Unit: Cell signaling and activation
Director: Israël Alain
The laboratory is involved in the study of two signaling pathways characterized by inducible proteolysis events that lead to the nuclear translocation of transcription factors. The first project deals with the NF-κB family of transcription factors, whose activity is controled by cytoplasmic retention through inhibitory molecules (IκB's) which get degraded in response to multiple signals. We have pursued our characterization of the events associated in T cells with TCR-mediated NF-κB activation. Another project involves the functional characterization of the protein NRP/optineurin, which shows very strong homologies with NEMO, the core element of the NF-κB signaling cascade.
The second signaling cascade we study centers around the Notch receptor whose signaling requires a series of three proteolytic steps which lead to the nuclear translocation of the intracellular part of the receptor, which behaves as a transcriptional co-activator. We have pursued 3 directions of research: first we undertook the functional characterization of a series of ubiquitin-ligases known to be involved in the early steps of the pathway. Then we have set up a genetic screen to identify new components of the γ-secretase activity, responsible for the last proteolysis step that affects the receptor. Finally we have pursued the study of the cell-autonomous function of the ligands of the Notch receptor.
TCR-mediated NF-κB activation (R. Weil, C Lobry, T. Lopez)
Our results allowed us to identify the first phosphorylation event by the IKK kinase complex which leads to the negative regulation of the NF-κB cascade. IKK is the central element of the cascade, containing 2 kinase (IKKα and IKKβ) and a regulatory subunit (NEMO/IKKγ). IKK normally phosphorylates the NF-κB inhibitors, leading to their degradation and to the nuclear transport of the NF-κB subunits. We have shown that following TCR stimulation of T cells, the IKK complex induces the phosphorylation of the Bcl10 protein, leading to its degradation and to the interruption of NF-κB activation. We have also identifed the phosphorylation sites as well as the ubiquitin-ligase involved in this degradation event.
Study of the NRP/optineurin protein (R. Weil, E. Laplantine, T. Lopez)
Despite strong homologies with the NEMO protein, NRP is not involved in the NF-κB signaling cascade, at least by the commonly used stimuli. We have demonstrated that it is associated (probably indirectly) with the Golgi apparatus, together with 2 unidentified kinases, and that it seems to be involved in Golgi to plasma membrane transport. We are now trying through genetic and biochemical approaches to better understand its function, and maybe to explain why mutations in the optineurin gene are linked to familial forms of primary open-angle glaucoma.
The Notch signaling cascade (F. Logeat, C. Brou, N. Gupta, E. Six, A. Olry, P. Chastagner, D. Ndiaye)
We have proposed a model according to which the activation of the Notch receptor involves a series of three proteolytic steps. The first cleavage is due to a furin-like convertase, and is required for surface expression of a functional Notch receptor. A second cleavage step takes place in the extracellular region of the receptor and involves the membrane metalloprotease TACE. The third and last cleavage is due to an activity called γ-secretase and results in the liberation of the intracellular region of the receptor, which is then transported to the nucleus where it behaves as a transcriptional coactivator.
One of our projects involves identifying new components or regulators of the γ-secretase activity, responsible for the last cleavage that affects Notch following ligand binding. For this we have designed a genetic screen based on the generation of cellular mutants which have lost γ-secretase activity. Complementation of these mutants with a cDNA libray will allow us to identify the affected gene. We have validated this approach by identifying cellular clones mutated in nicastrin, one of the 4 known components of the γ-secretase complex, and we are currently analyzing other types of mutants.
The second project aims at understanding the function of certain ubiquitin-ligases involved (on the basis of genetic approaches in invertebrates) in the Notch pathway, but whose actual function remains unknown. We focussed on the proteins Deltex (dtx) and Itch/AIP4, which seem to behave respectively as a positive and a negative regulator of the Notch pathway. We have shown that AIP4 is responsible for dtx degradation, through atypical polyubiquitin chains that lead to lysosomal degradation. We now try to understand the relationships that exist between these 2 proteins and the Notch receptor.
Finally we are dissecting the cell-autonomous function of the Notch ligands. The study of Delta1, one of these ligands, allowed us to show that this molecule, in a manner reminiscent of the Notch receptor, undergoes a series of cleavages resulting in the liberation of the intracelular region, which is partially found in the nucleus. We therefore study the events that regulate these cleavages, in particular the ubiquitination steps, as well as the consequences of the interactions between Delta1 and scaffolding proteins involved in cellular adhesion and motility. In parallel we analyze the transcriptome of cells that express the Delta1 molecule or mutants of it.
Keywords: Signalisation, phosphorylation, proteolysis, ubiquitination, NF-kappaB, Notch