Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver

Kimball, Scot R.; Karinch, Anne M.; Feldhoff, Richard C.; Mellor, Harry; Jefferson, Leonard S.

doi:10.1016/0304-4165(94)90079-5

Cited by 57 publications

(62 citation statements)

References 23 publications

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“…For this analysis, samples were resolved by electrophoresis using a 12.5% SDS-polyacrylamide gel and the proteins in the gel were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The mem-branes were incubated with the rabbit polyclonal antibody that specifically recognizes eIF2(␣P), and blots were developed as described previously (18). The horseradish peroxidase coupled to the anti-rabbit secondary antibody was then inactivated by incubating the membrane in 15% hydrogen peroxide for 30 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

Kimball

Horetsky

Ron

et al. 2003

American Journal of Physiology-Cell Physiology

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255

185

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cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA i (Met-tRNAi) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2 ⅐ GTP ⅐ Met-tRNA i complex is thought to bind to the 40S ribosomal subunit before the eIF4G ⅐ eIF4E ⅐ mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the ␣-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes. eIF4E; eIF4G; eIF3; unfolded protein response; PERK ONE OF THE BEST-CHARACTERIZED mechanisms for regulating mRNA translation in eukaryotic cells involves phosphorylation of the ␣-subunit of eukaryotic initiation factor, eIF2 (reviewed in Ref. 16). During initiation, eIF2 forms a complex with GTP and initiator methionyl-tRNA i (met-tRNA i ), and this ternary complex subsequently binds to the 40S ribosomal subunit to form the 43S preinitiation complex (reviewed in Refs. 10 and 24). Through the action of a translation initiation factor complex referred to as eIF4F, which is comprised of eIF4A, eIF4E, and eIF4G, mRNA is bound to the 43S preinitiation complex, resulting in formation of the 48S preinitiation complex (reviewed in Ref. 20). During a later step in initiation, the GDP bound to eIF2 is hydrolyzed in an eIF5-mediated process and initiation factors are released from the ribosome. Before binding Met-tRNA i and reforming the ternary complex, the GDP bound to eIF2 must be exchanged for GTP, a reaction that is catalyzed by the guanine nucleotide exchange factor, eIF2B. One mechanism for regulating the activity of eIF2B involves phosphorylation of the ␣-subunit of eIF2 on Ser51, an event that converts eIF2 from a substrate into a competitive inhibitor of eIF2B (reviewed in Ref. 10). Thus, by inhibiting eIF2B, phosphorylation of eIF2␣ results in a global inhibition of protein synthesis.Hyperphosphorylation of eIF2␣ occurs under a variety of conditions that result in disruption of normal cell homeostasis. For example, conditions that impede correct folding of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER), i.e., the so-called unfolded protein response, result in activation of the eIF2␣ kinase, PERK (also referred to as PEK) (28). PERK is a trans-ER membrane protein with a luminal domain...

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Section: Methodsmentioning

confidence: 99%

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

Kimball

Horetsky

Ron

et al. 2003

American Journal of Physiology-Cell Physiology

Self Cite

255

185

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“…Blots were developed using an Amersham enhanced chemiluminescence (ECL) Western blotting kit, as described previously (33). Chemiluminescence was measured using a Gene Genome imaging system (Syngene) and quantitated using GeneTools software (Syngene).…”

Section: Animals Eleven Multiparous Crossbred (Yorkshire ϫmentioning

confidence: 99%

“…Aliquots of muscle homogenates (supernatants) were heated at 100°C for 10 min, cooled to room temperature, and then centrifuged at 10,000 g for 10 min at 4°C. The supernatants were diluted with SDS sample buffer and then subjected to protein immunoblot analysis, as described previously (12,33). The membranes were incubated with a polyclonal antibody that specifically recognizes phosphorylation of 4E-BP1 at Thr 70 .…”

Section: Animals Eleven Multiparous Crossbred (Yorkshire ϫmentioning

confidence: 99%

“…The first important regulatory step in translation initiation is the binding of initiator methionyl-tRNA i (met-tRNA i ) to the 40S ribosomal subunit, a reaction that is mediated by eukaryotic initiation factor (eIF)2 and results in the formation of the 43S preinitiation complex (33,46). The eIF2-mediated binding of met-tRNA i to the 40S subunit is regulated by modulation of the activity of eIF2B, which exchanges GDP for GTP on eIF2 (34).…”

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confidence: 99%

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Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs

O'Connor

Kimball²,

Suryawan

et al. 2003

American Journal of Physiology-Endocrinology and Metabolism

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109

113

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Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs ( n = 8–12/group), insulin was infused to achieve plasma levels of ∼0, 2, 6, and 30 μU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.

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“…Solubilization of cell membranes was facilitated by rocking extracts on an orbital rocker for an additional 20 min. Cell lysates were then clarified by centrifugation at 14,000 ϫ g for 20 min at 4°C and subjected to Western analysis as described previously (38,39). For each immunoblot presented, samples from each condition were pooled from either duplicate or triplicate assays prior to electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K

Shah

Iñiguez‐Lluhí

Romanelli

et al. 2002

Journal of Biological Chemistry

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Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.

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Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver

Cited by 57 publications

References 23 publications

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs

The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K

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