The first known downstream component of TLR4 and TLR2 signaling is the adaptor MyD88. Another adapter MyD88-adaptor-like (Mal; also known as TIR-domain-containing adaptor protein or TIRAP) has also been described for TLR4 and TLR2 signaling. MyD88 comprises an N-terminal Death Domain (DD) and a C-terminal TIR, whereas Mal lacks the DD. The TIR homotypic interactions bring adapters into contact with the activated TLRs, whereas the DD modules recruit serine/threonine kinases such as interleukin-1-receptor-associated kinase (IRAK). Recruitment of these protein kinases is accompanied by phosphorylation, which in turn results in the interaction of IRAKs with TNF-receptor-associated factor 6 (TRAF6). The oligomerization of TRAF6 activates TAK1, a member of the MAP3-kinase family, and this leads to the activation of the IkB kinases. These kinases, in turn, phosphorylate IkB, leading to its proteolytic degradation and the translocation of NF-kB to the nucleus. Concomitantly, members of the activator protein-1 (AP-1) transcription factor family, Jun and Fos, are activated, and both AP-1 transcription factors and NF-kB are required for cytokine production, which in turn produces downstream inflammatory effects.
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
Jiang Z, Ninomiya-Tsuji J, Qian Y, Matsumoto K, Li X.; ''Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol.''; PubMedEurope PMCScholia
Rothwarf DM, Zandi E, Natoli G, Karin M.; ''IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex.''; 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
Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ.; ''TAK1 is a ubiquitin-dependent kinase of MKK and IKK.''; PubMedEurope PMCScholia
Banerjee A, Gerondakis S.; ''Coordinating TLR-activated signaling pathways in cells of the immune system.''; 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
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
Tao N, Wagner SJ, Lublin DM.; ''CD36 is palmitoylated on both N- and C-terminal cytoplasmic tails.''; PubMedEurope PMCScholia
Loiarro M, Gallo G, Fantò N, De Santis R, Carminati P, Ruggiero V, Sette C.; ''Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.''; PubMedEurope PMCScholia
da Silva Correia J, Ulevitch RJ.; ''MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.''; PubMedEurope PMCScholia
Dong C, Davis RJ, Flavell RA.; ''MAP kinases in the immune response.''; 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
Moynagh PN.; ''The Pellino family: IRAK E3 ligases with emerging roles in innate immune signalling.''; PubMedEurope PMCScholia
Brown K, Vial SC, Dedi N, Long JM, Dunster NJ, Cheetham GM.; ''Structural basis for the interaction of TAK1 kinase with its activating protein TAB1.''; PubMedEurope PMCScholia
Dunne A, Carpenter S, Brikos C, Gray P, Strelow A, Wesche H, Morrice N, O'Neill LA.; ''IRAK1 and IRAK4 promote phosphorylation, ubiquitination, and degradation of MyD88 adaptor-like (Mal).''; PubMedEurope PMCScholia
Sakurai H, Miyoshi H, Mizukami J, Sugita T.; ''Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1.''; PubMedEurope PMCScholia
Mansell A, Brint E, Gould JA, O'Neill LA, Hertzog PJ.; ''Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4.''; PubMedEurope PMCScholia
Kulathu Y, Akutsu M, Bremm A, Hofmann K, Komander D.; ''Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain.''; 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
Verstak B, Nagpal K, Bottomley SP, Golenbock DT, Hertzog PJ, Mansell A.; ''MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses.''; 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
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
Gangloff M, Gay NJ.; ''MD-2: the Toll 'gatekeeper' in endotoxin signalling.''; PubMedEurope PMCScholia
Newton K, Matsumoto ML, Wertz IE, Kirkpatrick DS, Lill JR, Tan J, Dugger D, Gordon N, Sidhu SS, Fellouse FA, Komuves L, French DM, Ferrando RE, Lam C, Compaan D, Yu C, Bosanac I, Hymowitz SG, Kelley RF, Dixit VM.; ''Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.''; PubMedEurope PMCScholia
Valkov E, Stamp A, Dimaio F, Baker D, Verstak B, Roversi P, Kellie S, Sweet MJ, Mansell A, Gay NJ, Martin JL, Kobe B.; ''Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction and disease protection.''; PubMedEurope PMCScholia
Moncrieffe MC, Grossmann JG, Gay NJ.; ''Assembly of oligomeric death domain complexes during Toll receptor signaling.''; 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
Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV.; ''TRAF6 is a signal transducer for interleukin-1.''; PubMedEurope PMCScholia
Jefferies CA, Doyle S, Brunner C, Dunne A, Brint E, Wietek C, Walch E, Wirth T, O'Neill LA.; ''Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4.''; 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
Ono K, Ohtomo T, Sato S, Sugamata Y, Suzuki M, Hisamoto N, Ninomiya-Tsuji J, Tsuchiya M, Matsumoto K.; ''An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK.''; PubMedEurope PMCScholia
Shibuya H, Yamaguchi K, Shirakabe K, Tonegawa A, Gotoh Y, Ueno N, Irie K, Nishida E, Matsumoto K.; ''TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction.''; PubMedEurope PMCScholia
Lee KG, Xu S, Kang ZH, Huo J, Huang M, Liu D, Takeuchi O, Akira S, Lam KP.; ''Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response.''; 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
Gottipati S, Rao NL, Fung-Leung WP.; ''IRAK1: a critical signaling mediator of innate immunity.''; 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
Gray P, Dunne A, Brikos C, Jefferies CA, Doyle SL, O'Neill LA.; ''MyD88 adapter-like (Mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction.''; PubMedEurope PMCScholia
Bardwell AJ, Frankson E, Bardwell L.; ''Selectivity of docking sites in MAPK kinases.''; 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
Suzuki N, Suzuki S, Yeh WC.; ''IRAK-4 as the central TIR signaling mediator in innate immunity.''; 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
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
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
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
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
Yamamoto M, Sato S, Hemmi H, Sanjo H, Uematsu S, Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T, Takeda K, Akira S.; ''Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4.''; 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
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
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
Muroi M, Tanamoto K.; ''TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB.''; 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
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
Xiong Y, Qiu F, Piao W, Song C, Wahl LM, Medvedev AE.; ''Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-beta-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor-associated factor 6, and IkappaB kinase gamma and increases A20 expression.''; PubMedEurope PMCScholia
Cheung PC, Nebreda AR, Cohen P.; ''TAB3, a new binding partner of the protein kinase TAK1.''; 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
Piao W, Song C, Chen H, Wahl LM, Fitzgerald KA, O'Neill LA, Medvedev AE.; ''Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance.''; 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
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
Flannery SM, Keating SE, Szymak J, Bowie AG.; ''Human interleukin-1 receptor-associated kinase-2 is essential for Toll-like receptor-mediated transcriptional and post-transcriptional regulation of tumor necrosis factor alpha.''; PubMedEurope PMCScholia
Fitzgerald KA, Palsson-McDermott EM, Bowie AG, Jefferies CA, Mansell AS, Brady G, Brint E, Dunne A, Gray P, Harte MT, McMurray D, Smith DE, Sims JE, Bird TA, O'Neill LA.; ''Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.''; PubMedEurope PMCScholia
Lin SC, Lo YC, Wu H.; ''Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling.''; 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
Towb P, Sun H, Wasserman SA.; ''Tube Is an IRAK-4 homolog in a Toll pathway adapted for development and immunity.''; PubMedEurope PMCScholia
Ohnishi T, Muroi M, Tanamoto K.; ''N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.''; PubMedEurope PMCScholia
Dunne A, Ejdeback M, Ludidi PL, O'Neill LA, Gay NJ.; ''Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88.''; 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
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
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
Brunner C, Müller B, Wirth T.; ''Bruton's Tyrosine Kinase is involved in innate and adaptive immunity.''; 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
Wesche H, Gao X, Li X, Kirschning CJ, Stark GR, Cao Z.; ''IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family.''; 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
Núñez Miguel R, Wong J, Westoll JF, Brooks HJ, O'Neill LA, Gay NJ, Bryant CE, Monie TP.; ''A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.''; PubMedEurope PMCScholia
Qian Y, Commane M, Ninomiya-Tsuji J, Matsumoto K, Li X.; ''IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFkappa B.''; PubMedEurope PMCScholia
Nagpal K, Plantinga TS, Wong J, Monks BG, Gay NJ, Netea MG, Fitzgerald KA, Golenbock DT.; ''A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling.''; PubMedEurope PMCScholia
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.
The listed studies describe an activation of IRAK-TRAF6-TAK1 axes downstream of IL1 receptor signaling cascade, which is mediated by its cytosolic domain called Toll/IL1R (TIR) domain. TLRs and IL1R are thought to share a similar downstream signaling pathway due to a high homology of their C-terminal TIR domains.
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).
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).
MAL(TIRAP) undergoes tyrosine phosphorylation mediated by Bruton's tyrosine kinase (BTK). BTK-specific inhibitor, LFM-A13, blocked the phosphorylation of MAL in human HEK293 stimulated with LPS or macrophage-activating lipopeptide-2. LFM-A13 also inhibited activation of NF-kB in LPS-treated human monocytic cell line THP-1 [Gray P et al 2006; Jefferies CA et al 2003]. Tyr-86, Tyr-106 and Tyr-187 were identified as possible phosphorylation sites [Gray P et al 2006]. An additional study has shown that Tyr-86, Tyr-106, and Tyr-159 are important residues, as mutagenesis of these residues impaired MAL phosphorylation, affected its interaction with BTK and also impaired downstream signaling [Piao W et al 2008].
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.
TIRAP/Mal-deficient mice showed normal responses to the TLR3, TLR5, TLR7, and TLR9 ligands, but were defective in TLR4 and TLR2 ligand-induced proinflammatory cytokine production (Horng et al. 2002,Yamamoto et al. 2002). In contrast, TLR4 ligand-induced activation of IRF-3 and expression of IFN-inducible genes were not impaired in TIRAP/Mal knockout macrophages or in mice lacking both MyD88 and TIRAP/Mal (Horng et al. 2002,Yamamoto et al. 2002). Thus, TIRAP/Mal is an essential adapter that is involved in the MyD88-dependent pathway via TLR4 and TLR2, but not in the MyD88-independent pathway. Mal contains a phosphatidylinositol 4,5-bisphosphate-binding domain required for retention in the plasma membrane. The intracellular TIR domains of TLR2 or 4 associate with Mal at the cytoplasmic side of the plasma membrane, which in turn facilitates the binding of MyD88 to the activated TLR, leading to NF-kB and MAPK activation [Nunez Miguel et al 2007].
Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase, which plays an essential role in B cell receptor (BCR) signaling [Brunner C et al 2005]. BTK has been also implicated in TLR signaling [Lee KG et al 2012, Jefferies CA et al 2003]. Interaction studies revealed that BTK can associate with intracellular TIR-domains of TLRs 4, 6, 8 and 9. Furthermore, BTK was found to interact with other proteins involved in TLR2/4 signaling - MyD88, MAL and IRAK-1 [Jefferies CA et al 2003].
IL1R/TLR induces the Lys48- polyubiquitination and proteosomal degradation of IRAK1. IRAK1 has been shown to undergo Lys63-linked polyubiquitination which induced activation of NFkB (Windheim et al 2008; Conze et al 2008). These two forms of ubiquitination are not mutually exclusive for a protein (Newton K et al 2008). Upon stimulation Lys63-linked ubiquitination may occur first to activate NFkB, but at later time Lys48-linked ubiquitination occurs to target the proteins for proteosomal degradation.
IRAK1 is ubiquitinated on Lys134 and Lys180; 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 a 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 Lys48-linked polyubiquitin chains; the type of linkage is controlled by the collaborating E2 enzyme. All the Pellino proteins can combine with the E2 heterodimer UbcH13/Uev1a to catalyze Lys63-linked ubiquitylation (Ordureau et al 2008).
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.
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.
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.
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.
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.
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.
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).
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).
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.
The TAK1 complex consists of the transforming growth factor-? (TGF-beta)-activated kinase (TAK1) and the TAK1-binding proteins TAB1, TAB2 and TAB3. TAK1 requires TAB1 for its kinase activity (Sakurai H et al 2000; Shibuya H et al 2000). TAB1 promotes autophosphorylation of the TAK1 kinase activation lobe, likely through an allosteric mechanism (Sakurai H et al 2000 ; Kishimoyo K et al 2000). The TAK1 complex is regulated by polyubiquitination. The TAK1 complex consists of the transforming growth factor-? (TGF- ?)-activated kinase (TAK1) and the TAK1-binding proteins TAB1, TAB2 and TAB3. TAK1 requires TAB1 for its kinase activity (Shibuya H et al 1996; Sakurai H et al 2000). TAB1 promotes autophosphorylation of the TAK1 kinase activation lobe, likely through an allosteric mechanism (Brown K et al 2005; Ono K 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 onto 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 leads to the activation of TAK1. 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).
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.
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.
Hyperphosphorylated IRAK1, still within the receptor complex, binds TRAF6 through multiple regions including the death domain, the undefined domain and the C-terminal C1 domain (Li et al. 2001). The C-terminal region of IRAK-1 contains three potential TRAF6-binding sites; mutation of the amino acids (Glu544, Glu587, Glu706) in these sites to alanine greatly reduces activation of NFkappaB (Ye et al. 2002).
Phosphorylation of IRAK-1 is due to three sequential phosphorylation steps, which leads 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 activated receptor complex. 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), It has been suggested that IRAK1 primarily acts as an adaptor for TRAF6 (Conze et al. 2008).
IRAK1 is recruited to the TLR complex through binding with IRAK4[Lin SC et al 2010].
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 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.
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].
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2 or TLR 2
6 heterodimers or TLR4 homodimerIKKB
NEMOp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 MyD88 oligomer Mal
activated TLRp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRoligo-MyD88 Mal
activated TLRBTK
activated TLR2/4There 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.
K63-linked polyUb p-IRAK1
IKK complexhp-IRAK1
Pellinohp-IRAK1 p-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK2 p-IRAK4 oligo-MyD88 Mal
activated TLRp-4Y-MAL
BTKMal
activated TLRp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 oligo-MyD88 Mal
activated TLRK63-linked pUb oligo-TRAF6 free K63 pUb
TAK1 complexK63-linked pUb oligo-TRAF6 free K63-linked pUb
p-TAK1complexp-IRAK4 oligo-MyD88 Mal
activated TLRhp-IRAK1
TRAF6oligo-MyD88 Mal
activated TLR receptorp-IRAK4 oligo-MyD88 Mal
activated TLRAnnotated Interactions
2 or TLR 2
6 heterodimers or TLR4 homodimerIKKB
NEMOp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRoligo-MyD88 Mal
activated TLRBTK
activated TLR2/4Phosphorylated 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.
IRAK1 is ubiquitinated on Lys134 and Lys180; 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 a 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 Lys48-linked polyubiquitin chains; the type of linkage is controlled by the collaborating E2 enzyme. All the Pellino proteins can combine with the E2 heterodimer UbcH13/Uev1a to catalyze Lys63-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.
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.
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.
hp-IRAK1
Pellinop-4Y-MAL
BTKMal
activated TLRp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRp-S,2T-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 oligo-MyD88 Mal
activated TLRK63-linked pUb oligo-TRAF6 free K63 pUb
TAK1 complexK63-linked pUb oligo-TRAF6 free K63-linked pUb
p-TAK1complexp-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 oligo-MyD88 Mal
activated TLRhp-IRAK1
TRAF6hp-IRAK1
TRAF6oligo-MyD88 Mal
activated TLR receptoroligo-MyD88 Mal
activated TLR receptoroligo-MyD88 Mal
activated TLR receptoroligo-MyD88 Mal
activated TLR receptorp-IRAK4 oligo-MyD88 Mal
activated TLRp-IRAK4 oligo-MyD88 Mal
activated TLR