Biochem. J. (2008) 409, 275–287 (Printed in Great Britain) doi:10.1042/BJ20070519 275 The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists Raj RAJAGOPAL, Andrew S. WALLER, James D. MENDOZA and Paul D. WIGHTMAN 1 Department of Pharmacology, 3M Pharmaceuticals, 3M Center, St. Paul, MN 55144, U.S.A. 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- κ B (nuclear factor κ B)/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, Tyr 898 and Tyr 904 , reside in the Box 1 motif and the third, Tyr 1048 , 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). Key words: covalent modification, immune response modifier, Toll/interleukin-1 receptor (TIR) domain, Toll-like receptor (TLR), tyrosine phosphorylation, ubiquitination. INTRODUCTION The survival of vertebrate species is dependent in part on their ability to mount a rapid defence to the sea of potential pathogens to which they are exposed. One evolutionary survival strategy has been for host species to reduce the complexity of the microbial world to recognition of a series of far more simple molecular patterns, which are recapitulated across multiple types of pathogens. This process of reductive defence is mediated through a family of receptors for such PAMPs (pathogen- associated molecular patterns) [1]. The mammalian TLRs (Toll-like receptors), homologues of the Drosophila Toll receptor, are among the PAMP receptors of the innate immune system. The ten different hTLRs (human TLRs) (TLR1–10) identified are type I transmembrane proteins. They are composed of an N-terminal extracellular domain with leucine-rich repeats involved in PAMP recognition, a single trans- membrane domain, and a cytoplasmic domain largely made up of the TIR (Toll/interleukin-1 receptor) homology domain required for downstream signalling. TLR1, TLR2, TLR4, TLR5 and TLR6 are involved in recognizing PAMPs associated with bacterial membranes. TLR3, TLR7, TLR8 and TLR9 recognize oligonucleotide (RNA and DNA)-based molecular patterns from both bacteria and viruses. TLR7 and TLR8 also respond to 3M Pharmaceuticals imidazoquinoline IRMs (immune-response modifiers). The role of TLR10 has not been established and its corresponding PAMP has not been identified [1]. Much has been learned of the signalling pathways for the TLRs and the understanding of these pathways has been broadened beyond the initial assumptions that the TLRs shared a common signalling pathway, both with the IL-1R [IL-1 (inter- leukin-1) receptor] and with each other. It is now appreciated that there exists a great deal of heterogeneity among the indivi- dual TLR signalling pathways [2]. This heterogeneity includes the spectrum of adaptor proteins and downstream effector proteins utilized to access either the pathways to pro-inflammatory cytokine or those to type 1 IFN (interferon) (IFNαβ ) synthesis. Whereas the TLR3 and TLR4 type 1 IFN synthetic pathways are TRIF (TIR domain-containing adaptor protein inducing IFNβ )-dependent [TLR4 requires TRAM (TRIF-related adaptor molecule) as well], TLR7- and TLR9-stimulated IFNα is, instead, MyD88 (myeloid differentiation factor 88)-dependent. While TLR3 and TLR4 stimulation of type 1 IFN is mediated through a TBK1 [TANK (tumour-necrosis-factor-receptor-associated factor-associated nuclear factor κ B activator)-binding kinase 1]– IKKi {Iκ B [inhibitor of NF-κ B (nuclear factor κ B)] kinase i}– IRF3 (IFN regulatory factor-3) effector pathway, TLR7 and TLR9 stimulation of IFNα occurs through an IRAK1 Abbreviations used: Akt, thymoma viral proto-oncogene; ERK, extracellular-signal-regulated kinase; HA, haemagglutinin; HEK-293 cells, human embryonic kidney cells; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; TLR, Toll-like receptor; hTLR, human TLR; IFN, interferon; NF-κB, nuclear factor κB; IκB, inhibitor of NF-κB; IKK, IκB kinase; IL-1, interleukin-1; IL-1R, IL-1 receptor; IL-1RAP1, IL-1R accessory protein 1; IRAK, IL-1R-associated kinase; IRF, IFN regulatory factor; IRM, immune-response modifier; ITIM, i mmunoreceptor t yrosine-based i nhibitory m otif; JNK, c-Jun N-terminal kinase; LC, liquid chromatography; MAPK, mitogen-activated protein kinase; MEKK, MAPK/ERK kinase kinase; MS/MS, tandem MS; MyD88, myeloid differentiation factor 88; nDHII, nuclear DNA helicase II; NFAT, nuclear factor of activated T-cells; PAMP, pathogen-associated molecular pattern; PBMC, peripheral blood mononuclear cell; PI3K, phosphoinositide 3-kinase; PTK, protein tyrosine kinase; RhoGAP, Rho GTPase-activating protein; RT, reverse transcriptase; SH domain, Src homology domain; SIGIRR, single immunoglobulin domain-containing IL-1R-related protein; ssRNA, single-stranded RNA; ST2, suppressor of tumorigenicity 2; TAK1, TGF (transforming growth factor)-β-activated kinase 1; TIR, Toll/IL-R; TNF, tumour necrosis factor; TRAF, TNF-receptor-associated factor; TRIF, TIR domain-containing adaptor protein inducing IFNβ; UIM, ubiquitin interaction motif; wt, wild-type. 1 To whom correspondence should be addressed (email pdwightman@mmm.com). c  The Authors Journal compilation c  2008 Biochemical Society