The N-Terminal Domain of IκBα Masks the Nuclear Localization Signal(s) of p50 and c-Rel Homodimers

Abstract

Members of theTranscription factors of the Rel/NF-B family are present in most or all mammalian and avian cells and influence the expression of many genes (for a review, see references 1 and 19). These factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding and dimerization. Although each protein has a nuclear localization signal (NLS), the various homo-and heterodimers are cytoplasmic by virtue of an inhibitor protein whose binding masks the NLSs. Several different inhibitors (called IBs) are known, and they constitute a family of related proteins. Each IB contains five or six so-called ankyrin repeats, motifs of about 33 amino acids which are abundantly represented in the protein ankyrin. In ankyrin, these repeats apparently function in groups of about six (18) and constitute a protein-protein interaction domain. In addition, the inhibitors contain a C-terminal region which tends to be highly acidic and to resemble PEST sequences associated with rapid protein turnover (22). The IB N-terminal region contains two serine residues which, upon phosphorylation, trigger ubiquitination and proteasomal degradation of the inhibitor, thus freeing the dimer to enter the nucleus (4,5,7,(26)(27)(28)(29).While several crystal structures of Rel family members are known (6, 6a, 10, 20), none is known for the IB family. Nevertheless, studies with various mutants of the Rel family and/or IB proteins have led to some general conclusions about their interaction. First, a Rel family dimer binds a single IB molecule (8,12,14). Second, at least some of the contacts with IB occur in the dimerization domains of the Rel proteins. Based on the crystal structure, each monomer of p50 and RelA consists of two separate domains connected by a short hinge region. The N-terminal domain contains some (but not all) of the residues that contact DNA, while the C-terminal domain contains the remaining DNA contacts and governs dimerization. Partial deletion of the N-terminal domain of RelA did not abolish binding of IB␣ (9). Similar results were obtained with Cactus and N-terminal deletion mutants of Dorsal (Drosophila relatives of IB and Rel proteins, respectively) (11, 25). In addition, mutation of two residues in the dimerization domain of Dorsal prevented interaction with Cactus but did not affect DNA binding or dimerization (16). These studies suggest contact between IB and the C-terminal part of the RHD. The third general conclusion is that the dimer NLSs, which were not resolved in the crystal structures but are immediately downstream of the dimerization domain in the primary structure, are somehow involved in binding to IB. While in at least some cases an intact NLS may not be required for binding to IB (11,23), several studies have shown that altering the NLS can abolish binding (2,9,15,25). Involvement of the NLS would correlate nicely with the fact that the dimer NLSs are masked in a dimer-IB complex. The final generalization is that most of the IB contacts oc...

Determinants of 5-Lipoxygenase Nuclear Localization Using Green Fluorescent Protein/5-Lipoxygenase Fusion Proteins

“…fibroblast growth factor 3) (41). Examples of NLS "masking" by structural alterations and/or other cellular proteins, a prime example being the transcription factor NF-B/Rel bound by its inhibitor IB are known (42). The data herein could be consistent with some sort of unmasking of an NLS to gain nuclear entry and also do not rule out the possibility that 5-lipoxygenase is "piggybacked" to the nucleus by some other chaperone protein.…”

IκBα and IκBα/NF-κB Complexes Are Retained in the Cytoplasm through Interaction with a Novel Partner, RasGAP SH3-binding Protein 2

“…Two distinct but not exclusive mechanisms have been proposed to account for the cytoplasmic retention of IB␣/NF-B complexes: (i) an active nuclear export mediated by the N-terminal NES promotes continuous export from the nucleus and (ii) the N-terminal region of IB␣ is thought to mask the p50 NLS either directly or indirectly. These dual functions of the N-terminal region are mediated by the same sequence located between residues 45 and 54, but differences are observed in the nature of residues essential for each function (42)(43)(44)58). Data reported here indicate that the same N-terminal domain (residues 37-55) displays properties of a CRS that promotes anchoring of IB␣ in the cytoplasm and on the outer membrane of the nuclear envelope.…”

“…It has been recently reported that an NES located in the N-terminal domain of IB␣ (which was not in the NF-B/IB␣ structure) participates to the cytoplasmic localization of inactive NF-B/IB␣ complexes (43,44) by inducing an efficient export of the complex out of the nucleus. In addition, the N-terminal region of IB␣ has also been proposed to directly or indirectly affect the p50 NLS accessibility and function, thus limiting nuclear import of NF-B/IB␣ complexes (43,58).…”

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

“…In the resting cell, the heterodimer generates a ternary complex with a member of the I B family of inhibitory proteins. I B binding sterically hinders a nuclear localization site, and consequently, the complex is retained in the cytosol (20,24). Following cellular stimulation by a wide array of activators, such as cytokines (tumor necrosis factor alpha and interleukin 1, for example), viral and bacterial products, UV light, and oxidants, specific I B kinases that phosphorylate the protein on two specific Ser residues, 32 and 36 (8), are activated (30,41,43).…”