David Baltimore

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-κB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3′ UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.

Studies on mice deficient in nuclear factor kappa B (NF-κB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-α (TNF-α)-dependent genes. Treatment of RelA-deficient (RelA−/−) mouse fibroblasts and macrophages with TNF-α resulted in a significant reduction in viability, whereas RelA+/+ cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA−/− fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-α. These results have implications for the treatment of inflammatory and proliferative diseases.

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

To characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all Ig variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the kappa enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the kappa enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells.

Two cDNAs were isolated whose dimerized products bind specifically to a DNA sequence, kappa E2, located in the immunoglobulin kappa chain enhancer. Both cDNAs share a region of extensive identity to the Drosophila daughterless gene and obvious similarity to a segment in three myc proteins, MyoD, and members of the Drosophila achaete-scute and twist gene family. The homologous regions have the potential to form two amphipathic helices separated by an intervening loop. Remarkable is the stringent conservation of hydrophobic residues present in both helices. We demonstrate that this new motif plays a crucial role in both dimerization and DNA binding.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.

In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210 bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210 bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160 gag /v- abl , which induces acute lymphomas. To determine whether P210 bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210 bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210 bcr/abl . These results demonstrate that P210 bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-κB is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-κB with a 60- to 70-kilodalton inhibitory protein (IκB). The fractionated inhibitor can inactivate NF-κB from various sources—including the nuclei of phorbol ester-treated cells—in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-κB and IκB was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase C, cause dissociation of a cytoplasmic complex of NF-κB and IκB by modifying IκB. This releases active NF-κB which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.

NF-κB is a nuclear protein, found only in cells that transcribe immunoglobulin light chain genes, that interacts with a defined site in the κ immunoglobulin enhancer. This protein can be induced in pre-B cells by stimulation with bacterial lipopolysaccharide (LPS). The induction involves a posttranslational activation, and the combined action of LPS and cycloheximide causes a superinduction. An active phorbol ester also induces this factor, and with kinetics more rapid than those for LPS stimulation. Phorbol-ester-mediated induction of NF-κB was observed in a T cell line (Jurkat) and a nonlymphoid cell line (HeLa), and is therefore not restricted to B-lymphoid cells. We interpret these results to indicate that factors that control transcription of specific genes in specific cells may be activated by posttranslational modification of precursor factors present more widely.

Single-cell mouse embryos were infected in vitro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter . Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene(s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte–specific promoters expressed high levels of GFP only in the appropriate cell types. We have also generated transgenic rats that express GFP at high levels, suggesting that this technique can be used to produce other transgenic animal species.

NF-kappa B, which consists of two polypeptides, p50 (M(r) 50K) and p65/RelA (M(r) 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-kappa B. Disruption of the relA locus leads to embryonic lethality at 15-16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for I kappa B alpha and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-kappa B-regulated pathways.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.

Nuclear localization of the transcriptional activator NF-kappaB (nuclear factor kappaB) is controlled in mammalian cells by three isoforms of NF-kappaB inhibitor protein: IkappaBalpha, -beta, and - epsilon. Based on simplifying reductions of the IkappaB-NF-kappaB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-kappaB activation by the coordinated degradation and synthesis of IkappaB proteins. The model demonstrates that IkappaBalpha is responsible for strong negative feedback that allows for a fast turn-off of the NF-kappaB response, whereas IkappaBbeta and - epsilon function to reduce the system's oscillatory potential and stabilize NF-kappaB responses during longer stimulations. Bimodal signal-processing characteristics with respect to stimulus duration are revealed by the model and are shown to generate specificity in gene expression.

The mammalian inflammatory response to infection involves the induction of several hundred genes, a process that must be carefully regulated to achieve pathogen clearance and prevent the consequences of unregulated expression, such as cancer. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that has also been linked to cancer. However, the relationship between inflammation, innate immunity, and miRNA expression is just beginning to be explored. In the present study, we use microarray technology to identify miRNAs induced in primary murine macrophages after exposure to polyriboinosinic:polyribocytidylic acid or the cytokine IFN-beta. miR-155 was the only miRNA of those tested that was substantially up-regulated by both stimuli. It also was induced by several Toll-like receptor ligands through myeloid differentiation factor 88- or TRIF-dependent pathways, whereas up-regulation by IFNs was shown to involve TNF-alpha autocrine signaling. Pharmacological inhibition of the kinase JNK blocked induction of miR-155 in response to either polyriboinosinic:polyribocytidylic acid or TNF-alpha, suggesting that miR-155-inducing signals use the JNK pathway. Together, these findings characterize miR-155 as a common target of a broad range of inflammatory mediators. Importantly, because miR-155 is known to function as an oncogene, these observations identify a potential link between inflammation and cancer.

Le NF-kB est un mediateur pleiotropique du controle des genes inductibles et tissu-specifiques. Cette revue indique comment intervient le NF-kB et quels sont les systemes dans lesquels il joue un role, tels que les messages intracellulaires, l'activation des cellules C, la regulation de la cytokinine, ou encore l'utilisation par les virus

Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene begins at a single nucleotide, but no TATA box is present. We have identified a 17 bp element that is sufficient for accurate basal transcription of this gene both in vitro and in vivo. This motif, the initiator (Inr), contains within itself the transcription start site. Homology to the Inr is found in many TATA-containing genes, and specific mutagenesis influences both the efficiency and accuracy of initiation. Moreover, in the presence of either a TATA box or the SV40 21 bp repeats, a greatly increased level of transcription initiates specifically at the Inr. Thus, the Inr constitutes the simplest functional promoter that has been identified and provides one explanation for how promoters that lack TATA elements direct transcription initiation.

NF-κB was discovered 30 years ago as a rapidly inducible transcription factor. Since that time, it has been found to have a broad role in gene induction in diverse cellular responses, particularly throughout the immune system. Here, we summarize elaborate regulatory pathways involving this transcription factor and use recent discoveries in human genetic diseases to place specific proteins within their relevant medical and biological contexts.

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3′ untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.

In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.

The Src homology 3 (SH3) region is a small protein domain present in a very large group of proteins, including cytoskeletal elements and signaling proteins. It is believed that SH3 domains serve as modules that mediate protein-protein associations and, along with Src homology 2 (SH2) domains, regulate cytoplasmic signaling. The SH3 binding sites of two SH3 binding proteins were localized to a nine- or ten-amino acid stretch very rich in proline residues. Similar SH3 binding motifs exist in the formins, proteins that function in pattern formation in embryonic limbs of the mouse, and one subtype of the muscarinic acetylcholine receptor. Identification of the SH3 binding site provides a basis for understanding the interaction between the SH3 domains and their targets.

NF-kappa B, a heterodimeric transcription factor composed of p50 and p65 subunits, can be activated in many cell types and is thought to regulate a wide variety of genes involved in immune function and development. Mice lacking the p50 subunit of NF-kappa B show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production. Mice lacking p50 are unable effectively to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae, but are more resistant to infection with murine encephalomyocarditis virus. These data support the role of NF-kappa B as a vital transcription factor for both specific and nonspecific immune responses, but do not indicate a developmental role for the factor.

The RAG-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when introduced into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a RAG-1 genomic probe that detects a single 6.6-7.0 kb mRNA species in transfectants and pre-B and pre-T cells. RAG-1 genomic and cDNA clones were biologically active when introduced into NIH 3T3 cells. Nucleotide sequencing of human and mouse RAG-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence identity. RAG-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. RAG-1 may activate V(D)J recombination indirectly, or it may encode the V(D)J recombinase itself.

Although a small decrease in survival and increase in tumor incidence was observed in ATR(+/-) mice, ATR(-/-) embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR(-/-) blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR(-/-) cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53.

The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.

Foxp3(+) regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR-146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of the IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity.

Over the past few years, the transcription factor nuclear factor (NF)-kappaB and the proteins that regulate it have emerged as a signaling system of pre-eminent importance in human physiology and in an increasing number of pathologies. While NF-kappaB is present in all differentiated cell types, its discovery and early characterization were rooted in understanding B-cell biology. Significant research efforts over two decades have yielded a large body of literature devoted to understanding NF-kappaB's functioning in the immune system. NF-kappaB has been found to play roles in many different compartments of the immune system during differentiation of immune cells and development of lymphoid organs and during immune activation. NF-kappaB is the nuclear effector of signaling pathways emanating from many receptors, including those of the inflammatory tumor necrosis factor and Toll-like receptor superfamilies. With this review, we hope to provide historical context and summarize the diverse physiological functions of NF-kappaB in the immune system before focusing on recent advances in elucidating the molecular mechanisms that mediate cell type-specific and stimulus-specific functions of this pleiotropic signaling system. Understanding the genetic regulatory circuitry of NF-kappaB functionalities involves system-wide measurements, biophysical studies, and computational modeling.

To avoid detection by CTL, HIV encodes mechanisms for removal of class I MHC proteins from the surface of infected cells. However, class I downregulation potentially exposes the virus-infected cell to attack by NK cells. Human lymphoid cells are protected from NK cell cytotoxicity primarily by HLA-C and HLA-E. We present evidence that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E. We then identify the residues in HLA-C and HLA-E that protect them from HIV downregulation. This selective downregulation allows HIV-infected cells to avoid NK cell–mediated lysis and may represent for HIV a balance between escape from CTL and maintenance of protection from NK cells. These results suggest that subpopulations of CTL and NK cells may be uniquely suited for combating HIV.

ATM, the gene mutated in the inherited human disease ataxia-telangiectasia, is a member of a family of kinases involved in DNA metabolism and cell-cycle checkpoint control. To help clarify the physiological roles of the ATM protein, we disrupted the ATM gene in mice through homologous recombination. Initial evaluation of the ATM knockout animals indicates that inactivation of the mouse ATM gene recreates much of the phenotype of ataxia-telangiectasia. The homozygous mutant (ATM-/-) mice are viable, growth-retarded, and infertile. The infertility of ATM-/- mice results from meiotic failure. Meiosis is arrested at the zygotene/pachytene stage of prophase I as a result of abnormal chromosomal synapsis and subsequent chromosome fragmentation. Immune defects also are evident in ATM-/- mice, including reduced numbers of B220+CD43- pre-B cells, thymocytes, and peripheral T cells, as well as functional impairment of T-cell-dependent immune responses. The cerebella of ATM-/- mice appear normal by histologic examination at 3 to 4 months and the mice have no gross behavioral abnormalities. The majority of mutant mice rapidly develop thymic lymphomas and die before 4 months of age. These findings indicate that the ATM gene product plays an essential role in a diverse group of cellular processes, including meiosis, the normal growth of somatic tissues, immune development, and tumor suppression.

To examine the DNA sequences involved in regulation of kappa immunoglobulin gene expression, an assay was developed whereby transcription of a cloned, rearranged kappa gene could be detected following its transfection into antibody-secreting mouse myeloma cells. In a transient expression assay, RNA transcripts initiating 20 to 25 bp before the AUG translation initiator of the kappa gene were readily detected. The constant region (C kappa) and part of the J kappa-C kappa intron was then deleted from the rearranged gene and a downstream SV40 polyadenylylation signal was provided. No transcription was detected from the deleted gene upon transfection into myeloma cells, suggesting that after rearrangement the kappa variable region promoter is activated by sequences more than 2.6 kb downstream of J kappa. The rearranged kappa gene was also transfected into a nonlymphoid line of monkey cells and RNA transcripts were detected. Transcription in these cells, however, was at a relatively lower level than in myeloma cells, did not depend on the presence of downstream sequences, and initiated from a site about 10 bp downstream of the myeloma site. These experiments define a sequence downstream of the promoter that is necessary for accurate transcription of the kappa chain gene.

The mammalian inflammatory response is a rapid and complex physiological reaction to noxious stimuli including microbial pathogens. Although inflammation plays a valuable role in combating infection, its dysregulation often occurs in people and can cause a variety of pathologies, ranging from chronic inflammation, to autoimmunity, to cancer. In recent years, our understanding of both the cellular and molecular networks that regulate inflammation has improved dramatically. Although much of the focus has been on the study of protein regulators of inflammation, recent evidence also points to a critical role for a specific class of noncoding RNAs, called microRNAs (miRNAs), in managing certain features of the inflammatory process. In this review, we discuss recent advances in our understanding of miRNAs and their connection to inflammatory responses. Additionally, we consider the link between perturbations in miRNA levels and the onset of human inflammatory diseases.

Chronic myelogenous leukemia (CML) is a human disease associated with a consistent chromosomal translocation that results in sequences from the c- abl locus on chromosome 9 being fused to sequences in a breakpoint cluster region ( bcr ) on chromosome 22. CML cells have two novel products: an 8.5-kilobase RNA transcript containing both abl and bcr and a 210-kilodalton phosphoprotein (P210) recognized by v- abl -specific antisera. To test whether the P210 is the product of the novel 8.5-kilobase bcr / abl fusion transcript, antibodies were prepared against c- abl and bcr determinants. By using these reagents and v- abl -specific antisera, it was demonstrated that the P210 in CML cells is indeed the protein product of the 8.5-kilobase transcript. By analogy to the gag / abl fusion protein of Abelson murine leukemia virus, the replacement of amino terminal c- abl sequences by bcr sequences in P210 may create a transforming protein involved in CML. A 190-kilodalton phosphoprotein that is a candidate for the normal bcr protein was identified in both HeLa and K562 cells.

Excessive or inappropriate activation of the immune system can be deleterious to the organism, warranting multiple molecular mechanisms to control and properly terminate immune responses. MicroRNAs (miRNAs), ∼22-nt-long noncoding RNAs, have recently emerged as key posttranscriptional regulators, controlling diverse biological processes, including responses to non-self. In this study, we examine the biological role of miR-146a using genetically engineered mice and show that targeted deletion of this gene, whose expression is strongly up-regulated after immune cell maturation and/or activation, results in several immune defects. Collectively, our findings suggest that miR-146a plays a key role as a molecular brake on inflammation, myeloid cell proliferation, and oncogenic transformation.

Mammalian noncoding microRNAs (miRNAs) are a class of gene regulators that have been linked to immune system function. Here, we have investigated the role of miR-155 during an autoimmune inflammatory disease. Consistent with a positive role for miR-155 in mediating inflammatory responses, Mir155−/− mice were highly resistant to experimental autoimmune encephalomyelitis (EAE). miR-155 functions in the hematopoietic compartment to promote the development of inflammatory T cells including the T helper 17 (Th17) cell and Th1 cell subsets. Furthermore, the major contribution of miR-155 to EAE was CD4+ T cell intrinsic, whereas miR-155 was also required for optimum dendritic cell production of cytokines that promoted Th17 cell formation. Our study shows that one aspect of miR-155 function is the promotion of T cell-dependent tissue inflammation, suggesting that miR-155 might be a promising therapeutic target for the treatment of autoimmune disorders.

<h2>Abstract</h2> The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas.

MicroRNA-155 (miR-155) has emerged as a critical regulator of immune cell development, function, and disease. However, the mechanistic basis for its impact on the hematopoietic system remains largely unresolved. Because miRNAs function by repressing specific mRNAs through direct 3'UTR interactions, we have searched for targets of miR-155 implicated in the regulation of hematopoiesis. In the present study, we identify Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) as a direct target of miR-155, and, using gain and loss of function approaches, show that miR-155 represses SHIP1 through direct 3'UTR interactions that have been highly conserved throughout evolution. Repression of endogenous SHIP1 by miR-155 occurred following sustained over-expression of miR-155 in hematopoietic cells both in vitro and in vivo, and resulted in increased activation of the kinase Akt during the cellular response to LPS. Furthermore, SHIP1 was also repressed by physiologically regulated miR-155, which was observed in LPS-treated WT versus miR-155(-/-) primary macrophages. In mice, specific knockdown of SHIP1 in the hematopoietic system following retroviral delivery of a miR-155-formatted siRNA against SHIP1 resulted in a myeloproliferative disorder, with striking similarities to that observed in miR-155-expressing mice. Our study unveils a molecular link between miR-155 and SHIP1 and provides evidence that repression of SHIP1 is an important component of miR-155 biology.

The POU domain: a large conserved region in

Abstract Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.

It was previously shown that the chromosome 19 breakpoint of the t(1;19)(q23;p13.3) translocation, found in human pre-B cell acute lymphoblastic leukemias, is within the E2A transcription factor gene on chromosome 19. A cell line with this translocation contains two novel chimeric mRNAs, both with the same 5'E2A sequences but with different lengths of 3' sequence from a previously unrecognized gene dubbed prl, located on chromosome 1. The chimeric RNAs encode a protein that lacks 171 amino acids of E2A, including its DNA binding and dimerization motifs, but have instead a homeobox-related sequence from prl. Therefore, the production of a chimeric E2A-Prl protein may contribute to the acute lymphoblastic phenotype by directly altering the expression of genes normally responsive to the Prl homeoprotein.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Correction of gene defects in human somatic cells by targeting as has been used in murine embryonic stem cells (1, 2) has been precluded by the low spontaneous rate of gene targeting (3). However, creation of a DNA double-stranded break (DSB) in the genomic target (DSB-GT) can stimulate homologous recombination by over 1000-fold (4). We can rapidly and quantitatively measure gene targeting by correcting a mutation in a green fluorescent protein (GFP) gene that has been stably integrated into the genome (5) (fig. S1). With an optimized GFP gene targeting system, the introduction of a DSB by I–Sce I (Sce) stimulated GT >40,000-fold and the absolute rate of gene targeting reached 3 to 5% (fig. S2). Such a system, however, depends on the prior introduction of a Sce binding site into the target gene and cannot be used for endogenous genes. Chimeric nucleases (CNs) have the potential to create sequence-specific DSBs (6). CNs—fusions between zinc finger binding DNA binding domains and the endonuclease domain of Fok I—can sitespecifically cleave naked DNA in vitro (6), extrachromosomal DNA in Xenopus oocytes (7), and chromosomal DNA in Drosophila (8). CNs work as dimers, and their efficiency depends on the spacing and orientation of the zinc finger binding sites with respect to the length of the amino acid linker between the DNA binding and endonuclease domains (7, 9). QQR is an artificial zinc finger DNA binding domain that recognizes the sequence 5′-GGGGAAGAA-3′ with nanomolar affinity (10). We modified QQR chimeric nucleases (QQR-CNs) (7, 9) (Fig. 1A) and tested whether they stimulated gene targeting (Fig. 1B). The background rate of gene targeting was 0.71 events per million transfected cells (fig. S1C). QQRL18-CN stimulated gene targeting 17-fold on target QQR6 and 260-fold on target QQR8 (Fig. 1B). QQRL0-CN did not stimulate gene targeting on target QQR8, but it was as efficient as Sce in stimulating gene targeting by over 2000-fold on target QQR6 (Fig. 1B). QQRL18-CN showed some preference for an 8–base pair (bp) spacing between binding sites whereas QQRL0-CN preferred 6-bp spacing. Thus, removing the linker between the zinc spacfinger and the nuclease domains increased the activity and specificity of the fusion protein in mammalian cells. As controls, we showed that the CNs did not stimulate gene targeting if (i) they lacked a nuclear localization signal, (ii) there was a single binding site rather than an inverted repeat binding site in the target, and (iii) the cognate binding site was changed. Thus, homodimers of CNs are potent stimulators of gene targeting in human somatic cells.

Double-stranded RNAs approximately 21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

<h2>Abstract</h2> To examine germ line genes of the heavy chain variable region (V_H) that might contribute to formation of antibodies of the NP^b family, we have derived cDNA clones from two hybridomas making NP^b antibodies. One, B1–8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG_2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the V_H region, had very different D segments and had identical J segments (J₂). A set of closely related germ line V_H genes was then cloned from a partial Eco RI library of C57BI/6 DNA. By comparing the germ line V_H regions to the cDNA V_H regions, we identified seven potential candidates for encoding the V_H regions of NP^b antibodies. The seven V_H regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1–8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1–8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other V_H genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their V_H regions from the same germ line gene. Certain of the sequenced V_H genes contain crippling mutations; the repertoire of germ line V_H genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hybridization.

A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322. Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus. Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment. However, the miR-155-induced GM populations displayed pathological features characteristic of myeloid neoplasia. Of possible relevance to human disease, miR-155 was found to be overexpressed in the bone marrow of patients with certain subtypes of acute myeloid leukemia (AML). Furthermore, miR-155 repressed a subset of genes implicated in hematopoietic development and disease. These data implicate miR-155 as a contributor to physiological GM expansion during inflammation and to certain pathological features associated with AML, emphasizing the importance of proper miR-155 regulation in developing myeloid cells during times of inflammatory stress.

The His274-->Tyr274 (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007-2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness. Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by "permissive" mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.

A model of reverse transcription has been devised by which the detailed architecture of ten molecular structures is predicted. The model includes a number of novel features for which experimental evidence is presented. First, growing minus DNA strand is copied from the viral RNA only up to a position about 150 nucleotides from the 5' end of the RNA. Second, plus-strand DNA, after being copied from approximately 600 nucleotides at the 5' end of the minus-strand DNA, then transcribes the first approximately 20 nucleotides of the tRNApro primer (which is covalently attaced to the 5' end of the minus DNA strand). The 3' ends of the minus and plus DNA probably form a hybrid through the homology conferred by the primer binding site sequences. Third, the minus and plus DNA strands are elongated in a continuous fashion resulting in a linear double-stranded DNA molecule containing a 600 nucleotide direct repeat at both ends. The most of the features of the model have experimental support, and it appears to provide a credible description of reverse transcription.

Individual protein-binding sites within the mouse immunoglobulin heavy chain and kappa light chain gene enhancers were altered, making it possible to examine the functional role of the sites during transcription. The E motifs, which bind factors that are present in many if not all cells, mostly behave as transcriptional activating sites. The only known heavy chain enhancer site that binds a lymphocyte-specific factor, the "octamer" site, plays a critical role in transcription but only in a truncated form of the enhancer. In the full enhancer, no one site is crucial because of an apparent functional redundancy. The site in the kappa enhancer that binds a factor specific to mature B cells, kappa B, was crucial to the constitutive activity of the enhancer in B cells. This factor is also inducible in pre-B cells, and the site was necessary for inducibility of the kappa enhancer. Thus, the sites defined by protein binding are important for the functional activity of immunoglobulin enhancers, with the sites that bind proteins restricted in their cellular distribution playing the most important roles.

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65.

The Fas death receptor can activate the Jun NH2-terminal kinase (JNK) pathway through the receptor-associated protein Daxx. Daxx was found to activate the JNK kinase kinase ASK1, and overexpression of a kinase-deficient ASK1 mutant inhibited Fas- and Daxx-induced apoptosis and JNK activation. Fas activation induced Daxx to interact with ASK1, which consequently relieved an inhibitory intramolecular interaction between the amino- and carboxyl-termini of ASK1, activating its kinase activity. The Daxx-ASK1 connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.

The activation dynamics of the transcription factor NF-κB exhibit damped oscillatory behavior when cells are stimulated by tumor necrosis factor–α (TNFα) but stable behavior when stimulated by lipopolysaccharide (LPS). LPS binding to Toll-like receptor 4 (TLR4) causes activation of NF-κB that requires two downstream pathways, each of which when isolated exhibits damped oscillatory behavior. Computational modeling of the two TLR4-dependent signaling pathways suggests that one pathway requires a time delay to establish early anti-phase activation of NF-κB by the two pathways. The MyD88-independent pathway required Inferon regulatory factor 3–dependent expression of TNFα to activate NF-κB, and the time required for TNFα synthesis established the delay.

The P210bcr/abl protein is associated with virtually every case of human chronic myelogenous leukemia. Unlike the related P160gag/v-abl oncogene product of Abelson murine leukemia virus, P210bcr/abl does not transform NIH 3T3 fibroblasts. To assess whether P210bcr/abl might transform hematopoietic cell types, retroviral constructs encoding P210bcr/abl were used to infect the bone marrow-derived interleukin 3-dependent Ba/F3 cell line. As for P160gag/v-abl, cell lines expressing P210bcr/abl were growth factor independent and tumorigenic in nude mice. No evidence for autocrine production of interleukin 3 by factor-independent cell lines was found. These experiments establish that P210bcr/abl can transform hematopoietic cell types to tumorigenicity.

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.

Research Article1 February 1995free access Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. K. Saksela K. Saksela Rockefeller University, New York, NY 10021. Search for more papers by this author G. Cheng G. Cheng Rockefeller University, New York, NY 10021. Search for more papers by this author D. Baltimore D. Baltimore Rockefeller University, New York, NY 10021. Search for more papers by this author K. Saksela K. Saksela Rockefeller University, New York, NY 10021. Search for more papers by this author G. Cheng G. Cheng Rockefeller University, New York, NY 10021. Search for more papers by this author D. Baltimore D. Baltimore Rockefeller University, New York, NY 10021. Search for more papers by this author Author Information K. Saksela1, G. Cheng1 and D. Baltimore1 1Rockefeller University, New York, NY 10021. The EMBO Journal (1995)14:484-491https://doi.org/10.1002/j.1460-2075.1995.tb07024.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding. Previous ArticleNext Article Volume 14Issue 31 February 1995In this issue RelatedDetailsLoading ...

The mRNA isolated from B lymphocyte tumor cell lines directs synthesis of two forms of μ heavy chain, one with a molecular weight of 67K and one of 64K. When these cell lines are converted to IgM-secreting cells by fusion with a myeloma cell, the 64K form of μ predominates; thus it is designated μs (μ-secreted). The 67K form correlates with the presence of surface IgM; thus it is designated μm (μ-membrane). Cells that make both forms of μ chain have two mRNAs, one of 2.4 kb that encodes μs and one of 2.7 kb that encodes μm. The difference between the μs and μm mRNAs can be localized to their 3′ ends by hybridizing 32P-cDNA copies of the mRNA to a cloned copy of μs mRNA, treating the mixtures with SI nuclease, and resolving the nuclease-resistant duplexes by electrophoresis. By probing the separated species of RNA with a DNA copy of the 3′ untranslated region of μs mRNA, it was shown that the 3′ ends of the two μ mRNAs do not cross-hybridize. The difference between the two RNAs was mapped to the 3′ edge of the Cμ4 domain. Apparently two separate 3′ terminal sequences for μ mRNA are encoded in the genome, one that specifies an amino acid sequence appropriate for membrane-binding and a second that is involved in secretion. At different stages of immunocyte development, different μ mRNAs predominate: μm during the lymphocyte stages and μs during the secretion stages.

A Src homology 3 (SH3) region is a sequence of approximately 50 amino acids found in many nonreceptor tyrosine kinases and other proteins. Deletion of the SH3 region from the protein encoded by the c-abl proto-oncogene activates the protein's transforming capacity, thereby suggesting the participation of the SH3 region in the negative regulation of transformation. A complementary DNA was isolated that encoded a protein, 3BP-1, to which the SH3 region of Abl bound with high specificity and to which SH3 regions from other proteins bound differentially. The sequence of the 3BP-1 protein is similar to that of a COOH-terminal segment of Bcr and to guanosine triphosphatase-activating protein (GAP)-rho, which suggests that it might have GAP activity for Ras-related proteins. The 3BP-1 protein may therefore be a mediator of SH3 function in transformation inhibition and may link tyrosine kinases to Ras-related proteins.

The interplay between proteins of the NF-kappa B/rel and I kappa B families is a tightly regulated process that ensures appropriate responses to specific environmental and developmental signals. Various mechanisms are utilized in regulating NF-kappa B/rel and I kappa B activities, some unique to this transcription factor system. All of these regulatory strategies converge towards one purpose, namely the controlled nuclear translocation of activated NF-kappa B/rel protein complexes. The variety of rel-related and ankyrin repeat containing subunits makes regulation of this system both rich and complicated.

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.

The complete 7410 nucleotide sequence of poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.

The world of animal viruses appears to offer an unfathomable diversity of specimens but, as the molecular biology of the replication of many viruses has been studied, a pattern of behavior has emerged. The viruses can be divided into classes, each of which has its own method of transmitting its genetic information from one generation to the next and its own style of expressing its genetic information. Although in some cases the data are still fragmentary it is possible to outline the behavior of these systems and to place them in a formal scheme. In this paper I will present such a scheme and I will discuss in some detail the behavior of three viral systems which have been investigated in my laboratory. Furthermore, I will discuss some of the implications of the existence of these viral systems in the context of the behavior of normal cells.

The DNA binding activities of some basic region and putative helix-loop-helix (bHLH)-containing transcriptional factors can be inhibited by the Id protein. Because Id contains the HLH motif for dimerization but not the basic amino acid region for DNA binding, heterodimers of Id with bHLH transcriptional factors may not bind to DNA. We have isolated and characterized the gene and cDNA clones for a new Id protein, designated Id2. The Id2 protein contains a helix-loop-helix motif similar to that of the previously described Id protein (referred to here as Id1), but the two proteins are different elsewhere. Id1 and Id2 are encoded by two unlinked genes, as shown by chromosome mapping. The two Id proteins have similar inhibitory activities. They selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3, but not that of the other set, including TFE3, USF, and AP4. The Id proteins also homodimerize poorly. Expression of both Id genes is down-regulated during differentiation in a variety of cell types.

Despite tremendous efforts, development of an effective vaccine against human immunodeficiency virus (HIV) has proved an elusive goal. Recently, however, numerous antibodies have been identified that are capable of neutralizing most circulating HIV strains. These antibodies all exhibit an unusually high level of somatic mutation, presumably owing to extensive affinity maturation over the course of continuous exposure to an evolving antigen. Although substantial effort has focused on the design of immunogens capable of eliciting antibodies de novo that would target similar epitopes, it remains uncertain whether a conventional vaccine will be able to elicit analogues of the existing broadly neutralizing antibodies. As an alternative to immunization, vector-mediated gene transfer could be used to engineer secretion of the existing broadly neutralizing antibodies into the circulation. Here we describe a practical implementation of this approach, which we call vectored immunoprophylaxis (VIP), which in mice induces lifelong expression of these monoclonal antibodies at high concentrations from a single intramuscular injection. This is achieved using a specialized adeno-associated virus vector optimized for the production of full-length antibody from muscle tissue. We show that humanized mice receiving VIP appear to be fully protected from HIV infection, even when challenged intravenously with very high doses of replication-competent virus. Our results suggest that successful translation of this approach to humans may produce effective prophylaxis against HIV.

The DNA binding protein NF-kappa B has been implicated in gene regulation in B and T lymphocytes. We have found that NF-kappa B also has a central role in virus induction of human beta-interferon (beta-IFN) gene expression. A critical virus-inducible element of this gene, PRDII, behaves interchangeably with the NF-kappa B binding site from the Ig kappa enhancer in both B lymphocytes and virus-infected fibroblasts. Single base substitutions that impair inducibility of the beta-IFN gene in vivo also reduce the binding of NF-kappa B to PRDII in vitro. Virus infection potently activates the binding and nuclear localization of NF-kappa B and, in pre-B lymphocytes, results in the expression of both the beta-IFN gene and the Ig kappa gene. The wide variety of cell types in which beta-interferon can be induced and the divergent set of gene induction processes involving NF-kappa B suggest that this transcription factor plays a broad role in gene regulation as a mediator of inducible signal transduction.

Caveolae are flask-shaped non-clathrin-coated invaginations of the plasma membrane. In addition to the demonstrated roles for caveolae in potocytosis and transcytosis, caveolae may regulate the transduction of signals from the plasma membrane. Transformation of NIH 3T3 cells by various oncogenes leads to reductions in cellular levels of caveolin, a principal component of the protein coat of caveolae. The reduction in caveolin correlates very well with the size of colonies formed by these transformed cells when grown in soft agar. Electron microscopy reveals that caveolae are morphologically absent from these transformed cell lines. These observations suggest that functional alterations in caveolae may play a critical role in oncogenic transformation, perhaps by disrupting contact inhibition in transformed cells.

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5' end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cpro and 3Dpol. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cpro or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production.

Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

We have investigated RNA recombination among poliovirus genomes by analyzing both intratypic and intertypic recombinant crosses involving the same defined genetic markers. Sequence analysis of the recombinant junctions of 13 nonsibling intertypic recombinants showed that intertypic RNA recombination is not site-specific, nor does it require extensive homology between the recombining parents at the crossover site. To discriminate between breaking-rejoining and copy choice mechanisms of RNA recombination, we have inhibited the replication of the recombining parents independently and found opposite effects on the frequency of genetic recombination in intratypic crosses. The results strongly support a copy choice mechanism for RNA recombination, in which the viral RNA polymerase switches templates during negative strand synthesis.

More than a decade ago, the transcriptional activator NF-κB was described as a protein that bound to a specific DNA site in the intronic enhancer of the immunoglobulin κ light chain gene (16Sen R. Baltimore D. Cell. 1986; 47: 921-928Abstract Full Text PDF PubMed Scopus (1464) Google Scholar). Following the cloning of genes encoding the p50 and p65 subunits of NF-κB, it became evident that both subunits are members of the larger NF-κB/Rel family of transcriptional regulator proteins. Since its initial description, our view of the role of NF-κB in immune and inflammatory responses has broadened significantly (for reviews, see1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). NF-κB regulation is part of a cellular response system to many different noxious stimuli. NF-κB is activated by a vast number of agents including cytokines like tumor necrosis factor α (TNFα) and interleukin-1 (IL-1), bacterial LPS, viral infection and expression of certain viral proteins like Tax of human T-cell leukemia virus, (HTLV-1), antigen receptor cross-linking of T and B cells, calcium ionophores, phorbol esters, UV radiation, free radicals, endoplasmic reticulum overloading, and others (for reviews, see17Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1660) Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). The genes regulated by the NF-κB family of transcription factors are diverse and include those involved in immune function, inflammatory response, cell adhesion, and growth control (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar). Recently, the activation of NF-κB has also been linked to the regulation of cell death (2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). NF-κB was initially believed to be lymphoid-specific because of its constitutive presence in the nuclei of mature B cells. In almost all other cells, however, NF-κB is sequestered in the cytoplasm by tightly bound inhibitory proteins called IκBs (17Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1660) Google Scholar). In the family of IκBs, the most important appear to be IκBα, IκBβ, and the newly discovered IκBε. Many of the signals known to activate NF-κB result in phosphorylation and subsequent degradation of the IκBs, allowing NF-κB to translocate into the nucleus and activate target genes. Early studies implicated the phosphorylation of IκB as a central event of this activation and identified potential kinases that were able to phosphorylate the inhibitor protein in vitro (17Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1660) Google Scholar, 1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar). A significant step toward understanding the mechanism of phosphorylation and degradation of IκB was the mapping of the sites phosphorylated in response to NF-κB inducers. Using site-directed mutagenesis, both serine residues S32 and S36 in IκBα were implicated in IκB phosphorylation and degradation in response to TNFα, phorbol 12-myristate 13-acetate (PMA) and ionomycin, as well as a number of other known NF-κB stimuli (17Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1660) Google Scholar, 1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar). Interestingly, replacement of S32 and S36 by threonine residues significantly decreases phosphorylation and degradation of the IκBα protein (5DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). Another member of the IκB family, IκBβ, is phosphorylated at homologous sites, S19 and S23, upon stimulation with extracellular agents, although with kinetics slower than those of IκBα phosphorylation (5DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). There are homologous putative phosphorylation sites on IκBε as well (2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). In vivo, phosphorylation of IκB occurs when it is complexed with NF-κB and does not cause dissociation of the complex. Rather, it signals ubiquitination of IκB, which in turn leads to proteasome-mediated degradation of the inhibitor, releasing free NF-κB (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). This understanding of the sites and roles of phosphorylation has allowed a systematic search for the kinase(s) responsible for IκB phosphorylation, a major component of the signal transduction pathways leading to NF-κB activation. In 1996, Maniatis' laboratory was the first to report the identification of a high molecular weight kinase complex that specifically phosphorylated IκBα at S32 and S36 (3Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). The 700 kDa complex was purified from an unstimulated HeLa cell cytoplasmic extract and required ubiquitin and the ubiquitination enzymes for activity. Although it was shown that a component of the complex is ubiquitinated in vitro and that this event is required for activating the kinase, the target of ubiquitination remains unclear. Recently, the long efforts of many researchers in the identification of the IκB kinase(s) have been remarkably successful in the molecular cloning and functional analysis of components of an IκB kinase complex (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar). One approach that led to the molecular identification of two polypeptides of a cytokine-induced IκB kinase complex was the direct biochemical purification of an activity induced by TNFα, which specifically phosphorylates IκBα at S32 and S36 (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar). The inducible kinase activity was found in a complex reported to be 500–900 kDa, the B inase or IKK. The IKK complex is composed of several polypeptides, two of which, 85 and 87 kDa in size, copurify with the IκB kinase activity on several columns (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar). Through peptide sequencing, the 85 kDa polypeptide was identified as a previously cloned serine-threonine kinase called CHUK (4Connelly M.A. Marcu K.B. Cell. Mol. Biol. Res. 1995; 41: 537-549PubMed Google Scholar). The 87 kDa protein was cloned using the same approach (13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar). Overall, the two polypeptides are 52% identical. They contain an amino-terminal catalytic domain and several putative protein interaction motifs, including a leucine zipper and a helix-loop-helix domain at their carboxyl terminus (Figure 1). In light of its new function, CHUK has been renamed IKKα and the second polypeptide has been designated IKKβ. A convergent approach, which successfully led to the independent discovery of IKKα, used the known signal transduction pathways downstream of TNF and IL-1 receptors to screen for interacting components. TNF and IL-1 activate NF-κB via distinct families of cell-surface receptors (Régnier et al., 1997 and references therein; Figure 2). However, both pathways utilize members of the TNF receptor–associated factor (TRAF) family of adaptor proteins as signal transducers. The TRAF proteins share homology at their carboxyl terminus domain, but their binding properties and activities differ. For example, whereas TRAF2 participates in the NF-κB activation by TNF, TRAF6 is involved in NF-κB activation by IL-1 (Figure 2). Recently, it was shown that these different pathways converge at the NF-κB-inducing kinase, NIK (12Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1164) Google Scholar). NIK has homology to the MAP kinase kinase kinase (MAP3K) family and was first identified by its interaction with TRAF2 (12Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1164) Google Scholar). NIK activates NF-κB when overexpressed and kinase-inactive mutants of NIK act as dominant-negative inhibitors for both TNF- and IL-1-mediated NF-κB activation. In a yeast two-hybrid screen for NIK-interacting proteins14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar identified IKKα, the same subunit of the kinase complex found by others to phosphorylate IκBα on serines S32 and S36. By searching for IKKα-related kinases18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar identified IKKβ. The recent reports assessed the specificity of the IKKα- and IKKβ-associated kinase activities by in vitro phosphorylation assays. Immunoprecipitates of epitope-tagged IKKα and IKKβ, produced either by translation in reticulocyte lysate or expression from transfected plasmids in mammalian cell lines, were employed to phosphorylate IκBα or IκBβ. Based on this assay, it was concluded that an activity associated with IKKα phosphorylates both S32 and S36 of IκBα with the same efficiency (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar), whereas the phosphorylation of IκBβ seems to be less efficient (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar), and takes place mainly at S23 (14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar). IKKβ-associated kinase activity seems to be more potent in general (13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar) and phosphorylates S19 and S23 of IκBβ equally well (18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar). In agreement with previous in vivo data, the IKK complex has a strong preference for serine residues. Replacing S32 and S36 of IκBα protein with threonine residues significantly decreased the efficiency of phosphorylation (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar). The in vitro results were confirmed by in vivo experiments where the transfection of one of the catalytic subunits of the IKK complex, IKKα, into a cell line with low endogenous IκB kinase activity increased the rate of phosphorylation and degradation of IκBα (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar). Another line of evidence that both IKKα and IKKβ are involved in TNF and IL-1 signaling pathways comes from experiments where anti-sense IKKα (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar) or kinase-inactive IKKα or IKKβ were used to inhibit NF-κB transcriptional activation mediated by TNF and IL-1, or the known downstream components such as TRAF2, TRAF6, or NIK (14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar). However, it should be pointed out that the extent to which the kinase-inactive mutants inhibit NF-κB activation differs between studies. First, whereas Régnier et al. and Woronicz et al. report that catalytically inactive versions of both IKKα and IKKβ independently block TNF- and IL-1-induced NF-κB-dependent gene activation, Mercurio et al. and Zandi et al. observe little inhibitory effect for the IKKα kinase mutant. This may be due to differences in the mutation that renders the kinase inactive (lysine 44 was mutated to alanine in the former studies, and methionine in the latter). Second, both Mercurio et al. and Zandi et al. report a potent inhibition of nuclear translocation of p65 by the IKKβ mutant, in TNF-treated cells. However, the effect of the IKKα mutant in this assay seems to vary. It is possible that these differences reflect the sensitivity of the assay, or they may indicate a more potent effect of IKKβ. However, it appears that both IKKα and IKKβ are essential contributors to the IKK activity. The fact that the IKK complex activity is rapidly stimulated by TNF, IL-1, or PMA and the kinetics of activation match those of IκBα phosphorylation and degradation in intact cells (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar) suggests that IKK is a critical kinase complex in vivo. The mechanism by which the complex becomes active in cells is not yet clear. The IKK activity appears sensitive to treatment with the phosphatase PP2A (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar), suggesting that phosphorylation may control its activity. Coexpression of IKKα with NIK seems to lead to the phosphorylation of IKKα and to an increase in its associated kinase activity (14Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar). However, more evidence is needed to conclude whether NIK is directly phosphorylating IKKα. IKKα and IKKβ can undergo both homotypic and heterotypic interactions as indicated by immunoprecipitation experiments (13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar), and their oligomerization appears to be mediated by the leucine zipper motif (18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar, 19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar; Table 1). Mutational analyses were employed to further assess the role of the motifs present in IKKα/β proteins, as well as a first attempt to understand their regulation. The helix-loop-helix domain seems to be important for the kinase activity (19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar; Table 1) but does not affect the interaction between the two subunits, or between IKKα/β and NIK (19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar; Table 1). Both IKKα and IKKβ contain a consensus MAP kinase kinase activation loop motif (SxxxS). Interestingly, mutations of this motif appear to affect mainly the activity of IKKβ (13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar; Table 1).Table 1Mutational Analyses of IKKα and IKKβSubunitMutationEffectReferenceIKKαIn the MAP kinase kinase motif (SxxxS)SS > EEMinimal enhancement of kinase activity. Low induction of13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholarnuclear translocation of p65.SS > AANo inhibition of TNF-induced translocation of p65.13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google ScholarIn the leucine zipper regionGreatly decreased kinase activity. Decreased association with IKKβ.19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google ScholarIn the helix-loop-helix regionGreatly decreased kinase activity. Still able to associate with IKKβ.19Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google ScholarIKKβIn the MAP kinase kinase motif (SxxxS)SS > EEIncreased kinase activity. Induced nuclear translocation of p65.13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google ScholarSS > AABlocked TNF-induced translocation of p65.13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google ScholarDeletion of the leucine zipper regionNo interaction with IKKα/β, but interaction with NIK unaffected.18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google ScholarDeletion of the helix-loop-helix regionNo effect on interaction with either IKKα/β or NIK.18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 281: 866-870Crossref Scopus (1068) Google Scholar Open table in a new tab These data suggest that IKKα and IKKβ are crucial for IκB phosphorylation. However, it is still unclear whether these kinases directly phosphorylate IκB. The in vitro studies that apparently show that IKKα or IKKβ can specifically phosphorylate the key serines in IκB have used kinases made either by overexpression in mammalian cells or in vitro translation in reticulocyte lysate. Because of the affinity of the kinases for other components of the IKK complex, especially for NIK, the in vitro analyses may have used IKKα/β complexed to other proteins. It is therefore possible that IKKα/β are not the kinases that directly phosphorylate IκB but rather that they participate in the activation of a kinase that has the true IκB specificity. Identification of the other subunits of the IKK complex and reconstitution of the IκB phosphorylation pathway in vitro from pure components will resolve this issue. The relationship between the initially described 700 kDa IκB kinase complex of 3Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar and the IKK complex characterized by others remains unclear. In particular, Chen et al. found that ubiquitin and ubiquitination enzymes are necessary for kinase activation; however, no requirement for a ubiquitination step in the activation of IKK has been described. It is possible that ubiquitination and phosphorylation may constitute alternative pathways of activation of an IκB kinase complex. Thus, the TNF-induced IKK complex may already be activated, possibly by phosphorylation, making the ubiquitination dispensable. By altering the manner in which the cell extracts are prepared, an IκB kinase complex, similar to the 700 kDa ubiquitination-inducible one, was reported to be activated by TNF treatment of the cells, with the same kinetics as IKK (10Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). The same complex isolated from unstimulated cells could alternatively be activated in vitro by addition of mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1), a MAP3K-related kinase of the c-Jun N terminus kinase (JNK) pathway (10Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar; Figure 2). In cells, overexpression of MEKK1 led to phosphorylation of IκBα at S32/S36 (10Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). The effect of dominant-negative MEKK1 mutants on the inhibition of NF-κB activation pathway is still ambiguous (8Hirano M. Osada S. Aoki T. Hirai S. Hosaka M. Inoue J. Ohno S. J. Biol. Chem. 1996; 271: 13234-13238Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 11Liu Z-g. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). Interestingly, the IKK complex contains MEKK1 as one of its tightly associated subunits, together with another protein involved in the MAP cascade, MAP kinase phosphatase-1 (13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B. Li J.W. Young D. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 281: 860-866Crossref Scopus (1852) Google Scholar). However, the functional significance of the presence of MAP kinase family proteins in the IKK remains to be established. Thus, only molecular identification of the components of the various complexes will precisely establish the relationship between the kinase activities found to phosphorylate the IκB. These recent studies elucidate the mechanism of IκB phosphorylation by a cytokine-induced kinase complex, which is able to phosphorylate the IκB at the sites critical for its degradation, causing the subsequent translocation and activation of NF-κB. However, it is still unclear whether all of the diverse stimuli known to activate NF-κB also lead to the activation of IKKα and IKKβ. Two recent reports suggest that the mitogen-activated ribosomal S6 protein kinase, pp90rsk, functions as an IκB kinase (7Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Crossref PubMed Scopus (182) Google Scholar, 15Schouten G.J. Vertegaal A.C.O. Whiteside S.T. Israël A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (207) Google Scholar; Figure 2). pp90rsk is activated by phorbol ester, a known inducer of NF-κB, associates in vivo with IκBα, and phosphorylates it mainly at S32 both in vivo and in vitro. Moreover, a dominant-negative form of pp90rsk inhibits IκBα degradation in response to mitogenic stimuli (15Schouten G.J. Vertegaal A.C.O. Whiteside S.T. Israël A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (207) Google Scholar). NF-κB activation can also be triggered by double-stranded RNA (dsRNA), and the double-stranded RNA–dependent protein kinase (PKR) can phosphorylate IκB in vitro (1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5569) Google Scholar). Furthermore, it was shown that in cells lacking PKR, there is a defect in NF-κB activation in response to interferon and dsRNA (9Kumar A. Yang Y.-L. Flati V. Der S. Kadereit S. Deb A. Haque J. Reis L. Weissmann C. Williams B.R.G. EMBO J. 1997; 16: 406-416Crossref PubMed Scopus (315) Google Scholar). However, the phosphorylation sites on IκB targeted by PKR and the mechanism of this pathway remain to be established. These results suggest that IKKα/β may not be unique integrators of the NF-κB response, and that certain stimuli may follow other pathways to IκB phosphorylation and NF-κB activation. The interesting observation that IKKα and IKKβ differ in their phosphorylation efficiency between IκBα and IκBβ inhibitory proteins, and possibly even between phosphorylation sites on the same molecule, raises the intriguing possibility that the tight regulation of IκB degradation may be achieved by a network of kinases, with different regulation and different preferences for the IκB family members. The identification of the IKKα and IKKβ kinases is a major step forward in the 10 year effort to understand NF-κB regulation. However, it is by no means the end of the story—at a minimum, identifying the other constituents of the IKK complex will help illuminate the mechanisms involved in control of NF-κB activity. It is likely that a screen for specific IKK inhibitors, led by the pharmaceutical industry, will provide new modulators of inflammatory responses. Such inhibitors could be very valuable as probes for the functions of the individual components of the IKK complex.

The structure of immunoglobulin-related gene was analyzed in individual Abelson murine leukemia virus (A-MuLV)-transformed lymphoid cell lines. Essentially all of these lines, whether immunoglobulin-containing or null, had DNA rearrangements in the vicinity of the JH regions on both chromosomes as well as deletions of at least 5 kb of DNA 5' to JH. None of these lines, however, except rare light chain producers, had detectable rearrangement at either their kappa or lambda light chain loci. In contrast to A-MuLV-transformants derived from bone marrow. Those from early fetal liver frequently contained more than two and sometimes 12 or more distinct, rearranged JH-containing fragments. Cellular subclones derived from these lines had a subset, usually two, of the fragments found in the parent line. Therefore, heavy chain gene rearrangement appears to precede that of light chain gene rearrangement and is still continuing in certain cultured A-MuLV transformants.

The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

A Cre/lox-conditional mouse line was generated to evaluate the role of ATR in checkpoint responses to ionizing radiation (IR) and stalled DNA replication. We demonstrate that after IR treatment, ATR and ATM each contribute to early delay in M-phase entry but that ATR regulates a majority of the late phase (2-9 h post-IR). Double deletion of ATR and ATM eliminates nearly all IR-induced delay, indicating that ATR and ATM cooperate in the IR-induced G2/M-phase checkpoint. In contrast to the IR-induced checkpoint, checkpoint delay in response to stalled DNA replication is intact in ATR knockout cells and ATR/ATM and ATR/p53 double-knockout cells. The DNA replication checkpoint remains intact in ATR knockout cells even though the checkpoint-stimulated inhibitory phosphorylation of Cdc2 on T14/Y15 and activating phosphorylation of the Chk1 kinase no longer occur. Thus, incomplete DNA replication in mammalian cells can prevent M-phase entry independently of ATR and inhibitory phosphorylation of Cdc2. When DNA replication inhibitors are removed, ATR knockout cells proceed to mitosis but do so with chromosome breaks, indicating that ATR provides a key genome maintenance function in S phase.

Transcription factors belonging to the NF-kappa B family are controlled by inhibitory I kappa B proteins, mainly I kappa B alpha and I kappa B beta. Apparently normal at birth, I kappa B alpha-/- mice exhibit severe runting, skin defects, and extensive granulopoiesis postnatally, typically dying by 8 days. Hematopoietic tissues from these mice display elevated levels of both nuclear NF-kappa B and mRNAs of some, but not all, genes thought to be regulated by NF-kappa B. NF-kappa B elevation results in these phenotypic abnormalities because mice lacking both I kappa B alpha and the p50 subunit of NF-kappa B show a dramatically delayed onset of abnormalities. In contrast to hematopoietic cells, I kappa B alpha-/- embryonic fibroblasts show minimal constitutive NF-kappa B, as well as normal signal-dependent NF-kappa B activation that is concomitant with I kappa B beta degradation. Our results indicate that I kappa b beta, but not I kappa B alpha, is required for the signal-dependent activation of NF-kappa B in fibroblasts. However, I kappa B alpha is required for the postinduction repression of NF-kappa B in fibroblasts. These results define distinct roles for the two forms of I kappa B and demonstrate the necessity for stringent control of NF-kappa B.

Circular double-stranded DNA produced after infection of mouse cells with Abelson murine leukemia virus (A-MuLV) was isolated and cloned in the phage vector Charon 21A. The resulting clones of the A-MuLV genome show homology to the ends of Moloney MuLV and to a 3.5 kb central region containing sequences unique to Abelson virus. A 2.3 kb restriction fragment containing only A-MuLV-specific sequences was subcloned in the plasmid vector pBR322 and used as a probe for the cellular gene that had been acquired by the virus. DNA from all inbred mouse lines examined contains an identical region of homology spread out over 11 to 20 kb. The cellular gene contains intervening sequences which are lacking in the viral genome. Rat, Chinese hamster, rabbit, chicken and human DNA also show homology to the viral probe.

The inflammatory response plays out over time in a reproducible and organized way after an initiating stimulus. Here we show that genes activated in cultured mouse fibroblasts in response to the cytokine tumor necrosis factor could be categorized into roughly three groups, each with different induction kinetics. Although differences in transcription were important in determining the grouping of these genes, differences in mRNA stability also exerted a strong influence on the temporal order of gene expression, in some cases overriding that of transcriptional control elements. Transcripts of mRNA expressed early had abundant AU-rich elements in their 3' untranslated regions, whereas those expressed later had fewer. Thus, mRNA stability and transcriptional control, two intrinsic characteristics of genes, control the kinetics of gene expression induced by proinflammatory cytokines.

CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-α (TNF-α) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-α receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.

IL-2 is an important growth and survival factor for T lymphocytes but also sensitizes these cells to Fas-mediated activation-induced cell death (AICD). The molecular basis of these different effects of IL-2 was studied by introducing wild-type and mutant forms of the IL-2 receptor beta (IL-2Rbeta) chain that lacked specific signaling capacities into receptor-deficient T cells by retroviral gene transfer. Activation of Stat5 by IL-2 was found to be involved in T cell proliferation and promoted Fas ligand (FasL) expression and AICD. T cell survival was dependent on a receptor region that activated Akt and the expression of Bcl-2. Thus, distinct IL-2Rbeta chain signaling modules regulate T cell fate by stimulating growth and survival or by promoting apoptosis.