Multi-functional regulation of cGAS by the nuclear localization signal2 (NLS2) motif: Nuclear localization, enzyme activity and protein degradation.

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.