Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein chain initiation in reticulocyte lysates is inhibited by (a) heme-deficiency, (b) low levels of double-stranded RNA, and (c) a purified translational inhibitor isolated from heme-deficient lysates. Previous studies have shown that the inhibitions produced by heme-deficiency and double-stranded RNA are prevented by 3': 5'-cyclic AMP, and that GTP, but not ATP, prevents the inhibition of heme-deficiency. In view of the recent finding that the inhibitor purified from heme-deficient lysates is associated with a protein kinase which appears to be involved in the mechanism of inhibition, the effects of cyclic AMP, GTP, and ATP on the three modes of inhibition were examines. In all three types of inhibition, cyclic AMP or GTP (a) prevents the onset of inhibition when added at zero time, and (b) restores protein synthesis in inhibited lysates. In contrast to these effects, ATP potentiates each inhibition, and blocks reversal of inhibition by cyclic AMP or GTP. On the basis of these and earlier findings, we propose that (a) these inhibitions involve the phosphorylation by protein kinases of the Met-tRNAf binding factor and/or a related site(s) on the 40S ribosomal subunit; and (b) cyclic AMP, GTP, and ATP exert their effects by their actions on this phosphorylation mechanism.
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PMID:Control of protein synthesis in reticulocyte lysates: effects of 3':5'-cyclic AMP, ATP, and GTP on inhibitions induced by hemedeficiency, double-stranded RNA, and a reticulocyte translationa inhibitor. 17 76

Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-indenpendent protein kinase activity that phosphorylates the low-molecular-weight (about 38,000) polypeptide chain of the initiation factor that forms a ternary complex with GTP and Met-tRNAf. These preparations also phosphorylate several polypeptide components of reticulocyte 40S ribosomal subunits. However, no significant levels of phosphorylation are observed when casein, histones, Artemia salina 40S ribosomal subunits, or other initiation factor fractions are used as substrates although high levels of phosphorylation are obtained with cruder preparations of the repressor. An antibody to these highly purified preparations of repressor has been obtained from the serum of immunized goats. Preincubation with immune goat IgG results in the neutralization of the inhibitory activity of the repressor, while normal IgG has no effect. Preincubation with immune IgG also abolishes the protein kinase activity responsible for the phosphorylation of the initiation factor and reticulocyte 40S subunits. Histone phosphorylation by crude repressor preparations, on the other hand, is unaffected by preincubation with immune IgG.
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PMID:Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte. 18 58

A previous study demonstrated that the translational inhibitor from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields two distinct protein kinase activities, PC1 and PC2. PC1, which consitutes about 90% of the activity in the unresolved preparation, does not inhibit protein synthesis in lysates, but actively phosporylates calf thymus histone II in a 3':5'-cyclic AMP-denpendent reaction. PC2 contains the translational inhibitor, phosphorylates histone poorly, and is not cyclic AMP-dependent. While [gamma-32P]ATP as the phosphate donor, the two kinase fractions were analyzed with the putative substrates, salt-washed 40S ribosomal subunits, and the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit. PC1 is inactive with the initiation factor, but phosphorylates 40S subunits at a single major site that migrates as a 31,000-dalton band in sodium dodecyl sulfate-acrylamide gels; phosphorylation requires cyclic AMP. Similar phosphorylation of the reticulocyte 40S site (31,000 daltons) can be demonstrated with other cyclic AMP-dependent kinases from reticulocytes, rat liver, and bovine heart muscle. PC2 phosphorylates the small subunit (38,000 daltons) but not the large subunit(s) of the initiation factor; the reaction does not require cyclic AMP. PC2 does not phosphorylate 40S subunits. In the presence of 40S subunits, the initiation factor appears to be rapidly bound in a manner that effectively blocks phosphorylation of the initiation factor by PC2; under the same conditions phosphorylation of the 40S subunit by PC1 is not affected. The initiation factor has been shown to reverse the inhibitions of protein chain initiation induced in lysates by heme deficiency, double-stranded RNA, oxidized glutathione, or the purified translational inhibitor. The observation that the Met-tRNAf binding factor is phosphorylated by PC2 supports the hypothesis that this initiation factor is a target for the action of the translational inhibitor activated in heme deficiency.
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PMID:Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates. 18 60

Preparations of the hemin-controlled repressor (HCR) from rabbit reticulocytes contain 3':5'-cyclic-AMP-independent protein kinase activity for the smallest subunit of the peptide initiation factor eIF-2 and for proteins of reticulocyte 40S ribosomal subunits. Binding of the ternary complex formed between Met-tRNAf, GTP, and eIF-2 to 40S ribosomal subunits is shown to be inhibited by phosphorylation of either the ribosomal subunits or eIF-2. The protein kinase activity responsible for phosphorylation of eIF-2 has been separated from the activity for phosphorylation of 40S ribosomal subunits and shown to independently block the same partial reaction of peptide initiation. It appears that different enzymes are involved, each capable of regulating peptide initiation at the same step but by a different mechanism.
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PMID:Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins. 19

The initiation inhibitor of reticulocyte lysates has been shown by others to be associated with a 3':5'-cyclic-AMP-independent protein kinase that catalyzes the phosphorylation of the small (38,000 daltons) subunit of the polypeptide chain initiation factor eIF-2. This factor forms a ternary complex with Met-tRNAi and GTP which, on interaction with a 40S ribosome, gives rise to a 40S complex. Ternary complex formation is inhibited by prior incubation of partially purified eIF-2 with reticulocyte inhibitor and ATP. The relation between phosphorylation and inactivation of eIF-2 is indicated by the lack of inhibition when ATP is omitted. Translation in hemin-containing reticulocyte lysates is also inhibited by cyclic-AMP-dependent protein kinases or their catalytic subunits. They act by converting proinhibitor (inactive eIF-2 kinase) present in lysates to inhibitor (active eIF-2 kinase). This reaction is analogous to the conversion of inactive phosphorylase kinase to active phosphorylase kinase.
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PMID:Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates. 19 1

The eukaryotic initiation factor eIF-2 forms a ternary complex with Met-tRNAf and GTP. This complex binds to the 40S ribosomal subunit in the absence of mRNA and mRNA binding factors. Highly purified eIF-2 from rabbit reticulocytes was labeled with 125I by using the Bolton-Hunter reagent or with [gamma-32P]ATP by using the heme-regulated translational inhibitor protein kinase. The labeled eIF-2 was bound, together with equimolar amounts of Met-tRNAf and GTP, to the 40S subunit. In the presence of mRNA, mRNA binding factors, and 60S ribosomal subunits (complete initiation assay), eIF-2 was released from the 40S initiation complex in the subunit joining reaction. GTP also was released in this step and probably was hydrolyzed in the reaction that is dependent upon eIF-5 and the 60S subunit. The function of phosphorylated eIF-2 in initiation of protein synthesis is discussed.
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PMID:Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation. 27 35

Despite the finding that the hemin-controlled translational inhibitor in reticulocyte lysates is a cyclic AMP-independent protein kinase that phosphorylates the small subunit of the initiation factor eIF-2, the mechanism of inhibition of translation remained unexplained. Whereas treatment of hemin-containing lysates with inhibitor in the presence of ATP inhibited translation, the same treatment of highly purified eIF-2 did not affect its ability to form a ternary complex with initiator Met-tRNA and GTP or a 40S initiation complex. We have isolated from ribosomal salt washes a protein (eIF-2 stimulating protein) that enhances the capacity of unphosphorylated eIF-2 to form ternary or 40S initiation complexes but has no effect on the phosphorylated factor. At low concentrations, eIF-2 is virtually inactive without this stimulating protein. Therefore, the translational inhibitor acts by converting eIF-2 to a form that is not stimulated by the stimulating protein.
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PMID:Mode of action of the hemin-controlled inhibitor of protein synthesis. 27 39

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet.40S ribosomal subunit complexes. 27 38

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase. 28 94

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex (Met-tRNAfMet.eIF-2.GTP) and AUG-dependent transfer of Met-tRNAfMet to 40S ribosomal subunits. A factor (sRF) that reverses protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleimide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF.
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PMID:Protein synthesis in rabbit reticulocytes: characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (Met-tRNAfMet.eIF-2.GTP) formation by heme-regulated inhibitor. 29 57


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