I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention.
NF-kappa B is an inducible transcription factor comprised of a 50-kD (p50) and a 65-kD (p65) subunit. Induction of NF-kappa B activity, which is a critical event in many signal transduction pathways, involves release from a cytoplasmic inhibitory protein, I kappa B, followed by translocation of the active transcription factor complex into the nucleus. Earlier studies suggested that I kappa B targets the p65 subunit of NF-kappa B. However, we demonstrate by in vitro and in vivo methods that the recently cloned I kappa B/MAD-3 interacts with both the p50 and p65 subunits of NF-kappa B, as well as c-Rel. Furthermore, an alternatively spliced, dimerization-deficient transforming variant of p65 (p65 delta) interacts extremely weakly with I kappa B/MAD-3, suggesting that dimerization is important for interaction. We demonstrate that the conserved nuclear localization sequences (NLSs) of NF-kappa B and c-Rel are the targets for I kappa B/MAD-3 interaction. Indirect immunofluorescence experiments demonstrate that I kappa B/MAD-3 expression retains both p65 and p50 in the cytoplasm. Furthermore, and most important, a p65 that contains an SV40 large T antigen NLS in addition to its own NLS is no longer retained in the cytoplasm in the presence of I kappa B/MAD-3. We propose that I kappa B/MAD-3 masks the NLSs of NF-kappa B and c-Rel and that this constitutes the mechanism for cytoplasmic retention of these proteins.
Beg
A A
AA
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599.
Ruben
S M
SM
Scheinman
R I
RI
Haskill
S
S
Rosen
C A
CA
Baldwin
A S
AS
D017398:Alternative Splicing; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051841:Transcription Factor RelB; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D051841:Transcription Factor RelB; D015500:Chloramphenicol O-Acetyltransferase