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Studies of the NF-kB/Rel Transcription Factors
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| Long considered to be the pivotal transcription factor regulating both immune and inflammatory responses, NF-kB/Rel has recently been shown to regulate such diverse cellular processes as proliferation, differentiation, and apoptosis. Targeted gene disruption studies have confirmed the importance of NF-kB/Rel factors in a wide variety of developmental and environmental responses. NF-kB is activated through specific phosphorylation of the cytoplasmic inhibitory protein, IkB. This modification triggers the rapid ubiquitination and proteasome-mediated degradation of this NF-kB inhibitor. The newly liberated NF-kB complex (p50/RelA heterodimer) then rapidly translocates into the nucleus, where it engages cognate kB enhancer elements and regulates the transcription of various target genes. The kinase complex responsible for phosphorylating IkB contains at least a pair of kinases, IKK1 (IKKa) and IKK2 (IKKb), and a third noncatalytic component termed NEMO (or IKKg/IKKAP1). Targeted gene disruption studies have clearly identified IKK2 as the key kinase responsible for phosphorylating IkBa. Our studies have further shown that IKK1 mediates the activation of IKK2, whereas others have shown that IKK1 regulates posttranslational processing of NF-kB2 p100 to p52 and participates in controlling keratinocyte differentiation by a nonenzymatic mechanism. Reversible Acetylation of RelA and the Regulation of Nuclear NF-kB Action.
 As noted above, activation of the NF-kB transcription factor is mediated through signal-coupled phosphorylation and degradation of IkBa and related inhibitors. However, the subsequent molecular events that regulate the duration of NF-kB action after its translocation into the nucleus remain poorly defined. Our previous finding that NF-kB activates de novo expression of the IkBa gene (see Sun et al., Science 259:1912, 1993) suggests that this shuttling inhibitor likely plays a key role in terminating this transcriptional response. Our studies now demonstrate that the RelA subunit of NF-kB is subject to inducible and reversible acetylation. Both p300 and CBP, but not PCAF, can mediate this posttranslational modification when coexpressed with RelA in cells. We further find that acetylated RelA binds with higher affinity to the kB enhancer, exhibits greater transcriptional activity than unacetylated forms of Rel, and displays little or no binding to IkBa (Figure 3). These acetylated forms of RelA are deacetylated through specific interaction with histone deacetylase 3 (HDAC3). As such, RelA is the first nonhistone substrate identified for HDAC3. Deacetylation of RelA promotes its effective binding to IkBa and leads to IkBa-dependent nuclear export of the NF-kB complex by a CRM-1-dependent pathway. In agreement with this notion, HDAC3-induced nuclear export of RelA does not occur in murine embryo fibroblasts lacking the IkBa gene. However, in these cells, reconstitution of IkBa by transfection restores the response. Thus, reversible acetylation of RelA functions as an intranuclear molecular switch that both controls the duration of the NF-kB transcriptional response and contributes to replenishment of the depleted cytoplasmic pool of latent NF-kB/IkBa complexes, thereby readying the cell for the NF-kB-inducing signal. A Link Between Translation and NF-kB p50 Biogenesis.
We have explored the biochemical basis for production of the p50 subunit of the prototypical NF-kB complex. Both the p50 and p105 proteins are products of the NFKB1 gene, with p50 corresponding to an amino-terminal portion of p105. Previously, it was assumed that p50 was produced by posttranslational processing of a p105 precursor by the 26S proteasome. We have found that p50 is principally generated during the translation of the NFKB1 gene before synthesis of p105 is completed. Specifically, if the translating chain is sufficiently unfolded, it forms a suitable substrate for processing by the proteasome, thereby generating p50. However, if sufficient cotranslational folding occurs, the peptide is no longer an acceptable proteasome substrate and results in production of the larger p105 protein. The p50 protein functions as a transcription factor, while p105 primarily functions as an inhibitor of p50. More recently, using a biochemical criterion that distinguishes between monomeric and dimeric states of the Rel homology domain in the NFKB1 gene product, we have discovered a second critical cotranslational event in p50 biogenesis, namely cotranslational dimerization. Cotranslational dimerization involves protein products generated on the same polysome and yields p50/p105 heterodimers. This cotranslational cellular strategy places p50 under the control of its p105 inhibitor early in its biogenesis and establishes a homeostasis between p50 and p105 in resting cells. Furthermore, selective cotranslational production of p50/p105 heterodimers indicates that p50 homodimer formation in vivo requires additional posttranslational steps possibly involving the action of Bcl-3 or the proteasome. This work provides the first example of cotranslational dimerization and proteasome-mediated processing in mammalian cells and offers significant new insights into how one set of NF-kB/Rel dimers is assembled.
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