Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

Sudhakar, Akulapalli; Krishnamoorthy, T.M.; Jain, Anjali; U, Chatterjee; Hasnain, Seyed E.; Kaufman, Randal J.; Konakanchi, Ramaiah

doi:10.1021/bi991211n

Cited by 27 publications

(38 citation statements)

References 39 publications

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“…Alanine substitution of Ser-48 has a similar effect in mammalian cells (4,7,15,18). Moreover, addition of recombinant human eIF2␣-S48A to rabbit reticulocyte lysates reduced the abundance of 15S complexes containing eIF2, thought to represent inactive eIF2B-eIF2(␣P)-GDP complexes stabilized by Ser-51 phosphorylation (26). This last finding suggests that mutation of Ser-48 to Ala reduces the affinity of eIF2(␣P)-GDP for eIF2B as a means of overcoming the inhibition by Ser-51 phosphorylation.…”

Section: Metmentioning

confidence: 90%

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Krishnamoorthy

Pavitt

Zhang

et al. 2001

Molecular and Cellular Biology

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326

316

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Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNAMet to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its ␣ subunit [eIF2(␣P)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the ␣ subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2␣ (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(␣P) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(␣P) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(␣P). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the ␤ and ␥ subunits of eIF2 in the manner required for GDP-GTP exchange.

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Section: Metmentioning

confidence: 90%

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Krishnamoorthy

Pavitt

Zhang

et al. 2001

Molecular and Cellular Biology

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326

316

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“…Conversely, substitution of an aspartic acid at this position mimics the effect of phosphorylation (Choi et al 1992), presumably as a result of the additional negative charge. Mutations at other positions dose to Ser 51 , induding Ser 48 and the sequence GYID downstream of Ser 51 , can also reduce the effects of eIF2a phosphorylation (Vazquez de Aldana et al 1993;Sharp et al 1997;Sudhakar et al 1999). These findings suggest that this region of eIF2a makes phosphorylation-sensitive contacts with eIF2B, probably involving the a, ß and Ö subunits of the latter protein (Pavitt et al 1997;Kimball et al 1998a, b;Asano et al 1999).…”

Section: Introductionmentioning

confidence: 93%

Initiation Factor eIF2α Phosphorylation in Stress Responses and Apoptosis

Clemens

2001

Progress in Molecular and Subcellular Biology

202

146

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Protein synthesis in eukaryotes is a complex process which can be regulated at many points in the pathway. In re cent years it has become dear that both the overall rate of translation and the relative rates of synthesis of individual proteins can be controlled post-transcriptionally through changes in the activities or levels of a small number of key components, and that such regulation usually takes pi ace at the level of polypeptide chain initiation. A large body of evidence indicates that the essential polypeptide chain initiation factor eIF2 is a frequent target for regulation, and that its activity is often rate-limiting for protein synthesis. The phosphorylation of the smallest (a) subunit of eIF2 is a widely used mechanism of translational control in many organisms, and there are numerous physiologically important situations where eIF2a kinases are activated or inhibited. This chapter provides a review of our knowledge concerning the mechanisms by which eIF2 is controlled by reversible pro tein phosphorylation.A great variety of intracellular and extraceHular influences can alter rates of protein synthesis. One important dass of such influences can be grouped under the general heading of ceHular stresses. In the broadest sense, these range from normal physiological changes such as variations in nutrient availability or the actions of growth factors and cytokines to pathological conditions such as virus infection, hyperthermia or the presence of toxic compounds. In some cases, these conditions can result in cell death by apoptosis. Modulation of eIF2a phosphorylation is often involved in these situations. The present state of knowledge of how this is brought about and what the physiological consequences may be for the activities of the ceH, induding its survival or death, are reviewed here.Limitations of space predude a detailed account of the mechanism of polypeptide chain initiation and the reader is referred to recent comprehensive reviews (Pain 1996; Kozak 1999;Preiss and Hentze 1999) for such information. Briefly, initiation factor eIF2 catalyses the binding of the initiator

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“…The recombinant clones were digested with NdeI and BglII to release the coding sequence for ␣3(IV)NC1, which was ligated into pAcHLT-A transfer vector (BD Biosciences PharMingen, San Diego, CA) predigested with the same restriction enzymes and the resulting recombinant transfer vector, pAcHLT-A/␣3(IV)NC1, was cotransfected into Sf-9 cells as previously reported. 15,24,34,35 …”

Section: Expression Of Recombinant ␣3(iv)nc1mentioning

confidence: 99%

Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

Boosani

Mannam

Cosgrove

et al. 2007

Blood

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Human ␣3 chain, a noncollagenous domain of type IV collagen [␣3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of ␣3(IV)NC1 to ␣V␤3 and ␣3␤1 integrins. ␣3(IV)NC1 binds ␣V␤3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/ mTOR/4E-BP1 pathways. In the present study, we evaluated the role of ␣3␤1 and ␣V␤3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on ␣3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by ␣3(IV)NC1 in endothelial cells, only ␣3␤1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of ␣3(IV)NC1 to ␣3␤1 integrin leads to inhibition of COX-2-mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IB␣/NFB axis, and is independent of ␣V␤3 integrin. Furthermore, ␤3 integrin-null endothelial cells, when treated with ␣3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in ␣3 integrin-null endothelial cells, indicating that regulation of COX-2 by ␣3(IV)NC1 is mediated by integrin ␣3␤1. Our in vitro and in vivo findings demonstrate that ␣3␤1 integrin is critical for ␣3(IV)NC1-mediated inhibition of COX-2-dependent angiogenic signaling and inhibition of tumor progression.

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Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

Cited by 27 publications

References 39 publications

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Initiation Factor eIF2α Phosphorylation in Stress Responses and Apoptosis

Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

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