Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, 443-721, South Korea; Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, 443-721, South Korea.
Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, 443-721, South Korea; Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, 443-721, South Korea.
Biochem Biophys Res Commun. 2023 Sep 17;673:1-8. doi: 10.1016/j.bbrc.2023.06.066. Epub 2023 Jun 19.
Cyclic GMP-AMP synthase (cGAS), which recognizes double-stranded DNA (dsDNA) and activates the innate immune system, is mainly localized in the cytosol, but also shows nuclear localization. Here, we sought to determine the role of nuclear cGAS by mutating known nuclear localization signal (NLS) motifs in cGAS and assessing its functionality by monitoring phosphorylation of the downstream target, interferon regulatory factor-3 (IRF3). Interestingly, NLS2-mutated cGAS failed to promote phosphorylation of IRF3, reflecting the loss of its ability to produce cyclic GMP-AMP (cGAMP). We further found that insertion of an NLS from SV40 large T antigen could not restore this loss of activity, indicating that this loss was attributable to the mutation of NLS2 itself, but not dependent on the inability of cGAS to enter the nucleus. NLS2-mutant cGAS protein also showed decreased stability dependent on polyubiquitination, an effect that was independent of both its loss of catalytic function and its inability to enter into the nucleus. Collectively, these findings indicate that the NLS2 motif of cGAS is not only involved in regulating the subcellular localization of cGAS protein but also influences its stability and enzymatic activity through independent mechanisms, highlighting the novel roles of NLS2 in regulating the intracellular functions of cGAS.
环鸟苷酸-腺苷酸合酶 (cGAS) 识别双链 DNA (dsDNA) 并激活先天免疫系统，主要定位于细胞质，但也显示核定位。在这里，我们通过突变 cGAS 中已知的核定位信号 (NLS) 基序来确定核 cGAS 的作用，并通过监测下游靶标干扰素调节因子 3 (IRF3) 的磷酸化来评估其功能。有趣的是，NLS2 突变的 cGAS 未能促进 IRF3 的磷酸化，反映出其产生环鸟苷酸-腺苷酸 (cGAMP) 的能力丧失。我们进一步发现，插入来自 SV40 大 T 抗原的 NLS 不能恢复这种活性丧失，表明这种丧失归因于 NLS2 本身的突变，而不是 cGAS 无法进入细胞核的原因。NLS2 突变的 cGAS 蛋白也表现出依赖多泛素化的稳定性降低，这种效应既不依赖于其催化功能的丧失，也不依赖于其无法进入细胞核。总之，这些发现表明 cGAS 的 NLS2 基序不仅参与调节 cGAS 蛋白的亚细胞定位，而且通过独立的机制影响其稳定性和酶活性，突出了 NLS2 在调节 cGAS 细胞内功能方面的新作用。
