The α-chain of the IL-15R (IL-15Rα) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Rα-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Rα and IL-2Rγ chains, but lacks the IL-2Rβ chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Rα chain. Upon association, the activated Syk kinase phosphorylates the IL-15Rα chain as well as phospholipase Cγ, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Rα and phospholipase Cγ phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Rα (Tyr227) abrogates both the IL-15Rα/Syk association and IL-15Rα phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Rα chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

Interleukin-15 is a potent growth factor for T and B lymphocytes and NK cells (1, 2, 3, 4, 5, 6, 7, 8), a chemoattractant for T cells (9), and an activator of the cytolytic program in T and NK cells (10, 11). Although IL-15 shares many biological activities with IL-2, there is increasing appreciation of a large spectrum of activities in which IL-2 and IL-15 differ (8, 12, 13, 14, 15). Also, despite their partially redundant functional properties, IL-2 and IL-15 differ substantially in their patterns of expression and secretion (16, 17, 18). Furthermore, in contrast to IL-2, IL-15 is transcribed by a broad variety of different tissues and cells (e.g., activated macrophages, keratinocytes, muscle cells, endothelial cells, and neural cells) (13, 16, 19, 20, 21, 22) and is expressed in a functionally active, membrane-bound form on monocytes (14).

The existing similarities in the action between IL-2 and IL-15 on the same cell type (12, 19) can be explained in part by the sharing of receptor subunits. Both IL-2 and IL-15 bind to a heterodimeric receptor complex, which shares the IL-2Rβ and IL-2Rγ chains (23, 24, 25), and which is thought to be responsible for intracellular signal transduction (25, 26). IL-2 and IL-15 signaling pathways in lymphocytes involve Janus kinases (Jaks)3 and the STATs (27). In fact, activation of the γ-chain leads to coprecipitation of Jak1 and Jak3 (27).

Each α-chain of the IL-2R and IL-15R recognizes only its cognate cytokine. IL-15Rα alone binds IL-15 with high affinity (Kd ∼ 10−11 M) (7) on human activated B cells and several B lymphoma cell lines such as Raji and SKW 6.4 cells (4). Therefore, it is reasonable to assume that the IL-15Rα subunit is responsible for the differential effects of IL-15 and IL-2 on cells of the same type.

It has been claimed that IL-15Rα, like IL-2Rα, is incapable of signaling when it is expressed in the absence of IL-2Rβ or IL-2Rγ (4, 20, 23, 28). Instead, Jurkat cells which lack the IL-15Rα chain can signal via the IL-2Rβ and IL-2Rγ chains upon IL-15 stimulation (4). However, a colon epithelial cell line reportedly signals upon IL-15 stimulation even though it expresses only the IL-15Rα, but no IL-2Rβ chain (29). Most recently, we also noted that IL-15 signals through the IL-15Rα chain in the murine fibrosarcoma cell line L929, which expresses the β-chain only at marginal levels and lacks the γ-chain component of the IL-2R complex (30). This strongly suggests that, contrary to conventional wisdom (4, 26, 28), the IL-15Rα chain can transduce a signal even in the absence of the β- and/or γ-chains.

Therefore, it is important to understand how IL-15Rα may transduce an intracellular signal in cells which lack expression of the IL-2Rβ and/or IL-2Rγ chains. To investigate the role of IL-15Rα in intracellular signaling we selected the B lymphoblastoid cell line Raji as a model, because it expresses the IL-15Rα and IL-2Rγ, but lacks the IL-2Rβ chain (4). To show the association of intracellular proteins with the IL-15Rα chain, immunoprecipitation and Western blotting techniques were used. Mutational analysis was performed to study the importance of a selected tyrosine residue in the intracellular part of IL-15Rα. Collectively, the data presented in this work suggest that IL-15Rα signals via recruiting Syk kinase which then phosphorylates IL-15Rα and phospholipase Cγ (PLCγ).

Human rIL-2 was purchased from PeproTech (London, U.K.) and IL-15 was purchased from Genzyme (Cambridge, MA). The mouse anti-human IL-15Rα Ab (IgG1, clone M161) was generously provided by Immunex (Seattle, WA). Rabbit anti-human Syk (N-19), anti-IL-2Rγ (N-20), anti-IL-2Rβ (S-20), anti-Jak3 (C-21), anti-PLCγ1 (1249), anti-β-actin (H-196), and goat anti-mouse IL-15Rα (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the mouse anti-phosphotyrosine (anti-p-Tyr) Ab (RC20) was purchased from Transduction Laboratories (Lexington, KY). Goat anti-mouse, goat anti-rabbit, and rabbit anti-goat HRP conjugates (Amersham Life Science, Little Chalfont, U.K.) were used as secondary Abs.

Raji, HUT 102, Akata, K562, Jurkat, and J558 cells were maintained in RPMI 1640, and COS-7 cells were maintained in DMEM. Culture medium was supplemented with 10% FCS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Before treatment cells were washed twice with Dulbecco’s PBS and incubated in RPMI 1640 without FCS at 37°C for 2 h. For each assay 5 × 106 cells/ml were stimulated with IL-15 or IL-2 (final concentration of 100 ng/ml) for 15 or 30 min at 37°C. Activation was interrupted by adding 8–10 volumes of ice-cold PBS with 10 mM EDTA and 100 mM sodium vanadate. Piceatannol (Calbiochem, London, U.K.), an inhibitor of Syk (31) was used to block Syk activities. Cells were treated with 50 μM piceatannol for 10 min before activation and then were stimulated as above. Anti-IL-15Rα Abs were used at a concentration of 1 μg/ml. For apoptosis induction, cells were treated for 48 h with active C2-ceramide or an inactive analog, C2-dihydroceramide (Calbiochem), at a final concentration of 20 μM (32). Human B and T lymphoblasts were obtained as previously described (3), stimulated with 10 μg/ml of LPS (for B lymphoblasts) or 10 μg/ml of Con A (for T lymphoblasts) for 48 h, and serum-starved for 2 h before cytokine treatment.

Tyrosine at position Y227 of murine IL-15Rα was mutated to phenylalanine (Y227F) using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The identity of mutations was verified by standard DNA sequencing. Mutant IL-15Rα was cloned into the pcDNA 3.1 expression vector (Invitrogen, Carlsbad, CA). Rat Syk cDNA in pSVL vector was generously provided by Dr. R. Siraganian (National Institutes of Health, Bethesda, MD) (33).

COS cells were transfected using the DEAE-dextran method (34), harvested after 48 h, and analyzed by immunoprecipitation and Western blotting. J558 cells were transfected by electroporation (960 μF, 300 V), using a Gene-Pulser (Bio-Rad, Munich, Germany). After 48 h they were serum-starved for 3 h and treated for 15 min with IL-15, lysed, and analyzed by Western blotting.

Stem-loop Syk oligonucleotides (ODNs) were used to block the transcription of Syk kinase (35). The sequence of ODNs is 5′-GGGGGGGCTGTCGTCAGCCATGCCGTGTCTTGTCTTTGTCGCTTCTTGAGGAGCCCCCCC-3′, and (for the scrambled-control) 5′-GGGGGGGATGGAATCATCTTGGGCATTCATTCGTTCCTCAAAGAAGAATATGCCCCCCC-3′. Both sequences were modified by phosphothioates at 5′ and 3′ termini. A total of 100 μl of ODN-liposome complexes (2 μg of lipofectAMINE (Life Technologies, Eggenstein, Germany) and 1 μg of ODNs) were added to 200 μl of Raji cells (1 × 106) in 24-well plates in RPMI 1640 without FCS and incubated for 4 h at 37°C. After transfection, the culture medium was adjusted to 10% FCS in a final volume of 1 ml. An additional 100 μl of ODN-liposome complexes were added, and cells were incubated for 24 h at 37°C before assays.

RNA was extracted using the RNA Clean reagent (AGS, Heidelberg, Germany) according to the manufacturer’s instructions. cDNA was synthesized from 5 μg of total RNA using random hexanucleotide primers and the Superscript II preamplification kit (Life Technologies, Paisley, U.K.). The PCR mixture (20 μl) contained 1.5 mM MgCl2, 250 μM dNTPs, 200 nM 5′ and 3′ ODN primers, and 1 U of Taq DNA polymerase (AmpliTaq; PerkinElmer/Cetus, Norwalk, CT).

The human primers used were IL-15Rα sense, 5′-GCCAGCGCCACCCTCCACAGTAA-3′; IL-15Rα antisense, 5′-GCCAGCGGGGGAGTTTGCCTTGAC-3′; IL-2Rα sense, 5′-AAGCTCTGCCACTCGGAACACAAC-3′; IL-2Rα antisense, 5′-TGATCAGCAGGAAAACACAGC-3′; IL-2Rβ sense, 5′-GAATTCCCTGGAGAGATGGCCACGGTCCCA-3′, IL-2Rβ antisense, 5′-GAATTCGAGGTTTGGAAATGGATGGACCAAGT3′; IL-2Rγ sense, 5′-AGCCCCAGCCTACCAACCTCACT-3′; IL-2Rγ antisense, 5′-TTAAAGCGGCTCCGAACACGAA-3′; β-actin sense 5′-GTGGGGCGCCCCAGGCACCA-3′; β-actin antisense, 5′-CTCCTTAATGTCACGCACGATTTC-3′; Syk sense 5′-GGTGTGTGCCCTCCGGCC-3′; and Syk antisense, 5′-CTGCAGGTTCCATGT-3′.

All primers used were purchased from TIB Molbiol (Berlin, Germany). Samples were amplified in a DNA Thermocycler (PerkinElmer/Cetus) for 35 cycles (94°C for 1 min, 60°C for 2 min, and 72°C for 2 min). Aliquots of PCR products were then electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining.

Proliferation of Raji cells was assessed by [3H]thymidine incorporation. Cells (1 × 105/ml) were cultured in triplicates in 96-well flat-bottom plates, in a final volume of 100 μl for 48 h, and then incubated with [3H]thymidine (1 μCi/well) for an additional 4 h. Cells were harvested onto glass filters, and incorporation of thymidine was determined by liquid scintillation counting (12).

Raji cells were incubated for 30 min with fura 2 (Molecular Probes, Eugene, OR) at 37°C. After three washes, cells were placed in a Hitachi F-2500 spectrophotometer (Hitachi, Tokyo, Japan) and Ca2+ influx was measured using 350/385 excitation filters. After recording of background for 20 s, cells were stimulated with 10 ng of IL-15 or IL-2 for comparison and induced Ca2+ influx was calculated using the equation of Grynkiewicz et al. (36). As positive control for Ca2+ influx 12-O-tetradecanoyl phorbol-13-acetate (TPA; Sigma-Aldrich, St. Louis, MO) was used.

Cell cycle analysis was performed as described earlier (32), with minor modifications. Briefly, after treatment, 1 × 106 Raji cells/ml were washed twice with PBS, resuspended in 300 μl of 0.1% sodium citrate with 0.1% Triton X-100 and 50 μg/ml propidium iodide, and incubated for at least 4 h at 4°C before FACS analysis. The latter was performed by FACSort (BD Biosciences, Mountain View, CA), using a linear mode.

Cell pellets were lysed for 15 min on ice in 1% N-octyl-β-d-thioglucopyranoside (ODGP; Calbiochem) cell extraction buffer (20 mM Tris-HCl buffer, pH 8.0, 15 mM NaCl, 10% glycerol, 2 mM EDTA, 10 mM sodium fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 10 mM PMSF, and 100 μM sodium vanadate) or in 1% Nonidet P-40 buffer. The detergent-insoluble materials were removed by centrifugation for 15 min at 13,000 rpm at 4°C. Protein concentration was determined (BSA protein assay kit; Bio-Rad), and 100-μg aliquots of proteins were analyzed by electrophoresis in 10% SDS-PAGE.

For immunoprecipitation studies, lysates containing 500 μg of proteins were precleared with the appropriate anti-human or anti-mouse IgG bound to protein A-agarose and immunoprecipitated overnight at 4°C by incubation with 2 μg/ml of Abs. Immunocomplexes were captured on protein A-agarose (with gentle mixing for 1 h at 4°C). After washing, pellets were resuspended in SDS-PAGE sample buffer (62.5 mM Tris-HCL, pH 8.0, 1% glycerol, 2% SDS, 5% 2-ME, and 0.01% bromphenol blue), boiled for 5 min, and analyzed in 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose (Bio-Rad) in buffer containing 25 mM Tris, 192 mM glycine, 1% SDS, and 20% methanol at 150 V for 40 min. Blots were blocked for 1 h in PBS with 0.05% Tween 20 (PBS-T) and 3% BSA (Sigma-Aldrich). After incubations with first and second Abs and washing with PBS-T, visualization of specific proteins was conducted by an ECL method using ECL Western blotting detection reagents (Amersham Life Science) according to the manufacturer’s instructions.

For kinase assay, immunocomplexes were washed once more with 25 mM HEPES (pH 7.4), 2 mM MnCl2, 10 mM MgCl2, and 1 mM Na3VO4 and incubated in 60 μl of 5 mM HEPES, 2 mM MnCl2, 10 mM MgCl2, 1 mM Na3VO4, 10 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham), 10 μM ATP, and 10 μg of GST-HS1 peptide (a Syk substrate; generously provided by Dr. U. Blank, Institut Pasteur, Paris, France) for 5 min at room temperature. The reaction was stopped by adding 20 μl of 4× sample buffer. Samples were boiled for 5 min and proteins were resolved in 12%SDS-PAGE. Phosphotyrosine-containing proteins were detected by autoradiography.

For GST precipitation, lysates were incubated with 5 μg of Syk-GST prebound to 20 μl of glutathione-agarose beads for 2 h at 4°C with rotation (construct, bearing two SH2 domains in pGEX-2TK expression vector, was generously provided by Dr. U. Blank). Beads were washed and precipitates were analyzed in 10% SDS-PAGE using anti-IL-15Rα Abs.

All experiments were performed in at least three independent assays, which yielded highly comparable results. Data are presented as mean values ± SD as indicated in the figure legends. Mann-Whitney U test was used to determine the level of statistical significance.

Raji cells reportedly express transcripts for the IL-15R complex (4). To confirm this and to analyze the expression of the corresponding proteins, RT-PCR, immunoprecipitation, and Western blotting techniques were used. As positive control we selected a T cell lymphoma line (HUT102), which expresses IL-15Rα as well as the β- and γ-chains (37, 38). RT-PCR was used for the detection of the IL-15Rα, IL-2Rβ, and IL-2Rγ cDNA in Raji and HUT102 cells.

As shown in Fig. 1 A, these assays revealed that Raji cells express the two alternatively spliced products of the IL-15Rα subunits as well as the γ-chain transcript but lack expression of IL-2Rβ chain transcripts.

FIGURE 1.

Raji cells express IL-15Rα and IL-2Rγ chains. A, RT-PCR analysis of IL-15Rα, IL-2Rα, IL-2Rβ, and IL-2Rγ expression in Raji cells. RNA extracted from Raji (lane 1) and HUT-102 (positive control, lane 2) was reverse transcribed and subjected to PCR amplification using specific primers for β-actin, IL-15Rα, IL-2Rα, IL-2Rβ, and IL-2Rγ (as indicated). The amplified products were electrophoresed through 1.5% agarose gel. A mock PCR (without cDNA) was used to exclude contamination (lane 3). The amount of cDNA analyzed was similar in different samples, as shown by PCR amplification of β-actin cDNA. B, Analysis of IL-15Rα protein expression in Raji cells. A total of 500 μg of proteins from Raji cell lysates were immunoprecipitated with 2 μg of mouse anti-human IL-15Rα, resolved in 10% SDS-PAGE, and Western blotted with anti-IL-15Rα Abs (lane 1). Akata cell extracts were used as negative control for IL-15Rα expression (lane 2). C, IL-2Rβ protein expression pattern in Raji cells. Proteins from Raji cell lysates were analyzed in 10% SDS-PAGE and assayed for IL-2Rβ expression (lane 1). Lysates from Jurkat cells were used as a positive control (lane 2). D, Analysis of IL-2Rγ protein expression in Raji cells. Total cell lysate (100 μg) was loaded into a 10% SDS-PAGE. Blots were assayed for IL-2Rγ expression using rabbit anti-IL-2Rγ Abs as first and HRP-conjugated goat anti-rabbit as second Abs (lane 1). Lysates from K562 cells were used as negative control (lane 2). The positions of IL-15Rα, IL-2Rβ, and IL-2Rγ are indicated on the left. The molecular mass standards (in kDa) are indicated on the right.

FIGURE 1.

Raji cells express IL-15Rα and IL-2Rγ chains. A, RT-PCR analysis of IL-15Rα, IL-2Rα, IL-2Rβ, and IL-2Rγ expression in Raji cells. RNA extracted from Raji (lane 1) and HUT-102 (positive control, lane 2) was reverse transcribed and subjected to PCR amplification using specific primers for β-actin, IL-15Rα, IL-2Rα, IL-2Rβ, and IL-2Rγ (as indicated). The amplified products were electrophoresed through 1.5% agarose gel. A mock PCR (without cDNA) was used to exclude contamination (lane 3). The amount of cDNA analyzed was similar in different samples, as shown by PCR amplification of β-actin cDNA. B, Analysis of IL-15Rα protein expression in Raji cells. A total of 500 μg of proteins from Raji cell lysates were immunoprecipitated with 2 μg of mouse anti-human IL-15Rα, resolved in 10% SDS-PAGE, and Western blotted with anti-IL-15Rα Abs (lane 1). Akata cell extracts were used as negative control for IL-15Rα expression (lane 2). C, IL-2Rβ protein expression pattern in Raji cells. Proteins from Raji cell lysates were analyzed in 10% SDS-PAGE and assayed for IL-2Rβ expression (lane 1). Lysates from Jurkat cells were used as a positive control (lane 2). D, Analysis of IL-2Rγ protein expression in Raji cells. Total cell lysate (100 μg) was loaded into a 10% SDS-PAGE. Blots were assayed for IL-2Rγ expression using rabbit anti-IL-2Rγ Abs as first and HRP-conjugated goat anti-rabbit as second Abs (lane 1). Lysates from K562 cells were used as negative control (lane 2). The positions of IL-15Rα, IL-2Rβ, and IL-2Rγ are indicated on the left. The molecular mass standards (in kDa) are indicated on the right.

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Immunoprecipitation and Western blotting documented the expression of the IL-15Rα (Fig. 1,B) and IL-2Rγ (Fig. 1,D), and the absence of IL-2Rβ protein (Fig. 1 C), in Raji cells. The B lymphoblastoid cell line Akata and the myeloid cell line K562, which lack IL-15Rα or IL-2Rγ chains, respectively (4, 18), were used as negative controls. The T cell leukemia line Jurkat, which expresses IL-2Rβ (4), was used as a positive control for this chain. Thus, Raji cells express the IL-15Rα and IL-2Rγ chains at both the gene and protein level, but lack the IL-2Rβ chain. As a consequence, we are confident to state that IL-2Rβ plays no role in IL-15-mediated signaling in these cells.

Next, we analyzed the effects of IL-15 stimulation on the proliferative activity of Raji cells. Cells were treated with IL-15 or IL-2 (for comparison) for 48 h, and proliferation was assessed by [3H]thymidine incorporation. These assays revealed that IL-15 moderately, but statistically significantly, stimulated the proliferation of Raji cells in a dose-dependent manner, while IL-2 had no proliferation-modulating effect (Fig. 2 A).

FIGURE 2.

IL-15 moderately stimulates proliferation of Raji cells and rescues them from C2-ceramide-induced apoptosis. A, A total of 1 × 105/ml cells were incubated with different concentrations of IL-15 or IL-2 for 48 h and [3H]thymidine incorporation in DNA was assayed. Proliferation of cells without cytokines is shown as point 0. ∗, p < 0.05 vs control and IL-2. B, Cells were incubated for 48 h with 10 ng/ml IL-15 or IL-2 in the presence or absence of anti-IL-15Rα or anti-IL-2Rγ Abs (both are in concentration 1 μg/ml) and [3H]thymidine incorporation in DNA was measured. Incubation of cells in the absence of cytokines was used as a control. ∗, p < 0.05 vs control and IL-15 plus anti-IL-15Rα. C, Raji cells were incubated with active cell-permeable C2-ceramide or with C2-dihydroceramide (an inactive analog) as control in combination with IL-2 or IL-15 for 48 h. Cell cycle analysis using propidium iodide staining was performed. M1 gate corresponds to apoptotic cells; M2, cells in G0/G1 phase; M3, cells in S phase; and M4, cells in G2/M phase respectively. D, Graphic representation of the percentage of cells in each phase of the cell cycle. Untreated cells are shown as hatched bar; IL-15-treated, open bar; and IL-2, filled bar. Shown is one representative experiment of three independent experiments, all of which yielded similar results (∗, p < 0.05 vs control and IL-2).

FIGURE 2.

IL-15 moderately stimulates proliferation of Raji cells and rescues them from C2-ceramide-induced apoptosis. A, A total of 1 × 105/ml cells were incubated with different concentrations of IL-15 or IL-2 for 48 h and [3H]thymidine incorporation in DNA was assayed. Proliferation of cells without cytokines is shown as point 0. ∗, p < 0.05 vs control and IL-2. B, Cells were incubated for 48 h with 10 ng/ml IL-15 or IL-2 in the presence or absence of anti-IL-15Rα or anti-IL-2Rγ Abs (both are in concentration 1 μg/ml) and [3H]thymidine incorporation in DNA was measured. Incubation of cells in the absence of cytokines was used as a control. ∗, p < 0.05 vs control and IL-15 plus anti-IL-15Rα. C, Raji cells were incubated with active cell-permeable C2-ceramide or with C2-dihydroceramide (an inactive analog) as control in combination with IL-2 or IL-15 for 48 h. Cell cycle analysis using propidium iodide staining was performed. M1 gate corresponds to apoptotic cells; M2, cells in G0/G1 phase; M3, cells in S phase; and M4, cells in G2/M phase respectively. D, Graphic representation of the percentage of cells in each phase of the cell cycle. Untreated cells are shown as hatched bar; IL-15-treated, open bar; and IL-2, filled bar. Shown is one representative experiment of three independent experiments, all of which yielded similar results (∗, p < 0.05 vs control and IL-2).

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To assess whether IL-15 stimulates the proliferation of Raji via IL-15Rα we used anti-IL-15Rα or anti-IL-2Rγ Abs to specifically block the binding of IL-15 to these subunits. Raji cells were stimulated for 48 h with 10 ng/ml of IL-15, IL-2, or in combination with 1 μg/ml anti-IL-15Rα or anti-IL-2Rγ Abs, and [3H]thymidine incorporation was measured. Cells not treated with cytokines were used as a control. As shown in Fig. 2 B, while anti-IL-15Rα Abs significantly inhibited the IL-15-induced proliferative response of Raji cells, anti-IL-2Rγ had no effect. Even though the proliferative stimulus provided by IL-15 is indeed modest and difficult to distinguish from antiapoptotic signals, it still appears to be IL-15Rα specific, because it is suppressed by anti-IL-15Rα Ab. This supports the notion that IL-15 mediates its stimulatory effects in Raji cells via the IL-15Rα.

As previously shown, IL-15 is capable of rescuing lymphocytes from programmed cell death (3, 39). Different agents were used to induce apoptosis in Raji cells, such as dexamethasone, TNF-α, anti-IgM, and C2-ceramide, yet without success (32, 40). However, only C2-ceramide was able to arrest Raji cells in the G0/G1 phase of the cell cycle without being able to induce apoptosis (32). To study the effect of IL-15 on C2-ceramide-induced cell cycle arrest, Raji cells were treated for 48 h with 20 μM active cell-permeable C2-ceramide, or with C2-dihydro-ceramide (an inactive analog) as negative control (41), in the presence or absence of IL-15, and were subsequently stained with propidium iodide as a marker for apoptosis. In contrast to a previous report (32), in our study, C2-ceramide significantly increased the percentage of apoptotic cells and decreased the percentage of cells in the G2/M phase (Fig. 2, C and D). Cotreatment with IL-15 reduced the amount of apoptotic cells significantly (p < 0.05), along with a simultaneous increase in the percentage of cells in G2/M phase. Furthermore, cotreatment of Raji cells with IL-15 and anti-IL-15Rα (but not with anti-IL-2Rγ) Abs abrogated the inhibitory effect of IL-15 on C2-ceramide-induced apoptosis (data not shown).

Thus, IL-15 modulates the ceramide-induced apoptosis and proliferation of Raji cells, most likely via binding to the IL-15Rα chain.

Many cytokine receptors use protein tyrosine phosphorylation for signaling (42, 43, 44, 45). Most of these receptors lack an intrinsic tyrosine kinase activity. Therefore, they recruit and activate cytoplasmic tyrosine kinases, such as Src, Syk, Zap-70, and Jak tyrosine kinases (27, 45, 46, 47, 48, 49). Thus, our next goal was to investigate which of the two IL-15R chains expressed by Raji cells (α or γ) is capable of signaling upon IL-15 stimulation, which tyrosine kinases are activated, and with which of the two IL-15R chains selected tyrosine kinases associate.

Raji cells were stimulated with IL-15 or IL-2 for 15 and 30 min and lysed with ODGP lysis buffer. Cell extracts were then immunoprecipitated with anti-IL-15Rα or anti-IL-2Rγ Abs, loaded onto an SDS-PAGE, and blotted on membranes which were subsequently probed with Abs against Syk and different members of the Src tyrosine kinase family expressed in B cells, namely, Lyn, Blk, and Fyn (45, 46, 49).

As shown in Fig. 3,A, stimulation of Raji cells with IL-15 for 15 min induced coprecipitation of Syk with the IL-15Rα, but not with the IL-2Rγ chain (Fig. 3 B). This association disappeared after 30 min of treatment. IL-2 stimulation was used as a negative control and did not coprecipitate Syk. Control isotype-matched Ab and anti-IL-2Rβ Abs did not precipitate Syk or any phosphorylated proteins (data not shown). The presence of trace amounts of IL-2Rβ in IL-15Rα precipitates was excluded by probing with anti-IL-2Rβ Abs (data not shown). Lyn, Blk, and Fyn kinases did not associate with IL-15Rα in ODGP lysates of IL-15-stimulated Raji cells (data not shown). Thus, Syk specifically associates with the intracellular domain of the IL-15Rα chain upon IL-15 treatment of Raji cells.

FIGURE 3.

IL-15 stimulation induces the association of Syk protein kinase to the IL-15Rα chain and enhances Syk kinase activity. Raji cells were stimulated with IL-15 (100 ng/ml) or IL-2 (100 ng/ml) or left untreated for 15 or 30 min at 37°C, lysed, and immunoprecipitated with anti-IL-15Rα Abs (A) or anti-IL-2Rγ Abs (B). After SDS-PAGE and blotting the membranes were probed with anti-Syk Abs. For control of loading, blots were stripped in 62.5 mM Tris-HCl buffer containing 2% SDS and 100 mM 2-ME at 4°C overnight and IL-15Rα (A) or IL-2Rγ (B) was detected. Syk (p72), IL-15Rα (p60–65), IL-2Rγ (p64), and H chains of IgG are indicated on the left; molecular mass standards (kDa) are indicated on the right. This figure shows one representative experiment of three independent experiments, which all gave comparable results. C, Raji cells were treated for 15 min with 100 ng of IL-15 or IL-2. Lysates from cells were precipitated with anti-Syk Abs. The activity of Syk was analyzed by kinase assay using GST-HS1 fusion protein as substrate followed by 12% SDS-PAGE and autoradiography (upper panel). Syk was immunoprecipitated from activated Raji cells and phosphorylation of Syk was detected using anti-p-Tyr Abs (middle panel). The amount of precipitated Syk was determined by immunoblotting with anti-Syk Abs (lower panel). The position of phosphorylated GST-SH1 protein (27 kDa) and Syk is indicated on the right.

FIGURE 3.

IL-15 stimulation induces the association of Syk protein kinase to the IL-15Rα chain and enhances Syk kinase activity. Raji cells were stimulated with IL-15 (100 ng/ml) or IL-2 (100 ng/ml) or left untreated for 15 or 30 min at 37°C, lysed, and immunoprecipitated with anti-IL-15Rα Abs (A) or anti-IL-2Rγ Abs (B). After SDS-PAGE and blotting the membranes were probed with anti-Syk Abs. For control of loading, blots were stripped in 62.5 mM Tris-HCl buffer containing 2% SDS and 100 mM 2-ME at 4°C overnight and IL-15Rα (A) or IL-2Rγ (B) was detected. Syk (p72), IL-15Rα (p60–65), IL-2Rγ (p64), and H chains of IgG are indicated on the left; molecular mass standards (kDa) are indicated on the right. This figure shows one representative experiment of three independent experiments, which all gave comparable results. C, Raji cells were treated for 15 min with 100 ng of IL-15 or IL-2. Lysates from cells were precipitated with anti-Syk Abs. The activity of Syk was analyzed by kinase assay using GST-HS1 fusion protein as substrate followed by 12% SDS-PAGE and autoradiography (upper panel). Syk was immunoprecipitated from activated Raji cells and phosphorylation of Syk was detected using anti-p-Tyr Abs (middle panel). The amount of precipitated Syk was determined by immunoblotting with anti-Syk Abs (lower panel). The position of phosphorylated GST-SH1 protein (27 kDa) and Syk is indicated on the right.

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Recently, the HS1 peptide was identified as a specific substrate for Syk kinase (50), and recombinant GST fusion protein containing this peptide has been successfully used for monitoring Syk kinase activity (51). Therefore, to study Syk kinase activity in IL-15-activated Raji cells we performed a kinase assay, using GST-HS1 fusion protein as a substrate. As shown in Fig. 3,C (upper panel), IL-15, but not IL-2, enhanced the Syk kinase activity against exogenous substrate. Syk did not induce phosphorylation of control GST protein (data not shown). In addition to the enhancement of Syk kinase activity against HS1 peptide, IL-15 also induced the Syk phosphorylation in vitro (Fig. 3,C, middle panel). Incubation of blots with anti-Syk Abs confirmed that equal amounts of Syk were precipitated from lysates of Raji cells (Fig. 3 C, lower panel).

To explore potential molecular targets of Syk kinase, which are phosphorylated upon IL-15 stimulation in Raji cells, proteins that physically and functionally associate with Syk were coprecipitated. For this purpose, cells were treated with IL-15 or IL-2, lysed in ODGP buffer, and precipitated with anti-Syk Abs. Immunoprecipitates were analyzed with anti-p-Tyr Abs so as to study proteins phosphorylated on tyrosine residues.

As shown in Fig. 4,A, stimulation of Raji cells with IL-15 induced the phosphorylation of at least three proteins with molecular masses of 60–65, 70–72, and 120 kDa. The IL-15Rα itself is detectable by Western blotting as a protein of 60–65 kDa (26, 30) and Syk as a protein of 72 kDa (51). Therefore, the same membranes were stripped and reprobed with anti-IL-15Rα, anti-Syk, and anti-PLCγ1 Abs. This suggested that the 60- to 65-, 70- to 72-, and 120-kDa proteins were indeed IL-15Rα, Syk, and PLCγ1, respectively (Figs. 4 B and 5C).

FIGURE 4.

Syk kinase activation induces phosphorylation of intracellular substrates. Raji cells were stimulated for 15 min with IL-15 (lane 2) or IL-2 (lane 3) or left unstimulated (lane 1), and lysed in ODGP lysis buffer. Proteins were immunoprecipitated with anti-Syk Abs. Blots were developed with anti-p-Tyr (RC-20) Abs. Arrows indicate the position of phosphorylated substrates (A). Membranes were stripped overnight and reprobed with IL-15Rα Abs (B). C, PLCγ1 detection. To prove the equal amount of proteins in each sample, blots were reprobed with anti-Syk Abs after stripping (D).

FIGURE 4.

Syk kinase activation induces phosphorylation of intracellular substrates. Raji cells were stimulated for 15 min with IL-15 (lane 2) or IL-2 (lane 3) or left unstimulated (lane 1), and lysed in ODGP lysis buffer. Proteins were immunoprecipitated with anti-Syk Abs. Blots were developed with anti-p-Tyr (RC-20) Abs. Arrows indicate the position of phosphorylated substrates (A). Membranes were stripped overnight and reprobed with IL-15Rα Abs (B). C, PLCγ1 detection. To prove the equal amount of proteins in each sample, blots were reprobed with anti-Syk Abs after stripping (D).

Close modal

Recently, it has been shown that both PLCγ1 and PLCγ2 (120 kDa) are potential substrates for the Syk kinase (52, 53). PLCγ2 is the most abundant isoform in B cells. However, our data (not shown) indicate that in Raji cells the level of expression of PLCγ2 is substantially lower than that of the PLCγ1 isoform. These data are in accordance with published observations by Kang et al. (54), who showed a low expression level of PLCγ2 in Raji cells. Thus, in Raji cells, PLCγ2 is likely of minor importance, and the identification of PLCγ1 as a downstream signaling molecule after IL-15 action upon Raji cells had to be the logical target of our studies.

To confirm that IL-15Rα and PLCγ1 are actually tyrosine phosphorylated after IL-15 administration, activated Raji cells were lysed and immunoprecipitated with anti-IL-15Rα or anti-PLCγ1 Abs, and Western blotting was performed with anti-p-Tyr Abs. As demonstrated in Fig. 5, A and C, IL-15 induced phosphorylation of these proteins. Moreover, IL-2 also induced the phosphorylation of PLCγ1. Furthermore, after depletion of PLCγ1 from the IL-15-activated cell lysates, neither PLCγ1 nor Syk were detectable anymore. The same results were obtained when IL-15Rα was depleted from the lysates (data not shown).

FIGURE 5.

Piceatannol inhibits IL-15-induced phosphorylation of IL-15Rα and PLCγ. Raji cells were serum-starved for 3 h and treated with 50 μM piceatannol for 10 min before activation. Nontreated cells were used as controls. Cells treated with medium, IL-15, or IL-2 for 15 min were lysed, immunoprecipitated with anti-IL-15Rα (A) or with anti-PLCγ (C) Abs, and analyzed for tyrosine phosphorylation patterns in Western blotting using RC20 Abs. Position of specific phosphorylated proteins is indicated on the left. As control of loading, membranes were stripped and reprobed with anti-IL-15Rα (B) or with anti-PLCγ (D) Abs, respectively.

FIGURE 5.

Piceatannol inhibits IL-15-induced phosphorylation of IL-15Rα and PLCγ. Raji cells were serum-starved for 3 h and treated with 50 μM piceatannol for 10 min before activation. Nontreated cells were used as controls. Cells treated with medium, IL-15, or IL-2 for 15 min were lysed, immunoprecipitated with anti-IL-15Rα (A) or with anti-PLCγ (C) Abs, and analyzed for tyrosine phosphorylation patterns in Western blotting using RC20 Abs. Position of specific phosphorylated proteins is indicated on the left. As control of loading, membranes were stripped and reprobed with anti-IL-15Rα (B) or with anti-PLCγ (D) Abs, respectively.

Close modal

To prove that Syk, and not other kinases (Lyn, Blk, Jaks, etc.), is responsible for the phosphorylation of IL-15Rα and PLCγ1, piceatannol, an inhibitor of Syk activity, was used (31, 55). Nevertheless, because piceatannol at high concentrations (30–50 μg/ml; 100–200 μM) inhibits Lyn, Fak, and Scr kinases (56, 57) in addition to Syk, in our experiments we added piceatannol at a lower concentration (50 μM). Raji cells were serum-starved for 3 h and treated for 10 min with 50 μM piceatannol before activation. As shown in Fig. 5, piceatannol inhibited IL-15-induced phosphorylation of IL-15Rα (Fig. 5,A) and PLCγ1 (Fig. 5 C) in Raji cells.

The association between Syk and IL-15Rα as well as downstream phosphorylation of IL-15Rα and PLCγ1 were confirmed in two other B lymphoblastoid cell lines, DG75 and Ramos (data not shown).

Thus, IL-15 induces physical and functional association of Syk kinase with IL-15Rα and PLCγ1, both molecules are phosphorylated by Syk, and this phosphorylation is abrogated by piceatannol.

To clarify whether the observed phenomenon of IL-15-mediated signaling takes place not only in the transformed B cell lines studied, but also in primary B lymphocytes, peripheral human B cells were stimulated for 48 h with LPS and T cells were stimulated with Con A. Lymphoblasts were activated thereafter with IL-15 or IL-2. Activated B and T cells express all subunits of the IL-2R complex as well as the IL-15Rα chain (1). As shown in Fig. 6 A, immunoprecipitation with anti-Syk Abs after IL-15 and IL-2 stimulation induced phosphorylation of Syk kinase. Thus, Syk is activated in peripheral LPS-activated B lymphoblasts and Con A-activated T lymphoblasts upon IL-15 or IL-2 stimulation. The ability of Syk to associate with the IL-2Rβ chain has previously been documented (45, 46).

FIGURE 6.

IL-15 stimulation induces Syk activation and association to the IL-15Rα and IL-2Rβ chain in activated human B and T cells. LPS-activated peripheral human B lymphocytes and Con A-activated T lymphocytes were obtained as described in Materials and Methods. Cells were serum-starved for 2 h and treated for 15 min with IL-15 or IL-2. Then the cells were lysed with ODGP buffer. A, Lysates were precipitated with anti-Syk, and phosphorylated Syk kinase was detected using anti-p-Tyr Abs (upper panel). The position of phosphorylated Syk is indicated on the left. Proteins from lysates were also precipitated with anti-IL-15Rα (B), anti-IL-2Rβ (C), or anti-IL-2Rγ (D) Abs, and Syk detection in precipitates was performed. For loading control, blots were stripped and subjected to Syk, IL-15Rα, IL-2Rβ, or IL-2Rγ detection, respectively (lower panels). Their position is indicated on the left.

FIGURE 6.

IL-15 stimulation induces Syk activation and association to the IL-15Rα and IL-2Rβ chain in activated human B and T cells. LPS-activated peripheral human B lymphocytes and Con A-activated T lymphocytes were obtained as described in Materials and Methods. Cells were serum-starved for 2 h and treated for 15 min with IL-15 or IL-2. Then the cells were lysed with ODGP buffer. A, Lysates were precipitated with anti-Syk, and phosphorylated Syk kinase was detected using anti-p-Tyr Abs (upper panel). The position of phosphorylated Syk is indicated on the left. Proteins from lysates were also precipitated with anti-IL-15Rα (B), anti-IL-2Rβ (C), or anti-IL-2Rγ (D) Abs, and Syk detection in precipitates was performed. For loading control, blots were stripped and subjected to Syk, IL-15Rα, IL-2Rβ, or IL-2Rγ detection, respectively (lower panels). Their position is indicated on the left.

Close modal

To confirm that not only IL-2Rβ, but also IL-15Rα can recruit Syk, we performed a series of immunoprecipitation and blocking experiments. As shown in Fig. 6,B (upper panel), IL-15 specifically induced association of Syk with IL-15Rα. Both IL-15 and IL-2 are capable of inducing the association of Syk with the IL-2Rβ chain (Fig. 6,C, upper panel). Neither IL-15 nor IL-2 induced coprecipitation of Syk with IL-2Rγ (Fig. 6,D). Preincubation of cells with IL-15Rα-blocking Abs significantly decreased the amount of Syk associated with IL-15Rα after IL-15 treatment, while the association of Syk kinase with IL-2Rβ was not affected (data not shown). Furthermore, IL-15Rα/Syk association upon IL-15 stimulation could also be detected in Con A-activated T lymphoblasts (Fig. 6). Thus, in activated primary human B cells and T cells, IL-15 induces Syk phosphorylation and its association with both the IL-15Rα and IL-2Rβ subunits.

Despite the reported selectivity to Syk kinase shown by several groups (31, 55), piceatannol at high concentrations could also inhibit Lyn, Src, and Fak kinase activity (57). Taking this fact into account, it was still unclear whether IL-15 directly signals through Syk or recruits other intracellular kinases. Therefore, to prove that IL-15 signals specifically through Syk, and to study the importance of Syk in IL-15-mediated signaling and protection of Raji cells from C2-ceramide-induced apoptosis, we used Syk antisense ODNs (32, 35).

We examined the influence of stem-loop Syk antisense ODNs on Syk mRNA and Syk protein expression in Raji cells. Cells were transfected with 1 μg of stem-loop Syk antisense ODNs or scrambled-control ODNs in complex with lipofectAMINE. Transfected cells were harvested on the third day after transfection, and cells treated with lipofectAMINE alone were used for comparison. Transfection efficiency was ∼30–35%. RT-PCR with Syk primers from total cell RNA was performed. Stem-loop Syk antisense ODNs completely inhibited Syk RNA expression in Raji cells (Fig. 7,A). Scrambled-control ODNs as well as liposome treatment alone did not reduce the level of Syk mRNA. Treatment of cells with any ODNs or liposomes did not influence the β-actin mRNA level (Fig. 7 A).

FIGURE 7.

Stem-loop antisense Syk ODNs completely block Syk transcription and abrogate IL-15-induced protection from C2-ceramide apoptosis in Raji cells. Raji cells were incubated twice with complexes containing 2 μg of lipofectAMINE and 1 μg of stem-loop antisense Syk ODNs or control ODNs. Nontransfected cells were used for comparison. A, Total RNA was extracted from cells and RT-PCR was performed with Syk primers. The amplified products were analyzed in 1.5% agarose gel. A mock PCR (without DNA) was used to exclude contamination. The amount of cDNA analyzed was similar in different samples, as shown by PCR amplification of β-actin cDNA. B, Protein lysates from transfected or nontransfected cells were immunoblotted with anti-Syk Abs. The expression of IL-15Rα is shown to prove the equal amount of loaded proteins. C, Transfected and nontransfected but lipofectAMINE-treated cells were incubated with active cell-permeable C2-ceramide or with C2-dihydroceramide (as a control) in combination with IL-2 or IL-15 for 48 h. The percentage of apoptotic cells was assessed by propidium iodide staining. ∗, p < 0.05 compared with C2-ceramide and C2-ceramide plus IL-2.

FIGURE 7.

Stem-loop antisense Syk ODNs completely block Syk transcription and abrogate IL-15-induced protection from C2-ceramide apoptosis in Raji cells. Raji cells were incubated twice with complexes containing 2 μg of lipofectAMINE and 1 μg of stem-loop antisense Syk ODNs or control ODNs. Nontransfected cells were used for comparison. A, Total RNA was extracted from cells and RT-PCR was performed with Syk primers. The amplified products were analyzed in 1.5% agarose gel. A mock PCR (without DNA) was used to exclude contamination. The amount of cDNA analyzed was similar in different samples, as shown by PCR amplification of β-actin cDNA. B, Protein lysates from transfected or nontransfected cells were immunoblotted with anti-Syk Abs. The expression of IL-15Rα is shown to prove the equal amount of loaded proteins. C, Transfected and nontransfected but lipofectAMINE-treated cells were incubated with active cell-permeable C2-ceramide or with C2-dihydroceramide (as a control) in combination with IL-2 or IL-15 for 48 h. The percentage of apoptotic cells was assessed by propidium iodide staining. ∗, p < 0.05 compared with C2-ceramide and C2-ceramide plus IL-2.

Close modal

We then examined the effect of stem-loop Syk antisense ODNs on the protein level of Syk in Raji cells. Total cell lysates were analyzed in Western blotting with anti-Syk Abs. Whereas scrambled-control ODNs had no effect, stem-loop Syk antisense ODNs blocked Syk protein expression in Raji cells (Fig. 7,B). Stem-loop Syk antisense ODNs had no effect on IL-15Rα expression (Fig. 7 B).

Thus, antisense ODNs dramatically inhibit Syk expression at the mRNA and protein level in Raji cells.

Next, we used stem-loop Syk antisense ODNs to study the influence of Syk on IL-15-mediated protection from C2-ceramide-induced apoptosis in Raji cells. Stem-loop Syk antisense ODNs have shown the ability to abrogate the antiapoptotic effect of IL-15 on Raji cells (Fig. 7 C). Transfection with control ODNs as well as liposome treatment did not affect IL-15 activities.

An inhibitor of Syk, piceatannol also blocked the ability of IL-15 to protect Raji cells from C2-ceramide-induced apoptosis (data not shown).

Taken together, these data suggest that Syk activity is required for the inhibition of C2-ceramide-induced apoptosis by IL-15 in Raji cells.

We analyzed the IL-15-mediated association of Syk with IL-15Rα in stem-loop Syk antisense ODN-transfected Raji cells. Cells were transfected with stem-loop Syk antisense ODNs or with control ODNs and were then activated with IL-15 for 15 min. Nontransfected and nonactivated cells were used as controls. Lysates were precipitated with anti-IL-15Rα Abs and analyzed for Syk association by Western blotting (Fig. 8 A, upper panel). Whereas control ODNs had no effect, stem-loop Syk antisense ODNs abrogated the IL-15-induced association of Syk kinase with IL-15Rα.

FIGURE 8.

Stem-loop antisense Syk ODNs abrogate the IL-15-mediated signaling in Raji cells. Nontransfected Raji cells or Raji cells transfected with stem-loop antisense or with scrambled-control ODNs were activated with IL-15 for 15 min and lysed, and proteins were immunoprecipitated with anti-IL-15Rα (A) or anti-PLCγ (B) Abs. Immunoblots were probed with anti-Syk Abs (A, upper panel), anti-phosphotyrosine (A, middle panel, and B, upper panel). For control of equal amount of specific protein in precipitates blots were stripped and reprobed with anti-IL-15Rα (A, lower panel) or anti-PLCγ (B, lower panel) Abs.

FIGURE 8.

Stem-loop antisense Syk ODNs abrogate the IL-15-mediated signaling in Raji cells. Nontransfected Raji cells or Raji cells transfected with stem-loop antisense or with scrambled-control ODNs were activated with IL-15 for 15 min and lysed, and proteins were immunoprecipitated with anti-IL-15Rα (A) or anti-PLCγ (B) Abs. Immunoblots were probed with anti-Syk Abs (A, upper panel), anti-phosphotyrosine (A, middle panel, and B, upper panel). For control of equal amount of specific protein in precipitates blots were stripped and reprobed with anti-IL-15Rα (A, lower panel) or anti-PLCγ (B, lower panel) Abs.

Close modal

Next, we examined the changes of intracellular protein phosphorylation in IL-15-activated Raji cells in the absence of Syk kinase. Lysates from transfected cells were immunoprecipitated with anti-IL-15Rα or anti-PLCγ1 Abs, and precipitated proteins were analyzed for patterns of tyrosine phosphorylation. As shown in Fig. 8, IL-15 induced IL-15Rα (Fig. 8,A, middle panel) as well as anti-PLCγ1 (Fig. 8,B, upper panel) phosphorylation in nontransfected, as well as in transfected with scrambled-control, ODN cells. Transfection of cells with stem-loop Syk antisense ODNs led to the abrogation of IL-15-induced phosphorylation of both proteins in Raji cells. Blots were stripped and reprobed with anti-IL-15Rα Abs (Fig. 8,A, lower panel) or with anti-PLCγ1 (Fig. 8 B, lower panel) Abs to prove that equal amounts of respective proteins had been precipitated by the specific Abs.

These data indicate that Syk kinase is required for the intracellular signaling mediated by IL-15. IL-15-induced phosphorylation of IL-15Rα and PLCγ is dependent on Syk kinase.

The involvement of PLCγ in cytokine-induced signaling suggests that IL-15 binding to the receptor leads to calcium influx and lipid turnover (53). We studied the influence of IL-15 on Ca2+ influx in Raji cells. As shown in Fig. 9, IL-15, but not IL-2, stimulation was able to modestly enhance the Ca2+ influx in Raji cells. As a positive control for Ca2+ influx triggering, Raji cells were stimulated with TPA. Transfection of Raji cells with stem-loop Syk antisense ODNs abolished the influence of IL-15 on Ca2+ signal.

FIGURE 9.

Stem-loop antisense Syk ODNs suppress the IL-15-mediated Ca2+ influx in Raji cells. Nontransfected Raji cells or Raji cells transfected with stem-loop Syk antisense or with control ODNs were incubated with 2 μM fura 2 for 30 min before Ca2+ measurement and treated with IL-15 or IL-2. Stimulation of Raji cells with TPA was used as positive control. The figure shows the average of three independent experiments.

FIGURE 9.

Stem-loop antisense Syk ODNs suppress the IL-15-mediated Ca2+ influx in Raji cells. Nontransfected Raji cells or Raji cells transfected with stem-loop Syk antisense or with control ODNs were incubated with 2 μM fura 2 for 30 min before Ca2+ measurement and treated with IL-15 or IL-2. Stimulation of Raji cells with TPA was used as positive control. The figure shows the average of three independent experiments.

Close modal

These data indicate that Syk might mediate IL-15-mediated Ca2+ influx in Raji cells.

The intracellular domain of murine and human IL-15Rα is very short (∼40 amino acid residues); it contains only one tyrosine residue located at amino acid position 227 (4). To further confirm the concept of tyrosine phosphorylation of IL-15Rα as a result of IL-15 stimulation, we wished to evaluate the requirement of this tyrosine residue for IL-15Rα-Syk binding and for IL-15-induced phosphorylation of Syk substrates. For this purpose, constructs of murine IL-15Rα in which Tyr227 had been replaced with phenylalanine (Y227F) were generated by site-directed mutagenesis. To study the role of this tyrosine residue in binding Syk kinase, COS cells were transfected with wild-type (WT) or mutated IL-15Rα constructs in combination with Syk-expressing constructs using the DEAE-dextran method. Lysates from transfected COS cells were immunoprecipitated with anti-IL-15Rα Abs and blotted with anti-Syk Abs. As seen in Fig. 10, Syk selectively precipitated with WT -IL-15Rα and failed to associate with Y227F-IL-15Rα mutant.

FIGURE 10.

Influence of Y227F-IL-15Rα mutation on the association with Syk kinase. COS-7 cells were transfected by DEAE-dextran method as indicated or left untransfected but DEAE treated. Forty-eight hours after transfection cells were harvested, lysed, and immunoprecipitated with anti-IL-15Rα Abs. A, Precipitates were analyzed in 10% SDS-PAGE for Syk. B, Stripped membranes were reprobed for IL-15Rα as a loading control. C, Western blotting of lysates with anti-Syk Abs was performed to prove the efficacy of transfection.

FIGURE 10.

Influence of Y227F-IL-15Rα mutation on the association with Syk kinase. COS-7 cells were transfected by DEAE-dextran method as indicated or left untransfected but DEAE treated. Forty-eight hours after transfection cells were harvested, lysed, and immunoprecipitated with anti-IL-15Rα Abs. A, Precipitates were analyzed in 10% SDS-PAGE for Syk. B, Stripped membranes were reprobed for IL-15Rα as a loading control. C, Western blotting of lysates with anti-Syk Abs was performed to prove the efficacy of transfection.

Close modal

Next, we studied the effect of this mutation on IL-15-induced phosphorylation of IL-15Rα and PLCγ. For these experiments, IL-15Rα- and Syk-negative murine plasmocytoma J558 cells were transiently transfected with Syk in combination with WT- or Y227F-IL-15Rα constructs. Forty-eight hours after transfection, cells were stimulated with IL-15, lysed, immunoprecipitated with anti-Syk Abs, and analyzed for phosphorylation patterns.

As shown in Fig. 11,A, IL-15 stimulated the phosphorylation of several proteins that coprecipitate with Syk in J558 cells transfected with WT-IL-15Rα, while it failed to induce such effects in cells expressing Y227F mutants. For subsequent analysis of these proteins, membranes were stripped and reprobed with Abs against IL-15Rα (Fig. 11,B) and PLCγ1 (Fig. 11,C). Among these phosphorylated proteins anti-IL-15Rα Abs detected IL-15Rα migrating as 60- to 65-kDa protein, while anti-PLCγ1 Abs demarcated p120 PLCγ1. Anti-Syk Abs specifically detected Syk (p72) expressed in Syk-transfected clones (Fig. 11 D).

FIGURE 11.

Influence of Y227F IL-15Rα mutation on IL-15-induced phosphorylation of Syk substrates. J558 cells were transfected by electroporation with different constructs as indicated. Forty-eight hours after transfection cells were harvested and serum-starved for 3 h. Untreated cells and cells treated with 100 ng/ml IL-15 for 15 min were lysed in ODGP buffer and immunoprecipitated with anti-Syk Abs. A, Precipitates were analyzed in 10% SDS-PAGE for phosphorylation pattern using anti-p-Tyr Abs (position of phosphorylated proteins is indicated on the right). Membranes were stripped and reprobed with anti-IL-15Rα (B) or anti-PLCγ (C) as well as with anti-Syk (D) Abs for control of loading. Western blotting of lysates with anti-IL-15Rα Abs was performed to prove the efficacy of transfection (D, lower panel).

FIGURE 11.

Influence of Y227F IL-15Rα mutation on IL-15-induced phosphorylation of Syk substrates. J558 cells were transfected by electroporation with different constructs as indicated. Forty-eight hours after transfection cells were harvested and serum-starved for 3 h. Untreated cells and cells treated with 100 ng/ml IL-15 for 15 min were lysed in ODGP buffer and immunoprecipitated with anti-Syk Abs. A, Precipitates were analyzed in 10% SDS-PAGE for phosphorylation pattern using anti-p-Tyr Abs (position of phosphorylated proteins is indicated on the right). Membranes were stripped and reprobed with anti-IL-15Rα (B) or anti-PLCγ (C) as well as with anti-Syk (D) Abs for control of loading. Western blotting of lysates with anti-IL-15Rα Abs was performed to prove the efficacy of transfection (D, lower panel).

Close modal

Thus, Tyr227 in the intracellular portion of IL-15Rα is critical not only for the reported association of IL-15Rα with Syk, but also for mediating the effects of Syk on the phosphorylation of IL-15Rα and PLCγ1.

It has recently been shown that the region of Syk kinase that contains two SH2 homology domains is important for the interaction between Syk and the receptors, as in the case of EpoR and FcεRI (51, 58). To test the hypothesis that this region could also mediate the interaction of Syk with IL-15Rα, we precipitated proteins from IL-15-activated Raji lysates using Syk-GST fusion protein containing both the SH2 domains and we then analyzed them for IL-15Rα presence using specific Abs. Fig. 12 shows that Syk-GST from IL-15- but not from IL-2-activated cells binds the IL-15Rα. GST alone did not bind any proteins from IL-15-activated cell lysates. As positive control we used anti-IL-15Rα immunoprecipitates from total lysates of IL-15-stimulated cells.

FIGURE 12.

Activated IL-15Rα associates with Syk kinase via Syk SH2 domains. Raji cells were stimulated with IL-15 or IL-2, lysed, and precipitated with GST alone (lane 1), Syk (SyK-GST)-GST fusion protein (lanes 2–4), or anti-IL-15Rα Abs as positive control (lane 5). Precipitates were analyzed for IL-15Rα presence using anti-IL-15Rα Abs. Position of IL-15Rα is indicated on right.

FIGURE 12.

Activated IL-15Rα associates with Syk kinase via Syk SH2 domains. Raji cells were stimulated with IL-15 or IL-2, lysed, and precipitated with GST alone (lane 1), Syk (SyK-GST)-GST fusion protein (lanes 2–4), or anti-IL-15Rα Abs as positive control (lane 5). Precipitates were analyzed for IL-15Rα presence using anti-IL-15Rα Abs. Position of IL-15Rα is indicated on right.

Close modal

These data indicate that Syk can directly associate with activated IL-15Rα via a region containing the two SH2 domains.

This study shows that the IL-15Rα chain alone is capable of mediating a signal upon activation by IL-15 through selective association with Syk kinase in human B cells. Mutational analysis showed the importance of Tyr227, localized in the intracellular part of IL-15Rα, for Syk binding and for the phosphorylation of the IL-15Rα chain itself and of PLCγ. To the best of our knowledge, this study provides the first evidence that IL-15Rα can directly signal in lymphoid cells without the requirement of the IL-2Rβ and IL-2Rγ chains.

Recently, it was reported that Raji cells are resistant to apoptosis induction by dexamethasone, cycloheximide, actinomycin D, TNF-α, or anti-IgM (32, 40). However, C2-ceramide reportedly is capable of inducing a G0/G1 cell cycle arrest in these cells (32). In this study, we confirm that C2-ceramide induces a G0/G1 cell cycle arrest in Raji cells, but we also document that Raji cells are indeed sensitive to C2-ceramide-induced apoptosis, which is inhibited by IL-15. This fact is in agreement with the previously documented ability of IL-15 to rescue different lymphoid and nonlymphoid cells from apoptosis (3, 26, 39, 59, 60).

Despite several reports on the functional activity of IL-4Rα, IL-7Rα, IL-9Rα, and IL-15Rα subunits in lymphoid (61) and nonlymphoid cells (27, 29, 30), the molecular mechanisms of intracellular signaling via these receptor chains are still poorly understood. In this work, we demonstrate that upon IL-15 stimulation, Syk kinase is activated in Raji cells, binds to the intracellular part of IL-15Rα, and phosphorylates at least two substrates, PLCγ1 and IL-15Rα itself.

In LPS-activated peripheral B cells and Con A-activated T cells, IL-15 induces the association of Syk with IL-15Rα and with IL-2Rβ, while IL-2 induces an association of Syk only with IL-2Rβ. Preliminary data from our laboratory show that anti-IL-15Rα chain Abs are able to significantly diminish the IL-15-induced association of Syk with IL-15Rα and do not affect the association of Syk with IL-2Rβ (E. Bulanova and S. Bulfone-Paus, unpublished data). Although these data require further confirmation, IL-15-mediated signaling pathways in B cells expressing the IL-15Rα/IL-2Rβ receptor complex seem to require Syk association. Because the multiple associations between Syk and IL-15 receptor chains in activated B lymphoblasts designates these a rather complicated system to be studied, we chose as a working model the B lymphoblastoid cell line Raji, which does not express the β subunit of the receptor.

Considering that a similar Syk association as in Raji cells was also observed in other B lymphoblastoid cell lines (DG57 and Ramos) as well as in primary B and T lymphoblasts, this signal transduction scenario after IL-15Rα stimulation seems to reflect general principles of IL-15Rα chain-mediated signaling (62, 63). Other kinases usually activated in B lymphocytes (Lyn, Blk, Fyn) (42, 44, 64) were not found to be involved in signal transduction through IL-15Rα, at least in the three cell lines studied here.

The role of Syk kinase in apoptosis control is not yet clear, but there is evidence of an essential role for Syk in the activation of the antiapoptotic pathways that are stimulated through the IL-3/IL-5/GM-CSF receptor β subunit in human eosinophils (65). Recently stem-loop Syk antisense ODNs that eliminate Syk from monocytes and affect FcγRII-mediated signal transduction and phagocytosis were generated (33). Transfection of Raji cells with stem-loop Syk antisense ODNs abrogated the protective effect of IL-15 on C-2-ceramide-treated cells and affected the IL-15-mediated association between Syk and IL-15Rα and IL-15-induced phosphorylation of intracellular proteins.

Moreover, piceatannol, a Syk inhibitor, also is able to abolish the protective effect of IL-15 on C2-ceramide-induced apoptosis in Raji cells (data not shown). These results further support the hypothesis of the important role of Syk in preventing cells from undergoing apoptosis.

The fact that IL-15 fails to induce downstream signaling in the B lymphoblastoid cell line SKW 6.4, which expresses IL-15Rα and is Syk deficient (E. Bulanova and S. Bulfone-Paus, unpublished data), is consistent with the concept that Syk plays an important role in mediating IL-15Rα signaling. This is further supported by preliminary evidence from our laboratory that IL-15 is incapable of rescuing SKW 6.4 cells from apoptosis induced by anti-APO-1 Abs (E. Bulanova, and S. Bulfone-Paus, unpublished observation).

Unlike the src family of protein tyrosine kinases, Syk carries no N-terminal myristylation site but bears two src homology (SH2) domains capable of interacting with tyrosine-phosphorylated protein (66, 67) The usual way of Syk binding to intracellular parts of cellular receptors involves the immunoreceptor tyrosine-based activation motif (ITAM) domain of the receptor as well as two SH2 domains of the Syk molecule (58, 62). The ITAM is based on two repeated YXX(L/I) sequences separated by six to eight amino acids (68, 69). An example of such association is the binding of Syk to B cell Ag receptor and to FcεRI receptor (58, 62). Syk apparently can also associate with the phosphorylated intracellular part of the erythropoietin receptor, which does not contain an ITAM but has several tyrosine residues (51). Our data show that the IL-15Rα from Raji cells binds to Syk-GST fusion protein bearing two SH2 domains. The IL-15Rα also does not contain an ITAM but has one tyrosine residue (4); thus, only one of the two SH2 domains of Syk is capable of binding the intracellular part of IL-15Rα. However, it is still not clear whether the IL-15Rα binds IL-15 as a homodimer. Therefore, we are currently exploring the possibility of Syk/cytokine receptor association without the involvement of an ITAM domain, and we are performing experiments designed to establish the minimal region(s) of Syk sufficient for its binding to IL-15Rα.

The mechanism by which IL-15Rα recruits Syk is currently under investigation. Association of Syk kinase to transmembrane receptor molecules is preceded by the phosphorylation of ITAM sequences or Tyr residues contained in their intracellular tails (58, 62). Recently, several adaptor proteins (LAT, DAP12, etc.) which are involved in the association of Syk to immunoreceptors in NK cells have been identified (70, 71). We are currently investigating the involvement of such adaptor proteins in the IL-15Rα/Syk interaction. Because IL-15Rα contains a single Tyr residue, only one SH2 domain of Syk is supposed to bind the intracellular region of the IL-15Rα chain. Thus, further investigations are necessary to define which one of the SH2 domains might be responsible for this association. In addition, we are currently studying whether the binding of IL-15 to the receptor might induce the formation of homodimeric IL-15Rα complexes, binding, as a consequence, both SH2 domains.

In summary, our data indicate that the IL-15Rα chain is capable of functioning independently of other components of the IL-15R complex, and offer important advances in our understanding of IL-15Rα-mediated signaling events. In light of the importance of the IL-15Rα/Syk association for mediating lymphoid cell growth and preventing apoptosis in these cells, targeted mutations in IL-15Rα and Syk seems to be a valuable approach for selectively disrupting these interactions. Because both IL-15Rα- and Syk-deficient mice have recently been generated (8, 62), these mutants may be instructively used in future investigations to dissect which cell types use the IL-15Rα/Syk signaling pathways in which specific context and how IL-15 modulates B cell functions in vivo. Given the emerging role of IL-15 in the regulation of numerous physiological and pathological processes, including autoimmunity, chronic infections, and cancer (7, 19, 26, 28, 72), the development of pharmacological agents designed to disrupt IL-15Rα/Syk interactions offers a particularly attractive tool for the therapeutic inhibition of clinically undesired IL-15Rα-mediated signaling events.

We are grateful to Dr. Reuben Siraganian for generously providing rat Syk cDNA, to Dr. Ulrich Blank for the GST-HS1 and GST-Syk cDNA, and to Dr. Tiziana Musso for critical reading of the manuscript.

1

This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (to S.B.P.; SFB415/A10).

3

Abbreviations used in this paper: Jak, Janus kinase; ODN, oligonucleotide; PLCγ, phospholipase Cγ; ODGP, N-octyl-β-d-thioglucopyranoside; TPA, 12-O-tetradecanoyl phorbol-13-acetate; WT, wild type; ITAM, immunoreceptor tyrosine-based activation motif.

1
Armitage, R. G., B. M. Macduff, J. Eisenman, R. Paxton, K. H. Grabstein.
1995
. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation.
J. Immunol.
154
:
483
2
Warren, H. S., B. F. Kinnear, R. L. Kastelein, L. L. Lanier.
1996
. Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells.
J. Immunol.
156
:
3254
3
Bulfone-Paus, S., D. Ungureanu, T. Pohl, G. Lindner, R. Paus, R. Rückert, H. Krause, U. Kunzeldorf.
1997
. Interleukin-15 protects from lethal apoptosis in vivo.
Nat. Med.
3
:
1124
4
Anderson, D. M., S. Kumaki, M. Ahdieh, J. Bertles, M. Tometsko, A. Loomis, J. G. Giri, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, et al
1995
. Functional characterization of the human interleukin-15 receptor α chain and close linkage of IL15RA and IL2RA genes.
J. Biol. Chem.
270
:
29862
5
Grabstein, K. H., J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. A. Schoenborn, M. Ahdieh, et al
1994
. Cloning of a T cell factor that interacts with the β chain of the interleukin-2 receptor.
Science
264
:
965
6
Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton, D. M. Anderson, J. Eisenman, K. H. Grabstein, M. A. Caligiuri.
1994
. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of IL-2 receptor.
J. Exp. Med.
180
:
1395
7
Carson, W., M. A. Caligiuri.
1998
. Interleukin-15 as a potential regulator of the innate immune response.
Braz. J. Med. Biol. Res.
31
:
1
8
Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trentin, A. Ma.
1998
. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation.
Immunity
9
:
669
9
Wilkinson, P. C., F. Y. Liew.
1995
. Chemoattraction of human blood T lymphocytes by IL-15.
J. Exp. Med.
181
:
1255
10
Bamford, R., A. Grant, J. Burton, C. Peters, G. Kurys, C. Goldman, J. Brennan, E. Roessler, T. Waldmann.
1994
. The interleukin (IL) 2 receptor β chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.
Proc. Natl. Acad. Sci. USA
91
:
4940
11
Burton, J. D., R. N. Bamford, C. Peters, A. J. Grant, G. Kuris, C. K. Goldman, J. Brennan, E. Roessler, T. A. Waldmann.
1994
. A lymphokine, provisionally designated interleukin T and produced by T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.
Proc. Natl. Acad. Sci. USA
91
:
4935
12
Bulfone-Paus. S, H., R. Dürkop, H. Paus, T. Krause, T. Pohl, A. Onu.
1997
. Differential regulation of human T lymphoblast functions by IL-2 and IL-15.
Cytokine
9
:
507
13
Satoh, J., K. Kurohara, M. Yukitake, Y. Kuroda.
1998
. Interleukin-15, a T-cell growth factor, is expressed in human neural cell lines and tissues.
J. Neurol. Sci.
155
:
170
14
Musso, T., L. Calosso, M. Zucca, M. Millesimo, D. Ravarino, M. Giovarelli, F. Malavasi, A. Negro Ponzi, R. Paus, S. Bulfone-Paus.
1999
. Human monocytes constitutively express membrane-bound, biologically active, and interferon-γ-upregulated interleukin-15.
Blood.
93
:
3531
15
Kobayashi, H., J. A. Carrasquillo, C. H. Paik, T. A. Waldmann, Y. Tagaya.
2000
. Differences of biodistribution, pharmacokinetics and tumor targeting between interleukins 2 and 15.
Cancer Res.
60
:
3577
16
Doherty, T. M., R. A. Seder, A. Sher.
1996
. Induction and regulation of IL-15 expression in murine macrophages.
J. Immunol.
156
:
735
17
Krause, H., B. Jandrig, C. Wernicke, S. Bulfone-Paus, T. Pohl, T. Diamanstein.
1996
. Genomic structure and chromosomal localization of the human interleukin 15 gene (IL-15).
Cytokine
8
:
667
18
Onu, A., T. Pohl, H. Krause, S. Bulfone-Paus.
1997
. Regulation of IL-15 secretion via the leader peptide of two IL-15 isoforms.
J. Immunol.
158
:
255
19
Trentin, L., R. Zambello, M. Facco, R. Sancetta, C. Agostini, G. Semenzato.
1997
. Interleukin-15: a novel cytokine with regulatory properties on normal and neoplastic B lymphocytes.
Leukemia and Lymphoma
27
:
35
20
Giri, J. G., M. Ahdieh, J. Eisenman, K. Shanebeck, K. Grabstein, A. Namen, L. S. Park, D. Cosman, D. Anderson.
1994
. Utilization of β and γ chains of the IL-2 receptor by the novel cytokine IL-15.
EMBO J.
13
:
2822
21
Ruckert, R., K. Asadullah, M. Seifert, V. Budagian, R. Arnold, C. Trombotto, R. Paus, S. Bulfone-Paus.
2000
. Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?.
J. Immunol.
165
:
2240
22
Quinn, L. S., K. L. Haugk, K. H. Grabstein.
1995
. IL-15: a novel anabolic cytokine for skeletal muscle.
Endocrinology
136
:
3669
23
Leonard, W. J., M. Noguchi, S. M. Russel, O. W. McBride.
1994
. The molecular basis of X-linked severe combined immunodeficiency: the role of the interleukin-2 receptor γ chain, γc.
Immunol. Rev.
138
:
61
24
Sugamura, K., H. Asao, M. Kondo, N. Tanaka, N. Ishii, M. Nakamura, T. Takeshita.
1995
. The common γ-chain for multiple cytokine receptors.
Adv. Immunol.
59
:
225
25
Lehours, P., S. Racher, S. Dubois, J. Guo, A. Godard, Y. Jacques.
2000
. Subunit structure of the high and low affinity human interleukin-15 receptors.
Eur. Cytokine Network
11
:
207
26
Waldman, T., Y. Tagaya.
1999
. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular patogenes.
Annu. Rev. Immunol.
17
:
19
27
Johnston, J. A., C. M. Bacon, D. S. Finbloom, R. C. Rees, D. Kaplan, K. Shibuya, J. R. Ortaldo, S. Gupta, Y. Q. Chen, J. D. Giri, J. J. O’Shea.
1995
. Tyrosine phosphorylation and activation of STAT 5, STAT 3, and Janus kinases by interleukin 2 and 15.
Proc. Natl. Acad. Sci. USA
92
:
8705
28
Giri, J. G., D. M. Anderson, S. Kumaki, L. S. Park, K. H. Grabstein, D. Cosman.
1995
. IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2.
J. Leukocyte Biol.
57
:
763
29
Stevens, A. C., J. Matthews, P. Andres, V. Baffis, X. X. Zheng, D.-W. Chae, J. Smith, T. B. Strom, W. Maslinski.
1997
. Interleukin-15 signals T84 colonic epithelial cells in the absence of the interleukin-2 receptor β-chain.
Am. J. Physiol.
272
:
G1201
30
Bulfone-Paus, S., E. Bulanova, T. Pohl, V. Budagian, H. Dürkop, R. Rückert, U. Kunzendorf, U. Paus, . Krause.
1999
. Death deflected: IL-15 inhibits TNF-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15Rα chain.
FASEB. J.
13
:
1575
31
Keely, P. J., L. V. Parise.
1996
. The α2β1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cγ2 in platelets.
J. Biol. Chem.
271
:
26668
32
Kuroki, J., M. Hirokawa, A. Kitabayashi, M. Lee, T. Horiuchi, Y. Kawabata, A. B. Miura.
1996
. Cell-permeable ceramide inhibits the growth of B lymphoma Raji cells lacking TNF-α-receptors by inducing G0/G1 arrest but not apoptosis: a new model for dissecting cell-cycle arrest and apoptosis.
Leukemia
10
:
1950
33
Zhang, J., T. Kimura, R. P. Siraganian.
1998
. Mutation in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity.
J. Immunol.
161
:
4366
34
R. F. Selden. 1994. In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. C. Strober, eds. New York, 1994.
35
Matsuda, M., J.-G. Park, D.-C. Wang, S. Hunter, P. Chien, A. D. Schreiber.
1996
. Abrogation of the Fcγ receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides.
Mol. Biol. Cell
7
:
1095
36
Grynkiewicz, G., M. Poenie, R. Y. Tsien.
1985
. A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260
:
3440
37
Gazdar, A. F., D. N. Carney, P. A. Bunn, E. K. Russell, E. S. Jaffe, G. P. Schechter, J. G. Guccion.
1980
. Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas.
Blood
55
:
409
38
Angiolillo, A. L., H. Kanegane, C. Sgadari, G. H. Reaman, G. Tosato.
1997
. Interleukin-15 promotes angiogenesis in vivo.
Biochem. Biophys. Res. Commun.
233
:
231
39
Dooms, H., M. Desmedt, S. Vancaeneghem, P. Rottiers, V. Goossens, W. Fiers, J. Grooten.
1998
. Quiescence-inducing and antiapoptotic activities of IL-15 enhance secondary CD4+ T cell responsiveness to antigen.
J. Immunol.
161
:
2141
40
Kaptein, J. S., C.-K. E. Lin, C. L. Wang, T. T. Nguyen, C. I. Kalunta, E. Park, F.-S. Chen, P. M. Lad.
1996
. Anti-IgM-mediated regulation of c-myc and its possible relationship to apoptosis.
J. Biol. Chem.
271
:
18875
41
Obeid, L. M., C. M. Linardic, L. A. Karolak, Y. A. Hannun.
1993
. Programmed cell death induced by ceramide.
Science
259
:
1769
42
Bolen, J. B., R. B. Rowley, C. Spana, A. Y. Tsygankov.
1992
. The src family of tyrosine protein kinases in hemopoietic signal transduction.
FASEB J.
6
:
3403
43
Kolanus, W., C. Romeo, B. Seen.
1993
. T cell activation by clustered kinases.
Cell
74
:
171
44
Reth, M., J. Wienands.
1997
. Initiation and processing of signal from the B cell antigen receptor.
Annu. Rev. Immunol.
15
:
453
45
Baird, A. M., R. M. Gerstein, L. J. Berg.
1999
. The role of cytokine receptor signaling in lymphocyte development.
Curr. Opin. Immunol.
11
:
157
46
Ihle, J. N., W. Thierfelder, S. Teglund, D. Stravapodis, D. Wang, J. Feng, E. Parganas.
1998
. Signaling by the cytokine receptor superfamily.
Ann. NY Acad. Sci.
865
:
1
47
Hatakeyama, M., T. Kono, N. Kobayashi, A. Kawahara, S. D. Levin, R. M. Perlmutter, T. Taniguchi.
1991
. Interaction of the IL-2 receptor with the sck-family kinase p56lck: identification of novel intermolecular association.
Science
252
:
1523
48
Minami, Y., Y. Nkagawa, A. Kawahara, T. Miyazaki, K. Sada, H. Yamamura, T. Taniguchi.
1995
. Protein tyrosine kinase Syk is associated with and activated by IL-2 receptor: possible link with c-myc induction pathway.
Immunity
2
:
89
49
Baixeras, E., G. Kroemer, E. Cuende, C. Marquez, L. Bosca, J. E. Ales Martinez, C. Martinez.
1993
. Signal transduction pathways involved in B-cell induction.
Immunol. Rev.
132
:
5
50
Brunati, A. M., A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna.
1995
. Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by src homology 2 domains of the Src family.
FEBS Lett.
367
:
149
51
Duprez, V., U. Blank, S. Chretien, S. Gisselbrecht, P. Mayeux.
1998
. Physical and functional interaction between p72syk and erythropoietin receptor.
J. Biol. Chem.
273
:
33985
52
Law, C.-L., K. A. Chandran, S. P. Sidorenko, E. A. Clark.
1996
. Phospholipase C-γ1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.
Mol. Cell. Biol.
16
:
1305
53
Kurosaki, T., S. Tsukada.
2000
. BLNK: connecting Syk and Btk to calcium signals.
Immunity
12
:
1
54
Kang, J. S., F. Kohlhuber, H. Hug, D. Marme, D. Eick, M. Ueffing.
1996
. Cloning and functional analysis of hematopoietic cell-specific phospholipase Cγ2 promoter.
FEBS Lett.
399
:
14
55
Raeder, M. B., P. J. Mansfield, V. Hinkovska-Galcheva, J. Shayman, L. Boxer.
1999
. Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis.
J. Immunol.
163
:
6785
56
Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, R. L. Geahlen.
1994
. Inhibition of mast cell FcεR1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol.
J. Biol. Chem.
269
:
29697
57
Law, D. A., L. Nannizzi-Alaimo, K. Ministri, P. I. Hughes, J. Forsyth, M. Turner, S. J. Shattil, M. H. Ginsberg, V. L. J. Tybulewicz, D. R. Phillips.
1999
. Genetic and pharmacological analyses of Syk function in αIIbβ3 signaling in platelets.
Blood
93
:
2645
58
Shiue, L., J. Green, O. M. Green, J. L. Karas, J. P. Morgenstern, M. K. Ram, M. K. Taylor, M. J. Zoller, L. D. Zydowsky, J. B. Bolen, J. S. Brugge.
1995
. Interaction of p72syk with the γ and β subunits of the high-affinity receptor for immunoglobulin E, FcεRI.
Mol. Cell. Biol.
15
:
272
59
Hjorth-Hansen, H., A. Waage, M. Borset.
1999
. Interleukin-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells.
Br. J. Haematol.
106
:
28
60
Marks-Konczalik, J., S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldman, Y. Tagaya.
2000
. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice.
Proc. Natl. Acad. Sci. USA
97
:
11445
61
Higushi, M., H. Asao, N. Tanaka, K. Oda, T. Takeshita, M. Nakamura, J. Van Snick, K. Sugamura.
1996
. Dispensability of Jak1 tyrosine kinase for interleukin-2-induced cell growth signaling in a human T cell line.
Eur. J. Immunol.
26
:
1322
62
Turner, M., E. Schweighoffer, F. Colucci, J. P. Di Santo, V. L. Tubulewicz.
2000
. Tyrosine kinase Syk: essential functions for immunoreceptor signaling.
Immunol. Today
21
:
148
63
Chu, D. H., N. S. van Oers, M. Malissen, J. Harris, M. Elder, A. Weiss.
1999
. Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression.
J. Immunol.
163
:
2610
64
Tanigushi, T..
1995
. Cytokine signaling through nonreceptor protein tyrosine kinases.
Science
268
:
251
65
Yousefi, S., D. C. Hoessli, K. Blaser, G. B. Mills, H. U. Simon.
1996
. Requirement of Lyn and Syk tyrosine kinases for the prevention of apoptosis by cytokines in human eosinophils.
J. Exp. Med.
183
:
1407
66
Ghazizadeh, S., J. B. Bolen, H. D. Fleit.
1995
. Tyrosine phosphorylation and association of Syk with FcγRII in monocytic THP-1 cells.
Biochem. J.
305
: (Pt. 2):
669
67
Law, C.-L., S. P. Sidorenko, K. A. Chandran, K. E. Draves, A. Chan, A. Weiss, S. Edeloff, C. M. Disteche, E. A. Clark.
1994
. Molecular cloning of human Syk, a B cell protein-tyrosine kinase associated with the surface immunoglobulin M-receptor complex.
J. Biol. Chem.
269
:
12310
68
Reth, M..
1989
. Antigen receptor tail clue.
Nature
338
:
383
69
Chu, D. H., C. T. Morita, A. Weiss.
1998
. The Syk family of protein kinases in T-cell activation and development.
Immunol. Rev.
165
:
167
70
Jevremovic, D., D. D. Billadeau, R. A. Schoon, C. J. Dick, B. J. Irvin, W. Zhang, L. E. Samelson, R. T. Abraham, P. J. Leibson.
1999
. Cutting edge: a role for the adaptor protein LAT in human NK cell-mediated cytotoxicity.
J. Immunol.
162
:
2453
71
Lanier, L. L., A. B. Bakker.
2000
. The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function.
Immunol. Today
21
:
611
72
Di Carlo, E., A. Comes, S. Baso, A. De Ambrosis, R. Meazza, P. Musiani, K. Moelling, A. Albini, S. Ferrini.
2000
. The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement.
J. Immunol.
165
:
3111