NF-κB: Ten Years After

Almost exactly ten years following the first publication on NF-κB (24Sen R. Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences.Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1842) Google Scholar), researchers working on transcriptional regulation by NF-κB/Rel and IκB proteins gathered for the third time to discuss recent developments in the field (Madrid, July 8-10, 1996). The first meeting of its kind was a Howard Hughes workshop at the NIH in November 1992 and the second one a Banbury Conference held at Cold Spring Harbor in October 1993. This year's meeting was organized by R. Bravo (Bristol-Myers Squibb, Princeton) and P. S. Lazo (Universidad de Oviedo) and held at the Juan March Foundation in Madrid, Spain. NF-κB is the prototype of a family of dimeric transcription factors (Figure 1) made from monomers that have approximately 300 amino-acid Rel regions which bind to DNA, interact with each other, and bind the IκB inhibitors (for most recent reviews, see27Verma I.M. Stevenson J.K. Schwarz E.M. Antwerp D.V. Miyamato S. Rel/NF-κB/IκB family intimate tales of association and dissociation.Genes Dev. 1995; 9: 2723-2735Crossref PubMed Google Scholar, 6Baldwin A.S. The NF-κB and IκB proteins new discoveries and insights.Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5379) Google Scholar). The inhibitors all have 5–7 ankyrin (Ank) repeat domains, each about 30 amino acids, which form a unit able to interact with Rel regions. Even before the meeting, it was evident that the importance of Rel-related transcription factors is continuing to grow, as reflected by the ever-increasing number of publications in the field (now, more than 1,200 in total) and the never-ending revelations from a large number of effective laboratories. By the end of the meeting, the participants appreciated that this transcription factor system still is a hot discovery zone and far from having reached a state of clean-up experimentation. The high level of interest in Rel-based transcription factors is founded on their broad role in inducibly and coordinately controlling genes of significant biomedical importance, such as those encoding inflammatory cytokines, chemokines, interferons, MHC proteins, growth factors, cell adhesion molecules, and viruses (reviewed by5Baeuerle P.A. Henkel T. Function and activation of NF-κB in the immune system.Annu. Rev. Immunol. 1995; 12: 141-179Crossref Scopus (4509) Google Scholar). The workshop mainly concentrated on understanding the functional roles of NF-κB and IκB proteins in immune regulation and development, using knock-out and transgenic animals models, and on deciphering the specificities and molecular control mechanisms by which the multiple DNA binding and inhibitory subunits that make up the Rel/Ank family of proteins interact with each other in various cell types. Some of the Rel proteins, notably c-Rel, were first identified by their involvement in abnormal growth of B and T cells (reviewed by13Gilmore T.D. Koedood M. Piffat K.A. White D.W. Rel/NF-κB/IκB proteins and cancer.Oncogene, in press. 1996; Google Scholar). However, they do not readily transform cells upon transfection. An exception is v-Rel which, however, can acutely transform only avian cells in culture. The transforming potential of a RelA splice variant called p65Δ in rodent cells remains a matter of debate (21Narajanan R. Klement J.F. Ruben S.M. Higgins K.A. Rosen C.A. Identification of a naturally occuring splice variant of the p65 subunit of NF-κB.Science. 1992; 256: 367-370Crossref PubMed Scopus (58) Google Scholar, 14Grimm S. Baeuerle P.A. Failure of splicing variant p65Δ of the p65 NF-κB subunit to transform fibroblasts.Oncogene. 1994; 9: 2391-2398PubMed Google Scholar). T. Gilmore (Boston University, Boston) reviewed the properties of v-Rel that contribute to its transforming potential. Weak transactivating potential, DNA binding, and homodimer formation are required whereas interaction with IκB is not critical. In an effort to understand its mechanism of transformation, two v-Rel-interacting proteins were cloned by yeast two hybrid screening and shown to have transactivating potential in yeast. Strong evidence that NF-κB/Rel proteins can be proto-oncogenes in mammals came from studies in Bravo's laboratory. v-Rel expressed in transgenic mice under T cell-specific control (lck proximal promoter) caused early death of animals from multicentric aggressive T cell lymphomas. A similarly expressed C-terminally truncated c-Rel did not have this affect, indicating that v-Rel is not oncogenic simply because it lacks most of the C-terminal transactivation domain of its cellular counterpart c-Rel. The v-Rel-transformed murine T cells were of immature phenotype and dependent on IL-2. In these cells, v-Rel was predominantly associated with p50 and partially with IκB-α. The prevalence of constitutively active v-Rel/p50 may explain the upregulation of NF-κB target genes such as IL-6 and ICAM-1. Crossing the v-Rel transgenic mice with mice overexpressing IκB-α under lck control caused a delay in death from leukemia. In mice with a disrupted p105/p50 gene, T cell leukemia appeared at an earlier stage, suggesting that v-Rel homodimer formation (due to a lack of the alternative p50 heterodimerization partner) may be the key issue in transforming potential, as observed in the chicken system. Aberrant p52 proteins are found in T cell lymphomas, as a result of chromosome rearrangements at the NFKB2 locus (22Neri A. Chang C.C. Lombardi L. Salina M. Corradini P. Maiolo A.T. Chaganti R.S. Dalla-Favera R. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-κB p50.Cell. 1991; 67: 1075-1087Abstract Full Text PDF PubMed Scopus (323) Google Scholar ). p52 is normally produced as p100, an inactive precursor harbouring IκB-like Ank-containing sequences in its C-terminal half. R. Bravo reported on transgenic mice in which the C-terminal portion of p100 is disrupted leading to production of constitutive p52. The phenotype is neonatal lethality, apparently because of reduced food intake. The reason appears to be hyperplasia of the stomach lining. There was no abnormality of B or T cell development but their T cell response was increased while macrophage activities were reduced. Increased constitutive NF-κB activity was detectable in several organs by anti-p52 antibodies. These data suggest a role of p52 in epithelial growth control. R. Bravo also mentioned a partial disruption of p105 that inactivates its C-terminal portion and would lead to the expression of constitutive p50; such mice were apparently healthier than IκB-δ knockouts. A major cause of embryonic death of mice lacking the RelA subunit is a massive apoptosis of liver cells (8Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Emryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB.Nature. 1995; 376: 167-170Crossref PubMed Scopus (1578) Google Scholar), suggesting a role for RelA-containing NF-κB dimers in protecting cells from pro-apoptotic stimuli produced in the embryonic liver, perhaps by resident haematopoetic cells. Data from several presentations supported this notion by showing that the absence or inhibition of NF-κB/Rel in cultured cells potentiated apoptosis in response to TNF or T cell activation. D. Baltimore (MIT, Cambridge) showed that the RelA subunit played such a role in macrophages and a fibroblast line established from RelA−/− mice. Compared to wild type cells, TNF readily killed these cells through TNF receptor type 1 and could be prevented by transfected RelA. A similar phenotype was observed by I. M. Verma (Salk Institute, La Jolla) and D. W. Ballard (Vanderbilt University, Memphis) using cell lines stably transfected with dominant negative IκB-α proteins produced, respectively, by point mutants in all known phosphorylation sites or deletion of the regulatory N-terminus. Stable expression of such IκB mutants would correspond to a knockout of all five NF-κB/Rel subunits as evident from the complete absence of any κB-specific DNA binding activity in extracts from cell lines stimulated with cytokines. Although the contribution of a particular subunit cannot easily be deciphered by this approach, the phenotype of cells with respect to apoptotic resistance was very similar to that of RelA−/− cells. When a dominant negative IκB mutant was expressed in transgenic mice under control of the T cell-specific lck promoter, a loss of CD8+ cells was observed in the thymus (D. W. Ballard). Moreover, the activation-induced apoptosis of T cells derived from the transgenic mice was enhanced. These various results all suggest that pro-apoptotic extracellular signals can also induce NF-κB which in turn probably induces expression of genes that are anti-apoptotic. Viruses that can induce NF-κB could therefore protect against apoptotic elimination of infected cells. There is some evidence, albeit less direct, of a pro-apoptotic aspect of RelA activity. 15Grimm S. Bauer M. Baeuerle P.A. Schulze-Osthoff K. Bcl-2 down-regulates the activity of transcription factor NF-κB induced upon apoptosis.J. Cell. Biol. 1996; 134: 13-23Crossref PubMed Scopus (332) Google Scholar recently showed that serum starvation of 293 cells causes cell death accompanied by the activation of RelA-containing NF-κB. Bcl-2 or a dominant negative RelA mutant could prevent both cell death and κB- dependent reporter gene activation. A pro-apoptotic role of NF-κB/Rel was also suggested by the study of radiation-sensitive fibroblasts from ataxia telangiectasia (AT) patients (A. Dritschilo, Georgetown University, Washington D. C.). Radiation-induced cell apoptosis in AT cells was reduced by a dominant negative IκB-α protein (16Jung M. Zhang Y. Lee S. Dritschilo A. Correction of radiation sensitivity in ataxia telengiectasia by a truncated IκB-α.Science. 1995; 268: 1619-1621Crossref PubMed Scopus (171) Google Scholar). Perhaps RelA can control apoptosis in quite opposite manners depending on both cell type and the nature of the apoptotic stimulus. c-Rel may also play a role in inducing apoptosis, as was earlier suggested by 1Abbadie C. Kabrun N. Bouali F. Smardova J. Stehelin D. Bandenbunder B. Enrietto P.J. High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro.Cell. 1993; 75: 899-912Abstract Full Text PDF PubMed Scopus (217) Google Scholar. C. Gelinas (UMDNJ, Piscataway) observed apoptosis upon expression of c-Rel in a HeLa cell line stably expressing the c-Rel gene under inducible control by the tet repressor. c-Rel-induced apoptosis involved cell cycle arrest at G1/S correlating with inhibition of E2F DNA binding activity, accumulation of hypophosphorylated Rb, inhibition of Cdk2 kinase, and accumulation of transcripts encoding the Cdk inhibitor p21Waf1. It will be interesting to investigate whether the κB motifs in the p21 promoter are functional and can bind c-Rel. It remains to be understood how RelA can protect from apoptosis while its potential dimerization partner c-Rel induces apoptosis. One explanation could be that the κB motifs in pro- and anti-apoptotic genes selectively bind complexes with c-Rel or RelA, respectively. It is also conceivable that overexpression of c-Rel prevents formation of RelA dimer combinations that are required for inducing anti-apoptotic genes. In contrast to c-Rel, its retroviral counterpart v-Rel does not induce cell death. On the contrary, C. Gelinas observed in a tetracycline-dependent system that downregulation of v-Rel causes apoptosis in chicken spleen cells, consistent with results obtained by T. Gilmore using a temperature sensitive v-Rel mutant. To study the impact of the RelA null mutation on the development of the immune system, hematopoietic cells from fetal livers of RelA−/− mice were transferred into lethally irradiated mice (D. Baltimore). The recipient mice survived and developed a functional immune system. T and B cells as well as macrophages appeared to develop normally and showed unaltered responses. However, an increase in granulocytes in peripheral blood and the bone marrow was apparent that developed into an inflammation of distal extremities, such as swelling of paws, suggesting a negative control function of RelA in granulopoiesis. Among all the single knockouts of NF-κB/Rel subunits, c-Rel−/− mice showed a phenotype that is most consistent with the notion that the transcription factor plays a key role in cytokine regulation and proper immune cell responses. S. Gerondakis (Walter and Eliza Hall Institute, Victoria) reviewed the properties of c-Rel−/− mice (18Koentgen F. Grumont R.J. Strasser A. Metcalf D. Li R. Tarlinton D. Gerondakis S. Mice lacking the c-Rel proto-oncogene exhibit defects in lymphocyte proiliferation, humoral immunity and interleukin-2 expression.Genes Dev. 1995; 9: 1965-1977Crossref PubMed Scopus (622) Google Scholar). These fail to mount an effective humoral and cellular immune response to various pathogens, including Leishmania major. Proliferation defects as well as decreased production of cytokines and immunoglobulins were seen in T and B cells, respectively. His studies of macrophages revealed a difference between the properties of resident cells, which killed bacteria poorly, and those elicited by thioglycollate, which showed normal killing. The resident macrophages showed diminished NO production, which correlated with reduced LPS-inducible iNOS expression. While TNF-α production in response to LPS was also reduced, the expression of GM-CSF, G-CSF, and IL-6, three other NF-κB/Rel-regulated genes, was elevated. The animals were more susceptible to influenza virus and Leishmania than wild type animals. RelB knockout mice are characterized by multiorgan inflammation due to infiltration of mixed inflammatory cells accompanied by myeloid hyperplasia and splenomegaly (29Weih F. Carrasco D. Durham S.K. Barton D.S. Rizzo C.A. Ryseck R.P. Lira S.A. Bravo R. Multiorgan inflammation and hematopoetic abnormalities in mice with targeted disruption of RelB, a member of the NF-κB/Rel family.Cell. 1995; 80: 331-340Abstract Full Text PDF PubMed Scopus (692) Google Scholar). R. Bravo reported that T cells were involved because crossing the RelB knockout mice with mice deficient in B and T cells (RAG-1 knockout) or T cells alone (Nur77/N10TG knockout) suppressed their RelB−/− phenotype. A RelB/p105 double knockout showed an increased mortality due to a more penetrant phenotype despite the absence of B cells in infiltrates. RelB−/− mice could not clear infections with Listeria monocytogenes or lymphocytic choriomeningitis virus. Serum IgM and IgE levels were elevated while antigen-specific IgG production was down, indicating impaired T helper cell function. NFKB1 knockout mice, unable to produce p105 or its cleavage product p50, show multiple defects in B cell function (25Sha W.C. Liou H.C. Tuomanen E. Baltimore D. Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses.Cell. 1995; 80: 321-330Abstract Full Text PDF PubMed Scopus (1030) Google Scholar). J. Caamano from Bravo's laboratory reported on a knockout of NFKB2, the gene that produces p100 and its cleavage product p52. Like the NFKB1 knockout mice, these develop normally. However, again like the animals lacking p50, these mice have a serious defect in the B cell lineage. They have fewer B cells and even fewer mature B cells, a problem that becomes more severe as the mice age. The spleen and lymph nodes have diffuse B cell areas with no follicles or germinal centers. B cell proliferation was significantly impaired in response to LPS, anti-δ-dextran, or CD40 ligand. The phenotype of p100/p52−/− mice was similar to that of a Bcl-3 knockout (see below), supporting a functional relationship between the two proteins. None of the individual knockouts showed a defect in B cell development but experiments in preB cell lines with an overexpressed dominant negative IκB-α—able to prevent the presence of all nuclear Rel proteins— gave a different answer (D. W. Ballard). Transcription and rearrangement of the germ line Igκ genes induced in these cells by LPS was entirely abrogated by the IκB. The same dominant IκB-mediated suppression was found in a temperature-sensitive Abelson virus transformant which ordinarily is induced to transcribe and rearrange κ by temperature shift. These results point to a key role for the κB site in the intronic κ enhancer (the other κ enhancer, 3′ to the coding region, is unresponsive to LPS). No effect on RAG-1 and -2 levels was evident. Either the cell lines show a different dependence on NF-κB than do cells in intact mice, or when multiple Rel proteins are eliminated the results on B cell development will be different from those with single knockouts. To study the effect of the IκB-α dominant mutant in T cells, the protein was expressed under control of the proximal lck promoter in the thymus of transgenic mice. Thymocytes had most of their NF-κB/Rel proteins complexed to and inactivated by the signal-resistant IκB mutant. As a consequence, there were fewer peripheral T cells and their proliferative response to a variety of T cell-activating conditions was totally blocked and could not be restored by exogenous IL-2. This provided good evidence that the NF-κB/Rel signaling pathway is as important as the NFAT/AP-1 pathway in T cell activation. A potentially important aspect of NF-κB function emerged from the study of demethylation of the Igκ locus in a construct dependent on the intronic κ enhancer (T. Wirth, ZMBH, Heidelberg). In the S107 myeloma cell line, there is no nuclear content of Rel-related proteins and no demethylation of this Igκ construct. However, demethylation could be restored upon transfection of a RelB expression vector that puts the RelB protein into the nucleus. This suggests that the intronic enhancer can mediate demethylation but, as seen for transcription and rearrangement, only if its κB site is occupied by a Rel protein. None of the null mutants for the five known murine Rel subunits showed a detectable defect in embryonic development. The same was true for the few double knockouts produced to date. It was thus surprising to learn from the presentation of L. D. Kerr (Vanderbilt University, Memphis) that c-Rel appeared to be important for proper limb bud development in mice. Using a c-Rel promoter-driven LacZ gene, high transgene expression was seen in the developing limb bud, correlating with elevated c-Rel transcript levels. Normal murine limb bud development was impaired if a recombinant Adenovirus expressing a transdominant negative IκB-α gene was injected (it is relevant to note that all Rel proteins would have their action blocked in this way, not just c-Rel). Evidence was presented that in embryos FGF-4 could induce c-Rel, which may regulate the expression of the homeodomain protein Msx-1. The Msx-1 promoter contains functional κB motifs that may be required for activation of the gene through c-Rel in response to morphogens. A. Israel (Pasteur Institute, Paris) pointed out that no limb bud staining was evident in transgenic mice where the LacZ gene is driven by three κB sites. A c-Rel/RelA/RelB tripple knockout may be the best means to investigate a role of κB-dependent gene activation in early vertebrate development. Bcl-3 is an unusual IκB protein in that it can not only inhibit nuclear NF-κB complexes but can bind to p50 and p52 dimers on DNA and provide the complexes with transactivating activity (9Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. The oncoprotein Bcl-3 directly transactivates through κB motifs via association with DNA-binding p50B homodimers.Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 11Fujita T. Nolan G.P. Liou H.C. Scott M.L. Baltimore D. The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-κB p50 homodimers.Genes Dev. 1993; 7: 1354-1363Crossref PubMed Scopus (339) Google Scholar). Its highest expression is found in lymph nodes and spleen. Mice deficient for Bcl-3 showed normal development and seemed healthy until challenged with either Toxoplasma gondii (U. Siebenlist, NIH, Bethesda) or Staphylococcus pneumonia (I.M. Verma). In contrast to wild type mice, the null mutants could not clear these pathogens and rapidly succumbed to the infections. U. Siebenlist reported that, while many immunological parameters of B and T cells seem normal, less cells could be extracted from Bcl-3−/− lymph nodes, which lacked germinal centers and B cell follicles. Apart from these histologically-apparent deficiencies, TH2 cells from null mutants could not produce significant amounts of γ-interferon. This explained the reduced NO production in macrophages and a decreased CTL activity, showing that Bcl-3 plays a critical role in antigen-specific T cell immunity. S. Whiteside and A. Israel (Pasteur Institute, Paris) reported on the characterization of IκB-ε, a novel protein with six ankyrin repeats belonging to the IκB family. Its cDNA was isolated through a yeast two-hybrid screen with p52 as bait. By using two different start codons, in principle it can be translated into 38 and 55 kDa species with N-terminal domains of different length. IκB-ε shares many properties with IκB-α: its 2.2 kB transcript is induced by phorbol esters; it is a potent inhibitor of κB-dependent gene expression; the constitutively phosphorylated protein undergoes signal-induced hyperphosphorylation and subsequent degradation which is blocked by proteasome inhibitors and antioxidants; and, lastly, its N-terminus harbors potential phosphorylation sites similar to those in IκB-α (see Figure 2). In collaboration with N. Rice (NCI, Frederick), IκB-ε was found to be a major IκB protein associated with RelA in all cell lines investigated. In THP-1 macrophages, it even constituted more than 90% of the IκB proteins. A major difference from IκB-α and β was that IκB-ε was exclusively found in association with RelA and c-Rel. Provided there are stimuli that selectively degrade or spare IκB-ε, a selective regulation of a certain NF-κB–Rel dimer combination (c-Rel–RelA and respective homodimers) would be possible. NF-κB plays an important role in the antiviral response as a virus-inducible transcriptional regulator of β-interferon, MHC class I, and inflammatory cytokine genes (reviewed by5Baeuerle P.A. Henkel T. Function and activation of NF-κB in the immune system.Annu. Rev. Immunol. 1995; 12: 141-179Crossref Scopus (4509) Google Scholar). Hence it is not too surprising that, during evolution, a large virus acquired an IκB-like protein which could attenuate the cell's antiviral response, although the virus might then have to worry about the cell's apoptotic response. Such a viral IκB-like protein was found in the African Swine Fever Virus, a pathogen receiving particular attention in Spain perhaps because of the dietary importance of marvelous sausages and air-dried ham from pork (M. Fresno, Universidad Autonoma de Madrid). The large DNA virus encodes a 28.2 kDa multiple Ank domain-containing polypeptide (A238L) with functional characteristics of a bona fide IκB protein. In virus-infected cells, A238L was found associated with NF-κB, and cells become deficient for NF-κB activation in response to TNF. Until recently only two NF-κB/Rel proteins, Dif and Dorsal, and one IκB family member, Cactus, were known to occur in insects. This number is now increased in both families through the discovery of a novel p100/p105-like protein, amusingly called Relish, with both a Rel homology domain and an Ank repeat domain (D. Hultmark, Stockholm University). Relish was identified as a gene that was highly upregulated upon bacterial infection of Drosophila. It is also expressed in early embryos, suggesting a possible role in embryogenesis as well, and is now awaiting further biochemical and functional characterization. Screening of a λ expression library with p52 as probe led to the re-isolation of a κB-specific DNA-binding protein, called RκB (C. Scheidereit, MDC, Berlin), originally isolated by G. Nabel and colleagues by screening a library with a DNA probe containing the IL2 receptor α-chain κB site (2Adams B.S. Leung K. Hanley E.W. Nabel G.J. Cloning of RκB, a novel DNA-binding protein that recognizes the interleukin-2 receptor a chain κB site.New Biol. 1991; 3: 1063-1073PubMed Google Scholar). Despite its similar DNA binding specificity, RκB is structurally completely unrelated to NF-κB/Rel proteins. C. Scheidereit showed that RκB has a C-terminal inhibitory domain, cannot bind IκB proteins but selectively associates with p50, p52, and c-Rel, presumably via a coiled-coil region. The central paradigm of NF-κB activation, which involves the removal of IκB proteins from a cytoplasmic complex with NF-κB (3Baeuerle P.A. Baltimore D. Activation of DNA-binding in an apparently cytoplasmic precursor of the NF-κB transcription factor.Cell. 1988; 53: 211-217Abstract Full Text PDF PubMed Scopus (779) Google Scholar4Baeuerle P.A. Baltimore D. IκB a specific inhibitor of the NF-κB transcription factor.Science. 1988; 242: 540-546Crossref PubMed Scopus (1647) Google Scholar) (Figure 1), has now been subjected to very detailed molecular analyses. The major pathway used by a variety of stimuli to activate NF-κB involves the phosphorylation of IκB-α at its regulatory N-terminus on serines 32 and 36, an event leading to subsequent conjugation with ubiquitin and proteasome-mediated degradation of the inhibitor (reviewed in28Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Rel/NF-κB/IκB family intimate tales of association and dissociation.Genes Dev. 1996; 9: 2723-2735Crossref Scopus (1621) Google Scholar). It is now evident that IκB-α (reviewed by27Verma I.M. Stevenson J.K. Schwarz E.M. Antwerp D.V. Miyamato S. Rel/NF-κB/IκB family intimate tales of association and dissociation.Genes Dev. 1995; 9: 2723-2735Crossref PubMed Google Scholar), the novel IκB-ε (S. Whiteside), and the Drosophila IκB homologue Cactus (S. Wasserman, Southwestern Medical Center, Dallas) all undergo a phosphorylation-induced proteolytic degradation in response to extracellular stimuli. As shown in Figure 2, the regulatory phosphorylation sites in these four IκB proteins show a high degree of similarity, suggesting that related kinases may modify the inhibitors in response to stimulation of cells. Mutational analyses showed earlier that both sites in IκB-α need to be phosphorylated for efficient degradation. An IκB kinase that modifies both sites has not been characterized in detail yet. A recent study by 10Chen Z. Parent L. Maniatis T. Site-specific phosphorylation of IκB-α by a novel ubiquitin-dependent kinase activity.Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar described such a kinase as being part of a large 700 kDa complex and requiring polyubiquitination for activity. A similarly sized complex was described by F. Mercurio (Signal Pharmaceutics, San Diego), while D. Baltimore found a 300 kDa kinase complex with an activity for the serines in the N-terminal region of IκB-α that was not evident if they were replaced by threonine residues. In the two latter cases, no requirement of the kinase for ubiquitin was evident. W. C. Greene (Gladstone Institute, San Francisco) showed that the mitogen-activated kinase pp90rsk can phosphorylate IκB-α on serine 32 (but not serine 36) and is able to physically associate with IκB-α in intact cells. Other IκB proteins were not modified by pp90rsk. This finding could explain why transient overexpression of kinases upstream of pp90rsk, such as MAPK, MEKK-1, and MEKK-3, cause IκB-α degradation and NF-κB activation (U. Siebenlist). The observation that pp90rsk is activated by PMA, okadaic acid, and LPS but not by TNF or HTLV-I Tax suggests that this kinase may be predominantly used by mitogenic signaling pathways. It remained an open question how serine 36 would be phosphorylated in such a scenario but such a phosphorylation is apparently necessary for efficient degradation of IκB-α. An attractive idea is that the synergistic effect between calcium- and protein kinase C (PKC)/Ras/Raf-dependent pathways in NF-κB activation observed in T cells may have its molecular basis in the action of two distinct kinases on IκB-α. Data from U. Siebenlist showed that a constitutive Raf kinase could potently activate NF-κB only in combination with a constitutive form of calcineurin. Future studies will be needed to examine whether the contribution from the PKC/Ras/Raf kinase pathway is via serine 32 phosphorylation by pp90rsk whereas the calcium/calcineurin pathway leads to phosphorylation on serine 36. Small Ras-like GTPases belonging to the Rho family are known regulators of protein kinases, such as JNK/SAPKs and PAK, and other enzymes including phospholipases and phosphotidylinositol kinases. R. Perona (Instituto de Investigaciones Biomedicas, Madrid) showed that three members of the Rho family, RhoA, Cdc42, and Rac1, potently activated NF-κB upon transient transfection in various cell types. The induction by the Rho proteins selectively depleted IκB-α and was not affected by trans-dominant negative mutants of Ras and Raf. The Rho GTPases seemed to play an important role in inflammatory cytokine signaling because the activation of an NF-κB reporter gene by TNF was impaired by transdominant negative forms of Cdc42 and RhoA though not Rac1. A number of studies have shown that IκB-α is constitutively phosphorylated in its C-terminal PEST domain by casein kinase II. S. Wasserman showed that Cactus also undergoes this modification and he purified and identified the responsible kinase as the Drosophila casein kin