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

Abstract

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...

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

“…Analysis of autopsied brain tissues from Alzheimer's disease patients showed accumulation of activated PKR in nuclei. Many of the stimuli that trigger PKR-dependent apoptosis in the absence of viral infection rely on PACT/RAX activation; PACT/RAX mediates PKR activation and subsequent apoptosis in response not only to cytokines and serum withdrawal but also to chemotherapy, ethanol, and viral infection (20). Alterations in levels of any of the array of PKR viral (and cellular) inhibitors (see the next section) also have a profound impact on apoptosis induction (64,351).…”

“…Although in vitro analysis shows that domain 3 of PACT is sufficient to activate PKR, all three domains are needed for efficient PKR activation in vivo (282). As mentioned above, different stresses trigger PACT phosphorylation (20), and only then does PACT bind and activate PKR (165,278,282). PACT might thus be involved in PKR activation in uninfected cells.…”

“…PACT and its mouse orthologue RAX are cell proteins that bind to and activate PKR independently of dsRNA or other molecules (165,279). RAX levels do not vary during cell stress, but RAX is phosphorylated, after which it binds to and activates PKR (20). PACT/RAX therefore acts a physiological mediator that links a wide range of different cell stresses to PKR.…”

The dsRNA-dependent protein kinase, PKR and cell death

“…[84][85][86][87] PACT/RAX is member of the DRBD family, as described earlier, and has been shown to induce apoptosis following activation of PKR, in response to stress such as serum starvation and arsenite treatment. 31,88 Similar studies aimed at seeking novel PKRinteracting proteins lead to the isolation of nucleophosmin (NPM), a protein often expressed at elevated levels in tumors. 89 NPM was shown to bind to PKR and inhibit its activity, thus preventing apoptotic responses and potentially promoting sporadic malignancies.…”

The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor κB and Akt Activation

“…We also investigated the role of the pro-survival Akt pathway because of data suggesting that TNF-a -induced PKR activation led to Akt phosphorylation only in PKR +/+ cells. 8 PKR +/+ MEFs exhibited dose-and time-dependent increases in phosphorylation of Akt after radiation, whereas PKR -/-MEFs did not show Akt phosphorylation ( Fig. 5A and B).…”