Constitutive phosphorylation of I kappa B alpha by casein kinase II.

The NF-kappa B/Rel proteins are sequestered in the cytoplasm in association with the phosphorylated form of I kappa B alpha. Upon induction with a wide variety of agents, the activity of NF-kappa B/Rel proteins is preceded by the rapid degradation of I kappa B alpha protein. We report the identification and partial purification of a cellular kinase from unstimulated or stimulated murine cells, which specifically phosphorylates the C terminus of I kappa B alpha. There are several consensus sites for casein kinase II (CKII) in the C-terminal region of I kappa B alpha. Additionally, the activity of the cellular kinase is blocked by antibodies against the alpha subunit of CKII. No phosphorylation of the C-terminal region of I kappa B alpha can be detected if the five possible serine and threonine residues that can be phosphorylated by CKII are mutated to alanine. A two-dimensional tryptic phosphopeptide map of I kappa B alpha from unstimulated cells was identical to that obtained by in vitro phosphorylation of I kappa B alpha with the partially purified cellular kinase. We propose that constitutive phosphorylation of I kappa B alpha is carried out by CKII.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls.

The human hepatitis B virus (HBV) HBx protein is a small transcriptional activator that is essential for virus infection. HBx is thought to be involved in viral hepatocarcinogenesis because it promotes tumorigenesis in transgenic mice. HBx activates the RAS-RAF-mitogen-activated protein (MAP) kinase signaling cascade, through which it activates transcription factors AP-1 and NF-kappa B, and stimulates cell DNA synthesis. We show that HBx stimulates cell cycle progression, shortening the emergence of cells from quiescence (G0) and entry into S phase by at least 12 h, and accelerating transit through checkpoint controls at G0/G1 and G2/M. Compared with serum stimulation, HBx was found to strongly increase the rate and level of activation of the cyclin-dependent kinases CDK2 and CDC2, and their respective active association with cyclins E and A or cyclin B. HBx is also shown to override or greatly reduce serum dependence for cell cycle activation. Both HBx and serum were found to require activation of RAS to stimulate cell cycling, but only HBx could shorten checkpoint intervals. HBx therefore stimulates cell proliferation by activating RAS and a second unknown effector, which may be related to its reported ability to induce prolonged activation of JUN or to interact with cellular p53 protein. These data suggest a molecular mechanism by which HBx likely contributes to viral carcinogenesis. By deregulating checkpoint controls, HBx could participate in the selection of cells that are genetically unstable, some of which would accumulate unrepaired transforming mutations.

The hepatitis B virus X protein targets the basic region-leucine zipper domain of CREB.

The X gene product encoded by the hepatitis B virus, termed pX, is a promiscuous transactivator of a variety of viral and cellular genes under the control of diverse cis-acting elements. Although pX does not appear to directly bind DNA, pX-responsive elements include the NF-kappa B, AP-1, and CRE (cAMP response element) sites. Direct protein-protein interactions occur between viral pX and the CRE-binding transcription factors CREB and ATF. Here we examine the mechanism of the protein-protein interactions occurring between CREB and pX by using recombinant proteins and in vitro DNA-binding assays. We demonstrate that pX interacts with the basic region-leucine zipper domain of CREB but not with the DNA-binding domain of the yeast transactivator protein Gal4. The interaction between CREB and pX increases the affinity of CREB for the CRE site by an order of magnitude, although pX does not alter the rate of CREB dimerization. Methylation interference footprinting reveals differences between the CREB DNA and CREB-pX DNA complexes. These experiments demonstrate that pX titers the way CREB interacts with the CRE DNA and suggest that the basic, DNA-binding region of CREB is the target of pX. Transfection assays in PC12 cells with the CREB-dependent somatostatin promoter demonstrate a nearly 15-fold transcriptional induction after forskolin stimulation in the presence of pX. These results support the significance of the CREB-pX protein-protein interactions in vivo.

Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.

NF-kappa B/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an I kappa B-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.

TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling.

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned. TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B. Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.

Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40- and TNF-mediated activation of the transcription factor NF-kappa B. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-kappa B activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Interactions of a Rel protein with its inhibitor.

Cactus, a Drosophila homologue of I kappa B, binds to and inhibits Dorsal, a homologue of the p50 and p65 components of NF-kappa B. We describe experiments in yeast with various Dorsal and Cactus derivatives showing that Cactus blocks the DNA binding and nuclear localization functions of Dorsal. In contrast, Dorsal's transcriptional activating region is functional in the Dorsal-Cactus complex. We identify two Dorsal mutants, Dorsal C233R and Dorsal S234P, that escape Cactus inhibition in vivo, and we show that these mutants fail to interact with Cactus in vitro. From this and data of others, we identify the likely surface of Dorsal that binds Cactus. We also describe a modified PCR mutagenesis procedure, easier to use than conventional methods, that produces a library of high complexity.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

V(D)J recombination is not activated by demethylation of the kappa locus

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.