Regulation of the CD95-dependent apoptosis by Protein Phosphatases

Read Also

Protein Tyrosine Phosphatases

Tyrosine phosphorylation of proteins is achieved by protein tyrosine kinases (PTK). This reversible protein post-translational modification regulates many transduction pathways in eukaryotic cells, like those involved in embryogenesis, development, cell proliferation and motility. Protein tyrosine phosphatases (PTP) act by removing phosphates from tyrosine residues, thereby counteracting PTK effects [29, 30]. PTPs contain a signature motif [I/V]HCXXGXXR[S/T] where the invariant cysteine residue is the nucleophile during catalysis and the arginine serves as phosphate binding [31]. Classical PTP are divided into two sub-groups, the cytoplasmic (non-receptor) and transmembrane proteins, also called receptor PTP (RPTP) [32]. Here, we will present the reported effects of classical PTPs on CD95 signaling pathway, focusing our attention particularly on the early events of this pathway.

FAP-1

FAP-1 (for Fas-associated phosphatase-1, also called PTPL1, PTP-BAS or PTP1E) is a non-receptor PTP of 270 kDa encoded by the PTPN13 gene. This huge protein contains a protein tyrosine phosphatase domain located at the extreme C-terminus part of the protein and several protein-protein interaction motifs in the N-terminus and central regions called respectively KIND, FERM and PDZ domains (Fig. 2) [33]. KIND is located at the extreme N-terminus and contains a kinase noncatalytic C-lobe domain showing homologies with the regulatory C-lobe of protein kinases, but lacking catalytic activity [34]. The functional role of this domain is yet unknown. The Four-point-one/Ezrin/Radixin/Moesin (FERM) domain follows the KIND domain. FERM domains are important mediators between plasma membrane receptors and cytoskeleton [35]. FAP-1 also contains five PDZ (PSD-95/Drosophila discs-large/Zonula occludens) domains which are located in the central region of the protein and are involved in the formation of supramolecular protein complexes [36]. The exhaustive description of FAP-1 interacting proteins is beyond the scope of this article and has been presented elsewhere [33, 37]. FAP-1 was reported to directly interact with the cytoplasmic domain of human CD95 via its PDZ 2 and 4 domains [38-41]. FAP-1 binds the C-terminal 15 amino acids of CD95, and the deletion of these 15 amino acids enhances apoptosis induced by CD95-L [38, 42]. The complementation of Jurkat T cells (which do not express FAP-1) with wt FAP-1, but not with a phosphatase inactive form, protects them from CD95-mediated apoptosis, suggesting that FAP-1 is involved in the negative regulation of the CD95 pathway [38]. However, this interaction does not seem to be evolutionary conserved, since the mouse CD95 does not interact with PTP-BL (the mouse homolog of FAP-1), and that PTP-BL does not inhibit CD95-induced apoptosis in mice [43]. Nonetheless, there is a clear correlation between the expression of FAP-1 and the survival of several human tumor models, including ovarian, colon, head and neck cancers, hepatocellular carcinoma, hepatoblastoma and pancreatic adenocarcinoma [41, 44-49]. Accordingly, stable introduction of FAP-1 in FAP-1 negative pancreatic and melanoma cell lines or in squamous cell carcinoma of the head and neck was reported to inhibit CD95-mediated apoptosis [46, 50, 51]. FAP-1 is also important for the regulation of immune cells apoptosis. A down-regulation of FAP-1 mRNA was observed in IL-2-activated T cells, accounting for a higher sensitivity to CD95-induced apoptosis [52]. Enhanced apoptosis in T helper 1 (Th1) comparing to Th2 cells is due to unequal FAP-1 expression between these two populations [53]. In the same way, up-regulation of FAP-1 is responsible for the escape of HTLV-1 infected T cells from CD95-induced apoptosis [54]. At the molecular level, it appears that FAP-1 is able to regulate cell surface localization of CD95. Forced expression of FAP-1 increases the intracellular pool of CD95, and siRNA against FAP-1 up-regulates CD95 membrane expression [46, 51]. Confocal microscopy studies revealed that FAP-1 is mainly associated with the Golgi complex where it appears to sequestrate CD95, thereby decreasing its membrane localization [50]. These observations suggest that tyrosine phosphorylation is involved in the localization of CD95 at the membrane. Indeed, it was shown that tyrosine kinases inhibitors prevent CD95-induced apoptosis [55, 56]. Moreover, CD95 interacts with p59^fyn and p56^lck tyrosine kinases, and this interaction enhances CD95-induced DISC formation and apoptosis [57, 58]. Recently, it was nicely shown that CD95L stimulation of hepatocytes (which do not express CD95 at the cell surface under basal conditions) induces a local production of reactive oxygen species resulting in a Yes-dependent activation of the EGF-R. This leads to the association between EGF-R and CD95 already in the cytosol and catalyses CD95 tyrosine phosphorylation [59, 60]. This tyrosine phosphorylation is a prerequisite for CD95 membrane targeting, oligomerization and DISC formation [61]. Tyrosine phosphorylation occurs at positions Y232 and Y291 (also named Y216 and Y275[1]) in the death domain, and mutation of these residues to F or D prevents or increases the targeting of CD95 to the plasma membrane, respectively [51, 61]. It has also been reported that intact CD95 Y291 is required for CD95L-induced internalization of CD95, a prerequisite for DISC assembly and apoptotic signal (Fig. 3) [62]. In that context, it is likely that FAP-1 regulates CD95 localization via tyrosine dephosphorylation of CD95. Indeed, a direct dephosphorylation of CD95 Y291 by FAP-1 was reported in astrocytoma cells (Fig. 3) [63]. All these results suggest that FAP-1 is a powerful negative regulator of CD95-induced apoptosis implicated in oncogenesis. This implies that FAP-1 expression must be tightly controlled in normal tissues to avoid oncogenic transformation. As already mentioned, FAP-1 transcription is down-regulated in activated T cells, and increased FAP-1 mRNA correlates with CD95 resistance in some leukemia cell lines [52, 64]. The molecular events underlying the control of PTPN13 (FAP-1) transcription has been recently clarified in myeloid cells. It was shown that the interferon consensus sequence-binding protein (ICSBP or IRF8) interacts with a cis element in the proximal PTPN13 promoter and repress transcription during myeloid differentiation, accounting for an increased CD95 sensitivity [65]. Accordingly, ICSBP-deficient mice develop a myeloproliferative disorder [66].         

  

SHP-1

SHP-1 (encoded by PTPN6, also called HCP, SH-PTP1) contains two tandem SH2 domains positioned at the N-terminus of the protein followed by a central catalytic region. The C-terminus region contains multiple phosphorylation sites and plays regulatory functions (Fig. 2) [67]. Mutation in the SHP-1 gene cause severe immunodeficiency accompanied by systemic autoimmune disease and chronic inflammation in mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (me^v) on chromosome 6 [68, 69]. This highlights the key role of this phosphatase in the negative regulation of cell function. Studies performed on viable motheaten mice reported that SHP-1 defect reduces lymphoid cells apoptosis induced by CD95, suggesting that SHP-1 is involved in the delivery of CD95-apoptosis signal in lymphocytes [70]. In neutrophils, SHP-1 binds a highly conserved Y₂₉₁xxL motif located in the death domain of CD95. Mutation of Y291 to A prevents SHP-1 binding upon CD95-L stimulation and inhibits cell death [71]. Since Y291 phosphorylation was shown to induce CD95 membrane targeting and internalization [61, 62], one can speculate that SHP-1 would be involved in that process (Fig. 3). In the same way, it was recently shown that SHP-1 binds caspase-8 via an Y₃₁₀xxL motif located in the pro-domain of caspase-8, and Y310F mutation disrupts this interaction. In neutrophils, caspase-8 is basally tyrosine phosphorylated on Y397 and 465, and its dephosphorylation by SHP-1 results in its activation and progression of the apoptotic cascade [72]. These two observations suggest that SHP-1, on the contrary of FAP-1, controls positively the CD95 pathway. However, discrepant results were obtained. Hepatocyte apoptosis remained unchanged in me^v mice compared to wt mice, highlighting some cell-type specificities in SHP-1 pro-apoptotic activity [70]. On the contrary to me^v mice, no involvement of SHP-1 in CD95-mediated T cell death was reported using me mice [73]. The me mutant carries a deletion of one base-pair in the SHP-1 gene, resulting in the absence of SHP-1 protein. On the contrary, me^v mice express two variants of the SHP-1 protein lacking phosphatase activity [68, 69, 74]. The discrepancy between results obtained with me versus me^v mice is still unexplained, even if it is attractive to speculate that SHP-1 inhibits the CD95 pathway independently of its phosphatase activity. In B cells, recent results reported that SHP-1 plays a negative role in CD95-induced apoptosis by blocking actin-dependent CD95 internalization, a prerequisite for DISC formation [75]. Therefore, the exact involvement of SHP-1 in the CD95 pathway is still matter of debate in the literature, and appears to be highly cell-type specific (Fig. 3).     

PTP-1B

PTP-1B (encoded by PTPN1) contains an N-terminal catalytic domain followed by tandem proline-rich motifs and a small hydrophobic endoplasmic reticulum-targeting sequence at its C-terminus [76]. PTP-1B modulates various growth factors-induced signaling pathways by dephosphorylating receptors, such as insulin, IGF-1, EGF, PDGF and erythropoietin receptors [77-80]. Particularly, PTP-1B has a crucial role in negatively regulating insulin signaling, since PTP-1B deficient mice have increased insulin sensitivity and obesity resistance [81]. It was also recently reported that PTP-1B deficiency protects against liver apoptosis and fulminant hepatic failure induced by CD95, suggesting that PTP-1B is also a key modulator of the CD95 pathway [82]. PTP-1B deficient mice exhibit no caspase-8, -9 and -3 cleavage upon injection of CD95 antibody due to elevated anti-apoptotic proteins such as FLIP_L, ERK1/2 and NF-κB. The HGF/Met receptor, a potent hepatoprotective molecule, was also found hyperphosphorylated in PTP-1B KO mice, accounting for CD95-resistance. It is noteworthy that, despite the ubiquitous PTP-1B expression, resistance to CD95-apoptosis is limited to hepatocytes. Indeed, thymocytes from PTP-1B KO mice exhibit equal response to CD95-induced apoptosis when compared to wt mice [82].   

Protein Serine/Threonine Phosphatases

The Protein Serine/threonine phosphatases superfamily is divided into two subgroups. The PPP (phospho protein phosphatases) group includes notably types 1, 2A, 2B, PP4, PP5, PP6 phosphatases. PPM (protein phosphatases magnesium-dependent) require Mg²⁺ or Mn²⁺ for their activity and comprise notably PP2C [83, 84]. The involvement of serine/threonine phosphatases in the CD95 pathway is poorly known. Using pharmacological inhibitors, it has been shown that inhibition of PP1 and PP2A suppresses CD95-induced apoptosis by preventing DISC formation [85, 86]. Even if the exact molecular mechanism is unknown, it appears that an increased MAPK/ERK activity would account for apoptosis resistance [86].  In neutrophils, PP2A regulates apoptosis by dephosphorylating both the pro-survival p38 MAPK and caspases-3, a p38 substrate.  Since phosphorylation of caspases-3 impairs its activity, PP2A appears to promote neutrophils apoptosis.  Accordingly, a rapid increase in PP2A activity is observed upon spontaneous or CD95-L-induced neutrophils apoptosis [87, 88]. Protein serine/threonine phosphatases are also implicated in the control of the mitochondrial apoptosis pathway [89-91].   

---

[1] There is some confusion regarding amino acid #1 for CD95: some people indicate as aa #1 Methionine (M) of the signal peptide, while the others indicate Arginine (R) from the mature protein without signal peptide. So, there is a shift of 16 aa if the full length CD95 is considered (V. Ivanov, personal communication).