Identification of cGAS acetylation and phosphorylation

DNA derived from the genetic material of pathogens or from cellular DNA damage provides a molecular pattern that can be sensed by pattern-recognition receptors of the mammalian innate immune system. In recent years, the cyclic GMP-AMP synthase (cGAS) protein has been characterized as a primary cytosolic DNA sensor during infection with bacteria, DNA viruses, or retroviruses. While the role of cGAS in downstream immune signaling through STING-TBK1-IRF3 proteins is well-defined, regulatory mechanisms of cGAS activity, such as through post-translational modifications (PTMs), are still an active area of research. Here, we report a comprehensive characterization of cGAS phosphorylations and acetylations in three different cell types. Data-dependent proteomic analyses of immunoaffinity purified cGAS was performed in HEK293T cells under control and DNA-challenged conditions (N = 3 each) and human fibroblasts (HFF) cells under control and HSV-1 infected conditions (N = 4 each) to generate candidate cGAS PTM sites. Using parallel reaction monitoring, a total of 11 PTMs (4 phosphorylations and 7 acetylations) were validated in HEK293T, HFF, and THP-1 cells. Of these, 3 phosphorylations and 5 acetylations have not been previously identified. The functions of these modifications were by generating a series of mutants and measuring cGAS-dependent apoptotic and immune signaling activities.