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RIIB1 Activates Src Homology 2-Containing Protein Tyrosine Phosphatase-11
The John P. Robarts Research Institute and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB1 (CD32) is a receptor that binds the Fc domain of
Ag-complexed IgG. Coaggregation of B cell receptor (BCR) and FcRIIB1
generates a dominant negative signal that inhibits B cell activation.
In Ag-specific Id-positive B cells, the co-cross-linking of BCR and
FcRIIB1 by anti-Id Ab resulted in the association of both Src
homology 2-containing protein tyrosine phosphatase (SHP-1) and Src
homology 2-containing inositol phosphatase (SHIP) with the FcRIIB1;
however, only SHIP activity was detected. "Superclustering" of the
BCR and FcRIIB1 complex induced by stimulation with anti-Id Ab
plus polyvalent Ag synergistically activated SHP-1. The degree of
co-cross-linking between BCR and FcRIIB1 may determine the
activation status of SHP-1 and SHIP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB1 receptor, CD22, and more
recently PIR-B (paired Ig-like receptor B) generate inhibitory signals
that antagonize BCR-mediated activation signals (4, 5, 6, 7, 8, 9, 10, 11). FcRIIB1
inhibitory signaling is initiated by binding of IgG immune complexes,
thus making such immune complexes a potent immunosuppressive reagent in
vivo (12, 13).
The studies of FcRIIB1-deficient mice showed that humoral and
anaphylactic responses to antigenic challenge were augmented in these
mice (14). These observations reinforced the importance of dominant
negative signals transduced by this receptor in vivo. Upon
coaggregating FcRIIB1 with
BCR,3 SHP-1 and SHIP were
found to associate with the tyrosine-phosphorylated immunoreceptor
tyrosine-based inhibitory motif (pITIM) of the FcRIIB1 receptor (7, 15), suggesting that both SHP-1 and SHIP contribute to generation of
the dominant negative signals. Indeed, B cells of mice carrying
noncatalytic SHP-1 (motheaten mutations) were hyper-responsive to BCR
stimulation (16).
Previous studies of SHP-1 in FcRIIB1-stimulated B cell lines
paradoxically demonstrated that coaggregation of FcRIIB1 with BCR
only marginally altered the tyrosine phosphorylation levels of cellular
proteins while simultaneously promoting the association of SHP-1 with
FcRIIB1 (7, 17, 18, 19). Interestingly, coaggregation of FcRIIB1 with
BCR resulted in the activation of p53/56lyn
and Syk, which plays an obligatory role in B cell activation (20).
Recent studies of SHP-1-deficient B cells further indicated that SHP-1
is unlikely to be an essential molecule for FcRIIB1-mediated
inhibitory signaling (21, 22). Accordingly, the role of SHP-1 in
dominant negative signals induced by FcRIIB1 remains largely
elusive.
In this communication we have employed A20/2J B lymphoma cells
transfected with TNP-specific BCR, termed A20/2J HLTNP, as
the B cell model to investigate the function of SHP-1 and SHIP in
FcRIIB1 inhibitory signaling. An anti-Id Ab was used to
coaggregate TNP-specific BCR with FcRIIB1. To enhance the
co-cross-linking of BCR and FcRIIB1, cells were also incubated with
the polyvalent Ag, TNP22-BSA. Our data indicate that activation of
SHP-1 is dependent upon a high degree of clustering
("superclustering") induced by coaggregation of FcRIIB1 with BCR
using anti-Id Ab and additional co-cross-linking with TNP22-BSA. In
A20/2J HLTNP cells dually stimulated with anti-Id Ab
and TNP22-BSA, tyrosine phosphorylation of cellular proteins was
greatly reduced. Immunoprecipitated FcRIIB1 of these same cells
dephosphorylated activated p53/56lyn. These
data show that SHP-1 is potentially a molecular element of FcRIIB1
signaling that inhibits the BCR-complexed protein tyrosine kinases
(PTKs; e.g., p53/56lyn). The observation
provides further insight into FcRIIB1 signaling, in that the degree
of clustering of FcRIIB1-coaggregated BCR determines the extent of
dominant negative signaling in B cells: superclustering activates both
SHP-1 and SHIP-mediated signaling, whereas lower levels of clustering
only stimulate SHIP signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A20/2J B lymphoma (2a+, 
+,
H-2d, FcRIIB1+) (23) and its
FcRIIB1- mutant, IIA1.6 (24) were stably transfected
with a plasmid encoding the TNP-specific µ heavy chain and light
chain Ig genes (25) as described previously (26). The preparation of
TNP7-BSA, TNP22-BSA, anti-Id Ab, and F(ab')2 of
anti-Id Ab was described previously (26).
Immunoblotting
A20/2J HLTNP and IIA1.6 HLTNP cells
(107) were incubated with 10 µg/ml TNP22-BSA in the
presence or the absence of 50 µg/ml anti-Id Ab or control IgG for
5 min. The cells were washed twice in cold PBS and resuspended in 200
µl of lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 137 mM
NaCl, 10% glycerol, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate). The nuclei and
the insoluble cell debris were removed by centrifugation at 4°C for
10 min at 14,000 x g. The postnuclear extracts were
collected and used as total cell lysates. Total cell lysates were used
as is or subjected to immunoprecipitation with
anti-p53/56lyn Ab or 2.4G2 mAb
(anti-FcRIIB1). Each sample for SDS-PAGE contained
105 cells extract when total cell lysate was used as is. To
prepare immunoprecipitated samples, Ab-Ag complexes were collected by
protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and
washed three times with lysis buffer. Total cell lysates or
immunoprecipitates were suspended in 2x SDS sample buffer (313 mM
Tris-HCl (pH 6.8), 10% SDS, 2% 2-ME, 50% glycerol, and 0.01%
bromophenol blue) and heated at 95°C for 3 min. In some experiments,
postimmunoprecipitation supernatants were used as Ag-precleared total
cell lysates. Protein samples were fractionated by 10 or 12% SDS-PAGE,
and transferred onto polyvinylidene difluoride (PVDF) membranes
(Millipore, Bedford, MA). Nonspecific Ab binding on the membrane was
blocked with 1% BSA and 0.1% Tween-20 in Tris-saline (10 mM Tris-HCl
(pH 7.4) and 100 mM NaCl) for 20 min at 37°C. The membrane was then
incubated for 1 h at room temperature with HRP-conjugated
anti-phosphotyrosine Ab (or the relevant Abs as required, followed
by HRP-conjugated secondary Abs) and washed for 15 min with 0.5%
Tween-20 in Tris-saline. The blot was visualized by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL). For some
experiments, membranes were stripped of primary Ab with 100 mM glycine
(pH 2.5) and 1 M NaCl, washed, and reprobed.
In vitro kinase assay
Cells (107) were incubated as described previously
(14), lysed with 200 µl of RIPA buffer (1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl,
10% glycerol, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1
mM PMSF, and 1 mM sodium orthovanadate), and the lysates were subjected
to immunoprecipitation with
anti-p53/56lyn Ab. To test the
specificity of anti-p53/56lyn Ab,
immunizing peptide (Santa Cruz Biotechnology) was used to block
immunoprecipitation. The samples were washed three times with RIPA
buffer and twice with kinase buffer (50 mM Tris-HCl (pH 7.4), 50 mM
NaCl, 10 mM MnCl2, 10 mM MgCl2, and 0.1 mM
sodium orthovanadate) before resuspension in 20 µl of kinase buffer
containing 10 µCi of [-32P]ATP (3000
Ci/mmol-1; DuPont-New England Nuclear, Boston, MA). The
mixture was incubated at 30°C for 10 min. Reactions were terminated
by addition of 20 µl of 2x SDS sample buffer. Samples were heated at
95°C for 3 min, separated by 10% SDS-PAGE, dried, and exposed to
film at -80°C for 2 h. To quantitate changes in activity, bands
corresponding to the autophosphorylated
p53/56lyn were scanned and analyzed using a
Molecular Imager (Bio-Rad, Richmond, CA).
SHP-1 assay
For analysis of protein tyrosine phosphatase activity of SHP-1, 2.4G2 mAb immunoprecipitates (prepared in lysis buffer without sodium orthovanadate) were incubated at 37°C for 30 min in 200 µl of phosphatase buffer (62 mM HEPES (pH 5.0), 6.25 mM EDTA, and 12.5 mM DTT) containing 25 mM p-nitrophenylphosphate (Sigma, St. Louis, MO). Reactions were terminated by addition of 0.8 ml of 1 N NaOH, and the absorbance measured at 410 nm by spectrophotometry. To assay dephosphorylation of autophosphorylated p53/56lyn by 2.4G2 mAb immunoprecipitates, anti-p53/56lyn Ab immunoprecipitates prepared from Ag-stimulated A20/2J HLTNP cells (108) were subjected to in vitro kinase reaction as described. The reaction was terminated by addition of 1 ml of cold phosphatase buffer, and the precipitates were recovered by centrifugation and resuspended in 250 µl of phosphatase buffer. 32P-phosphorylated anti-p53/56lyn Ab immunoprecipitates (107 cell equivalents) were mixed with 2.4G2 immunoprecipitates prepared from a separate group of untreated or stimulated A20/2J HLTNP cells. Addition of protein A/G agarose beads alone in the absence of 1 mM sodium orthovanadate was also performed as a control. After an overnight incubation at 37°C, the reaction was stopped by addition of 20 µl of 2x SDS sample buffer, and the protein samples were resolved by 10% SDS-PAGE, transferred onto PVDF membranes, and detected by autoradiography. In a similar experiment, nonradiolabeled anti-p53/56lyn Ab immunoprecipitates were incubated with 2.4G2 mAb immunoprecipitates for 24 h at 37°C, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was subsequently subjected to anti-phosphotyrosine Ab (RC20) Western blotting. To ensure equivalent amounts of p53/56lyn in each sample, the same membrane was probed with anti-p53/56lyn Ab and developed with HRP-conjugated goat F(ab')2 anti-rabbit IgG Ab by ECL.
SHIP assay
The inositol phosphatase activity of anti-SHIP Ab or 2.4G2 mAb immunoprecipitates was assayed by hydrolysis of [3H]inositol 1,3,4,5-tetrakisphosphate ([3H]Ins(1, 3, 4, 5)P4) as described previously (15) with some modifications. Anti-SHIP or 2.4G2 mAb immunoprecipitates prepared from untreated or stimulated cells (5 x 107) or protein A/G agarose alone controls were washed twice with inositol phosphatase buffer (50 mM Tris-HCl (pH 7.4) and 10 mM MgCl2) and resuspended in 25 µl of inositol phosphatase buffer containing 25 pCi of [3H]Ins(1, 3, 4, 5)P4 (21 Ci/mmol; DuPont-New England Nuclear). The reaction mixtures were incubated at 37°C for 30 min, and incubation was stopped by adding 500 µl of cold 2 mM LiCl. Samples were subjected to AG1-X8 formate column chromatography (Bio-Rad) with the column pre-equilibrated with 50 mM ammonium formate. Serial elutions were collected, starting at 0.4 M ammonium formate/0.1 M formic acid and ending at 1.2 M ammonium formate/0.1 M formic acid. Radioactivity in eluted fractions was measured in a liquid scintillation counter. Fractions containing [3H]InsP3 were detected according to an InsP3 standard sample ([3H]inositol 1,4,5-trisphosphate; 21 Ci/mmol; DuPont-New England Nuclear). The amounts (picomoles) of Ins(1, 3, 4)P4 were calculated based on the counts per minute values.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB1-dependent
fashion (26). To investigate whether the inhibitory effects associated
with anti-Id Ab correlated with modification of intracellular
signaling induced by Ag stimulation, we assessed total cellular
tyrosine phosphorylation in whole cell lysates (Fig. 1
). TNP22-BSA or anti-Id Ab
stimulation resulted in increased cellular tyrosine phosphorylation;
however, following dual stimulation with TNP22-BSA and anti-Id Ab,
the degree of tyrosine phosphorylation of cellular proteins was
dramatically weakened. The synergistic inhibition of cellular protein
tyrosine phosphorylation by TNP22-BSA and anti-Id Ab was contingent
upon the expression of FcRIIB1, as identical stimulation of an
FcRIIB1-negative mutant A20/2J transfectant, IIA1.6
HLTNP, resulted in protein tyrosine phosphorylation
equivalent to that observed in TNP22-BSA-stimulated cells. The data
suggest the presence of a unique inhibitory mechanism against PTKs in
TNP22-BSA- and anti-Id Ab-stimulated A20/2J HLTNP
cells.
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, A and B) and
autokinase assay (Fig. 2, C and D) of
p53/56lyn demonstrated that
p53/56lyn is inactive in TNP22-BSA- and
anti-Id Ab-stimulated A20/2J HLTNP cells, while
TNP22-BSA or anti-Id Ab alone resulted in tyrosine phosphorylation
and kinase activity of p53/56lyn. The
results indicated that anti-Id Ab simulates activation of
p53/56lyn as reported previously (20).
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RIIB1 (data not shown). As tyrosine phosphorylation of FcRIIB1
is a key step in triggering the inhibition of Ag-stimulated BCR
signaling, we hypothesized that FcRIIB1 may possess elevated
tyrosine phosphorylation levels in A20/2J HLTNP cells after
TNP22-BSA and anti-Id Ab stimulation. We therefore examined
FcRIIB1 tyrosine phosphorylation levels (Fig. 3A). In a manner similar to
previous reports that used anti-Ig Ab as the stimulating reagent by
coaggregating FcRIIB1 with BCR (7, 17, 18, 19), we observed an induction
of FcRIIB1 tyrosine phosphorylation in anti-Id Ab-stimulated
cells. The position of FcRIIB1 (
66 kDa) was determined by Western
blotting with an anti-FcRIIB1 Ab (14) (Fig. 3B).
Concomitant stimulation with TNP22-BSA caused a slight increase in
tyrosine phosphorylation. Furthermore, the data showed an increase
in the level of tyrosine phosphorylation of the proteins associated
with FcRIIB1 immunoprecipitated from dually stimulated A20/2J
HLTNP cells. Therefore, dual stimulation with TNP22-BSA and
anti-Id Ab exerts an additive effect that increased the tyrosine
phosphorylation of FcRIIB1 as well as recruitment of
tyrosine-phosphorylated proteins to the FcRIIB1.
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RIIB1 signaling (7, 15, 28).
Accordingly, the increased phosphorylation of FcRIIB1 observed in
TNP22-BSA- and anti-Id Ab-stimulated A20/2J HLTNP cells
may represent enhanced phosphorylation of ITIM, thereby augmenting the
degree of association between FcRIIB1 and these phosphatases. To
test this possibility, we have examined the association of SHP-1 with
FcRIIB1 (Fig. 3
C). While stimulation with anti-Id Ab
induced SHP-1 to associate with FcRIIB1, additive stimulation with
TNP22-BSA increased the association about twofold. Thus, the increase
in tyrosine phosphorylation of FcRIIB1 paralleled the increased
association of SHP-1 with FcRIIB1. We also investigated another
phosphatase, SHIP, which potentially complexes with the FcRIIB1 of
A20/2J HLTNP cells stimulated with anti-Id Ab or
TNP22-BSA and anti-Id Ab (Fig. 3D). We observed that
anti-Id Ab induced association of SHIP with FcRIIB1, and that
the association was enhanced by additive stimulation with TNP22-BSA.
The FcRIIB1-associated SHP-1 must be activated for this association
to be an effector mechanism in FcRIIB1 dominant negative signaling.
However, when we examined tyrosine-specific phosphatase activity in
FcRIIB1 immunoprecipitates, we consistently failed to demonstrate
SHP-1 activity in samples stimulated with anti-Id Ab alone (Fig. 4, top). Strikingly,
tyrosine-specific phosphatase activity was recorded in samples dually
stimulated with TNP22-BSA and anti-Id Ab. Preclearing of whole cell
lysates with anti-SHP-1 Ab, but not anti-SHP-2 Ab, eliminated
the phosphotyrosine-specific phosphatase activity from FcRIIB1
immunoprecipitates (Fig. 4, middle). Furthermore, TNP7-BSA
was less efficient than TNP22-BSA in the activation of SHP-1,
indicating that the degree of BCR clustering is a crucial factor in
determining FcRIIB1-associated phosphatase activity (Fig. 4, bottom). These results indicate that SHP-1 is not activated
by association with FcRIIB1, but a mechanism subsequently triggers
FcRIIB1-associated SHP-1 activity. The data strongly suggest that
FcRIIB1-associated SHP-1 is activated in A20/2J HLTNP
cells only by high levels of co-cross-linking between BCR and
FcRIIB1.
|
) and
FcRIIB1-associated SHP-1 activity, we have performed in vitro
phosphatase assays using 32P-autophosphorylated
p53/56lyn as a substrate. Both
autoradiography (Fig. 5, A and
B) and phosphotyrosine-specific Western blotting (Fig. 5, C and D) clearly indicated that only dual
stimulation with TNP22-BSA and anti-Id Ab caused the FcRIIB1
immunoprecipitate to tyrosine dephosphorylate
p53/56lyn. This result suggests that SHP-1 is
directly involved in the suppression of
p53/56lyn activity, a key PTK in BCR
activation signaling. In parallel studies of other PTKs, including Syk
and Blk, we found that the tyrosine phosphorylation and the activities
of these PTKs are also suppressed in A20/2J HLTNP cells
stimulated with TNP22-BSA and anti-Id Ab, but not in those
stimulated with anti-Id Ab alone (data not shown). SHP-1,
therefore, may be involved in the inactivation of multiple PTKs.
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RIIB1
immunoprecipitates after anti-Id Ab stimulation, while additive
stimulation with TNP22-BSA further increased SHIP activity (Fig. 6). Thus, there was a strong correlation
between the degree of association of SHIP with FcRIIB1 and its
resultant inositol phosphate-specific phosphatase activity.
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RIIB1 and BCR
co-cross-linking induced by dual stimulation with TNP22-BSA and
anti-Id Ab activated FcRIIB1-complexed SHP-1 (Fig. 4). It seems
evident from these observations that the activation requirements of
SHP-1 are different from those of SHIP in vivo. In fact, a molecular
study of SHP-1 indicated that of the two tandem SH2 domains, the
N-terminal SH2 domain regulated phosphatase activity, while the
C-terminal SH2 domain mediated docking with pITIM (28). In comparison,
cytosolic SHIP is constitutively active and possesses a single SH2
domain that regulates docking to tyrosine-phosphorylated proteins such
as FcRIIB1 or Shc (15, 17, 18, 19). Accordingly, upon docking of SHP-1
to the pITIM of FcRIIB1, the coupling of a second
tyrosine-phosphorylated motif to the N-terminal SH2 domain is required
for the induction of phosphatase activity. As tested in vitro, pITIM of
FcRIIB1 was a potential ligand for binding to the N-terminal SH2 of
SHP-1 (7); however, this SH2 domain appeared to possess a lower
affinity for pITIM of FcRIIB1 compared with the C-terminal SH2
domain (28). Therefore, a protein that is newly recruited to FcRIIB1
may possess a phosphotyrosine motif with a higher affinity for the
N-terminal SH2 of SHP-1. Indeed, the pY429 peptide of the
erythropoietin receptor cytoplasmic domain binds to N-terminal SH2 with
a greater affinity than pITIM of FcRIIB1 (28).
The mechanism of the observed superclustering of BCR and FcRIIB1 in
vivo is an intriguing question. In general, the coupling of BCR with
Ag-Ig immune complexes will be a cumulative event. Thus, it is probable
that in the initial stages of an encounter with immune complexes, SHIP
will be the sole mediator of FcRIIB1 signaling, whereas in later
stages, when numerous immune complexes occupy BCR and FcRIIB1 on the
B cell surface, both SHIP and SHP-1 will synergistically generate
dominant negative signals. It is noteworthy that treatment with
anti-Id Ab alone, which probably represents a low level clustering,
inhibited the internalization of BCR and Ag complex (26). The
inhibition of BCR internalization is likely to increase the amount of
Ag bound to the membrane BCR and may cause the subsequent
superclustering that triggers both SHP-1 and SHIP in B cells.
It is also probable that the concentrations of the Ag, the specific Ab (or anti-Id Ab), and the Ab isotype are all key factors in the generation of dominant negative signals in B cells. The potential role of SHP-1 and SHIP activation in B cell tolerance has important implications for immune regulation and could be further investigated using Ag-specific B cell transgenic mice.
Phosphatases have also been suggested as repressors of T cell and NK cell activation (29, 30, 31). Upon defining the mechanism of phosphatase-mediated repression of B cells, the fundamental rules for regulating the balance among activation, anergy, and differentiation by membrane-expressed receptors may be elucidated.
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Acknowledgments |
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RIIB-specific Western blotting, M. Ono for helpful suggestions in
the measurement of the SHIP activity, G. Krystal and N.
R. StC. Sinclair for helpful advice and comments, and S. Ragg
(Robarts Research Institute) for assistance in the preparation
of this manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Atsuo Ochi, The John P. Robarts Research Institute, 1400 Western Rd., London, Ontario, Canada N6G 2V4. E-mail address:
3 Abbreviations used in this paper: BCR, B cell receptor; SHP, Src homology 2-containing protein tyrosine phosphatase; SHIP, Src homology 2-containing inositol phosphatase; pITIM, tyrosine-phosphorylated immunoreceptor tyrosine inhibitory motif; PTK, protein tyrosine kinase; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakis-phosphate; SH2, Src homology 2.
Received for publication June 25, 1997. Accepted for publication May 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB modulates B-cell receptor signalling. Nature 368:70.[Medline]
RIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation. Immunity 3:635.[Medline]
RIIB1. Science 268:293.RII-deficient mice. Nature 379:346.[Medline]
RIIB. Nature 383:263.[Medline]
2 tyrosine phosphorylation but not Syk or Lyn activation. J. Biol. Chem. 271:20182.RIIB-mediated inhibition of B cell antigen receptor activation. J. Biol. Chem. 272:20038.RIIB1 signals. Immunol. Lett. 61:135.[Medline]
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