Phosphoinositide-dependent kinase-1 inhibits TRAF6 ubiquitination by interrupting the formation of TAK1-TAB2 complex in TLR4 signaling.

Phosphoinositide-dependent protein kinase 1 (PDK1) plays a key role in the phosphoinositide 3-kinase (PI3K)-PDK1-Akt pathway that induces cell survival and cardiovascular protections through anti-apoptosis, vasodilation, anti-inflammation, and anti-oxidative stress activities. Although several reports have proposed the negative role of PDK1 in Toll-like receptor 4 (TLR4) signaling, the molecular mechanism is still unknown. Here we show that PDK1 inhibits tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) ubiquitination by interrupting the complex between transforming growth factor beta-activated kinase 1 (TAK1) and TAK1 binding protein 2 (TAB2), which negatively regulates TAK1 activity. The overexpression of PDK1 in 293/TLR4 cells resulted in suppressions of nuclear factor kappa B (NF-κB) activation and production of proinflammatory cytokines including interleukin (IL)-6 and TNF-α in response to lipopolysaccharide stimulation. Conversely, THP-1 human monocytes transiently cultured in low glucose medium displayed down-regulated PDK1 expression, and significantly enhanced TLR4-mediated signaling for the activation of NF-κB, demonstrating a negative role of PDK1. Biochemical studies revealed that PDK1 significantly interacted with TAK1, resulting in the inhibition of the association of TAB2 with TAK1, which led to the attenuation of TRAF6 ubiquitination. Moreover, PDK1-knockdown THP-1 cells displayed enhancement of downstream signals, activation of NF-κB, and increased production of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α, which potentially led to the up-regulation of NF-κB-dependent genes in response to TLR4 stimulation. Collectively, the results demonstrate that PDK1 inhibits the formation of the TAK1-TAB2-TRAF6 complex and leads to the inhibition of TRAF6 ubiquitination, which negatively regulates the TLR4-mediated signaling for NF-κB activation.

TAK1-ECSIT-TRAF6 complex plays a key role in the TLR4 signal to activate NF-κB.

ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different signals, including Toll-like receptors (TLRs), TGF-ß, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSIT(KD) THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1(KD) or TRAF6(KD) THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling. Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated signals to activate NF-κB.

S6K1 negatively regulates TAK1 activity in the toll-like receptor signaling pathway.

Transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1(-/-) and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1(-/-) mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.