From the Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U.255, Institut Curie, 75005 Paris, France
Received for publication, July 21, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Fc Fc To inhibit cell activation, Fc A general property of ITIMs is to have an affinity for cytoplasmic SH2
domain-containing phosphatases when tyrosyl- phosphorylated (17).
Phosphatases that are recruited to the membrane antagonize with
activation signals transduced by ITAM-bearing receptors. ITIM-bearing
receptors were found to recruit two classes of phosphatases which exert
markedly different effects: protein-tyrosine phosphatases and inositol
5-phosphatases. Protein-tyrosine phosphatases are SHP-1 and SHP-2. They
have two SH2 domains and their substrates are tyrosyl-phosphorylated
proteins (18). SHP-1 is thought to dephosphorylate tyrosines in ITAMs
(19), protein tyrosine kinases and/or adapter proteins such as SLP-76
(20) whose phosphorylation is critical for activation signals. SHP-1
thus stops the initial steps of transduction. The possible role of
SHP-2 is not clear, as both positive and negative effects have been
assigned to this phosphatase. Inositol 5-phosphatases are SHIP1 and
SHIP2. They have a single SH2 domain and they remove 5'-phosphate
groups in inositol phosphates and phosphatidylinositol phosphates that
are 3'-phosphorylated (21). The preferred substrate of SHIP1 is phosphatidylinositol (3,4,5)-trisphosphate which enables the membrane translocation of the Bruton's tyrosine kinase via its pleckstrin homology domain (22). Bruton's tyrosine kinase is mandatory for
phospholipase C The in vitro binding specificity of ITIMs was analyzed using
phosphorylated synthetic peptides to precipitate phosphatases from cell
lysates. Under these conditions, all known ITIMs, including the
Fc In the present work, we aimed at clarifying these questions by
analyzing the conditions required for SHP-1 to bind to phosphorylated ITIM-coated beads in vitro and to be recruited by
phosphorylated Fc Cells--
The rat mast cells RBL-2H3 (34) were cultured in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Fc Transfectants--
Clones of RBL-2H3 cells, stably transfected
with cDNA encoding murine Fc Antibodies--
The mouse IgE mAb 2682-I was used as culture
supernatant of a subclone of DNP-H1- ITIM Peptides and in Vitro Binding of
Phosphatases--
Nonphosphorylated or tyrosyl-phosphorylated
Fc GST Fusion Proteins--
cDNA encoding the intracytoplasmic
domain of Fc In Vitro Phosphorylation of GST-ITIM and GST-ICIIB1' and in Vitro
Binding of Phosphatases--
Ten µl of glutathione-agarose beads
coated with GST-ITIM or GST-ICIIB1' were washed in kinase buffer
containing 100 mM Tris-HCl, pH 7.4, 125 mM
MgCl2, 2 mM EGTA, 0.25 mM
Na3VO4, and 2 mM dithiothreitol, and incubated for the indicated periods at 30 °C with 20 µl of kinase buffer containing 2 units of the Src kinase Lyn (Chemicon) and
100 µM ATP. Kinase reaction was stopped on ice; beads
were immediately washed in lysis buffer, and incubated for 2 h at
4 °C with lysates from 1 × 107 RBL-2H3 cells.
Cell Stimulation and Immunoprecipitation--
RBL transfectants,
resuspended at 5 × 106/ml, were incubated or not for
1 h at 37 °C with IgE anti-DNP (culture supernatant diluted
1/10) and with 3 µg/ml 2.4G2 F(ab')2, washed, and
resuspended at 1 × 107 cells/ml. Cells were
challenged or not for 5 min or for the indicated periods of time at
37 °C with 10 µg/ml TNP-MAR F(ab')2. Unless otherwise
specified, IIA1.6 and K46µ transfectants, resuspended at 5 × 107/ml, were stimulated at 37 °C for 5 min with 0.3 µM RAM IgG (45 µg/ml) or RAM F(ab')2 (30 µg/ml). BMMCs were sensitized as RBL-2H3 cells with the indicated
dilutions of IgE anti-DNP, and resuspended at 1 × 107/ml. BMMCs were stimulated at 37 °C for 5 min with
preformed immune complexes made with the indicated final concentrations
of DNP25-BSA and mAb mouse IgG1 anti-DNP. K46µ
transfectants, resuspended at 5 × 107/ml, were
stimulated with preformed immune complexes made with the indicated
final concentrations of NP9-BSA-TNP12 and of
polyclonal rabbit IgG anti-DNP. Immune complexes were preformed at
37 °C for 10 min immediately before stimulation.
Pervanadate was generated by mixing 1 ml of 20 mM
Na3VO4 with 330 µl of 30%
H2O2 followed by a 5-min incubation at room
temperature, yielding a solution of 6 mM pervanadate.
Unless otherwise specified, cells were incubated with a final
concentration of 100 µM pervanadate for 15 min at
37 °C.
Following stimulation, cells were lysed as described above, and cell
lysates were used for immunoprecipitation. Protein G-Sepharose (Amersham Pharmacia Biotech) was used to precipitate 2.4G2-bound Fc Western Blot Analysis--
Adsorbents were washed in lysis
buffer and boiled for 3 min in sample buffer. Eluted material was
fractionated by SDS-PAGE and transferred onto Immobilon-P membranes
(Millipore, Bedford, MA). In most experiments, immunoprecipitates were
fractionated and transferred onto two membranes. These were used to
assess: 1) tyrosyl phosphorylation and Fc Tyrosyl-phosphorylated Fc In Vitro Binding of SHP-1 Requires a Higher Phosphorylation Level
of Fc
Another difference between the two types of experiments is that intact
receptors are used for in vivo recruitment whereas 13-amino
acid peptides are used for in vitro binding. One must therefore consider the possibility that flanking sequences might affect
the recruitment of phosphatases by the phosphorylated ITIM. To examine
this possibility, we constructed GST fusion proteins containing either
the whole intracytoplasmic domain of the Fc The Density of Phosphorylated ITIM Critically Determines the
Cooperative Binding of the Two SHP-1 SH2 Domains--
A major
difference between SHIP1 and SHP-1 is that the phosphatidylinositol
phosphatase contains one SH2 domain whereas the protein tyrosine
phosphatase contains two tandem SH2 domains. We investigated the
possible role of this difference in the differential binding of these
phosphatases to pITIM-coated beads. Agarose beads were coated with
increasing concentrations of Fc
When using increasing amounts of pITIM to coat beads, one increases not
only the quantity of pITIM bound to beads, but also the density of
pITIM on beads. To investigate whether pITIM density may determine the
binding of the two SH2 domain-containing SHP-1, the same amounts of
pITIM were used to coat different amounts of beads. These were
incubated in RBL cell lysate, and phosphatase binding was then examined
as above. For a given amount of pITIM, the binding of SHIP1 did not
vary with the amount of beads. By contrast, for a given amount of
pITIM, the binding of SHP-1 dramatically decreased when the amount of
beads increased (Fig. 3B). SHP-1 binding, but not SHIP1
binding, therefore depends on the density of pITIM coated to beads.
Increasing Fc
We first tried to increase Fc
This negative result led us to use IgG immune complexes, which are the
natural ligands of Fc
The co-precipitation of phosphatases was next examined in BMMCs and in
Fc Fc
Fc We show here that murine Fc Evidence that, when tyrosyl phosphorylated, the Fc In the same 1995 paper, D'Ambrosio et al. (30) reported
that SHP-1 co-precipitated with Fc That SHP-1 was found by two groups to co-precipitate with Fc Due to the different experimental conditions used for the two assay
systems many differences can possibly explain this discrepancy. One
difference could bear on the level of ITIM phosphorylation. Indeed, all
ITIMs are phosphorylated on beads used for in vitro binding
assay whereas an unknown proportion of Fc Based on data previously reported by others (43, 44), the binding of
SHP-1 is likely to involve the two tandem SH2 domains of this
phosphatase. Compared with GST fusion proteins containing the two SH2
domains of SHP-1, no GST fusion proteins containing the N-terminal
domain of SHP-1 and minute amounts of GST fusion proteins containing
the C-terminal SH2 domain of SHP-1 bound to pITIM-coated beads,
whatever the concentration of peptides on beads. This suggests that,
since there is one tyrosine only in pITIM, GST fusion proteins
containing the two SHP-1 SH2 domains bound to adjacent pITIMs on the
same bead. The same holds for the binding of SHP-1 when incubating
pITIM-coated beads with a cell lysate. If so, variations in the
affinity of SHP-1 to beads coated with increasing amounts of pITIM
might depend on the density of peptides coated to beads. To examine
this possibility, we used a constant amount of pITIM to coat variable
numbers of beads that were incubated in a cell lysate, and we compared
the ability of these beads to bind SHIP1 and SHP-1. The binding of
SHIP1 was proportional to the amount of pITIM on beads, and did not
vary with the pITIM density. By contrast, the binding of SHP-1 depended not only on the amount of pITIM but also, critically, on the density of
pITIM bound to beads. The in vitro binding of SHP-1
therefore requires a cooperative binding of its two SH2 domains to two
adjacent pITIMs in trans, and this feature explains that
pITIMs need to be closer to each other for enabling the binding of
SHP-1 than for enabling the binding of SHIP1.
Another difference that might explain the discrepancy between the
in vitro binding and the in vivo recruitment of
SHP-1 is that isolated ITIMs are used in vitro whereas whole
receptors are used in vivo. One cannot exclude that the
recruitment of SHP-1 might be hampered by non-ITIM sequences or by
molecules that could possibly bind to these sequences. Supporting this
possibility, the N-terminal KIR2DL3 ITIM that could recruit SHP-2
in vivo, when kept in its original context, failed to
recruit this phosphatase, when transposed in the intracytoplasmic
domain of Fc Based on the latter result, we searched for experimental conditions
that would induce a Fc In both mast cells and B cells, pervanadate treatment indeed induced a
higher degree of Fc The latter interpretation of the effect of pervanadate may explain our
failure to co-precipitate SHP-1 with FcRIIB are IgG receptors that inhibit
immunoreceptor tyrosine-based activation motif
(ITAM)-dependent cell activation. Inhibition depends on an
immunoreceptor tyrosine-based inhibition motif (ITIM) that is
phosphorylated upon Fc
RIIB coaggregation with ITAM-bearing receptors
and recruits SH2 domain-containing phosphatases. Agarose bead-coated
phosphorylated ITIM peptides (pITIMs) bind in vitro the
single-SH2 inositol 5-phosphatases (SHIP1 and SHIP2) and the two-SH2
protein tyrosine phosphatases (SHP-1 and SHP-2). Phosphorylated Fc
RIIB, however, recruit selectively SHIP1/2 in vivo. We
aimed here at explaining this discordance. We found that beads coated with low amounts of pITIM bound in vitro SHIP1, but not
SHP-1, i.e. behaved as phosphorylated Fc
RIIB in
vivo. The reason is that SHP-1 requires its two SH2 domains to
bind on adjacent pITIMs. Consequently, the binding of SHP-1, but not of
SHIP1, increased with pITIM density on beads. When trying to increase
Fc
RIIB phosphorylation in B cells and mast cells, we found that
concentrations of ligands optimal for Fc
RIIB phosphorylation failed
to induce SHP-1 recruitment. SHP-1 was, however, recruited by Fc
RIIB
when hyperphosphorylated following cell treatment with pervanadate. Our
data suggest that Fc
RIIB phosphorylation may not be sufficient
in vivo to enable the recruitment of SHP-1 but that
(pathological?) conditions that would hyperphosphorylate Fc
RIIB
might enable SHP-1 recruitment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIB are single-chain low-affinity receptors for the Fc
portion of IgG antibodies that bind multivalent immune complexes. They
exist as two (Fc
RIIB1 and B2 in humans) or three (Fc
RIIB1, B1',
and B2 in mice) alternatively spliced products of the FcgR2b gene (1). All murine and human Fc
RIIB isoforms were shown to
negatively regulate cell activation induced by all receptors bearing
intracytoplasmic immunoreceptor tyrosine-based activation motifs
(ITAMs)1 (2). Fc
RIIB also
negatively regulate cell proliferation induced by growth factor
receptors with an intrinsic protein tyrosine kinase activity (3).
Confirming these results, Fc
RIIB-deficient mice were found: 1) to
exhibit enhanced antibody responses (4); 2) to develop exaggerated IgE-
(5) and IgG-dependent anaphylactic reactions (4); 3) to
have an enhanced susceptibility to experimental murine models of
IgG-dependent autoimmune diseases (6-8); and 4) to exhibit
enhanced antibody-dependent cell-mediated cytotoxic responses to the injection of therapeutic antibodies to tumor antigens
(9).
RIIB need to be coaggregated with
ITAM-bearing receptors by immune complexes or by any extracellular ligand capable of interacting with the two receptors simultaneously (10-12). Coaggregation indeed enables Fc
RIIB to be
tyrosyl-phosphorylated by Lyn (13), a Src family protein
tyrosine kinase provided by ITAM-bearing receptors. Fc
RIIB isoforms
contain a variable number of tyrosine residues in their
intracytoplasmic domain, one of which proved to be critical (2, 14).
This tyrosine stands within a 13-amino acid sequence that was found to
be necessary (2, 15) and sufficient (14) for inhibition. Related
sequences subsequently found in a large number of transmembrane
molecules with negative regulatory properties provided the molecular
basis for the definition of an immunoreceptor tyrosine-based inhibition motif (ITIM) having the (I/V/L)xYxxL consensus
sequence (16, 17).
to be activated and to hydrolyze
phosphatidylinositol (4,5)-bisphosphate into inositol
(1,4,5)-trisphosphate, which induces a Ca2+ response (23),
and diacylglycerol, which activates protein kinase C. SHIP1 was
recently shown to inhibit the Ras pathway by acting as an
adapter molecule in B cells. When phosphorylated by Lyn, SHIP1 recruits
Dok by its protein tyrosine-binding domain. Dok is phosphorylated and
recruits RasGAP which inactivates Ras by exchanging GTP for GDP on the
latter molecule (24). SHIP1 therefore arrests the propagation of
intracellular signals leading to the Ca2+ response and to
the activation of the Ras pathway. The possible role of SHIP2 is not
yet known.
RIIB ITIM, bound SHP-1 and SHP-2 (25). Remarkably, the Fc
RIIB
ITIM also bound SHIP1 (26) and SHIP2 (27). We previously identified two
hydrophobic residues, at positions Y-2 and Y+2, that determine the
binding of SHPs (28) and SHIPs (29), respectively. The in
vivo recruitment of phosphatases by ITIM-bearing receptors was
analyzed by co-precipitation, following their tyrosyl phosphorylation upon coaggregation with ITAM-bearing receptors. Tyrosyl-phosphorylated Fc
RIIB were initially reported to recruit SHP-1 in B cells following their coaggregation with B cell receptors (BCR) (30, 31). Fc
RIIB,
however, were found to recruit selectively SHIP1, when coaggregated
with high-affinity IgE receptors (Fc
RI) in mast cells (26, 32).
SHIP1, but not SHP-1, was subsequently demonstrated to be necessary for
Fc
RIIB-dependent inhibition of cell activation in
SHIP1-deficient DT40 B cells (33) and in SHP-1-deficient mast cells
derived from motheaten mice (26, 32). These results altogether
generated some confusion, and whether Fc
RIIB indeed recruit SHP-1
in vivo remains unclear. Depending on the answer, the
following two issues may be addressed: 1) if they do, do they recruit
SHP-1 exclusively in B cells or also in other cell types, and 2) if
they do not, how can one reconcile the apparent discordance between the
in vitro binding of phosphatases to ITIM peptides and the
in vivo recruitment of phosphatases by Fc
RIIB.
RIIB in mast cells and in B cells. We failed to
detect SHP-1 recruitment by Fc
RIIB in vivo when
coaggregated either with Fc
RI in mast cells, or with BCR in B cells.
We found that the in vitro binding of SHP-1 required a
higher level of Fc
RIIB phosphorylation than SHIP1 binding. Indeed,
the two SH2 domains of SHP-1 were required to bind phosphorylated ITIMs
and, as a consequence, SHP-1 binding, but not SHIP1 binding, depended
on the density of phosphorylated ITIMs. In vivo, SHP-1
recruitment also required a higher level of Fc
RIIB phosphorylation
than SHIP1 recruitment. The level of Fc
RIIB phosphorylation that
enabled the recruitment of SHP-1 was reached after treating cells with
pervanadate, but not following coaggregation of Fc
RIIB with BCR or
Fc
RI, in B cells and mast cells, respectively.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIB-deficient murine
lymphoma B cells K46µ, expressing an anti-NP BCR (35), and IIA1.6
(36) were cultured in RPMI supplemented with 10% fetal calf serum, 0.5 µM 2-mercaptoethanol, 2 mM sodium pyruvate,
100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine, and 20 mM Hepes. Bone
marrow-derived mast cells (BMMC) were obtained from BALB/c bone marrow
cells as described (3). After 4 weeks, cultures contained more than
90% mast cells. Culture reagents were from Life Technologies, Inc.
(Paisley, Scotland, United Kingdom).
RIIB1 (37) or Fc
RIIB1' (38) and
clones of IIA1.6 cells, stably transfected with cDNA encoding
Fc
RIIB1 (15) were described previously. cDNA encoding
Fc
RIIB1, inserted in a NT-neo vector (39), was stably transfected in
K46µ cells by electroporation. Transfectants were selected with
neomycin (Cayla, Toulouse, France) and by cell sorting using a
FACSCalibur (Becton Dickinson, Mountain View, CA).
Fc
RIIB1-transfected K46µ cells were cloned as described (37). The
expression of recombinant receptors on clones remained stable over the
duration of experiments.
-26 hybridoma cells (40). The
rat anti-mouse Fc
RIIB 2.4G2 mAb (41) was purified by affinity
chromatography on Protein G-Sepharose from ascitic fluid of nude mice
inoculated with 2.4G2 hybridoma cells intraperitoneally.
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. The mouse IgG1 mAb anti-DNP was provided
by Dr. Jacques Couderc (Institut Curie, Paris, France). The rabbit
polyclonal IgG anti-DNP were purchased from Sigma. F(ab')2
fragments of polyclonal mouse anti-rat Ig (MAR) were purchased from
Jackson ImmunoResearch Laboratories (West Grove, PA). MAR
F(ab')2 were trinitrophenylated by incubation for 1 h
at room temperature with trinitrobenzene sulfonic acid (Eastman Kodak,
Rochester, NY) in borate-buffered saline, pH 8.0. TNP10-MAR F(ab')2 were obtained after
purification on Sephadex G-25 (Amersham Pharmacia Biochemicals,
Uppsala, Sweden). Polyclonal rabbit anti-mouse immunoglobulins (RAM)
IgG and F(ab')2 were purchased from Cappel Laboratories
(West Chester, PA). Rabbit antibodies against soluble recombinant
extracellular domains of Fc
RIIB and mouse mAb anti-GST were gifts
from Prof. Catherine Sautès-Fridman and Dr. Jean-Luc Teillaud
(Institut Curie, Paris, France), respectively. Horseradish peroxidase
(HRP)-conjugated mouse monoclonal anti-phosphotyrosine antibodies
(pY20) were purchased from Chemicon (Temecula, CA), mouse monoclonal
anti-SHP-1 from Transduction Laboratories (Lexington, KY), anti-SHIP1
antibodies from Upstate Biotechnology (Lake Placid, NY), and
HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-SHP-1 antibodies were a gift from Dr. John
Cambier (National Jewish Medical and Research Center, Denver, CO).
RIIB ITIM-biotinylated peptides with the amino acid sequence
KTAENTITYSLLK were synthesized by Neosystems. Biotinylated ITIM
peptides were coupled to streptavidin-coated agarose beads (Pierce,
Rockford, IL). Beads were saturated with 1 mg/ml D-biotin
(Sigma), washed 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
phenylmethylsulfonyl fluoride (lysis buffer) and incubated for 2 h
at 4 °C with lysates from 1 × 107 RBL-2H3 cells or
with SH2 domain-containing GST fusion proteins. Cell lysates were
prepared by incubating 1 × 107 RBL-2H3 cells for 10 min on ice in lysis buffer. Post-nuclear supernatants were collected
after centrifugation at 12,000 × g for 20 min at
4 °C, and used as a source of phosphatases.
RIIB1' (ICIIB1') was generated by polymerase chain
reaction using as a template cDNA encoding murine Fc
RIIB1' (38).
The following primers were used: sense
5'-CGTAGGATCCAAGAAAAAGCAGGTTCCA-3'; antisense
5'-TACGGTCGACCTAAATGTGGTTCTGGTA-3'. Nucleotides corresponding to the
cDNA sequence encoding amino acids 273-285 of Fc
RIIB1',
i.e. the same 13 amino acids as synthetic ITIM peptides,
were purchased from Amersham Pharmacia Biotech (Little Chalfont,
Buckinghamshire). cDNA encoding the SH2 domain of SHIP1 was
amplified by polymerase chain reaction, using as a template cDNA
generated from RNA extracted from RBL-2H3 cells. The following primers
were used: sense 5'-CTGACCCAGTCTAGAGGATCCATGCCTGCCATGGTCCCTG-3'; antisense 5'-GACACCTCGAGCTCTCAGGGAGGCAGCTCA-3'. The sequence was checked on the two strands by dideoxynucleotide sequencing. ICIIB1' cDNA, ITIM-encoding nucleotides, and SHIP1 SH2 domain cDNA were inserted into the pGEX-4T-2 vector (Amersham Pharmacia Biotech) and
transfected into DH5
Escherichia coli. Bacteria producing SHP-1 SH2 domain-containing GST fusion proteins were a gift from Dr.
Eric Vivier (Center d'Immunologie de Marseille-Luminy, Marseille, France). All GST fusion proteins were produced in DH5
E. coli following
isopropohy-1-thio-
-D-galactopyranoside induction,
purified on glutathione-agarose (Sigma), and analyzed by SDS-PAGE.
Soluble SH2 domain-containing GST fusion proteins were eluted from
glutathione-agarose beads with a solution of 50 mM Tris and
25 mM glutathione, pH 8.0.
RIIB in lysates from RBL transfectants and 2.4G2-coated Sepharose beads were used to precipitate Fc
RIIB in lysates from BMMCs or IIA1.6 and K46µ transfectants.
RIIB or GST on one
membrane, and 2) SHIP1 and SHP-1 phosphatases on the other membrane.
Membranes were saturated with either 5% BSA (Sigma) or 5% skimmed
milk (Régilait, Saint-Martin-Belle-Roche, France) diluted in 10 mM Tris buffer, pH 7.4, containing 0.5% Tween 20 (Merck,
Schuchardt, Germany). Membranes Western blotted with HRP-conjugated
anti-phosphotyrosine antibodies were stripped and reblotted with
anti-Fc
RIIB or anti-GST antibodies followed by HRP-conjugated GAR or
GAM. In all experiments, the same membrane was used for blotting with
anti-SHIP1 and anti-SHP-1 antibodies after having been cut into two
pieces. The upper part, containing molecules with a molecular mass
higher than 100 kDa was hybridized with anti-SHIP1, and the lower part,
containing molecules with a molecular mass lower than 100 kDa was
hybridized with anti-SHP-1 antibodies. mAb anti-SHP-1 was used for mast
cells analysis while polyclonal antibodies was used for B cells
analysis. They were revealed with HRP-conjugated GAM or GAR,
respectively. Labeled antibodies were detected using the Amersham ECL kit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIB Recruit SHIP1 but Not SHP-1 Both
in Mast Cells and in B Cells--
Because phosphatases were reported
to be differentially recruited by Fc
RIIB, when coaggregated with BCR
in B cells or with Fc
RI in mast cells, we compared the ability of
Fc
RIIB1 to recruit SHP-1 and SHIP1 in a rat mast cell line, RBL-2H3,
and in two Fc
RIIB-deficient murine B cell lines, IIA1.6 cells and
K46µ. The three cell lines were stably transfected with murine
Fc
RIIB1. Fc
RIIB1 were coaggregated with Fc
RI, that are
constitutively expressed in RBL cells, by challenging transfectants,
previously sensitized with mouse IgE anti-DNP and coated with
F(ab')2 fragments of the rat anti-Fc
RIIB mAb 2.4G2,
using TNP-MAR F(ab')2. Fc
RIIB1 were coaggregated with BCR, that are constitutively expressed in IIA1.6 cells and K46µ cells, using RAM IgG. Fc
RIIB1 were immunoprecipitated, their phosphorylation was assessed by Western blotting with
anti-phosphotyrosine antibodies, and phosphatases co-precipitated with
Fc
RIIB1 were examined by Western blotting with anti-SHP-1 and
anti-SHIP1 antibodies. As previously observed, Fc
RIIB1 became
tyrosyl phosphorylated following coaggregation with Fc
RI or with
BCR, in mast cells and in B cells, respectively. SHIP1, but not SHP-1,
co-precipitated with tyrosyl-phosphorylated Fc
RIIB1 in RBL cells,
but also in IIA1.6 cells and K46µ cells (Fig.
1A). Because the recruitment of SHIP1 and SHP-1 may not have identical kinetics, we repeated the
experiment at various time points in RBL transfectants. The induced
phosphorylation of Fc
RIIB1 was constant over the time range studied,
and comparable amounts of SHIP1 co-precipitated with phosphorylated
Fc
RIIB1. No co-precipitation of SHP-1 was detected between 15 s
and 15 min (Fig. 1B). SHIP1, but not SHP-1, was therefore
detectably recruited by tyrosyl-phosphorylated Fc
RIIB1, and we found
no differential in vivo recruitment of phosphatases in a
mast cell line and in two B cell lines.
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Fig. 1.
Recruitment of SHIP1, but not SHP-1, by
Fc RIIB1 in mast cells and in B cells after
coaggregation with Fc
RI or BCR,
respectively. A, co-precipitation of phosphatases with
Fc
RIIB1 in RBL-2H3, IIA1.6 and K46µ transfectants. 6.5 × 107 RBL-2H3 transfectants expressing Fc
RIIB1 were
incubated with 2.4G2 F(ab')2, sensitized or not with IgE
anti-DNP, and challenged or not with TNP-MAR F(ab')2 for 5 min. 6.5 × 107 IIA1.6 and K46µ transfectants
expressing Fc
RIIB1 were stimulated with 0.3 µM RAM
F(ab')2 or RAM IgG for 5 min. B,
kinetics of phosphatase recruitment by Fc
RIIB1 in RBL transfectants.
6.5 × 107 RBL-2H3 transfectants expressing
Fc
RIIB1, incubated with 2.4G2 F(ab')2 and sensitized or
not with IgE anti-DNP, were challenged or not with TNP-MAR
F(ab')2 for the indicated periods of time. Cells were lysed
and Fc
RIIB were immunoprecipitated. Immunoprecipitated material was
fractionated by SDS-PAGE, transferred onto Immobilon, and Western
blotted with anti-Fc
RIIB, anti-Tyr(P), anti-SHIP1, and anti-SHP-1
antibodies. Whole cell lysates (WCL) corresponding to 5 × 105 cells were used as positive controls for Western
blotting with anti-phosphatase antibodies.
RIIB ITIM Than Binding of SHIP1--
Such a selective in
vivo recruitment of phosphatases by tyrosyl-phosphorylated
Fc
RIIB1 was in marked contrast with the ability of phosphorylated
synthetic peptides corresponding to the Fc
RIIB ITIM to bind in
vitro not only to the two SHIPs, but also to the two SHPs, when
bound to agarose beads. In an attempt to understand this discrepancy,
we reasoned that, among other differences between the two types of
experiments, the proportion of Fc
RIIB1 whose ITIM was phosphorylated
in vivo was unknown, whereas all ITIM peptides used to coat
agarose beads for in vitro experiments were phosphorylated.
To evaluate the possibility that variations in the intensity of ITIM
phosphorylation might differentially affect the affinity for SHIPs and
for SHPs, we coated beads with a constant amount of ITIM peptides, in
which the proportion of phosphorylated peptides (pITIM) varied from 100 to 0%. These were incubated with cell lysate from RBL-2H3 cells, and
the binding of SHIP1 and SHP-1 was examined by Western blotting. As
previously observed, no phosphatase precipitated with beads coated with
nonphosphorylated ITIM (0% pITIM) and both phosphatases precipitated
with beads coated with 100% pITIM. SHIP1 precipitation did not
detectably decrease with the proportion of pITIM until beads were
coated with less than 12% pITIM, and a detectable amount of
phosphatase remained precipitated by beads coated with 6% pITIM. By
contrast, SHP-1 precipitation progressively decreased with the
proportion of pITIM coated to beads and it was not detected when beads
were coated with less than 25% pITIM (Fig.
2A). Beads coated with ITIM
peptides, a small proportion of which were phosphorylated, therefore
bound in vitro SHIP1 but not SHP-1, i.e.
displayed the same selectivity for SHIP1 as tyrosyl-phosphorylated
Fc
RIIB in vivo.
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Fig. 2.
Differential in vitro
binding of SHP-1 and SHIP1 to phosphorylated
Fc RIIB ITIM. A, SHIP1 and
SHP-1 binding to beads coated with Fc
RIIB ITIM peptides in which the
proportion of phosphorylated peptides (pITIM) varied. 12.5-µl agarose
beads were coated with 3.2 nmol of a mixture of nonphosphorylated and
phosphorylated ITIM peptides (pITIM), in variable proportions. Beads
were incubated with RBL-2H3 cell lysate corresponding to 1 × 107 cells. Precipitated material was fractionated by
SDS-PAGE, transferred onto Immobilon and Western blotted with
anti-SHIP1 and anti-SHP-1 antibodies. B, SHIP1 and
SHP-1 binding to GST-ITIM and GST-ICIIB1' phosphorylated in
vitro by Lyn for increasing periods of times. Agarose beads coated
with GST fusion proteins containing either the same ITIM containing 13 amino acids as synthetic peptides (GST-ITIM) or the whole
intracytoplasmic domain of the Fc
RIIB1' isoform (GST-ICIIB1') were
incubated for increasing periods of time with Lyn. Beads were incubated
with RBL-2H3 cell lysate corresponding to 1 × 107
cells. Precipitated material and whole cell lysate (WCL)
were fractionated by SDS-PAGE, transferred onto Immobilon, and Western
blotted with anti-GST, anti-Tyr(P), anti-SHIP1, and anti-SHP-1
antibodies.
RIIB1' isoform
(GST-ICIIB1') or only the same ITIM-containing 13 amino acids as
synthetic peptides (GST-ITIM). Purified proteins were in
vitro phosphorylated by purified Lyn for various periods of time,
incubated in RBL lysate, and their ability to precipitate SHIP1 and
SHP-1 was assessed by Western blotting. When submitted to in
vitro kinase assay with Lyn, GST alone failed to be phosphorylated and recruited no phosphatase (data not shown). GST-ICIIB1' and GST-ITIM
were phosphorylated by Lyn, and phosphorylation increased as a function
of time. No phosphatase was precipitated by nonphosphorylated proteins.
Both GST-ITIM and GST-ICIIB1' precipitated SHIP1 and SHP-1 when heavily
phosphorylated, but SHIP1 only when less phosphorylated (Fig.
2B). A tyrosyl-phosphorylated intact Fc
RIIB
intracytoplasmic domain could therefore bind not only SHIP1 but also
SHP-1 in vitro. SHP-1 binding, however, required a higher
degree of phosphorylation than SHIP1 binding. The degree of ITIM
phosphorylation seems therefore to determine the in vitro
binding for SHP-1 rather than the presence or absence of sequences
flanking the phosphorylated Fc
RIIB ITIM.
RIIB pITIM and incubated either with
RBL cell lysates or with purified SH2 domain-containing GST fusion
proteins. These were the SH2 domain of SHIP1 (GST-SH2 SHIP1), the two
SH2 domains of SHP-1 (GST-SH2 (N+C) SHP-1), the N-terminal SH2 domain
of SHP-1 (GST-SH2 (N) SHP-1), or the C-terminal SH2 domain of SHP-1
(GST-SH2 (C) SHP-1). Phosphatases precipitated from cell lysates were
identified by Western blotting with anti-SHIP1 and anti-SHP-1
antibodies. GST fusion proteins precipitated were identified by Western
blotting with anti-GST antibodies. SHIP1 precipitation was detected
with beads coated with as little as 0.2 nmol of pITIM and slowly
increased with the amount of pITIM coated to beads. SHP-1 precipitation
became detectable with beads coated with 0.4 nmol of pITIM and rapidly
increased with the amount of pITIM. Parallel variations in binding were
observed for GST-SH2 SHIP and GST-SH2 (N+C) SHP-1. No binding of
GST-SH2 (N) SHP-1 was detectable, whatever the amount of pITIM coated
to beads, and a faint binding of GST-SH2 (C) SHP-1 was observed for
beads coated with high amounts of pITIM only (Fig.
3A). The differential binding
of phosphatases, in a cell lysate, can therefore be reproduced using
GST-SH2 fusion proteins. The single SHIP1 SH2 domain bound readily to
ITIM-coated beads, but not isolated SHP-1 SH2 domains. The two SHP-1
SH2 domains, however, bound with a similar pattern as SHP-1 in a cell
lysate, indicating the requirement for a cooperative binding between
the two SHP-1 SH2 domains.
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Fig. 3.
Cooperative binding of the two SH2 domains of
SHP-1 and effect of the density of pITIMs on beads. A,
binding of SHIP1 and SHP-1 SH2 domains to beads coated with variable
amounts of pITIM. 12.5-ml agarose beads were coated with increasing
amounts of pITIM. pITIM-coated beads were incubated in the same
experiment either with RBL-2H3 cell lysate or with SH2
domain-containing GST fusion proteins. These were the SH2 domain of
SHIP1 (GST-SH2 SHIP), the two SH2 domains of SHP-1 (GST-SH2 (N+C)
SHP-1), the N-terminal SH2 domain of SHP-1 (GST-SH2 (N) SHP-1), or the
C-terminal SH2 domain of SHP-1 (GST-SH2 (C) SHP-1). Precipitated
material was fractionated by SDS-PAGE and transferred onto Immobilon.
Precipitates from cell lysates and whole cell lysate (WCL)
were Western blotted with anti-SHIP1 and anti-SHP-1. Precipitates from
fusion proteins were Western blotted with anti-GST antibodies. Aliquots
of GST fusion proteins used for incubation were electrophoresed and
Western blotted with anti-GST antibodies to control the relative
amounts of fusion proteins. B, binding of SHIP1 and SHP-1 to
beads coated with Fc RIIB pITIM at variable densities. 12.5 ml or 50 ml of agarose beads were incubated with the same amounts of pITIM.
Beads were incubated with RBL-2H3 cell lysates corresponding to 1 × 107 cells. Precipitated material and WCL were
fractionated by SDS-PAGE, transferred onto Immobilon, and Western
blotted with anti-SHIP1 and anti-SHP-1 antibodies.
RIIB Phosphorylation by Increasing the
Concentration of Immune Complexes Does Not Enable SHP-1 to Be
Recruited--
Based on the above results, we wondered 1) whether the
intensity of Fc
RIIB phosphorylation would increase with the
concentration of extracellular ligands used to coaggregate Fc
RIIB
with Fc
RI in mast cells, or with BCR in B cells, and 2) whether
phosphorylation levels induced under these conditions might enable
Fc
RIIB to recruit SHP-1 in vivo.
RIIB phosphorylation by stimulating
Fc
RIIB1-transfected IIA1.6 cells with increasing concentrations of
RAM IgG and, as negative controls, with the same molar concentrations of RAM F(ab')2. Fc
RIIB1 were immunoprecipitated, their
phosphorylation was assessed by Western blotting with
anti-phosphotyrosine antibodies, and co-precipitated phosphatases were
examined by Western blotting with anti-phosphatase antibodies.
Fc
RIIB1 phosphorylation induced by a RAM IgG concentration as high
as 2.4 µM (360 µg/ml) was not higher than Fc
RIIB1
phosphorylation induced by 0.3 µM (45 µg/ml) RAM IgG.
Comparable amounts of SHIP1 co-precipitated with phosphorylated Fc
RIIB1, whatever the concentration of RAM IgG, but no detectable SHP-1 (Fig. 4).
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Fig. 4.
Absence of co-precipitation of SHP-1 with
Fc RIIB1 in IIA1.6 transfectants stimulated
with increasing concentrations of RAM IgG. 6.5 × 107 IIA1.6-Fc
RIIB1 cells were stimulated with increasing
concentrations of RAM F(ab')2 or IgG. Cells were lysed and Fc
RIIB
were immunoprecipitated. Immunoprecipitated material and whole cell
lysate (WCL) were fractionated by SDS-PAGE, transferred onto
Immobilon, and Western blotted with anti-Fc
RIIB, anti-Tyr(P),
anti-SHIP1, and anti-SHP-1 antibodies.
RIIB, to coaggregate these receptors with
ITAM-bearing receptors in mast cells and B cells. Fc
RI and Fc
RIIB
expressed constitutively in BMMCs (42) were coaggregated by
challenging cells, sensitized with increasing concentrations of mouse
anti-DNP IgE, with immune complexes made with increasing concentrations
of DNP-BSA and of a monoclonal mouse IgG1 anti-DNP antibody. Fc
RIIB phosphorylation increased with the concentration of
IgE used for sensitization and with the concentration of IgG in immune
complexes. It also varied with the concentration of DNP-BSA in immune
complexes, and optimal concentrations depended on the concentration of
IgG antibodies (Fig. 5A).
Likewise, Fc
RIIB were coaggregated with BCR in
Fc
RIIB1-transfected K46µ B cells, which express an anti-NP BCR, by
incubating cells with immune complexes made of increasing
concentrations of polyclonal rabbit IgG anti-DNP and of NP-BSA-TNP.
Fc
RIIB1 phosphorylation varied with the concentration of IgG
antibody and antigen: it peaked with higher concentrations of antigen
as the antibody concentrations increased. Immune complexes that induced
the highest Fc
RIIB1 phosphorylation were made of 10 µg/ml IgG and
1 µg/ml NP-BSA-TNP (Fig. 5B).
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Fig. 5.
Absence of co-precipitation of SHP-1 with
Fc RIIB in cells stimulated with increasing
concentrations of immune complexes. A, Fc
RIIB
phosphorylation in BMMCs stimulated with increasing concentrations of
ligands. 1 × 107 BMMCs were sensitized with a
constant (1/10; left panel) or various dilutions of mouse
IgE anti-DNP supernatant (right panel). Cells were then
stimulated for 5 min with preformed immune complexes made with a
constant (20 µg/ml; right panel) or increasing
concentrations of DNP-BSA (left panel) and with increasing
concentrations of monoclonal mouse IgG1 anti-DNP antibody.
B, Fc
RIIB phosphorylation in K46µ-Fc
RIIB1
stimulated with increasing concentrations of ligands. 1 × 107 K46µ transfectants expressing both an anti-NP BCR and
Fc
RIIB1 (K46µ-Fc
RIIB1) were stimulated for 5 min with preformed
immune complexes made with increasing concentrations of NP-BSA-TNP and
with increasing concentrations of rabbit polyclonal IgG anti-DNP.
C, SHIP1 and SHP-1 co-precipitation when Fc
RIIB is
optimally phosphorylated by immune complexes. 6.5 × 107 BMMCs sensitized with mouse IgE anti-DNP supernatant
(diluted 1/10) were stimulated or not with immune complexes made with 5 µg/ml DNP-BSA and 50 µg/ml mouse IgG1 anti-DNP (final
concentrations). K46µ-Fc
RIIB1 cells were stimulated for 5 min with
10 µg/ml rabbit IgG anti-DNP or with immune complexes made with 1 µg/ml NP-BSA-TNP and 10 µg/ml rabbit IgG anti-DNP (final
concentrations). For comparison, K46µ transfectants were also
stimulated with 0.3 µM RAM IgG or F(ab')2.
BMMCs and K46µ transfectants were lysed after stimulation, and
Fc
RIIB were immunoprecipitated. Immunoprecipitated materials and
whole cell lysate from K46µ transfectants (WCL) were
fractionated by SDS-PAGE, transferred onto Immobilon, and Western
blotted with anti-Fc
RIIB, anti-Tyr(P) (A, B, and
C), anti-SHIP1 and anti-SHP-1 antibodies
(C).
RIIB1-transfected K46µ cells stimulated with concentrations of
antigen and antibodies in the range of those which induced maximal
Fc
RIIB phosphorylation. For comparison, K46µ transfectants were
also stimulated with RAM IgG or F(ab')2 under the same
conditions as in Fig. 1. In both cell types, Fc
RIIB were markedly
phosphorylated following stimulation with immune complexes. Fc
RIIB
phosphorylation was of comparable magnitude in K46µ cells stimulated
with immune complexes or with RAM IgG. SHIP1 co-precipitated with
phosphorylated Fc
RIIB both in mast cells and in B cells, but not
SHP-1 (Fig. 5C). The above results altogether
indicate that even when using concentrations of ligands that were
optimal for Fc
RIIB phosphorylation, SHIP1 but not SHP-1
co-precipitation was detectable, both in mast cells and in B cells.
RIIB Phosphorylation following Pervanadate Treatment Enables
SHP-1 Recruitment--
A possible reason explaining the absence of
detectable recruitment of SHP-1 by Fc
RIIB phosphorylated following
stimulation with high concentrations of extracellular ligands was that
Fc
RIIB phosphorylation levels reached under these conditions were
not high enough. We therefore compared the effect of coaggregating Fc
RIIB with Fc
RI, in RBL transfectants, or with BCR, in IIA1.6 and K46µ transfectants, and of treating the same three cells with pervanadate.
RIIB1'-expressing RBL cells were sensitized with mouse IgE
anti-DNP and incubated with 2.4G2 F(ab')2, treated or not
treated with pervanadate and challenged or not with
TNP-MAR-F(ab')2. Likewise, Fc
RIIB1-expressing IIA1.6 and
K46µ cells were treated or not with pervanadate and challenged with
RAM F(ab')2 or IgG. Fc
RIIB phosphorylation and
phosphatase recruitment were assessed as in previous experiments. As
expected, Fc
RIIB phosphorylation was induced by coaggregating
Fc
RIIB1' with Fc
RI in RBL transfectants, and by coaggregating
Fc
RIIB1 with BCR in IIA1.6 and K46µ transfectants. In all three
cells, pervanadate treatment alone induced a much higher level of
Fc
RIIB phosphorylation that did not further increase by
coaggregating Fc
RIIB with Fc
RI or with BCR. SHIP1 co-precipitated with Fc
RIIB phosphorylated following their coaggregation with Fc
RI or with BCR. SHIP1 co-precipitated also with Fc
RIIB
phosphorylated following treatment of cells with pervanadate (in higher
amounts than following coaggregation of Fc
RIIB with Fc
RI, in RBL
cells, or with BCR, in K46µ cells). SHP-1 did not co-precipitate with Fc
RIIB phosphorylated following their coaggregation with Fc
RI or
BCR. SHP-1, however, co-precipitated with Fc
RIIB phosphorylated following pervanadate treatment in all three cells (Fig.
6A). Treating
Fc
RIIB1-expressing RBL transfectants with decreasing concentrations
of pervanadate induced a dose-dependent tyrosyl phosphorylation of Fc
RIIB1. Interestingly, as Fc
RIIB1
phosphorylation decreased, the co-precipitation of SHP-1 was lost
before that of SHIP1 (Fig. 6B). Treating cells with
pervanadate, but not coaggregating Fc
RIIB with ITAM-bearing
receptors, could therefore induce a phosphorylation of Fc
RIIB that
was high enough to enable the recruitment of SHP-1.
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Fig. 6.
Co-precipitation of SHP-1 with
Fc RIIB phosphorylated following pervanadate
treatment. A, co-precipitation of phosphatases with
Fc
RIIB1 in mast cell and B cell transfectants. 6.5 × 107 Fc
RIIB1'-expressing RBL cells were sensitized with
mouse IgE anti-DNP and incubated with 2.4G2 F(ab')2 before
they were treated or not with 100 µM pervanadate for 15 min. During the last 5 min of pervanadate treatment, cells were
stimulated (coaggregation +) or not (coaggregation
) with TNP-MAR
F(ab')2. 6.5 × 107 IIA1.6-Fc
RIIB1 or
K46µ-Fc
RIIB1 transfectants were treated or not with pervanadate
and stimulated (coaggregation +) or not (coaggregation
) with RAM
IgG, under the same conditions as RBL cells. B,
dose-dependent co-precipitation of phosphatases
with Fc
RIIB1 in mast cell transfectants treated with pervanadate.
9.5 × 107 Fc
RIIB1-expressing RBL cells were
treated or not with the indicated concentrations of pervanadate for 15 min. Following stimulation, cells were lysed, and Fc
RIIB were
immunoprecipitated. Immunoprecipitated material from RBL, IIA1.6, and
K46µ transfectants were fractionated by SDS-PAGE, transferred onto
Immobilon, and Western blotted with anti-Fc
RIIB, anti-Tyr(P),
anti-SHIP1, and anti-SHP-1 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIB recruit the inositol
5-phosphatase SHIP1, but not the protein-tyrosine phosphatase SHP-1 in vivo, although the Fc
RIIB ITIM has an affinity for
both phosphatases in vitro, because the binding of SHP-1
requires a higher degree of Fc
RIIB phosphorylation than the binding
of SHIP1. The same phosphorylation-dependent preference for
SHIP1 was observed 1) in vitro using beads coated with
suboptimal concentrations of pITIM, 2) in vitro using
GST-ICIIB1' phosphorylated by Lyn for short periods of time, 3)
in vivo using phosphorylated Fc
RIIB precipitated from
cells treated with low concentrations of pervanadate, and 4) in
vivo, when Fc
RIIB was phosphorylated following coaggregation with BCR or Fc
RI, in B cells and in mast cells, respectively. Our
results suggest that, depending on their level of phosphorylation, Fc
RIIB could potentially use the two phosphatases, with different consequences.
RIIB ITIM has an
affinity for SH2 domain-containing phosphatases was first provided in
1995 by D'Ambrosio et al. (30) who demonstrated that
phosphorylated synthetic peptides containing the Fc
RIIB ITIM
precipitated several molecular species from
[35S]methionine-labeled cell lysates, one of which was
identified as SHP-1. Other molecules precipitated by these peptides
were subsequently shown to be SHP-2 (28) and SHIP1 (26). Similar experiments confirmed D'Ambrosio's results (28, 32). SHIP2, a second
SH2 domain-containing inositol phosphatase was recently found to bind
also to phosphorylated Fc
RIIB ITIM (27, 29). It follows that
phosphorylated Fc
RIIB ITIM peptides can bind all four known SH2
domain-containing phosphatases in vitro.
RIIB bearing an intact ITIM, following coaggregation with BCR in A20 and in IIA1.6 B cells reconstituted with Fc
RIIB, and that Fc
RIIB-dependent
inhibition of B cell proliferation was impaired in B cells from
SHP-1-deficient motheaten mice. In 1996, Ono et al. (26)
reported that SHIP1 co-precipitated with Fc
RIIB following
coaggregation with Fc
RI in BMMCs or with BCR in A20 cells, and that
Fc
RIIB-dependent inhibition of IgE-induced serotonin
release was unaffected in BMMCs derived from motheaten mice. Fong
et al. (32) reported that SHIP1, but not SHP-1 or SHP-2,
co-precipitated with Fc
RIIB following coaggregation with Fc
RI in
BMMCs. In 1997, Ono et al. (33) showed that
Fc
RIIB-dependent inhibition of Ca2+
responses and of NF-AT activity was abolished in SHIP1-deficient, but
not in SHP-1-deficient, DT40 chicken B cells, and that SHIP1, but not
SHP-1, was detectably co-precipitated with Fc
RIIB following coaggregation with BCR in A20 cells. In 1998, however, Sato et al. (31) observed the co-precipitation of both SHIP1 and SHP-1 with Fc
RIIB in A20 cells expressing an anti-TNP BCR following coaggregation with intact anti-idiotypic antibodies. Contrasting with
the consensus that Fc
RIIB recruit SHIP1 both in B cells and in mast
cells, their ability to recruit SHP-1 in vivo therefore remains controversial.
RIIB in
B cells, but not in mast cells, suggested the possibility that some
discrepancies might be accounted for by a cell type-specific differential in vivo recruitment. To address this issue, we
examined the co-precipitation of SHP-1 with recombinant Fc
RIIB1
stably expressed by transfecting the same cDNA into the rat mast
cells RBL-2H3, and into the two Fc
RIIB-deficient mouse lymphoma B
cells IIA1.6 and K46µ. The coaggregation of Fc
RIIB1 with Fc
RI
or with BCR induced a comparable tyrosyl phosphorylation of Fc
RIIB1
and the co-precipitation of SHIP1, but not of SHP-1, in all three cells. The same result, observed at 5 min in the three cell lines, was
also observed between 15 s and 15 min in RBL cells. Failure to
detect SHP-1 co-precipitation cannot be accounted for an insufficient sensitivity of Western blotting because traces of SHP-1 could be seen
on overexposed films, but in equal amounts in unstimulated and in
stimulated cells (data not shown). Due to experimental conditions
inherent to the co-precipitation technique, however, we cannot exclude
that SHP-1, possibly recruited by Fc
RIIB in vivo was
lost. Whatever the explanation, we observed no difference between the
three cells examined in which Fc
RIIB preferentially, if not
exclusively, recruited SHIP1. There is therefore a discrepancy between
the ability of SHP-1 and SHIP1 to bind in vitro to Fc
RIIB pITIM peptides and to co-precipitate with phosphorylated Fc
RIIB in vivo.
RIIB are phosphorylated following coaggregation with ITAM-bearing receptors. To explore the
possible role of quantitative differences in ITIM phosphorylation, we
studied the binding of SHIP1 and SHP-1 to beads coated with Fc
RIIB
ITIMs phosphorylated in varying proportions and incubated in a cell
lysate. We found that SHP-1 binding decreased more sharply with the
proportion of pITIM than SHIP1 binding, so that beads coated with 12%
pITIM bound selectively SHIP1. Comparable results were obtained when
incubating beads coated with increasing concentrations of pITIM with
GST fusion proteins containing the SH2 domain of SHIP1 or the two SH2
domains of SHP-1. The use of SH2 domains permitted comparison between
the binding of the two molecules using the same anti-GST antibodies for
blotting. It also excluded that phosphatase binding was mediated by
unknown intermediates present in cell lysates. Supporting these
results, the Fc
RIIB pITIM was reported to have a higher affinity for
the SH2 domain of SHIP1 than for the two SH2 domains of SHP-1, when
measured by Biacore analysis (43). Results obtained in these two sets of experiments are reminiscent of the selective in vivo
recruitment of SHIP1 by Fc
RIIB.
RIIB1 (29). To answer this question, we compared the
ability of GST fusion proteins containing the Fc
RIIB ITIM only or
the whole intracytoplasmic domain of Fc
RIIB1' to bind SHIP1 and
SHP-1, when incubated with a cell lysate, following their
phosphorylation with Lyn for various periods of time. No difference was
observed between the two fusion proteins and, like the isolated ITIM,
the intracytoplasmic domain of Fc
RIIB could bind SHP-1 when high
enough phosphorylated.
RIIB phosphorylation sufficient to enable them
to recruit SHP-1 in vivo. To this aim, we used several
extracellular ligands including IgG immune complexes that are the
physiological ligands of Fc
RIIB, at various concentrations, in mast
cells and in B cells. We found that indeed, the phosphorylation of
Fc
RIIB varied with the concentrations of antigen and antibody in
immune complexes but that ligands which induced a maximal
phosphorylation of Fc
RIIB readily induced the recruitment of SHIP1
but failed to induce a detectable recruitment of SHP-1. Based on our
results of in vitro binding with pITIM-coated beads, this
suggests that a small proportion (less than 12%?) of Fc
RIIB become
tyrosyl phosphorylated in vivo upon coaggregation with
ITAM-bearing receptors by physiological ligands. If so, we wondered
whether Fc
RIIB phosphorylation would reach a level high enough to
enable the recruitment of SHP-1 following treatment of cells with pervanadate.
RIIB phosphorylation than coaggregation with
Fc
RI or BCR, respectively, and under these conditions, not only
SHIP1 but also SHP-1 co-precipitated with phosphorylated Fc
RIIB.
This observation indicates that, in resting cells, Fc
RIIB are
tyrosyl phosphorylated but that protein-tyrosine phosphatases maintain
this phosphorylation below the detection level. This implies that
Fc
RIIB are the substrates of both protein-tyrosine kinases and
phosphatases and that, under resting conditions, the effect of
phosphatases is dominant over that of kinases. Fc
RIIB phosphorylation observed following their coaggregation with
ITAM-bearing receptors results from the additional effect of a Src
kinase, brought by activating receptors (13), leading to a displacement of the balance so that the effect of kinases becomes dominant over that
of phosphatases. It should be emphasized that the higher intensity of
Fc
RIIB phosphorylation induced by pervanadate, compared with
phosphorylation induced by coaggregation, may be due to the phosphorylation of a higher number of receptors and/or to the phosphorylation of a higher number of tyrosine residues in each receptor. The recruitment of SHP-1 by Fc
RIIB phosphorylated after pervanadate treatment may indeed simply be explained by a
quantitatively different phosphorylation, resulting in an increased
density of phosphorylated ITIMs that might permit the binding in
trans of the two SHP-1 SH2 domains. Supporting this
interpretation, the recruitment of SHP-1 was lost before that of SHIP1
when pervanadate-induced Fc
RIIB phosphorylation decreased following
treatment of cells with decreasing concentrations of pervanadate.
Alternatively, the recruitment of SHP-1 after pervanadate treatment may
be explained by a qualitatively different phosphorylation of Fc
RIIB,
enabling SHP-1 to be recruited through the binding in cis of
its two SH2 domains to two tyrosines borne by the same receptor. The
intracytoplasmic domain of Fc
RIIB1 contains four tyrosine residues.
Supporting this possibility, the recruitment of SHP-1 by KIR2DL3 was
found to require the conservation of its two ITIMs (39) and all
ITIM-bearing receptors that were shown to recruit SHPs in
vivo bear more than one ITIM. Finally, SHP-1 may co-precipitate
with Fc
RIIB phosphorylated following pervanadate treatment because,
under these conditions, the enzymatic activity of the phosphatase is
inhibited. Several ITIM-bearing receptors were shown both to recruit
and to be the substrates of SHP-1 (45) or SHP-2 (46, 47). If recruited by phosphorylated Fc
RIIB under physiological conditions, SHP-1 might
thus decrease Fc
RIIB phosphorylation, thereby giving an advantage
for the recruitment of SHIP over that of SHP-1.
RIIB phosphorylated upon
coaggregation with ITAM bearing receptors. This hypothesis would also
endow Fc
RIIB with additional regulatory properties. These receptors
could indeed transiently recruit SHP-1 which could dephosphorylate not
only ITIMs, but also ITAMs and other signaling molecules whose
phosphorylation is critical for positive signaling. This would have
important consequences. By displacing the balance between kinases and
phosphatases recruited to the receptor complex in favor of the latter,
it would increase the signaling threshold and/or dampen activation
signals. Fc
RIIB might thus use two different mechanisms,
i.e. SHIP-mediated and SHP-mediated, to adjust negative regulation to the intensity of extracellular signals. By decreasing positive signals, SHP-1 would in turn decrease ITIM phosphorylation bringing Fc
RIIB back to conditions under which they recruit SHIPs. It remains to be determined whether Fc
RIIB could be phosphorylated enough to recruit SHP-1 under physiological or pathological situations such as diseases associated with exaggerated antibody responses or
immune complexes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John C. Cambier for K46µ cells
expressing an anti-NP BCR, NP-BSA-TNP, and polyclonal anti-C-terminal
sequences of SHP-1 antibodies, Prof. Catherine Sautès-Fridman for
polyclonal antibodies to the extracellular domains of FcRIIB, Odile
Malbec (Institut Curie, Paris, France) for GST-ICIIB1', Dr. Eric Vivier for SHP-1 SH2 domain-containing GST fusion proteins, and Dr. Jean-Luc Teillaud for monoclonal anti-GST antibodies. We acknowledge the expert
assistance of Zosia Maciorowski for cells sorting.
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FOOTNOTES |
---|
* This work was supported in part by the Institut National de la Santé et de la Recherche Médicale, the Institut Curie, and the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Ministère de
l'Enseignement Supérieur et de la Recherche.
§ To whom correspondence should be addressed: Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U. 255, Institut Curie, 26 rue d'Ulm, 75005 Paris, France. Tel.: 33-1-4432-4223; Fax: 33-1-4051-0420; E-mail: Marc.Daeron@curie.fr.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M006537200
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ABBREVIATIONS |
---|
The abbreviations used are:
ITAM, immunoreceptor
tyrosine-based activation motif;
ITIM, immunoreceptor tyrosine-based
inhibition motif;
BCR, B cell receptor for antigen;
BMMCs, bone
marrow-derived mast cells;
BSA, bovine serum albumin;
DNP, dinitrophenyl;
FcRI, high-affinity receptors for the Fc portion of
IgE;
Fc
RIIB, low-affinity receptors for the Fc portion of IgG;
GAM, goat anti-mouse Ig;
GAR, goat anti-rabbit Ig;
GST, gluthatione
S-transferase;
HRP, horseradish peroxidase;
MAR, mouse
anti-rat Ig;
pITIMs, phosphorylated ITIMs;
NP, 3-nitro-4-hydroxyphenyl
acetic acid;
RAM, rabbit anti-mouse Ig;
SH2, Src homology-2 domains;
SHIPs, SH2 domain-containing inositol 5-phosphatases;
SHPs, SH2
domain-containing protein-tyrosine phosphatases. TNP, trinitrophenyl;
PAGE, polyacrylamide gel electrophoresis.
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