Interaction between RAX and PKR Modulates the Effect of Ethanol on Protein Synthesis and Survival of Neurons

Abstract

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the ␣-subunit of eukaryotic translation initiation factor-2 (eIF2␣) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2␣ in the developing cerebellum. The effect of ethanol on PKR/eIF2␣ phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2␣ phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2␣ phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2␣ activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system. Fetal alcohol syndrome is the most common non-hereditary cause of mental retardation (1). Prenatal exposure to alcohol disrupts many events of neuronal development, including neurogenesis, migration, cell survival, protein synthesis, axonal growth, and synaptogenesis (2-5). Neuronal death is a prominent pathologic effect of fetal alcohol exposure. This loss of neurons may underlie many of the behavioral deficits observed in fetal alcohol syndrome. The vulnerability of neurons to alcohol neurotoxicity differs among brain regions and changes with developmental stages (6, 7). The causes for ethanol-induced neuronal loss remain incompletely elucidated, as are the cellular and molecular mechanisms underlying the spatiotemporal window of susceptibility.The double-stranded RNA (dsRNA) 2 -activated protein kinase (PKR) is a serine/threonine protein kinase ubiquitously expressed in mammalian cells (8,9). PKR is initially identified as an interferon-induced protein that is activated in virus-infected cells by dsRNA produced during the virus life cycle (10, 11). PKR consists of two functionally distinct domains, an N-terminal dsRNA binding regulatory domain and a C-terminal catalytic domain. PKR is activated by dsRNA (11); interaction with dsRNA causes PKR to form homodimers and to autophosphoryl...

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“…Moreover, their interaction with PKR has functional implications in inflammation, stress response, viral infection, and oncogenesis (20,34,(42)(43)(44). Although TRBP and PACT directly interact with each other (Fig.…”

The Role of Glycogen Synthase Kinase 3β in the Transformation of Epidermal Cells

“…Stable transfectants expressing various GSK3h constructs were established as previously described (19). V5-tagged GSK3h constructs (wild-type, S9A, and K85R) carried by vector pcDNA3 were generous gifts from Dr. Thilo Hagen (University Hospital Nottingham).…”

The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation

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