Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419, Republic of Korea.
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin, 17035, Republic of Korea.
Biochem Biophys Res Commun. 2019 May 21;513(1):172-178. doi: 10.1016/j.bbrc.2019.03.171. Epub 2019 Apr 2.
The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.
NFAT 转录因子家族在免疫系统的发育和功能中发挥着重要作用。NFATc1 和 NFATc2 在周围 T 细胞中高度表达，并且通过使用不同的启动子和多聚腺苷酸化位点产生几种同工型。具有相对较长 C 末端的特定同工型，NFATc1/C 和 NFATc2/A，已被证明在其特定的 C 末端区域被 SUMO 修饰，这调节 NFAT 蛋白的定位和反式激活活性。在这里，我们证明了一种同工型 NFATc1/A，它具有短的 C 末端并且不包含在长同工型中发现的 SUMO 化位点，也被 SUMO 修饰。在共转染的细胞中，低水平表达 SUMO E3 连接酶，特别是 PIAS1、PIAS3 和 PIASy 时，NFATc1/A 的 SUMO 化增加。PIAS3 与 NFATc1/A 相互作用，并且活性位点突变体不能促进 NFATc1/A 的 SUMO 化，表明 PIAS3 作为 SUMO E3 连接酶的作用。在中央调节域内的 351 个赖氨酸残基被鉴定为在共转染和体外测定中的主要 SUMO 附着位点。NFATc1/A 的 SUMO 化在离子霉素和佛波醇 12-肉豆蔻酸 13-醋酸酯处理时不会影响核易位。然而，尽管 NFATc1/A 的 SUMO 化略微增加了蛋白质稳定性，但它抑制了由包含 NFAT 位点的启动子驱动的报告基因的反式激活活性。我们的结果表明，NFATc1/A 的反式激活活性受 PIAS 蛋白介导的 SUMO 化负调节，并且 SUMO 是具有长或短 C 末端的 NFAT 家族成员的通用调节剂。
