Timothy A. McKinsey

Members of the NF-kappaB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappaB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappaB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappaB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IkappaB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappaB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappaB via an IkappaB targeting mechanism. Functional coupling of NF-kappaB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.

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.