The E3 ligase RNF32 controls the IκB kinase complex and NF-κB signaling in intestinal stem cells

Nuclear factor κB (NF-κB) signaling is a central pathway regulating a plethora of cellular functions. Here, we find that RNF32, a RING E3 ubiquitin ligase whose expression is enriched in murine intestinal stem cells, regulates the activity of the IκB kinase (IKK) complex, the signal integration hub for NF-κB activation. The E3 ligase activity of RNF32 depends on calmodulin, the primary calcium sensor in eukaryotic cells. Increased levels of intracellular calcium ion (Ca 2+ ) induce RNF32 binding to calmodulin, RNF32 activation, and autoubiquitylation. In turn, polyubiquitin chains conjugated to RNF32 recruit NEMO, the regulatory subunit of the IKK complex. Moreover, Ca 2+ rise triggers RNF32 phase separation, which is required for the formation of NEMO condensates and IKK activation. Finally, we show that RNF32 is required for NF-κB activation triggered by bacterial lipopolysaccharides. Collectively, our findings uncover a mechanism controlling NF-κB signaling in the intestinal epithelium. • RNF32 is a Ca 2+ -calmodulin-dependent E3 ubiquitin ligase • Polyubiquitin chains conjugated to RNF32 recruit and activate the IκB kinase complex • Ca 2+ rise triggers RNF32 phase separation needed for the formation of NEMO condensates • RNF32 regulates the NF-κB signaling pathway in intestinal stem cells Lauriola, Enriqué Steinberg, et al. report that RNF32, a ubiquitin ligase enriched in intestinal stem cells, links calcium levels to NF-κB signaling by activating the IKK complex. This study uncovers a calcium-dependent mechanism involved in responses to bacterial stimuli, revealing how intestinal cells integrate signaling cues to regulate intestinal homeostasis.