Functional Characterization of pkr Gene Products Expressed in Cells from Mice with a Targeted Deletion of the N terminus or C terminus Domain of PKR

Baltzis, Dionissios; Li, Suiyang; Koromilas, Antonis E.

doi:10.1074/jbc.m203564200

Cited by 67 publications

(64 citation statements)

References 39 publications

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“…Furthermore, PKR does not appear to be responsible for RAX phosphorylation, because we found that RAX can be phosphorylated when stably expressed in PKR null mouse embryonic fibroblasts (data not shown). Although recent evidence indicates that these fibroblasts may retain "some" PKR kinase activity (32), these data are consistent with our previous observation that RAX phosphorylation is not blocked by the often used PKR inhibitor, 2-aminopurine (1). Thus, additional studies will be required to identify the RAX/ PACT kinase(s).…”

Section: Fig 4 Rax Phosphorylation Does Not Affect Protein Stabilitysupporting

confidence: 81%

Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition

Bennett¹,

Blalock²,

May

2004

Journal of Biological Chemistry

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It is now apparent that the double-stranded (ds)RNAdependent protein kinase, PKR, is a regulator of diverse cellular responses to stress. Recently, the murine dsRNA-binding protein RAX and its human ortholog PACT were identified as cellular activators of PKR. Previous reports demonstrate that following stress, RAX/ PACT associates with and activates PKR resulting in eIF2␣ phosphorylation, consequent translation inhibition, and cell death via apoptosis. Although RAX/PACT is phosphorylated during stress, any regulatory role for this post-translational modification has been uncertain. Now we have discovered that RAX is phosphorylated on serine 18 in both human and mouse cells. The non-phosphorylatable form of RAX, RAX(S18A), although still able to bind dsRNA and associate with PKR, fails to activate PKR following stress. Furthermore, stable expression of RAX(S18A) results in a dominant-negative effect characterized by deficiency of eukaryotic initiation factor 2 ␣ subunit phosphorylation, delay of translation inhibition, and failure to undergo rapid apoptosis following removal of interleukin-3. We propose that the ability of RAX to activate PKR is regulated by a sequential mechanism featuring RAX association with PKR, RAX phosphorylation at serine 18, and activation of PKR.The mouse protein RAX and its human ortholog PACT are the only known cellular activators for the double-stranded RNAdependent kinase, PKR 1 (1-3). RAX and PACT are 98% identical in amino acid sequence and contain three conserved dsRNA binding motifs. The N-terminal first and second motifs bind to dsRNA and are necessary for association with PKR, whereas the C-terminal third motif is apparently not important for either dsRNA or PKR interaction but is instead responsible for activating the kinase activity of PKR. It has been demonstrated that RAX/PACT can efficiently activate PKR in vitro (1, 2). However, in vivo, RAX/PACT-mediated PKR activation is dependent on stress application to cells (1, 3, 4). Although both RAX/PACT and PKR are dsRNA-binding proteins, PKR activation may not rely on dsRNA binding because in vitro activation by RAX/PACT does not require dsRNA (1-3, 5).Traditionally, PKR has been studied in the context of the host anti-viral response (6, 7). The serine-threonine kinase activity of PKR is activated by viral dsRNA, resulting in the phosphorylation of a physiological substrate of PKR, eIF2␣, and consequent translation inhibition. However, it has become clear recently that PKR is also an essential mediator of signaling by both cytokines and growth factors and may even function as a tumor suppressor (8 -10). In addition to its role in translation, PKR has been observed to participate in several transcription pathways (11)(12)(13)(14)(15). Under such circumstances, where there may be no apparent source of dsRNA necessary to activate PKR, it is believed that RAX/PACT may be responsible for PKR activation (1, 2).Our laboratory has studied the mechanism of apoptosis following withdrawal of growth factor IL-3 from factor-dependent hemato...

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Section: Fig 4 Rax Phosphorylation Does Not Affect Protein Stabilitysupporting

confidence: 81%

Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition

Bennett¹,

Blalock²,

May

2004

Journal of Biological Chemistry

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“…13 We examined the biological role of PKR in response to DNA damage in MEFs that are deficient in p53 due to spontaneous immortalization. For this purpose immortalized MEFs completely deficient in PKR activity (PKR À/À MEFs) 14,15 together with their isogenic wild-type counterparts were treated with the chemotherapeutic drug doxorubicin and subjected to analysis of cell death by flow cytometry. We noticed that PKR was required for optimal induction of cell death in response to doxorubicin (Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

et al. 2010

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The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the a-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2a phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2a kinases and the biological role of eIF2a phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2a kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2a phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2a phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2a phosphorylation. The eukaryotic cell has established intricate mechanisms to cope with environmental stress. It does so by changing protein expression in a manner that promotes adaptation to the assaulting stress. Protein expression within the cell is regulated at the transcriptional, translational and posttranslational levels. Translational control is often used under conditions of cellular stress because it allows immediate and selective changes in protein levels. 1 Translation itself is divided into three distinct phases: initiation, elongation and termination. By far the most explored step is that of initiation, which has been shown to be regulated by different extracellular stimuli. 1 One of the major pathways that control translation initiation is that of the phosphorylation of the a-subunit of translation initiation factor eIF2. 2 Phosphorylation of eIF2a at serine 51 (S51) leads to the inhibition of global protein synthesis providing the cell with the opportunity to elicit adaptive responses not only by saving energy but also by preventing the accumulation of unwanted proteins that could interfere with cellular functions. 1 There are four eIF2a kinases that share a homologous kinase domain (KD) but possess different regulatory domains that enable them to become activated by distinct stimuli. 2 The eIF2a kinases are the heme-regulated inhibitor, which is found mainly in erythroid cells and is activated by heme deficiency; the endoplasmic reticulum (ER)-resident kinase (PERK), which is activated by ER stress and inhibits protein synthesis as part of the unfolded protein response; the general aminoacid nonderepressing kinase 2 (GCN2), which responds to amino-acid deprivation and becomes activated by uncharged tRNA and the RNA-dependent protein kinase PKR, an interfer...

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“…Immunoprecipitation and immunoblotting were performed as described in ref. 32. The following Abs were used: anti-phosphotyrosine 4G10 mouse mAb (Upstate Biotechnology, Lake Placid, NY), antihemagglutinin 12CA5 mouse mAb (Roche), anti-human PKR mouse monoclonal F9 mAb (16), anti-GST rabbit polyclonal Ab (Amersham Pharmacia), anti-TC-PTP mouse mAb (33), antieIF2␣ rabbit polyclonal Ab (Cell Signaling Technology), antiphosphoS51 of eIF2␣ rabbit polyclonal Ab (16), anti-FLAG (M2) mouse mAb (Sigma), anti-phosphoT446 of human PKR (anti-PKRpT446) rabbit polyclonal Ab (Upstate Biotechnology), anti-phosphoY101 of human PKR (anti-PKRpY101) polyclonal Ab, anti-phosphoY162 of human PKR (anti-PKRpY162) polyclonal Ab, anti-phosphoY293 of human PKR (antiPKRpY293) polyclonal Ab, Ab against the catalytic D subunit of PKA (C-20; Santa Cruz Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2α RNA-dependent protein kinase

Wang

Baltzis

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of the ␣-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine͞threonine RNAdependent protein kinase (PKR) is an eIF2␣ kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2␣ phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2␣ kinase PKR.cell proliferation ͉ protein phosphorylation ͉ translational control ͉ virus infection

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Functional Characterization of pkr Gene Products Expressed in Cells from Mice with a Targeted Deletion of the N terminus or C terminus Domain of PKR

Cited by 67 publications

References 39 publications

Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition

Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2α RNA-dependent protein kinase

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