Protein Kinases IKKε Research Articles

Generation of inhibitory peptides for IKKε from a kinase‐focused phage library of helix‐loop‐helix peptides

AbstractConformationally constrained peptides are attracting attention as peptide‐based molecular tools in chemical biology and drug discovery because of their desirable properties such as high selectivity for target binding and stability toward proteolytic enzymes. We previously reported a protein kinase–focused helix‐loop‐helix (HLH) peptide library. The library was constructed by phage display and subsequent chemical modification with adenosine that was employed as an anchor molecule binding to the ATP‐binding pocket of kinase. Here, we discovered and characterized HLH peptides that bind to and inhibit the serine/threonine protein kinase IKKε, a member of the IκB kinase (IKK) family. Screening the library against IKKε identified de novo HLH peptides binding to the kinase. One of the peptides, IKK‐05, showed a high α‐helical content and inhibited IKKε with a mixed‐inhibition mechanism with respect to ATP. The adenosine‐tethering peptide Adc‐IKK‐05 demonstrated significantly enhanced enzyme inhibition activity, indicating a bivalency effect in binding to IKKε. In addition, Adc‐IKK‐05 showed the highest inhibitory activity against IKKε of the IKK family members and other families tested. The peptides obtained in this research can be developed as molecular tools to study the biological functions of IKKε and to obtain structural insights for the design of selective IKKε inhibitors.

FGF21 is required for the metabolic benefits of IKKε/TBK1 inhibition.

The protein kinases IKKε and TBK1 are activated in liver and fat in mouse models of obesity. We have previously demonstrated that treatment with the IKKε/TBK1 inhibitor amlexanox produces weight loss and relieves insulin resistance in obese animals and patients. While amlexanox treatment caused a transient reduction in food intake, long-term weight loss was attributable to increased energy expenditure via FGF21-dependent beiging of white adipose tissue (WAT). Amlexanox increased FGF21 synthesis and secretion in several tissues. Interestingly, although hepatic secretion determined circulating levels, it was dispensable for regulating energy expenditure. In contrast, adipocyte-secreted FGF21 may have acted as an autocrine factor that led to adipose tissue browning and weight loss in obese mice. Moreover, increased energy expenditure was an important determinant of improved insulin sensitivity by amlexanox. Conversely, the immediate reductions in fasting blood glucose observed with acute amlexanox treatment were mediated by the suppression of hepatic glucose production via activation of STAT3 by adipocyte-secreted IL-6. These findings demonstrate that amlexanox improved metabolic health via FGF21 action in adipocytes to increase energy expenditure via WAT beiging and that adipocyte-derived IL-6 has an endocrine role in decreasing gluconeogenesis via hepatic STAT3 activation, thereby producing a coordinated improvement in metabolic parameters.

Zika Virus Non-Structural Protein NS5 Inhibits the RIG-I Pathway and Interferon Lambda 1 Promoter Activation by Targeting IKK Epsilon.

The Zika virus (ZIKV) is a member of the Flaviviridae family and an important human pathogen. Most pathogenic viruses encode proteins that interfere with the activation of host innate immune responses. Like other flaviviruses, ZIKV interferes with the expression of interferon (IFN) genes and inhibits IFN-induced antiviral responses. ZIKV infects through epithelial barriers where IFN-λ1 is an important antiviral molecule. In this study, we analyzed the effects of ZIKV proteins on the activation of IFN-λ1 promoter. All ZIKV proteins were cloned and transiently expressed. ZIKV NS5, but no other ZIKV protein, was able to interfere with the RIG-I signaling pathway. This inhibition took place upstream of interferon regulatory factor 3 (IRF3) resulting in reduced phosphorylation of IRF3 and reduced activation of IFN-λ1 promoter. Furthermore, we showed that ZIKV NS5 interacts with the protein kinase IKKε, which is likely critical to the observed inhibition of phosphorylation of IRF3.

Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss.

The activity of protein kinases IKK-ε and TANK-binding kinase 1 (TBK1) has been shown to be associated with inflammatory diseases. As an inhibitor of IKK-ε and TBK1, amlexanox is an anti-inflammatory, anti-allergic, immunomodulator and used for treatment of ulcer, allergic rhinitis and asthma in clinic. We hypothesized that amlexanox may be used for treatment of osteoclast-related diseases which frequently associated with a low grade of systemic inflammation. In this study, we investigated the effects of amlexanox on RANKL-induced osteoclastogenesis in vitro and ovariectomy-mediated bone loss in vivo. In primary bone marrow derived macrophages (BMMs), amlexanox inhibited osteoclast formation and bone resorption. At the molecular level, amlexanox suppressed RANKL-induced activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPKs), c-Fos and NFATc1. Amlexanox decreased the expression of osteoclast-specific genes, including TRAP, MMP9, Cathepsin K and NFATc1. Moreover, amlexanox enhanced osteoblast differentiation of BMSCs. In ovariectomized (OVX) mouse model, amlexanox prevented OVX-induced bone loss by suppressing osteoclast activity. Taken together, our results demonstrate that amlexanox suppresses osteoclastogenesis and prevents OVX-induced bone loss. Therefore, amlexanox may be considered as a new therapeutic candidate for osteoclast-related diseases, such as osteoporosis and rheumatoid arthritis.

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation.

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. Here, we demonstrate that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. We found that Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F–IKKε complex during bristle cell elongation.

Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1

Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate β-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored β-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C). Conversely, overexpression of the kinases reduced induction of Ucp1, lipolysis, cAMP levels, and phosphorylation of hormone sensitive lipase in response to isoproterenol or forskolin. Noncanonical IKKs reduce catecholamine sensitivity by phosphorylating and activating the major adipocyte phosphodiesterase PDE3B. In vivo inhibition of these kinases by treatment of obese mice with the drug amlexanox reversed obesity-induced catecholamine resistance, and restored PKA signaling in response to injection of a β-3 adrenergic agonist. These studies suggest that by reducing production of cAMP in adipocytes, IKKε and TBK1 may contribute to the repression of energy expenditure during obesity. DOI: http://dx.doi.org/10.7554/eLife.01119.001.

The Protein Kinase IKKε Is a Potential Target for the Treatment of Inflammatory Hyperalgesia

Inhibitor-κB kinase ε (IKKε) was only recently identified as an enzyme with high homology to the classical I-κB kinase subunits, IKKα and IKKβ. Despite this similarity, it is mainly discussed as a repressor of viral infections by modulating type I IFNs. However, in vitro studies also showed that IKKε plays a role in the regulation of NF-κB activity, but the distinct mechanisms of IKKε-mediated NF-κB activation are not clear. Given the paramount role of NF-κB in inflammation, we investigated the regulation and function of IKKε in models of inflammatory hyperalgesia in mice. We found that IKKε was abundantly expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia. IKKε mRNA and protein levels rapidly increased in spinal cord and dorsal root ganglia during hind paw inflammation evoked by injection of zymosan or formalin. IKKε knockout mice showed normal nociceptive responses to acute heat or mechanical stimulation. However, in inflammatory pain models, IKKε-deficient mice exhibited a significantly reduced nociceptive behavior in comparison with wild type mice, indicating that IKKε contributed to the development of inflammatory hyperalgesia. Antinociceptive effects were associated with reduced activation of NF-κB and attenuated NF-κB-dependent induction of cyclooxygenase-2, inducible NO synthase, and metalloproteinase-9. In contrast, IRF-3, which is an important IKKε target in viral infections, was not regulated after inflammatory nociceptive stimulation. Therefore, we concluded that IKKε modulates inflammatory nociceptive sensitivity by activation of NF-κB-dependent gene transcription and may be useful as a therapeutic target in the treatment of inflammatory pain.

291 Polo-like kinase 1 (PLK1) and protein kinase IKKε, two markers of cancer, down-regulate IFN induction by MAVS

291 Polo-like kinase 1 (PLK1) and protein kinase IKKε, two markers of cancer, down-regulate IFN induction by MAVS

Regulation of Interferon Production by RIG-I and LGP2: A Lesson in Self-Control

The cytoplasmic CARD-containing DExD/H box RNA helicases RIG-I and MDA5 act as sensors of viral infections through recognition of viral double-stranded (ds) RNAs. They both associate with the mitochondrial adaptor IPS-1 (also referred to as MAVS, VISA, and CARDIF) through homotypic CARD-CARD interactions. IPS-1, in turn, triggers signaling pathways, including activation of the protein kinases TBK1 and IKKepsilon, responsible for the phosphorylation of IRF3, a key transcription factor involved in interferon (IFN) synthesis, one essential element of the innate immune response. RIG-I remains in an autoinhibited state in the absence of dsRNA, through an internal repressor domain (RD) that binds within both its CARD and its RNA helicase domains and therefore acts in cis to control its multimerization and interaction with IPS-1. Ectopic expression of the RD prevents signaling and increases cell permissiveness to viruses, including hepatitis C virus. LGP2, which is another DExD/H RNA helicase of the RIG-I and MDA5 family and which is devoid of CARD domain, negatively controls IFN induction at different levels: by sequestering dsRNA, by blocking RIG-I's multimerization in trans through a domain analogous to the RIG-I RD, and by competing with the protein kinase IKKepsilon for a common interaction site on IPS-1. The ability of RIG-I and LGP2 to exert such a feedback control at the earliest steps of IFN synthesis allows the cells to exert a tight regulation of the induction of the innate immune response.

The protein kinase IKKε can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers

During a viral infection, binding of viral double-stranded RNAs (dsRNAs) to the cytosolic RNA helicase RIG-1 leads to recruitment of the mitochondria-associated Cardif protein, involved in activation of the IRF3-phosphorylating IKKepsilon/TBK1 kinases, interferon (IFN) induction, and development of the innate immune response. The hepatitis C virus (HCV) NS3/4A protease cleaves Cardif and abrogates both IKKepsilon/TBK1 activation and IFN induction. By using an HCV replicon model, we previously showed that ectopic overexpression of IKKepsilon can inhibit HCV expression. Here, analysis of the IKKepsilon transcriptome profile in these HCV replicon cells showed induction of several genes associated with the antiviral action of IFN. Interestingly, IKKepsilon still inhibits HCV expression in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative STAT1alpha mutant. This suggests that good IKKepsilon expression levels are important for rapid activation of the cellular antiviral response in HCV-infected cells, in addition to provoking IFN induction. To determine the physiological importance of IKKepsilon in HCV infection, we then analyzed its expression levels in liver biopsy specimens from HCV-infected patients. This analysis also included genes of the IFN induction pathway (RIG-I, MDA5, LGP2, Cardif, TBK1), and three IKKepsilon-induced genes (IFN-beta, CCL3, and ISG15). The results show significant inhibition of expression of IKKepsilon and of the RNA helicases RIG-I/MDA5/LGP2 in the HCV-infected patients, whereas expression of TBK1 and Cardif was not significantly altered. In conclusion, given the antiviral potential of IKKepsilon and of the RNA helicases, these in vivo data strongly support an important role for these genes in the control of HCV infection.