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BANK1 interacts with TRAF6 and MyD88 in innate immune signaling in B cells

Abstract

Evidence supports a possible role of BANK1 in innate immune signaling in B cells. In the present study, we investigated the interaction of BANK1 with two key mediators in interferon and inflammatory cytokine production, TRAF6 and MyD88. We revealed by coimmunoprecipitation (CoIP) analyses the binding of BANK1 with TRAF6 and MyD88, which were mediated by the BANK1 Toll/interleukin-1 receptor (TIR) domain. In addition, the natural BANK1–40C variant showed increased binding to MyD88. Next, we demonstrated in mouse splenic B cells that BANK1 colocalized with Toll-like receptor (TLR) 7 and TLR9 and that after stimulation with TLR7 and TLR9 agonists, the number of double-positive BANK1–TLR7, –TLR9, –TRAF6, and –MyD88 cells increased. Furthermore, we identified five TRAF6-binding motifs (BMs) in BANK1 and confirmed by point mutations and decoy peptide experiments that the C-terminal domain of BANK1-full-length (-FL) and the N-terminal domain of BANK1–Delta2 (-D2) are necessary for this binding. Functionally, we determined that the absence of the TIR domain in BANK1–D2 is important for its lysine (K)63-linked polyubiquitination and its ability to produce interleukin (IL)-8. Overall, our study describes a specific function of BANK1 in MyD88–TRAF6 innate immune signaling in B cells, clarifies functional differences between the two BANK1 isoforms and explains for the first time a functional link between autoimmune phenotypes including SLE and the naturally occurring BANK1–40C variant.

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Acknowledgements

We wish to thank the Unit of Microscopy and Flow Cytometry of GENYO for their constant help and support during experiments. This work was supported by the MINECO and cofinanced by the FEDER funds of the European Union [SAF2016-78631-P entitled “The Role of BANK1 in B Cell signaling through TLRs and autoimmunity”]; by the Swedish Research Council of Medicine to M.E.A.R.; by the Instituto de Salud Carlos III grant to I.G. [CD11/00277]; by the Proyecto de Excelencia, Consejería de Economía, Innovación y Ciencia of Andalucía [CTS-2548]; and by the Fundación Ramón Areces.

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I.G. designed, analyzed, and provided overall guidance for the experiments and wrote the manuscript. A.D.B. designed, performed, and analyzed the experiments and wrote the manuscript. M.M. assisted with and performed experiments that involved mice. A.L.P. helped design the decoy peptide experiments. M.E.A.R. proposed the project, supervised the performance of the experiments and their analyses, revised the manuscript, and led the project.

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Correspondence to Ina Georg or Alejandro Díaz-Barreiro.

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Georg, I., Díaz-Barreiro, A., Morell, M. et al. BANK1 interacts with TRAF6 and MyD88 in innate immune signaling in B cells. Cell Mol Immunol 17, 954–965 (2020). https://doi.org/10.1038/s41423-019-0254-9

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