dsRNA-dependent Kinase PKR Research Articles

Activation of the Double-stranded RNA-dependent Protein Kinase PKR by Small Ubiquitin-like Modifier (SUMO)

The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.

IFN-γ-induced upregulation of Fcγ-receptor-I during activation of monocytic cells requires the PKR and NFκB pathways

Interferon (IFN)-γ is a potent activator of macrophages, increasing the cells capacity to perform specific functions during inflammation and immune response. In this report we use IFN-γ-induced upregulation of the high affinity receptor for IgG (FcγRI/CD64) in the human monocytic cell line U-937 as a model for monocytic activation. We show that upregulation of FcγRI is dependent on signals mediated by the dsRNA-dependent kinase PKR, and the transcription factor NFκB. Silencing of PKR expression by siRNA or inhibition of PKR by 2-aminopurine (2-AP) potently blocks the IFN-γ-induced transcriptional activation of the FcγRI promoter. We find that the serine 727 phosphorylation of Stat1, required for full IFN-γ-induced FcγRI promoter activity, is dependent on PKR. We further show that IFN-γ induction of FcγRI upregulation is dependent on the NFκB pathway, as evidenced by inhibition of NFκB using a phosphorylation defective IκBα (S32A/S36A) mutant, or inhibiting the IκB-kinase (IKK) by treatment with BMS345541. Our results suggest that IFN-γ-induced increase of FcγRI expression requires the integration of two signalling events: PKR-dependent Stat1 serine 727 phosphorylation, and activation of NFκB.

A FADD-dependent innate immune mechanism in mammalian cells

Vertebrate innate immunity provides a first line of defence against pathogens such as viruses and bacteria. Viral infection activates a potent innate immune response, which can be triggered by double-stranded (ds)RNA produced during viral replication. Here, we report that mammalian cells lacking the death-domain-containing protein FADD are defective in intracellular dsRNA-activated gene expression, including production of type I (alpha/beta) interferons, and are thus very susceptible to viral infection. The signalling pathway incorporating FADD is largely independent of Toll-like receptor 3 and the dsRNA-dependent kinase PKR, but seems to require receptor interacting protein 1 as well as Tank-binding kinase 1-mediated activation of the transcription factor IRF-3. The requirement for FADD in mammalian host defence is evocative of innate immune signalling in Drosophila, in which a FADD-dependent pathway responds to bacterial infection by activating the transcription of antimicrobial genes. These data therefore suggest the existence of a conserved pathogen recognition pathway in mammalian cells that is essential for the optimal induction of type I interferons and other genes important for host defence.