Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

Peters, Gregory A.; Hartmann, Rune; Qin, Jun; Sen, Ganes C.

doi:10.1128/mcb.21.6.1908-1920.2001

Cited by 153 publications

(202 citation statements)

References 36 publications

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“…Once activated, PKR then phosphorylates its downstream targets, including eIF2␣, thereby resulting in translation inhibition and subsequent apoptosis. These data extend recent reports with PACT deletion mutants indicating that the first two dsRNA binding motifs are responsible for both dsRNA and PKR binding but dispensable for PKR activation, whereas the third motif is required for PKR activation (5,30). Thus, we propose that the phosphorylation of serine 18 may lead to a conformational change in RAX/PACT that facilitates the interaction of its requisite C-terminal dsRNA binding motif with PKR thereby leading to PKR activation (Fig.…”

Section: Fig 4 Rax Phosphorylation Does Not Affect Protein Stabilitysupporting

confidence: 74%

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: 74%

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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“…Based on these observations it was proposed that the entire PKR regulatory domain (1~250), including the RBD and the linker that connects it to the kinase needs to be sequestered to achieve full activation. 24 The requirement for a spacer in the artificial PACT construct (RBD-spacer-d3) suggests that the RBD-KD linker of PKR is displaced by PACT-KD interactions rather than sequestered by direct binding to PACT d3. Analogous to PACT d3 domain, heparin has been shown to activate PKR by binding directly to the N-lobe of the KD.…”

Section: Discussionmentioning

confidence: 99%

Mapping of the Auto-inhibitory Interactions of Protein Kinase R by Nuclear Magnetic Resonance

Gelev

Aktaş

Marintchev

et al. 2006

Journal of Molecular Biology

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SummaryThe dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and antiproliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed 15 N-HSQC titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2α docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2α docking site, F495A displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2α binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.

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“…with PACT leading to PKR activation in the absence of dsRNA [45]. PACT consists of two redundant PKR binding domains and one distinct PKR activation domain [46] and is itself regulated by TAR DNA binding protein (TRBP). In the unstressed state, TRBP sequesters PACT and so prevents it from activating PKR.…”

Section: Dystonia- Parkinsonism (Dyt16)mentioning

confidence: 99%

Recent Advances in the Molecular Pathogenesis of Dystonia-Plus Syndromes and Heredodegenerative Dystonias

Casper

Kalliolia²,

Warner³

2013

Current Neuropharmacology

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The majority of studies investigating the molecular pathogenesis and cell biology underlying dystonia have been performed in individuals with primary dystonia. This includes monogenic forms such as DYT1and DYT6 dystonia, and primary focal dystonia which is likely to be multifactorial in origin. In recent years there has been renewed interest in non-primary forms of dystonia including the dystonia-plus syndromes and heredodegenerative disorders. These are caused by a variety of genetic mutations and their study has contributed to our understanding of the neuronal dysfunction that leads to dystonia These findings have reinforced themes identified from study of primary dystonia including abnormal dopaminergic signalling, cellular trafficking and mitochondrial function. In this review we highlight recent advances in the understanding of the dystonia-plus syndromes and heredodegenerative dystonias.

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Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

Cited by 153 publications

References 36 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

Mapping of the Auto-inhibitory Interactions of Protein Kinase R by Nuclear Magnetic Resonance

Recent Advances in the Molecular Pathogenesis of Dystonia-Plus Syndromes and Heredodegenerative Dystonias

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