Patrick A. Baeuerle

Nuclear Factor Kb: An Oxidative Stress-Responsive Transcription Factor of Eukaryotic Cells (A Review)

NF-kappa B is a multiprotein complex that can activate a great variety of genes involved in early defence reactions of higher organisms. In nonstimulated cells, NF-kappa B resides in the cytoplasm in an inactive complex with the inhibitor I kappa B. Pathogenic stimuli cause release of I kappa B and allow NF-kappa B to enter the nucleus, bind to DNA control elements and, thereby, induce the synthesis of mRNA. A puzzling feature of NF-kappa B is that its activation is triggered by a great variety of agents. These include the cytokines interleukin-1 and tumor necrosis factor, viruses, double-stranded RNA, endotoxins, phorbol esters, UV light and ionizing radiation. We recently found that also low concentrations of H2O2 activate NF-kappa B and that various antioxidants prevent the induction by H2O2. Subsequent analysis revealed that antioxidants not only suppress the activation of NF-kappa B by H2O2 but by all other inducers tested so far. In this review, we will discuss the evidences that NF-kappa B is an oxidative stress-responsive transcription factor of higher eukaryotic cells.

The DNA binding subunit of NF-κB is identical to factor KBF1 and homologous to the rel oncogene product

The major determinant in the transcriptional control of class I genes of the major histocompatibility complex is an enhancer sequence located around -170 from the transcription start site, which binds a factor named KBF1. We have isolated a complementary cDNA coding for KBF1 and identified the DNA binding and dimerization domain of the protein. Because KBF1 and the transcription factor NF-kappa B bind to similar sequences, we investigated the relationship between these two molecules. It appeared that KBF1 is, by all criteria used, identical to the 50 kd DNA binding subunit of NF-kappa B. KBF1 (and therefore p50) also displays extensive amino acid sequence homology with the v-rel oncogene and the Drosophila maternal morphogen dorsal. In vitro experiments suggest functional homologies between KBF1 and v-rel.

A role for oxygen radicals as second messengers

All cells seem to produce oxygen radicals. Recent results suggest that small nontoxic amounts of these radicals are released by various cell types in response to stimulation with tumour necrosis factor (TNF), interleukin 1 (IL-1) and phorbol esters, all of which activate a cytoplasmic form of the transcription factor NF-kappa B by releasing an inhibitory protein subunit. Activation is inhibited by agents that remove oxygen radicals, and mimicked by exposure to mild oxidant stress. This article proposes that oxygen radicals act as second messengers for a variety of agents, including the immunomodulatory cytokines TNF and IL-1, in at least one type of regulatory pathway activating NF-kappa B.

Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-κB subunit

We show that the non-DNA-binding precursor for the p50 subunit (p110), like NF-kappa B, is subject to control of nuclear uptake. In contrast to p50, p110 was excluded from nuclei and unable to associate detectably with p50 or p65 NF-kappa B subunits. The nuclear location signal in the N-terminal half of p110 was not accessible for monospecific antibodies. Removal of only 191 amino acids from the C-terminus of p110 restored antibody accessibility as well as nuclear uptake. The C-terminal half of p110, which is linked to the p50 portion via a glycine-rich hinge, could also noncovalently bind to p50. This helps to explain why p50, after cleavage of the precursor in intact cells, was still retained in an inactive form in the cytoplasm. Our study describes a novel mechanism of nuclear uptake control by masking of a nuclear location signal through a remote domain within a precursor molecule.

EpCAM (CD326) finding its role in cancer

Although epithelial cell adhesion/activating molecule (EpCAM/CD326) is one of the first tumour-associated antigens identified, it has never received the same level of attention as other target proteins for therapy of cancer. It is also striking that ever since its discovery in the late 1970s the actual contribution of EpCAM to carcinogenesis remained unexplored until very recently. With a First International Symposium on EpCAM Biology and Clinical Application this is now changing. Key topics discussed at the meeting were the frequency and level of EpCAM expression on various cancers and its prognostic potential, the role of EpCAM as an oncogenic signalling molecule for cancer cells, recent progress on EpCAM-directed immunotherapeutic approaches in clinical development and the interaction of EpCAM with other proteins, which may provide a basis for a therapeutic window and repression of its growth-promoting signalling in carcinoma. Future research on EpCAM may benefit from a unified nomenclature and more frequent exchange among those who have been working on this cancer target during the past 30 years and will do so in the future.Although epithelial cell adhesion/activating molecule (EpCAM/CD326) is one of the first tumour-associated antigens identified, it has never received the same level of attention as other target proteins for therapy of cancer. It is also striking that ever since its discovery in the late 1970s the actual contribution of EpCAM to carcinogenesis remained unexplored until very recently. With a First International Symposium on EpCAM Biology and Clinical Application this is now changing. Key topics discussed at the meeting were the frequency and level of EpCAM expression on various cancers and its prognostic potential, the role of EpCAM as an oncogenic signalling molecule for cancer cells, recent progress on EpCAM-directed immunotherapeutic approaches in clinical development and the interaction of EpCAM with other proteins, which may provide a basis for a therapeutic window and repression of its growth-promoting signalling in carcinoma. Future research on EpCAM may benefit from a unified nomenclature and more frequent exchange among those who have been working on this cancer target during the past 30 years and will do so in the future.

Purified human IκB can rapidly dissociate the complex of the NF-κB transcription factor with its cognate DNA

Abstract IκB is an inhibitory protein that stabilizes the inducible cytoplasmic form of the NF-κB transcription factor. We have purified IκB-α, a major form of IκB with an apparent molecular size of 37 kd, from cytosol of human placenta. A second chromatographically distinct form, IκB-β, was partially purified and found to be more basic and 3–8 kd larger than the α form. The occurrence of distinct forms of IκB could explain how NF-κB can be activated in response to various agents that signal via different intracellular messenger systems. Both IκB-α and -β exclusively inactivated NF-ϑB containing the non-DNA binding 65 kd subunit and, within minutes, could dissociate a high affinity complex of NF-κB with its cognate DNA. On the assumption that free IκB-α and -β can enter the nucleus, these proteins could rapidly release NF-κB from high affinity binding sites in enhancer and promoter elements, thereby terminating NF-κB-dependent initiation of gene expression.

Nuclear Import-Export: In Search of Signals and Mechanisms

E. A. Nigg,’ P. A. Baeuerle,t and R. LiihrmannS *Swiss Institute for Experimental Cancer Research CH-1066 Epalinges Switzerland TGenzentrum der Ludwig-Maximilians-Universitkt D-8033 Martinsried Germany *lnstitut fiir Molekularbiologie und Tumorforschung Universitat Marburg D-3550 Marburg Germany In eukaryotic cells, there is continuous exchange of macro- molecules between the nucleoplasm and the cytoplasm. Transport in both directions occurs through nuclear pore complexes (NPCs; Feldherr et al., 1984; Dworetzky and Feldherr, 1988). The bidirectional nature of the exchange at the nuclear envelope distinguishes it from transport across the membranes of other organelles such as mito- chondria and chloroplasts. The 3rd International Sympo- sium on Nuclear-Cytoplasmic Transport (Heidelberg, April 21-24, 1991) enabled discussion of the latest prog- ress toward elucidating the structures and mechanisms involved in nuclear import and export processes (for recent reviews on protein transport see Garcia-Bustos et al., 1991; Silver, 1991). The structural components of the nuclear envelope in- clude a double membrane, NPCs, and the nuclear lamina (Figure 1). NPCs are large supramolecular structures (Fig- ure 2A; Reichelt et al., 1990; Akey, 1991, and references therein). They provide aqueous channels with a functional diameter of 9-10 nm, allowing diffusion of ions, small mol- ecules, and proteins with molecular masses up to 40- 60 kd (Peters, 1986). The transport of larger karyophilic proteins through the pore is an active process: it is temper- ature dependent, requires ATP, and displays saturation

[14] Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-κB

Publisher Summary The eukaryotic transcription factor system specifically activated by peroxides is NF- κ B. Micromolar concentrations of H 2 O 2 can mobilize the sequestered cytoplasmic form of NF- κ B in cultured cells. This involves release of the regulatory subunit I κ B from a heterodimer of DNA-binding p50 and p65 (also called Rel-A) subunits and nuclear translocation of p50-p65. Oxidants activate protein kinases that trigger dissociation of the NF-KB-I κ B. Additional evidence that NF- κ B is an oxidative stress-responsive transcription factor comes from the inhibitory effects of various structurally unrelated antioxidants on the activation of NF- κ B in response to many diverse stimuli. This chapter describes how the activation of NF- κ B by oxidants and the inhibitory effects of antioxidants on activation of NF- κ B are investigated using intact cultured cells. The procedures described in the chapter are particularly useful in achieving two goals. The first goal is to find novel inhibitors of NF- κ B that may be potent antioxidative drugs interfering with the release of I κ B from the cytoplasmic complex of NF- κ B. The second goal is to understand molecular mechanisms underlying the oxidative stress response in higher eukaryotes—that is, to determine what molecules sense a disturbance of the intracellular levels of reactive oxygen intermediates (ROIs) and how they transmit their signals to the nucleus where genes are newly transcribed.

Proteins controlling the nuclear uptake of NF-κB, Rel and dorsal

Abstract The two DNA-binding subunits of the transcription factor NF-κB, the products of the rel oncogene family and the product of the developmental control gene dorsal of Drosophila are homologous within a 300 amino acid region. This sequence represents a novel DNA-binding and dimerization domain. The access of the NF-κB/Rel/dorsal (NRD) transcription factor family to the cell nucleus is regulated. There is now evidence that functionally and structurally related accessory proteins of NF-κB, Rel and dorsal control the nuclear entry as well as DNA-binding activity of the transcription factors. This review summarizes current knowledge about the nuclear-uptake regulatory proteins (NURPs) IκB-α, IκB-β/pp40 and cactus.

Endoplasmicreticulum-induced signal transduction and gene expression.

Cells can respond to perturbations in endoplasmic reticulum (ER) function by activating two distinct signal-transduction pathways: one responds to unfolded proteins, the other to an overload of the organelle with membrane proteins. A third pathway is activated upon sterol depletion of cells and involves the cleavage and subsequent nuclear translocation of an ER membrane-bound transcription factor. Thus, three distinct pathways each activated by a different signal are currently known to project from the ER into the nucleus. This review summarizes the current understanding of these three pathways.