The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists

Abstract

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).

Tyrosine phosphorylation in Toll-like receptor signaling

“…Human TLR8 has been shown to be tyrosine phosphorylated upon stimulation by an immunomodulator, 3M-003 (69). Mutational analyses revealed that among thirteen tyrosine residues in the cytoplasmic domain of TLR8, Tyr898, Tyr904 and Tyr1048 are critical for NF-κB activation.…”

Toll-like receptors, signaling adapters and regulation of the pro-inflammatory response by PI3K

“…74 Similarly, TLR3 and TLR8 had phosphorylated tyrosine residues upon ligand engagement, which led to the recruitment of PI3K-p85 subunit. 75,76 However, these responses to TLR4 stimulation, including enhanced production of Tnf, Il6 and Ccl3 (also known as MIP1α). 29 Mechanistically, the authors found that signals provided by Akt1 were critical for induction of several miRNA species, including miRNA-155.…”

TLR8-dependent TNF-α overexpression in Fanconi anemia group C cells

“…on May 9, 2018. by guest www.bloodjournal.org From Whether the major increase in TLR8 in antiubiquitin antibody immunoprecipitates reflects excessive ubiquitinylation of TLR8 or a TLR8 binding protein is not yet solved, but there is precedent for direct ubiquitinylation of TLR8 in response to the TLR7/8 agonist 3M-003. 41 However, the design of these previous studies involved overexpression of TLR8 in HEK-293 cells and detection of peptides by mass spectrometry. No ubiquitinylation sites or differential biologic function studies were reported by this group.…”