Study of the CD95-Mediated Non-apoptotic Signaling Pathway: PI3K.

CD95 is a plasma membrane receptor that belongs to the TNF receptor family (Itoh and Nagata, J Biol Chem 268(15):10932-10937, 1993; Trauth et al., Science 245(4915):301-305, 1989). Accumulating evidence indicate that this so-called death receptor can also trigger non-apoptotic signaling pathways promoting inflammation and oncogenesis (Barnhart et al., Embo J 23(15):3175-3185, 2004; Chen et al., Nature 465(7297):492-496, 2010; Legembre et al., Cell Cycle 3(10):1235-1239, 2004; Legembre et al., EMBO Rep 5(11):1084-1089, 2004; Malleter et al., Cancer Res 73(22):6711-6721, 2013; Tauzin et al., PLoS Biol 9(6):e1001090, 2011). We and others demonstrated that CD95 implements the PI3K signaling pathway through the formation of a molecular complex designated Motility Inducing Signaling Complex (MISC) contributing to cell survival, growth, proliferation, differentiation and motility (Malleter et al., Cancer Res 73(22):6711-6721, 2013; Tauzin et al., PLoS Biol 9(6):e1001090, 2011; Kleber et al., Cancer Cell 13(3):235-248, 2008). This chapter describes how to immunoprecipitate CD95 to characterize MISC involved in PI3K activation.