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)

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.
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PMID:A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation. 254 69

The GCN2 protein of Saccharomyces cerevisiae stimulates the expression of amino acid biosynthetic genes under conditions of amino acid starvation by derepressing GCN4, a transcriptional activator of these genes. GCN2 contains sequences homologous to the catalytic domain of protein kinases. We show here that substitution of a highly conserved lysine in the presumed ATP-binding site of this domain impairs the derepression of histidine biosynthetic genes under GCN4 control. This result supports the idea that protein kinase activity is required for GCN2 positive regulatory function. Determination of the nucleotide sequence of the entire GCN2 complementation unit, and measurement of the molecular weight of GCN2 protein expressed in vivo, indicate that GCN2 is a Mr approximately 180,000 protein and contains a Mr approximately 60,000 segment homologous to histidyl-tRNA synthetases (HisRSs) juxtaposed to the protein kinase domain. Several two-codon insertion mutations in the HisRS-related coding sequences inactivate GCN2 regulatory function. Based on these results, we propose that the GCN2 HisRS domain responds to the presence of uncharged tRNA by activating the adjacent protein kinase moiety, thus providing a means of coupling GCN2-mediated derepression of GCN4 expression to the availability of amino acids.
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PMID:Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. 266 Jan 41

The aminoacyl-tRNA synthetase complex from rabbit liver possesses an endogenous protein kinase activity. The associated protein kinase in the complex was defined as casein kinase I. Using FPLC, a fraction of the supramolecular complex with a high level of metabolic activity was isolated; this preparation was found to be enriched in the casein kinase activity. Incubation of this fraction with [gamma-32P] ATP leads to the intensive incorporation of labeled phosphate into 12 polypeptides of the complex, i.e., glutamyl-, isoleucyl-, leucyl-, methionyl-, lysyl-, arginyl- and aspartyl-tRNA synthetases. An addition of free homologous casein kinase I does not change the spectrum or level of phosphorylation of the complex substrates. The homologous casein kinase II phosphorylates polypeptides with Mr of 65, 43 and 20 kDa in the complex.
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PMID:[Phosphorylation of components of high molecular weight aminoacyl-tRNA-synthetase complex from the rabbit liver by associated casein kinase type I]. 275 79

Casein kinase II purified from nuclei of Xenopus laevis oocytes is inhibited by several specific nucleic acids. This kinase, the main phosphorylating activity of the oocyte nucleus, is markedly inhibited by poly U at 10 micrograms/ml, and this polymer is a competitive inhibitor of the phosphorylation of the substrate casein (Kiapp 80 nM). M 13 phage ssDNA and unfractionated yeast tRNA also inhibit between 50 and 200 micrograms/ml. Poly C, poly A, poly AG, dsDNA and Escherichia coli rRNA do not alter activity significantly at similar concentrations. Inhibitions are reversed by RNase (poly U, tRNA) or S1 nuclease (ssDNA). Oocyte casein kinase I or rabbit cAMP-dependent protein kinase are not inhibited by poly U at 200 micrograms/ml. The sensitivity of the casein kinase II to these inhibitors suggests a regulatory role for nucleic acids in nuclear phosphorylation reactions.
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PMID:Nucleic acids can regulate the activity of casein kinase II. 279 84

Threonyl-tRNA synthetase has been shown to be phosphorylated in reticulocytes (Dang, C. V., Tan, E. M., and Traugh, J. A., (1988) FASEB J. 2, 2376-2379). Upon incubation of reticulocytes with 8-bromo-cAMP, phosphorylation of threonyl-tRNA synthetase is stimulated approximately 2-fold, an increase similar to that observed with ribosomal protein S6. To analyze the effects of phosphorylation on activity, threonyl-tRNA synthetase has been purified to apparent homogeneity from rabbit reticulocytes utilizing a four-step purification procedure with the simultaneous purification of seryl-tRNA synthetase. Both synthetases are phosphorylated in vitro by the cAMP-dependent protein kinase. Prior to phosphorylation, the two synthetases produce significant amounts of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) in the presence of the cognate amino acid and ATP, with activities comparable to that of lysyl-tRNA synthetase. Phosphorylation has no effect on aminoacylation, but an increase in Ap4A synthesis of up to 6-fold is observed with threonyl-tRNA synthetase and 2-fold with seryl-tRNA synthetase. Thus, cAMP-mediated phosphorylation of specific aminoacyl-tRNA synthetases appears to be a potential mode of regulation of Ap4A synthesis in mammals.
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PMID:Phosphorylation of threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) synthesis. 292 38

Extensively purified EF-1H (EF-1 alpha beta beta' gamma) from wheat embryos had a protein kinase activity and phosphorylated EF-1 beta which is one of the two EF-Ts-like factors (EF-1 beta and EF-1 beta'). In this reaction ATP and GTP were equally effective as phosphate donors, and threonine residue was phosphorylated. At 10(-7)M, 3', 5' cyclic GMP stimulated both the phosphorylation and phe-tRNA binding reactions, whereas 3', 5' cyclic AMP inhibited both reactions. These findings indicate that phosphorylation of EF-1H may play an important role in the translational control of protein biosynthesis at the elongation step.
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PMID:Effect of cyclic AMP and cyclic GMP on the autophosphorylation of elongation factor 1 from wheat embryos. 298 81

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.
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PMID:Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes. 313 46

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.
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PMID:Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. 369 53

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.
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PMID:Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I. 386 66

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.
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PMID:The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells. 679 61


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