Antibody Cross-linking of the Glycosylphosphatidylinositol-linked Protein CD59 on Hematopoietic Cells Induces Signaling Pathways Resembling Activation by Complement*

CD59 is a glycosylphosphatidylinositol-anchored cell surface glycoprotein involved in protecting cells from host-mediated complement attack. Studies have shown that antibody cross-linking of CD59 induces a series of intracellular signaling events including the activation of protein-tyrosine kinases (PTK). To further characterize these events, antibodies and complement 8, one of the natural ligands of CD59, were used to activate CD59. Antibody-induced cross-linking of CD59 on the surface of THP-1 and U937 hematopoietic cell lines as well as exposure to complement 8 induces a rapid increase in the tyrosine phosphorylation of several proteins within the cell. Consistent with an early role for the Src family PTKs in these signaling events, we found that transient activation of Hck- and CD59-mediated signaling was abrogated in the presence of the Src family PTK-selective inhibitor PP1. Although the molecular mechanism by which CD59 communicates to Hck is unknown, cellular fractionation studies indicated that both CD59 and Hck are compartmentalized in plasma membrane microdomains. We also detected tyrosine phosphorylation of the adaptor proteins p120 and Shc, and the cytoplasmic nonreceptor tyrosine kinase Syk. The identification of CD59-mediated signaling events may help explain why paroxysmal nocturnal hemoglobinuria patients, who are deficient in glycosylphosphatidylinositol-linked proteins including CD59, are susceptible to proliferative disorders.

CD59 is one of a large family of proteins that are attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) 1 moiety attached to their C-terminal ends (1). It is abundantly expressed on a wide variety of cells, including many cells of hematopoietic lineage (2)(3)(4)(5), and functions to inhibit complement-mediated lysis by interfering with the assembly of the membrane attack complex and the insertion of complement 9 into the cell membrane (reviewed in Ref. 6). The natural ligands for CD59 include the ␣-chain of complement 8 (C8) and the "b" domain of complement 9 (7). Antibody-mediated cross-linking of CD59 on various hematopoietic cells induces a cascade of intracellular signaling activities including events such as calcium influx and release of calcium from intracellular stores, generation of reactive oxygen intermediates (8 -12), and induction of tyrosine phosphorylation (13,14). These events are not unique to CD59, since many other GPI-anchored proteins induce a series of similar intracellular signaling events when cross-linked or upon binding of appropriate ligand. Other GPI-linked molecules expressed on hematopoietic cells include the lipopolysaccharide receptor (CD14) (15,16) and THY-1, a regulatory molecule in T-cell activation (17,18).
Since GPI-anchored proteins adhere to cells through their acyl chains and do not span the membrane, the mechanism by which this family of proteins transduce their signal into the cell remains unknown. However, GPI-linked proteins have been shown to cluster in detergent-insoluble glycolipid-enriched domains (DIGs (19,20)), which may be analogous to caveolae in some cell types. In addition to the enrichment of GPI-linked proteins, DIGs are enriched in other molecules including glycosphingolipids, cholesterol, and the resident structural protein caveolin (20). In hematopoietic cells, DIGs have been isolated, but the absence of both caveolin and morphologically defined caveolae leaves their designation equivocal in these cells. DIGs contain other molecules involved in signal transduction, including the Src family protein-tyrosine kinases (PTKs) (21)(22)(23)(24)(25). The Src family PTKs may be involved in transmission of the GPI-linked signals, since they have been shown to co-immunoprecipitate with various GPI-anchored molecules (23,(25)(26)(27)(28). The ability of the Src family PTKs to co-precipitate with GPI-anchored proteins such as CD55 requires them to be palmitoylated (26), a fatty acid modification also required for the Src family PTKs to localize to caveolae or DIGs (21,24). However, since neither molecule spans the plasma membrane, their exact connection remains obscure.
In this report, we show that antibody-mediated cross-linking of CD59 on the surface of the human myelomonocytic cell lines U937 and THP-1 induced a rapid and robust increase in the total tyrosine-phosphorylated proteins within the cell. We have demonstrated for the first time that one of CD59's natural ligands, C8, induces the accumulation of phosphotyrosine-containing proteins virtually indistinguishable from that obtained with antibody cross-linking. Concomitant with the activation of the Src family PTK Hck, there is the tyrosine phosphorylation of several proteins including the signaling adaptor proteins, Cbl and Shc, and the cytoplasmic tyrosine kinase Syk. Consistent with Hck playing an early proximal role in CD59 signal transduction, the Src family PTK-selective inhibitor PP1 abrogated the downstream signaling events observed. The activa-tion of diverse signaling molecules may explain the phenotypic consequences of CD59 activation.

MATERIALS AND METHODS
Cell Lines-U937, K562, and THP-1 cells were obtained from the American Type Culture Collection. IVEE cells are derived from K562 cells and are deficient in GPI anchors, therefore lacking CD59 (29). All cells were maintained in RPMI 1640 supplemented with 25 mM HEPES, 2 mM glutamine, 10% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, and 5 ϫ 10 Ϫ5 M ␤-mercaptoethanol. To induce differentiation of the U937 cells to the more mature macrophage phenotype, cells were washed, suspended in fresh medium containing 12-O-tetradecanoylphorbol-13-acetate at a concentration of 20 ng/ml, and incubated for 48 h.
Cell Stimulations-Antibody cross-linking of CD59 receptors was performed by incubation of 2 ϫ 10 7 cells in 1 ml of PBS containing 10 g/ml of anti-CD59 antibody (mouse anti-human CD59 MEM43 or rat anti-human CD59 YTH53.1; Serotec) for 15 min on ice. Following low speed centrifugation, cells were resuspended in 1 ml of PBS containing 20 g/ml secondary antibody (goat anti-mouse IgG F(abЈ) 2 or mouse anti-rat Ig whole antibody, respectively (Jackson Laboratories, Mississauga, Canada), and incubated at 37°C from 1 to 60 min. Cells were centrifuged, washed, and lysed. C8 (Sigma-Aldrich Canada) stimulation of the cells was performed after cells were washed once in PBS. C8 was diluted in PBS, and 100 units was added to 1 ϫ 10 7 cells for varying times at 37°C in 1.0 ml of PBS. For experiments using the tyrosine kinase inhibitor PP1 (Calbiochem), U937 and THP-1 cells were preincubated with 10 M PP1 for 20 min at 37°C and then stimulated with anti-CD59 antibodies or complement 8 as described above.
Immunoprecipitations and Analysis of Cell Lysates-Following Nonidet P-40 lysis of cells, whole cell lysates were used for immunoprecipitations with polyclonal antibodies to Syk (31), Hck (24), and Shc (32) or a monoclonal antibody to Cbl. 2 Whole cell lysates prepared from 0.4 ϫ 10 7 cells in 0.2 ml of lysis buffer were immunoprecipitated using 5 g of anti-Shc or anti-Syk, 2 g of anti-Hck, or 3 g of Cbl monoclonal antibody for 1 h at 4°C. Protein A-Sepharose (50 l of a 50% slurry in PBS) was added, and the samples were rocked at 4°C for 30 min. The Protein A-Sepharose beads were washed three times in Nonidet P-40 lysis buffer and were boiled for 5 min in reducing buffer before electrophoresis.
All samples were analyzed by Western blotting after electrophoresis and transfer onto Immobilon-P polyvinylidene fluoride membranes (Millipore Corp., Mississauga, Canada). For CD59 Western blots, membranes were blocked for 1 h in 5% bovine serum albumin in PBS with 0.05% Tween 20 (PBSTw) and probed with mouse anti-human CD59 MEM43/5 (MONOSAN Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) diluted 1:100 in blocking solution. Following several washes in PBSTw, blots were incubated in horseradish peroxidase-conjugated sheep anti-mouse IgG secondary antibody (Amersham Pharmacia Biotech, Oakville, Canada) diluted in blocking buffer for 30 min and were washed extensively in PBSTw before incubation in enhanced chemiluminescence detection reagents (ECL; Amersham Pharmacia Biotech) and exposure to x-ray film. Anti-phosphotyrosine blots were blocked in 5% bovine serum albumin in TBS-Tw (10 mM Tris, pH 7.5-8.0, 135 mM NaCl, 5 mM KCl, 0.5% Nonidet P-40, 0.1% Tween 20) and washed in TBS-Tw before probing with anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) followed by horseradish peroxidase-conjugated sheep anti-mouse secondary antibody and washing prior to visualization as described above. All other Western blots were blocked in 5% skim milk powder in TBS-Tw before probing with the appropriate antibodies and visualization.

CD59 Is Expressed and Compartmentalized on the Cell Surface of Hematopoietic Cell
Lines-To examine cell surface expression of CD59, we used indirect immunofluorescence with CD59-specific antibodies on several human hematopoietic cell lines. Representative results with antibody YTH53.1 are shown in Fig. 1A. THP-1 (a human myelomonocytic cell line) and K562 (a human erythroleukemia cell line) cells stained positively for CD59 in contrast to IVEE cells (a GPI-defective cell line derived from K562 cells (29)), which were negative for CD59. We used two different anti-CD59 antibodies, YTH53.1 and MEM43, and found no substantial difference in the detection of CD59 in THP-1 cells. In contrast to many reports that have described U937 cells (a human myelomonocytic cell line) as being defective in (33) or absent of CD59 (11, 33, 34), we found by indirect immunofluorescence that they stained positively for both CD59-specific antibodies used ( Fig. 1A and data not shown). In addition, there is an equivalent level of fluorescence regardless of whether the cells were differentiated toward a macrophage-like lineage or not (Fig. 1A).
GPI-linked proteins have been shown to localize to DIGs of the plasma membrane, which may represent caveolae in some cell types. To examine whether CD59 was compartmentalized in various hematopoietic cells, we used linear sucrose gradients to isolate caveolar-like Triton X-100-insoluble microdomains and looked for the presence of CD59 by Western blotting using anti-CD59 antibody MEM43/5 (Fig. 1B). We found that several glycosylated isoforms of CD59 were present in small amounts in Triton X-100-soluble fractions of the cell lysates but were highly enriched in the caveolar-like fractions in K562 and THP-1 cells without prior cross-linking of the receptor. The GPI-defective cells (IVEE) were negative for CD59. We were unable to detect CD59 by Western blotting in differentiated or undifferentiated U937 cells although expression could be detected by indirect immunofluorescence (Fig. 1A). This probably reflects a difference in the expression levels of CD59, since K562 and THP-1 cells were the most positive for CD59 by indirect immunofluorescence (Fig. 1A).
Cross-linking of Cell Surface CD59 Induces Intracellular Tyrosine Phosphorylation-The activation of many GPI-linked proteins has successfully been achieved through binding of specific monoclonal antibodies followed by dimerization of the GPI-linked proteins using anti-Ig secondary antibodies (8 -12). In order to assess receptor activation, we used monoclonal antibodies to cross-link CD59. The human myelomonocytic cells THP-1 show a robust increase in the tyrosine phosphorylation state of several cellular proteins upon cross-linking of CD59 ( Fig. 2A), including, most noticeably, protein bands of approximately 160, 120, 70, and 50 kDa in size. Phosphorylation changes also occurred in U937 cells but only after they were differentiated to the macrophage-like phenotype ( Fig. 2A). This difference in signaling competence of U937 cells did not appear to be at the receptor level, since flow cytometry analysis suggested that there was no change in the level of receptor expression during the differentiation process (Fig. 1A). We observed the same patterns in tyrosine phosphorylation regardless of primary antibody used (MEM43 versus YTH53.1, data not shown). To eliminate the possibility of activation of the Fc␥R due to the presence of the cross-linking antibodies, we repeated the experiments with the F(abЈ) 2 fragment of the secondary antibody and confirmed that all phosphorylation events were due to cross-linking of CD59 (data not shown).
To test whether Src family PTKs were involved in the phosphorylation events observed after CD59 cross-linking, we preincubated the cells for 20 min with the Src family PTK-selective inhibitor PP1 (35) and then repeated the antibody crosslinking procedure ( Fig. 2A). PP1 inhibited the tyrosine phosphorylation of all proteins activated upon CD59 stimulation, suggesting that Src family PTKs play an early and essential role in the signaling cascades activated by CD59.
A time course of cross-linking with CD59 antibody YTH53.1 was performed to evaluate the temporal phosphorylation patterns in THP-1 cells (Fig. 2B). THP-1 cells showed a rapid increase in phosphorylation with the peak occurring around 3 min followed by a decrease in phosphorylation to below initial background levels, consistent with the activation of a phosphatase.
Tyrosine Phosphorylation of Substrate Proteins after C8 Stimulation of THP-1 and U937 Cells Resembles the Phosphorylation Pattern Obtained by Cross-linking with Anti-CD59 Antibodies-To evaluate whether antibody cross-linking experiments reflected the cellular responses to the native ligand of CD59, we stimulated THP-1 cells with purified C8 and evaluated the tyrosine phosphorylation patterns at various time points (Fig. 3A). The pattern of phosphorylation upon activation of CD59 by C8 and antibody cross-linking were similar (Fig. 3A), suggesting that antibody cross-linking resembles at least some portion of the signaling cascade involved in the inhibition of complement attack by hematopoietic cells and is a relevant readout for CD59 function. However, we found that the peak of phosphorylation resulting from C8 stimulation was sustained for more than 15 min, unlike the cross-linking antibodies, which gave a peak of phosphorylation at 3 min (data not shown and Fig. 2B). Phosphorylation resulting from stimulation by either C8 or cross-linking antibodies returned to background levels by 60 min (Fig. 2B and data not shown). In addition, phosphorylation resulting from stimulation with either reagent was inhibited by the Src family PTK-selective inhibitor PP1 (Fig. 3B).
The Src Family PTK Hck Is Phosphorylated upon CD59 Cross-linking-Our previous experiments showed that the Src family PTK-selective inhibitor PP1 inhibited the accumulation of the phosphotyrosine-containing proteins upon CD59 crosslinking ( Fig. 2A). Since the Src family PTK Hck is abundantly expressed in myelomonocytic cells, we examined whether Hck was phosphorylated upon CD59 cross-linking (Fig. 4) in THP-1 cells. Both Hck isoforms (p59 hck and p61 hck ) were phosphorylated after stimulation with either CD59 antibody (YTH53.1 or MEM43) with kinetic profiles similar to that seen for total tyrosine phosphorylation (Figs. 2B).
The Adaptor Proteins p120 cbl and Shc and the Nonreceptor Tyrosine Kinase Syk Become Tyrosine-phosphorylated upon CD59 Cross-linking-To further identify the substrates of CD59 activation, we performed immunoprecipitations of various signaling proteins and evaluated their phosphorylation status upon CD59 activation (Fig. 5). Cbl has been shown to be phosphorylated in response to a wide array of extracellular signals in hematopoietic cells (36 -41). We also found that Cbl is phosphorylated in response to CD59 activation (Fig. 5) and that this activation could be blocked by the inhibitor PP1 ( Fig.  2A and data not shown).
The nonreceptor tyrosine kinase Syk has been shown to play a role in a number of signaling events in hematopoietic cells (32,40,(42)(43)(44). We evaluated the phosphorylation status of Syk following CD59 cross-linking (Fig. 5) and found that Syk was phosphorylated in a manner that appears to be dependent on Src family PTKs, since its phosphorylation status could be inhibited by PP1 ( Fig. 2A and data not shown).
Next we evaluated the phosphorylation of Shc, an adaptor protein that plays a role in the activation of the Ras/Raf signaling pathway resulting in the downstream activation of the MAP kinases. In v-Src-transformed cells, Shc was found to be phosphorylated, suggesting that Src family PTKs can phosphorylate Shc (45). We found that Shc is also phosphorylated in a Src family PTK-dependent manner in response to CD59 crosslinking (Fig. 5). In addition, the anti-Shc antibody immunoprecipitated a protein of about 145 kDa that is also phosphorylated upon CD59 activation. DISCUSSION In order to define the mechanism by which GPI-anchored proteins mediate signal transduction in hematopoietic cells, we used the complement receptor CD59 as a model. CD59 activation was achieved by cross-linking antibodies or by stimulation with one of CD59's natural ligands, C8. CD59 is an 18 -20-kDa GPI-anchored glycoprotein, which, because of its method of cell surface anchoring, does not span the plasma membrane (1). Despite this, we have shown that antibody-mediated crosslinking of CD59 on the surface of human hematopoietic cells resulted in a rapid and robust increase in the level of tyrosine phosphorylation of several intracellular proteins. In addition, we showed for the first time that the binding of one of the natural ligands, C8, to CD59 mimics the tyrosine phosphorylation pattern obtained with antibody-mediated cross-linking, suggesting that this technique may be a relevant indicator of  anti-CD59 antibody for 15 min followed by secondary antibody for the times indicated. Cells were lysed in Nonidet P-40 lysis buffer, immunoprecipitated with anti-Hck-specific antibody, electrophoresed, and then Western blotted followed by probing with anti-phosphotyrosine antibody 4G10. 2°, incubation with the secondary antibody alone. 0Ј, incubation with the primary antibody alone. CD59 function. As well, we have identified several of the major proteins that become tyrosine-phosphorylated upon CD59 activation.
Many groups have reported an association of GPI-linked proteins with Src family PTKs (25,26,46,47), so we initially examined the activation of one of the Src family PTKs that is abundantly expressed in hematopoietic cells, Hck (48). The levels of tyrosine phosphorylation of both isoforms of Hck (p59 hck and p61 hck ) transiently increased upon CD59 crosslinking, indicating activation of Hck and establishing its role in CD59-mediated signal transduction.
In addition to the activation of Src family PTKs, CD59 activation also initiates a cascade of tyrosine kinase activation including the phosphorylation of the cytoplasmic tyrosine kinase p72 syk . Syk and its close relative Zap-70 have both been shown to be downstream of the activation of the Src family PTKs (43,49).
We have shown previously that p120 cbl is a downstream target of Src family PTKs including Hck, 2 and we find that c-Cbl is tyrosine-phosphorylated in conjunction with the activation of Hck upon CD59 cross-linking. Although Cbl is phosphorylated in response to a number of extracellular ligands (36 -41, 50), its function in signal transduction remains unknown, but it probably functions as an adaptor protein coupling to a number of signaling molecules. Shc also functions as an adaptor protein and is also phosphorylated in response to diverse extracellular signals. By virtue of its phosphorylation-dependent interaction with Grb2, Shc is one means to activating the MAP kinase pathway (reviewed in Ref. 51). We find that two isoforms of Shc, p46 and p52, are phosphorylated upon CD59 activation, and we have evidence that the MAP kinase (ERK1/ERK2) pathway is activated. We also detected phosphorylation of a 145-kDa protein, consistent with the inositol-5phosphatase SHIP (52), in immunoprecipitations of Shc upon CD59 activation. SHIP is phosphorylated in response to various growth factors and cytokines (52) and may also play a role in apoptosis and growth regulation (53).
The cellular consequences of the signaling events we observe are unknown except that CD59 activation results in the stimulation of a cascade of tyrosine kinases that serves, in part, to recruit a variety of adaptor molecules and potentially may activate at least one negative regulator. These signaling events were similar for both THP-1 cells and U937 cells. We have also observed a similar increase of cellular phosphotyrosine levels upon CD59 cross-linking in freshly isolated human neutrophils, indicating that this effect is not an artifact of the hematopoietic cell lines (data not shown).
The mechanism by which GPI-linked CD59 sends messages is unknown. It is possible that CD59 needs a transmembranespanning co-receptor to send signals. Two reports have described the presence of an 80-kDa glycoprotein present in immunoprecipitations of the activated complement-binding proteins CD55 and CD59, a possible transmembrane link of the intracellular kinases to the extracellular receptor (13,28). The association of a transmembrane component with a GPI-linked receptor is not uncommon. Both ciliary neurotrophic factor and glial-cell-line-derived neurotrophic factor are GPI-linked receptors complexed with transmembrane receptors to transmit signals upon binding of their specific ligands (54 -58).
We found that U937 cells express CD59 on their cell surfaces regardless of their differentiation status; however, only those cells differentiated to the more mature macrophage phenotype were capable of transmitting a signal upon CD59 activation. The up-regulation of Hck in differentiated U937 cells (48) may be critical to the acquisition of CD59 signal responsiveness in these cells. Alternatively, differentiating U937 cells may upregulate an accessory receptor subunit that couples CD59 with nonreceptor tyrosine kinases.
Controversy exists regarding the localization of GPI-linked proteins into microdomains such as caveolae and DIGs (59). We did not cross-link CD59 to induce clustering on these cells prior to microdomain isolation; thus, our results would contradict those who suggest that GPI-linked proteins translocate to these microdomains only after antibody cross-linking. However, the use of nonionic detergents to isolate these membrane microdomains may impart some effects on localization. It is unclear what role clustering of GPI-linked proteins and their co-localization with cytoplasmic tyrosine kinases may play in GPI-linked protein signaling, but, regardless of the mechanism used by GPI-linked proteins to send signals across a membrane, the signals they send appear to be unique for the different GPI-linked proteins. For example, we see phosphorylation of Cbl upon CD59 activation, yet activation of CD14 (the GPIlinked lipopolysaccharide receptor) by bacterial lipopolysaccharide does not result in Cbl phosphorylation. 3 In addition, PP1 inhibits phosphorylation from CD59 activation but has no effect on CD14 signaling, despite their colocalization in detergent-insoluble microdomains.
Paroxysmal nocturnal hemoglobinuria is an acquired somatic defect of the PIG-A gene in hematopoietic stem cells that results in a defect in the expression of GPI-linked proteins (including CD59) on the cell surface, giving rise to blood cells that are more susceptible to host complement-mediated lysis (60,61). Despite this, these patients are more susceptible to leukemia (62), and it is unclear why the loss of GPI-linked proteins can lead to proliferative disorders. Recently, it was shown that the mutations in paroxysmal nocturnal hemoglobinuria cells confer resistance to apoptosis, thereby allowing clonal expansion of the GPI-defective cells (63). Consistent with that, upon activation of CD59 we see phosphorylation of a 145-kDa protein presumed to be the inositol phosphatase SHIP, a negative regulator of cell signaling that is involved in the induction of apoptosis (53). It will be important to investigate the role CD59 and other GPI-linked proteins have in regulating hematopoietic cell growth, proliferation, and apoptosis and how this may be altered in various malignant diseases.  5. CD59 cross-linking induces the tyrosine phosphorylation of p120 cbl , p72 syk , and p46/52 shc . THP-1 cells were lysed in Nonidet P-40 lysis buffer without stimulation (Ϫ) or following stimulation with secondary antibody alone for 3 min (2°) or primary (15 min) plus secondary (3 min) (1°ϩ 2°) antibody. Lysates were electrophoresed without immunoprecipitation (Ϫ) or were immunoprecipitated with specific antibodies to Cbl, Syk, or Shc followed by electrophoresis Western blotting and probing with the anti-phosphotyrosine antibody 4G10. The migration of the immunoprecipitated proteins is indicated in kDa on the right.