Novel splice variants of human IKKε negatively regulate IKKε‐induced IRF3 and NF‐kB activation

Abstract

The inhibitor of jB kinase e (IKKe) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKe involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-jB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKe, designated IKKe-sv1 and IKKe-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type-and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKe (IKKe-wt) leads to the activation of NF-jB-as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKe-sv1 still activates NF-jB, whereas IKKe-sv2 is also defective in NF-jB activation. Both splice variants form dimers with IKKe-wt and inhibit IKKe-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKe.Key words: IFN . Innate immunity . NF-kB pathway . Protein/protein interactions . Signal transductionSupporting Information available online IntroductionViral infections are recognized by the innate immune system, which is essential for the subsequent initiation of adaptive immunity. Invading viruses are sensed by pattern-recognition receptors (PRR) recognizing pathogen-associated molecular patterns such as singleor double-stranded RNA. These PRR comprise TLR with endosomal/lysosomal localization like TLR3 and cytoplasmic receptors such as the retinoic acid-inducible protein I and melanoma differentiation-associated gene 5. Activation of these PRR engages intracellular signaling cascades leading to the secretion of type I IFN, which are important anti-viral cytokines ultimately facilitating viral clearance [1,2]. The signal transduction pathways leading to type I IFN expression involve activation of the serine/threonine kinases TANK-binding kinase 1 (TBK-1), also known as NF-kB activating kinase NAK [3], and inhibitor of kB kinase e (IKKe), also known as IKKi [4]. After virus infection, both kinases are activated and phosphorylate the transcription factor IFN regulatory factor 3 (IRF3), leading to homo-dimerization, translocation into the nucleus, and activation of promoters containing the IRF3 binding site termed IFN-stimulated response element [5,6]. For IRF3 activation after triggering of different PRR, the three related scaffold proteins NAP1, TANK, and SINTBAD are essential [7][8][9], whereas the use of a distinct scaffold protein depends on the respective stimulus activating the TBK1/IKKe pathway [10]. Ultimately, the formation of a multisubunit complex c...

Arenavirus Nucleoproteins Prevent Activation of Nuclear Factor Kappa B

“…7B). In agreement with previous findings, we observed that transient transfection with either TBK-1 or IKKε promoted activation of both IFN-␤ (9, 17, 26, 38, 67, 81)-and NF-B (3,18,40,62,69,70,77,79,82,87)-dependent promoters ( Fig. 7C and D).…”

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML

“…Notch2 splice variants may act in a dominant-negative manner that compromises the functions of Notch2-FL and are likely to block corresponding signaling pathways in AML cells. 7,[37][38][39] Based on these discoveries and the findings presented here, we suggest that silencing of the Notch2 signaling pathway could be a due to dominantnegative effects of Notch2 splice variants on Notch2-FL. It is thus possible to speculate that Notch2 pathway reactivation could be achieved by the using of blocking antibodies against Notch2-Va and Notch2-Vb splice variants.…”

Human DEAD Box Helicase 3 Couples IκB Kinase ε to Interferon Regulatory Factor 3 Activation

“…1, we have demonstrated that DDX3 enhances IKKε autoactivation. Other adaptor proteins that contribute to IKKε and TBK1 activation in cells are NAP1 (NAK-associated protein 1), TANK (TRAF family member-associated NF-B activator) and Sintbad (similar to NAP1 TBK1 adaptor) (28), which have recently been shown to bind to the C-terminal region of IKKε (aa 684 to 716) and the corresponding region in TBK1 (29,30). We therefore asked whether DDX3 interacts with IKKε in a similar manner.…”

“…These adaptor proteins have also been shown to be involved in activation of TBK1 and IKKε downstream of PRRs (28). However, as they interact with a different region of IKKε, they appear to have a distinct mode of action compared to DDX3 (29,30). It was suggested that they might recruit TBK1/IKKε to distinct subcellular locations for activation (29).…”