Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.
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PMID:The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. 762 40

Phosphorylation of translation initiation factor 2 alpha is a highly conserved mechanism for down-regulating protein synthesis in response to starvation or stress. The yeast eIF-2 alpha kinase GCN2 is stimulated by deprivation for amino acids or purines. In addition to inhibiting general protein synthesis, GCN2 specifically stimulates translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. HRI is an eIF-2 alpha kinase that is activated in rabbit reticulocytes by heme-deprivation and stress conditions that elicit the heat-shock response. The eIF-2 alpha kinase DAI is activated by double-stranded RNA during viral infections and is an important component of the interferon response. DAI has also been implicated as a tumor suppressor. These protein kinases provide an important means of coupling the rate of protein synthesis and cell division to environmental conditions.
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PMID:The eIF-2 alpha kinases: regulators of protein synthesis in starvation and stress. 771 Dec 90

The GCN2 (general control kinase 2) protein is an eIF2-alpha (eukaryotic initiation factor alpha) kinase which mediates translational derepression of the yeast general control transcriptional activator, GCN4, upon amino-acid starvation. We isolated and characterized GCN2 mutations differentially affecting GCN2 function. Mutations mapping in, or close to, the ATP-binding site of the kinase moiety result in constitutively activated GCN2 molecules. A C-terminal regulatory mutation dramatically affects translation initiation rates resulting in pleiotropic phenotypes. The effect of mutations in both regions were found to depend on eIF2-alpha phosphorylation. We have demonstrated that GCN2 mutants have altered autophosphorylation activities in vitro, depending on the presence or absence of a wild-type GCN2 gene and that GCN2 elutes in gel-filtration chromatography fractions with high apparent molecular mass. Both these genetic and biochemical findings suggest that GCN2 functioning might involve polymerization to form dimers or tetramers.
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PMID:Genetic and biochemical evidence for yeast GCN2 protein kinase polymerization. 820 May 34

The transcriptional activator protein GCN4 is responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. This induction depends on increased expression of GCN4 at the translational level. We show that starvation for purines also stimulates GCN4 translation by the same mechanism that operates in amino acid-starved cells, being dependent on short upstream open reading frames in the GCN4 mRNA leader, the phosphorylation site in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the protein kinase GCN2, and translational activators of GCN4 encoded by GCN1 and GCN3. Biochemical experiments show that eIF-2 alpha is phosphorylated in response to purine starvation and that this reaction is completely dependent on GCN2. As expected, derepression of GCN4 in purine-starved cells leads to a substantial increase in HIS4 expression, one of the targets of GCN4 transcriptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitors of purine biosynthesis, suggesting that derepression of GCN4 is required for maximal expression of one or more purine biosynthetic genes under conditions of purine limitation. Analysis of mRNAs produced from the ADE4, ADE5,7, ADE8, and ADE1 genes indicates that GCN4 stimulates the expression of these genes under conditions of histidine starvation, and it appeared that ADE8 mRNA was also derepressed by GCN4 in purine-starved cells. Our results indicate that the general control response is more global than was previously imagined in terms of the type of nutrient starvation that elicits derepression of GCN4 as well as the range of target genes that depend on GCN4 for transcriptional activation.
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PMID:Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2. 833 37

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.
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PMID:Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae. 844 23

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.
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PMID:GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. 849 69

In yeast, starvation for amino acids stimulates GCN2 phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2). Phosphorylation of eIF-2alpha induces the translational expression of GCN4, a transcriptional activator of the general amino acid control pathway. It has been proposed that GCN2 sequences containing homology to histidyl-tRNA synthetases (HisRS) bind uncharged tRNA that accumulate during amino acid limitation and stimulate the activity of GCN2 kinase. In this report we address whether the HisRS-related sequences are required for GCN2 phosphorylation of eIF-2alpha in an in vitro assay. To measure the activity of GCN2 kinase in cellular extracts, we expressed and purified a truncated form of yeast eIF-2alpha. Phosphorylation of the recombinant eIF-2alpha substrate was dependent on both GCN2 kinase activity and the eIF-2alpha phosphorylation site, serine 51. Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2alpha in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2alpha phosphorylation in the in vitro assay. These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function.
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PMID:Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2. 879 80

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.
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PMID:Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. 923 5

The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.
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PMID:Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain. 956 89

Based on characteristic amino acid sequences of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc-3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells.
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PMID:cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains required for general amino acid control. 968 94


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