Dok3–protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity
Jia Tong Loh, … , Yue Wang, Kong-Peng Lam
Jia Tong Loh, … , Yue Wang, Kong-Peng Lam
Published June 10, 2019
Citation Information: J Clin Invest. 2019;129(7):2717-2729. https://doi.org/10.1172/JCI126341.
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Research Article Immunology Infectious disease

Dok3–protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity

Abstract

Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.

Authors

Jia Tong Loh, Shengli Xu, Jian Xin Huo, Susana Soo-Yeon Kim, Yue Wang, Kong-Peng Lam

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Figure 5

Dok3 mediates Card9 dephosphorylation through PP1 recruitment.

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Dok3 mediates Card9 dephosphorylation through PP1 recruitment. (A) Purif...
(A) Purified Dok3+/+ and Dok3–/– neutrophils were treated with zymosan (10 μg/ml) for 30 minutes. Whole cell lysates (WCL) were IP with a Card9-specific antibody, and precipitates were probed for p-Thr and Card9. Whole cell lysates were also probed for Card9 and β-actin. (B) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a Dok3-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, PP1, PP2A, and Dok3. (C) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated for 30 minutes with zymosan (10 μg/ml), and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Dok3, PP1, PP2A, and Card9. (D) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a PP1-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, Dok3, and PP1. (E) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for indicated periods of time and IP with a Dok3-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, PP1, and Dok3. (F) Co-IP analysis of cell lysates from purified Dok3+/+ and Dok3–/– neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for PP1, Dok3, and Card9. (G) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for PP1α, PP1β, PP1γ, Dok3, and Card9. All images are representative of 3 independent experiments.