Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling

The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation.     

Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4(IRAK-1-K239S)). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4(IRAK-1-K239S) cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.     

Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.     

Contribution of globular death domains and unstructured linkers to MyD88·IRAK-4 heterodimer formation: An explanation for the antagonistic activity of MyD88s

Homotypic interactions of death domains (DD) mediate complex formation between MyD88 and IL-1 receptor-associated kinases (IRAKs). A truncated splice variant of MyD88, MyD88s, cannot recruit IRAK-4 and fails to elicit inflammatory responses. We have generated recombinant DD of MyD88 and IRAK-4, both alone and extended by the linkers to TIR or kinase domains. We show that both MyD88 DD variants bind to the linker-extended IRAK-4 DD and pull-down full-length IRAK-4 from monocyte extracts. By contrast, residues up to Glu¹¹⁶ from the DD-kinase connector of IRAK-4 are needed for strong interactions with the adaptor. Our findings indicate that residues 110-120, which form a C-terminal extra helix in MyD88, but not the irregular linker between DD and TIR domains, are required for IRAK-4 recruitment, and provide a straightforward explanation for the negative regulation of innate immune responses mediated by MyD88s.     

Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation

Oxidative stress and inflammation are fundamental for the onset of aging and appear to be causatively linked. Previously, we reported that hepatocytes from aged rats, compared with young rats, are hyperresponsive to interleukin-1beta (IL-1beta) stimulation and exhibit more potent c-Jun N-terminal kinase (JNK) activation and attenuated interleukin-1 receptor-associated kinase-1 (IRAK-1) degradation. An age-related increase in the activity of neutral sphingomyelinase-2 (NSMase-2), a plasma membrane enzyme, was found to be responsible for the IL-1beta hyperresponsiveness. The results reported here show that increased NSMase activity during aging is caused by a 60-70% decrease in hepatocyte GSH levels. GSH, at concentrations typically found in hepatocytes from young animals, inhibits NSMase activity in a biphasic dose-dependent manner. Inhibition of GSH synthesis in young hepatocytes activates NSMase, causing increased JNK activation and IRAK-1 stabilization in response to IL-1beta, mimicking the hyperresponsiveness typical for aged hepatocytes. Vice versa, increased GSH content in hepatocytes from aged animals by treatment with N-acetylcysteine inhibits NSMase activity and restores normal IL-1beta response. Importantly, the GSH decline, NSMase activation, and IL-1beta hyperresponsiveness are not observed in aged, calorie-restricted rats. In summary, this report demonstrates that depletion of cellular GSH during aging plays an important role in regulating the hepatic response to IL-1beta by inducing NSMase-2 activity.     

Identification and characterization of murine IRAK-2

Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.     

IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family.

The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family.     

Activation of c-Jun-N-terminal kinase is critical in mediating lipopolysaccharide-induced changes in the rat hippocampus

Lipopolysaccharide (LPS) exerts a myriad of effects in rat hippocampus; it increases the concentration of the proinflammatory cytokine, interleukin-1beta (IL-1beta), and signalling via the IL-1 type I receptor (IL-1RI) resulting in phosphorylation of the stress-activated protein kinase, c-jun-N-terminal kinase (JNK) and impairment in long-term potentiation (LTP). This study was designed to establish whether activation of JNK is a pivotal event in mediating the effects of LPS in hippocampus and therefore LPS-treated rats were injected intracerebroventricularly with saline, the JNK inhibitor D-JNKI1, or with the anti-inflammatory cytokine IL-4, which antagonizes the effects of IL-1beta upstream of JNK activation. We report that IL-4 blocked the LPS-induced increase in IL-1RI expression and associated increases in phosphorylation of JNK and c-jun, whereas D-JNKI1 inhibited the LPS-induced phosphorylation of c-jun. Both IL-4 and D-JNKI1 inhibited the increase in caspase-3 staining which was associated with LPS treatment, and both abrogated the LPS-induced inhibition of LTP in perforant path-granule cell synapses. The data presented are consistent with the proposal that JNK activation, probably as a result of increased IL-1RI activation, is a critical step in mediating the detrimental effects of LPS in hippocampus.     

Cross-talk between IRAK-4 and the NADPH oxidase

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.     

IRAK-4-dependent degradation of IRAK-1 is a negative feedback signal for TLR-mediated NF-kappaB activation

The activation of interleukin 1 receptor-associated kinase (IRAK)-1 is a key event in the transmission of signals from Toll-like receptors (TLRs). The catalytic activity of the protein kinase is not essential for its ability to activate nuclear factor (NF) kappaB, because transfection of a kinase-dead mutant of IRAK-1 (IRAK-1KD) is able to activate NF-kappaB in HEK293T cells. In the present study, we observed that the effect of IRAK-1KD was impaired by simultaneous expression of IRAK-4. The effect of IRAK-4 was accompanied by the phosphorylation and degradation of IRAK-1KD. Expression of IRAK-4KD instead of IRAK-4 did not cause these events. In IRAK-4-deficient Raw264.7 macrophages that were prepared by introducing short-hairpin RNA probes, the basal level of IRAK-1 was increased markedly. Stimulation of these cells with TLR ligands did not cause the degradation of IRAK-1, which was clearly observed in the parent cells. These results suggested that the expression of IRAK-4 alone is sufficient to cause the degradation of IRAK-1; the autophosphorylation of IRAK-1 is not necessary to terminate the TLR-induced activation of NF-kappaB. IRAK-4 has an ability to induce the degradation of IRAK-1 in addition to its role as an activator of IRAK-1.     

Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members

Interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for the proinflammatory cytokine interleukin-1 (IL-1) and was later implicated in signal transduction of other members of the Toll-like receptor (TLR)/IL-1 receptor (IL-1R) family. In the meantime, four different IRAK-like molecules have been identified: two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. All IRAKs mediate activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways. Although earlier observations suggested that IRAKs have redundant functions, this hypothesis is now challenged by knockout studies. Furthermore, recent data imply a role for IRAK-1 in tumor necrosis factor receptor (TNFR) superfamily-induced signaling pathways as well. The scope of this review is to highlight the specific role of different IRAKs and to discuss several mechanisms that contribute to their activation and regulation.     

PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes

This study examined the role of interleukin (IL)-1 receptor-associated kinase (IRAK) and protein kinase C (PKC) in oxidized LDL (Ox-LDL)-induced monocyte IL-1β production. In THP1 cells, Ox-LDL induced time-dependent secretory IL-1β and IRAK1 activity; IRAK4, IRAK3, and CD36 protein expression; PKCδ-JNK1 phosphorylation; and AP-1 activation. IRAK1/4 siRNA and inhibitor (INH)-attenuated Ox-LDL induced secreted IL-1β and pro-IL-1β mRNA and pro-IL-1β and mature IL-1β protein expression, respectively. Diphenyleneiodonium chloride (NADPH oxidase INH) and N-acetylcysteine (free radical scavenger) attenuated Ox-LDL-induced reactive oxygen species generation, caspase-1 activity, and pro-IL-1β and mature IL-1β expression. Ox-LDL-induced secretory IL-1β production was abrogated in the presence of JNK INH II, Tanshinone IIa, Ro-31-8220, Go6976, Rottlerin, and PKCδ siRNA. PKCδ siRNA attenuated the Ox-LDL-induced increase in IRAK1 kinase activity, JNK1 phosphorylation, and AP-1 activation. In THP1 macrophages, CD36, toll-like receptor (TLR)2, TLR4, TLR6, and PKCδ siRNA prevented Ox-LDL-induced PKCδ and IRAK1 activation and IL-1β production. Enhanced Ox-LDL and IL-1β in systemic inflammatory response syndrome (SIRS) patient plasma demonstrated positive correlation with each other and with disease severity scores. Ox-LDL-containing plasma induced PKCδ and IRAK1 phosphorylation and IL-1β production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes. Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKCδ-IRAK1-JNK1-AP-1 axis in Ox-LDL-induced IL-1β production.     

MyD88-induced downregulation of IRAK-4 and its structural requirements

IRAK-4 plays an essential role in Toll-like receptor (TLR)/IL-1 receptor signaling. However, its signaling and regulation mechanisms have remained elusive. We have reported previously that stimulation of TLR2, TLR4 or TLR9, but not TLR3, leads to downregulation of IRAK-4 protein. Here, we show that expression of MyD88 leads to downregulation of endogenous as well as exogenously expressed IRAK-4 protein in HEK293 cells. Expression of TRIF did not cause IRAK-4 downregulation although it induced NF-kappaB activation. Expression of either a deletion mutant of MyD88 lacking its death domain or MyD88s, neither of which induced NF-kappaB activation, did not lead to IRAK-4 downregulation. MyD88-induced downregulation was observed in an IRAK-4 mutant lacking the kinase domain, but not in another mutant lacking the death domain. These results demonstrate that downregulation of IRAK-4 requires activation of the MyD88-dependent pathway and that the death domains of both MyD88 and IRAK-4 are important for this downregulation.     

Tumor-produced, active interleukin-1β regulates gene expression in carcinoma-associated fibroblasts

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial–mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR.SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression.     

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and toll-like receptor 7-mediated signaling and gene expression

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.     

IRAK4 kinase activity is redundant for interleukin-1 (IL-1) receptor-associated kinase phosphorylation and IL-1 responsiveness

Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFkappaB-dependent reporter gene expression, the activation of NFkappaB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.     

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.