Coupling of a Signal Response Domain in IκBα to Multiple Pathways for NF-κB Activation

TLDR

NF‑κB is a key transcription factor for HIV‑1 expression and T‑cell activation, and its nuclear activity is tightly controlled by the cytoplasmic inhibitor IκBα, which is rapidly phosphorylated and degraded in response to diverse stimuli. The authors generated IκBα mutants that separate its inhibitory role from signal transduction to investigate NF‑κB induction mechanisms in human T lymphocytes. Deletion of the N‑terminal 36 residues or alanine substitutions at two serines in IκBα blocked its Tax‑induced phosphorylation, degradation, and release, whereas phosphoserine mimetics restored turnover, showing that this domain is essential for coordinating IκBα stability and NF‑κB activation by viral and immune signals.

Abstract

The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli.