Natural loss-of-function mutation of myeloid differentiation protein 88 disrupts its ability to form Myddosomes

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knock-out mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wild-type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y loss-of-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.     

MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.     

Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88

MyD88 is an adaptor protein that is involved in interleukin-1 receptor (IL-1R)- and Toll-like receptor (TLR)-induced activation of NF-kappaB. It is composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD), which mediate the interaction of MyD88 with the IL-1R/TLR and the IL-1R-associated kinase (IRAK), respectively. The interaction of MyD88 with IRAK triggers IRAK phosphorylation, which is essential for its activation and downstream signaling ability. Both domains of MyD88 are separated by a small intermediate domain (ID) of unknown function. Here, we report the identification of a splice variant of MyD88, termed MyD88(S), which encodes for a protein lacking the ID. MyD88(S) is mainly expressed in the spleen and can be induced in monocytes upon LPS treatment. Although MyD88(S) still binds the IL-1R and IRAK, it is defective in its ability to induce IRAK phosphorylation and NF-kappaB activation. In contrast, MyD88(S) behaves as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced, NF-kappaB activation. These results implicate the ID of MyD88 in the phosphorylation of IRAK. Moreover, the regulated expression and antagonistic activity of MyD88(S) suggest an important role for alternative splicing of MyD88 in the regulation of the cellular response to IL-1 and LPS.     

Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88_E52A) and 58 (MyD88_Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88_K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu⁵² and Tyr⁵⁸ residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growth-inhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an amino-terminal death domain (DD),³ found in proteins involved in cell death (3, 4), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7, 8) and TLR4 (9). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10).By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11, 12). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13), IRAK2 (7), IRAK-M (15), and IRAK4 (16), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment of IRAK-4 appears to be rather unusual. Burns et al. (6) first demonstrated that an alternatively spliced variant of MyD88 (MyD88s), lacking the ID domain, failed to interact with IRAK-4, suggesting that residues located in both the DD and ID of MyD88 are crucially involved in the recruitment of IRAK-4. Nevertheless, no information is available on the specific residues in the DD in MyD88 required for its interaction with either IRAK1 or IRAK4.The DD was initially defined as the region of homology between the cytoplasmic tails of the FAS/Apo1/CD95 and TNF receptors required for their induction of cytotoxic signaling (18, 19). In analogy with other DD-containing proteins, this domain in MyD88 is also involved in the formation of homomeric and heteromeric interactions. Herein, we have undertaken an alanine-scanning mutational analysis to identify amino acids that are required for downstream signaling and might participate in the homomeric and heteromeric interactions. Our studies revealed that MyD88_E52A and MyD88_Y58A mutants are strongly impaired in the recruitment of both IRAK1 and IRAK4, whereas the MyD88_K95A mutant is deficient in recruiting IRAK4. These findings identify residues within the DD of MyD88 crucially involved in the formation of higher order complexes containing IRAK1 and IRAK4 and required for the propagation of the TLR/IL1-R signaling pathways.     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R-associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88- IL-1R-associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-β signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.     

IRAK1 and IRAK4 Promote Phosphorylation,Ubiquitination, and Degradation of MyD88 Adaptor-like (Mal)

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.     

FADD negatively regulates lipopolysaccharide signaling by impairing interleukin-1 receptor-associated kinase 1-MyD88 interaction

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.     

The Brucella TIR-like protein TcpB interacts with the death domain of MyD88

The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface.     

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

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.     

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.     

Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family

The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.     

R753Q Polymorphism Inhibits Toll-like Receptor (TLR) 2 Tyrosine Phosphorylation,Dimerization with TLR6, and Recruitment of Myeloid Differentiation Primary Response Protein 88

The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.     

Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.     

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.