Bruton's Tyrosine Kinase Is a Toll/Interleukin-1 Receptor Domain-binding Protein That Participates in Nuclear Factor κB Activation by Toll-like Receptor 4

Caroline A Jefferies,Sarah Doyle,Cornelia Brunner,Aisling Dunne,Elizabeth Brint,Claudia Wietek,Eva Walch,Thomas Wirth,Luke A.J O'Neill

doi:10.1074/jbc.m301484200

Caroline A Jefferies, Sarah Doyle + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m301484200

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Abstract

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.

Highlights

The Tec family of protein tyrosine kinases, of which Bruton’s tyrosine kinase (Btk)1 is a prototypical member, is involved in a vast array of signaling pathways in cells of hematopoietic lineage
Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction
We investigated the involvement of Btk in LPS signaling via TLR4 and found that LPS activated Btk and that inactive mutants of Btk inhibited LPS signaling to nuclear factor ␬B (NF␬B) activation

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screening—Full-length Btk was cloned into pPCH1 [19] and used as a bait. A total of 2 ϫ 107 cells were used per point and stimulated with LPS (1 ␮g/ml) for the time points indicated, washed twice in ice-cold phosphate-buffered saline, and lysed in 1 ml of lysis buffer (150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM Na3VO4, 2 ␮g/ml aprotonin, and 1 ␮g/mlϪ1 leupeptin) as described above. Endogenous Btk-containing immunocomplexes were obtained and either blotted for tyrosine phosphorylation using monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) or incubated with [␥-32P]ATP (2 ␮Ci/sample) in kinase buffer (20 mM HEPES, 2 mM dithiothreitol, 10 mM MgCl2, 100 ␮M Na3VO4, 20 mM ␤-glycerol phosphate, and 20 ␮M ATP) for 30 min at 37 °C, washed with 1 ml of lysis buffer, and analyzed by SDS-PAGE. DNAprotein complexes were resolved on native (5%) polyacrylamide gels that were subsequently dried and autoradiographed

RESULTS

Btk Is a Transducer of TLR Signaling

DISCUSSION