Poly(dI·dC)-induced Toll-like Receptor 3 (TLR3)-mediated Activation of NFκB and MAP Kinase Is through an Interleukin-1 Receptor-associated Kinase (IRAK)-independent Pathway Employing the Signaling Components TLR3-TRAF6-TAK1-TAB2-PKR

Abstract

Recent studies show that a member of the interleukin-1 (IL-1)/Toll receptor superfamily, Toll-like receptor 3 (TLR3), recognizes double-stranded RNA (dsRNA). Because of the similarity in their cytoplasmic domains, IL-1/Toll receptors share signaling components that associate with the IL-1 receptor, including IL-1 receptorassociated kinase (IRAK), MyD88, and TRAF6. However, we find that, in response to dsRNA, TLR3 can mediate the activation of both NFB and mitogen-activated protein (MAP) kinases in IL-1-unresponsive mutant cell lines, including IRAK-deficient I1A and I3A cells, which are defective in a component that is downstream of IL-1R but upstream of IRAK. These results clearly indicate that TLR3 does not simply share the signaling components employed by the IL-1 receptor. Through biochemical analyses we have identified an IRAK-independent TLR3-mediated pathway. Upon binding of dsRNA to TLR3, TRAF6, TAK1, and TAB2 are recruited to the receptor to form a complex, which then translocates to the cytosol where TAK1 is phosphorylated and activated. The dsRNA-dependent protein kinase (PKR) is also detected in this signal-induced TAK1 complex. Kinase inactive mutants of TAK1 (TAK1DN) and PKR (PKRDN) inhibit poly(dI⅐dC)-induced TLR3-mediated NFB activation, suggesting that both of these kinases play important roles in this pathway.The interleukin-1 (IL-1) 1 /Toll receptors play essential roles in inflammation and innate immunity. The defining feature of members of the superfamily is a TIR (Toll/IL-1 receptor) domain on the cytoplasmic side of the receptors (1, 2). The first described members of this superfamily are in the IL-1R family. These receptors contain three Ig domains in their extracellular regions (3-5). The second group includes only one receptor so far, the single Ig IL-1R-related molecule (SIGIRR), which has only a single extracellular Ig domain (6). The last group in the superfamily is the recently identified pattern recognition receptors, the Toll-like receptors (TLRs), 10 members of which contain two major domains characterized by extracellular leucine-rich repeats and an intracellular Toll-like domain (2, 7-10). TLR4 has been genetically identified as a signaling molecule essential for the responses to LPS, a feature of Gramnegative bacteria (11). Mice with targeted disruption of the TLR4 gene are LPS-unresponsive. Unlike TLR4, TLR2 responds to mycobacteria, yeast, and Gram-positive bacteria (12-15). TLR9 has been shown to recognize bacterial DNA (10), whereas TLR5 mediates the induction of the immune response by bacterial flagellins (16). Recent studies show that TLR3 recognizes dsRNA (17).Much progress has been made in understanding the IL-1R-mediated signaling. Upon IL-1 stimulation, the cytosolic proteins and Tollip (21) are recruited to the receptor complex, which then recruits serine-threonine kinases IRAK4 (IL-1 receptor-associated kinase 4) 2 (22, 23) and IRAK (24). Although IRAK is hyperphosphorylated, mediating the recruitment of TRAF6 to the receptor complex (25), IRAK4 is probably the ki...

RIP1, a kinase on the crossroads of a cell's decision to live or die

“…48 The dsRNA-activated protein kinase PKR has also been implicated in polyIC-induced NF-kB activation, 49 raising the possibility that dsRNA-induced NF-kB activation may involve PKR and RIP1. Consistent with this model, Jiang et al 50 reported that PKR was recruited to a TRAF6/TAK1/TAB2 complex upon polyIC stimulation. Besides activating NF-kB, this TRAF6/TAK1/TAB2/PKR complex could also activate MKK6 and consequently MAPKs.…”

“…Besides activating NF-kB, this TRAF6/TAK1/TAB2/PKR complex could also activate MKK6 and consequently MAPKs. 50 Another RIP member, RIP2, also contributes to immune responses upon TLR3 and TLR4 activation. 51 TLR3-and TLR4-induced cell death.…”

The caspase-generated fragments of PKR cooperate to activate full-length PKR and inhibit translation

“…5,13,14 Together with TRAF6, TAK1 and TAB2, PKR is also recruited to TLR3 upon binding of dsRNA. 25 As seen before with RIP1, 34 the catalytic activity of PKR is not required for NF-kB activation. 35 Similar to RIP1, PKR can activate NF-kB via interaction with TRAFs and the I k B kinase (IKK) complex.…”

“…Similar to RIP1, PKR was suggested to participate in TNFRI and TLR3 signaling complex formation leading to NF-kB activation. 25,26 We demonstrated recently the formation of a large molecular weight complex, including RIP1 in Hek293T cell lysates upon incubation at 371C. 27 Therefore, we compared the molecular weights of complexes containing either RIP1 or PKR in the Jurkat E cell extracts following incubation for 1 h at 4 or 371C.…”

“…Indeed several reports demonstrated that the kinase is recruited to different complexes including those initiated by ligand-mediated activation of TLR3 and TNFR1. 25,26 In the current report, we studied the fate of PKR in cells during dsRNA-induced apoptosis and necrosis. Our results suggest a new and alternative mode of activation of this protein kinase in apoptosis that may shed light on the mode of PKR activation in cells in general.…”