Specific phosphorylation in vitro of a protein associated with ribosomes of interferon‐treated mouse L cells

Zilberstein, Asher; Federman, P.; Shulman, Lester M.; Revel, Michel

doi:10.1016/0014-5793(76)80418-8

Cited by 235 publications

(84 citation statements)

References 18 publications

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“…The hemin-controlled inhibitor of protein synthesis has been studied in reticulocytes and is activated by various stimuli including hemin deprivation and heat treatment (6,19). The double-stranded RNA (dsRNA)-activated inhibitor (DAI) is induced by interferon, and its activity is dependent on dsRNA (12,18,20,31). Translational control mediated by DAI protein kinase has also been studied in adenovirusinfected cells, in which it has been found that two RNA polymerase III transcripts from the adenovirus genome are expressed at high levels and are required for efficient translation of adenovirus mRNAs late after infection (27).…”

mentioning

confidence: 99%

Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.

Kaufman¹,

Murtha²

1987

Mol. Cell. Biol.

127

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The translational efficiency of mRNA molecules transcribed from plasmid DNA transfected into COS-1 monkey cells can be increased 10-to 20-fold by the coexpression of the adenovirus virus-associated RNAs I and II. Experiments described here demonstrate a similar increase in translational efficiency by the addition of 2-aminopurine, an inhibitor of double-stranded RNA-activated protein kinase, to the culture medium. Both virus-associated RNA and 2-aminopurine presumably exert their effect by alteration of the functional level of eucaryotic initiation factor-2. The translational stimulation mediated by both means is shown to be restricted to the plasmid-derived mRNAs because there is no qualitative or quantitative alteration in host protein synthesis. The results are consistent with models invoking a localized activation of double-stranded RNAactivated kinase leading to a translational block.To initiate polypeptide chain synthesis, eucaryotic initiation factor-2 (eIF-2) forms a ternary complex with GTP and initiator Met-tRNA (for reviews, see references 14 and 15). The ternary complex binds a 40S ribosomal subunit, forming a 40S initiation complex. Subsequently, an mRNA molecule and the 60S ribosomal subunit are added to form the 80S initiation complex, with the concomitant hydrolysis of GTP to GDP. To reinitiate, GDP bound to eIF-2 must be replaced by GTP, a reaction catalyzed by the guanine nucleotide exchange factor. The phosphorylation of the alpha subunit of eIF-2 results in stabilizing the guanine exchange factor-eIF-2-GDP complex, thereby inhibiting recycling. This depletes the amount of functional eIF-2 competent for translation initiation.Two protein kinases have been shown to regulate initiation by phosphorylation of the alpha subunit of eIF-2 (15). The hemin-controlled inhibitor of protein synthesis has been studied in reticulocytes and is activated by various stimuli including hemin deprivation and heat treatment (6,19). The double-stranded RNA (dsRNA)-activated inhibitor (DAI) is induced by interferon, and its activity is dependent on dsRNA (12,18,20,31). Translational control mediated by DAI protein kinase has also been studied in adenovirusinfected cells, in which it has been found that two RNA polymerase III transcripts from the adenovirus genome are expressed at high levels and are required for efficient translation of adenovirus mRNAs late after infection (27). The virus-associated (VA) RNA I is involved in the control of the level of active eIF-2 (17, 21-23). VA RNA I can inhibit the activation of DAI kinase by dsRNA in vitro (11,16). It has been proposed that dsRNA which may result from asymmetric transcription of the replicating adenoviral genome in infected cells can activate DAI kinase to phosphorylate the alpha subunit of eIF-2, resulting in inhibition of translation initiation. VA RNA may prevent the translational block by inhibiting activation of DAI kinase. In addition to the role of VA RNA in adenovirus-infected cells, VA RNA can potentiate the translation of mRNA in transient transfecti...

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mentioning

confidence: 99%

Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.

Kaufman¹,

Murtha²

1987

Mol. Cell. Biol.

127

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“…A protein kinase(s), 2'-5'A synthetase [responsible for the formation of a series of 2'-5'-linked oligo(adenylic acid) triphosphate (2'-5'A) inhibitors of protein synthesis in which the trimer pppA2'p5'A2'p5'A is predominant], and a nuclease may all be involved in this inhibition (4)(5)(6)(7)(8)(9)(10)(11)(12). The kinase is thought to phosphorylate the smallest subunit of initiation factor eIF2, as appears to be the case in rabbit reticulocyte lysates, on inhibition of protein synthesis by a number of agents, including dsRNA (12)(13)(14)(15)(16).…”

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confidence: 99%

Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A.

Hovanessian¹,

Wood²,

Meurs³

et al. 1979

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

144

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A series of 2'-5'-linked oligo(adenylic acid) triphosphate (2'-5'A) inhibitors of protein synthesis were described recently. These inhibitors are synthesized from ATP by an enzyme activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA. We show here that 2'-'A is a potent inhibitor of protein synthesis in intact cells of different origin (human, monkey, hamster, and mouse). At a concentration of 10 nM (in AMP equivalents), protein synthesis is inhibited by 50-85%. There is also a secondary effect on the total RNA synthesis which becomes evident several hours after inhibition of protein synthesis. All of these effects, however, are transient and, after a recovery period, both RNA and protein synthesis resume rates comparable to the appropriate controls.

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“…Two protein kinases from rabbit reticulocytes have been characterized that phosphorylate eIF-2a, apparently at the same serine residues (5). One is a ribosome-bound enzyme that is activated by double-stranded RNA (5,6) and appears to be similar or identical to the enzyme that is induced by interferon in other cells (7)(8)(9)(10). The other protein kinase is activated in the absence of heme (2,5,11,12) and is associated with the heme-controlled repressor, HCR (13).…”

mentioning

confidence: 99%

“…The reaction mixture (300 sl) contained 20 mM Tris-HCl at pH 7.5, 2.5 mM dithioerythritol, 5 mM MgCl2, 0.1 mM [y-32P]ATP (4)(5)(6)(7)(8) Ci/mmol; 1 Ci = 3.7 x 1010 becquerels), 1 mg of eIF-2, and 720 mg ofprotein ofthe eIF-2a kinase fraction. After incubation at 37°C for 5 min, the mixture was loaded on phosphocellulose that had been packed in a Pasteur pipette and equilibrated with 20 mM Tris-HCI, pH 7.5/100 mM KCV5 mM 2-mercaptoethanol/1 mM dithioerythritoV0.…”

mentioning

confidence: 99%

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

Zardeneta¹,

Kramer²,

Hardesty³

1982

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

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A procedure is described by which the 38,000-dalton a subunit of native eukarfotic peptide initiation factor 2 (eIF-2) can be cleaved by trypsin to yield a 34,000-dalton fragment and a peptide of about 4,000 daltons after elimination of the (3 subunit. Under nondenaturingconditions the 4,000-dalton peptide remains bound to the modified eIF-2 and still can be phosphorylated by the heme-controlled eIF-2a Idnase from reticulocytes. All of the phosphorylation sites for this protein Idnase are located on the 4,000-dalton peptide. The ability ofeIF-2 to form a ternary complex with GTP and Met-tRNAf and the ability to promote binding of Met-tRNAf to 40S ribosomal subunits are lost differentially during the proteolysis. Loss of the latter activity occurs rapidly and appears to be correlated with loss of the (3 subunit. Loss of activity for ternary complex formation is correlated with the appearance of the 4,000-dalton peptide.

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Specific phosphorylation in vitro of a protein associated with ribosomes of interferon‐treated mouse L cells

Cited by 235 publications

References 18 publications

Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.

Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.

Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A.

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

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