Human Gene Profiling in Response to the Active Protein Kinase, Interferon-induced Serine/threonine Protein Kinase (PKR), in Infected Cells

Abstract

The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were downregulated. The ATF-3 transcription factor, involved in stress-induced ␤-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VV⌬E3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR ؊/؊ cells. Using null cells for ATF-3 and for the p65 subunit of NF-B, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-B activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis.The double-stranded RNA (dsRNA) 4 -dependent protein kinase (PKR) is a key mediator in the antiviral effects of interferon (IFN) and a dynamic participant in apoptosis induced by various stimuli (1) (reviewed in Ref.2). PKR controls cell processes, such as growth (3) differentiation (4), apoptosis (5, 6), stress response (7), anti-tumor activity (8, 9), and antiviral functions (10, 11). After PKR activation, the initiation of protein synthesis is inhibited by phosphorylation of the ␣ subunit of the eukaryotic initiation factor 2 (eIF-2␣) by PKR (12). This kinase regulates the activation of several transcription factors involved in virus-induced apoptosis (13), including p53 (14), IRF-1 (15), and IRF-3, and activation of c-Jun (16). By regulating the expression of genes involved in cell proliferation, NF-B induction is important in mediating PKR function (16). NF-B activation by PKR also triggers production of 17), an essential component in PKR functionality.The genes involved in PKR-induced apoptosis in infected human cells have not been defined. We previously described an isopropyl-␤-D-thiogalactopyranoside-inducible VV system in which PKR expression triggers apoptosis (5) (for a review, see Ref. 18). When PKR was expressed, the antiviral action against VV and vesicular stomatitis virus provoked a translational block by phosphorylation of eIF-2␣ and triggered NF-B activation through the IB kinase complex (5,10,11,19,20)....

Activating transcription factor 3: a hormone responsive gene in the etiology of hypospadias

“…It has traditionally been related to cell survival and/or apoptosis, stress, DNA damage, homeostasis, wound healing, cell adhesion, inflammation, nutrient limitation, and chemical exposure (32)(33)(34)(35)(36). Moreover, overexpression of ATF3 suppresses cell growth, by slowing the progression of cells from G1 to S phase, and it has been related to pro-apoptotic events and in the regulation of cell fate (36,37). This is a relevant aspect due to the importance of cell proliferation and apoptosis regulation during development (38,39).…”

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

“…Although translation of most cell and viral mRNAs is inhibited by eIF-2␣ phosphorylation, translation of few mRNAs involved in the stress response is enhanced by limited eIF-2␣ phosphorylation. This is the case for yeast GCN4 and mammalian activating transcription factor 4 (ATF-4), ATF-3, and CAT-1 mRNAs (79,134,232,386). Under nonstress conditions, translation of these mRNAs is inhibited by the presence of upstream short open reading frames (ORFs) that attract ribosomes to translate short peptides, restricting the flow of scanning ribosomes to the bona fide GCN4 and ATF-4 ORFs (78).…”

“…Overexpression of full-length ATF-3 protein in colorectal cancer has antitumorigenic properties, whereas an antisense RNA targeting ATF-3 has the opposite effect (27). Using microarray analysis of human cells infected with a VV recombinant expressing wt PKR or the catalytically inactive form of PKR-K296R under inducible conditions, ATF-3 was recently identified as a gene that is selectively upregulated by the active PKR enzyme (134). Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VV⌬E3L) triggered an increase in ATF-3 expression that was not observed in PKR Ϫ/Ϫ cells.…”

Cigarette smoke selectively enhances viral PAMP– and virus-induced pulmonary innate immune and remodeling responses in mice

“…After activation, it plays an important role in cell signaling and regulates gene transcription, at least in part, via its ability to interact with known signaling pathways, including NF-κB, MAP kinases, p53, STATs, and IFN regulatory factor 1 (IRF1) (41). As a result, it is now appreciated that it also plays an important role in the regulation of cell proliferation and differentiation, tumor suppression, and cellular apoptosis (40)(41)(42). Our studies demonstrate that CS interacts with viral PAMPs and live virus to activate PKR and eIF2α and that this induction is mediated via a MAVS-, IL-18Rα-, and IFN-γ-dependent mechanism.…”