Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates

Delaunay, Jean; Ranu, Rajinder Singh; Levin, Daniel H.; Ernst, Vivian; London, Irving M.

doi:10.1073/pnas.74.6.2264

Cited by 58 publications

(21 citation statements)

References 37 publications

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“…This difference is most likely due to species-specific variation. Inhibitors of protein synthesis initi-ation with properties similar to those of HRI have also been reported in some nonerythroid cell types such as Ehrlich ascites cells (9), rat liver cells (12), and HeLa cells (11). Most recently, Olmsted et al (39) purified two eIF-2a kinases from Ehrlich ascites cells which they believe to be distinct from HRI and the double-stranded RNA-dependent eIF-2ot kinase (PKR).…”

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confidence: 99%

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

et al. 1994

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The role of heme-regulated eIF-2a kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2o kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. In heme deficiency, inhibition of protein synthesis occurs as a result of the activation of the heme-regulated inhibitor HRI (5,21,23,31). HRI is a cyclic AMP-independent protein kinase which specifically phosphorylates the 38-kDa ot subunit (eIF-2a) of eukaryotic initiation factor 2 (eIF-2) (18,26,28,46). Phosphorylation of eIF-2ao in reticulocyte lysates results in the binding and sequestration of eIF-2B in an eIF2(cP)-eIF-2B complex. The unavailability of eIF-2B, which is required for the exchange of GTP for GDP in the recycling of eIF-2 and the formation of the ternary complex (eIF-2/GTP/Met-tRNAf), results in the cessation of the initiation of protein synthesis (reviewed in references 21, 23, and 31).HRI is present in an inactive form in hemin-supplemented reticulocyte lysates (33, 34) and becomes active under conditions of heme deficiency (33,34) Ehrlich ascites cells which they believe to be distinct from HRI and the double-stranded RNA-dependent eIF-2ot kinase (PKR). Furthermore, increased eIF-2ot phosphorylation has been observed in HeLa and Ehrlich ascites cells under conditions of heat shock and nutrient starvation (11,13,14,39,50), in GH3 pituitary cells treated with calcium-mobilizing agents (45), and in vasopressin-treated rat liver (24). It has not yet been determined whether the eIF-2ot kinase activities measured in these cells are mediated by HRI or one or more as yet uncharacterized eIF-2a kinases.Previous studies using a monoclonal ant...

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Erythroid expression of the heme-regulated eIF-2 alpha kinase.

et al. 1994

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“…In erythroid cells, HRI plays a critical role in hemoglobin synthesis through coordinated regulation of the synthesis of its heme and globin moieties Chen, 2007). Although HRI eIF2␣ kinase was once viewed as erythroid-specific (Pal et al, 1991;Crosby et al, 1994;Chen, 2000), the existence of HRI mRNA in nonerythroid tissues and the characterization of a mouse liver HRI protein have been reported (Delaunay et al, 1977;Mellor et al, 1994;Berlanga et al, 1998;Igarashi et al, 2004). However, a plausible reason for overlooking HRI in the liver is its apparently low abundance (per milligram of protein) relative to erythroid HRI.…”

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confidence: 99%

RETRACTION: Hepatic Heme-Regulated Inhibitor (HRI) Eukaryotic Initiation Factor 2α Kinase: A Protagonist of Heme-Mediated Translational Control of CYP2B Enzymes and a Modulator of Basal Endoplasmic Reticulum Stress Tone

Acharya

Chen²,

Correia³

2010

Mol Pharmacol

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We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2␣ (eIF2␣) kinase is activated in acute heme-deficient states, resulting in translational shut-off of global hepatic protein synthesis, including phenobarbital (PB)-mediated induction of CYP2B enzymes in rats. These findings revealed that heme regulates hepatic CYP2B synthesis at the translational level via HRI. As a proof of concept, we have now employed a genetic HRI-knockout (KO) mouse hepatocyte model. In HRI-KO hepatocytes, PB-mediated CYP2B protein induction is no longer regulated by hepatic heme availability and proceeds undeterred even after acute hepatic heme depletion. It is noteworthy that genetic ablation of HRI led to a small albeit significant elevation of basal hepatic endoplasmic reticulum (ER) stress as revealed by the activation of ER stress-inducible RNA-dependent protein kinase-like ERintegral (PERK) eIF2␣-kinase, and induction of hepatic protein ubiquitination and ER chaperones Grp78 and Grp94. Such ER stress was further augmented after PB-mediated hepatic protein induction. These findings suggest that HRI normally modulates the basal hepatic ER stress tone. Furthermore, because HRI exists in both human and rat liver in its heme-sensitive form and is inducible by cytochrome P450 inducers such as PB, these findings are clinically relevant to acute heme-deficient states, such as the acute hepatic porphyrias. Activation of this exquisitely sensitive heme sensor would normally protect cells by safeguarding cellular energy and nutrients during acute heme deficiency. However, similar HRI activation in genetically predisposed persons could lead to global translational arrest of physiologically relevant enzymes and proteins, resulting in the severe and often fatal clinical symptoms of the acute hepatic porphyrias.Suppression of global protein synthesis through translational control is an effective way to preserve cellular energy and nutrients after various forms of cellular stress and injury. Through a rapid and reversible control of gene expression, it critically regulates various vital cellular processes, including growth stimulation, cell cycle progression, differentiation, hypoxia, ER stress, and heme deficiency

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“…Little is known about eIF-2 a-subunit kinases in cells other than reticulocytes, but the presence of eIF-2 a-subunit kinases or related enzymes has been reported in Ehrlich ascites tumor cells (24), Friend leukemia cells (25), rat liver (26), and bovine adrenal cortex (27) …”

Section: Resultsmentioning

confidence: 99%

Activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

Haro¹,

Herreros²,

Ochoa³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Hemin-supplemented reticulocyte lysates can be activated for translational inhibition by addition of Ca2+ or phospholipid. The fact that this inhibition is prevented or decreased in both cases either by the Ca2+ chelator EGTA or by polymyxin B, an inhibitor of the recently described Ca2+-and phospholipiddependent protein kinases, suggests the involvement of both Ca2+ and phospholipid in this activation. The inhibition by Ca2+ or phospholipid is accompanied by phosphorylation of the 38-kilodalton subunit of the eukaryotic initiation factor 2 (eIF-2) and the 90-kilodalton band of the heme-controlled translational inhibitor (HCI) and can be reversed by high concentrations of eIF-2 or GTP. When incubation is conducted at 30°C, the inhibition produced by Ca2+ is not reversed by EGTA after 15 min. However, at 20°C, Ca2+ inhibition can be fully reversed as late as 90 min from the start of incubation and phosphorylation of the eIF-2 a-subunit is correspondingly decreased. These results are consistent with the idea that, like heme deprivation, the activation by Ca2+ and phospholipid promotes the first step of the reaction proinhibitor reversible inhibitor -* irreversible inhibitor and suggest that, in the presence of hemin albeit by a different mechanism, this activation affects the same inhibitor that is activated in the absence of heme-namely, HCI. Whether this activation is direct or indirect-e.g., via a separate Ca2+-and phospholipid-dependent protein kinase-remains to be determined.While isolating a heat-stable factor (HS) from reticulocyte lysates that, like a factor described from bovine heart (1), inhibits translation in hemin-supplemented lysates and promotes phosphorylation of the eukaryotic initiation factor 2 (eIF-2) a-subunit, we used Ca2" for a negative control. Ca2" is known to inhibit protein synthesis, but we did not expect this metal ion to promote eIF-2 a-subunit phosphorylation. However, much to our surprise, it did so. It also clearly promoted the phosphorylation of a polypeptide that comigrates with the 90-kilodalton (kDa) band of the heme-controlled translational inhibitor (HCI) on electrophoresis in dissociating polyacrylamide gels (2). At 30°C, translational inhibition by Ca2+ was not reversed by the Ca2+ chelator EGTA after 15 min of incubation, but at 20°C, the inhibition was fully reversed by EGTA, even after incubation for 90 min, and phosphorylation of the eIF-2 a-subunit was correspondingly decreased (2). Other metal ions, including Mg2 , Mn2 , Cd2 , Co2+, and Cu2+, inhibited the hemin-dependent translation but of these only Co2+ promoted significant phosphorylation of the eIF-2 a-subunit. The effect of Co2+ has not been further investigated.We first thought that the Ca2+-promoted activation of HCI might be mediated by calmodulin. This hypothesis had some support in the fact that reticulocyte HS, unlike the one from bovine heart (1), resembles calmodulin in molecular weight and other properties: However, calmodulin antibody had no effect on the inhibition produced by Ca2", and auth...

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Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates

Cited by 58 publications

References 37 publications

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

RETRACTION: Hepatic Heme-Regulated Inhibitor (HRI) Eukaryotic Initiation Factor 2α Kinase: A Protagonist of Heme-Mediated Translational Control of CYP2B Enzymes and a Modulator of Basal Endoplasmic Reticulum Stress Tone

Activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

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