This review describes the structure and function of the interferon (IFN)-inducible, double-stranded RNA-activated protein kinase PKR. This protein kinase has been studied extensively in recent years, and a large body of evidence has accumulated concerning its expression, interaction with regulatory RNA and protein molecules, and modes of activation and inhibition. PKR has been shown to play a variety of important roles in the regulation of translation, transcription, and signal transduction pathways through its ability to phosphorylate protein synthesis initiation factor eIF2, I-kappaB (the inhibitor of NF-kappaB), and other substrates. Expression studies involving both the wild-type protein and dominant negative mutants of PKR have established roles for the enzyme in the antiviral effects of IFNs, in the responses of uninfected cells to physiologic stresses, and in cell growth regulation. The possibility that PKR may function as a tumor suppressor and inducer of apoptosis suggests that this IFN-regulated protein kinase may be of central importance to the control of cell proliferation and transformation.

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the wellcharacterized ATR (Ataxia telangiectasia and Rad-3 related)/ CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the Sphase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3 0 UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Objective: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. Methods:White matter postmortem MS (n ϭ 10) and control tissue (n ϭ 11) was analyzed for the expression of the proinflammatory cytokine interferon ␣ (IFN␣) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. Results:We detected overexpression of IFN␣ in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFN␣ in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFN␣ was overexpressed in these preselected active MS lesions. EBERϩ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFN␣ production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFN␣ production in vitro. Conclusion:These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFN␣ production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases. Neurology ® 2012;78:15-23 GLOSSARY ds ϭ double-stranded; EBER ϭ EBV-encoded RNA; EBNA-1 ϭ EBV nuclear antigen 1; EBV ϭ Epstein-Barr virus; FCS ϭ fetal calf serum; H&E ϭ hematoxylin & eosin; HEK ϭ human embryonic kidney; HLA ϭ human leukocyte antigen; HSE ϭ herpes simplex encephalitis; IFN␣ ϭ interferon ␣; IgG ϭ immunoglobulin G; ISH ϭ in situ hybridization; LFB ϭ Luxol fast blue; mAb ϭ monoclonal antibody; MS ϭ multiple sclerosis; OHL ϭ oral hairy leukoplakia; PBS ϭ phosphate-buffered saline; pDC ϭ plasmacytoid dendritic cell; PLP ϭ protein lipid protein; SLE ϭ systemic lupus erythematosus; SSPE ϭ subacute sclerosing panencephalitis; TLR3 ϭ Toll-like receptor-3.The innate immune response is an essential part of the host response to many infections and type I interferon (IFN) is produced within hours of systemic infection.1 Our previous findings showed that Toll-like receptor-3 (TLR3) stimulation of human microglia with the viral-mimic polyIC led to IFN␣ production and downstream Th1 polarization of CD4ϩ helper T cells, 2 which may impact on CNS immunity.

Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb⅐GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb⅐GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/ mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acidreplete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyltRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA Leu does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecularweight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecularweight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m 7 GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.