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Chain Signals Through Association with Syk in Human B Cells1
*
Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany; Departments of
Urology and
Pathology, University Hospital Benjamin Franklin, Free University, Berlin, Germany; and
Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of the IL-15R (IL-15R) 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-15R-mediated signaling in activated primary
B cells and in Raji cells, a human B-lymphoblastoid cell line which
expresses the IL-15R and IL-2R
chains, but lacks the IL-2R
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-15R chain. Upon
association, the activated Syk kinase phosphorylates the IL-15R
chain as well as phospholipase C, which coprecipitates with Syk.
Furthermore, transfection of Raji cells with stem-loop Syk antisense
oligonucleotides prevents IL-15R and phospholipase C
phosphorylation as well as the inhibition of apoptosis by IL-15.
Mutation of a defined region of the intracellular signaling portion of
IL-15R (Tyr227) abrogates both the IL-15R/Syk
association and IL-15R phosphorylation. Taken together, this
suggests that Syk kinase physically and functionally associates with
the IL-15R 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-2R and IL-2R
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 -chain leads to
coprecipitation of Jak1 and Jak3 (27).
Each -chain of the IL-2R and IL-15R recognizes only its cognate
cytokine. IL-15R 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-15R 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-15R, like IL-2R, is incapable of
signaling when it is expressed in the absence of IL-2R or IL-2R
(4, 20, 23, 28). Instead, Jurkat cells which lack the
IL-15R chain can signal via the IL-2R and IL-2R 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-15R, but no IL-2R chain (29). Most
recently, we also noted that IL-15 signals through the IL-15R chain
in the murine fibrosarcoma cell line L929, which expresses the
-chain only at marginal levels and lacks the -chain component of
the IL-2R complex (30). This strongly suggests that,
contrary to conventional wisdom (4, 26, 28), the IL-15R
chain can transduce a signal even in the absence of the - and/or
-chains.
Therefore, it is important to understand how IL-15R may transduce an
intracellular signal in cells which lack expression of the IL-2R
and/or IL-2R chains. To investigate the role of IL-15R in
intracellular signaling we selected the B lymphoblastoid cell line Raji
as a model, because it expresses the IL-15R and IL-2R, but
lacks the IL-2R chain (4). To show the association of
intracellular proteins with the IL-15R 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-15R. Collectively, the data presented in
this work suggest that IL-15R signals via recruiting Syk kinase
which then phosphorylates IL-15R and phospholipase C
(PLC).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-2 was purchased from PeproTech (London, U.K.) and
IL-15 was purchased from Genzyme (Cambridge, MA). The mouse
anti-human IL-15R Ab (IgG1, clone M161) was generously provided
by Immunex (Seattle, WA). Rabbit anti-human Syk (N-19),
anti-IL-2R (N-20), anti-IL-2R (S-20), anti-Jak3
(C-21), anti-PLC1 (1249), anti--actin (H-196), and goat
anti-mouse IL-15R (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.
Cell culture and stimulation condition
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 x 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-15R 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.
Plasmid construction and cell transfection
Tyrosine at position Y227 of murine IL-15R 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-15R 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 x 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.
RT-PCR
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-15R sense,
5'-GCCAGCGCCACCCTCCACAGTAA-3'; IL-15R antisense,
5'-GCCAGCGGGGGAGTTTGCCTTGAC-3'; IL-2R sense,
5'-AAGCTCTGCCACTCGGAACACAAC-3'; IL-2R antisense,
5'-TGATCAGCAGGAAAACACAGC-3'; IL-2R sense,
5'-GAATTCCCTGGAGAGATGGCCACGGTCCCA-3', IL-2R antisense,
5'-GAATTCGAGGTTTGGAAATGGATGGACCAAGT3'; IL-2R sense,
5'-AGCCCCAGCCTACCAACCTCACT-3'; IL-2R antisense,
5'-TTAAAGCGGCTCCGAACACGAA-3'; -actin sense
5'-GTGGGGCGCCCCAGGCACCA-3'; -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 assay
Proliferation of Raji cells was assessed by [3H]thymidine incorporation. Cells (1 x 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).
Intracellular Ca2+ measurements
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
Cell cycle analysis was performed as described earlier (32), with minor modifications. Briefly, after treatment, 1 x 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.
Immunoprecipitation, Western blotting, and kinase reaction
Cell pellets were lysed for 15 min on ice in 1%
N-octyl--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
[-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
4x 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-15R Abs.
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2R chains but lack
IL-2R
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-15R as well as the -
and -chains (37, 38). RT-PCR was used for the detection
of the IL-15R, IL-2R, and IL-2R 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-15R subunits as well as the -chain transcript
but lack expression of IL-2R chain transcripts.
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(Fig. 1
B) and IL-2R (Fig. 1D),
and the absence of IL-2R protein (Fig. 1C), in
Raji cells. The B lymphoblastoid cell line Akata and the myeloid cell
line K562, which lack IL-15R or IL-2R chains, respectively
(4, 18), were used as negative controls. The T cell
leukemia line Jurkat, which expresses IL-2R (4), was
used as a positive control for this chain. Thus, Raji cells express the
IL-15R and IL-2R chains at both the gene and protein level, but
lack the IL-2R chain. As a consequence, we are confident to state
that IL-2R plays no role in IL-15-mediated signaling in these
cells. IL-15 induces modest proliferation of Raji cells and rescues them from C2-ceramide-induced apoptosis
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. 2A).
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we used anti-IL-15R or anti-IL-2R 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-15R or anti-IL-2R Abs,
and [3H]thymidine incorporation was measured.
Cells not treated with cytokines were used as a control. As shown in
Fig. 2B, while anti-IL-15R Abs significantly
inhibited the IL-15-induced proliferative response of Raji cells,
anti-IL-2R 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-15R specific,
because it is suppressed by anti-IL-15R Ab. This supports the
notion that IL-15 mediates its stimulatory effects in Raji cells via
the IL-15R.
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-,
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-15R (but not with
anti-IL-2R) 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-15R chain.
Syk physically associates with the IL-15R chain after IL-15
treatment
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
( or ) 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-15R or anti-IL-2R 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. 3A,
stimulation of Raji cells with IL-15 for 15 min induced coprecipitation
of Syk with the IL-15R, but not with the IL-2R chain (Fig. 3B). 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-2R Abs
did not precipitate Syk or any phosphorylated proteins (data not
shown). The presence of trace amounts of IL-2R in IL-15R
precipitates was excluded by probing with anti-IL-2R Abs (data
not shown). Lyn, Blk, and Fyn kinases did not associate with IL-15R
in ODGP lysates of IL-15-stimulated Raji cells (data not shown). Thus,
Syk specifically associates with the intracellular domain of the
IL-15R chain upon IL-15 treatment of Raji cells.
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and PLC1 via Syk kinase
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. 3C (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. 3C,
middle panel). Incubation of blots with anti-Syk Abs
confirmed that equal amounts of Syk were precipitated from lysates of
Raji cells (Fig. 3C, 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. 4A,
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-15R 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-15R, anti-Syk, and
anti-PLC1 Abs. This suggested that the 60- to 65-, 70- to 72-,
and 120-kDa proteins were indeed IL-15R, Syk, and PLC1,
respectively (Figs. 4B and 5C).
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1 and PLC2 (120 kDa) are
potential substrates for the Syk kinase (52, 53). PLC2
is the most abundant isoform in B cells. However, our data (not shown)
indicate that in Raji cells the level of expression of PLC2 is
substantially lower than that of the PLC1 isoform. These data are in
accordance with published observations by Kang et al.
(54), who showed a low expression level of PLC2 in Raji
cells. Thus, in Raji cells, PLC2 is likely of minor importance, and
the identification of PLC1 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-15R and PLC1 are actually tyrosine
phosphorylated after IL-15 administration, activated Raji cells were
lysed and immunoprecipitated with anti-IL-15R or
anti-PLC1 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 PLC1. Furthermore, after depletion of PLC1
from the IL-15-activated cell lysates, neither PLC1 nor Syk were
detectable anymore. The same results were obtained when IL-15R was
depleted from the lysates (data not shown).
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and PLC1,
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-15R (Fig. 5A) and PLC1 (Fig. 5C) in Raji cells.
The association between Syk and IL-15R as well as downstream
phosphorylation of IL-15R and PLC1 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-15R and PLC1, both molecules are phosphorylated by Syk,
and this phosphorylation is abrogated by piceatannol.
IL-15 induces Syk association with the IL-15R and IL-2R
chains in primary activated B and T cells
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-15R chain (1). As shown in Fig. 6A, 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-2R chain has previously been documented (45, 46).
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, but also IL-15R can recruit Syk,
we performed a series of immunoprecipitation and blocking experiments.
As shown in Fig. 6B (upper panel), IL-15
specifically induced association of Syk with IL-15R. Both IL-15 and
IL-2 are capable of inducing the association of Syk with the IL-2R
chain (Fig. 6C, upper panel). Neither IL-15 nor
IL-2 induced coprecipitation of Syk with IL-2R (Fig. 6D).
Preincubation of cells with IL-15R-blocking Abs significantly
decreased the amount of Syk associated with IL-15R after IL-15
treatment, while the association of Syk kinase with IL-2R was not
affected (data not shown). Furthermore, IL-15R/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-15R and IL-2R subunits. Stem-loop Syk antisense ODNs inhibit IL-15-induced protection from apoptosis and signaling in Raji cells
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. 7A).
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 -actin mRNA level (Fig. 7A).
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B). Stem-loop Syk antisense
ODNs had no effect on IL-15R expression (Fig. 7B). 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. 7C). 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.
Stem-loop Syk antisense ODNs abrogate IL-15-mediated Syk/IL-15R
association and phosphorylation of intracellular protein
We analyzed the IL-15-mediated association of Syk with IL-15R
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-15R Abs and analyzed for Syk association by Western
blotting (Fig. 8A, upper
panel). Whereas control ODNs had no effect, stem-loop Syk
antisense ODNs abrogated the IL-15-induced association of Syk kinase
with IL-15R.
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or anti-PLC1 Abs, and precipitated proteins were analyzed for
patterns of tyrosine phosphorylation. As shown in Fig. 8, IL-15 induced
IL-15R (Fig. 8A, middle panel) as well as
anti-PLC1 (Fig. 8B, 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-15R Abs (Fig. 8A, lower panel) or
with anti-PLC1 (Fig. 8B, 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-15R
and PLC is dependent on Syk kinase.
Stem-loop Syk antisense ODNs block IL-15-mediated Ca2+ influx in Raji cells
The involvement of PLC 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.
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IL-15R mutation abrogates both the association with Syk and the
phosphorylation of IL-15R and PLC
The intracellular domain of murine and human IL-15R 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-15R as a
result of IL-15 stimulation, we wished to evaluate the requirement of
this tyrosine residue for IL-15R-Syk binding and for IL-15-induced
phosphorylation of Syk substrates. For this purpose, constructs of
murine IL-15R 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-15R constructs in combination with Syk-expressing constructs
using the DEAE-dextran method. Lysates from transfected COS cells were
immunoprecipitated with anti-IL-15R Abs and blotted with
anti-Syk Abs. As seen in Fig. 10, Syk selectively precipitated with WT -IL-15R and failed to associate
with Y227F-IL-15R mutant.
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and PLC. For these experiments,
IL-15R- and Syk-negative murine plasmocytoma J558 cells were
transiently transfected with Syk in combination with WT- or
Y227F-IL-15R 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. 11A, IL-15
stimulated the phosphorylation of several proteins that coprecipitate
with Syk in J558 cells transfected with WT-IL-15R, 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-15R (Fig. 11B) and PLC1
(Fig. 11C). Among these phosphorylated proteins
anti-IL-15R Abs detected IL-15R migrating as 60- to 65-kDa
protein, while anti-PLC1 Abs demarcated p120 PLC1. Anti-Syk
Abs specifically detected Syk (p72) expressed in Syk-transfected clones
(Fig. 11D).
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is critical not only for the reported association of
IL-15R with Syk, but also for mediating the effects of Syk on the
phosphorylation of IL-15R and PLC1.
IL-15R associates with Syk kinase via Syk SH2 domains
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 Fc
RI
(51, 58). To test the hypothesis that this region could
also mediate the interaction of Syk with IL-15R, 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-15R
presence using specific Abs. Fig. 12
shows that Syk-GST from IL-15- but not from IL-2-activated cells binds
the IL-15R. GST alone did not bind any proteins from IL-15-activated
cell lysates. As positive control we used anti-IL-15R
immunoprecipitates from total lysates of IL-15-stimulated cells.
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via a region containing the two SH2 domains. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-15R, for Syk binding and for the
phosphorylation of the IL-15R chain itself and of PLC. To the
best of our knowledge, this study provides the first evidence that
IL-15R can directly signal in lymphoid cells without the requirement
of the IL-2R and IL-2R chains.
Recently, it was reported that Raji cells are resistant to apoptosis
induction by dexamethasone, cycloheximide, actinomycin D, TNF-, 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-4R,
IL-7R, IL-9R, and IL-15R 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-15R, and phosphorylates at least two
substrates, PLC1 and IL-15R itself.
In LPS-activated peripheral B cells and Con A-activated T cells, IL-15
induces the association of Syk with IL-15R and with IL-2R, while
IL-2 induces an association of Syk only with IL-2R. Preliminary data
from our laboratory show that anti-IL-15R chain Abs are able to
significantly diminish the IL-15-induced association of Syk with
IL-15R and do not affect the association of Syk with IL-2R (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-15R/IL-2R 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
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-15R stimulation seems to reflect general principles of
IL-15R 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-15R, 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 subunit in human eosinophils (65). Recently
stem-loop Syk antisense ODNs that eliminate Syk from monocytes and
affect FcRII-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-15R 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-15R 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-15R 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 FcRI 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-15R from Raji cells
binds to Syk-GST fusion protein bearing two SH2 domains. The IL-15R
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-15R. However, it is
still not clear whether the IL-15R 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-15R.
The mechanism by which IL-15R 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-15R/Syk
interaction. Because IL-15R contains a single Tyr residue, only one
SH2 domain of Syk is supposed to bind the intracellular region of the
IL-15R 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-15R
complexes, binding, as a consequence, both SH2 domains.
In summary, our data indicate that the IL-15R chain is capable of
functioning independently of other components of the IL-15R complex,
and offer important advances in our understanding of IL-15R-mediated
signaling events. In light of the importance of the IL-15R/Syk
association for mediating lymphoid cell growth and preventing apoptosis
in these cells, targeted mutations in IL-15R and Syk seems to be a
valuable approach for selectively disrupting these interactions.
Because both IL-15R- 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-15R/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-15R/Syk interactions offers a
particularly attractive tool for the therapeutic inhibition of
clinically undesired IL-15R-mediated signaling events.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Silvia Bulfone-Paus, Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail address: sbulfone{at}fz-borstel.de
3 Abbreviations used in this paper: Jak, Janus kinase; ODN, oligonucleotide; PLC, phospholipase C; ODGP, N-octyl--D-thioglucopyranoside; TPA, 12-O-tetradecanoyl phorbol-13-acetate; WT, wild type; ITAM, immunoreceptor tyrosine-based activation motif.
Received for publication January 19, 2001. Accepted for publication October 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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