We have developed the first assays that measure the protein kinase activities of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 reliably in human cell extracts, by employing Pellino1 as a substrate in conjunction with specific pharmacological inhibitors of IRAK1 and IRAK4. We exploited these assays to show that IRAK4 was constitutively active and that its intrinsic activity towards Pellino1 was not increased significantly by stimulation with interleukin-1 (IL-1) in IL-1R-expressing HEK293 cells, Pam3CSK4-stimulated human THP1 monocytes or primary human macrophages. Our results, in conjunction with those of other investigators, suggest that the IL-1-stimulated trans-autophosphorylation of IRAK4 is initiated by the myeloid differentiation primary response gene 88-induced dimerization of IRAK4 and is not caused by an increase in the intrinsic catalytic activity of IRAK4. In contrast with IRAK4, we found that IRAK1 was inactive in unstimulated cells and converted into an active protein kinase in response to IL-1 or Pam3CSK4 in human cells. Surprisingly, the IL-1-stimulated activation of IRAK1 was not affected by pharmacological inhibition of IRAK4 and not reversed by dephosphorylation and/or deubiquitylation, suggesting that IRAK1 catalytic activity is not triggered by a covalent modification but by an allosteric mechanism induced by its interaction with IRAK4.

The interleukin-6 -type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia-inducible factor 1␣ (HIF1␣) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1␣ under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1␣ increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogenactivated protein kinase kinase/extracellular signal-regulated kinase 1/2 pathways. OSMmediated HIF1␣ up-regulation did not result from an increase in HIF1␣ protein stability but from increased transcription from the HIF1␣ gene. In addition, we show that the OSM-induced HIF1␣ gene transcription and the resulting enhanced HIF1␣ protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1␣ levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1␣ contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration. (HEPATOLOGY 2009;50:253-260.)

PROteolysis TArgeting Chimeras (PROTACs) targeting the degradation of MEK have been designed based on allosteric MEK inhibitors. Inhibition of the phosphorylation of ERK1/2 was less effective with the PROTACs than a small-molecule inhibitor; the best PROTACs, however, were more effective in inhibiting proliferation of A375 cells than an inhibitor.

Background The airway epithelium is a major target tissue in respiratory infections, and its antiviral response is mainly orchestrated by the interferon regulatory factor-3 (IRF3), which subsequently induces type I (β) and III (λ) interferon (IFN) signalling. Dual specificity mitogen-activated protein kinase kinase (MEK) pathway contributes to epithelial defence, but its role in the regulation of IFN response in human primary airway epithelial cells (AECs) is not fully understood. Here, we studied the impact of a small-molecule inhibitor (MEKi) on the IFN response following challenge with two major respiratory viruses rhinovirus (RV2) and respiratory syncytial virus (RSVA2) and a TLR3 agonist, poly(I:C). Methods The impact of MEKi on viral load and IFN response was evaluated in primary AECs with or without a neutralising antibody against IFN-β. Quantification of viral load was determined by live virus assay and absolute quantification using qRT-PCR. Secretion of cytokines was determined by AlphaLISA/ELISA and expression of interferon-stimulated genes (ISGs) was examined by qRT-PCR and immunoblotting. A poly(I:C) model was also used to further understand the molecular mechanism by which MEK controls IFN response. AlphaLISA, siRNA-interference, immunoblotting, and confocal microscopy was used to investigate the effect of MEKi on IRF3 activation and signalling. The impact of MEKi on ERK and AKT signalling was evaluated by immunoblotting and AlphaLISA. Results Here, we report that pharmacological inhibition of MEK pathway augments IRF3-driven type I and III IFN response in primary human AECs. MEKi induced activation of PI3K-AKT pathway, which was associated with phosphorylation/inactivation of the translational repressor 4E-BP1 and activation of the protein synthesis regulator p70 S6 kinase, two critical translational effectors. Elevated IFN-β response due to MEKi was also attributed to decreased STAT3 activation, which consequently dampened expression of the transcriptional repressor of IFNB1 gene, PRDI-BF1. Augmented IFN response translated into inhibition of rhinovirus 2 replication in primary AECs but not respiratory syncytial virus A2. Conclusions Our findings unveil MEK as a key molecular mechanism by which rhinovirus dampens the epithelial cell’s antiviral response. Our study provides a better understanding of the role of signalling pathways in shaping the antiviral response and suggests the use of MEK inhibitors in anti-viral therapy against RV. Electronic supplementary material The online version of this article (10.1186/s12964-019-0378-7) contains supplementary material, which is available to authorized users.