A glycosphingolipid binding domain controls trafficking and activity of the mammalian notch ligand delta-like 1.

The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization.

The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.

Genetic studies have shown that ubiquitination and endocytosis of the Drosophila ligand Delta in signal-sending cells are required for activation of Notch signaling, but how these events promote Notch activation remains poorly understood. Here, we show that an ubiquitination-defective mutant of the murine Delta-homologue Dll1 is endocytosed but, in contrast to the wild-type Dll1, is unable to subsequently recycle back to the cell surface or to bind Notch1 efficiently. These results demonstrate that ubiquitination, although not required for endocytosis, is essential for Dll1 recycling and that recycling is required to acquire affinity for the receptor.

The notch ligand Delta1 recruits Dlg1 at cell-cell contacts and regulates cell migration.

Delta1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. By using peptide affinity chromatography followed by mass spectrometry, we have identified Dlg1 as a partner of the Delta1 C-terminal region. Dlg1 is a human homolog of the Drosophila Discs large tumor suppressor, a member of the membrane-associated guanylate kinase family of molecular scaffolds.

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor.

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu.

The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase.

Notch signaling is involved in numerous cell fate decisions in invertebrates and vertebrates. The Notch receptor is a type I transmembrane (TM) protein that undergoes two proteolytic steps after ligand binding, first by an ADAM (a distintegrin and metalloprotease) in the extracellular region, followed by gamma-secretase-mediated cleavage inside the TM domain. We demonstrate here that the murine ligand Delta1 (Dll1) undergoes the same sequence of cleavages, in an apparently signal-independent manner.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.