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

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.
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PMID:The substrate specificity of adenosine 3':5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle. 19 23

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

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNA(f) binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: (i) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; (ii) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition-i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; (iii) both inhibitions are prevented or reversed by eIF-2; (iv) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; (v) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of elF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.
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PMID:Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates. 19 85

In this study, we used two-dimensional gel electrophoresis to analyze the responses of cultured S49 mouse lymphoma cells to incubation with analogs or inducers of cyclic AMP (cAMP). Putative phosphorylations were detected by charge alterations in proteins labeled with 35S--methionine and, in some cases, confirmed by labeling with 32P--phosphate. We assessed the relative stabilities of proteins affected by cAMP, the periods of susceptibility of proteins to cAMP-dependent modification and any cAMP-mediated changes in protein synthesis or stability. Five proteins (of about 650 resolved) behave as expected for "orthodox" substrates of a cAMP-activated protein kinase: both newly synthesized and prelabeled forms of these proteins are subject to modification; this modification involves an acidic charge shift of about one unit; and cAMP-mediated conversion of these proteins to their modified forms is virtually complete. The acidic forms of at least three of these proteins also exhibit cAMP-mediated increases in 32P--phosphate incorporation. Each protein comprised less than approximately 0.005% of cellular protein. Under basal conditions they appear to be phosphorylated to an extent about 20--30% of that found in fully stimulated cells. Nine proteins show cAMP-dependent changes in rates of synthesis with six inductions and three repressions. Most of these changes are of a magnitude of about 3 to 5 fold, and reach their maximal extents after about 4--5 hr of exposure to dibutyryl cAMP. In addition to the phosphorylations, inductions and repressions mentioned above, approximately 12 other reproducible cAMP-dependent changes in protein patterns are observed. Mutant cell lines deficient in catalytic activity of cAMP-dependent protein kinase show none of the changes in protein pattern attributable to cAMP.
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PMID:Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells: protein modifications, inductions and repressions. 22 61

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


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