Dataset Information

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.

ABSTRACT: TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-beta in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFkappaB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKepsilon at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKepsilon at Ser-172 in response to LPS, poly(I:C), interleukin-1alpha (IL-1alpha), or tumor necrosis factor alpha and actually enhanced the LPS, poly(I:C), and IL-1alpha-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKepsilon are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKepsilon control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1alpha (but not tumor necrosis factor alpha) to prevent the hyperactivation of these enzymes.

SUBMITTER: Clark K

PROVIDER: S-EPMC2682862 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.

Clark Kristopher K Plater Lorna L Peggie Mark M Cohen Philip P

The Journal of biological chemistry 20090322 21

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activ ...[more]

PMID: 19307177

Similar Datasets

Project description:Abstract：DNA transfection is widely used in biomedical research. However, transfection efficiency is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the possible involvement of the cGAS-STING signaling pathway, which induces type-I interferons in response to cytosolic DNA. We found deletion of cGAS enhances the expression of transfected genes. This enhancement is inversely correlated with the expression of type I interferons and interferon stimulated genes (ISGs). We provide evidence that the interferon responses can suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. As a result, administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression levels of exogenous genes in multiple cell lines and in primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression and this strategy may be applied to gene therapy. Goal：Analysis of mouse fibroblast cell line L929 transfected with pEGFP-N1 plasmid in the presence of DMSO or TBK1 inhibiter (BX795) . Results provide insight into molecular mechanisms underlying a inhibitor effect of BX795 on DNA-induced cGAS signaling which improves expression of transfected genes. Methods： L929 cells were pretreated with TBK1 inhibitor BX795 or mock treated with DMSO for 1 hour, then transfected with pEGFP-N1 plasmid. There are four groups in this experiment. S1, L929 cells mock-treated; S2, L929 cells transfected with pEGFP-N1 plasmid; S3, L929 treated with 0.5 uM BX795; S4, L929 treated with 0.5 uM BX795 and transfected with pEGFP-N1 plasmid. Each group has three replicates. There are total 12 samples in this experiment. Six hours later, total RNA of 12 samples was extracted using Trizol reagent from cells according to the manufacturer’s instruction. cDNA library construction and sequencing were performed by BGI Genomics (BGI-SHENZHEN, China) using BGISEQ-500 platform. High-quality reads were aligned to the Mus_musculus reference genome (Mus musculus GRCm38.p5) using Bowtie2 (version:2.2.5, parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200). The expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM) (version:1.2.12, default parameters). For the identification of differentially expressed genes (DEGs), we used PossionDis, which is developed by BGI Genomics, implementing the passion distribution method. We analyzed up-expression genes in S2 other than S1, S3 and S4.

Dataset Information

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.

Publications

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets