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Receptor I-Associated lyn-Dependent Phosphorylation of Fc
Receptor IIB During Negative Regulation of Mast Cell Activation1
*
Laboratoire d’Immunologie Cellulaire et Clinique, INSERM U.255, Institut Curie, Paris, France;
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206; and
Division of Cellular Immunology, National Institute for Medical Research, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB are low-affinity receptors for IgG whose intracytoplasmic
domain contains an immunoreceptor tyrosine-based inhibition motif
(ITIM). FcRIIB inhibit cell activation triggered by receptors that
signal via immunoreceptor tyrosine-based activation motifs. This
inhibition requires ITIM tyrosyl phosphorylation and is correlated with
the binding of SH2 domain-containing phosphatases that may mediate the
inhibitory signal. In the present work, we investigated the mechanism
of FcRIIB phosphorylation and its consequences in mast cells. We
demonstrate that the phosphorylation of FcRIIB requires
coaggregation with Fc
RI and that, once phosphorylated, FcRIIB
selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in
vivo. In vitro, however, the phosphorylated FcRIIB ITIM binds not
only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and
SHP-2. We show that the coaggregation of FcRIIB with FcRI does
not prevent FcRI-mediated activation of lyn and
syk. Both kinases can phosphorylate FcRIIB in vitro.
However, when coaggregated with FcRI, FcRIIB was in vivo
phosphorylated in syk-deficient mast cells, but not in
lyn-deficient mast cells. When FcRI are coaggregated
with FcRIIB by immune complexes, FcRI-associated lyn
may thus phosphorylate FcRIIB. By this mechanism, FcRI initiate
ITIM-dependent inhibition of intracellular propagation of their own
signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB, have long been
known to inhibit B cell activation when coaggregated with
BCR3 by immune complexes
(1, 2). FcRIIB are single-chain low-affinity IgG receptors (3, 4, 5)
encoded by a single gene in which a separate exon encodes the
transmembrane domain and three other exons the intracytoplasmic (IC)
domain (6). This enables three membrane isoforms to be generated by
alternative splicing of corresponding sequences. FcRIIB1 retain
sequences encoded by all four exons; FcRIIB2 lack sequences encoded
by the first IC exon; FcRIIB1' are generated by the use of a cryptic
splice donor site located in the murine first IC exon (7). Mouse B
cells express FcRIIB1 (8) and FcRIIB1' (7). A 13-amino acid IC
sequence containing a tyrosine residue, followed by a leucine at
position +3, was found to be necessary (9) and sufficient (10) for
inhibition. This sequence, present in all three murine FcRIIB
isoforms (7, 9), is conserved in the two human FcRIIB isoforms,
FcRIIB1 and FcRIIB2, and both human isoforms were reported to
inhibit mouse (11) and human (12) B cell activation. FcRIIB were
subsequently shown to negatively regulate not only BCR-mediated B cell
activation, but also TCR-mediated T cell activation (13) and
high-affinity IgE receptor (FcRI)-mediated mast cell activation
(14). FcRIIB also inhibited cell activation by chimeric molecules
whose IC domain consisted of that of the Ig
(10), TCR-
, or FcR
(13) transduction subunits, providing evidence that FcRIIB functions
as a general negative coreceptor for all receptors containing an
immunoreceptor tyrosine-based activation motif (ITAM). The same
sequence was found to account for the regulatory effects of FcRIIB
on cell activation by the three ITAM-containing immunoreceptors, and
mutation of the tyrosine residue was sufficient to abrogate
inhibition (10, 13). Because the YxxL motif present in this sequence was reminiscent of the double YxxL motif constitutive of ITAMs, this sequence was referred to as an immunoreceptor tyrosine-based inhibition motif (ITIM), and occurrence of ITIMs was searched for in other negative coreceptors. ITIMs were found in the IC domain of human (15) and murine (16) killer cell inhibitory receptors (KIRs), which inhibit the cytotoxic activity of NK cells when they bind to MHC class I molecules on target cells (17); in the IC domain of CD22 (18) and of CTLA-4 (19), expressed on B and T cells, respectively, and which inhibit B cell (20) and T cell activation (21); in the IC domain of gp49B1 (22) and MAFA (23), expressed on mast cells, and which inhibit IgE-induced mediator release (24, 25); and in the IC domain of ILT3, expressed on monocytes and dendritic cells, and which can inhibit the activation of these cells (26).
One general property of ITIMs is their tyrosine phosphorylation
and recruitment of SH2 domain-bearing phosphatases. FcRIIB was shown
to be phosphorylated both in B cells (10) and in mast cells (27, 28).
FcRIIB was reported to recruit the tyrosine phosphatase SHP-1 in B
cells (29), and the inositol phosphatase SHIP in both B cells (27, 30, 31) and mast cells (27, 28). In the present work, we devised an
experimental model to study the mechanism of inhibition of mast cell
activation by FcRIIB. We show that the coaggregation of FcRI with
FcRIIB not only does not inhibit the activation of protein tyrosine
kinases (PTK) associated with FcRI, but enables one of these
kinases, lyn, to phosphorylate FcRIIB ITIM.
Phosphorylated FcRIIB then selectively recruit SHIP. We propose a
model according to which FcRI contribute to the extinction of the
intracellular propagation of their own signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RBL-2H3 cells (32) were cultured in DMEM supplemented with 10%
FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Only adherent
cells, recovered with trypsin-EDTA, were used. All culture reagents
were from Life Technologies (Paisley, Scotland, U.K.). Previously
described cDNAs encoding murine FcRIIB1, FcRIIB1', FcRIIB2,
and FcRIIB(IC1) (7, 33) were inserted into an expression vector
under the control of the SR promoter in pBR322 (34) and in which a
resistance gene to zeocin was introduced (NT-zeo). Transfectants were
selected by culture with 500 µg/ml of zeocin (Cayla, Toulouse,
France). RBL transfectants recovered after selection were cloned with
steel cylinders, as described (33). The expression of recombinant
receptors by cloned cells was assessed by indirect immunofluorescence.
The expression of recombinant receptors on clones remained stable over
the duration of experiments. Several clones of each transfectant
expressing comparable amounts of recombinant receptors were selected
and used for experiments. They gave similar results. Transfectants
expressing FcRIIB2(Y26G) were previously described (35).
Mast cells were derived by culturing fetal liver cells from wild-type
and syk-deficient 16.5-day embryos in culture medium
supplemented with 10% WEHI 3B-conditioned medium, as previously
described (36). Bone marrow-derived mast cells (BMMC) were obtained by
culturing bone marrow cells from wild-type and lyn-deficient
mice under the same conditions. After 4 wk, the cultures were found to
contain more than 90% mast cells, as assessed by microscopic
examination. Based on indirect immunofluorescence, all cells expressed
FcRI and FcRIIB, and the deficient cells expressed comparable
levels of both receptors as wild-type counterpart cells.
Abs and immunoadsorbents
The mouse IgE mAb 2682-I was used as culture supernatant of a
subclone of DNP-H1--26 hybridoma cells (37). The rat anti-mouse
FcRIIB 2.4G2 mAb (38) was purified by affinity chromatography on
protein G-Sepharose (Pharmacia, Uppsala, Sweden) from ascitic fluid of
nude mice inoculated with 2.4G2 hybridoma cells i.p.
F(ab')2 fragments were obtained by pepsin digestion
for 48 h. The purity of IgG and F(ab')2 fragments was
assessed by SDS-PAGE analysis. F(ab')2 fragments of
polyclonal mouse anti-rat Ig (MAR), and F(ab')2
fragments and intact IgG of polyclonal rabbit anti-mouse Ig (RAM)
were purchased from Jackson ImmunoResearch Laboratories (West Grove,
PA). MAR F(ab')2 were trinitrophenylated by incubation for
2 h at room temperature with trinitrobenzene sulfonic acid
(Eastman Kodak, Rochester, NY) in borate-buffered saline, pH 8.
TNP10-MAR F(ab')2 obtained were purified on
Sephadex G25 (Pharmacia). Rabbit Abs against recombinant extracellular
domains of mouse FcRIIB (39) were gifts from Dr. Catherine
Sautès (Institut Curie, Paris, France). Horseradish
peroxidase-conjugated anti-phosphotyrosine (PY) mouse mAbs PY-20
were purchased from Chemicon (Temecula, CA). Mouse monoclonal
anti-SHP-1 and anti-SHP-2 Abs were purchased from Transduction
Laboratories (Lexington, KY). Rabbit anti-SHIP Abs were a gift from
Dr. Gerald Krystal (University of British Columbia, Vancouver, Canada).
Rabbit Abs against FcR were a gift from Dr. Jean-Pierre Kinet
(Harvard Medical School, Boston, MA). Rabbit anti-lyn
Abs used for immunoprecipitation were purchased from Upstate
Biotechnology (Lake Placid, NY), and mouse anti-lyn Abs
used for Western blotting were purchased from Transduction
Laboratories. Rabbit anti-syk Abs used for
immunoprecipitation were gifts from Dr. Ulrich Blank (Institut Pasteur,
Paris, France), and rabbit anti-syk Abs used for Western
blotting were purchased from Santa Cruz Biotechnologies (Santa Cruz,
CA). Horseradish peroxidase-conjugated polyclonal goat anti-mouse
(GAM) Ig Abs and polyclonal goat anti-rabbit (GAR) Ig Abs were
purchased from Santa Cruz Biotechnologies.
Protein G-Sepharose (50 µl beads diluted 1/2) was used to precipitate
2.4G2-bound FcRIIB. Synthetic nonphosphorylated or
tyrosine-phosphorylated peptides with the FcRIIB ITIM sequence
EAENTIT(p)YSLLKH were purchased from Macromolecular Resources (Fort
Collins, CO). They were coupled to CNBr-activated Sepharose beads
(Pharmacia) at 1 mg/ml. Anti-FcR, anti-lyn, and
anti-syk Abs were adsorbed onto protein A-Sepharose
(Sigma Chemical Co., St. Louis, MO) (5 µl serum or 5 µg purified
Abs/50 µl beads diluted 1/2) by an overnight incubation at 4°C.
Washed beads were used as immunoadsorbents to precipitate FcRI
complexes, lyn or syk.
GST fusion proteins
cDNA encoding the IC domain of FcRIIB1' (ICIIB1') was
generated by PCR and inserted in the pGEX-4T-2 vertor (Pharmacia). The
sequence was determined on the two strands by dideoxynucleotide
sequencing. GST-ICIIB1' was produced in DH5- bacteria by
isopropylthiogalactopyranoside induction, purified on glutathione
agarose (Sigma Chemical Co.), and analyzed by SDS-PAGE. Soluble
GST-ICIIB1' was obtained by incubating the beads with 50 mM Tris, 25 mM
glutathione, pH 8. GST-HS1 was a gift from Dr. Ulrich Blank. GST-p62
(amino acids 331–443 of the mouse GTPase-activating
protein-associated p62) was purchased from Santa Cruz
Biotechnologies.
Serotonin release and assay
Transfected RBL cells, resuspended in RPMI medium supplemented with 10% FCS (RPMI-FCS) at 1 x 106 cells/ml, were incubated at 37°C for 1 h with 2 µCi/ml [3H]serotonin (Amersham, Les Ulis, France), washed, resuspended in RPMI-FCS, incubated for another hour at 37°C to remove excess [3H]serotonin, washed again, resuspended in the same medium, distributed in 96-well microculture plates at 2 x 105 cells/well, and incubated for 1 h at 37°C with IgE and 2.4G2 F(ab')2 or not in a final volume of 50 µl. Adherent cells were washed four times with 200 µl HBSS; 25 µl culture medium was added to each well and cells were warmed at 37°C for 15 min before challenge. Cells were challenged for 30 min at 37°C with 25 µl TNP-MAR F(ab')2 or RAM F(ab')2 or intact RAM IgG, previously warmed at 37°C for 15 min. Reactions were stopped by adding 50 µl ice-cold medium and by placing plates on ice. Fifty microliters of supernatants were mixed with two hundred microliters of Aqualuma-Plus scintillation fluid (Lumac, The Netherlands) and counted in a beta plate counter (Pharmacia). The percentage of [3H]serotonin release was calculated, using as 100% cpm in 50 µl harvested from wells containing the same number of cells that were lysed in 100 µl of 0.5% SDS and 0.5% Nonidet P-40.
Measurement of intracellular Ca2+ concentration
Transfected RBL cells were resuspended at 1 x 106 cells/ml in IMDM containing 5% FBS, 5 µM Indo-1 AM (Molecular Probes, Eugene, OR), and IgE, incubated for 1 h at 37°C, and washed twice. Cells were resuspended in IMDM containing 5% FBS and stimulated at 1 x 106 cells/ml/sample with either 10 µg/ml RAM F(ab')2 or 15 µg/ml intact RAM IgG. Intracellular Ca2+ was monitored for 15 min using a flow cytometer (model 50H; Ortho Diagnostic Systems, Raritan, NJ). The percentage of cells responding was calculated using the data acquisition system ACQ-CYTE and MTIME software (Phoenix Flow Systems, Phoenix, AZ).
[35S]methionine labeling
RBL cells were washed twice in methionine-free DMEM (Sigma Chemical Co.), resuspended at 1 x 106 cells/ml in methionine-free DMEM supplemented with 5% dialyzed FBS, and incubated for 4 h with 0.5 mCi/sample of [35S]methionine (DuPont NEN, Wilmington, DE) at 37°C. Cells were washed twice in PBS and lysed, as described below.
Immunoprecipitation and Western blot analysis
Transfected RBL cells, resuspended at 5 x
106/ml, were incubated for 1 h at 37°C with IgE
anti-DNP (culture supernatant diluted 1/10) and 2.4G2
F(ab')2 (3 µg/ml) in complete culture medium, washed
three times, resuspended in the same medium at 107
cells/ml, and challenged for 2 min or for the indicated periods of time
at 37°C with 10 µg/ml or indicated concentrations of TNP-MAR
F(ab')2. They were centrifuged, and pellets containing
107 cells for FcRIIB immunoprecipitation or 3 x
107 cells for FcRI immunoprecipitation were lysed for 10
min at 0°C at 2 x 108 cells/ml in lysis buffer
containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM
Na3VO4, 5 mM NaF, 5 mM sodium pyrophosphate,
0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, and 1 mM PMSF. Lysates were centrifuged at 10,000 rpm for 10
min at 4°C, and supernatants were incubated with protein G-Sepharose
for 30 min at 4°C or with anti-FcR-bound protein A-Sepharose
for 1 h at 4°C. Immunoadsorbents were washed three times in 1 ml
lysis buffer and boiled for 3 min in reducing sample buffer. Eluted
material was submitted to SDS-PAGE and transferred onto Immobilon-P
membranes (Millipore, Bedford, MA). Membranes were saturated with
either 5% BSA or 5% skimmed milk (Régilait,
Saint-Martin-Belle-Roche, France) diluted in a buffer containing 10 mM
Tris, 150 mM NaCl, pH 7.4, and 0.5% Tween-20 (Merk, Schuchardt,
Germany) (Western buffer), and blotted with appropriate dilutions of
either horseradish peroxidase-conjugated anti-PY Abs or
anti-FcRIIB, anti-SHP-1, anti-SHP-2, and anti-SHIP
Abs, followed by horseradish peroxidase-conjugated GAR or GAM IgG.
Peroxidase-labeled Abs were detected using the Amersham enhanced
chemoluminescence kit.
Immunoprecipitation and in vitro kinase assay
RBL cells or transfectants sensitized with IgE anti-DNP and
2.4G2 F(ab')2 were washed three times, resuspended at
107 cells/ml, and challenged for 2 min at 37°C with 10
µg/ml TNP-MAR F(ab')2 or 1 µg/ml DNP-BSA. Pellets
containing 107 cells for anti-lyn and
anti-syk immunoprecipitations or 3 x
107 cells for anti FcR immunoprecipitation were lysed
for 10 min at 0°C at 2 x 108 cells/ml in TNE buffer
containing 50 mM Tris, pH 8, 1% Nonidet P-40, 20 mM EDTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM
Na3VO4. Lysates were centrifuged at 10,000 rpm
for 10 min at 4°C, and incubated for 1 h at 4°C with protein
A-Sepharose coated with the indicated Abs. Immunoadsorbents were washed
three times for anti-FcR immunoprecipitation or seven times for
anti-lyn and anti-syk
immunoprecipitations in 1 ml TNE buffer and once in kinase buffer
containing 30 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM
MnCl2, and 0.1 mM Na3VO4.
Immunoadsorbents were incubated in 40 µl reaction buffer containing 1
µM ATP, 10 µCi [-32P]ATP or not, and 1 µg of the
indicated GST fusion proteins for 3 min at 30°C. Reactions were
stopped by boiling for 3 min with 20 µl 3x concentrated reducing
sample buffer. Eluted material was subjected to SDS-PAGE and either
stained with Coomassie blue and autoradiographed or transferred onto
Immobilon-P membranes. Membranes were saturated with 5% BSA diluted in
Western buffer and blotted with an appropriate dilution of horseradish
peroxidase-conjugated anti-PY Abs. In these experiments, the
controls for immunoprecipitation were treated in the same conditions,
but were not submitted to in vitro kinase assay (IVKA). Eluted material
was run on SDS-PAGE, transferred onto Immobilon-P. Membranes were
saturated with 5% skimmed milk diluted in Western buffer and blotted
with the appropriate dilutions of anti-lyn or
anti-syk Abs, followed by horseradish
peroxidase-conjugated GAM or GAR IgG.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB with FcRI inhibits IgE-dependent
mast cell activation
To analyze the mechanism of FcRIIB-mediated inhibition in mast
cells, we used the rat RBL-2H3 mastocytoma cells, which express
endogenous FcRI and which we stably transfected with cDNA encoding
wild-type mouse FcRIIB of the three isoforms or FcRIIB without IC
domain.
In a first set of experiments, transfectants expressing FcRIIB were
sensitized with mouse IgE and challenged with F(ab')2
fragments of polyclonal RAM Ig Abs to aggregate FcRI, or with intact
RAM IgG to coaggregate FcRI with FcRIIB. As expected, RAM
F(ab')2 induced an IgE dose-dependent serotonin release.
Serotonin release of a lower magnitude was observed when cells were
challenged with RAM IgG (Fig. 1
A). Likewise, RAM
F(ab')2 induced a rapid increase of intracellular
Ca2+ concentration that persisted for several minutes. RAM
IgG induced a rapid increase of intracellular Ca2+ with a
similar magnitude, but which declined sharply within a few seconds. By
contrast, the same sustained Ca2+ response was observed
when RBL transfectants expressing FcRIIB whose IC domain was deleted
(FcRIIB(IC1)) were challenged with RAM F(ab')2 or IgG.
The sustained Ca2+ response was restored in
FcRIIB1-expressing transfectants sensitized with mouse IgE, if the
IgG binding site of FcRIIB1 was blocked with 2.4G2 before challenge
with RAM IgG (Fig. 1B). When coaggregated with
FcRI, FcRIIB can therefore inhibit IgE-induced serotonin release
and extracellular Ca2+ influx.
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RIIB1 were
sensitized with murine IgE anti-DNP and incubated or not with 2.4G2
F(ab')2 before they were challenged with TNP-conjugated
F(ab')2 fragments of polyclonal mouse anti-rat Ig Abs
(TNP-MAR F(ab')2). This reagent was recognized as a
multivalent Ag by FcRI-bound mouse IgE anti-DNP and recognized
FcRIIB-bound 2.4G2 F(ab')2. It therefore aggregated
FcRI in cells sensitized with IgE, it aggregated FcRIIB in cells
incubated with 2.4G2 F(ab')2, and it coaggregated FcRI
with FcRIIB in cells sensitized with IgE and incubated with 2.4G2
F(ab')2. Unless otherwise specified, these conditions were
used in all experiments and, whenever possible, they are referred to as
aggregation of FcRI, aggregation of FcRIIB, and coaggregation of
FcRI with FcRIIB, respectively. We first assessed the kinetics of
FcRIIB-dependent inhibition of mast cell activation. Serotonin
release in response to FcRI aggregation was complete after 10 min of
stimulation. The coaggregation of FcRIIB1 with FcRI induced an
inhibition of serotonin release that was visible as soon as serotonin
was detectable and that persisted over a 30-min stimulation period
(Fig. 2A). The
coaggregation of FcRIIB with FcRI, therefore, does not affect the
kinetics of mediator release, but induces an early and long-lasting
inhibition of mast cell activation. We next compared the inhibition
induced by the three isoforms of murine FcRIIB, FcRIIB1,
FcRIIB1', or FcRIIB2, expressed in RBL cells. Serotonin release
induced by FcRI aggregation was inhibited when FcRI were
coaggregated with FcRIIB1, with FcRIIB1', or with FcRIIB2
(Fig. 2B). All three isoforms of FcRIIB can
therefore inhibit IgE-mediated mast cell activation.
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RIIB with FcRI induces the
phosphorylation of FcRIIB ITIM, enabling the selective recruitment
of SHIP by phosphorylated FcRIIB
FcRIIB were found previously to become tyrosine phosphorylated
when coaggregated with BCR in B cells (10, 29) or with FcRI in
BMMC (27, 28). To determine conditions required for FcRIIB
phosphorylation, transfectants expressing FcRIIB1 were incubated
with 2.4G2 F(ab')2 and sensitized or not with IgE
anti-DNP before they were challenged with TNP-MAR
F(ab')2 for various periods of time. FcRIIB were
immunoprecipitated and analyzed by Western blotting with anti-PY
and anti-FcRIIB Abs. The weak phosphorylation of resting (data
not shown) or aggregated FcRIIB1 increased dramatically upon
coaggregation with FcRI (Fig. 3A). The increase of
FcRIIB1 phosphorylation was maximal at 15 s and persisted for
several minutes. To identify FcRIIB tyrosine residues that become
phosphorylated upon coaggregation with FcRI, we used RBL
transfectants expressing FcRIIB2 bearing a point mutation of the
ITIM tyrosine. FcRIIB1 IC domain contains four tyrosine residues,
and FcRIIB2 IC domain only two. Like FcRIIB1, FcRIIB2 became
strongly tyrosine phosphorylated upon coaggregation with FcRI. There
was, however, no detectable tyrosine phosphorylation in FcRIIB2
whose tyrosine residue, in the ITIM (Y26), was replaced by a glycine
(Fig. 3B). When coaggregated with FcRI, FcRIIB
are therefore rapidly phosphorylated, and phosphorylation is abolished
when the ITIM tyrosine is mutated.
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RIIB were shown previously to bind to
the SH2 domain-containing protein tyrosine phosphatases SHP-1 and
SHP-2, and to the inositol polyphosphate 5-phosphatase SHIP. These
three phosphatases, however, were differentially recruited in B cells
(29, 30) and in BMMC (28), as judged by their ability to coprecipitate
with phosphorylated FcRIIB. To determine whether phospho-ITIM could
bind to SHP-1, SHP-2, SHIP, and/or to other SH2-containing molecules in
RBL cells, synthetic peptides consisting of the ITIM sequence,
phosphorylated or not on the tyrosine residue and coupled to Sepharose
beads, were used to precipitate lysates from
[35S]methionine-labeled RBL cells. As detected by
autoradiography, no material was precipitated by nonphosphorylated ITIM
peptides. Phosphorylated ITIM peptides precipitated three major
proteins, whose apparent m.w. were 150, 69, and 66 kDa. These proteins
were identified, by Western blot, as the various isoforms of SHIP,
SHP-2, and SHP-1, respectively (Fig. 4A).
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RIIB, after coaggregation with FcRI.
Transfectants expressing FcRIIB1 were incubated with or without
2.4G2 F(ab')2 and sensitized, or not, with mouse IgE
anti-DNP, before they were challenged with TNP-MAR
F(ab')2 for 2 min. FcRIIB immunoprecipitates were
analyzed by Western blotting with anti-FcRIIB, anti-PY,
anti-SHP-1, anti-SHP-2, and anti-SHIP Abs. FcRIIB1
was not phosphorylated when aggregated, but became strongly tyrosine
phosphorylated when coaggregated with FcRI. Neither SHP-1 nor SHP-2
was coprecipitated with phosphorylated FcRIIB1. However, SHIP was
coprecipitated with phosphorylated FcRIIB1 (Fig. 4
B). It was reported recently that SHIP can bind
several cytoplasmic molecules involved in cell activation via FcRI
(40). To determine whether SHIP seen in FcRI-coaggregated FcRIIB
immunoprecipitates was associated with FcRIIB, RBL transfectants
expressing FcRIIB whose IC domain was deleted were used under the
same conditions in the same experiment. No SHP-1 and no SHP-2, but also
no SHIP, were coprecipitated with IC domain-deleted FcRIIB (Fig. 4B). SHIP is therefore selectively recruited by
phosphorylated FcRIIB, and recruitment requires the IC domain of
FcRIIB.
These results altogether indicate that the coaggregation of FcRIIB
with FcRI induces the phosphorylation of FcRIIB on the ITIM
tyrosine and this phosphorylation leads to the selective recruitment
of SHIP.
The coaggregation of FcRI with FcRIIB does not affect the
phosphorylation of FcRI ITAMs and the activation of syk
To assess the effects of FcRIIB on signals transduced by
FcRI, we compared the pattern of tyrosine phosphorylation in whole
cell lysates after aggregation of FcRIIB, aggregation of FcRI, or
coaggregation of the two receptors in transfectants expressing
FcRIIB1. No tyrosine phosphorylation was induced after FcRIIB1
aggregation, whereas FcRI aggregation induced the tyrosine
phosphorylation of a number of intracellular proteins. A comparable
pattern of phosphorylation was induced when FcRI were coaggregated
with FcRIIB (Fig. 5A). These results
indicate that FcRIIB neither induce phosphorylation, when
aggregated, nor inhibit IgE-induced phosphorylation, when coaggregated
with FcRI.
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RI with FcRIIB would affect the
tyrosine phosphorylation of ITAMs in the FcR and FcRß subunits of
FcRI. RBL transfectants expressing FcRIIB1 were sensitized with
mouse IgE anti-DNP, incubated or not with 2.4G2
F(ab')2, and challenged with increasing concentrations of
TNP-MAR F(ab')2. FcRI complexes were immunoprecipitated
with anti-FcR Abs and analyzed by Western blotting with
anti-PY Abs. The aggregation of FcRI induced a TNP-MAR
F(ab')2 dose-dependent tyrosine phosphorylation of FcRß
and FcR. Neither phosphorylation of FcRß nor that of FcR was
reduced significantly upon coaggregation of FcRI with FcRIIB
(Fig. 5B). ITAM phosphorylation was therefore not
prevented by coaggregation, suggesting that the activity of PTK
activated following FcRI aggregation was not affected by
coaggregation.
To analyze the next steps of signal transduction, we examined the
phosphorylation of lyn and syk and their kinase
activities. Transfectants expressing FcRIIB1 were sensitized with
mouse IgE anti-DNP and incubated or not with 2.4G2
F(ab')2 before they were challenged or not with TNP-MAR
F(ab')2 for 2 min. lyn and syk were
immunoprecipitated with anti-lyn or
anti-syk Abs, and immunoprecipitates were submitted or
not to an IVKA in the presence of ATP and, as exogenous substrates,
GST-p62 and GST-HS1, respectively. GST-HS1 was phosphorylated by
syk, but not by lyn, whereas GST-p62 was
phosphorylated by both kinases (data not shown). Reaction mixtures were
analyzed by Western blotting with anti-PY, anti-lyn,
or anti-syk Abs. As previously reported (41, 42),
lyn was phosphorylated constitutively in
anti-lyn immunoprecipitates, and lyn
phosphorylation was neither significantly increased upon FcRI
aggregation nor significantly decreased upon coaggregation of FcRI
with FcRIIB. Likewise, the in vitro kinase activity of
lyn on GST-p62 neither increased after FcRI aggregation
nor decreased after FcRI-FcRIIB coaggregation (data not shown).
Therefore, no conclusion can be drawn regarding inhibition of
lyn activation by FcRIIB. By contrast, no tyrosine
phosphorylation of syk was seen in nonstimulated cells, but
syk phosphorylation was induced after the aggregation of
FcRI. syk phosphorylation was also observed when
FcRI were coaggregated with FcRIIB, and this phosphorylation,
although appearing slightly reduced in this experiment, was not
reproducibly decreased (Fig. 5C). GST-HS1 was in
vitro phosphorylated by syk immunoprecipitated from
nonstimulated cells. The aggregation of FcRI strongly increased the
in vitro phosphorylation of GST-HS1. A comparable in vitro
phosphorylation of GST-HS1 was mediated by syk
immunoprecipitated from transfectants whose FcRI were coaggregated
with FcRIIB1 (Fig. 5C). The coaggregation of
FcRIIB with FcRI therefore does not detectably affect either the
phosphorylation or the kinase activity of syk induced upon
FcRI aggregation by IgE and Ag.
Taken together, these results indicate that FcRIIB-mediated
inhibition does not prevent FcRI-mediated transduction of signals
leading to receptor phosphorylation and syk activation.
The coaggregation of FcRIIB with FcRI enables
FcRI-associated lyn to phosphorylate FcRIIB
Since FcRIIB: 1) does not become phosphorylated when
aggregated, but only when coaggregated with FcRI, and 2) does not
prevent FcRI-associated kinases from being activated when
coaggregated with FcRI, we hypothesized that FcRIIB might be a
substrate for FcRI-associated PTK.
To test this hypothesis, we made a GST fusion protein consisting of the
IC domain of FcRIIB1' (GST-ICIIB1'), which was used as an exogenous
substrate for FcRI-associated kinases in IVKA. Nontransfected RBL
cells were sensitized with mouse IgE anti-DNP and challenged or not
with DNP-BSA for 2 min. FcRI complexes were precipitated with
anti-FcR Abs and submitted to an IVKA in the presence of
[-32P]ATP and GST-ICIIB1' or, as a negative
control, GST. Reaction mixtures were fractionated by SDS-PAGE and
analyzed by Coomassie blue staining and by autoradiography. The
aggregation of FcRI increased the in vitro phosphorylation of a
number of proteins that coprecipitated with FcRI, including a
protein with the same apparent m.w. as FcRß. GST-ICIIB1' was weakly
phosphorylated when added to FcRI complexes precipitated from
nonstimulated cells and heavily phosphorylated when added to FcRI
complexes precipitated from stimulated cells. Under the same
conditions, GST was not phosphorylated (Fig. 6). Active kinase(s) that associates with
FcRI complexes following receptor aggregation can therefore
phosphorylate the IC domain of FcRIIB in vitro.
|
RI aggregation (41, 42). Indeed, when cell lysates from RBL
sensitized with IgE and challenged with Ag were immunoprecipitated with
anti-FcR and analyzed by IVKA on GST-p62 and GST-HS1 used as
exogenous substrates for lyn and syk,
respectively, both substrates were phosphorylated (data not shown). To
investigate whether lyn and/or syk may
phosphorylate GST-ICIIB1' in vitro, RBL cells were sensitized with IgE
anti-DNP and challenged with DNP-BSA for 2 min. lyn and
syk were immunoprecipitated and submitted to IVKA with
GST-ICIIB1' as an exogenous substrate. GST-p62 and GST-HS1 were used as
positive control substrates for lyn and syk,
respectively. Reaction mixtures were analyzed by Western blotting with
anti-PY, anti-lyn, and anti-syk Abs.
Equivalent in vitro phosphorylation of GST-ICIIB1' was observed when
GST-ICIIB1' was added to anti-lyn and
anti-syk immunoprecipitates (Fig. 7). Both lyn and
syk can therefore phosphorylate GST-ICIIB1' in vitro.
|
RIIB, when coaggregated with
FcRI in mast cells derived from syk-deficient or
lyn-deficient mice. Mast cells derived from both knockout
mice expressed similar amounts of FcRI and FcRIIB as mast cells
derived from wild-type mice, as assessed by indirect immunofluorescence
(data not shown). As expected, no syk was detected in
syk-deficient mast cells (Fig. 8A), and no
lyn was detected in lyn-deficient mast cells
(Fig. 8B), as assessed by Western blotting in whole
cell lysates. To analyze kinase activities present in
syk-deficient and lyn-deficient mast cells, cells
were sensitized with mouse IgE and challenged with RAM
F(ab')2. syk and lyn were
immunoprecipitated and submitted to IVKA in the presence of GST-HS1 or
GST-p62, respectively. Reaction mixtures were analyzed by Western
blotting with anti-PY Abs. The same phosphorylation of GST-p62 was
observed when added to anti-lyn immunoprecipitates from
wild-type and syk-deficient mast cells, whereas GST-HS1 was
phosphorylated when added to anti-syk immunoprecipitates
from wild-type mast cells, but not from syk-deficient mast
cells (Fig. 8A). The same phosphorylation of GST-HS1
was observed when added to anti-syk immunoprecipitates
from wild-type and lyn-deficient mast cells, whereas GST-p62
was phosphorylated when added to anti-lyn
immunoprecipitates from wild-type mast cells, but not from
lyn-deficient mast cells (Fig. 8B). To
study the in vivo phosphorylation of FcRIIB,
syk-deficient and lyn-deficient mast cells were
sensitized with mouse IgE and challenged or not with RAM
F(ab')2 or RAM IgG. FcRIIB were immunoprecipitated and
analyzed by Western blotting with anti-PY and anti-FcRIIB
Abs. FcRIIB phosphorylation was seen in immunoprecipitates from
wild-type and syk-deficient mast cells, and the level of
phosphorylation was correlated with the amount of immunoprecipitated
FcRIIB (Fig. 8A). FcRIIB phosphorylation was
seen in immunoprecipitates from wild-type mast cells, but not in
immunoprecipitates from lyn-deficient mast cells, although
comparable amounts of FcRIIB were precipitated from wild-type and
lyn-deficient mast cells (Fig. 8B). The
phosphorylation of FcRIIB was therefore not impaired in
syk-deficient mast cells, but completely inhibited in
lyn-deficient mast cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RIIB, following
coaggregation with FcRI. We found that: 1) phosphorylated FcRIIB
selectively recruit the inositol polyphosphate 5 phosphatase, SHIP, in
vivo; 2) the phosphorylation of FcRIIB requires the coaggregation
with FcRI; 3) the coaggregation of FcRIIB with FcRI does not
prevent FcRI from transducing signals leading to FcRI
phosphorylation and syk activation; 4) when activated by the
aggregation of FcRI, lyn and syk can both
phosphorylate FcRIIB in vitro; but 5) lyn, but not
syk, is required for in vivo phosphorylation of
FcRIIB.
To study the mechanism of FcRIIB-mediated inhibition of IgE-induced
mast cell activation, we devised an experimental model in which FcRI
and murine recombinant FcRIIB, expressed in RBL-2H3 cells, could be
independently aggregated, or coaggregated at the cell surface. Using
this model, we confirmed that the three mouse FcRIIB isoforms, which
all contain an ITIM, are able to inhibit IgE-induced serotonin
release. Inhibition was observed as soon as serotonin release was
detectable, and it did not result from a delay in secretion.
An early event observed when coaggregating FcRI with FcRIIB
was the tyrosine phosphorylation of FcRIIB. FcRIIB
phosphorylation occurred within 15 s, i.e., as early as that of
the ITAM-containing FcRI subunits (43). FcRIIB phosphorylation
required the conservation of the ITIM tyrosine. This result does not
formally prove that phosphorylation occurs on the ITIM tyrosine since
FcRIIB2 contains two tyrosines. It nevertheless indicates that this
residue is mandatory for FcRIIB phosphorylation. Supporting the
assumption that the ITIM tyrosine is indeed phosphorylated, FcRIIB
was found to recruit SH2 domain-bearing phosphatases (27, 28, 29, 30). We
observed that, when incubated with lysates from
[35S]methionine-labeled RBL cells, synthetic peptides
corresponding to FcRIIB phospho-ITIM bound in vitro to the same
three phosphatases as when incubated with B cell (29) or BMMC (27, 28)
lysates. These were two tyrosine phosphatases, SHP-1 and SHP-2, and the
inositol polyphosphate 5 phosphatase, SHIP. Remarkably, only SHIP was
coprecipitated with phosphorylated FcRIIB in RBL cells. The same was
observed in BMMC (28). The structural bases and biologic significance
of selective recruitment remain to be established. Supporting a
selective in vivo recruitment of SHIP in mast cells, FcRIIB were
found to inhibit IgE-mediated activation in mast cells derived from
bone marrow of the SHP-1-deficient motheaten mice (27, 28), indicating
that SHP-1 is not required for inhibition of serotonin release. Whether
SHIP is directly responsible for FcRIIB-mediated inhibition of mast
cell activation remains to be demonstrated. The coprecipitation of SHIP
with phosphorylated FcRIIB is simply correlated with inhibition of
secretion. We observed that the coaggregation of FcRI with FcRIIB
in RBL cells inhibits the late Ca2+ flux, without affecting
the early phase of Ca2+ mobilization. The same was observed
in B cells (44) and in BMMC (28). How SHIP can possibly block the
influx of extracellular Ca2+ is unclear. SHIP, indeed,
dephosphorylates 5'-phosphate groups in inositol (1, 3, 4, 5)-tetraphosphate and in phosphatidylinositol (3, 4, 5)-trisphosphate
(45). These substrates are not known to be directly involved in the
opening of Ca2+ channels. SHIP can however interfere with
the phospholipid metabolism: phosphatidylinositol (3, 4, 5)-trisphosphate was suggested to enhance the translocation of PLC-2
to the plasma membrane (46), where it hydrolyzes phosphatidylinositol
(4, 5)-bisphosphate into inositol (1, 4, 5)-trisphosphate. SHIP may
thus ultimately decrease the sustained production of inositol (1, 4, 5)-trisphosphate, which is needed for generating the signals leading to
the entry of extracellular Ca2+ (47), as it was observed in
B cells (48). Alternatively and not exclusively, SHIP can bind,
directly or indirectly, to other molecules implicated in signal
transduction, such as shc (49) or SHP-2 (50), respectively.
Finally, one cannot exclude that not yet identified molecules may
contribute to FcRIIB-mediated inhibition.
Compatible with our finding that no tyrosine phosphatase is recruited
by phosphorylated FcRIIB in mast cells, we observed that the
coaggregation with FcRIIB did not prevent FcRI from activating
PTK. The pattern of phosphorylation of intracellular substrates was not
affected by coaggregation. The phosphorylation of FcRß and FcR
ITAMs was not inhibited, and the activation of syk was not
decreased, as judged by its in vivo phosphorylation and by its in vitro
kinase activity on a syk-specific substrate. This indicates
that the initial steps of signal transduction following FcRI
aggregation are not impaired by FcRIIB, and that inhibition acts on
second messengers rather than on early phosphorylation events.
FcRIIB must be phosphorylated to inhibit mast cell activation: the
point mutation of the ITIM tyrosine was sufficient to abrogate
inhibition of mediator release (13), and this mutation prevented
FcRIIB phosphorylation. Inhibition of serotonin release required
that FcRI be coaggregated with FcRIIB (14), and FcRIIB became
tyrosine phosphorylated only when coaggregated with FcRI. This
suggests that FcRIIB may not be constitutively associated with a
tyrosine kinase. Supporting this hypothesis, FcRIIB aggregation
induced no phosphorylation of intracellular substrates. We therefore
hypothesized that FcRIIB phosphorylation depends on
FcRI-associated kinases that become activated and/or recruited in
the receptor complex. We provide evidence supporting this possibility.
PTK that were coprecipitated with activated FcRI could phosphorylate
in vitro a GST fusion protein consisting of the FcRIIB IC domain.
lyn and syk were shown previously to
coprecipitate with aggregated FcRI under these conditions (41, 42),
and we found in vitro kinase activities on both lyn and
syk substrates in immunoprecipitates of activated FcRI
(data not shown). When immunoprecipitated from RBL cells sensitized
with IgE and challenged with Ag, both lyn and syk
phosphorylated GST-FcRIIB IC in vitro. To validate these in vitro
data, we examined wether FcRIIB could be phosphorylated in vivo when
coaggregated with FcRI in mast cells derived from
syk-deficient mice or lyn-deficient mice. We
found that FcRIIB became phosphorylated in syk-deficient
mast cells. This indicates that syk is not mandatory for
FcRIIB phosphorylation. By contrast, FcRIIB phosphorylation was
abrogated in lyn-deficient mast cells, although the kinase
activity of syk was conserved in these cells. This indicates
that lyn is required for FcRIIB phosphorylation in vivo,
and that it cannot be replaced by another kinase. Whether
lyn directly phosphorylates FcRIIB or whether it
activates another kinase that phosphorylates FcRIIB cannot be
determined. Our data altogether suggest that, upon coaggregation of the
two receptors, lyn is normally activated and brought by
FcRI in the vicinity of the FcRIIB ITIM, enabling FcRIIB to be
phosphorylated, to recruit SHIP and, ultimately, to inhibit mast cell
responses. Such a mechanism seems to be unique, in view of previous
studies on FcRIIB-mediated inhibition of B cell activation and on
inhibition mediated by other ITIM-bearing negative coreceptors such as
human KIRs.
In B cells, phosphorylated FcRIIB were reported to recruit SHP-1,
and FcRIIB-mediated inhibition of cell activation was abrogated in B
cells from motheaten mice (29). CD19 was proposed to be a potential
substrate of SHP-1, as its phosphorylation was inhibited upon
coaggregation of BCR with FcRIIB. As a consequence, the association
of CD19 with phosphatidylinositol 3-kinase seen upon BCR aggregation
was prevented (51, 52). No equivalent of CD19 is known in mast cells.
Neither the phosphorylation pattern of intracellular substrates nor the
phosphorylation of specific proteins involved in signal transduction,
including Ig, Igß, lyn, syk, and PLC-,
was affected (51, 52) upon coaggregation of BCR with FcRIIB. Kinases
associated with BCR might therefore phosphorylate FcRIIB in B cells,
like we found for kinases associated with FcRI in mast cells, as
suggested by the recent observation that FcRIIB-mediated inhibition
is impaired in B cells from lyn-/- mice (53). Finally,
the apparent discrepancy between studies made in B cells and in mast
cells may be reconciled by the recent finding that phosphorylated
FcRIIB could recruit SHIP also in B cells (30, 31). By contrast with
FcRIIB, phosphorylated KIR ITIM peptides were shown to bind in vitro
to SHP-1 (54) and SHP-2, but not to SHIP (55) and, when coaggregated
with FcRI in RBL cells (56), p58 KIRs recruited SHP-1, whereas
FcRIIB recruited SHIP (our unpublished results). The recruitment of
tyrosine phosphatases by phosphorylated KIRs was correlated with the
dephosphorylation of , ZAP-70, and PLC-1/2 in NK cells
(57).
In conclusion, this study provides evidence that FcRI are involved
in FcRIIB-mediated inhibition of IgE-dependent mast cell activation.
FcRIIB (14) and KIRs (56) both must be coaggregated with
ITAM-bearing receptors to inhibit cell activation. The biologic
significance of coaggregation seems, however, to be different for the
two ITIM-bearing receptors. In KIR-mediated inhibition, coaggregation
makes phosphorylated tyrosines of cell-activating receptors and/or
kinases the substrates of protein tyrosine phosphatases recruited by
phosphorylated KIRs. In FcRIIB-mediated inhibition, coaggregation
makes the ITIM a substrate for FcRI-associated lyn,
enabling FcRIIB to recruit SHIP. As a consequence, cell-activating
receptors may be passive targets in KIR-mediated inhibition, whereas
they actively participate to their own inhibition, by enabling ITIM
phosphorylation, in FcRIIB-mediated inhibition.
|
Acknowledgments |
|---|
RIIB
Abs, Dr. Jean-Pierre Kinet for rabbit anti-FcR Abs, Dr. Gerald
Krystal for rabbit anti-SHIP Abs, and Dr. Ulrich Blank for
anti-syk Abs and for GST-HS1. We are grateful to Drs.
Siegmund Fischer and Paco Romero (Institut Cochin de
Génétique Moléculaire, Paris) for their help in the
preparation of GST-ICIIB1'. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr Marc Daëron, Laboratoire d’Immunologie Cellulaire & Clinique, INSERM U.255, Institut Curie, 26 rue d’Ulm, 75005 Paris, France. E-mail address:
3 Abbreviations used in this paper: BCR, B cell receptor; BMMC, bone marrow-derived mast cell; GST, gluatathione S-transferase; IC, intracytoplasmic; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; IVKA, in vitro kinase assay; KIR, killer cell inhibitory receptor; MAR, mouse anti-rat; PTK, protein tyrosine kinase; PY, phosphotyrosine; RAM, rabbit anti-mouse; GAR, goat anti-rabbit; TNP, trinitrophenyl; IMDM, Iscove’s modified Dulbecco’s medium; SHIP, SH2-bearing inositolphosphate phosphatase; SHP, SH2-bearing protein tyrosine phosphatase.
Received for publication July 9, 1997. Accepted for publication October 27, 1997.
|
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H. Lienard, P. Bruhns, O. Malbec, W. H. Fridman, and M. Daeron Signal Regulatory Proteins Negatively Regulate Immunoreceptor-dependent Cell Activation J. Biol. Chem., November 5, 1999; 274(45): 32493 - 32499. [Abstract] [Full Text] [PDF]
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O. Malbec, W. H. Fridman, and M. Daeron Negative Regulation of c-kit-Mediated Cell Proliferation by Fc{gamma}RIIB J. Immunol., April 15, 1999; 162(8): 4424 - 4429. [Abstract] [Full Text] [PDF]
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P. Bruhns, P. Marchetti, W. H. Fridman, E. Vivier, and M. Daeron Differential Roles of N- and C-Terminal Immunoreceptor Tyrosine-Based Inhibition Motifs During Inhibition of Cell Activation by Killer Cell Inhibitory Receptors J. Immunol., March 15, 1999; 162(6): 3168 - 3175. [Abstract] [Full Text] [PDF]
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S. Hiraoka, Y. Furumoto, H. Koseki, Y. Takagaki, M. Taniguchi, K. Okumura, and C. Ra Fc receptor ß subunit is required for full activation of mast cells through Fc receptor engagement Int. Immunol., February 1, 1999; 11(2): 199 - 207. [Abstract] [Full Text] [PDF]
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P. Bruhns, F. Vely, O. Malbec, W. H. Fridman, E. Vivier, and M. Daeron Molecular Basis of the Recruitment of the SH2 Domain-containing Inositol 5-Phosphatases SHIP1 and SHIP2 by Fcgamma RIIB J. Biol. Chem., November 22, 2000; 275(48): 37357 - 37364. [Abstract] [Full Text] [PDF]
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R. Lesourne, P. Bruhns, W. H. Fridman, and M. Daeron Insufficient Phosphorylation Prevents Fcgamma RIIB from Recruiting the SH2 Domain-containing Protein-tyrosine Phosphatase SHP-1 J. Biol. Chem., February 23, 2001; 276(9): 6327 - 6336. [Abstract] [Full Text] [PDF]
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O. Malbec, C. Schmitt, P. Bruhns, G. Krystal, W. H. Fridman, and M. Daeron Src Homology 2 Domain-containing Inositol 5-Phosphatase 1 Mediates Cell Cycle Arrest by Fcgamma RIIB J. Biol. Chem., August 3, 2001; 276(32): 30381 - 30391. [Abstract] [Full Text] [PDF]
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A. J. Smith, Z. Surviladze, E. A. Gaudet, J. M. Backer, C. A. Mitchell, and B. S. Wilson p110beta and p110delta Phosphatidylinositol 3-Kinases Up-regulate Fcepsilon RI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production J. Biol. Chem., May 11, 2001; 276(20): 17213 - 17220. [Abstract] [Full Text] [PDF]
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