Proapoptotic protein PACT is expressed at high levels in colonic epithelial cells in mice

2002 ◽  
Vol 283 (3) ◽  
pp. G801-G808 ◽  
Author(s):  
Vishal Gupta ◽  
Rekha C. Patel
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Initiation Factor ◽  
Colonic Epithelial Cells ◽  
Α Subunit ◽  
Proapoptotic Protein ◽  
Mouse Tissues ◽  
In Cells

The protein activator of RNA-activated protein kinase (PKR) is a proapoptotic protein called PACT. PKR is an interferon (IFN)-induced serine-threonine protein kinase that plays a central role in IFN's antiviral and antiproliferative activities. PKR activation in cells leads to phosphorylation of the α-subunit of the eukaryotic protein synthesis initiation factor (eIF)2α, inhibition of protein synthesis, and apoptosis. In the absence of viral infections, PKR is activated by its activator PACT, especially in response to diverse stress signals. Overexpression of PACT in cells causes enhanced sensitivity to stress-induced apoptosis. We examined PACT expression in different mouse tissues and evaluated its possible role in regulating apoptosis. PACT is expressed at high levels in colonic epithelial cells, especially as they exit the cell cycle and enter an apoptotic program. PACT expression also coincides with the presence of active PKR and phosphorylated eIF2α. These results suggest a possible role of PACT-mediated PKR activation in the regulation of epithelial cell apoptosis in mouse colon. In addition, transient overexpression of PACT in a nontransformed intestinal epithelial cell line leads to induction of apoptosis, further supporting PACT's role in inducing apoptosis.

scholarly journals eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jennifer Deborah Wuerth ◽  
Matthias Habjan ◽  
Markus Kainulainen ◽  
Besim Berisha ◽  
Damien Bertheloot ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Nonstructural Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mammalian Host ◽  
Eukaryotic Initiation Factor ◽  
Α Subunit ◽  
Immune Factor

ABSTRACT RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α. IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

scholarly journals Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells

2009 ◽  
Vol 83 (23) ◽  
pp. 12462-12472 ◽  
Author(s):  
Xiaoxing Wang ◽  
Ying Liao ◽  
Pei Ling Yap ◽  
Kim J. Png ◽  
James P. Tam ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Viral Replication ◽  
De Novo ◽  
Initiation Factor ◽  
Animal Cells ◽  
Protein Kinase R ◽  
Α Subunit ◽  
Infected Cells ◽  
De Novo Protein Synthesis

ABSTRACT A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the α subunit of eukaryotic initiation factor 2 (eIF-2α). In this report, we show that phosphorylation of eIF-2α was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2α, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2α, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2α, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

scholarly journals Bile acids modulate the Golgi membrane fission process via a protein kinase Cη and protein kinase D-dependent pathway in colonic epithelial cells

2010 ◽  
Vol 31 (4) ◽  
pp. 737-744 ◽  
Author(s):  
Anne-Marie Byrne ◽  
Eilis Foran ◽  
Ruchika Sharma ◽  
Anthony Davies ◽  
Ciara Mahon ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Bile Acids ◽  
Protein Kinase D ◽  
Golgi Membrane ◽  
Colonic Epithelial Cells ◽  
Fission Process ◽  
Membrane Fission ◽  
Dependent Pathway

scholarly journals Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2α

1999 ◽  
Vol 342 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Shinya SATOH ◽  
Makoto HIJIKATA ◽  
Hiroshi HANDA ◽  
Kunitada SHIMOTOHNO
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Caspase 3 ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation

Eukaryotic translation initiation factor 2α (eIF-2α), a target molecule of the interferon-inducible double-stranded-RNA-dependent protein kinase (PKR), was cleaved in apoptotic Saos-2 cells on treatment with poly(I)˙poly(C) or tumour necrosis factor α. This cleavage occurred with a time course similar to that of poly(ADP-ribose) polymerase, a well-known caspase substrate. In addition, eIF-2α was cleaved by recombinant active caspase-3 in vitro. By site-directed mutagenesis, the cleavage site was mapped to an Ala-Glu-Val-Asp300 ↓ Gly301 sequence located in the C-terminal portion of eIF-2α. PKR phosphorylates eIF-2α on Ser51, resulting in the suppression of protein synthesis. PKR-mediated translational suppression was repressed when the C-terminally cleaved product of eIF-2α was overexpressed in Saos-2 cells, even though PKR can phosphorylate this cleaved product. These results suggest that caspase-3 or related protease(s) can modulate the efficiency of protein synthesis by cleaving the α subunit of eIF-2, a key component in the initiation of translation.

Intravenous administration of amino acids during anesthesia stimulates muscle protein synthesis and heat accumulation in the body

2006 ◽  
Vol 290 (5) ◽  
pp. E882-E888 ◽  
Author(s):  
Ippei Yamaoka ◽  
Masako Doi ◽  
Mitsuo Nakayama ◽  
Akane Ozeki ◽  
Shinji Mochizuki ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Intravenous Administration ◽  
Translation Initiation ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Mammalian Target Of Rapamycin ◽  
The Body ◽  
Initiation Factor ◽  
Heat Accumulation

The present study was conducted to determine the contribution of muscle protein synthesis to the prevention of anesthesia-induced hypothermia by intravenous administration of an amino acid (AA) mixture. We examined the changes of intraperitoneal temperature (Tcore) and the rates of protein synthesis ( Ks) and the phosphorylation states of translation initiation regulators and their upstream signaling components in skeletal muscle in conscious (Nor) or propofol-anesthetized (Ane) rats after a 3-h intravenous administration of a balanced AA mixture or saline (Sal). Compared with Sal administration, the AA mixture administration markedly attenuated the decrease in Tcore in rats during anesthesia, whereas Tcore in the Nor-AA group became slightly elevated during treatment. Stimulation of muscle protein synthesis resulting from AA administration was observed in each case, although Ks remained lower in the Ane-AA group than in the Nor-Sal group. AA administration during anesthesia significantly increased insulin concentrations to levels ∼6-fold greater than in the Nor-AA group and enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 protein kinase relative to all other groups and treatments. The alterations in the Ane-AA group were accompanied by hyperphosphorylation of protein kinase B and the mammalian target of rapamycin (mTOR). These results suggest that administration of an AA mixture during anesthesia stimulates muscle protein synthesis via insulin-mTOR-dependent activation of translation initiation regulators caused by markedly elevated insulin and, thereby, facilitates thermal accumulation in the body.

scholarly journals Varicella-Zoster Virus IE63, a Major Viral Latency Protein, Is Required To Inhibit the Alpha Interferon-Induced Antiviral Response

2007 ◽  
Vol 81 (15) ◽  
pp. 7844-7851 ◽  
Author(s):  
Aruna P. N. Ambagala ◽  
Jeffrey I. Cohen
Keyword(s):  
Varicella Zoster Virus ◽  
Melanoma Cells ◽  
Alpha Interferon ◽  
Parental Virus ◽  
Initiation Factor ◽  
Viral Gene ◽  
Α Subunit ◽  
Varicella Zoster ◽  
In Cells ◽  
Hsv 1

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is the most abundant transcript expressed during latency in human sensory ganglia. VZV with ORF63 deleted is impaired for replication in melanoma cells and fibroblasts and for latency in rodents. We found that replication of the ORF63 deletion mutant is fully complemented in U2OS cells, which have been shown to complement the growth of herpes simplex virus type 1 (HSV-1) ICP0 mutants. Since HSV-1 ICP0 mutants are hypersensitive to alpha interferon (IFN-α), we examined the effect of IFN-α on VZV replication. Replication of the ORF63 mutant in melanoma cells was severely inhibited in the presence of IFN-α, in contrast to other VZV mutants that were similarly impaired for replication or to parental virus. The VZV ORF63 mutant was not hypersensitive to IFN-γ. IFN-α inhibited viral-gene expression in cells infected with the ORF63 mutant at a posttranscriptional level. Since IFN-α stimulates gene products that can phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF-2α) and inhibit translation, we determined whether cells infected with the ORF63 mutant had increased phosphorylation of eIF-2α compared with cells infected with parental virus. While phosphorylated eIF-2α was undetectable in uninfected cells or cells infected with parental virus, it was present in cells infected with the ORF63 mutant. Conversely, expression of IE63 (encoded by ORF63) in the absence of other viral proteins inhibited phosphorylation of eIF-2α. Since IFN-α has been shown to limit VZV replication in human skin xenografts, the ability of VZV IE63 to block the effects of the cytokine may play a critical role in VZV pathogenesis.

scholarly journals Phosphorylation of the α subunit of initiation factor 2 correlates with the inhibition of translation following transient cerebral ischaemia in the rat

1994 ◽  
Vol 302 (2) ◽  
pp. 335-338 ◽  
Author(s):  
J Burda ◽  
M E Martín ◽  
A García ◽  
A Alcázar ◽  
J L Fando ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cerebral Ischaemia ◽  
Ternary Complex ◽  
Initiation Factor ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Vessel Occlusion ◽  
Α Subunit ◽  
Free System ◽  
Initiation Factor 2

Rats were subjected to the standard four-vessel occlusion model of cerebral transient ischaemia (vertebral and carotid arteries) for 15 and 30 min. After a 30 min recirculation period, protein synthesis rate, initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) activities, and the level of phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) were determined in the neocortex region of the brain from sham-operated controls and ischaemic animals. Following reversible cerebral ischaemia, the protein synthesis rate, as measured in a cell-free system, was significantly inhibited (70%) in the ischaemic animals. eIF-2 activity, as measured by its ability to form a ternary complex, also decrease parallel to the decrease in protein synthesis. As eIF-2 activity was assayed in the presence of Mg2+ and GTP-regenerating capacity, the decrease in ternary-complex formation indicated the possible impairment of GEF activity. Since phosphorylated eIF-2 [eIF-2(alpha P)] is a powerful inhibitor of GEF, the levels of phosphorylated eIF-2 alpha were determined, and an increase from 7% phosphorylation in sham control rats to 20% phosphorylation in 15 min and 29% phosphorylation in 30 min in ischaemic rats was observed, providing evidence for a tight correlation of phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Moreover, GEF activity measured in the GDP-exchange assay was in fact inhibited in the ischaemic animals, proving that protein synthesis is impaired by the presence of eIF-2(alpha P), which blocks eIF-2 recycling.

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats

2001 ◽  
Vol 281 (1) ◽  
pp. C179-C187 ◽  
Author(s):  
Troy A. Hornberger ◽  
R. Bridge Hunter ◽  
Susan C. Kandarian ◽  
Karyn A. Esser
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Muscle Atrophy ◽  
Protein Translation ◽  
Hindlimb Unloading ◽  
Initiation Factor ◽  
Skeletal Muscle Atrophy ◽  
Α Subunit ◽  
Translation Factors ◽  
Ribosomal S6 Kinase

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 α-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).

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