2α Phosphorylation

ABSTRACT The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (α, β, and γ) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2α. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2α phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, MalawiΔNL and E70ΔNL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2α phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2α during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2α phosphorylation and the subsequent protein synthesis inhibition.

scholarly journals The translation initiation factor eIF2β is an interactor of protein phosphatase-1

2006 ◽  
Vol 400 (2) ◽  
pp. 377-383 ◽  
Author(s):  
Paulina Wakula ◽  
Monique Beullens ◽  
Aleyde van Eynde ◽  
Hugo Ceulemans ◽  
Willy Stalmans ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Protein Phosphatase ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Homology Search ◽  
Initiation Factors ◽  
Initiation Of Translation ◽  
Rvxf Motif

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2α, eIF2Bϵ and eIF4E. Other initiation factors, including eIF2β, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2β contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2β-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2β contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2β functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51of eIF2α by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bϵ by this phosphatase. Strikingly, eIF2β emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2β was decreased by the mere mutation of its RVxF-motif. These results make eIF2β an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2β or eIF2β with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.

scholarly journals Growth Arrest and DNA Damage-Inducible Protein GADD34 Targets Protein Phosphatase 1α to the Endoplasmic Reticulum and Promotes Dephosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2

2003 ◽  
Vol 23 (4) ◽  
pp. 1292-1303 ◽  
Author(s):  
Matthew H. Brush ◽  
Douglas C. Weiser ◽  
Shirish Shenolikar
Keyword(s):  
Protein Phosphatase ◽  
Growth Arrest ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Inducible Protein ◽  
Translation Initiation Factor 2

ABSTRACT The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the α subunit of eukaryotic translation initiation factor 2, (eIF-2α). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2α phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2α phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2α phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2α dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the α isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2α phosphatase that regulates protein translation in mammalian cells.

The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence

1994 ◽  
Vol 14 (4) ◽  
pp. 2307-2316
Author(s):  
N Méthot ◽  
A Pause ◽  
J W Hershey ◽  
N Sonenberg
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Recognition Motif ◽  
Helicase Activity ◽  
Binding Region ◽  
Stimulation Of

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.

scholarly journals SARP, a new alternatively spliced protein phosphatase 1 and DNA interacting protein

2007 ◽  
Vol 402 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Gareth J. Browne ◽  
Margarida Fardilha ◽  
Senga K. Oxenham ◽  
Wenjuan Wu ◽  
Nicholas R. Helps ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Transforming Growth Factor Β ◽  
Ankyrin Repeat ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Alternative Mrna Splicing

PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP–PP1 activity in mammalian cells. This SARP–PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M110/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor β inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92–95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1γ1 and PP1γ2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1α and PP1γ1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

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