Upon binding of their ligands, TLR7/8 and TLR9 recruit a cytoplasmic adaptor MyD88 and IRAKs, downstream of which the signaling pathways are divided to induce either inflammatory cytokines or type I IFNs.
View original pathway at Reactome.
Li S, Strelow A, Fontana EJ, Wesche H.; ''IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase.''; PubMedEurope PMCScholia
Ordureau A, Smith H, Windheim M, Peggie M, Carrick E, Morrice N, Cohen P.; ''The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1.''; PubMedEurope PMCScholia
Gottipati S, Rao NL, Fung-Leung WP.; ''IRAK1: a critical signaling mediator of innate immunity.''; PubMedEurope PMCScholia
Kollewe C, Mackensen AC, Neumann D, Knop J, Cao P, Li S, Wesche H, Martin MU.; ''Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling.''; PubMedEurope PMCScholia
Schauvliege R, Janssens S, Beyaert R.; ''Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases.''; PubMedEurope PMCScholia
Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, Matsumoto K, Takeuchi O, Akira S.; ''Essential function for the kinase TAK1 in innate and adaptive immune responses.''; PubMedEurope PMCScholia
Lee FS, Hagler J, Chen ZJ, Maniatis T.; ''Activation of the IkappaB alpha kinase complex by MEKK1, a kinase of the JNK pathway.''; PubMedEurope PMCScholia
Cui J, Zhu L, Xia X, Wang HY, Legras X, Hong J, Ji J, Shen P, Zheng S, Chen ZJ, Wang RF.; ''NLRC5 negatively regulates the NF-kappaB and type I interferon signaling pathways.''; PubMedEurope PMCScholia
Xia ZP, Sun L, Chen X, Pineda G, Jiang X, Adhikari A, Zeng W, Chen ZJ.; ''Direct activation of protein kinases by unanchored polyubiquitin chains.''; PubMedEurope PMCScholia
Wan Y, Xiao H, Affolter J, Kim TW, Bulek K, Chaudhuri S, Carlson D, Hamilton T, Mazumder B, Stark GR, Thomas J, Li X.; ''Interleukin-1 receptor-associated kinase 2 is critical for lipopolysaccharide-mediated post-transcriptional control.''; PubMedEurope PMCScholia
Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, Takeshita F, Matsuda M, Coban C, Ishii KJ, Kawai T, Takeuchi O, Akira S.; ''Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction.''; PubMedEurope PMCScholia
Cheng H, Addona T, Keshishian H, Dahlstrand E, Lu C, Dorsch M, Li Z, Wang A, Ocain TD, Li P, Parsons TF, Jaffee B, Xu Y.; ''Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop.''; PubMedEurope PMCScholia
Ning S, Campos AD, Darnay BG, Bentz GL, Pagano JS.; ''TRAF6 and the three C-terminal lysine sites on IRF7 are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein 1.''; PubMedEurope PMCScholia
Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ.; ''TAK1 is a ubiquitin-dependent kinase of MKK and IKK.''; PubMedEurope PMCScholia
Takaesu G, Kishida S, Hiyama A, Yamaguchi K, Shibuya H, Irie K, Ninomiya-Tsuji J, Matsumoto K.; ''TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway.''; PubMedEurope PMCScholia
Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV.; ''TRAF6 is a signal transducer for interleukin-1.''; PubMedEurope PMCScholia
Smith H, Peggie M, Campbell DG, Vandermoere F, Carrick E, Cohen P.; ''Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4.''; PubMedEurope PMCScholia
Keating SE, Maloney GM, Moran EM, Bowie AG.; ''IRAK-2 participates in multiple toll-like receptor signaling pathways to NFkappaB via activation of TRAF6 ubiquitination.''; PubMedEurope PMCScholia
Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z.; ''MyD88: an adapter that recruits IRAK to the IL-1 receptor complex.''; PubMedEurope PMCScholia
Cheung PC, Nebreda AR, Cohen P.; ''TAB3, a new binding partner of the protein kinase TAK1.''; PubMedEurope PMCScholia
Lin SC, Lo YC, Wu H.; ''Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling.''; PubMedEurope PMCScholia
Shim JH, Xiao C, Paschal AE, Bailey ST, Rao P, Hayden MS, Lee KY, Bussey C, Steckel M, Tanaka N, Yamada G, Akira S, Matsumoto K, Ghosh S.; ''TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo.''; PubMedEurope PMCScholia
Windheim M, Stafford M, Peggie M, Cohen P.; ''Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase.''; PubMedEurope PMCScholia
Thiefes A, Wolter S, Mushinski JF, Hoffmann E, Dittrich-Breiholz O, Graue N, Dörrie A, Schneider H, Wirth D, Luckow B, Resch K, Kracht M.; ''Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.''; PubMedEurope PMCScholia
Lamothe B, Besse A, Campos AD, Webster WK, Wu H, Darnay BG.; ''Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation.''; PubMedEurope PMCScholia
Gangloff M, Gay NJ.; ''MD-2: the Toll 'gatekeeper' in endotoxin signalling.''; PubMedEurope PMCScholia
Kawagoe T, Sato S, Jung A, Yamamoto M, Matsui K, Kato H, Uematsu S, Takeuchi O, Akira S.; ''Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling.''; PubMedEurope PMCScholia
Fraczek J, Kim TW, Xiao H, Yao J, Wen Q, Li Y, Casanova JL, Pryjma J, Li X.; ''The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation.''; PubMedEurope PMCScholia
Yang K, Puel A, Zhang S, Eidenschenk C, Ku CL, Casrouge A, Picard C, von Bernuth H, Senechal B, Plancoulaine S, Al-Hajjar S, Al-Ghonaium A, Maródi L, Davidson D, Speert D, Roifman C, Garty BZ, Ozinsky A, Barrat FJ, Coffman RL, Miller RL, Li X, Lebon P, Rodriguez-Gallego C, Chapel H, Geissmann F, Jouanguy E, Casanova JL.; ''Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses.''; PubMedEurope PMCScholia
Motshwene PG, Moncrieffe MC, Grossmann JG, Kao C, Ayaluru M, Sandercock AM, Robinson CV, Latz E, Gay NJ.; ''An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4.''; PubMedEurope PMCScholia
Cherfils-Vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro R, Magdeleinat P, Mami-Chouaib F, Dieu-Nosjean MC, Fridman WH, Damotte D, Sautès-Fridman C, Cremer I.; ''Triggering of TLR7 and TLR8 expressed by human lung cancer cells induces cell survival and chemoresistance.''; PubMedEurope PMCScholia
Rao N, Nguyen S, Ngo K, Fung-Leung WP.; ''A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling.''; PubMedEurope PMCScholia
Krappmann D, Hatada EN, Tegethoff S, Li J, Klippel A, Giese K, Baeuerle PA, Scheidereit C.; ''The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component.''; PubMedEurope PMCScholia
George J, Motshwene PG, Wang H, Kubarenko AV, Rautanen A, Mills TC, Hill AV, Gay NJ, Weber AN.; ''Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly.''; PubMedEurope PMCScholia
Ross K, Yang L, Dower S, Volpe F, Guesdon F.; ''Identification of threonine 66 as a functionally critical residue of the interleukin-1 receptor-associated kinase.''; PubMedEurope PMCScholia
Moynagh PN.; ''The Pellino family: IRAK E3 ligases with emerging roles in innate immune signalling.''; PubMedEurope PMCScholia
Kanayama A, Seth RB, Sun L, Ea CK, Hong M, Shaito A, Chiu YH, Deng L, Chen ZJ.; ''TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains.''; PubMedEurope PMCScholia
Conze DB, Wu CJ, Thomas JA, Landstrom A, Ashwell JD.; ''Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation.''; PubMedEurope PMCScholia
Banerjee A, Gerondakis S.; ''Coordinating TLR-activated signaling pathways in cells of the immune system.''; PubMedEurope PMCScholia
Rothwarf DM, Zandi E, Natoli G, Karin M.; ''IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex.''; PubMedEurope PMCScholia
Butler MP, Hanly JA, Moynagh PN.; ''Kinase-active interleukin-1 receptor-associated kinases promote polyubiquitination and degradation of the Pellino family: direct evidence for PELLINO proteins being ubiquitin-protein isopeptide ligases.''; PubMedEurope PMCScholia
Honda K, Yanai H, Mizutani T, Negishi H, Shimada N, Suzuki N, Ohba Y, Takaoka A, Yeh WC, Taniguchi T.; ''Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling.''; PubMedEurope PMCScholia
Chockalingam A, Brooks JC, Cameron JL, Blum LK, Leifer CA.; ''TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA.''; PubMedEurope PMCScholia
Muroi M, Tanamoto K.; ''TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB.''; PubMedEurope PMCScholia
Suzuki N, Suzuki S, Yeh WC.; ''IRAK-4 as the central TIR signaling mediator in innate immunity.''; PubMedEurope PMCScholia
Kawagoe T, Sato S, Matsushita K, Kato H, Matsui K, Kumagai Y, Saitoh T, Kawai T, Takeuchi O, Akira S.; ''Sequential control of Toll-like receptor-dependent responses by IRAK1 and IRAK2.''; PubMedEurope PMCScholia
Dong C, Davis RJ, Flavell RA.; ''MAP kinases in the immune response.''; PubMedEurope PMCScholia
Wu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD.; ''Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected].''; PubMedEurope PMCScholia
Kishimoto K, Matsumoto K, Ninomiya-Tsuji J.; ''TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop.''; PubMedEurope PMCScholia
Kopp E, Medzhitov R, Carothers J, Xiao C, Douglas I, Janeway CA, Ghosh S.; ''ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway.''; PubMedEurope PMCScholia
Marié I, Smith E, Prakash A, Levy DE.; ''Phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks DNA binding and a bipartite transactivation domain.''; PubMedEurope PMCScholia
Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, Yamamoto M, Terai K, Matsuda M, Inoue J, Uematsu S, Takeuchi O, Akira S.; ''Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6.''; PubMedEurope PMCScholia
Bardwell AJ, Frankson E, Bardwell L.; ''Selectivity of docking sites in MAPK kinases.''; PubMedEurope PMCScholia
Ye H, Arron JR, Lamothe B, Cirilli M, Kobayashi T, Shevde NK, Segal D, Dzivenu OK, Vologodskaia M, Yim M, Du K, Singh S, Pike JW, Darnay BG, Choi Y, Wu H.; ''Distinct molecular mechanism for initiating TRAF6 signalling.''; PubMedEurope PMCScholia
Co-immunoprecipitation studies and size exclusion chromatography analysis indicate that the high molecular weight (around 700 to 900 kDa) IKK complex is composed of two kinase subunits (IKK1/CHUK/IKBKA and/or IKK2/IKBKB/IKKB) bound to a regulatory gamma subunit (IKBKG/NEMO) (Rothwarf DMet al. 1998; Krappmann D et al. 2000; Miller BS & Zandi E 2001). Variants of the IKK complex containing IKBKA or IKBKB homodimers associated with NEMO may also exist. Crystallographic and quantitative analyses of the binding interactions between N-terminal NEMO and C-terminal IKBKB fragments showed that IKBKB dimers would interact with NEMO dimers resulting in 2:2 stoichiometry (Rushe M et al. 2008). Chemical cross-linking and equilibrium sedimentation analyses of IKBKG (NEMO) suggest a tetrameric oligomerization (dimers of dimers) (Tegethoff S et al. 2003). The tetrameric NEMO could sequester four kinase molecules, yielding an 2xIKBKA:2xIKBKB:4xNEMO stoichiometry (Tegethoff S et al. 2003). The above data suggest that the core IKK complex consists of an IKBKA:IKBKB heterodimer associated with an IKBKG dimer or higher oligomeric assemblies. However, the exact stoichiometry of the IKK complex remains unclear.
The mitogen activated protein kinase (MAPK) cascade, one of the most ancient and evolutionarily conserved signaling pathways, is involved in many processes of immune responses. The MAP kinases cascade transduces signals from the cell membrane to the nucleus in response to a wide range of stimuli (Chang and Karin, 2001; Johnson et al, 2002).
There are three major groups of MAP kinases
the extracellular signal-regulated protein kinases ERK1/2,
the p38 MAP kinase
and the c-Jun NH-terminal kinases JNK.
ERK1 and ERK2 are activated in response to growth stimuli. Both JNKs and p38-MAPK are activated in response to a variety of cellular and environmental stresses. The MAP kinases are activated by dual phosphorylation of Thr and Tyr within the tripeptide motif Thr-Xaa-Tyr. The sequence of this tripeptide motif is different in each group of MAP kinases: ERK (Thr-Glu-Tyr); p38 (Thr-Gly-Tyr); and JNK (Thr-Pro-Tyr).
MAPK activation is mediated by signal transduction in the conserved three-tiered kinase cascade: MAPKKKK (MAP4K or MKKKK or MAPKKK Kinase) activates the MAPKKK. The MAPKKKs then phosphorylates a dual-specificity protein kinase MAPKK, which in turn phosphorylates the MAPK.
The dual specificity MAP kinase kinases (MAPKK or MKK) differ for each group of MAPK. The ERK MAP kinases are activated by the MKK1 and MKK2; the p38 MAP kinases are activated by MKK3, MKK4, and MKK6; and the JNK pathway is activated by MKK4 and MKK7. The ability of MAP kinase kinases (MKKs, or MEKs) to recognize their cognate MAPKs is facilitated by a short docking motif (the D-site) in the MKK N-terminus, which binds to a complementary region on the MAPK. MAPKs then recognize many of their targets using the same strategy, because many MAPK substrates also contain D-sites.
The upstream signaling events in the TLR cascade that initiate and mediate the ERK signaling pathway remain unclear.
NF-kappaB is sequestered in the cytoplasm in a complex with inhibitor of NF-kappaB (IkB). Almost all NF-kappaB activation pathways are mediated by IkB kinase (IKK), which phosphorylates IkB resulting in dissociation of NF-kappaB from the complex. This allows translocation of NF-kappaB to the nucleus where it regulates gene expression.
This is the hyperphosphorylated, active form of IRAK1. The unknown coordinate phosphorylation events are to symbolize the multiple phosphorylations that likely take place in the ProST domain (aa10-211).
TRAF6 binding to MAPK kinase kinase 1 (MEKK1) is mediated by the adapter protein evolutionarily conserved signaling intermediate in Toll pathway or in short ECSIT (Kopp E et al 1999). Induced MEKK1 can activate both IKK alpha and IKK beta thus leading to induction of NF-kappa-B activation. MEKK1 was also shown to induce ERK1/2 and JNK activation [Yujiri T et al 1998].
Although TRAF6 interacts with several upstream mediators (IRAK1, IRAK2, TRIF), there is no data showing MEKK1 participating in the interaction with the TRAF6 activators. Therefore this reaction is simplified to include only TRAF6 and MEKK1.
Polyubiquitinated TRAF6 (as E3 ubiquitin ligase) generates free K63 -linked polyubiquitin chains that non-covalently associate with ubiquitin receptors of TAB2/TAB3 regulatory proteins of the TAK1 complex, leading to the activation of the TAK1 kinase.
p-IRF7 dimers are then transported into the nucleus and assemble with the coactivator CBP/p300 to activate transcription of type I interferons and other target genes.
Phosphorylation stimulates the C-terminal autoinhibitory domain of IRF7 to attain a highly extended conformation triggering dimerization through extensive contacts to a second IRF7 subunit.
TAK1-binding protein 2 (TAB2) and/or TAB3, as part of a complex that also contains TAK1 and TAB1, binds polyubiquitinated TRAF6. The TAB2 and TAB3 regulatory subunits of the TAK1 complex contain C-terminal Npl4 zinc finger (NZF) motifs that recognize with Lys63-pUb chains (Kanayama et al. 2004). The recognition mechanism is specific for Lys63-linked ubiquitin chains [Kulathu Y et al 2009]. TAK1 can be activated by unattached Lys63-polyubiquitinated chains when TRAF6 has no detectable polyubiquitination (Xia et al. 2009) and thus the synthesis of these chains by TRAF6 may be the signal transduction mechanism.
Structural analysis of MyD88:IRAK4 and MyD88:IRAK4:IRAK2 suggested that upon MyD88 recruitment to an activated dimerized TLR the MyD88 death domains clustering induces the formation of Mydosome, a large oligomeric signaling platform (Motshwene PG et al 2009, Lin SC et al 2010). Assembly of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. The oligomer complex stoichiometry was reported as 7:4 and 8:4 for MyD88:IRAK4 (Motshwene PG et al 2009), and 6:4:4 in the complex of MyD88:IRAK4:IRAK2(Lin SC et al 2010).
Hyperphosphorylated IRAK1 and TRAF6 are thought to dissociate from the activated receptor. (Gottipati et al. 2007) but the IRAK1:TRAF6 complex may remain associated with the membrane (Dong et al. 2006).
Phosphorylated IRAK2, like its paralog IRAK1, possibly dissociates from the activated receptor as shown here, although mechanism of IRAK2 activation by IRAK4 followed by TRAF6 binding remains to be deciphered.
The TAK1 complex consists of Transforming growth factor-beta (TGFB)-activated kinase (TAK1) and TAK1-binding protein 1 (TAB1), TAB2 and TAB3. TAK1 requires TAB1 for its kinase activity (Shibuya et al. 1996, Sakurai et al. 2000). TAB1 promotes TAK1 autophosphorylation at the kinase activation lobe, probably through an allosteric mechanism (Brown et al. 2005, Ono et al. 2001). The TAK1 complex is regulated by polyubiquitination. Binding of TAB2 and TAB3 to Lys63-linked polyubiquitin chains leads to the activation of TAK1 by an uncertain mechanism. Binding of multiple TAK1 complexes to the same polyubiquitin chain may promote oligomerization of TAK1, facilitating TAK1 autophosphorylation and subsequent activation of its kinase activity (Kishimoto et al. 2000). The binding of TAB2/3 to polyubiquitinated TRAF6 may facilitate polyubiquitination of TAB2/3 by TRAF6 (Ishitani et al. 2003), which might result in conformational changes within the TAK1 complex that lead to TAK1 activation. Another possibility is that TAB2/3 may recruit the IKK complex by binding to ubiquitinated NEMO; polyubiquitin chains may function as a scaffold for higher order signaling complexes that allow interaction between TAK1 and IKK (Kanayama et al. 2004).
IRF7 is phosphorylated on Ser477 and Ser479 residues [Lin R et al 2000] . IRAK1[Uematsu et al 2005] and IKK alpha[Hoshino et al 2006] are thought to mediate the phosphorylation upon TLR7/8/9 activation.
IRAK-2 has two TRAF6 binding motifs that are responsible for initiating TRAF6 signaling transduction (Ye H et al 2002). IRAK2 point mutants with mutated TRAF6-binding motifs abrogate NFkB activation and are incapable to stimulate TRAF6 ubiquitination (Keating SE et al 2007).
IRAK2 has been implicated in IL1R and TLR signaling by the observation that IRAK2 can associate with MyD88 and Mal (Muzio et al. 1997). Like IRAK1, IRAK2 is activated downstream of IRAK4 (Kawagoe et al. 2008). It has been suggested that IRAK1 activates IRAK2 (Wesche et al. 1999) but IRAK2 phosphorylation is observed in IRAK1–/– mouse macrophages while IRAK4 deficiency abrogates IRAK2 phosphorylation (Kawagoe et al. 2008), suggesting that activated IRAK4 phosphorylates IRAK2 as it does IRAK1. IL6 production in response to IL1beta is impaired in embryonic fibroblasts from IRAK1 or IRAK2 knockout mice and abrogated in IRAK1/2 dual knockouts (Kawagoe et al. 2007) suggesting that IRAK1 and IRAK2 are both involved in IL1R signaling downstream of IRAK4.
TRAF6 E3 ubiquitin ligase activity was shown to be essential for IRF7 activation, although the role of TRAF6-dependent ubiquitination remains unclear [Kawai T et al 2004]. It has been demonstrated that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo[Ning et al 2008].It has been also shown that K63-linked ubiquitination of IRF7 is independent of its C-terminal functional phosphorylation sites.
NF-kappa-B essential modulator (NEMO, also known as IKKG abbreviated from Inhibitor of nuclear factor kappa-B kinase subunit gamma) is the regulatory subunit of the IKK complex which phosphorylates inhibitors of NF-kappa-B leading to dissociation of the inhibitor/NF-kappa-B complex. NEMO binds to K63-pUb chains (Ea et al. 2006; Wu et al. 2006), linking K63-pUb-hp-IRAK1 with the IKK complex. Models of IL-1R dependent activation of NF-kappaB suggest that the polyubiquitination of both TRAF6 and IRAK1 within a TRAF6:IRAK1 complex and their subsequent interactions with the TAK1 complex and IKK complex respectively brings these complexes into proximity, facilitating the TAK1-catalyzed activation of IKK (Moynagh, 2008).
IL1 induces the poly-ubiquitination and degradation of IRAK1. This was believed to be K48-linked polyubiquitination, targeting IRAK1 for proteolysis by the proteasome, but recently IL-1R signaling has been shown to lead to K63-linked polyubiquitination of IRAK1 (Windheim et al. 2008; Conze et al. 2008), and demonstrated to have a role in the activation of NF-kappaB. IRAK1 is ubiquitinated on K134 and K180; mutation of these sites impairs IL1R-mediated ubiquitylation of IRAK1 (Conze et al. 2008). Some authors have proposed a role for TRAF6 as the E3 ubiquitin ligase that catalyzes polyubiquitination of IRAK1 (Conze et al. 2008) but this view has been refuted (Windheim et al. 2008; Xiao et al. 2008). There is stronger agreement that Pellino proteins have a role as IRAK1 E3 ubiquitin ligases. Pellino1-3 possess E3 ligase activity and are believed to directly catalyse polyubiquitylation of IRAK1 (Xiao et al. 2008; Butler et al. 2007; Ordureau et al. 2008). They are capable of catalysing the formation of K63- and K48-linked polyubiquitin chains; the type of linkage is controlled by the collaborating E2 enzyme. All the Pellino proteins can combine with the E2 heterodimer UBE2N:UBE2V1 (Ubc13:Uev1a) to catalyze K63-linked ubiquitylation (Ordureau et al. 2008).
A series of sequential phosphorylation events lead to full or hyper-phopshorylation of IRAK1. Under in vitro conditions these are all autophosphorylation events. First, Thr-209 is phosphorylated resulting in a conformational change of the kinase domain. Next, Thr-387 in the activation loop is phosphorylated, leading to full enzymatic activity. Several additional residues are phosphorylated 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 IRAK1 from the upstream adapters MyD88 and Tollip. The significance of these phosphorylation events is not clear; the kinase activity of IRAK1 is dispensable for IL1-induced NFkB and MAP kinase activation (Knop & Martin, 1999), unlike that of IRAK4 (Suzuki et al. 2002; Kozicak-Holbro et al. 2007), so IRAK1 is believed to act primarily as an adaptor for TRAF6 (Conze et al. 2008).
Both IRAK1 and IRAK4 were shown to phosphorylate Pellino isoforms in vitro. The phosphorylation of Pellino proteins is a necessary step in enhancing of their E3 ubiquitin ligase activity. It remains unclear whether IRAK1(as shown here), IRAK4, or both protein kinases mediate the activation of Pellino isoforms in vivo.
Pellino isoforms -1, 2 and 3 have been shown to interact with IRAK1 and IRAK4 (Jiang et al. 2003, Strellow et al. 2003, Butler et al. 2005, 2007). It has been also reported that Pellino-1 forms a complex with TRAF6, but not TAK1 or IL1R (Jiang et al. 2003), suggesting that Pellino-1 function as intermediate complex with IRAK1 in the propagation of signal from the activated receptor to activation of TAK1.
All Pellino isoforms function as E3 ubiquitin ligases in conjunction with several different E2-conjugating enzymes - Ubc13-Uev1a, UbcH4, or UbcH5a/5b.(Schauvliege R et al. 2006, Butler MP et al. 2007, Ordureau A et al. 2008). Their C-terminus contains a RING-like domain which is responsible for IL1-induced Lys63-linked polyubiquitination of IRAK1 in vitro.
TRAF6 possesses ubiquitin ligase activity and undergoes K-63-linked auto-ubiquitination after its oligomerization. In the first step, ubiquitin is activated by an E1 ubiquitin activating enzyme. The activated ubiquitin is transferred to a E2 conjugating enzyme (a heterodimer of proteins Ubc13 and Uev1A) forming the E2-Ub thioester. Finally, in the presence of ubiquitin-protein ligase E3 (TRAF6, a RING-domain E3), ubiquitin is attached to the target protein (TRAF6 on residue Lysine 124) through an isopeptide bond between the C-terminus of ubiquitin and the epsilon-amino group of a lysine residue in the target protein. In contrast to K-48-linked ubiquitination that leads to the proteosomal degradation of the target protein, K-63-linked polyubiquitin chains act as a scaffold to assemble protein kinase complexes and mediate their activation through proteosome-independent mechanisms. This K63 polyubiquitinated TRAF6 activates the TAK1 kinase complex.
IRAK4 is the mammalian homolog of Drosophila melanogaster Tube [Towb P et al 2009; Moncrieffe MC et al 2008]. Like Tube, IRAK4 possesses a conserved N-terminal death domain (DD), which mediates interactions with MyD88 at one binding site and a downstream IRAK kinase at the other, thereby bridging MyD88 and IRAK1/2 association [Towb P et al 2009; Lin SC e al 2010]. IRAK-4 plays a critical role in Toll receptor signaling - any interference with IRAK-4's kinase activity virtually abolishes downstream events. This is not the case with other members of the IRAK family [Suzuki N et al 2002; Li S et al 2002].
IRAK4 deficient macrophages fail to induce IRAK2 phosphorylation (Kawagoe et al. 2008), suggesting that activated IRAK4 phosphorylates IRAK2 as it does IRAK1.
Phosphorylation sites of IRAK2 remain to be characterized.
MyD88 binds to IRAK (IL-1 receptor-associated kinase) and the receptor heterocomplex (the signaling complex) and thereby mediates the association of IRAK with the receptor. MyD88 therefore couples a serine/threonine protein kinase to the receptor complex.
First, IRAK1 is phosphorylated at Thr209 by IRAK4. This 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 IRAK1.
The mechanism by which IRAK-2 induces TRAF6 E3 ligase activity remains to be deciphered, but one possibility is that IRAK-2 may direct TRAF6 oligomerization.
Upon TLR7/8 or 9 stimulation IRF7, but not IRF3, forms a signaling complex with MyD88, TRAF6[Honda K et al 2004, Kawai T et al 2004], IRAK1 [Uematsu S et al 2005], Also IRAK4 was shown to mediate IRF7 activation[Honda K et al 2004].
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IRAK2
There are three major groups of MAP kinases
ERK1 and ERK2 are activated in response to growth stimuli. Both JNKs and p38-MAPK are activated in response to a variety of cellular and environmental stresses. The MAP kinases are activated by dual phosphorylation of Thr and Tyr within the tripeptide motif Thr-Xaa-Tyr. The sequence of this tripeptide motif is different in each group of MAP kinases: ERK (Thr-Glu-Tyr); p38 (Thr-Gly-Tyr); and JNK (Thr-Pro-Tyr).
MAPK activation is mediated by signal transduction in the conserved three-tiered kinase cascade: MAPKKKK (MAP4K or MKKKK or MAPKKK Kinase) activates the MAPKKK. The MAPKKKs then phosphorylates a dual-specificity protein kinase MAPKK, which in turn phosphorylates the MAPK.
The dual specificity MAP kinase kinases (MAPKK or MKK) differ for each group of MAPK. The ERK MAP kinases are activated by the MKK1 and MKK2; the p38 MAP kinases are activated by MKK3, MKK4, and MKK6; and the JNK pathway is activated by MKK4 and MKK7. The ability of MAP kinase kinases (MKKs, or MEKs) to recognize their cognate MAPKs is facilitated by a short docking motif (the D-site) in the MKK N-terminus, which binds to a complementary region on the MAPK. MAPKs then recognize many of their targets using the same strategy, because many MAPK substrates also contain D-sites.
The upstream signaling events in the TLR cascade that initiate and mediate the ERK signaling pathway remain unclear.
the activated TLR
receptorby phosphorylation and activation of
IKKs complexpolyUb p-IRAK1:IKK
complexor p-IRAK2 bound to the
pIRAK4:MyD88:activated TLR7/8 or 9 complexpUb oligo-TRAF6:free K63 pUb:TAK1
complexpUb oligo-TRAF6:free K63-linked
pUb:p-TAK1complexAnnotated Interactions
IRAK2
the activated TLR
receptorthe activated TLR
receptorAlthough TRAF6 interacts with several upstream mediators (IRAK1, IRAK2, TRIF), there is no data showing MEKK1 participating in the interaction with the TRAF6 activators. Therefore this reaction is simplified to include only TRAF6 and MEKK1.
Phosphorylated IRAK2, like its paralog IRAK1, possibly dissociates from the activated receptor as shown here, although mechanism of IRAK2 activation by IRAK4 followed by TRAF6 binding remains to be deciphered.
Pellino1-3 possess E3 ligase activity and are believed to directly catalyse polyubiquitylation of IRAK1 (Xiao et al. 2008; Butler et al. 2007; Ordureau et al. 2008). They are capable of catalysing the formation of K63- and K48-linked polyubiquitin chains; the type of linkage is controlled by the collaborating E2 enzyme. All the Pellino proteins can combine with the E2 heterodimer UBE2N:UBE2V1 (Ubc13:Uev1a) to catalyze K63-linked ubiquitylation (Ordureau et al. 2008).
All Pellino isoforms function as E3 ubiquitin ligases in conjunction with several different E2-conjugating enzymes - Ubc13-Uev1a, UbcH4, or UbcH5a/5b.(Schauvliege R et al. 2006, Butler MP et al. 2007, Ordureau A et al. 2008). Their C-terminus contains a RING-like domain which is responsible for IL1-induced Lys63-linked polyubiquitination of IRAK1 in vitro.
Phosphorylation sites of IRAK2 remain to be characterized.
polyUb p-IRAK1:IKK
complexor p-IRAK2 bound to the
pIRAK4:MyD88:activated TLR7/8 or 9 complexpUb oligo-TRAF6:free K63 pUb:TAK1
complexpUb oligo-TRAF6:free K63 pUb:TAK1
complexpUb oligo-TRAF6:free K63-linked
pUb:p-TAK1complex