title: Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity.
abstract: DNA binding of the homodimeric p50 subunit of NF-kappa B was inhibited by a bacterially expressed protein containing the ankyrin repeats present in the C-terminus of the p105 precursor but not by the I kappa B protein MAD-3. However p50 was retained on protein affinity matrices containing either the C-terminal ankyrin repeats of p105 or MAD-3. To investigate the interaction between p50 and proteins containing ankyrin repeats we have used a number of approaches to probe the accessibility of the p50 nuclear localisation signal in the protein complex. A monoclonal antibody recognising a linear epitope either very close to, or including, the nuclear localisation signal of the p50 protein could immunoprecipitate p50 homodimers but was unable to precipitate the protein when it was bound to the C-terminal region of p105. A close association between the nuclear localisation signal of p50 and the C-terminal region of p105 was also suggested by protease accessibility experiments. While the nuclear localisation signal of free p50 is extremely susceptible to cleavage with trypsin the same site is masked in the presence of the C-terminal ankyrin repeats of p105 and, to a lesser extent MAD-3. Removal of the nuclear localisation signal by trypsin digestion generates a protein that is fully competent for DNA binding but is refractile to inhibition by the C-terminal ankyrin repeats of p105. Addition of DNA destabilises complexes between p50 and ankyrin repeat containing proteins, increasing the susceptibility of the nuclear localisation signal to trypsin cleavage. The data suggest that there is a rapid exchange of p50 between complexes containing DNA or I kappa B proteins via a metastable complex containing DNA, p50 and I kappa B.
authors.affiliation: School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
authors.forename: J R
authors.initials: JR
authors.lastname: Matthews
authors.forename: E
authors.initials: E
authors.lastname: Watson
authors.forename: S
authors.initials: S
authors.lastname: Buckley
authors.forename: R T
authors.initials: RT
authors.lastname: Hay
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002451:Cell Compartmentation; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D051841:Transcription Factor RelB; D004247:DNA; D014357:Trypsin