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)

Incubation of 80S ribosomes with a substoichiometric amount of [alpha-32P]GTP and with eEF-2 resulted in the specific labeling of one ribosomal protein which migrated very close to the position of the acidic phosphoprotein P2 from the 60S subunit in two-dimensional isofocusing-SDS gel electrophoresis. Localization of protein P2 in this electrophoretic system was ascertained by correlation with its position in the standard two-dimensional acidic-SDS gel electrophoresis after its specific phosphorylation by casein kinase II. Labeling of the ribosomal protein was dependent on the presence of eEF-2, and could be attributed to [alpha-32P]GDP binding from the results of chase experiments and HPLC identification, this binding being very likely responsible for the slight shift in the electrophoretical position of the protein. Incubation of ribosomes with tRNA(Phe) in the absence of mRNA induced the release of the bound GDP.
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PMID:Binding of GDP to a ribosomal protein after elongation factor-2 dependent GTP hydrolysis. 142 Mar 8

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.
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PMID:Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. 144 7

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys). Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.
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PMID:Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms. 150 29

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10.7 kb. The sequence was determined using a double-strand sequencing strategy adapted from the random-clone strategy. The segment contains seven non-overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumably a protein kinase.
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PMID:Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases. 156 35

The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.
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PMID:Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae. 203 14

The polypeptide with a mobility of the tryptophanyl-tRNA-synthetase subunit can be labeled in bovine pancreas extracts from [gamma-32P]ATP. Immunoprecipitation analysis with monospecific polyclonal antibodies against the enzyme as well as identification of [32P]phosphoamino acids in the immunoprecipitate revealed that in bovine pancreas extracts tryptophanyl-tRNA-synthetase undergoes phosphorylation at serine residues. The level of phosphorylation does not change in the presence of activity modulators of cAMP-, cGMP- and Ca2(+)-dependent protein kinases, decreases after addition of phosphoseryl/phosphothreonyl-protein phosphatase inhibitors and increases in the presence of their activators. It was supposed that phosphorylation of tryptophanyl-tRNA-synthetase catalyzed by seryl/threonyl-specific protein kinase depends on the activity of phosphoseryl/phosphothreonyl-phosphatase.
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PMID:[Phosphorylation of tryptophanyl-tRNA-synthase in extracts of bovine pancreas]. 212 Dec 90

GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.
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PMID:Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. 218

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.
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PMID:Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP. 222 78

Sephadex G-200 gel filtration of DNA cellulose-treated crude extracts of rat skeletal muscle, revealed a broad peak-fraction of tRNA-inhibitory protein kinases (PK) coeluted endogenous substrates. In comparison, the elution profile of baker's yeast exhibited multiple peak-fractions of tRNA-inhibiting PK. Various tRNA all showed inhibition to PK. In the presence of regulatory subunit of cyclic AMP-dependent protein kinase, tRNA did not exert synergetic inhibition on PK. Moreover, the interaction of tRNA with active muscle PK fractions could not be monitored by the increment of absorbance at 340 nm. tRNA had no significant regulatory effect on the phosphorylation of actin and myosin.
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PMID:Inhibitory effect of transfer RNA on protein kinases from baker's yeast and rat skeletal muscle. 225 56

Five aminoacyl-tRNA synthetases found in the high molecular weight core complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The synthetases were isolated by affinity chromatography on tRNA-Sepharose followed by immunoprecipitation. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, and aspartyl-tRNA synthetases and, to a lesser extent, the methionyl-tRNA synthetase. In addition, a 37,000-dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and 3-isobutyl-1-methylxanthine resulted in a 6-fold increase in phosphorylation of the glutaminyl-tRNA synthetase and a 2-fold increase in phosphorylation of the aspartyl-tRNA synthetase. When the high molecular weight core complex was isolated by gel filtration/affinity chromatography, the profile of phosphorylation was similar to that observed by immunoprecipitation with a 9- and 3-fold stimulation of the glutaminyl- and aspartyl tRNA-synthetase, respectively. From this data it was concluded that the increased phosphorylation of the glutaminyl- and aspartyl-tRNA synthetases obtained with 8-bromo-cAMP did not appear to be involved in dissociation of the high molecular weight core complex.
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PMID:Regulation of phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight core complex in reticulocytes. 243 10


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