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

Area of Science:

• Cellular Biology
• Molecular Biology
• Immunology

Background:

• Nuclear factor κB (NF-κB) signaling regulates critical cellular functions.
• The IκB kinase (IKK) complex integrates signals for NF-κB activation.
• RNF32 is an E3 ubiquitin ligase found in intestinal stem cells.

Purpose of the Study:

• To investigate the role of RNF32 in NF-κB signaling.
• To elucidate the mechanism by which RNF32 regulates IKK complex activity.
• To understand the influence of calcium ions on RNF32 function.

Main Methods:

• Investigated RNF32 expression in murine intestinal stem cells.
• Assessed the dependence of RNF32 E3 ligase activity on calmodulin.
• Analyzed the effect of intracellular calcium ion (Ca2+) levels on RNF32 binding, activation, and autoubiquitylation.
• Examined the recruitment of NEMO to RNF32 polyubiquitin chains.
• Studied Ca2+-triggered RNF32 phase separation and its role in NEMO condensate formation and IKK activation.
• Assessed RNF32's requirement for NF-κB activation by bacterial lipopolysaccharides.

Main Results:

• RNF32 regulates IKK complex activity, a key hub for NF-κB activation.
• RNF32's E3 ligase activity is dependent on calmodulin and activated by increased intracellular Ca2+.
• Ca2+ induces RNF32 binding to calmodulin, activation, and autoubiquitylation.
• Polyubiquitin chains on RNF32 recruit NEMO, a subunit of the IKK complex.
• Ca2+ triggers RNF32 phase separation, essential for NEMO condensate formation and IKK activation.
• RNF32 is necessary for NF-κB activation induced by bacterial lipopolysaccharides.

Conclusions:

• RNF32 acts as a crucial regulator of NF-κB signaling in the intestinal epithelium.
• Calcium signaling modulates NF-κB activation through the RNF32-calmodulin-NEMO axis.
• This study uncovers a novel mechanism controlling NF-κB signaling in response to cellular stimuli.