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 3

USP16 promotes the deubiquitination of CNA.

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USP16 promotes the deubiquitination of CNA. (A–C) HEK293T cells were tra...
(A–C) HEK293T cells were transfected with HA-tagged ubiquitin along with the HA-tagged USP16-WT or USP16-CI expression plasmids. 3CA, 3CB, and 3CC were isolated by IP with anti-FLAG antibody under denaturing conditions, and followed by the detection of ubiquitin levels with anti-HA. The cell lysates were also subjected to direct IB analyses (bottom 3 panels). (D) In vitro deubiquitination assays were performed to detect the USP16-mediated deubiquitination of 3CB. In vitro translated WT or catalytically inactive USP16 was mixed with ubiquitinated 3CB, which was isolated from transfected HEK293T cells. Deubiquitination of 3CB was detected by direct IB of HA-tagged ubiquitin. (E) HEK293T cells were transfected with multiple HA-tagged ubiquitin mutants, including K6, K11, K48, K63, K29 and K33, along with the indicated expression plasmids. FLAG-tagged 3CB was isolated by IP, and followed by IB detection of ubiquitin. (F) IB analyses of the indicated proteins in WL of CD4+ T cells from WT and USP16-KO mice stimulated with αCD3/αCD28. (G) IB analyses of TCR-triggered deubiquitination of CNA in WT and USP16-deficient CD4+ T cells. CNA was isolated by IP and followed by IB detection of ubiquitin. (H) USP16-deficient CD4+ T cells were reconstituted with USP16-WT or USP16-CI using nucleofection. After stimulation with αCD3/αCD28 as indicated, CNA in these reconstituted T cells was isolated by IP and followed by IB detection of ubiquitin. Immunoprecipitated CNA in panels G and H was detected by Trueblot. Data are representative of 3 independent experiments with 3 mice in each group (F–H) and 3 independent experiments (A–E).