Thèse de doctorat en Aspects moléculaires et cellulaires de la biologie
Sous la direction de Patrick Auberger.
Soutenue en 2004
à Nice , dans le cadre de École doctorale Sciences de la vie et de la santé (Sophia Antipolis, Alpes-Maritimes) , en partenariat avec Université de Nice-Sophia Antipolis. Faculté des sciences (autre partenaire) .
Apoptosis is an active form of cell death that plays a fundamental role in normal development, tissue homeostasis and pathological situations. Alterations of this normal process can result in the disruption of the delicate balance between cell proliferation and cell death and can lead to a variety of diseases such as cancers. Apoptosis is executed through the activation and function of cystein proteinases called caspases which cleave key substrates that in turn orchestrate the death process. Bcl-2 family is the central regulator of caspase activation, and its opposing functions of anti and pro-apoptotic members arbitrate the life-or-death decision. Phorbol esters are tumor promoters that bind and activate both conventional and new PKC isoforms. In various circumstances, PKC-dependent signaling athways can promote cell survival and protect against cell death. In the first part of my work, we have undertaken experiments to analyse the molecular events underlying PMA-induced inhibition of apoptosis. This was first analyzed in Jurkat T cells where PMA was found to inhibit Fas-mediated apoptosis as judged by DiOC6(3) staining, caspase activation and DNA fragmentation, indicating that PMA exerts its protective effect upstream or at the mitochondrial level in these cells. PMA activated most of the main kinase pathways in T cells such as PKCs, p42/44MAPK, P38MAPK and p90Rsk but not JNK and Akt. A pharmacological approach allowed us to identify that nPKCs are both necessary and likely sufficient to promote T cell survival. Besides this post-transcriptional regulation, nPKCs may also regulate apoptosis at the transcriptional level. CDNA arrays were used to identify a set of genes whose expression was modulated in death versus survival conditions. Following PMA treatment, expression of Mcl-1 increased while that of c-Myc was significantly reduced. C-Myc modulation seems to be regulated at the transcriptional level while decrease in Mcl-1 protein in CH11-treated cells resulted especially from a caspase-dependent proteolysis. Taken together, our data demonstrate that PMA-mediated inhibition of apoptosis is a complex process that involves both transcriptional and post-transcriptional controls and point out to the potential role of Mcl-1 and c-Myc in this process. The second axis of my work focused on the regulation of Mcl-1, an anti-apoptotic member of the Bcl-2 family that promote cell viability. We report here that Mcl-1 is a new substrate for caspases during induction of apoptosis. Mcl-1 cleavage occurs after Asp127 and Asp157 and generates four fragments of 24, 19, 17 and 12 kDa in both intact cells and in vitro, an effect prevented by selective caspase inhibitors. As a consequence, the resulting protein that lacks the first 127 or 157 amino-acids contains only the BH1, BH2 and BH3 domains of Bcl-2 family members. Mutation of Asp127 and Asp157 abolishes the generation of the 24 and 12 kDa fragments and that of the 19 and 17 kDa fragments, respectively. Interestingly, when expressed in HeLa cells Mcl-1 wild-type and Mcl-1 D127 showed marked different intracellular distribution. Mcl-1 wild-type colocalised with a-Tubulin near the internal face of the plasma membrane, while Mcl-1 D127 coassociated with Bim at the mitochondrial level. Coimmunoprecipitation experiments also demonstrated that Mcl1 D127 exhibited increased binding to Bim as compared to Mcl-1 wild-type. Finally, Mcl-1 wild-type unlike Mcl-1 D127 inhibited Bim-induced caspase activation. Altogether, the findings presented in this manuscrit describe a new post-translational mechanism for the regulation of Mcl-1 that involves caspase-dependent cleavage.
Roles of PKC and Mcl-1 in the life-or-death decision
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