SHIP-1 inhibits CD95/APO-1/Fas-induced apoptosis in primary T
lymphocytes and T leukemic cells by promoting CD95
glycosylation independently of its phosphatase activity
Running title: SHIP-1 inhibits CD95-induced apoptosis in T lymphocytes
Edith Charlier1,2, Claude Condé1,2, Jing Zhang4, Laurence Deneubourg4, Emmanuel Di Valentin1,2, Souad Rahmouni1,3, Alain Chariot1,2, Patrizia Agostinis5, Poh-Choo Pang6, Stuart M. Haslam6, Anne Dell6, Josef Penninger7, Christophe Erneux4, Jacques Piette1,2 and Geoffrey Gloire1, 2*.
1GIGA-Research, 2Signal Transduction and 3Infection, Immunity and Inflammation units, University of Liège, Liège, Belgium.4IRIBHM, Free University of Brussels, Brussels, Belgium.5Department of Molecular and Cell Biology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium.6Division of Molecular Biosciences, Imperial College London, London SW72AZ, UK. 7IMBA Institute of Molecular Biotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria.
*Corresponding author: Dr. Geoffrey Gloire University of Liège GIGA-Research (B34) 1, Avenue de l’hôpital 4000 Liège, Belgium. Tel: +32 4 366 24 26 Fax: +32 4 366 45 34 geoffrey.gloire@ulg.ac.be
ABSTRACT
SHIP-1 functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by PI 3-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B cell lymphoma. On the other hand, SHIP-1 deficient mice have a reduced T cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1-/- mice and T leukemic cell lines, we report here that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum where it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired DISC formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1 negative leukemic T cells.
INTRODUCTION
T lymphocytes homeostasis is tightly regulated by CD95-mediated apoptosis (1, 2). Triggering the CD95 pathway by CD95 ligand or agonist antibodies induces its oligomerization and the subsequent formation of the death inducing signaling complex (DISC), having as main components oligomerized CD95, the adaptor protein FADD and pro-caspases-8 and -10 (3). The recruitment of pro-caspase-8 to the DISC leads to its dimerization-induced auto-proteolytic cleavage and subsequent activation. Activated caspase-8 allows the activation of downstream effectors caspases-3 and -7, which in turn cleave cellular substrates, such as PARP, resulting in cellular changes defining apoptosis (1). Depending on the cell model, two pathways of CD95 signaling have been described (4). Type I cells (Hut78 and H9 T leukemic cells and SKW6.4 B lymphoblastoid cells) exhibit high DISC formation upon CD95 stimulation, and activated caspase-8 directly activates caspase-3. In type II cells (Jurkat and CEM T leukemic cells), low DISC formation is observed and the apoptotic signal is amplified by the release of cytochrome c from the mitochondria through the cleavage of Bid (5, 6). SH2-containing inositol 5’-phosphatase-1 (SHIP-1) is a 145 kDa inositol 5’-phosphatase mainly expressed in hematopoietic cells (7). SHIP-1 harbors a central phosphatase domain, an N-terminal SH2 domain as well as a C-terminal domain that includes NPXY phosphorylation sites and proline-rich sequences. The SH2 domain and NPXY sites mediate the binding of SHIP-1 with a series of consensus motifs or adapter proteins (8). Through its inositol phosphatase activity, SHIP-1 antagonizes the PI 3-kinase pathway by hydrolyzing phosphatidylinositol 3,4,5-triphosphate into phosphatidylinositol 3,4-diphosphate, thereby negatively regulating downstream survival pathways, such as Akt activation (7). In support of this, germ-line deletion of SHIP-1 in mice or its downregulation
by miR-155 which targets SHIP-1, results in myeloproliferation as well as B cell lymphoma (9-12). For this reason, SHIP-1 is often considered as a tumor-suppressor protein (13-15). Although several lines of evidence indicate its emerging importance, the role of SHIP-1 in T cell fate is still unclear (16, 17). In mice, SHIP-1 deficiency leads to a decrease in the T cell population, particularly in the periphery (9, 10, 18, 19). Peripheral T cells from SHIP-1-/- mice are also constitutively activated and behave like TReg cells (18-20). Given the phenotype of SHIP-1-/- mice, we hypothesized that SHIP-1 could have different roles depending on cell-type. We postulate that, unlike its well-known pro-apoptotic activity in myeloid and B cells, SHIP-1 has an anti-apoptotic function in T cells. Here, we report for the first time that SHIP-1 negatively regulates the CD95 signaling pathway in primary mouse T lymphocytes and in various T cell human leukemic cell lines. We observed that SHIP-1 localizes to the endoplasmic reticulum where it increases the presence of high molecular weight forms of CD95 due to glycosylation. Evidence is provided to implicate SHIP-1 docking properties in this mechanism. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired DISC formation and apoptotic cascade.
MATERIALS AND METHODS
Murine T lymphocytes isolation and cell culture. Cell suspensions of spleen and lymph
nodes from wt and SHIP-1-/- mice were prepared and pooled. T cells were enriched by the Magnetic Activated Cell Sorting device (StemCell Technologies) using the mouse CD3+ T cell negative selection kit according to the manufacturer’s instructions (StemCell Technologies). The purity of T cells was assessed by flow cytometry using an anti-CD3ɛ-PE antibody (BD Pharmingen) and was estimated to be 90%. After isolation, primary murine T lymphocytes were maintained in RPMI medium containing 10% fetal bovine serum, 50 µM 2-mercaptoethanol, 1% L- glutamine, 25 mM HEPES, 50 µg/mL gentamycin and 50 U/mL IL-2. Jurkat, CEM and HuT78 T leukemic cells were cultured as previously described (21).
Antibodies and reagents. Antibodies anti-SHIP-1 (clone P1C1) and anti-CD95 (clone C-20)
were from Tebu Bio (Santa Cruz, CA, USA). Anti-actin was purchased from Sigma (Bornem, Belgium) and anti-CD2 from Serotec. Anti-Akt and anti-P-Akt (T308) were from Cell Signaling (Beverly, MA, USA). Anti-PARP was purchased from BD Pharmingen (USA). Anti-caspase-8 (clone C-15), anti-caspase-3, monoclonal anti-APO-1-3, soluble recombinant human CD95 ligand (SuperFas Ligand) and soluble recombinant human TRAIL ligand (SuperKiller TRAIL) were from Alexis Biochemicals (Germany). The monoclonal anti-CD95 antibody (clone 7C11) was from Beckman Coulter. Tetracycline and camptothecin were purchased from Sigma. Biotinylated lectins were purchased from Vector Laboratories (Peterborough, United Kingdom). Agarose-linked streptavidin were obtained from Sigma. Glycoprotein Deglycosylation Kit (Cat. No. 362280) and benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (b-GalNAc, Cat. No. 200100) were from Calbiochem (La Jolla, CA, USA).
Lentiviral vectors and transduction protocol. Myc-tagged SHIP-1 wt, R41G (SH2 mutant)
coding sequence was subcloned in the BamHI/XhoI sites of the TRIPΔU3-EF1α lentiviral vector (23). Vector particles production and Jurkat cells infection were performed as previously described (23).
siRNA electroporation. 1x107 cells were incubated with 300nM of SHIP-1 siRNA duplex (5′-GCUAAGUGCUUUACGAACA-3′, Applied Biosystems) (24) or 300nM of control firefly luciferase siRNA (ref. AM4627, Applied Biosystems) for 10 min prior electroporation (25 ms, 293V) with a Biorad gene pulser (BioRad Laboratories Inc.). The cells were then incubated during 20 min at room temperature and then maintained in IMDM (Gibco) complemented with 20% FBS.
CD95 stimulation. Jurkat, CEM and HuT78 T leukemic cells were treated with anti-CD95
antibodies (clone 7C11, Beckman Coulter or APO-1-3, Alexis) or SuperFas Ligand (Alexis) at concentration and times indicated. Primary murine T cells were activated by co-stimulation with anti-CD3/CD28-coated beads (Dynabeads, Invitrogen) for 18h prior to be stimulated with SuperFas Ligand at concentration and time indicated.
DISC analysis. Cells (1x108) were treated with 1µg/mL anti-APO-1-3 for the indicated times at 37°C. The CD95 DISC was immunoprecipitated from protein lysates at 4°C for 2 h with Protein-A Sepharose beads. The immunoprecipitates were resolved by SDS PAGE followed by Western blotting.
CD95 surface expression. Cell surface staining for CD95 was performed using a one-sep
procedure with PE-conjugated anti-human CD95 (clone DX2, BD-Pharmingen, Germany) or PE-conjugated mouse IgG1 as a control (BD-Pharmingen, Germany). Cells were analyzed by flow cytometry (FACScanto Becton Dickinson, San Diego, CA, USA).
Glycosidase treatment and inhibition of glycosylation. Enzymatic removal of glycans was
performed on cell lysates with a glycoprotein deglycosylation kit from Calbiochem (Cat. No. 362280), according to manufacturer’s instructions. Tunicamycin (Sigma) and
benzyl-a-GalNAc (Calbiochem) were added to the cell culture for 48h at final concentrations of 2µg/mL and 4mM, respectively. After 24h incubation, fresh inhibitor was added at the same final concentration.
Lectin pull-down assay. Lysates from Jurkat and Jurkat SHIP-1 cells were mixed with 40 µg
of biotinylated lectins and incubated under rotation at 4°C overnight. Then lectins-glycoproteins complexes were immunoprecipitated using Agarose-coupled streptavidin (Sigma).
Fractionation of subcellular organelles. Fractionation of subcellular organelles was
performed essentially according to the Axis Shield NS11 protocol allowing isolation of mitochondria, peroxisomes, ER and lysosomes. Isolation of the ER fraction was obtained by nonlinear Nycodenz density gradient ultracentrifugation.
RESULTS
SHIP-1 protects primary T lymphocytes and T leukemic cells from CD95-induced apoptosis
Given the phenotype of SHIP-1-/- mice, we hypothesized that SHIP-1 could exert an anti-apoptotic role in T lymphocytes. We isolated CD3+ T cells from wt and SHIP-1-/- mice and evaluated their ability to undergo apoptosis after activation, a prerequisite for their sensitivity to CD95-L, the major ligand regulating T cell apoptosis in vivo (1, 2). Deficiency in SHIP-1 expression in KO mice was confirmed by Western blotting analysis on thymus extracts (Fig.
1a). AnnexinV/PI staining revealed that activated SHIP-1-/- T cells display similar basal cell death compared to wt T cells (Fig. 1b). Upon CD95-L (SuperFas Ligand) treatment, cell death in SHIP-1-/- T cells reached ~90%, whereas only ~60% of wt T cells died, suggesting that SHIP-1 indeed protects T lymphocytes from CD95-induced cytotoxicity (Fig. 1b). In order to explore the molecular mechanism involved, we used the Jurkat T leukemic cell line. Jurkat cells do not expressed SHIP-1 at the protein level (21, 25, 26) due to a bi-allelic null mutation and a frame-shift deletion (27) and we restored SHIP-1 expression in these cells using lentiviral transduction method. As a negative control, Jurkat cells were transduced with an empty vector (hereafter referred as Jurkat Ev). SHIP-1 expression and catalytic activity were evaluated by Western blotting using SHIP-1 and P-Akt (T308) antibodies, respectively. The constitutive P-Akt signal is elevated in Jurkat Ev, reflecting the absence of inositol phosphatase activity and the subsequent high basal level of PtdIns(3,4,5)P3, the docking site for Akt (25, 26) (Fig. 1c). In contrast, P-Akt was not detected in Jurkat expressing SHIP-1 exogenously (hereafter referred as Jurkat SHIP-1) or in the Jurkat JR cell line, a subclone that naturally expresses SHIP-1 (21) (Fig. 1c). To test whether SHIP-1 expression protects Jurkat cells from CD95-induced apoptosis, Jurkat Ev and Jurkat SHIP-1 cells were treated with CD95 antibodies (clones APO-1 and 7C11) or CD95-L for 24h. As shown in Fig. 1d, Jurkat
SHIP-1 cells were significantly resistant to CD95-induced cell death when compared with Jurkat Ev. Similarly, we found that Jurkat SHIP-1 were also resistant to camptothecin and TRAIL-induced apoptosis (Fig. 1d). Moreover, the analysis of pro-caspase-8, pro-caspase-3 and PARP cleavages in both cell lines treated with 7C11 Ab confirmed this result. In Jurkat Ev cells, pro-caspase-8, -3 and PARP cleavage is observed at any time point of 7C11 Ab treatment, while the Jurkat SHIP-1 exhibited no processing of pro-caspase-8 and a delayed processing of pro-caspase-3 and PARP (Fig. 1e). Altogether, these data suggest that SHIP-1 negatively regulates CD95-mediated apoptosis in primary mouse T lymphocytes and in Jurkat leukemic cells. To further validate these results, we transiently knocked down the expression of SHIP-1 by RNAi in various SHIP-1 positive T leukemic cell lines. SHIP-1 depleted Jurkat JR exhibited caspase-3 and PARP cleavages upon stimulation with 7C11 Ab, whereas no cleavage was observed in control cells (Fig. 2a). Similar results were obtained with Hut78 and H9 type I cells (Fig. 2b and data not shown). SHIP-1 depletion in CEM, a type II T leukemic cell line, enhanced the kinetics of caspases and PARP cleavages (Fig. 2c). These results indicate that SHIP-1 protects primary T lymphocytes and various type I and type II T leukemic cell lines against CD95-induced cell death.
The catalytic activity of SHIP-1 is not required for the protection against CD95-induced apoptosis.
To determine whether the catalytic activity of SHIP-1 is involved in the protection against CD95-mediated apoptosis, we transduced Jurkat cells with a mutated form of SHIP-1 lacking phosphatase activity (SHIP-1 D672A). Western blotting analysis showed that SHIP-1 D672A was correctly expressed in Jurkat cells and was unable to reduce constitutive P-Akt (Fig. 3a). A slightly lower level of P-Akt is observed with the Jurkat D672A compared to the parental Jurkat cells, most probably due to a slightly lower level of total Akt. We then compared the percentage of apoptosis between Jurkat Ev, Jurkat SHIP-1 wt and Jurkat SHIP-1 D672A cells
upon CD95 Ab treatment. As shown in Fig. 3b, expression of SHIP-1 D672A still protects Jurkat cells from CD95-induced apoptosis. Study of pro-caspases-8, -3 and PARP cleavage confirmed these findings (Fig. 3c). To further confirm this result, we used Jurkat cells stably transfected with a CD2:SHIP-1 construct under the control of a Tet-Off expression system (26). The resulting protein is a fusion of the rat CD2 transmembrane domain with the human SHIP-1 catalytic domain and thus exhibits constitutive inositol phosphatase activity at the membrane (26). The Tet-regulated expression of CD2:SHIP-1 as well as its catalytic activity were evaluated by Western blotting analysis using anti-CD2 and P-Akt antibodies, respectively (Fig. 3d). The effect of Tet-regulated expression of the CD2:SHIP-1 chimeric protein on CD95-mediated cell death in Jurkat cells was next investigated. We did not observe any significant difference in apoptotic cell death whether the cells expressed or not the catalytic core of SHIP-1 (Fig. 3e). Altogether, these data suggest that the enzymatic activity of SHIP-1 is not involved in its anti-apoptotic function upon CD95 stimulation in Jurkat T cells.
The SH2 domain of SHIP-1 is required for its anti-apoptotic activity upon CD95 induction.
In addition to a catalytic domain, SHIP-1 encompasses numbers of protein-protein interaction domains, including the SH2 sequence (8). In order to investigate the involvement of this region in the anti-apoptotic function of SHIP-1, we generated the Jurkat SHIP-1 R41G cell line, which expresses a mutated form of SHIP-1 lacking functional SH2 domain but still able to inhibit P-Akt (Fig. 3f). Strikingly, we observed that expression of SHIP-1 R41G restored the sensitivity to 7C11 Ab-induced apoptosis to levels similar to those observed with parental Jurkat cells (Fig. 3g). This was supported by the pro-caspase-8, -3 and PARP cleavage profiles (Fig. 3h). These data suggest that SHIP-1 negative regulation of CD95-mediated apoptosis involves the docking properties of SHIP-1 SH2 domain.
SHIP-1 does not affect CD95 membrane expression but increases the presence of high molecular weight forms of the receptor.
We next wondered whether SHIP-1 could modulate the expression of CD95 on the cell surface by flow cytometry. A normal, and even higher expression of CD95 at the surface of Jurkat SHIP-1 compared to Jurkat EV or Jurkat SHIP-1 R41G was observed (Fig.4a), indicating that SHIP-1 does not inhibit the trafficking of CD95 to the plasma membrane. However, Western blotting analysis revealed the presence of higher molecular weight forms of CD95 upon SHIP-1 expression in Jurkat cells (Fig. 4b). This electrophoretic mobility shift corresponds to approximately 10 kDa and is no more induced upon SHIP-1 R41G expression
(Fig. 4b). Importantly, this CD95 shift was also detected in all the SHIP-1 positive leukemic
cell lines tested in this study (Fig. 4c). These results suggest that SHIP-1 induces an SDS-resistant post-translational modification of CD95 in a SH2 domain-dependent manner. However, despite having a higher molecular weight, the modified CD95 routed normally to the cell surface.
SHIP-1 promotes CD95 glycosylation.
It has been shown that CD95 is a glycoprotein whose molecular weight can vary depending on the level of N- or O-linked carbohydrates residues added (28-30). We therefore studied the effect of oligosaccharide chain removal on CD95 molecular weight in Jurkat Ev and Jurkat SHIP-1. We used a set of enzymes that remove all N-linked and O-linked oligosaccharides from glycoproteins and observed a decrease of CD95 molecular weight to ~35 kDa in both Jurkat Ev and SHIP-1, which corresponds to the predicted size of CD95 without any modification (29) (Fig. 5a). This result indicates that CD95 is basally glycosylated in Jurkat Ev and that the CD95 shift induced by SHIP-1 expression corresponds to an additional carbohydrate moiety. We then separately analyzed N- and O-glycosylation of CD95 from
both cell lines, using pharmacological inhibitors of either N-glycosylation (i.e. tunicamycin) or O-glycosylation (i.e. benzyl-GalNAc, which competitively inhibits the extension of the Tn antigen (GalNAc-Ser/Thr) to more complex O-glycans) (Supplementary Materials &
Methods). As shown in Supplementary Fig. 1a, addition of tunicamycin to Jurkat Ev cells
generated the shortest form of CD95 (~35kDa), whereas in Jurkat SHIP-1 cells, the use of tunicamycin made CD95 detection size only felt to the ~40 kDa form observed in non treated Jurkat Ev cells. However, addition of benzyl-GalNAc provoked a partial decrease of CD95 molecular weight only in Jurkat SHIP-1 cells (Supplementary Fig. 1b). Altogether, these results suggest that CD95 from Jurkat EV is basally N-glycosylated, and that the CD95 shift induced by SHIP-1 corresponds to additional O-glycans. However, from these western-blotting experiments, we cannot rule out firmly that minor changes in N-glycosylation also occurred. In order to better evaluate the differences in CD95 glycans content, we performed a lectin pull-down assay. CD95 from both Jurkat and Jurkat SHIP-1 cells interacted with SNA and RCA120 lectins, indicating the presence of respectively sialic acid and galactose residues in both cell lines (Fig. 5b). However, CD95 from Jurkat SHIP-1 also reacted with other lectins, in particular UEA (fucose) or WGA (N-acetylglucosamine), indicating a more complex glycosylation pattern than in parental Jurkat cells (Fig. 5b). A recapitulative table is shown in Fig. 5c. Obviously, structural characterization of CD95 glycosylation would be the best mean to rigorously compare the differences between CD95 from Jurkat Ev vs Jurkat SHIP-1, but this is a very challenging task which is outside the scope of the current work. It was, nevertheless, possible to gain some insight into the glycosylation changes occurring upon SHIP-1 expression by using mass spectrometric methodologies to fingerprint the N- and O-glycans present in Jurkat EV, Jurkat SHIP-1 and CEM cells (Supplementary Material &
Methods). All three cell types expressed the same repertoire of N-glycans, albeit with some
dramatic difference was observed in O-glycosylation of Jurkat cells with and without SHIP-1. Thus, in accordance with the known absence of a functional core 1 synthase in Jurkat cells, the Tn antigen (GalNAc) was the only significant glycan observed in these cells
(Supplementary Table 2) (31). In contrast, this antigen was not detectable in Jurkat SHIP-1
and CEM cells. Instead these cells expressed a mixture of core type-1 and core type-2 O-glycans which are typical of leukocytes (32). Together these results show that the major effect of SHIP-1 expression on Jurkat glycosylation is the restitution of the normal O-glycosylation pathway which stops at Tn (GalNAc-Ser/Thr) synthesis in wildtype Jurkat cells. Further evidence that the core 1 synthase is active in the Jurkat cells expressing SHIP-1 was obtained by treating the CD95 samples with sialidase and O-glycanase. This combination of enzymes removes intact core type-1 sequences but not the Tn antigen. A substantial shift in molecular weight of Jurkat SHIP-1 CD95 was observed using these enzymes (Supplementary Figure
2) confirming the presence of sialylated core type-1 glycans on this sample.
Inhibition of N-, but not O-glycosylation, restores CD95-sensitivity in Jurkat SHIP-1
We next investigated whether inhibition of N- or O-glycosylation could sensitize Jurkat SHIP-1 to CD95-induced cell death. Pre-treatment of Jurkat SHIP-1 with the N-glycosylation inhibitor tunicamycin significantly increased 7C11 Ab-induced cell death when compared with non pre-treated cells (Fig. 6a). PARP cleavage detection by Western blotting confirmed this result (Fig. 6b). By contrast, pre-treatment of Jurkat SHIP-1 with benzyl-GalNAc, an inhibitor of O-glycosylation, did not modify 7C11 Ab-induced cell death compared with non pre-treated cells (Fig. 6a). However SDS-PAGE showed that benzyl-GalNAc treatment reduced the molecular weight of CD95 to an intermediate position between that of Jurkat EV CD95 and Jurkat SHIP-1 CD95 suggesting that only partial inhibition of O-glycan biosynthesis had occurred (Supplementary Figure 1b). Therefore no firm conclusions concerning the importance of O-glycosylation in SHIP-1 negative regulation of
CD95-induced apoptosis could be drawn from this experiment. On the other hand, the tunicamycin experiment suggested that the N-glycosylation status of CD95 is important for apoptotic activity. Whether the N-linked oligosaccharides themselves are directly involved, or whether their absence in the tunicamycin experiment results in altered O-glycosylation leading to the observed effects, remains to be established.
SHIP-1 is localized to the endoplasmic reticulum
To understand the mechanism involved in SHIP-1-induced CD95 glycosylation, SHIP-1 subcellular localization was investigated. Nascent polypeptide chains become initially N-glycosylated with a mannose-rich branched oligosaccharide in the endoplasmic reticulum (ER) (33) and it was reported that some phosphatases like PTP-1B are localized to ER membranes (34) where they control glycosylation state of receptor tyrosine kinases (35). A biochemical analysis of subcellular fractionation performed on Jurkat SHIP-1 revealed that ~15% of the SHIP-1 pool was detected in the fraction of membrane/organelle that coincides with the ER marker Grp78 (Fig. 7a). Similarly, a nonlinear Nycodenz density gradient ultracentrifugation performed on the membrane/organelle fraction revealed that SHIP-1 and CD95 are present in fractions corresponding to the ER (Fig. 7b, fractions 5 to 8). Collectively, these data suggest that SHIP-1 is localized to the ER together with CD95.
Glycosylated CD95 fails to oligomerize and to recruit the DISC upon stimulation.
To further establish a relationship between SHIP-1 expression and the apoptosis inhibition, we explored the impact of SHIP-1-induced CD95 glycosylation on CD95 oligomerization and DISC formation, two early events required for efficient downstream signaling (1). Jurkat Ev and Jurkat SHIP-1 cells were stimulated with the APO-1 antibody. Anti-APO-1 immunoprecipitations followed by anti-CD95 Western blotting were carried out. Stimulation of Jurkat Ev cells induced the presence of SDS and 2-mercaptoethanol-resistant CD95
aggregates of high molecular weight corresponding to oligomerized forms of CD95 (36), while Jurkat SHIP-1 cells exhibited much less CD95 aggregates upon stimulation (Fig. 8a). We also analyzed DISC formation by blotting caspase-8 and FADD after CD95 immunoprecipitations. We observed that FADD and caspase-8 were strongly recruited to CD95 5 min to 15 min after stimulation of Jurkat Ev cells (Fig. 8a). p18 cleavage product of caspase-8 was also detected, reflecting caspases-8 activation (37). By contrast, FADD and caspase-8 were only slightly recruited in Jurkat SHIP-1 cells, and no p18 was detected at these time points (Fig. 8a). Interestingly, CD95 oligomerization and DISC formation were nearly completely restored in Jurkat expressing SHIP-1 R41G (Fig. 8a). Noteworthy, no SHIP-1 was detected at the DISC (Fig. 8a). Corresponding lysates before immunoprecipitation are shown in Fig. 8b. Based on our data, we conclude that hyper-glycosylation of CD95 prevents its oligomerization and subsequent DISC formation upon stimulation.
DISCUSSION
Lymphopenia observed in SHIP-1 deficient mice suggests that SHIP-1 is involved in T lymphocytes survival, which is in contradiction with the well-established inhibitory function of this phosphatase in the myeloid lineage (7). CD95 is the prototypic regulator of T cell homeostasis that induces T cell apoptosis, thereby terminating the immune response (1, 2). We report here for the first time that SHIP-1 protects both primary T lymphocytes and type I and II T leukemic cell lines against CD95-induced apoptosis. Interestingly, we have shown that SHIP-1 anti-apoptotic role is independent of its inositol phosphatase activity, but requires an intact SH2 domain. This implies that, in T cells, despite the fact that SHIP-1 can downregulate P-Akt, its prominent role is not related to its phosphatase activity, but relies on the anti-apoptotic activity of its SH2 domain. In contrast, lymphocytes from PTEN+/- mice are unresponsive to CD95-mediated apoptosis, and these mice develop lymphoproliferative disorders (38). T-cell specific loss of PTEN gives rise to the same phenotype (39). This suggests that PTEN and SHIP-1 inositol phosphatases play non-redundant roles in T lymphocyte apoptosis. The role of SHIP-1 in T lymphocyte biology is still poorly understood and debated. SHIP-1 deficiency in mice leads to myeloproliferation and B cell lymphoma but also to a decrease in the percentage of circulating lymphocytes, particularly in the periphery (9, 10, 18, 19). Peripheral T cells from SHIP-1-/- mice are also constitutively activated and behave like TReg cells (18-20). However, T-cell specific deletion of SHIP-1 does not lead to altered T cell number, activation or emergence of TReg cells, but instead revealed a role for SHIP-1 in the regulation of Th1/Th2 and cytotoxic responses (40). It was proposed that the emergence of TReg cells in mice bearing a germ-line mutation of SHIP-1 might be a consequence of the inflammatory environment in those mice rather than a T-cell defect as a result of SHIP-1 deletion (40). Nevertheless, the influence of SHIP-1 deficiency on CD95-induced apoptosis was never examined upon T cell activation, a condition necessary for their
sensitivity to CD95-mediated cell death, likely to occur in the inflammatory environment present in SHIP-1-/- mice. Our results suggest that, in the course of an immune/inflammatory response, SHIP-1 is a key negative regulator of CD95-induced T cell death. On the other hand, it is noteworthy that freshly isolated TReg are highly sensitive toward CD95-induced apoptosis, similarly to T cells from SHIP-1-/- mice, thereby reinforcing the existence of a potential link between SHIP-1, TReg cells development and apoptosis sensitivity (41).
In this work, we also demonstrated for the first time SHIP-1 association to the ER. Several protein phosphatases, including PTP-1B and SHP-1, were also found to be localized in the ER or be enriched in a perinuclear compartment, where they regulate receptor maturation or trafficking (35). Our observation of SHIP-1 localization to the ER opens new avenues of research about its function in cellular mechanisms probably unrelated to its control of PI 3-kinase signaling. Notably, we found that SHIP-1 promotes CD95 glycosylation through the involvement of its SH2 domain. Remarkably, SHIP-1 Jurkat cells express a similar repertoire of O-glycans to those found in CEM cells, which express SHIP-1 naturally. These glycans are mono-and disialylated core type-1 and core type-2 O-glycans (Supplementary Table 2). In wildtype Jurkat cells O-glycosylation is restricted to the formation of the Tn antigen (GalNAc-Ser/Thr). This is because they lack the active T synthase that is required for the addition of galactose to the Tn antigen to form the core type-1 sequence (Galb1-3GalNAc-Ser/Thr), which is the precursor for all O-glycans in normal leukocytes. T synthase, which resides in the Golgi apparatus, requires a chaperone called Cosmc for correct folding in the ER. Because Jurkat cells lack functional Cosmc, due to a mutation which introduces a stop codon near the middle of its gene, their T synthase is incorrectly folded in the ER and is routed to the proteasome for degradation (31, 42). Our data indicate that SHIP-1 expression rescues Cosmc function via a yet unknown mechanism, perhaps through docking with the cytoplasmic domain of T synthase and/or the truncated Cosmc. Previous cloning of CD95 has
suggested the protein to be glycosylated, but structural information is sparse and the influence of glycosylation on CD95 signaling is poorly understood. We report here that Jurkat SHIP-1 CD95 carrying sialylated complex O-glycans, that are absent in wildtype Jurkat CD95, was routed normally to the cell surface. However, it failed to oligomerize upon stimulation, resulting in an impaired apoptotic signaling cascade.
Several studies suggest that SHIP-1 inactivation can participate in the leukemogenic process in T cells. Indeed, recent findings by Lo et al. report near universal inactivation of SHIP-1 in childhood T-cell Acute Lymphoblastic Leukemia (T-ALL) (27). It has been postulated that this inactivation could play a role in the deregulation of Akt pathway and tumorigenesis in T-ALL, probably in conjunction with PTEN inactivation. Accordingly, restoration of SHIP-1 expression in Jurkat T leukemic cells downregulates constitutively activated PI 3-K and AKT signaling pathway and leads to an increased transit time through the G1 phase of the cell cycle, thereby down-regulating the proliferation of these cells (43, 44). We now propose that SHIP-1, via its SH2 domain, also acts as a potent regulator of T cells homeostasis by inhibiting CD95-mediated cell death. Taken together, these results may provide a new basis for therapeutic interventions using compounds engaging the CD95 pathway on SHIP-1 negative leukemic T cells. Our data also raise serious concerns about possible complications associated with SHIP-1 gene transfer for the treatment of T-ALL.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).
ACKNOWLEDGEMENTS
This work was supported by grants from the Belgian National Fund for Scientific Research (FNRS, Brussels, Belgium), the Interuniversity Attraction Pole (IAP6/18, Brussels, Belgium), the concerted research action program (ARC04/09-323) and the “fond anticancéreux près l’Université de Liège”. CE was supported by grants from the IAP6/28 and the FNRS. AD and SMH are grateful for financial support from the Biotechnology and Biological Sciences Research Council (BBF0083091). We thank Dr. S.Ormenese from the imaging and flow cytometry GIGA-Research technological platform for FACS analysis, Dr. S. Ward for providing Tet-regulated Jurkat CD2:SHIP-1 cells, Dr. E. Ravet for providing TRIPΔU3 lentiviral vector and for technical advices and Drs. I. Lavrik and P. Krammer for technical advices and helpful comments. EC was supported by the Télévie (FNRS, Brussels). GG, SR, AC and JP are Postdoctoral Researcher, Research Associate, Senior Research Associate and Research Director from the FNRS, respectively.
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TITLES AND LEGENDS TO FIGURES
Figure 1. SHIP-1 protects primary T lymphocytes and Jurkat leukemic cells from CD95-mediated apoptosis. (a) SHIP-1 expression deficiency in thymus extracts from SHIP-1 WT and KO
mice. (b) Primary murine T lymphocytes from WT and SHIP-1-/- spleen and lymph nodes were activated for 18h with CD3/CD28-coated beads prior to be stimulated by CD95-L for 16h. Cell death was assessed by staining using AnnexinV-FITC/PI (mean ± SD, (n=3); *p< 0.01). (c) SHIP-1 expression and P-Akt (T308) in Jurkat transduced with an empty lentiviral vector (Ev) or a vector expressing SHIP-1. Jurkat JR expresses SHIP-1 naturally. (d) Jurkat expressing SHIP-1 are more resistant to CD95 Abs (7C11 and APO-1; 50 ng/mL), CD95-L (25 ng/mL), camptothecin (campto; 1µM) and TRAIL-L (100ng/mL) -induced apoptosis (mean ± SD, (n=4); *p< 0.003). (e) 7C11-induced pro-caspases-8, -3 and PARP cleavage is delayed upon SHIP-1 expression in Jurkat cells.
Figure 2. SHIP-1 knock-down sensitizes type I and type II T leukemic cells to CD95 Ab-induced apoptosis. (a) Jurkat JR cells were electroporated without siRNA or with siRNA control (LUC) and
anti-SHIP-1. 24h later, cells were stimulated with 7C11 Ab (50 ng/mL) for the indicated times. Cell lysates were analyzed by western-blotting using anti-SHIP-1, caspase-3 and PARP antibodies. (b) Knock-down of SHIP-1 in Hut78 (type I) and (c) CEM (type II) sensitizes them to APO-1 and 7C11-induced pro-caspase-3 and PARP cleavages, respectively.
Figure 3. The anti-apoptotic function of SHIP-1 is independent of its phosphatase activity but requires an intact SH2 domain. (a) Expression of SHIP-1 and level of P-Akt (T308) in Jurkat
transduced with vectors expressing SHIP-1 and SHIP-1 D672A. (b) Expression of SHIP-1 D672A does not restore apoptosis sensitivity in Jurkat cells. (mean ± SD, (n=2); *p≤ 0.03). The difference between Jurkat SHIP-1 and Jurkat SHIP-1 D672A is not statistically significant (p= 0.1577). (c) 7C11 (50 ng/mL)-induced-pro-caspases-8, -3 and PARP cleavage is similar whether cells express SHIP-1 wt or D672A. (d) Expression and catalytic activity of tetracycline-regulated CD2:SHIP-1 chimera in Jurkat cells. (e) Sensitivity of Jurkat expressing CD2:SHIP-1 or not to 7C11-induced apoptosis. p= 0.5632, not significant. (f) Expression of SHIP-1 and level of P-Akt (T308) in Jurkat transduced with
vector expressing SHIP-1 and SHIP-1 R41G. (g) Expression of SHIP-1 R41G restores 7C11-induced apoptosis sensitivity in Jurkat cells. (mean ± SD, (n=4); *p≤ 0.003) (h) 7C11 (50 ng/mL)-induced-pro-caspases-8, -3 and PARP cleavage is restored upon SHIP-1 R41G expression in Jurkat cells.
Figure 4. SHIP-1 does not affect CD95 membrane expression but increases the presence of high molecular weight forms of the receptor. (a) Jurkat Ev, SHIP-1 and SHIP-1 R41G cells were
analyzed by flow cytometry to monitor their respective cell surface content of CD95. (b) SDS extracts from Jurkat EV, SHIP-1 and SHIP-1 R41G and from various SHIP-1 positive T leukemic cells (c) were analyzed for the presence of CD95 by Western blotting analysis. *n.s: non-specific bands.
Figure 5. SHIP-1 promotes CD95 glycosylation. (a) O- and N-linked glycans were enzymatically
removed from cell lysates of Jurkat Ev and Jurkat SHIP-1 cells prior Western blotting analysis. (b) Glycosylated proteins were affinity purified from lysates of Jurkat Ev and Jurkat SHIP-1 cells with the indicated biotinylated lectins and agarose-linked streptavidin. The lectins from the following species were used: Dolichos biflorus (DBA), Ricinus communis (RCA120), Glycine max (SBA), Sambucus
nigra (SNA), Ulex europaeus (UEA), Wheat Germ Agglutinin (WGA) and Peanut Agglutinin (PNA).
The presence of CD95 in the precipitates was detected by immunoblotting with anti-CD95 antibody.
(c) Recapitulative panel of lectin selectivity and reactivity. Reactivity is given in a semiquantitative
scale ranging from – (no reactivity) to ++++ (very strong reactivity).
Figure 6. Tunicamycin restores CD95-sensitivity in Jurkat SHIP-1. (a) Tunicamycin (TN), but not
b-GalNAc, restores 7C11-induced apoptosis sensitivity in Jurkat SHIP-1 cells. 7C11 was added for the last 16h of TN or b-GalNAc treatment. (mean ± SD, (n=4); *p< 0.001) (b) 7C11 (50 ng/mL)-induced cleavage of PARP in Jurkat Ev and SHIP-1 upon tunicamycin pre-treatment.
Figure 7. SHIP-1 is localized to the ER. (a) Cytosolic (cyto) and membrane/organelles (MO)
compartments were isolated from Jurkat SHIP-1 cells prior detection of SHIP-1 and CD95 by Western blotting analysis. Anti-IκBα and anti-Grp78 antibodies served as cytosolic and ER markers, respectively. SHIP-1 corresponding bands intensities were quantified using Quantity One 4.6.5 software (BioRad Laboratories Inc.). (b) Nicodenz gradient centrifugation was performed on the MO
fraction. The individual fractions from the top to the bottom were subjected to Western blotting analysis, using specific antibodies directed against SHIP-1, Grp78 and CD95.
Figure 8. Glycosylated CD95 fails to oligomerize and to recruit DISC upon stimulation. (a) DISC
formation after APO-1 (1µg/mL) stimulation of Jurkat Ev, Jurkat SHIP-1 and Jurkat SHIP-1 R41G was visualized after CD95 immunoprecipitations by Western blotting using anti-caspase-8, -FADD and -CD95 antibodies. To test the presence of SHIP-1 at the DISC, anti-SHIP-1 antibody was also used. (b) Caspase-8, FADD, CD95 and SHIP-1 were detected using specific antibodies in the lysates prior to immunoprecipitations.
