IκBβ, but Not IκBα, Functions as a Classical Cytoplasmic Inhibitor of NF-κB Dimers by Masking Both NF-κB Nuclear Localization Sequences in Resting Cells

Shiva Malek,Yi Chen,Tom Huxford,Gourisankar Ghosh

doi:10.1074/jbc.m105865200

Shiva Malek, Yi Chen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m105865200

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Abstract

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.

Highlights

NF-␬B represents a paradigm for inducible transcription factors that are regulated, in large part, through their subcellular compartmentalization [1,2,3]
We show that the presence of one free nuclear localization signals (NLSs) in the NF-␬B1⁄7I␬B␣ complex renders it a dynamic nucleocytoplasmic complex, whereas NF-␬B1⁄7I␬B␤ complexes are localized to the cytoplasm of resting cells
NF-␬B dimers reside in the cytoplasm in complex with I␬B proteins, which function by masking the nuclear localization signals (NLSs)1 of NF-␬B

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cloning, expression, and purification of the NF-␬B subunits and amino-terminal deletion mutants has been described previously [27, 28]. Protease Digestion Experiments—NF-␬B p65-(191–321)1⁄7E5-I␬B␤ complex at 50 mg/ml was diluted with 20 mM Tris (pH 8.0), 50 mM NaCl, 50% glycerol, and 1 mM dithiothreitol to a final concentration of 8 mg/ml. 0.1 ␮g of thermolysin was added to a 50-␮l reaction containing 1.5 mg/ml p65-(191–321)1⁄7E5-I␬B␤ complex or p65-(191–321) alone in 10 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 0.145 mM ZnSO4, and 1 mM dithiothreitol. The reaction was quenched by the addition of 10 mM EDTA and 1ϫ SDS-polyacrylamide gel electrophoresis loading dye (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol) followed by 1 min of boiling and immediate storage at Ϫ20 °C. Cells were incubated with rabbit antibody alone or in a mixture as required for the experiment in PBS containing 5 mg/ml bovine serum albumin and 0.2% Nonidet P-40 at room temperature for 2 h. Cells were washed three times with PBS with 0.1% Tween 20 and covered with a drop of mounting solution (Vector)

RESULTS

Relative KI nM

DISCUSSION