Frédérique Logeat

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.

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.

Delta-1 Activation of Notch-1 Signaling Results in HES-1 Transactivation

ABSTRACT The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown inDrosophila melanogaster that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Splitgenes. Genetic evidence has also implicated the kuzbaniangene, 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 aHES-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-inducedHES-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 endogenousHES-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.

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.

Monoubiquitination and endocytosis direct γ-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFα-converting enzyme–dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent γ-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for γ-secretase processing of a constitutively active Notch derivative, ΔE, which mimics the TNFα-converting enzyme–processing product. PS interacts with this modified form of ΔE, ΔEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other γ-secretase substrates.

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.

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. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.

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

SummaryThe 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).

Control of Notch Activity by the Ubiquitin-Proteasome Pathway

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. Though Notch-induced transcription of genes has been extensively reported, its endogenous nuclear form has seldom been visualized. We report that the nuclear intracellular domain of Notch 1 is stabilized by proteasome inhibitors, suggesting an involvement of the ubiquitin-proteasome pathway. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Linl2/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 Notch 1 stability and activity. This molecule could stabilize intracellular Notch 1 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 Notchl 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, following a nuclear phosphorylation event.