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

Abstract

The eukaryotic transcription factor NF-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-B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called IB␣. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-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 IB␣-dependent mechanisms for NF-B induction, we identified novel mutants of IB␣ that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of IB␣ failed to disrupt its ability to form latent complexes with NF-B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of IB␣ from NF-B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of IB␣ also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-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 IB␣ that coordinately controls the biologic activities of IB␣ and NF-B in response to viral and immune stimuli.

Dual role of TMS1/ASC in death receptor signaling

“…The IkBa super-repressor contains alanines instead of serines at positions 32 and 36, and as a consequence cannot be phosphorylated by the IKK complex or degraded by the 26S proteasome (Brockman et al, 1995;Mayo et al, 2003). Expression of the dominant-negative IkBa thus sequesters NF-kB in the cytoplasm, thereby preventing NF-kB dependent transcription (Brockman et al, 1995;Mayo et al, 2003).…”

p38 protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-κB activity and Fas expression

“…The expression vector pCMV4-IkBDN (Brockman et al, 1995) plus pcDNA3-neo was also co-transfected by electroporation (230 V, 1050 mF) into LU1205 cells as previously described (Chan et al, 1999). The expression constructs, pcDNA3-FLAG-MKK7b1 (Tournier et al, 1999), pcDNA3-JNK1-APF (Butter®eld et al, 1997) p38-ASP (Enslen et al, 1998) MKK6-DD (Han et al, 1996), and ASK1DN (Saitoh et al, 1998) were also used in these experiments.…”

NF-κB is required for H-ras oncogene induced abnormal cell proliferation and tumorigenesis

“…DN-IkBa lacks the N-terminal 36 amino acids encompassing two serine residues (S32, 36) required for signal-induced phosphorylation and degradation of this inhibitor (Brockman et al, 1995). Five days following viral infection and drug selection, pools of infected cells were counted.…”

“…Electrophoretic mobility shift assay, luciferase reporter assay, and apoptosis analysis Preparation of nuclear extracts and gel mobility assay were carried out as previously described (Brockman et al, 1995;Schreiber et al, 1989). a 32 P-dATP-labeled double stranded NF-kB consensus oligonucleotides, 5'-CAACGGCAGGG-GAATTCCCCTCTCCTT-3' (Brockman et al, 1995) was incubated with 7 mg of nuclear extract.…”

“…a 32 P-dATP-labeled double stranded NF-kB consensus oligonucleotides, 5'-CAACGGCAGGG-GAATTCCCCTCTCCTT-3' (Brockman et al, 1995) was incubated with 7 mg of nuclear extract. For luciferase reporter assay, 7610 6 cells were plated in 65 mm culture plate 24 h prior to transfection.…”