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PMID: 1493333 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.

Molecular biology of the cell ·Vol. 3 ·No. 12 ·1992-12-00 ·Pages 1339-52

Ganchi PA, Sun SC, Greene WC, Ballard DW

Abstract

The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65.

Related Genes
v-rel
MeSH Terms
Animals Base Sequence Binding Sites Cell Line Cell Nucleus/metabolism Chloramphenicol O-Acetyltransferase/genetics,metabolism Cloning, Molecular DNA Probes DNA-Binding Proteins/metabolism Humans I-kappa B Proteins Macromolecular Substances Molecular Sequence Data Mutagenesis, Site-Directed NF-KappaB Inhibitor alpha NF-kappa B/genetics,metabolism Oligodeoxyribonucleotides/metabolism Oncogene Proteins v-rel Oncogenes Protein-Tyrosine Kinases/genetics Recombinant Proteins/metabolism Retroviridae Proteins, Oncogenic/genetics Sequence Homology, Amino Acid Signal Transduction T-Lymphocytes Transcriptional Activation Transfection Tumor Cells, Cultured
Chemicals
DNA Probes DNA-Binding Proteins I-kappa B Proteins Macromolecular Substances NF-kappa B NFKBIA protein, human Oligodeoxyribonucleotides Oncogene Proteins v-rel Recombinant Proteins Retroviridae Proteins, Oncogenic NF-KappaB Inhibitor alpha Chloramphenicol O-Acetyltransferase Protein-Tyrosine Kinases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Ganchi P A
Gladstone Institute of Virology and Immunology, University of California, San Francisco.
Sun S C
Greene W C
Ballard D W
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.3.12.1339
Published
1992-12-00
Pages
1339-52
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC275704
Subset
IM
Grants
NIAID NIH HHS · 5RO-1AI-29117-05 · United States
NCI NIH HHS · 5T32CA-09111 · United States
External Links
PubMed source DOI: 10.1091/mbc.3.12.1339 PMC Full Text View journal details
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