Immunoglobulin heavy-chain switching is effected by a DNA recombination event that replaces the C,, gene

The transcription factor NF-cB is retained in the cytoplasm by its inhibitor 1KB-a. Upon cellular stimulation with a variety of pathogen-or stress-related agents, 1KB-a is functionally inactivated and NF-cB translocates to the nucleus to trigger transcription of a large array of genes, many of which encode proteins critical for immune or stress responses. Here, we demonstrate that signal-induced proteolysis of I1cB-a is an obligatory step for activation of NF-cB:calpain inhibitors I and IH, which inhibit cysteine proteases, block activation of NF-KcB by blocking degradation of IcB-a without affecting signal-induced phosphorylation of this inhibitor. This contrasts with previous models in which phosphorylation of licB-a was postulated to be sufficient for activation. We demonstrate further that signal-induced phosphorylation of IcB-a does not by itself lead to dissociation of the inhibitor from NF-cB, providing a rationale for and confirmation of the need to proteolyze I1cB-a in order to activate NF-cB. Signal-controlled, target-specific proteolysis is an unexpected, yet likely more general, mechanism for regulating transcription factors.The transcription factor NF-KB has been implicated as an essential component of pathogen-and stress-related responses of host organisms. Signals directly or indirectly related to pathogens or stress potently activate NF-KB, which then transcriptionally induces many genes encoding defense-related proteins (reviewed in refs. 1-3). NF-KB is a family of dimers, all of which are composed of members of the Rel/NF-KB family of polypeptides; typically, NF-KB activity is due primarily to p5O/p65 (NF-KB1/RelA) heterodimers, although other dimeric combinations often coexist, such as p50/Rel or p52/ p65 (NF-KB2/RelA) (3).The mechanisms leading to activation of NF-KB are of intense interest. In unstimulated cells, NF-KB is normally held in the cytoplasm by the inhibitory protein IKB-a, which avidly binds to most dimers (in particular p50/p65), thereby shielding their nuclear translocation signals. In addition, IKB-a prevents binding of most NF-KB dimers to DNA-exceptions are homodimers of p50 and p52, which lack recognizable transactivation domains (2, 3). Appropriate cellular stimuli inactivate 1KB-a, at least transiently, to allow NF-KB to translocate to the nucleus and induce gene transcription through cis-acting KB elements. The prevalent model for activation holds that phosphorylation of IKB-a in response to signals dissociates the inhibitor from the NF-KB dimer, thereby activating the transcription factor (4). The model is based on early experiments in which NF-KB was activated by kinases added to extracts in vitro. The activation was apparently mediated by phosphorylation of IKB-a (5-7). This hypothesis was called into question by the recent and unexpected observation that activation of NF-KB correlates with rapid proteolytic degradation of IKB-a in vivo, regardless of signal or cell (8-10). In addition, it was reported that inhibitors of chymotrypsin-like proteases blocked act...

IB␣ retains the transcription factor NF-B in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IB␣ by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-B translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IB␣ to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IB␣ is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IB␣ is controlled by transferable N-terminal and C-terminal domains.The transcription factor NF-B is present in the cytoplasm of most animal cells. Much of it is retained there in an inactive form by the inhibitory protein IB␣, which binds to it and masks the nuclear localization signal (7,22,23). When cells are stimulated by a wide variety of agents, including phorbol esters, cytokines, viruses, and stress, IB␣ is first rapidly phosphorylated, then ubiquitinated, and finally degraded by proteasomes (1, 8, 12-18, 34, 35, 38, 42-45, 47; reviewed in references 3, 5, and 40). This allows translocation of NF-B to the nucleus, where it transactivates a large array of genes important in the defense of the organism. We previously showed by mutational analysis and two-dimensional electrophoresis that phosphorylation of serines 32 and 36, the two most N-terminal serine residues of IB␣, occurred upon stimulation and that induced phosphorylation was essential for proteolysis (13). Direct, independent proof is now presented that in fact both serines must be phosphorylated in order for signal-induced proteolysis to occur. We also show that mutation of either serine does not necessarily block induced phosphorylation of the other. However, such singly phosphorylated forms are very efficiently dephosphorylated by endogenous phosphatases unless appropriate phosphatase inhibitors are present. These observations confirm and extend recently published data on the subject (18, 44, 47).Previously we showed that, in addition to N-terminal phosphorylation and ubiquitination (primarily on lysine residues 21 and 22 [4,34,38]), the C-terminal 41 amino acids of IB␣ also contribute to rapid signal-induced proteolysis (13). Similar conclusions have been reached by others (47), but there have also been reports suggesting a less critical role for the C terminus during induced degradation in different cells (2,43,46). Now we report that the signal-induced phosphorylation response of IB␣ can be transferred to heterologous polypeptides by their fusion to the N-terminal domain of IB␣...