Amplification of the Fas-mediated apoptotic signal by lipid rafts – PIKFAS

The PI3K (phosphatidylinositol 3-kinase) signaling pathway is involved in the inhibition of the Fas-mediated apoptotic pathway by an yet poorly define mechanism. This signal plays an important role in tumorigenesis and in relapse of transformed cells after chemotherapy. Fas belongs to the TNF-R (Tumor necrosis factor Receptor) superfamily. Fas engagement upon binding of its cognate ligand, FasL triggers an apoptotic signal. Microdomains or lipid rafts are compact proteo-lipidic structures floating in the bi-layer of the cytoplasmic membrane whose inner leaflet concentrates numerous signaling effector proteins. Cross-linking of Fas with a microdomain-concentrated protein induces the Fas redistribution into lipid rafts and amplifies dramatically the Fas-mediated apoptotic signal in tumoral cell lines. Our preliminary data show that inhibition of the PI3K signaling pathway in activated T lymphocytes leads to the redistribution of Fas into the lipid rafts and triggers apoptosis. Aims and Methodology: Aim 1) Caspase-8 activation is amplified through the redistribution of Fas into the lipid rafts. To determine the signaling pathways responsible of this amplification, we will carry out the following experiments : - Kinases of the Src family are concentrated into the lipid rafts. The p56Lck and p59fyn are members of this family and they have been described as pro-apoptotic factors. Using p56Lck-deficient cell line, chemical inhibitors or small interfering RNA, we will determine the implication of these kinases in the pro-apoptotic signal. To fully characterize the role of kinases in this signal, we will examine the phosphorylation state of signaling proteins upon Fas and Fas/CD28 cross-linking. We will use protein micro-arrays developed by Panomics (CA, USA) and we will confirmed and improved these findings by the utilization of the multiplexed reverse phase protein assay developed by Dr Utz P.J. (Stanford, CA, USA). Indeed using this protein chips, we will accurately characterize the activation pattern of 62 different kinases. - To determine the early molecular ordering involved in the activation of the proximal caspase-8, we will co-immunoprecipitate Fas alone and Fas/CD28. Fas-associated proteins will be resolved in a SDS-PAGE and the different bands will be analyzed using the liquid chromatography/mass spectrometry method. This study will be achieved in collaboration with the Proteomic facility core of the University of Bordeaux-2 . Identified proteins and signaling pathways will be studied in details using classical methods (over-expression, dominant negative expression, siRNA knock-down, etc.). The purpose of this study is to determine the molecular mechanism responsible of the microdomain-mediated amplification of Fas apoptotic signal. Aim 2) We observed that inhibition of this signal induces 1) Fas relocalisation into the lipid rafts and 2) induction of a Fas-dependent cell death pathway. Our goals will be to determine i) the molecular mechanism responsible of the Fas redistribution into lipid rafts (cytoskeleton) and ii) the different signaling pathways induced when Fas is relocalised into the lipid rafts. a) Actin cytoskeleton has been involved in the Fas-mediated apoptotic signal. Ezrin belongs to the family of ERM proteins (Ezrin/moesin/Radixin) and connects actin to Fas. Firstly, we will determine if 1) ezrin effectively links Fas to actin, and if actin and ezrin are essential to transmit a potent Fas apoptotic signal. To confirm these results, we will use also chemical inhibitors such as Latrunculin A and Cytochalasin D which disrupt the actin filament network. Secondly, we will focus on the role of ezrin and actin cytoskeleton in the redistribution of Fas into the lipid rafts upon PI3K inhibition. We have generated different ezrin dominant negative mutants and using confocal microscopy, we will determine if the disconnection of Fas from actin impairs the Fas redistribution into lipid rafts upon PI3K inhibition. b) The phospho-protein chip assays described above will be performed in the context of PI3K inhibition to determine whether the src kinases or other kinases favor the transmission of the Fas signal in the context of PI3K inhibition. c) The potentially implicated signaling pathways in the Fas-mediated apoptotic amplification will be next studied in details using classical methods (over-expression, dominant negative expression, siRNA knock-down, etc.). Aim 3) We will screen a large panel of primary leukemia cells to characterize PI3K activity and Fas sensitivity. Next we will focus on leukemia cells with a highly activated PI3K pathway and resistant to Fas-induced cell death, we will attempt to resensitize these cells to death by simultaneously triggering Fas and the characterized pro-apoptotic signals. This study will be realized in collaboration with the Service des Maladies du Sang du CHU Bordeaux (Prof Milpied N.).