Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells

JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY

卷:142

Article

Kim, Do-Kyun¹  Beaven, Michael A.²  Metcalfe, Dean D.¹  Olivera, Ana¹ 

[1] NIAID, Mast Cell Biol Sect, Lab Allerg Dis, Bethesda, MD 20892 USA

[2] NHLBI, Lab Mol Immunol, NIH, Bldg 10, Bethesda, MD 20892 USA

关键词: Human mast cells;    IgE receptor;    Lyn activation;    DJ-1;    signaling;    reactive oxygen species;    spleen tyrosine kinase;   

DOI  :  10.1016/j.jaci.2017.08.030

来源: Elsevier

【 摘 要 】

Background: DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown. Objective: We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation. Methods: DJ-1 was silenced in human CD34(+)-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of beta-hexosaminidase, prostaglandin D-2, and GM-CSF and changes in reactive oxygen species levels were measured after Fc epsilon RI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies. Results: DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following Fc epsilon RI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound toLyn but not to other Src kinases, and this interactionwas specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling. Conclusion: We demonstrate a novel role for DJ-1 in the early activation of Lyn by Fc epsilon RI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

【 授权许可】

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