B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell–independent and T cell–dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-κB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell–independent and T cell–dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.

No abstract

The inducible pleiotropic transcription factor NF-#cB is composed of two subunits, p50 and p65. The p5O subunit is encoded on the N-terminal half of a 105-kDa open reading frame and contains a rel-like domain. To date, no function has been described for the C-terminal portion. We show here that the C-terminal half of p105, when expressed as a separate molecule, binds to p50 and can rapidly disrupt protein-DNA complexes of p5O or native NF-jcB. Deletion analysis of this precursor-derived inhibitor activity indicated a domain containing ankyrin-like repeats as necessary for inhibition. The protooncogene bcl-3, which contains seven ankyrin repeats, can equally inhibit p50 DNA binding. These observations identify bd-3 as an inhibitor of NF-icB and strongly suggest that the ankyrin repeats in these factors are involved in protein-protein interactions with the rel-like domain of p50.Comparison with other ankyrin repeat-containing proteins suggests that a subclass of these proteins acts as regulators of rel-like transcription factors.The early response transcription factor NF-KB is involved in the regulation of a number of viral and cellular genes (for review, see refs. 1 and 2). Cloning of the p50 (3-6) and p65 (7, 8) subunits of NF-KB revealed a conserved domain with homology to the rel protooncogene and the Drosophila morphogen dorsal. The rel-homologous region contains the DNA-binding and dimerization domains as well as a conserved nuclear transfer signal (9). p50 is synthesized as a 105-kDa precursor protein, which must be processed in vivo to release active p50 (3)(4)(5)(6). In analogy to p105, p100 (10) represents a structurally closely related precursor for another NF-KB-like transcription factor (unpublished data). The C-terminal half of p105 contains seven repeated motifs of at least 33 amino acids. This motif is also found in fruitfly, nematode, and yeast cell cycle or differentiation control proteins (11-17), in human erythrocyte ankyrin (18), in the putative human protooncogene bcl-3 (19), and in the recently isolated human immediate-early gene . No function has yet been described for the ankyrin repeat domains, except for the human erythrocyte ankyrin and for the p-subunit of the transcription factor GABP (21). In human ankyrin, a region containing 23 tandem repeats has been shown to confer binding to the anion-exchanger protein (22). The ,8-subunit of GABP contains four imperfect ankyrin-like repeats, which mediate association with the a-subunit (21).NF-KB and c-rel have been shown to be associated with cytoplasmic proteins (23,24). For the cytoplasmic inhibitor of NF-KB, IKB, two forms have been described, both of which inhibit DNA binding in vitro and nuclear translocation of NF-KB in vivo through binding to p65 (25). One of these forms is presumably identical with . We show here that the C-terminal half of p105 encodes an IKB-like molecule with an affinity for p50, which has the potential to bind to NF-KB as a separate molecule and inhibit DNA binding. A region containing the ankyrin repeats i...

BiochemistryCloning of the DNA-binding subunit of human nuclear factor cB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor a (transcription factor/protein purifilcation/DNA sequencing/regulation of nuclear factor ucB expression/multigene family) RALF MEYER*, EUNICE N. HATADA*, HANS-PETER HOHMANNt, MONIKA HAIKERt, CORNELIA BARTSCH*, URS R6THLISBERGERt, HANS-WERNER LAHMt, ERNST J. SCHLAEGERt, ADOLPHUS P. G. M. VAN ABSTRACTThe DNA binding subunit of nuclear factor KB (NF-acB), a B-cell protein that interacts with the inmunoglobulin K light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor a (TNFa), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-cB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-KB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFa or phorbol ester. Thus, both factors not only activate NF-KB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-KB.

p105 (NFKB1) acts in a dual way as a cytoplasmic IB molecule and as the source of the NF-B p50 subunit upon processing. p105 can form various heterodimers with other NF-B subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKK␤ phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSG⌿ motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IB␣, -␤, and -. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules ␤TrCP1 and ␤TrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKK␥, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.The NF-B transcription factor family plays an evolutionarily conserved role in innate and adaptive immune responses, and its members are essential regulators of proinflammatory processes (12,18,33). NF-B is also an important regulator in cell fate decisions, such as programmed cell death and proliferation control, and is critical in tumorigenesis (32, 42). The vertebrate NF-B/Rel factors p50, p52, p65 (RelA), c-Rel, and RelB form various dimers and are under the control of a coevolved family of cytoplasmic IB molecules, IB␣, -␤, and -ε, and the nuclear IB homologue Bcl-3 (1). p50 and p52 are formed by processing from the precursor molecules p105 and p100, respectively. Prototypic heterodimeric NF-B p50-p65 is rapidly released from cytoplasmic complexes with IBs upon cellular stimulation with diverse agents, including tumor necrosis factor alpha (TNF-␣), interleukin-1␤, bacterial lipopolysaccharides (LPS), and phorbol myristate acetate, following viral infection or exposure to ␥-irradiation (31). All these agents induce an IB kinase (IKK) complex to phosphorylate IB␣, -␤, and -ε. The IKK complex contains the kinases IKK␣ and IKK␤ and the noncatalytic IKK␥ subunit, which is essential for signal responsiveness of the complex (see reference 17 for a review). Once phosphorylated by the IKK complex, the IBs are ubiquitinated and degraded by the 26S proteasome, resulting in nuclear translocation of NF-B. The ubiquitin ligase complex specif...