Co-ordinated control of the ADP-heptose/ALPK1 signalling network by the E3 ligases TRAF6, TRAF2/c-IAP1 and LUBAC.

ADP-heptose activates the protein kinase ALPK1 triggering TIFA phosphorylation at Thr9, the recruitment of TRAF6 and the subsequent production of inflammatory mediators. Here, we demonstrate that ADP-heptose also stimulates the formation of Lys63- and Met1-linked ubiquitin chains to activate the TAK1 and canonical IKK complexes, respectively. We further show that the E3 ligases TRAF6 and c-IAP1 operate redundantly to generate the Lys63-linked ubiquitin chains required for pathway activation, which we demonstrate are attached to TRAF6, TRAF2 and c-IAP1, and that c-IAP1 is recruited to TIFA by TRAF2. ADP-heptose also induces activation of the kinase TBK1 by a TAK1-independent mechanism, which require TRAF2 and TRAF6. We establish that ALPK1 phosphorylates TIFA directly at Thr177 as well as Thr9 in vitro. Thr177 is located within the TRAF6-binding motif and its mutation to Asp prevents TRAF6 but not TRAF2 binding, indicating a role in restricting ADP-heptose signalling. We conclude that ADP-heptose signalling is controlled by the combined actions of TRAF2/c-IAP1 and TRAF6.

Target protein localization and its impact on PROTAC-mediated degradation.

Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitin ligase, promoting POI ubiquitylation and proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V (dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome or lysosome. Differentially localized Halo or FKBP12F36V proteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation.

Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Orally Bioavailable, and Blood-Brain Barrier Penetrant PROTAC Degrader of Leucine-Rich Repeat Kinase 2.

Leucine-rich repeat kinase 2 (LRRK2) is one of the most promising targets for Parkinson's disease. LRRK2-targeting strategies have primarily focused on type 1 kinase inhibitors, which, however, have limitations as the inhibited protein can interfere with natural mechanisms, which could lead to undesirable side effects. Herein, we report the development of LRRK2 proteolysis targeting chimeras (PROTACs), culminating in the discovery of degrader XL01126, as an alternative LRRK2-targeting strategy. Initial designs and screens of PROTACs based on ligands for E3 ligases von Hippel-Lindau (VHL), Cereblon (CRBN), and cellular inhibitor of apoptosis (cIAP) identified the best degraders containing thioether-conjugated VHL ligand VH101. A second round of medicinal chemistry exploration led to qualifying XL01126 as a fast and potent degrader of LRRK2 in multiple cell lines, with DC50 values within 15-72 nM, Dmax values ranging from 82 to 90%, and degradation half-lives spanning from 0.6 to 2.4 h. XL01126 exhibits high cell permeability and forms a positively cooperative ternary complex with VHL and LRRK2 (α = 5.7), which compensates for a substantial loss of binary binding affinities to VHL and LRRK2, underscoring its strong degradation performance in cells. Remarkably, XL01126 is orally bioavailable (F = 15%) and can penetrate the blood-brain barrier after either oral or parenteral dosing in mice. Taken together, these experiments qualify XL01126 as a suitable degrader probe to study the noncatalytic and scaffolding functions of LRRK2 in vitro and in vivo and offer an attractive starting point for future drug development.

Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader.

SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation.  SGK3-PROTAC1 (0.3 µM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1-0.3 µM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.

Myeloid cell deficiency of p38γ/p38δ protects against candidiasis and regulates antifungal immunity.

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.

Differential control of Toll-like receptor 4-induced interleukin-10 induction in macrophages and B cells reveals a role for p90 ribosomal S6 kinases.

Increasing evidence has linked dysregulated interleukin (IL)-10 production by IL-10+ve B cells to autoimmunity, highlighting the importance of improving the understanding of the regulation of IL-10 production in these cells. In both B cells and myeloid cells, IL-10 can be produced in response to Toll-like receptor (TLR) agonists. In macrophages, previous studies have established that mitogen- and stress-activated protein kinases (MSKs) regulate IL-10 production via the phosphorylation of cAMP response element-binding (CREB) protein on the IL-10 promoter. We found here that although MSKs are activated in peritoneal B cells in response to TLR4 agonists, neither MSKs nor CREB are required for IL-10 production in these cells. Using a combination of chemical inhibitors and knockout mice, we found that IL-10 induction in B cells was regulated by an ERK1/2- and p90 ribosomal S6 kinase-dependent mechanism, unlike in macrophages in which p90 ribosomal S6 kinase was not required. This observation highlights fundamental differences in the signaling controlling IL-10 production in B cells and macrophages, even though these two cell types respond to a common TLR stimulus.

The hVps34-SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth.

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT-474 breast cancer cell-derived tumours in a xenograft model. Finally, we present the kinome-wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34-SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

Lys63/Met1-hybrid ubiquitin chains are commonly formed during the activation of innate immune signalling.

We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain.

Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes.

Transthiolation is a fundamental biological reaction and is utilized by many enzymes involved in the conjugation of ubiquitin and ubiquitin-like proteins. However, tools that enable selective profiling of this activity are lacking. Transthiolation requires cysteine-cysteine juxtaposition; therefore a method that enables irreversible "stapling" of proximal thiols would facilitate the development of novel probes that could be used to profile this activity. Herein, we characterize biocompatible chemistry that enables sequential functionalization of cysteines within proteins at a single atomic center. We use our method to develop a new class of activity-based probe that profiles transthiolation activity of human E1 activating enzymes. We demonstrate use in vitro and in situ and compatibility with competitive activity-based protein profiling. We also use the probe to gain insight into the determinants of transthiolation between E2 and a RING-in-between-RING (RBR) E3 ligase. Furthermore, we anticipate that this method of thiol functionalization will have broad utility by enabling simple redox-stable cross-linking of proximal cysteines in general.

Protein kinase IKKß-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in myeloid cells.

The siRNA knockdown of IFN Regulatory Factor 5 (IRF5) in the human plasmacytoid dendritic cell line Gen2.2 prevented IFNß production induced by compound CL097, a ligand for Toll-like receptor 7 (TLR7). CL097 also stimulated the phosphorylation of IRF5 at Ser462 and stimulated the nuclear translocation of wild-type IRF5, but not the IRF5[Ser462Ala] mutant. The CL097-stimulated phosphorylation of IRF5 at Ser462 and its nuclear translocation was prevented by the pharmacological inhibition of protein kinase IKKß or the siRNA knockdown of IKKß or its "upstream" activator, the protein kinase TAK1. Similar results were obtained in a murine macrophage cell line stimulated with the TLR7 agonist compound R848 or the nucleotide oligomerization domain 1 (NOD1) agonist KF-1B. IKKß phosphorylated IRF5 at Ser462 in vitro and induced the dimerization of wild-type IRF5 but not the IRF5[S462A] mutant. These findings demonstrate that IKKß activates two "master" transcription factors of the innate immune system, IRF5 and NF-κB.

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K.

Mechanism of action of compound-13: an α1-selective small molecule activator of AMPK.

AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, ß1, or ß2.

Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity.

The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser431) by PKA (cAMP-dependent protein kinase) and RSK (ribosomal S6 kinase) and farnesylated (Cys433) within a CAAX motif. To better define the role that these post-translational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1C433S/C433S mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1S431A/S431A animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1C433S/C433S mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1C433S/C433S mice. The activity of several of the AMPK-related kinases analysed [BRSK1 (BR serine/threonine kinase 1), BRSK2, NUAK1 (NUAK family, SNF1-like kinase 1), SIK3 (salt-inducible kinase 3) and MARK4 (MAP/microtubule affinity-regulating kinase 4)] was not affected in tissues derived from LKB1S431A/S431A or LKB1C433S/C433S mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the plasma membrane as a result of myristoylation of its regulatory AMPKß subunit. This raises the possibility that LKB1 farnesylation and myristoylation of AMPKß might promote the interaction and co-localization of these enzymes on a two-dimensional membrane surface and thereby promote efficient activation of AMPK.

The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system.

The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys48-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB.

Akt is efficiently activated by PIF-pocket- and PtdIns(3,4,5)P3-dependent mechanisms leading to resistance to PDK1 inhibitors.

Mutations leading to inappropriate activation of Akt isoforms contribute to proliferation and survival of a significant proportion of human cancers. Akt is activated by phosphorylation of its T-loop residue (Thr(308)) by PDK1 (3-phosphoinositide-dependent kinase-1) and its C-terminal hydrophobic motif (Ser(473)) by mTORC2 [mTOR (mammalian target of rapamycin) complex 2]. Potent PDK1 inhibitors such as GSK2334470 have recently been elaborated as potential anti-cancer agents. However, these compounds were surprisingly ineffective at suppressing Akt activation. In the present study we demonstrate that resistance to PDK1 inhibitors results from Akt being efficiently recruited to PDK1 via two alternative mechanisms. The first involves ability of Akt and PDK1 to mutually interact with the PI3K (phosphoinositide 3-kinase) second messenger PtdIns(3,4,5)P3. The second entails recruitment of PDK1 to Akt after its phosphorylation at Ser(473) by mTORC2, via a substrate-docking motif termed the PIF-pocket. We find that disruption of either the PtdIns(3,4,5)P3 or the Ser(473) phosphorylation/PIF-pocket mechanism only moderately impacts on Akt activation, but induces marked sensitization to PDK1 inhibitors. These findings suggest that suppression of Ser(473) phosphorylation by using mTOR inhibitors would disrupt the PIF-pocket mechanism and thereby sensitize Akt to PDK1 inhibitors. Consistent with this, we find combing PDK1 and mTOR inhibitors reduced Akt activation to below basal levels and markedly inhibited proliferation of all of the cell lines tested. Our results suggest further work is warranted to explore the utility of combining PDK1 and mTOR inhibitors as a therapeutic strategy for treatment of cancers that harbour mutations elevating Akt activity.

Essential role for IKKß in production of type 1 interferons by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKß signaling pathway to induce the production of IFNß via a pathway that is independent of the degradation of IκBα. We also show that IKKß activity, as well as the subsequent IFNß-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKß is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKß inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKß may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes.

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.

Comparison of the specificity of Trk inhibitors in recombinant and neuronal assays.

Neurotrophins are important mediators of neuronal development, survival and plasticity. They act via binding to Trk receptors, which results in the stimulation of the intracellular tyrosine kinase domain of the receptor leading to autophosphorylation of this domain. This in turn creates a scaffold that recruits various adapter proteins allowing the activation of intracellular signaling cascades including the PLCγ, MAPK and PI3K pathways. Compounds that specifically block the activity of the tyrosine kinase domain of Trk receptors would provide a powerful tool to study the role of these receptors in cells. K252a has previously been used for this purpose, however we show here that it can inhibit many tyrosine and serine/threonine kinases in vitro. Profiling of 3 newer inhibitors, referred to here as SHN-753, SHN-722 and GSK-Trk, demonstrate that they have significantly improved specificity for the kinase activity of TrkA in vitro compared to K252a. In addition these compounds were found to block the TrkB mediated activation of ERK1/2 by BDNF, but did not affect NMDA induced ERK1/2 activation. These compounds, while still not completely specific for Trk receptor kinase activity, do represent a considerable improvement over K252a and should prove valuable in the study of neurotrophin-mediated actions in the nervous system.