Inhibition of cytokine-mediated JNK signalling by purinergic P2Y11 receptors,a novel protective mechanism in endothelial cells

Previous research from our laboratory has demonstrated a novel phenomenon whereby GPCRs play a role in inhibiting cytokine-mediated c-Jun N-terminal kinase (JNK) signalling. So far this novel phenomenon seems to have been vastly overlooked, with little research in the area. Therefore, in this study we explored this further; by assessing the potential of P2YRs to mediate inhibition of cytokine-mediated JNK signalling and related functional outcomes in human endothelial cells. We utilised primary endothelial cells, and employed the use of endogenous activators of P2YRs and well characterised pharmacological inhibitors, to assess signalling parameters mediated by P2YRs, Interleukin-1β (IL-1β), TNFα and JNK. Activation of P2YRs with adenosine tri-phosphate (ATP) resulted in a time- and concentration-dependent inhibition of IL-1β-mediated phosphorylation of JNK and associated kinase activity. The effect was specific for cytokine-mediated JNK signalling, as ATP was without effect on JNK induced by other non-specific activators (e.g. sorbitol, anisomycin), nor effective against other MAPK pathways such as p38 and the canonical NFκB cascade. Pharmacological studies demonstrated a role for the P2Y₁₁ receptor in mediating this effect, but not the P2Y₁ nor the adenosine receptors (A1, A2A, A2B & A3). The novel Gα_q/11 inhibitor YM254890 and a protein kinase A (PKA) inhibitor H89 both partially reversed ATP-mediated inhibition of IL-1β-stimulated JNK indicating involvement of both Gα_q/11 and Gα_s mediated pathways. ATP also partially reversed IL-1β-mediated induction of cyclo‑oxygenase-2 (COX-2) and E-selectin. Collectively, these studies indicate the potential for activation of purinergic receptors to protect the endothelium from inflammatory driven JNK activation and may be a new target for inflammatory disease therapy.     

Docking interactions of the JNK scaffold protein WDR62

JNK (c-Jun N-terminal kinase) is part of a MAPK (mitogen-activated protein kinase) signalling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signalling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 (WD repeat domain 62) is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. In the present study we demonstrate the association of WDR62 with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7 (MAPK kinase 7). Association of WDR62 with JNK2 and MKK7 occurs via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lacking the putative JNK-binding domain fails to activate and recruit JNK to cellular granules. Furthermore, a synthetic peptide composed of the WDR62 docking domain inhibits JNK2 activity in vitro. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JIP1 (JNK-interacting protein 1). Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7β1 isoform independently of JNK binding, but fails to interact with MKK7α1. Furthermore, MKK7β1 recruits a protein phosphatase that dephosphorylates WDR62. Interestingly, a premature termination mutation in WDR62 that results in severe brain developmental defects does not abrogate WDR62 association with either JNK or MKK7. Therefore such mutations represent a loss of WDR62 function independent of JNK signalling.     

High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1 → MKK4 → JNK MAPK phosphorylation cascade

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His₆-tagged proteins and purified using urea refolding and Ni²⁺-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.     

Nonvisual arrestins function as simple scaffolds assembling the MKK4-JNK3α2 signaling complex

Arrestins make up a small family of proteins with four mammalian members that play key roles in the regulation of multiple G protein-coupled receptor-dependent and -independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated, and even the direct binding of arrestins with MAP kinases was never demonstrated. Here, using purified proteins, we show that both nonvisual arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of arrestin-3 for these kinases is higher than that of arrestin-2. Reconstitution of the MKK4-JNK3α2 signaling module from pure proteins in the presence of different arrestin-3 concentrations showed that arrestin-3 acts as a "true" scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both the level of JNK3α2 phosphorylation by MKK4 and JNK3α2 activity toward its substrate ATF2 increase at low and then decrease at high arrestin-3 levels, yielding a bell-shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of action of arrestin-3 on the MKK4-JNK3α2 signaling module.     

Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of beta-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1-/- mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1-/- cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.     

The Role of Reactive Oxygen Intermediates in the Regulation of Cytokine-Induced ICAM-1 Surface Expression on Endothelial Cells

ICAM-1 upregulation by endothelial cells plays a pivotal role in many disease processes, but signalling mechanisms leading to increased expression are poorly understood. In the current study we investigated the regulatory capacity of reactive oxygen intermediates (ROIs) in ICAM-1 activation by stimulating endothelial cells with the pro-inflammatory cytokines IL-1β, TNFα, IFNγ, IL-2, and IL-4 prior to antioxidant treatment. ICAM-1 was expressed constitutively and upregulated on ECV304 by IL1-β, IL2, and IFNγ and on SKHEP-1 by IFNγ, IL1-β, and TNFα. Phenanthroline (PHE) and disulfiram (DIS) showed the greatest ability to inhibit cytokine-stimulated ICAM-1 expression and in a dose-dependent manner. The α,α-diphenyl-β-picrylhydrazyl (DPPH) conversion assay showed that PHE and DIS had zero ability to scavenge free radicals and thus no known antioxidant activity. However, both are known metal chelators and our findings therefore suggest a unique role for metal ions in the control of cytokine-induced ICAM-1 expression on endothelial cells.     

Ba2+ currents in inner and outer hair cells of mice lacking the voltage-dependent Ca2+ channel subunits β3 or β4

Voltage-activated Ca2+ channels comprise complexes of a pore-forming Cavα1 and auxiliary subunits Cavβ, Cavα2δ and sometimes Cavγ. The intracellular Cavβ subunit assists in trafficking and surface expression of the Cavα1 subunit and can modulate biophysical properties of the Ca2+ channel. Four genes, Cavβ1-4, exist which confer different properties to Ca2+ currents through the various Cavα1 subunits. Ca2+ currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L type Cavα1 subunit Cav1.3, but associated Cavβ subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Cavβ subunits including Cavβ2, but clear assignment of the Cavβ1 4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Cavβ3 knockout (Cavβ3 / ) and Cavβ4 mutant mice (Cavβ4lh/lh), which had normal hearing. Recording voltage-activated Ba2+ currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba2+ current properties compared with their wildtype controls. Neonatal Cavβ4lh/lh IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Cavβ4wt, altogether indicating the presence of Cavβ4 in IHCs. Ba2+ currents of Cavβ3 / OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba2+ current activation, and increased inactivation compared with Cavβ3wt, pointing to a role of Cavβ3 for OHCs. These results indicate that neither Cavβ3 nor Cavβ4 are indispensable for hair cell Ca2+ currents but contribute to the overall current properties.     

Role of plant-specific N-terminal domain of maize CK2β1 subunit in CK2β functions and holoenzyme regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.     

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1β, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

Discovery of potent and selective rhodanine type IKKβ inhibitors by hit-to-lead strategy

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα.