Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor.

The Notch signaling pathway is essential in many cell fate decisions in invertebrates as well as in vertebrates. After ligand binding, a two-step proteolytic cleavage releases the intracellular part of the receptor which translocates to the nucleus and acts as a transcriptional activator. Although Notch-induced transcription of genes has been reported extensively, its endogenous nuclear form has been seldom visualized. We report that the nuclear intracellular domain of Notch1 is stabilized by proteasome inhibitors and is a substrate for polyubiquitination in vitro. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Lin12/Notch-negative regulators in Caenorhabditis elegans. We isolated human and murine counterparts of SEL-10 and investigated the role of a dominant-negative form of this protein, deleted of the F-box, on Notch1 stability and activity. This molecule could stabilize intracellular Notch1 and enhance its transcriptional activity but had no effect on inactive membrane-anchored forms of the receptor. We then demonstrated that SEL-10 specifically interacts with nuclear forms of Notch1 and that this interaction requires a phosphorylation event. Taken together, these data suggest that SEL-10 is involved in shutting off Notch signaling by ubiquitin-proteasome-mediated degradation of the active transcriptional factor after a nuclear phosphorylation event.

A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE.

The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway.

Delta-1 activation of notch-1 signaling results in HES-1 transactivation.

The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown in Drosophila melanogaster that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Split genes. Genetic evidence has also implicated the kuzbanian gene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. By using a two-cell coculture assay, we show here that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1 a HES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Furthermore, we observed that Dl-1-induced HES-1 transactivation was dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. We also observed that exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenous HES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as the Jagged proteins do.

The Notch1 receptor is cleaved constitutively by a furin-like convertase.

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.

Phosphorylation of p105 PEST sequence via a redox-insensitive pathway up-regulates processing of p50 NF-kappaB.

The p105 Rel protein has dual functions; it is the precursor of the p5O subunit of NF-kappaB, and it acts as an IkappaB-like inhibitor to retain other Rel subunits in the cytoplasm. We have investigated the posttranslational regulation of p105 following activation of Jurkat T cells and find that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-kappaB, is correlated with an increase in proteolytic processing to p5O. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, as a 68-amino acid C-terminal deletion abolishes the response to stimulation. When compounds that block Ikappa B alpha phosphorylation and degradation were tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosyl-amido-7-amino-2-heptanone blocked inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol did not. Thus, while regulation of the p105 IkappaB resembles that of lkappaBa, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, it depends on a different, redox-insensitive, signaling pathway.

Signalling downstream of activated mammalian Notch.

Notch belongs to a family of transmembrane proteins that are widely conserved from flies to vertebrates and are thought to be involved in cell-fate decisions. In Drosophila, the Suppressor of hairless (Su(H)) gene and genes of the Enhancer of split (E(Spl)) complex, which encode proteins of the basic helix-loop-helix type have been implicated in the Notch signalling pathway. Mammalian homologues of E(Spl), such as the mouse Hairy enhancer of split (HES-1), have been isolated. Both HES-1 and the intracellular domain of murine Notch (mNotch) are able to block MyoD-induced myogenesis. Here we show that activated forms of mNotch associate with the human analogue of Su(H), KBF2/RBP-J kappa (refs 8,9) and act as transcriptional activators through the KBF2-binding sites of the HES-1 promoter.

The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein-Barr virus EBNA2 protein to its DNA responsive elements.

The Epstein-Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high-affinity binding site for the human recombination signal sequence binding protein RBP-J kappa. Here we provide evidence that RBP-J kappa binds to specific sequences in EBNA2-responsive elements. Our results also demonstrate that RBP-J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP-J kappa may mediate EBNA2 transactivation of both cellular and viral genes.

Genomic structure and chromosomal localization of processed pseudogenes for human RBP-Jk.

The functional gene for human recombination signal sequence-binding protein (RBP-Jk) and corresponding processed psudogenes have been isolated from various species, such as Drosophila, Xenopus, mouse, and human. Here we report the isolation of another two genomic pseudogenes of human RBP-Jk, named K2 and K7, from a cosmid library of Hela cells. The nucleotide sequences of both genes exhibited more than 95% homology to the functional human gene for RBP-Jk. Moreover, they did not contain any intron sequences and were interrupted by several stop codons in all frames. In situ hybridization demonstrated that the pseudogenes, K2 and K7, were localized at chromosomes 9p13 and 9q13, respectively. Their physical maps differed from those of the true functional gene and of the pseudogenes reported previously by Amakawa et al. (1993).

Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless.

We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed us to define a DNA-binding domain conserved during evolution. Because Su(H) mutant alleles exhibit dose-sensitive interactions with Hairless (H) loss-of-function mutations, we have investigated whether the RBP-J kappa or Su(H) proteins directly interact with the H protein in vitro. We show here that H can inhibit the DNA binding of both Su(H) and RBP-J kappa through direct protein-protein interactions. Consistent with this in vitro inhibitory effect, transcriptional activation driven by Su(H) in transfected Drosophila S2 cells is inhibited by H. These results support a model in which H acts, at least in part, as a negative regulator of Su(H) activity. This model offers a molecular view to the antagonistic activities encoded by the H and Su(H) genes for the control of sensory organ cell fates in Drosophila. We further propose that a similar mechanism might occur in mammals.

Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant.

The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.

The DNA binding subunit of NF-kappa B 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.

Regulatory elements involved in the liver-specific expression of the mouse MHC class I Q10 gene: characterization of a new TATA-binding factor.

The murine MHC genes code for the classical H-2K, D, and L transplantation antigens, and for other class I-like proteins called Qa and TIa molecules. Most of the latter have a restricted tissue distribution whereas classical transplantation antigens are virtually expressed by all somatic cells of the adult organism. Q10 is a Qa region gene, which was found to be expressed in liver and yolk sac, a regulatory pattern more evocative of the expression of a large set of serum proteins secreted by the liver than of a classical class I antigen. First, we have characterized several regions in the promoter of Q10 which bind factors present in liver nuclear extracts. Our most striking observation is that one of these factors, which we named TA-f, binds in the TATA box region of Q10 and Kb and displays tissue-specific expression, in that we found the activity only in liver and kidney. Secondly, we have performed a comparative analysis of the 5' upstream sequences of Q10 with those of H-2Kb, Ld, and other Qa genes. We have shown that most of the regulatory elements involved in the ubiquitous expression of H-2Kb are punctually altered and not functional in Q10. Similarly, most of the binding sequences for liver factors in the Q10 promoter, except the TA region, do not exist in the other H-2 class I genes which, however, have conserved most of the functional regions defined in H-2Kb and Ld. These results suggest that the tissue-specific expression of Q10 is associated with both alteration of the sequences conferring ubiquitous expression to other class I genes and/or creation of new sequences able to bind liver-specific regulatory factors. However, our observations also suggest that a unique sequence, the TATA box, may confer differential regulation in different tissues, since it binds a factor whose expression is restricted to the liver and kidney.

TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors.

We have dissected the mouse H-2Kb gene promoter in order to define the sequences responsible for induction by tumour necrosis factor (TNF-alpha). An enhancer element (-187 to -158) composed of two imperfect direct palindromic repeats has been shown to be necessary and sufficient for TNF-alpha induction of a heterologous promoter. A multimer of either repeat is also responsive, while a single copy is not: this is the situation in the beta 2-microglobulin (beta 2-m) promoter which contains a single palindrome and does not respond to TNF-alpha. We had previously found that the two repeats can bind a factor named KBF1. We show here that in the uninduced state the transcription factor AP2 binds to the interpalindromic region, while in TNF-treated cells an NF kappa B-like activity is induced which displaces both KBF1 and AP2 and binds to the two palindromes. This strongly suggests that induction of an NF kappa B-like activity is responsible for TNF-alpha stimulation of mouse MHC class I genes.

Two purified factors bind to the same sequence in the enhancer of mouse MHC class I genes: one of them is a positive regulator induced upon differentiation of teratocarcinoma cells.

The MHC class I murine and beta-2-microglobulin genes are silent in embryonal carcinoma (EC) cells but are induced upon differentiation of these cells. We have previously shown that enhancer-like sequences located in the promoter of the H-2Kb gene are non-functional in F9 and PCC3 cells. We have previously purified a 48 kd protein (KBF1) from a mouse T cell line which binds to a palindromic sequence located in this enhancer and to a similar sequence in the promoter of the beta-2-microglobulin gene. We describe here the purification of a second protein (KBF2, 58 kd) which also binds to this sequence. While both activities are present in differentiated cells, KBF1 binding activity is absent in undifferentiated EC cells, where the palindromic sequence shows no enhancer activity. Upon differentiation, KBF1 binding activity is induced and the palindromic sequence becomes active as an enhancer. Thus, the absence of KBF1 activity in undifferentiated EC cells is at least in part responsible for the lack of expression of H-2 class I and beta-2-microglobulin genes in these cells and suggests that KBF1 activity is regulated during differentiation.

Radioimmunoassay of progesterone receptor in human tissues: application to breast cancer.

A radioimmunoassay method to measure progesterone receptor in rabbit and human tissues was devised and applied to human breast cancer. A specific progesterone receptor antibody was prepared by purifying rabbit receptor by immunoaffinity chromatography, sodium dodecylsulphate-polyacrylamide gel electrophoresis and injection of the isolated 110,000 dalton receptor band into a goat. Immunoblot studies of progesterone target and non-target tissues showed the specificity of the antibody, which was used at a dilution of 1/45,000. The tracer consisted of 125I-labelled electroeluted 110,000 dalton receptor. The sensitivity of the method was 1 fmol/tube for the rabbit receptor and 3 fmol/tube for the human receptor. The intra-assay coefficient of variation was 11% for tumours positive for the progesterone receptor and 9.9% for those on the borderline (10-30 fmol receptor/mg protein). The interassay coefficients of variation were 20 and 19% respectively. The correlation between the radioimmunoassay and a steroid-binding assay was studied in 40 tumour biopsies. In 39 cases, very good correlation was found (r = 0.99); in a single case an immunoreactive protein was detected which apparently bound steroid poorly. One important feature of this method was that receptor immunoreactivity remained unchanged when either the tissue or the cytosol was exposed to a temperature of 20 degrees C for relatively long periods of time. Under the same conditions the steroid-binding capacity declined rapidly. This characteristic of the radioimmunoassay may prevent errors due to improper handling of tissue samples. Such stability was not observed for oestrogen receptors when measured by a sandwich immunoenzymatic method after incubation of tissue at 20 degrees C.

Characterization of a casein kinase which interacts with the rabbit progesterone receptor. Differences with the in vivo hormone-dependent phosphorylation.

Previous in vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatography. 1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods. 2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubilization of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestin-treated rabbits. 3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate (Km approximately equal to 0.15 mg/ml) and is not regulated by cyclic nucleotides, Ca2+ and calmodulin or phospholipids. Its classification as a casein kinase I or II is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate. 4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic "upshift" in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction.

Cloning and sequence analysis of rabbit progesterone-receptor complementary DNA.

Two lambda gt11 clones containing fragments of cDNA encoding the rabbit progesterone receptor were isolated with the aid of monoclonal and monospecific polyclonal antireceptor antibodies. RNA gel blot analysis showed that the corresponding mRNA was approximately equal to 5900 nucleotides in size and present in the uterus, where its concentration was increased by estrogen treatment, and in the vagina. This mRNA was not detected in liver, in spleen, in intestine, and in kidney where the receptor protein is known to be absent or present in very small concentration. Cross-hybridizing clones were isolated from a lambda 10 library. The DNA was sequenced, and the primary structure of the progesterone receptor was deduced. It consists of 930 amino acids and contains a basic, cysteine-rich region (residues 568-645) with extensive homology to the glucocorticoid and estrogen receptors and the v-erbA oncogene protein. This region is followed by a C-terminal domain that is similar in size to the corresponding domains of the other steroid receptors and v-erbA and shows striking amino acid homology with the glucocorticoid receptor and significant homology with the estrogen receptor. In contrast, the region extending from the cysteine-rich segment toward the N terminus differed in size and amino acid sequence from that of the other receptors and v-erbA. This region had a high proline content in the progesterone receptor.

Immunocytochemical localization of progesterone receptor in the guinea pig central nervous system.

The distribution of neurons containing progesterone receptors in the brain of guinea pigs ovariectomized and primed by estradiol was investigated immunohistochemically using monoclonal antibodies to the progesterone receptor. We found that the picric acid paraformaldehyde perfusion provided satisfactory conditions of fixation for visualizing the progesterone receptor in sections of frozen tissue. Among the different techniques of immunocytochemical detection used, the indirect antibody peroxidase-antiperoxidase method gave the best results. The immunostained neurons were mainly located in two specific regions of the hypothalamus: the preoptic area and the mediobasal hypothalamus. Within the preoptic region, the majority of immunoreactive cells were present in the nucleus preopticus periventricularis particularly at the level of the pars suprachiasmatica. Within the mediobasal hypothalamus, immunostained neurons were found in the nucleus periventricularis, arcuatus, ventromedialis and premamillaris. Differences in the intensity of immunoreactivity appeared from one region to another. A marked cellular heterogeneity was observed: in each neuroanatomical structure, labeled cells alternated with non-labeled cells. The receptor, even in absence of progesterone, was localized in the nucleus. The nucleolus was not stained and only neurons were labeled. There was no progesterone receptor immunoreactivity in other regions of the brain, especially in the amygdala, hippocampus and cortex where biochemical studies have shown the presence of a non-estrogen regulated protein binding the progestin [3H]R 5020. Control experiments with antibody pretreated with purified progesterone receptor or with mouse receptor-unrelated monoclonal antibody did not show fluorescent or immunoreactive cells.

Ultrastructural localization of the progesterone receptor by an immunogold method: effect of hormone administration.

The progesterone receptor has been localized in the rabbit uterus by immunocytochemistry at the electron microscopic level, using monoclonal antibodies and the protein A-gold technique. The progesterone receptor in uterine stromal cells was mainly localized in the nucleus; however, a small fraction of antigen was present in the cytoplasm, where it was associated with the rough endoplasmic reticulum and with free ribosomes. The plasma membrane was not labeled. In the nucleus, the receptor was always associated with condensed chromatin or areas surrounding condensed chromatin, whereas the nuceolus was not labeled. In the chromatin, receptor distribution varied according to the hormonal state: in the absence of progesterone, the receptor was randomly scattered over the clumps of condensed chromatin; after administration of the progestin R5020, it was mainly detected in the border regions between condensed chromatin and nucleoplasm and, to a lesser extent, over dispersed chromatin in the nucleoplasm. These areas have been shown to be the most active sites of gene transcription.