Regulation of the Initial Steps of CD95-mediated Signaling

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Lipid rafts

In addition to CD95 down-regulation or expression of a mutated allele of the receptor, alteration of the plasma membrane distribution of CD95 represents an additional mechanism by which tumor cells could develop resistance to CD95L-expressing immune cells. The plasma membrane is a heterogeneous lipid bilayer comprising compacted or liquid-ordered domains, called microdomains, lipid rafts, or detergent-resistant microdomains (DRMs). These domains, which are enriched in ceramides, have been described as floating in a more fluid or liquid-disordered two-dimensional (2-D) lipid bilayer [118]. A series of elegant experiments showed that although CD95 is mostly excluded from lipid rafts in activated T lymphocytes, TCR-dependent re-activation of these cells leads to rapid distribution of the death receptor into lipid rafts [119]. This CD95 compartmentalization contributes to a reduction in the apoptotic threshold, leading to clonotypic elimination of activated T lymphocytes through activation of the CD95-mediated apoptotic signal [119]. Similarly, the reorganization of CD95 into DRMs can occur independently of ligand upon addition of certain chemotherapeutic drugs (e.g., rituximab [120], resveratrol [121, 122], edelfosine [79, 123, 124], aplidin [125], perifosine [124], and cisplatin [126]). The molecular cascades underlying this process remain elusive. Nevertheless, a growing body of evidence leads us to postulate that alteration of intracellular signaling pathway(s), such as the aforementioned PI3K signal [79, 82], may change biophysical properties of the plasma membrane, such as membrane fluidity, which in turn may facilitate CD95 clustering into large lipid raft-enriched platforms, favoring DISC formation and induction of the apoptotic program [82].

3.4.2 Post-translational modifications

Accumulation of CD95 mutations is not the only mechanism by which malignant cells inhibit the extrinsic signaling pathway. Post-translational modifications in the intracellular tail of CD95, such as reversible oxidation or covalent attachment of palmitic acid, alter the plasma membrane distribution of CD95 and thereby its downstream signaling. For instance, S-glutathionylation of mouse CD95 at cysteine 294 promotes clustering of CD95 and its distribution into lipid rafts [127]. This amino acid is conserved in the human CD95 sequence and corresponds to cysteine 304 (or C288 when the 16 amino-acid signal peptide is taken into consideration [8, 128]). Interestingly, Janssen-Heininger and colleagues emphasize that death receptor gluthationylation occurs downstream of activation of caspase-8 and -3; the catalytic activities of these caspases damage the thiol transferase glutaredoxin 1 (Grx1) [127]. One consequence of Grx1 inactivation is accumulation of glutathionylated CD95, which clusters into lipid rafts, thereby sensitizing cells to CD95-mediated apoptotic signals. Based on these findings, caspase-8 activation occurs prior to aggregation of CD95 and redistribution into lipid rafts, both of which are required to form the DISC and subsequently activate larger amounts of caspase-8. In agreement with these observations, activation of caspase-8 occurs in a two-step process. First, a small amount of activated caspase-8 (<1%) is generated immediately when CD95L interacts with CD95, resulting in acid sphingomyelinase (ASM) activation, ceramide production, and CD95 clustering; these in turn promote DISC formation and the burst of caspase-8 processing that is essential for implementation of the apoptotic program [129].

S-glutathionylation consists of a bond between a reactive Cys-thiol and reduced glutathione (GSH), a tripeptide consisting of glycine, cysteine, and glutamate. Attachment of this group to a protein alters its structure and function in a manner similar to the addition of a phosphate [130]. S-glutathionylation is not the only post-translational modification of a cysteine in CD95: S-nitrosylation of cysteine 199 (corresponding to C183 after subtraction of signal peptide sequence) and 304 (C288) in colon and breast tumor cells also promotes the redistribution of CD95 into DRMs, formation of the DISC, and the transmission of the apoptotic signal [131].

Two reports have demonstrated that covalent coupling of a 16-carbon fatty acid (palmitic acid) to cysteine 199 (C183) elicits the redistribution of CD95 into DRMs, the formation of SDS-stable CD95 microaggregates resistant to denaturing and reducing treatments, and internalization of the receptor [132, 133]. Although the order of these events remains to be precisely determined, it is clear that these molecular steps play a critical role in the implementation of apoptotic signals.

As with S-nitrosylation, both the aforementioned S-glutathionylation at C304 (C288) and palmitoylation at C199 (C183) promote the partition of CD95 into lipid rafts and augment the subsequent apoptotic signal. Further investigation is required to determine whether these post-translational modifications are redundant, and occur simultaneously in dying cells, or instead are elicited in a cell-specific and/or in a microenvironment-specific manner. Understanding the molecular mechanisms controlling these post-translational modifications would be of great value in efforts to identify the mechanisms by which tumor cells block them, leading to resistance to the extrinsic signaling pathway.

Soon after CD95 was cloned, several groups investigated phosphorylation of this protein on serine/threonine and tyrosine and explored its biological role. Although serine/threonine phosphorylation may participate in the implementation of the CD95 signal, these authors mainly focused on the role of tyrosine phosphorylation in the cell death pathway. Phosphorylation can occur on two tyrosines located in the first (Y232, corresponding to Y216 starting from the first amino acid after the signal peptide) and fifth (Y291/Y275) α-helices of CD95-DD [134]. Y275 is located within a conserved YXXL motif reminiscent of the conserved ‘I/VxYxxL’ motif, termed the immunoreceptor tyrosine-based inhibitory motif (ITIM), which is responsible for the recruitment and activation of inhibitory phosphatases [135]. By recruiting the src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), Y275 phosphorylation promotes CD95-mediated cell death in T cells [136] and counteracts the GM-CSF–driven pro-survival signals in neutrophils [135]. Notably, this Y²⁷⁵DTL cytoplasmic domain is also a putative consensus YXXF sequence for AP-2 binding [137], which is instrumental in CD95 internalization (see below and [138]). Consistent with this, replacement of Y275 by a phenylalanine inhibits CD95 internalization and thereby blocks the induction of apoptosis, but does not affect non-apoptotic responses [138]. In addition, tyrosine phosphorylation of CD95 promotes the recruitment of the src kinases Fyn and Lyn through their SH2 domains, thereby promoting cell death [139, 140]. Accordingly, it is tempting to speculate that Y275 phosphorylation may guide the receptor through the induction of the apoptotic signal at the expense of non-apoptotic pathways. These data raise some questions about the identity of the tyrosine kinase involved in Y275 phosphorylation, the order of the molecular events leading to phosphatase and src kinase recruitment, and their respective roles in the CD95 signaling pathway.

CD95 internalization

A powerful magnetic method for isolating receptor-containing endocytic vesicles was used to show that CD95 promptly associates with endosomal and lysosomal markers upon incubation of cells with agonistic anti-CD95 mAb [138]. In addition, expression of a CD95 mutant in which the DD-located tyrosine 291 (Y275) is changed to phenylalanine does not seem to alter the capacity to bind FADD, but instead compromises CD95L-mediated CD95 internalization occurring through an AP-2/clathrin-driven endocytic pathway [138]. More strikingly, expression of the internalization-defective CD95 mutant Y291F abrogates the transmission of apoptotic signals, but fails to block the non-apoptotic signaling pathways (i.e., NF-κB and ERK); indeed, the mutant even promotes these pathways (Figure 3). These findings highlight the presence of a region in the DD, which interacts with AP2 and promotes a clathrin-dependent endocytic pathway in a FADD-independent manner. The role of palmitoylation in the AP2/clathrin-driven internalization of CD95 remains to be elucidated.

Ca²⁺ response

A recent study demonstrated that CD95 engagement evokes rapid and transient Ca²⁺ signaling, which stimulates the recruitment of protein kinase C-β2 (PKC-β2) from the cytosol to the DISC[141]. This kinase transiently halts DISC formation, providing a checkpoint before the irreversible commitment to cell death [142]. These findings raised two important questions: what are the Ca²⁺-dependent molecular mechanisms transiently inhibiting DISC formation, and do tumor cells use this signal to escape the immune response and/or resist chemotherapy?