Binding of the ligand CD95L to the death receptor CD95 causes receptor clustering, resulting in the formation of the CD95-Cap, which acts as a platform for the recruitment of adaptor proteins and initiator caspases, including caspase-8. The resulting death-inducing signaling complex (DISC) elicits apoptosis. Khadra et al. investigated a role for Ca2+ in CD95-induced cell death and found that CD95L stimulated a biphasic Ca2+ response in lymphocytes and T cell lines. The first phase consisted of Ca2+ release from intracellular stores, whereas the second phase involved store-operated Ca2+ entry mediated by Orai1 at the plasma membrane. Inhibition of CD95-dependent Ca2+ flux enhanced the extent of DISC formation compared with that in control cells and increased caspase-8 processing. Single-cell imaging showed that CD95L stimulated the movement of Orai1 into the area of the CD95-Cap, resulting in localized increases in Ca2+ concentration. Expression of a dominant-negative (DN) mutant form of Orai1 resulted in diminished Ca2+ flux compared with that in control cells but increased the extent of DISC formation and caspase-8 processing. CD95L stimulated the recruitment of protein kinase C β2 (PKCβ2) from the cytosol to the CD95-Cap, where it coimmunoprecipitated with the DISC. Expression of DN Orai1 blocked the recruitment of PKCβ2 to the DISC, and knockdown of PKCβ2 increased the sensitivity of cells to CD95-mediated apoptosis. Experiments in which mitochondrial membrane potential was measured in cells that were treated first with CD95L and then with a CD95-blocking antibody at various times indicated that the kinetics of cell death were delayed by overexpression of Orai1 and increased by expression of DN Orai1. Thus, the authors suggest that Orai1-mediated Ca2+ signaling transiently inhibits CD95 signaling as part of a “decision-making” process before commitment to cell death.
N. Khadra, L. Bresson-Bepoldin, A. Penna, B. Chaigne-Delalande, B. Ségui, T. Levade, A.-M. Vacher, J. Reiffers, T. Ducret, J.-F. Moreau, M. D. Cahalan, P. Vacher, P. Legembre, CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation. Proc. Natl. Acad. Sci. U.S.A. 108, 19072–19077 (2011). [Abstract] [Full Text]