USP16-mediated deubiquitination of calcineurin A controls peripheral T cell maintenance
Yu Zhang, … , Yi-yuan Li, Jin Jin
Yu Zhang, … , Yi-yuan Li, Jin Jin
Published May 28, 2019
Citation Information: J Clin Invest. 2019;129(7):2856-2871. https://doi.org/10.1172/JCI123801.
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Research Article Cell biology Immunology

USP16-mediated deubiquitination of calcineurin A controls peripheral T cell maintenance

Abstract

Calcineurin acts as a calcium-activated phosphatase that dephosphorylates various substrates, including members of the nuclear factor of activated T cells (NFAT) family, to trigger their nuclear translocation and transcriptional activity. However, the detailed mechanism regulating the recruitment of NFATs to calcineurin remains poorly understood. Here, we report that calcineurin A (CNA), encoded by PPP3CB or PPP3CC, is constitutively ubiquitinated on lysine 327, and this polyubiquitin chain is rapidly removed by ubiquitin carboxyl-terminal hydrolase 16 (USP16) in response to intracellular calcium stimulation. The K29-linked ubiquitination of CNA impairs NFAT recruitment and transcription of NFAT-targeted genes. USP16 deficiency prevents calcium-triggered deubiquitination of CNA in a manner consistent with defective maintenance and proliferation of peripheral T cells. T cell–specific USP16 knockout mice exhibit reduced severity of experimental autoimmune encephalitis and inflammatory bowel disease. Our data reveal the physiological function of CNA ubiquitination and its deubiquitinase USP16 in peripheral T cells. Notably, our results highlight a critical mechanism for the regulation of calcineurin activity and a novel immunosuppressive drug target for the treatment of autoimmune diseases.

Authors

Yu Zhang, Rong-bei Liu, Qian Cao, Ke-qi Fan, Ling-jie Huang, Jian-shuai Yu, Zheng-jun Gao, Tao Huang, Jiang-yan Zhong, Xin-tao Mao, Fei Wang, Peng Xiao, Yuan Zhao, Xin-hua Feng, Yi-yuan Li, Jin Jin

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Figure 1

T cell activation is associated with the deubiquitination of calcineurin A.

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T cell activation is associated with the deubiquitination of calcineurin...
(A) IB analysis of the deubiquitination of calcineurin A (CNA) in mouse splenic CD4+ T cells upon αCD3/αCD28 or PMA/ionomycin (P/I) stimulation. Whole-cell lysate (WL) was subjected to IP using anti-CNA or rabbit IgG control as nonspecific control (NC), followed by IB analysis of the ubiquitination (Ub) level. (B) Human CD4+ T cells isolated from the peripheral blood of healthy donors were activated with P/I. The same Ub assay was performed as described in A. Immunoprecipitated CNA was detected by Trueblot. (C) Calcineurin is composed of calcineurin B (CNB) and CNA, and the latter is encoded by PPP3CA, PPP3CB, or PPP3CC. (D) HEK293T cells were transfected with a distinct truncation mutant of CNA encoded by PPP3CB (3CB) along with HA-tagged Ub. WLs were subjected to IP using anti-FLAG antibody followed by Ub analysis. The WLs were also subjected to direct IBs (bottom 2 panels). (E) Schematic representation of the different domains of human 3CB. (F) Wild-type 3CB (3CB-W) and Ub site mutants were transfected into HEK293T cells. Whole-cell lysates were subjected to IP using anti-FLAG followed by Ub analysis. (G) Comparison of the amino acid sequences around potential Ub sites on human CNA catalytic subunit. (H) Sequence alignment of Ub sites on CNA orthologs of different species. (I) Crystal structure of the CNA: NFAT2 PxIxIT complex. The potential Ub sites are shown in yellow. (J) Interaction assay of in vitro translational NFAT2 with the ubiquitinated catalytic domain (CD) of WT or mutant 3CB, which was isolated from transfected HEK293T cells. Data are representative of 4 independent experiments with 4 mice in each group (A, B), 4 experiments (D, F), and 3 experiments (J).