IKKϵ negatively regulates RIG-I via direct phosphorylation.

Area of Science:

• Immunology
• Molecular Biology
• Cellular Signaling

Background:

• Inhibitor of nuclear factor kappa-B kinase Epsilon (IKKϵ) is an IKK-related kinase.
• IKKϵ is dispensable for interferon induction by viral infection, despite its relation to TBK-1.
• The precise role of IKKϵ in innate immune signaling remains incompletely understood.

Purpose of the Study:

• To investigate the direct role of IKKϵ in regulating RIG-I-mediated innate immune signaling.
• To identify the specific mechanism by which IKKϵ modulates cytosolic sensor activation.
• To elucidate the feedback regulatory role of IKKϵ in innate immunity.

Main Methods:

• Utilized IKKϵ-deficient mouse embryonic fibroblasts (MEFs) to assess cytokine production.
• Performed biochemical analyses to confirm physical association and phosphorylation of RIG-I by IKKϵ.
• Employed mass spectrometry to identify the specific phosphorylation site on RIG-I.

Main Results:

• Loss of IKKϵ in MEFs led to increased cytokine production upon activation of cytosolic sensors.
• IKKϵ was found to physically associate with and phosphorylate RIG-I.
• Mass spectrometry identified serine 855 within the RIG-I RNA-binding pocket as the phosphorylation site targeted by IKKϵ.

Conclusions:

• IKKϵ directly phosphorylates serine 855 of RIG-I, inhibiting its RNA-binding and downstream signaling.
• IKKϵ acts as a negative regulator of RIG-I-mediated innate immune responses.
• This phosphorylation represents a crucial feedback mechanism controlling innate immunity.