The p110δ isoform of the kinase PI(3)K controls the subcellular compartmentalization of TLR4 signaling and protects from endotoxic shock

Aksoy, Ezra; Taboubi, Salma; Torres, David; Delbauve, Sandrine; Hachani, Abderrahman; Whitehead, Maria A.; Pearce, Wayne; Berenjeno-Martin, Inma; Nock, Gemma; Filloux, Alain; Beyaert, Rudi; Flamand, Véronique; Vanhaesebroeck, Bart

doi:10.1038/ni.2426

Cited by 170 publications

(234 citation statements)

References 62 publications

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“…Thus, this data supports the requirement of hydrophobic contacts, such as the acyl chains of the lipid, for binding. TIRAP PBM binding to PtdIns(4,5)P 2 -enriched liposomes was with nanomolar affinity and displayed sigmoidal kinetics, resembling the binding traces reported for TIRAP 13 . Similar sets of NMR resonance perturbations of TIRAP PBM were also observed in the presence of PtdIns(3)P, emphasizing the promiscuity of the PI binding site of TIRAP.…”

Section: Discussionsupporting

confidence: 63%

“…Membrane binding of TIRAP is regulated by phosphatidylinositol-5 kinase (PI5K), an enzyme that generates intracellular PtdIns(4,5)P 2 and co-localizes with TIRAP at the plasma membrane 12 . Moreover, TIRAP interacts with PI3Ks, enzymes that convert PtdIns(4,5)P 2 into PtdIns(3,4,5)P 3 , impairing TIRAP's membrane targeting 13 . The activity of TIRAP upon TLR2 and TLR4 activation is tightly regulated by sequential events of phosphorylation and ubiquitination.…”

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confidence: 99%

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Membrane Targeting of TIRAP is Negatively Regulated by Phosphorylation in its Phosphoinositide-Binding Motif

Capelluto

Xiong

Zhao

et al. 2017

Biophysical Journal

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1Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.The innate immune system is composed of germline-encoded receptor proteins that recognize invading pathogens by what is referred to as pathogen associated molecular patterns (PAMPs)1 . Toll-like receptors (TLRs) are the best-studied group of innate immune receptors that are able to recognize various PAMPs, such as bacterial lipopolysaccharides (LPS) or double stranded RNA from viruses 2 . Ligand-activated TLRs self-associate, triggering the recruitment of cytoplasmic adaptor proteins. Through their TIR domains, TLR2 and TLR4 interact with the TIR domain-containing adaptor protein (TIRAP; also known as MAL) and the myeloid differentiation primary response gene 88 (MyD88) [reviewed in ref. 3]. TIRAP contains an N-terminal phosphoinositide (PI)-binding domain (PBD) followed by a TIR domain. Plasma membrane localization of TIRAP depends on the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 )-enriched regions 4 . TIRAP also serves as a bridge to recruit MyD88 through TIR-TIR domain interactions 5 . However, the presence of TIRAP at the plasma membrane is required even when there are low TLR ligand levels 6 . TIRAP's association triggers further recruitment of members of the IRAK family of kinases to promote formation of the myddosome, which in turn activates TRAF6 and NF-κ B nuclear translocation 7 . As a transcription factor, NF-κ B mediates pro-inflammatory and anti-microbial gene expression. The structure of the TIRAP TIR domain reveals two potential dimerization interfaces in a configuration that allows the two monomers of the N-terminal PBD to be oriented in the same direction, facilitating PtdIns(4,5)P 2 -mediated plasma membrane targeting 8,9 . Plasma membrane targeting of TIRAP is likely mediated by a short stretch of basic residues [amino acids 15-35; herein named PI-binding motif (PBM)] within the putative PBD 10 . Alanine mutagenesis of TIRAP residues Lys15, Lys16, Lys31, and Lys32 or hydrolysis of cellular PtdIns(4,5)P 2...

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Section: Discussionsupporting

confidence: 63%

mentioning

confidence: 99%

Membrane Targeting of TIRAP is Negatively Regulated by Phosphorylation in its Phosphoinositide-Binding Motif

Capelluto

Xiong

Zhao

et al. 2017

Biophysical Journal

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“…The small GTPase ADP-ribosylation factor 6 also regulates transport of the TLR4 adaptor TIRAP and TICAM-2 to distinct signaling platforms (33). In addition, multiple molecules such as CD14, MD-2, PI3K, and Raftlin regulate TLR4 internalization (16,17,31), which is important in shifting the TLR4 signaling axis from MyD88 to TICAM-1, leading to expression of IFN-b and IFNstimulated genes including caspase 4/11, a cytoplasmic LPS receptor (34,35). Hence LPS-induced dynamic redistribution and intracellular trafficking of TLR4 and its adaptor molecules control innate immune signaling against microbial infection.…”

Section: Discussionmentioning

confidence: 99%

“…Membrane-bound CD14 mediates the LPS-induced endocytosis of TLR4 and the tyrosine kinase Syk regulates this process (16). Moreover, the p110d isoform of PI3K acts as a regulator of TLR4 endocytosis in dendritic cells (DCs) through modulating phosphatidylinositol-(4, 5)-bisphosphate metabolism (17). However, the precise molecular mechanisms that control the translocation of LPS-TLR4 and spatiotemporal regulation of TLR4-mediated signaling are still unknown.…”

mentioning

confidence: 99%

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type–Specific Manner

Tatematsu

Yoshida

Morioka

et al. 2016

The Journal of Immunology

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The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll–IL-1R domain–containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2–TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1–mediated signaling including IFN-β production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-β promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-β production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein–2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein–2 in clathrin-mediated endocytosis of TLR3/4 ligands.

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“…It has been shown that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and its derivatives are vital for TLR4 signaling (Kagan and Medzhitov, 2006;Aksoy et al, 2012). PI(4,5)P 2 is confined almost exclusively to the inner leaflet of the plasma membrane and is generated mainly by phosphorylation of phosphatidylinositol 4-monophosphate [PI(4)P] catalyzed by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms Iα, Iβ and Iγ (encoded in humans by PIP5K1A, PIP5K1B and PIP5K1C, respectively) (Kunz et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

LPS-induced clustering of CD14 triggers generation of PI(4,5)P2

Płóciennikowska

Zdioruk

Traczyk

et al. 2015

Journal of Cell Science

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Bacterial lipopolysaccharide (LPS) induces strong pro-inflammatory reactions after sequential binding to CD14 protein and TLR4 receptor. Here, we show that CD14 controls generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] in response to LPS binding. In J774 cells and HEK293 cells expressing CD14 exposed to 10-100 ng/ml LPS, the level of PI(4,5)P 2 rose in a biphasic manner with peaks at 5-10 min and 60 min. After 5-10 min of LPS stimulation, CD14 underwent prominent clustering in the plasma membrane, accompanied by accumulation of PI(4,5)P 2 and type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms Iα and Iγ (encoded by Pip5k1a and Pip5k1c, respectively) in the CD14 region. Clustering of CD14 with antibodies, without LPS and TLR4 participation, was sufficient to trigger PI(4,5)P 2 elevation. The newly generated PI(4,5)P 2 accumulated in rafts, which also accommodated CD14 and a large portion of PIP5K Iα and PIP5K Iγ. Silencing of PIP5K Iα and PIP5K Iγ, or application of drugs interfering with PI(4,5)P 2 synthesis and availability, abolished the LPS-induced PI(4,5)P 2 elevation and inhibited downstream pro-inflammatory reactions. Taken together, these data indicate that LPS induces clustering of CD14, which triggers PI(4,5)P 2 generation in rafts that is required for maximal pro-inflammatory signaling of TLR4.

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The p110δ isoform of the kinase PI(3)K controls the subcellular compartmentalization of TLR4 signaling and protects from endotoxic shock

Cited by 170 publications

References 62 publications

Membrane Targeting of TIRAP is Negatively Regulated by Phosphorylation in its Phosphoinositide-Binding Motif

Membrane Targeting of TIRAP is Negatively Regulated by Phosphorylation in its Phosphoinositide-Binding Motif

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type–Specific Manner

LPS-induced clustering of CD14 triggers generation of PI(4,5)P2

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