scholarly journals Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2.

1993 ◽  
Vol 13 (8) ◽  
pp. 5099-5111 ◽  
Author(s):  
R J Rolfes ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Transcriptional Activation ◽  
Phosphorylation Site ◽  
Transcriptional Activator ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Single Amino Acid ◽  
Biosynthetic Genes ◽  
Transcriptional Activator Protein

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.

Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2

1993 ◽  
Vol 13 (8) ◽  
pp. 5099-5111
Author(s):  
R J Rolfes ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Transcriptional Activation ◽  
Phosphorylation Site ◽  
Transcriptional Activator ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Single Amino Acid ◽  
Biosynthetic Genes ◽  
Transcriptional Activator Protein

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.

scholarly journals A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation

2007 ◽  
Vol 402 (1) ◽  
pp. 163-173 ◽  
Author(s):  
Alex B. Lopez ◽  
Chuanping Wang ◽  
Charlie C. Huang ◽  
Ibrahim Yaman ◽  
Yi Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Basic Leucine Zipper

The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-β and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPβ activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPβ. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.

scholarly journals GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 21-27 ◽  
Author(s):  
J M Salmeron ◽  
K K Leuther ◽  
S A Johnston
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Activation Function ◽  
Single Amino Acid ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Transcriptional Activator Protein ◽  
Carboxy Terminal

Abstract The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.

Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases

1992 ◽  
Vol 12 (12) ◽  
pp. 5801-5815
Author(s):  
M Ramirez ◽  
R C Wek ◽  
C R Vazquez de Aldana ◽  
B M Jackson ◽  
B Freeman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translation Initiation ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Atp Binding ◽  
Sequence Motif ◽  
Trna Synthetases

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.

scholarly journals The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

2006 ◽  
Vol 394 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Franc Llorens ◽  
Anna Duarri ◽  
Eduard Sarró ◽  
Nerea Roher ◽  
Maria Plana ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Mutant Cell ◽  
Phosphorylation Site ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Main Site

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.

scholarly journals Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

1992 ◽  
Vol 12 (12) ◽  
pp. 5801-5815 ◽  
Author(s):  
M Ramirez ◽  
R C Wek ◽  
C R Vazquez de Aldana ◽  
B M Jackson ◽  
B Freeman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translation Initiation ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Atp Binding ◽  
Sequence Motif ◽  
Trna Synthetases

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.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

scholarly journals C-Terminal Region of STAT-1α Is Not Necessary for Its Ubiquitination and Degradation Caused by Mumps Virus V Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 12683-12690 ◽  
Author(s):  
Noriko Yokosawa ◽  
Shin-ichi Yokota ◽  
Toru Kubota ◽  
Nobuhiro Fujii
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Tandem Repeats ◽  
Mumps Virus ◽  
Terminal Region ◽  
Single Amino Acid ◽  
Luciferase Reporter ◽  
Enhancer Element ◽  
V Protein ◽  
Gene Assay

ABSTRACT Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-α) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-α-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.

scholarly journals Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5275-5284 ◽  
Author(s):  
Jun Lu ◽  
Qinghua Wang ◽  
Lianghu Huang ◽  
Huiyue Dong ◽  
Lingjing Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endoplasmic Reticulum ◽  
Mesenchymal Stem Cells ◽  
Protein Kinase ◽  
Er Stress ◽  
Human Mesenchymal Stem Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Human Umbilical Cord ◽  
Amp Activated Protein Kinase

Abstract Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative

1992 ◽  
Vol 12 (2) ◽  
pp. 444-454
Author(s):  
S M Ruben ◽  
R Narayanan ◽  
J F Klement ◽  
C H Chen ◽  
C A Rosen
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Functional Characterization ◽  
Transcriptional Activator ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Activation Domain ◽  
Transcriptional Activator Protein

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.

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