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Epac1 Regulates Cellular SUMOylation by Promoting The Formation of SUMO‐activating Nuclear Condensates

Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation remains elusive. We hypothesized that cAMP, a major stress‐response second messenger, acts through exchange protein directly activated by cAMP (Epac1) to regulate cellular SUMOylation. Proteomic analysis reveals that Epac1 associated proteome is highly‐enriched with major components of the SUMOylation machinery and well‐known SUMO target proteins. Activation of Epac1 by intracellular cAMP in Human Umbilical Vein Endothelial Cells (HUVECs) promotes cellular SUMOylation and activates cellular SUMO‐activating enzyme E1 (AOS1/UBA2). Conversely, silencing Epac1 leads to a reduced cellular SUMOylation and SUMO E1 activation. However, in vitro SUMOylation assay using purified recombinant components of SUMOylation machinery shows that Epac1 does not directly activate SUMO E1 and SUMO‐conjugating enzyme E2 (UBC9). Moreover, Epac1‐mediated cellular SUMOylation is independent on its guanine nucleotide exchange activity, suggesting that Epac1 promotes cellular SUMOylation through an unconventional mechanism. Co‐immunofluorescence staining and Structured Illumination Microscopy (SIM) super‐resolution imaging analyses reveal that Epac1 activation promotes the formation of Epac1 nuclear condensates that co‐localize with nuclear UBA2/UBC9/SUMO2/3. The discovery of Epac1‐based biomolecular condensates is consistent with the facts that Epac1 contains intrinsically disordered regions (IDRs) and undergoes salt‐ and cAMP‐dependent phase separation (PS). Importantly, the formation of Epac1 nuclear condensates is required for Epac1‐mediated cellular SUMOylation, as a Δ (1‐148) Epac1 mutant incapable of PS and forming nuclear condensates fails to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low‐density lipoprotein (oxLDL) induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. Our findings represent a major conceptual advance in our understanding of cell stress responses by providing a direct connection between protein SUMOylation and cAMP/Epac1 signaling pathway, two major cellular stress processes. The ability of cAMP to directly modulate the dynamics of cellular condensate provides the first experimental evidence that protein phase separation can be regulated by an endogenous ligand.