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The FASEB Journal Review

Beyond IBs: alternative regulation of NF-B activity

Manfred Neumann and Michael Naumann
Institute of Experimental Internal Medicine, Otto-von-Guericke University, Magdeburg, Germany

The transcription factor nuclear factorkappa B (NF-B) is a crucial regulator of many physiological and patho-physiological processes, including control of the adaptive and innate immune responses, inammation, proliferation, tumorigenesis, and apoptosis. Thus, the tight regulation of NF-B activity within a cell is extremely important. The central mechanism of NF- regulation is the signal-induced proteolytic degradation of a family of cytoplasmic inhibitors of NF-B, the IBs. However, with the discovery of an IB-independent noncanonical or alternative pathway of NF-B activation, the importance of other regulatory mechanisms responsible for the ne-tuning of NF-B became clear. Post-translational modication, especially phosphorylation, of the Rel proteins, of which dimeric NF-B is composed, are such alternative regulatory mechanisms. The best analyzed example is RelA phosphorylation, which takes place at specic amino acids resulting in distinct functional changes of this gene regulatory protein. The interaction of NF-B with other proteins such as glucocorticoid receptors is very important for the regulation of NF-B activity. Recently, exciting new concepts of IB-independent NF-B control like dimer exchange and nucleolar sequestration of RelA have been described, indicating that many aspects of NF-B control are waiting to be discovered.Neumann, M., Naumann, M. Beyond IBs: alternative regulation of NF-B activity. FASEB J. 21, 26422654 (2007) Key Words: RelA phosphorylation RelA acetylation GR/ NF-B cross-talk nucleolar sequestration dimer exchange nf-b is a pleiotropic transcription factor activated by a huge array of stimuli (1, 2), including inammatory cytokines recognized by specic membrane receptors such as tumor necrosis factor receptor (TNF-R) and microbial pathogens, which are recognized by the Toll-like receptors (TLRs) as well as receptors of the NACHT (domain present in NAIP, CIITA, HET-E, and TP1) -LRR (leucine-rich repeat) family (NLRs). The NLR family includes nucleotide binding oligomerization domain (NOD) proteins and NACHT-, LRR-, and pyrin-domain-containing proteins (NALPs). These receptor proteins function in a similar manner in that they recognize microbial components at the cell surface, in vesicles, and in the cytosol (3). Other important NF-B inducers are phorbol esters such as TPA, cytotoxic agents like chemotherapeutic drugs, oxidative stress, UV light, and ionizing radiation.

NF-B is a dimer composed of the ve mammalian Rel proteins p65/RelA, c-Rel, RelB, NF-B1/p50, and NFB2/p52 in almost any combination. The last two are synthesized as precursor molecules, p105 and p100, respectively, and processing of these precursors is needed to generate the mature transcription factors. Only RelA, c-Rel, and RelB, but not p50 or p52, possess C-terminal transactivation domains. All Rel proteins share the N-terminal Rel homology domain (RHD) mediating dimerization, DNA binding, and nuclear localization and interaction with the inhibitors of NFB, the IBs. This family of mainly cytoplasmic inhibitors currently has eight members: IB, IB, IB, IB, IB, Bcl-3, as well as p105 and p100. These precursors possess ankyrin repeats in their C terminus, the characteristic structural feature of all IBs (1, 2). The IB-dependent NF-B activation pathways outlined below certainly play the dominant role in controlling NF-B activity. However, the question remains: how is the specicity of NF-B function achieved under certain physiological conditions or in a specic cell type? It is the purpose of this review to demonstrate the complex mechanisms contributing to the ne-tuning of NF-B activity. IB-DEPENDENT NF-B ACTIVATION The IBs (inhibitors of NF-B) are the centerpiece of the canonical (classical) pathway (1, 4) (Fig. 1A). In unstimulated cells, NF-B is complexed with the IBs and thereby locked into the cytoplasm. Upon stimulation of the cell, multiple intracellular signaling pathways are activated that converge at the IB kinase (IKK) complex. The most common form of this complex consists of two functionally nonredundant kinases, IKK (IKK1) and IKK (IKK2), as well as a regulatory subunit, IKK, also called NEMO (NF-B essential modulator). Upon activation, the IKK complex phosphorylates the IBs at specic amino acid residues. The phosphorylation of IB at Ser-32 and Ser-36 is predominantly mediated by IKK. This site-specic phosphorylation is a prerequisite for a subsequent posttranslational modication, the ubiquitinylation of IB, which tags the NF-B inhibitor for degradation in
Correspondence: Institute of Experimental Internal Medicine, Otto-von-Guericke University, Medical Faculty, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail: doi: 10.1096/fj.06-7615rev
0892-6638/07/0021-2642 FASEB


Figure 1. Schematic representation of four IB-dependent NF-B pathways. A) The canonical (classical) pathway depends on the site-specic phosphorylation of IBs by activated IKK complexes. The TNF-R1-mediated NF-B activation is shown as an example. Activation of NF-B takes place near the membrane. One recently discovered critical stepthe interaction of NEMO with Lys-63 polyubiquitinylated RIP1is shown. It should be noted that the TNF-RI complex contains more components. Some of them, such as the transforming growth factor beta-activated kinase 1 (TAK1) and the TAK1 binding protein 2 (TAB2), which also bind polyubiquitinylated RIP1, are able to interact with and activate the IKK complex. B) The noncanonical (alternative) pathway based on the processing of the p100 precursor. NIK and IKK, but not IKK, are critical kinases. Mainly RelB/p52 NF-B dimers are generated. C) The unique NF-B activation pathway induced by UV light and a few select chemotherapeutic agents. The MAP kinase p38 and CKII are needed to phosphorylate IB at sites different from the amino acid residues used in the canonical pathway. These phosphorylation events are a prerequisite for signal-induced IB degradation. D) A stress-activated pathway based on the sequential, nuclear modication of NEMO. Nuclear NEMO rst becomes sumoylated, mediated by PIDD and RIP1, then phosphorylated by the stress sensor ATM kinase. This phosphorylation is a prerequisite for mono-ubiquitinylation of NEMO, which serves as nuclear export signal. ATM is also translocated to the cytoplasm along with NEMO interacting with and activating IKK.

the 26S proteasome. NF-B is now free to translocate into the nucleus, where it regulates the expression of genes involved in fundamental physiological and patho-physiological cellular processes such as control of the immune system, especially of the innate immune response, as well as the regulation of inammation and apoptosis. Various MAP3kinases (mitogen-activated protein kinase kinase kinases) located immediately upstream of the IKK complex have been thought to be key components of IKK activation. However, recent studies have demonstrated that the binding of NEMO to Lys-63-linked polyubiquitinylated RIP1 is the essential step in IKK activation mediated by TNF-R1 (57). This nding provided a key step to understanding the link between cytokine receptor proximal signaling and the activation of IKK/NF-B. The canonical NF-B activation pathway outlined above is generally regarded to be the main mechanism by which NF-B activity is regulated. However, this

fundamental pathway is not the only one controlling NF-B. Recently, a noncanonical (alternative) signaling pathway of NF-B activation was discovered that involves processing of the p52 precursor p100 (1, 4) (Fig. 1B). This pathway is completely independent of IKK or NEMO, whereas IKK and the NF-B-inducing kinase (NIK) are essential. NIK is positioned immediately upstream of the IKK homodimer. Cytokines such as lymphotoxin (LT-) and B cell-activating factor, CD40 ligand, as well as viruses such as the human T cell leukemia virus 1 (HTLV-1) or the Epstein-Barr virus (EBV), are among the few select stimuli able to activate the noncanonical pathway. The phosphorylation of p100 at two specic C-terminal serine residues by IKK homodimers is a key event in this pathway. Again, this site-specic phosphorylation is essential for polyubiquitinylation and proteasomal degradation. However, the entire molecule is not degraded; only the C terminus of p100 is destroyed. RelB/p52 heterodimers are

the main NF-B factors generated by p100 processing so that not only the signals inducing this pathway, but also its products, are specic. The mechanism of UV light-induced NF-B induction is unique (Fig. 1C). Exposure of cells to UV light leads to the degradation of IB, but this phosphorylation-dependent process is not mediated by the IKK complex (8). It could be demonstrated that activation of a p38-MAPkinase-dependent pathway is critical for UV-induced IB-degradation. One of the main targets of this pathway is casein kinase II (CKII), a serine/ threonine kinase that can phosphorylate IB at a cluster of C-terminal sites. Like the IKK-mediated Nterminal phosphorylation, this post-translational IB modication induces degradation of this NF-B inhibitor. Thus, CKII is a stress-activated rather than a constitutive IB kinase playing a crucial role in UV protection by inducing NF-B-dependent antiapoptotic gene expression (8). UV light can induce NF-B activation in a DNA damage-independent manner. In contrast, other DNAdamaging stimuli such as ionizing radiation or chemotherapeutic drugs also induce an NF-B response. However, this type of NF-B activation is not only induced by DNA damage, particularly the formation of double strand breaks (genotoxic stress), but is also found to be associated with other stress conditions, such as oxidative stress as well as heat or electric shock (9) (Fig. 1D). Recent studies have shed new light onto these nuclear-to-cytoplasmic pathways of NF-B activation (4, 10, 11). The regulatory IKK component NEMO and its tripartite post-translational modication stand in the center of this NF-B activation pathway. Upon cellular stress, NEMO is sumoylated within the nucleus. This means it is modied by covalent attachment of the small ubiquitin-like modier (SUMO). PIASy (protein inhibitor of activated STATy) was recently identied to be the NEMO-specic SUMO ligase (12). Sumoylation is increased in the presence of PIDD (p53-inducible death domain-containing protein), a protein implicated in cell cycle control. PIDD forms a complex with NEMO and RIP1 (11, 13). Then a second ataxia telangiectasia-mutated (ATM) -dependent pathway results in the phosphorylation of NEMO and subsequently its mono-ubiquitinylation. The last modication does not mark NEMO for proteasomal degradation, but rather is a signal for cytoplasmic translocation, where modied NEMO activates the IKK complex. The NF-B activation pathway originating from DNA damage described above is an excellent example of the fact that protein ubiquitinylation has emerged as an important intracellular mechanism regulating various biological processes (14, 15). Ubiquitin is a highly conserved 76 amino acid polypeptide that can be covalently attached to a lysine residue of a target protein by an E3-ubiquitin ligase, which is primarily responsible for providing substrate specicity. Subsequent to the attachment of the rst ubiquitin, polyubiquitin chains are often generated by the ligation of
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multiple ubiquitin moieties. Ubiquitin has seven lysine residues, of which Lys-48 and Lys-63 are used for generating most polyubiquitinylation chains. Whereas Lys-48-linked polyubiquitinylation is a marker for proteasomal degradation, the attachment of Lys-63-linked polyubiquitin chains regulates protein kinase activities and protein trafcking (7). The zinc nger protein A20 functions as a ubiquitin editor and a potent NF-B inhibitor; both functions are connected (16). The central role played by A20 in regulating NF-B is demonstrated by the fact that A20-decient mice develop severe inammation and cachexia, are hypersensitive to LPS as well as TNF-, and die prematurely (17). A20 interferes with TNF--induced NF-B activation upstream of the IKK complex (18). A20 interacts with TRAF2 (TNF receptor-associated factor 2) and NEMO, both critical signaling components of the pathway originating from the TNF receptor. Other A20 binding proteins such as ABIN-1 (A20 binding inhibitor of NF-B 1) also bind to NEMO and cooperate with A20 in inhibiting NF-B (19). However, the critical A20 target for inhibition of TNF-induced NF-B activation seems to be the serine/ threonine protein kinase RIP1 (receptor-interacting protein 1), which also interacts with A20. RIP1 is activated by TRAF2-driven Lys-63-linked polyubiquitinylation. A deubiquitinylation activity located in the so-called OTU (ovarian tumor) domain at the N terminus of A20 can remove these polyubiquitin chains. The subsequent attachment of Lys-48-linked polyubiquitin chains to RIP1 is mediated by the C terminus of A20, which encompasses seven novel zinc nger structures. This domain functions as an E3ubiquitin ligase. RIP1 is marked for proteasomal degradation by the latter post-translational modication interrupting the NF-B activation pathway (18). Thus, A20 is an astonishing molecule combining two distinct, but connected, functions of ubiquitin editing.

IB-INDEPENDENT REGULATION OF NF-B: POST-TRANSLATIONAL MODIFICATIONS OF REL PROTEINS Not only the IB inhibitors, but also the Rel proteins themselves, are subject to signal-induced post-translational modications (20, 21) (Fig. 2). These modications alter various physiological functions of the NF-B factors. The most important post-translational modication is phosphorylation, and the basal and signal-induced phosphorylation of RelA is by far the best-characterized modication. Site-specic phosphorylation is often a prerequisite for other modications regulating RelA activity. Such subsequent post-translational modications include acetylation, ubiquitinylation, and isomerization of specic amino acid residues.

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Figure 2. Compilation of post-translational modications of Rel proteins and their functional consequences. increase, up-regulation; decrease, down-regulation.

REGULATION OF NF-B ACTIVITY BY MODIFICATIONS OF RelA The rst description of an inducible phosphorylation of RelA in vivo already suggested an important mechanism in the control of NF-B activity (22, 23). Today, nine putative sites of RelA phosphorylation are known: six serine and three threonine residues (Fig. 2). Three of these sites (Ser-276, Ser-311, and Thr-254) are located within the N-terminal RHD, whereas six acceptor sites (Ser-468, Ser-529, Ser-535, Ser-536, Thr-435, and Thr-505) are found within the C-terminal transactivation domain. The effects of RelA phosphorylation at these specic sites vary dramatically, ranging from transcriptional activation to the complete repression of certain genes. The pattern of RelA phosphorylation is most likely stimulus- and cell type-specic. It becomes more and more apparent that RelA phosphorylation at different sites serves as an integrator for multiple incoming signals, which could control both the kinetics

and strength of the transactivating potency of RelA. We will now look at each RelA modication, its functional consequences, and biological signicance. Since many reviews comprehensively describe RelA phosphorylation (20, 21), the focus here is laid on the most recently discovered phosphorylation events. We start with serine phosphorylation, specically with the most C-terminal phosphorylation, followed by threonine phosphorylation, ubiquitinylation, and nally acetylation of RelA. The evolutionary conserved Ser-536 of RelA is the target of ve known protein kinases representing ve different signaling pathways in vitro and/or in vivo. In addition to IKK/ (24 26), these protein kinases include IKK and TBK1 (TANK binding kinase 1) (27, 28). Ser-536 can be phosphorylated by IKK after T cell costimulation (29). Both TBK1 and IKK show sequence homology to IKK/, but are not components of the IKK complex. DNA-damaging agents, such as the anticancer drugs doxorubicin or etoposide, activate NF-B through Ser-536 phosphorylation of RelA medi2645

ated by ribosomal S6 kinase 1 (RSK1) involving a p53-dependent pathway (30). The physiological consequence is a weaker interaction, with nuclear IB resulting in a drastic decrease of nuclear RelA export and thus enhanced RelA-dependent transcription. In line with this observation, a recent study claims that Ser-536-phosphorylated RelA does not interact at all with cytosolic IB and that this RelA species regulates a distinct set of target genes emphasizing the importance of the Ser-536 phosphorylation (31). Ser-535 of RelA is specically targeted by calmodulindependent kinase IV (CaMKIV) (32, 33). This phosphorylation results in the activation of gene expression, especially of antiapoptotic genes. This is in line with the observation that CaMKIV activity, like NF-B, has antiapoptotic and proproliferative effects, suggesting that NF-B is a potential effector of CaMKIV (33). CKII phosphorylates Ser-529 of RelA upon stimulation with TNF- or IL-1 (34, 35). Ser-529 phosphorylation is dependent on IB degradation, and thus takes place exclusively within the cytoplasm. This RelA modication appears to play a minor role in regulating RelA-dependent transactivation and appears to be relevant only for an optimal cellular response induced by HTLV-1 infection (25). Phosphorylation of the serine residue at position 468 located in the TAD2 of RelA appears to have opposing effects. RelA phosphorylated at Ser-468 is located predominantly within the nucleus, whereas most other versions of phosphorylated RelA are found mainly in the cytoplasm, indicating a function for this specic phosphorylation in the control of RelA-dependent transactivation. This site is the target of three protein kinases: IKK, IKK, and GSK-3 (36 38). GSK-3 mediates basal Ser-486 phosphorylation inhibiting the basal activity of RelA (38). In line with this, expression of a constitutive-active form of GSK-3 suppresses NF-B activity (39, 40). However, a contradictory nding appears to be of greater physiological relevance: NF-B cannot be activated in cells derived from GSK3-decient embryos despite an unaltered prole of IB degradation, which suggests a positive regulatory role of GSK-3 in NF-B activation (41). A positive impact on RelA activity was also observed for the IKK-mediated Ser-468 phosphorylation on T cell costimulation (37), whereas the IKK-dependent phosphorylation induced in HeLa cells stimulated with TNF- or IL-1 had a negative effect on RelA-mediated transactivation (36). This contradiction might be explained by the inuence of other phosphorylation events, which differ signicantly in both experimental systems. Phosphorylation of both serine residues 311 and 276 located within the N-terminal RHD of RelA results in an increase in transactivation potency. Ser-311 is phosphorylated by PKC upon TNF- stimulation, leading to an enhanced interaction of Ser-311-phosphorylated RelA with the cAMP response element binding (CREB) binding protein (CBP) and its recruitment to the interleukin 6 (IL-6) promoter together with RNA poly2646 Vol. 21 September 2007

merase II (42). Ser-276 is phosphorylated exclusively within the cytoplasm by the catalytic subunit of protein kinase A (PKAc) (43). Since PKAc is complexed to and inactivated by IB, the stimulus-induced degradation of IB is a prerequisite for the activation of PKAc. Ser-276 is also targeted by the mitogen- and stressactivated protein kinase-1 (MSK1) (44). In this case, the phosphorylation takes place exclusively within the nucleus. Thus, a single serine residue is phosphorylated by different protein kinases in distinct cellular compartments. In both cases, RelA phosphorylation results in an enhanced recruitment of histone acetyltransferases (HATs) such as CBP and p300 and to increased displacement of inhibitory p50/histone deacetylase-1 (HDAC1) complexes from the DNA (45). In addition, Ser-276-phosphorylated RelA can interact with RelB in the nucleus (46). These heterodimers cannot bind DNA, and thus represent inactive NF-B. The physiological consequence is the inhibition of RelB-mediated NF-B activity. RelA can also be phosphorylated at three specic threonine residues. Two are located within the Cterminal TADs (Thr-435 and Thr-505) and one (Thr254) is found at the N-terminal RHD of RelA (47 49). Phosphorylation of each of the two C-terminal amino acids has a suppressive effect on RelA activity. Phosphorylation of Thr-505 is induced by the ARF tumor suppressor (human p14ARF). ARF induces RelA phosphorylation via activation of the ATM/Rad3-related (ATR) checkpoint kinase and checkpoint kinase 1 (Chk1) in an p53-independent fashion. Phosphorylation of Thr-505 results in an increased association of RelA with HDAC1, and this complex formation is responsible for the ARF-induced inhibition of RelA activity. In osteosarcoma cells, an induction of the inhibitory Thr-505 phosphorylation of RelA is observed upon treatment with the chemotherapeutic drug cisplatin. Since it uses the same signaling pathways as ARF, cisplatin can be regarded as an ARF mimic (49). Phosphorylation of Thr-254 leads to the activation of RelA by a novel mechanism. The nuclear peptidylprolyl isomerase Pin1 [protein-interacting NIMA (never in mitosis arrest) 1] binds and isomerizes specic phosphorylated serine or threonine residues that precede a proline residue (50). These Pin1-induced conformational changes have profound functional effects on the target proteins. One of these targets is RelA, which is bound by Pin1 at phosphorylated Thr254-Pro-255. Binding precedes the isomerization of the proline residue. This alteration of RelA structure results in a strong decrease in its afnity to IB, whereas the binding to p50 remains unchanged. Further, phosphorylation-dependent Pin1/RelA interaction increases the nuclear translocation of RelA and, most important, results in stabilization of RelA, as demonstrated by the extreme instability of the T254A mutant of RelA. The counterpart of Pin1 in the regulation of RelA stability is the E3-ubiquitin ligase suppressor of cytokine signaling 1 (SOCS-1), which has been shown to ubiquitinylate RelA, thereby inducing its proteolysis.

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Pin1 and SOCS-1 bind RelA at sites extremely close to one another, making it likely that both proteins compete for the same binding site (50). The role of SOCS-1 in RelA ubiquitinylation was conrmed in a recent study demonstrating that COMMD1 (copper metabolism gene MURR1 domain containing 1) accelerates ubiquitinylation and degradation of RelA through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, cullin 2 (Cul2), and SOCS-1 (ECS(SOCS-1)) (51). COMMD1 binds to Cul2 in a stimulus-dependent manner, thereby strengthening the interaction between SOCS-1 and RelA. The important role of COMMD1 for RelA ubiquitinylation is stressed by the nding that RelA is stabilized in COMMD1-decient cells (51). However, it is still a matter of discussion whether SOCS-1 is the only ubiquitin ligase mediating ubiquitinylation of RelA (7). Functionally, RelA ubiquitinylation is a potential mechanism regulating the termination of NF-B-mediated inammatory responses (52) and may also contribute to the oscillation of nuclear NF-B activity (53).

The nding that the phosphorylation status of Rel proteins, especially of RelA, is the decisive parameter for their association with either activating HATs (e.g., CBP/p300) or suppressing HDACs was a hallmark discovery in the eld of NF-B regulation (45) (Fig. 3A). RelA is also reversibly modied by these enzymes. Upon TNF- stimulation, RelA is acetylated at ve specic lysine residues: Lys-122, -123, -218, -221, and -310 (54, 55). Modication of Lys-218 and Lys-221 weakens the interaction of RelA with IB and increases RelA DNA binding (56). Acetylation of Lys-310 strongly enhances the transactivation potency of RelA (56), whereas deacetylation by histone deacetylase 3 (HDAC3) or SIRT1 (sirtuin 1) inhibits the transcriptional activity of NF-B and augments TNF--mediated apoptosis (56, 57). Stimulation with TPA specically acetylates RelA at Lys-122 and -123. PCAF (p300/CBPassociated factor) and p300 are involved in these posttranslational modications, which decreases RelA-specic DNA binding and facilitates its nuclear export (58). The most important aspect of these studies of

Figure 3. Changes in NF-B-dependent transcription induced by the interaction with GR. A) All cytoplasmic and nuclear kinases that can phosphorylate RelA upon TNF-RI stimulation are shown. Only phosphorylated RelA is able to interact with coactivators such as CBP/p300. The recruitment of such coactivators changes the chromatin structure by histone acetylation and facilitates assembly of the basic transcription apparatus. DNA-bound RelA can induce an activating phosphorylation of RNA polymerase II. B) The interaction of activated, monomeric GR with DNA-bound NF-B results in the active recruitment of corepressors such as HDAC2, whose expression is also up-regulated. The activity of CBP/p300 is suppressed by the GR/NF-B complex. Further, histone 3 methylation adds to the inactive chromatin state. Finally, the activating RNA polymerase II phosphorylation is inhibited. hsp, heat shock protein.

RelA acetylation is that modication of specic lysine residues regulates distinct functions of NF-B. Thus, modication of RelA by site-specic acetylation is another mechanism whereby different signals controlling NF-B activity are integrated. It was recently demonstrated that decreased phosphorylation of Ser-276 or Ser-536 by ectopic expression of enzymatically inactive forms of PKAc/MSK1 (target: Ser-276) or IKK/IKK (target: Ser-536), respectively, drastically reduced RelA acetylation at Lys-310. In line with this, reconstitution of RelA-decient murine embryonic broblasts with mutated forms of RelA (RelA S276A or S536A) resulted in a sharp decrease of RelA acetylation. Reconstitution with wild-type RelA led to an increased assembly of phospho-RelA with p300 and, as a consequence, to an up-regulation of Lys-310 acetylation compared with cells expressing mutant RelA (59). Phosphorylation of DNA-bound RelA at Ser-536 occurs prior to RelA acetylation at lysine 310. In parallel, the repressor SMRT (silencing mediator for retinoic acid and thyroid hormone receptor), which is bound to RelA/p50 heterodimers, is also phosphorylated and thereby derepressed (55). Both phosphorylation events are mediated by nuclear IKK recruited to the chromatin. The derepression of SMRT potentiates RelA acetylation by p300, leading to a further increase in NF-B activity. In line with these ndings, histone deacetylase inhibitors induce phosphorylation and thus the activation of p300 (54).

of RelB is Ser-368 phosphorylation. A murine plasmacytoma cell line lacking endogenous RelB expression was substituted with wild-type and Ser-368-mutated RelB (62). The authors observed that Ser-368 phosphorylation is not required for the nuclear translocation of RelB, but rather appears to be critical for RelB dimerization with the p52 precursor p100. RelB phosphorylated at Ser-368 stabilizes p100, suggesting a role in regulating the noncanonical pathway of NF-B activation. As in the case of the two other RelB phosphorylation sites, the kinase (or kinases) mediating Ser-368 phosphorylation has not been identied (62).

REGULATION OF NF-B ACTIVITY BY MODIFICATIONS OF C-REL Regulation of c-Rel activity is achieved to a large extent by its signal-induced phosphorylation (Fig. 2). An initial hint stressing the importance of c-Rel phosphorylation came from a study reporting that tyrosine phosphorylation can be rapidly induced by G-CSF (granulocyte colony-stimulating factor) in human neutrophils (63). However, neither the protein tyrosine kinase mediating the G-CSF effect nor the target tyrosine residue (or residues) has been identied. Six years later, the complex formation of a 65 kDa serine/ threonine protein kinase with murine c-Rel was reported (64). This kinase apparently has two binding sites and mediated C-terminal phosphorylation of c-Rel. Again, the specic phospho-acceptor sites within c-Rel and the protein kinase responsible for c-Rel phosphorylation have not been identied. Two prominent serine/threonine signaling kinases, PKC and the C isoform of PKA, have been shown to phosphorylate c-Rel (65, 66). Both phosphorylation events have the same result: they enhance the transactivation potential of c-Rel. The target serine/threonine residue of PKA is still unknown, whereas PKC phosphorylates Ser-471 of c-Rel in Jurkat T cells stimulated by TNF-. The signaling pathway leading to this c-Rel modication also involves the PI3K/AKT kinase located upstream of PKC (67). The mutation of either Ser-471 or Ser-460 to a nonphosphorylatable residue resulted in a version of c-Rel with higher transforming potency, whereas the transactivation ability was not inuenced (68). However, the expression of selected genes was differentially affected by mutated vs. wildtype c-Rel, indicating that phosphorylation inhibits the transforming potency of this proto-oncoprotein by enabling it to correctly express target genes. A dysregulated phosphorylation of c-Rel might also contribute to the development of certain human B cell lymphomas. In two patients with different types of B cell lymphoma, point mutations affecting Ser-525 were detected (69). The mutant c-Rel proteins display altered transactivation properties depending on the cell type analyzed, and the potency of mutant c-Rel to transform chicken spleen cells was drastically enhanced.

REGULATION OF NF-B ACTIVITY BY MODIFICATIONS OF RelB RelB is in many respects an unusual member of the Rel/NF-B family. For example, RelB preferentially binds to p50 and p52, although an inhibitory interaction with RelA and homodimerization have been demonstrated. Further, it could be shown that the RelB monomer is an extremely unstable protein that is almost instantly degraded (60). RelB is phosphorylated at Thr-84 and Ser-552 (61) (Fig. 2). These phosphorylation events have functional consequences that are contrary to the effects of RelA phosphorylation. RelB phosphorylation is induced by specic signals and is a prerequisite for the subsequent proteasomal degradation of RelB. The inducing signals are T cell receptor activation by CD3/CD28-specic antibodies or TPA/ionomycin stimulation, which is supposed to mimic T cell receptor activation. At rst sight, induced proteolysis of RelB resembles the signalinduced degradation of the IBs. However, there are also differences between the two processes. The phosphorylation-dependent RelB degradation takes place in at least two distinct steps: an N-terminal processing is followed by the complete proteasomal degradation of the protein. Further, RelB degradation is stimulus specic and cannot be induced by cytokines like TNF- or IL-1 (61). Another prominent post-translational modication
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Phosphorylation of specic sites within c-Rel as well as of RelA appears to be crucial for the resolution of inammation. A clear division of tasks between IKK and IKK was noted in a study analyzing NF-B activation in macrophages during inammatory processes (52). IKK mediates NF-B activation in response to proinammatory cytokines and microbial products, whereas IKK contributes to the termination of inammation by accelerating the turnover of c-Rel as well as RelA in a phosphorylation-dependent manner. Both Rel proteins have been shown to be ubiquitinylated and subsequently proteolytically degraded. This study demonstrated that IKK is able to phosphorylate c-Rel and RelA, and thereby induce their degradation. The serine residue 536 of RelA appears to be one IKK target, whereas the phospho-acceptor site of c-Rel was not identied in detail. It is known only that phosphorylation takes place at the C terminus of c-Rel. Recent studies identied NIK, TBK1, and IKK as c-Rel-specic kinases (70, 71). TBK1 and IKK phosphorylate c-Rel at its C terminus with the functional consequence of an increased nuclear c-Rel accumulation. The degradation of IB is not affected, but rather IKK-mediated phosphorylation leads to dissociation of the IB-c-Rel complex. REGULATION OF NF-B ACTIVITY BY MODIFICATIONS OF p50/NF-B1 Similar to the IBs, p105, the precursor of the Rel protein p50, is site-specically phosphorylated, then polyubiquitinylated and proteolytically degraded in the 26S proteasome. Although it has been known for many years that p50 as well as its p105 precursor are inducibly phosphorylated upon stimulation of various cell types (23), reports about site-specic phosphorylation of the mature p50 and the functional consequences of such a post-translational modication are rare. So far, the nding that the catalytic subunit of PKA (PKAc) can inducibly phosphorylate Ser-337, which is located within the RHD of p50, stands alone. Since p50 has no transactivation domains, the only functional consequence appears to be an enhanced p50 DNA binding (72).

IB-INDEPENDENT REGULATION OF NF-B: CROSS-TALK BETWEEN NF-B AND OTHER PROTEINS Complex formation of NF-B with transcriptional regulators such as AP-1 (activator protein 1), SP1 (specicity protein 1), and C/EBP, termed cross-coupling or cross-talk, is important for the control of NF-B activity. Cross-talk is dened as the interaction between different transcription factors resulting in either the synergistic activation or antagonistic inhibition of gene expression. The interaction of NF-B with AP-1 leads to activation of both transcription factors, whereas NFIB-INDEPENDENT NF-B REGULATION

B/C/EBP complex formation leads to the enhancement of C/EBP activity but to the repression of NF-B via inhibition of RelA phosphorylation (73). A recent study showed that interaction of RelA with breast cancer-associated protein 3 (BCA3) results in the transcriptional repression of NF-B (74). This complex formation is dependent on neddylation of BCA3. Neddylated BCA3 can recruit SIRT1, a histone deacetylase that might be responsible for the suppression of NF-B activity. The binding of NF-B to glucocorticoid receptors (GR), ligand-induced transcription factors belonging to the nuclear receptor superfamily, is an excellent example of the regulation of NF-B activity by crosscoupling to unrelated proteins (75, 76) (Fig. 3B). Glucocorticoids are potent immunosuppressants that work mainly by inhibiting cytokine gene expression. Surprisingly, it could be shown that the DNA binding and transactivating ability of GR are not needed to mediate most of these anti-inammatory effects. It is mainly the interaction with NF-B that nally leads to down-regulation of the transcription of cytokine genes due to GR-mediated NF-B repression. This inhibition results in decreased transactivation potency, DNA binding, and nuclear localization of NF-B. The precise molecular mechanisms mediating the inhibiting effects of glucocorticoids on NF-B vary with the cell type and depend on the availability of specic cofactors, the promoter context of a given target gene, the status of the chromatin, the so-called histone code, and other parameters. The general importance of cytoplasmic mechanisms of GR-mediated NF-B transrepression, such as an glucocorticoid-induced increase in IB levels, is still a matter of controversy (77). In most cases, gene repression by activated GR takes place through interaction with DNAbound NF-B. GR DNA binding and homodimerization are not required for this process (78). Evidence of a completely nuclear mechanism of glucocorticoid repression of specic promoters, such as the IL-8 or the ICAM promoter, came from in vivo footprinting and chromatin immunoprecipitation experiments showing that the pattern of RelA/NF-B binding remains unchanged under repression conditions (79, 80). One crucial nuclear mechanism is based on changes in the chromatin environment of the regulatory elements of the respective genes called chromatin remodeling (75). In the resting cell, DNA is tightly wrapped around a protein core. This chromatin structure is composed of nucleosomes, which are particles consisting of 146 bp DNA associated with an octamer of two molecules each of core proteins (histones H2A, H2B, H3, and H4). This closed chromatin structure can be loosened by modication of the N-terminal tails of the core histones enabling access of RNA polymerase II and the rest of the transcription machinery. These modications include methylation, ubiquitinylation, phosphorylation, and (of special importance) acetylation. It is known that NF-B activated by TNF- or IL-1 can induce histone acetylation preferentially on histone H4

at lysine residues 8 and 12. This takes place at NF-Bresponsive regulatory elements. Upon DNA binding, NF-B recruits large coactivator complexes containing the histone acetylase (HAT) proteins CBP and p300. In overexpression experiments, PCAF was also shown to be critical for the RelA-associated increase in HAT activity. Further, several other HATs are known to interact with NF-B such as transcriptional intermediary factor 2 (TIF-2), aka GR interacting protein 1, and steroid receptor coactivator 1 (SRC-1). In addition to the site-specic acetylation of histone H4, the phosphorylation of histone H3 at Ser-10 is associated with gene induction. Surprisingly, this phosphorylation was found to be mediated by nuclear IKK, which is recruited to the promoters of NF-B-regulated genes in association with CBP and RelA upon stimulation with TNF- (81, 82). The interaction between transcription factors, such as NF-B and GRs, may result in differential effects on histone acetylation/deacetylation (75). Several mechanisms, which are probably not exclusive, are currently discussed. Activated GRs can directly modify the chromatin structure. It is known that low glucocorticoid concentrations are able to repress TNF--induced histone H4 acetylation via direct inhibition of CBP-associated HAT activity. In addition, high concentrations of glucocorticoids lead to an increase in HDAC2 expression and the activated GR can be recruited to activated inammatory gene complexes, switching off NF-Bdriven gene expression (83). Using a bioluminiscence resonance energy transfer assay, it was shown that treatment with the GR antagonist RU486 efciently recruited the corepressor NcoR (nuclear corepressor) to GR (84). These and other ndings led to the conclusion that suppression of NF-B-driven gene expression may involve recruitment of a fully functional corepressor complex that minimally contains HDAC2 and NcoR (76). The GR/NF-B cross-talk can also change the chromatin structure by methylating histone H3. This change of the histone code, the sequence of histone modications within a distinct chromatin area, might be critical for switching off NF-B-mediated inammatory gene transcription. Another level of GR action seems to be the reduction of an activating C-terminal serine phosphorylation of RNA polymerase II induced by DNA-bound NF-B (80). This phosphorylation is mediated by the kinase pTEFb and promotes glucocorticoid-mediated repression (85). This indicates that the GR/NF-B cross-talk has effects downstream of NF-B DNA binding and even downstream of coactivator function (Fig. 3B).

NUCLEOLAR SEQUESTRATION AND DIMER EXCHANGE: NEW CONCEPTS IN IBINDEPENDENT REGULATION OF NF-B The compartmentalization of transcription-associated proteins within nuclear bodies is increasingly recog2650 Vol. 21 September 2007

nized as an important mechanism for the control of gene expression, proliferation, and apoptosis. Two prominent examples are regulation of the p53 tumor suppressor gene by the nucleolar sequestration of its inhibitor MDM2 (86, 87) and the inhibition of c-mycinduced cell cycle progression by sequestration of this proto-oncoprotein to the nucleolus (88). With regard to the NF-B system, NIK (89) and the transacting NF-B-repressing factor (NRF) (90) have been shown to shuttle between the nucleoplasm and nuclear bodies. Furthermore, two prominent nucleolar proteins, NF-B binding protein (NFBP) (91) and nucleophosmin/B32 (92), strongly interact with NF-B, indicating a possible role for nucleolar sequestration of NF-B in regulating NF-B activity. The nal proof of this hypothesis came from a study demonstrating that RelA translocates into the nucleolus of colon cancer cells in response to proapoptotic stimuli like aspirin, serum withdrawal, and UV-C radiation (93). In contrast, RelA is excluded from the nucleolus in response to the cytokines TNF- or TRAIL (TNF-related apoptosis-inducing ligand). Nucleolar RelA sequestration is functionally accompanied by a sharp decrease in NF-B transcriptional activity and a strong increase in apoptosis. An N-terminal motif seems to be responsible for nucleolar translocation of RelA, but the exact molecular mechanism has yet to be identied. The current model suggests the existence of an unknown nuclear cofactor (corepressor) that selectively associates with the cytoplasmic NF-B pool activated by proapoptotic stimuli such as aspirin. A second NF-B population, the basal pool, constitutively drives the transcription of antiapoptotic genes. The induced RelA/corepressor complex does not activate transcription but interacts with basal NF-B; subsequently, all nuclear RelA translocates into the nucleolus. As a consequence, NF-B is in a cellular location different from its target promoters, resulting in a decreased transcription of antiapoptotic genes and nally in apoptosis. In contrast to TNF- and TRAIL, proapototic stimuli induce NF-B with a delay, which might be responsible for the selective association of the hypothetical cofactor with this NF-B pool (93). Dimer exchange at specic NF-B-dependent promoters represents a new and intriguing concept for the modulation of NF-B activity. There are two types of NF-B-regulated promoters. One type is selectively controlled by only one specic dimer, whereas other promoters are regulated by more than one NF-B complex. Using dendritic cell maturation as a model system, it could be demonstrated that rapidly activated dimers like p50/RelA are replaced by more slowly activated dimers such as p52/RelB at a subset of promoters (94). p52/RelB dimers are less stringently controlled by IBs and are less sensitive to resynthesized IBs, enabling them to mediate a sustained activation. Thus, dimer exchange contributes to both the ne-tuning of the NF-B response as well as a prolonged activation at specic NF-B target promoters. Further, it was shown that each NF-B complex has a

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specic transcriptional activity at a specic promoter, which is not simply due to afnity variations of different NF-B dimers. At some promoters, a complete replacement of specic dimers in an ordered sequence was notable, whereas multiple dimers were recruited to others without an obvious temporal order. The occurrence of dimer exchange was also dependent on the applied stimulus. These ndings indicate that the regulated disassembly of existing promoter complexes and the controlled reassembly of new complexes containing other components would make the expression of specic genes responsive to changes in the concentration of Rel proteins as well as other cooperating transcription factors (94). The same research group discovered a mechanism that might promote the exchange of NF-B dimers at specic target genes (95). They found that RelA polyubiquitinylation and subsequent proteasomal degradation is a dominant control mechanism for the posttranscriptional repression of NF-B activity in the absence of IB. This polyubiquitinylation-dependent degradation specically affects DNA-bound RelA. Thus, it also provides an elegant mechanism for how the composition of NF-B dimers at specic promoters can be altered in an ordered, sequential manner.

indications that the regulation of phosphorylation of Rel proteins plays an important role in the pathogenesis of inammatory and malignant diseases (95, 97). Physiological and biological processes controlled by alternative regulatory mechanisms of NF-B are the specic NF-B-dependent gene expression by controlling the transactivating potency of NF-B, proliferation including tumorigenesis, apoptosis, immune responses, inammatory processes, and intracellular viral responses. The ultimate goal of research focusing on physiological effects of NF-B modications has to be the identication of novel targets to interfere with disease processes. Such targets could be enzymes (i.e., protein kinases) known to modify Rel proteins in pathological conditions. For example, the activity of these enzymes could be blocked by specic inhibitory drugs. The prerequisite for such an interference with disease processes is a profound understanding of the physiological role played by each NF-B control mechanism. Progress in this eld of research will become increasingly faster in the near future. The expected discoveries will certainly add to the fascination surrounding the eld of IB-independent regulation of NF-B and will provide new insights into the regulation of NF-B-dependent physiological processes.
The authors thank Dr. Jonathan Lindquist for critical reading of the manuscript. The work described from the authors laboratory was funded by grants of the Deutsche Forschungsgemeinschaft NE608/32 to Ma.N. and NA292/ 73 to Mi.N., respectively. The authors apologize to those colleagues whose work could not be cited due to space limitations.

PROSPECTS AND PREDICTIONS A few years ago most of the regulatory aspects in the NF-B system appeared to be solved. An elegant activation mechanism centering around the signal-induced degradation of the IBs had been elucidated in detail. However, research within the last few years has unraveled a far more sophisticated picture of NF-B activation. For example, IB-independent mechanisms of NF-B regulation exist and their importance for the control of nuclear NF-B activity is increasingly recognized. What can we expect in the future from this exciting research eld? First, new types of post-translational modication of Rel proteins important for regulating NF-B activity will be discovered; one strong candidate is sumoylation. The precise molecular mechanisms underlying the controlled nucleolar sequestration of Rel proteins should be discovered in the near future. The same is true for another exciting concept of NF-B regulation, the dimer exchange at specic promoters. Last, but not least, the research area of microRNAs (miRNAs) promises to become increasingly important for unraveling novel mechanisms regulating NF-B activity independent of IBs. miR-146 is such an inducible miRNA inhibiting NF-B-activating pathways involved in the innate immune response (96). Potential targets of miR-146 are TRAF6 and IRAK1 (interleukin-1 receptor-associated kinase 1). The expression of this miRNA is NF-B-dependent, suggesting a feedback regulatory loop. Analysis of the physiological signicance of alternative mechanisms of NF-B regulation will be another area of intense future research. There are already clear

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Vol. 21

September 2007

The FASEB Journal