Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis.

The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.

Cisplatin-induced apoptosis involves a Fas-ROCK-ezrin-dependent actin remodelling in human colon cancer cells.

In human colon cancer cells, cisplatin-induced apoptosis involves the Fas death receptor pathway independent of Fas ligand. The present study explores the role of ezrin and actin cytoskeleton in relation with Fas receptor in this cell death pathway. In response to cisplatin treatment, a rapid and transient actin reorganisation is observed at the cell membrane by fluorescence microscopy after Phalloidin-FITC staining. This event is dependent on the membrane fluidification studied by electron paramagnetic resonance and necessary for apoptosis induction. Moreover, early after the onset of cisplatin treatment, ezrin co-localised with Fas at the cell membrane was visualised by membrane microscopy and was redistributed with Fas, FADD and procaspase-8 into membrane lipid rafts as shown on Western blots. In fact, cisplatin exposure results in an early small GTPase RhoA activation demonstrated by RhoA-GTP pull down, Rho kinase (ROCK)-dependent ezrin phosphorylation and actin microfilaments remodelling. Pretreatment with latrunculin A, an inhibitor of actin polymerisation, or specific extinction of ezrin or ROCK by RNA interference prevents both cisplatin-induced actin reorganisation and apoptosis. Interestingly, specific extinction of Fas receptor by RNA interference abrogates cisplatin-induced ROCK-dependent ezrin phosphorylation, actin reorganisation and apoptosis suggesting that Fas is a key regulator of cisplatin-induced actin remodelling and is indispensable for apoptosis. Thus, these findings show for the first time that phosphorylation of ezrin by ROCK via Fas receptor is involved in the early steps of cisplatin-induced apoptosis.