From the Departments of Medicine, Immunology and
Medical Genetics and Microbiology, University of Toronto, the
Samuel Lunenfeld Research Institute, Mount Sinai Hospital and the
University Health Network Research Institute, Toronto, Ontario M5G 1X5,
Canada, and the
Department of Oncology Research of the
University Health Network Research Institute and the Canadian Blood
Services, Toronto, Ontario M5G 2M1, Canada
Received for publication, July 31, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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SHP-1 is a cytosolic tyrosine phosphatase
implicated in down-regulation of B cell antigen receptor signaling.
SHP-1 effects on the antigen receptor reflect its capacity to
dephosphorylate this receptor as well as several inhibitory
comodulators. In view of our observation that antigen receptor-induced
CD19 tyrosine phosphorylation is constitutively increased in B cells
from SHP-l-deficient motheaten mice, we investigated the possibility
that CD19, a positive modulator of antigen receptor signaling,
represents another substrate for SHP-1. However, analysis of CD19
coimmunoprecipitable tyrosine phosphatase activity in CD19
immunoprecipitates from SHP-1-deficient and wild-type B cells revealed
that SHP-1 accounts for only a minor portion of CD19-associated
tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is
modulated by the Lyn protein-tyrosine kinase, Lyn activity was
evaluated in wild-type and motheaten B cells. The results revealed both
Lyn as well as CD19-associated Lyn kinase activity to be constitutively
and inducibly increased in SHP-1-deficient compared with wild-type B
cells. The data also demonstrated SHP-1 to be associated with Lyn in
stimulated but not in resting B cells and indicated this interaction to
be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These
findings, together with cyanogen bromide cleavage data revealing that
SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.
B cell responses to antigen stimulation are transduced
intracellularly via the B cell antigen receptor
(BCR),1 a multimeric receptor
complex that comprises membrane immunoglobulin and the immunoglobulin
The initial events of BCR signal relay are characterized by the
activation of several PTKs, including Lyn, Fyn, Blk, Syk, and Btk, and
the subsequent recruitment of secondary signaling molecules, including
phosphatidylinositol 3-kinase (PI3K), Shc, BLNK/SLP-65, Vav, SOS1, and
phospholipase C Among the myriad of proteins implicated in the regulation of BCR
signaling, the cytosolic protein-tyrosine phosphatase (PTP) SHP-1 is
distinguished by its predominant role as an inhibitor of BCR-driven
activation events (5). The inhibitory effect of SHP-1 on BCR signaling
was initially revealed by the demonstration that BCR-evoked
proliferation of mature B cells and clonal deletion of self-reactive B
cell precursors are aberrantly increased in the context of SHP-1
deficiency (14, 15). These latter studies involved analysis of B cells
from motheaten (me/me) and viable motheaten
(mev/mev)
mice, animals in which expression of no SHP-1 or a catalytically inactive form of SHP-1 protein, respectively, is associated with increased levels of serum immunoglobulins, high autoantibody titer, and
a marked expansion of CD5+ B-1 cells in the periphery (16,
17). At present, the biochemical basis whereby SHP-1 exerts its
inhibitory effects on BCR-evoked responses is not entirely defined.
This PTP has, however, been shown to interact with the BCR complex in
resting B cells and likely acts in this context to maintain the
receptor in a tyrosine-dephosphorylated state (14). Following BCR
ligation, SHP-1 no longer associates with the BCR, but instead
interacts with a number of BCR-inducible tyrosine-phosphoryated
transmembrane coreceptors (18-20). These coreceptors, which include
CD22, PIR-B, and CD72, have all been implicated in the down-regulation
of BCR signaling (21-23) and have been shown to interact with the
SHP-1 SH2 domains via phosphorylated tyrosine residues embedded
within immunoreceptor tyrosine-based inhibitory motifs (ITIMs)
(18-20). The inhibitory effects of these receptors depend on their
binding to SHP-1 and appear to be realized via SHP-1-mediated
dephosphorylation of tyrosine residues within the receptor cytosolic
domains and/or other intracellular signaling effectors recruited to
these receptors following BCR engagement.
In contrast to the ITIM-containing coreceptor molecules, a number of B
cell transmembrane coreceptors modulate BCR signaling so as to amplify
the signal and promote its downstream propagation. Among these positive
modulatory receptors, the B lineage-specific CD19 molecule appears to
play a central role in enhancing BCR coupling to a spectrum of cellular
behaviors. CD19, which is expressed as a component of a multimeric
complex on the B cell surface (24), becomes rapidly
tyrosine-phosphorylated following BCR engagement (25) and consequently
interacts with SH2 domain-containing signaling effectors, such as Lyn,
Fyn, Syk, Vav, and PI3K, which play integral roles in BCR signal
delivery (25-28). Recent data suggest that CD19 effects on BCR
signaling reflect its capacity to not only interact with Src-family
PTKs but also to amplify the activities of these enzymes (29). As is
consistent with the positive role for CD19 in regulation of BCR
signaling, mice, which overexpress CD19 consequent to the expression of
a CD19 transgene, manifest augmented B cell proliferative responses to
BCR cross-linking and show markedly increased serum immunoglobulin
levels (30). These animals also display a dramatic increase in the
numbers of B-1 lineage cells and a proportionate decrease in the
numbers of conventional B cells within the periphery (31). These
observations, therefore, reveal the phenotype engendered by CD19
overexpression to be very similar to the B cell phenotype conferred by
SHP-1 deficiency, a finding that suggests that the influence of these respective proteins on BCR signaling thresholds reflects the modulation of a common signaling element or cascade. This hypothesis is further supported by our previous data revealing BCR-evoked CD19
phosphorylation to be markedly reduced in cells lacking both the CD45
and SHP-1 PTPs (32) and thus identifying CD19 as a possible target of SHP-1-mediated dephosphorylation.
In the current study, we have directly investigated the role for SHP-1
in modulating the tyrosine phosphorylation of CD19. The results of
these studies confirm that BCR-induced tyrosine phosphorylation of CD19
is enhanced in SHP-1-deficient mice but also suggest that the
contribution of SHP-1 to the direct dephosphorylation of CD19 is small.
Because of these observations, as well as data revealing CD19 to be
associated with the Lyn protein-tyrosine kinase following BCR
engagement (25, 26) and identifying a central role for Lyn in modifying
CD19 effects on B cell survival (33, 34), we next investigated the
possibility that SHP-1 influence on CD19 tyrosine phosphorylation is
mediated via the regulation of Lyn activity. The results of this
analysis indicate both BCR-induced tyrosine phosphorylation and
activation of the Lyn protein-tyrosine kinase to be markedly augmented
in me/me and
mev/mev compared with
wild-type B cells. In addition, Lyn inducibly associates with the SHP-1
N-terminal SH2 domain and is dephosphorylated at its
autophosphorylation site (Tyr-397) by incubation with SHP-1. These data
identify Lyn as a substrate for SHP-1-mediated
dephosphorylation/deactivation and suggest that SHP-1 inhibitory
effects on Lyn activity contribute to the down-regulation of CD19
tyrosine phosphorylation and may thereby provide an important mechanism
for disrupting CD19 interactions with downstream effectors involved in
the relay and amplification of BCR-initiated activation signal.
Reagents--
Antibodies used for these studies included the
following: PE-conjugated B220 antibody from PharMingen (Mississauga,
Ontario), rabbit polyclonal anti-Lyn antibody from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA), monoclonal anti-phosphotyrosine
antibody 4G10 from Upstate Biotechnology Inc. (Lake Placid, NY), and
goat F(ab')2 anti-mouse IgM antibody from Jackson
ImmunoResearch (West Grove, PA). Rat anti-mouse CD19 antibody was
produced by the 1D3 hybridoma (provided by Dr. D. Fearon, University of
Cambridge School of Medicine, Cambridge, UK) (35), and a rabbit
polyclonal anti-CD19 antibody was derived by immunization with a
polylysine-conjugated peptide corresponding to amino acids 504-523
within the CD19 cytosolic domain (SynPep Corp., Dublin, CA). Rabbit
polyclonal anti-SHP-1 antibody and monoclonal anti-Thy1.2 antibody from
the hybridoma clone J1j.10 (ATCCT1B184) were produced in our laboratory
as described previously (14, 16). Low-Tox rabbit complement was
purchased from Cedarlane (Hornby, ONT), and chemicals used for
immunoprecipitation/immunoblotting were purchased from Sigma Chemical
Co. (St. Louis, MO).
Cells and Cell Lines--
Single cell suspensions of splenocytes
were obtained from 10- to 14-day-old C3HeBFeJ-melme
(motheaten),
C57BL6-mev/mev (viable
motheaten), and congenic wild-type (+/+) mice derived at the Samuel
Lunenfeld Research Institute breeding facility by mating
C3HeBFeJ-me/+ and +/+ and
C57BL/6J-mev/+ and +/+ breeding
pairs. Purified populations of splenic B lymphocytes were obtained from
me/me,
mev/mev, and wild-type
congenic mice by subjecting splenic cell suspensions to erythrocyte
lysis in 0.8% ammonium chloride, followed by treatment with
anti-Thy1.2 antibody for 30 min on ice and a subsequent 45-min incubation with a 1:15 dilution of rabbit complement (Serotec Ltd.,
Toronto, Ontario). The cells were then washed and layered over a
Percoll gradient (Amersham Pharmacia Biotech, Baie d'Urfé, Province of Quebec) as described previously (14). The resulting cells
were >90% mIg and B220 positive as determined by
fluorescence-activated cell sorting (Becton Dickinson, Mountainview,
CA) analysis. The CD5+ murine B lymphoma line (36)
(provided by Dr. A. Kaushik, University of Guelph, Guelph, Ontario) and
the WEHI-231 B lymphoma line (purchased from ATCC, Rockville, MD) were
cultured at 37 °C in RPMI 1640 (Life Technologies, Inc., Grand
Island, NY) supplemented with 5% fetal bovine serum (Sterile System
Inc., Logan, UT), 50 µM 2- Cell Stimulation and Lysis--
WEHI-231, CH12, or purified
splenic B cells (2-5 × 107) were resuspended in 5 ml
of culture medium and stimulated with 40 µg/ml F(ab')2
antibody for varying periods of time. Stimulations were done on ice to
retard biochemical reactions when studying the kinetics of CD19
phosphorylation and Lyn kinase activation (37). For biotinylation,
5-6 × 107 cells/ml were suspended at 107
cells/ml in ice-cold PBS and mixed with 0.3 mg/ml sulfo-NHS-Biotin solution (Pierce Chemical Co., Rockford, IL). After 30-min incubation at room temperature, the reaction was quenched by 5-min incubation with
50 mg/ml glycine in PBS. Cells were than washed twice in cold PBS and
subjected to stimulation as above. Following stimulation, biotinylated
or nonbiotinylated cells were incubated in lysis buffer (50 mM Tris-HCI, pH 8.0, 150 mM NaCl, 50 µM NaF, 2 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 50 mM
ZnCl2, 50 µM o-phosphate, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin)
containing either 1% CHAPS (Sigma) or 1% Nonidet P-40. Cell lysates
were centrifuged at 14,000 × g for 10 min at 4 °C,
and protein concentrations were then determined using the bicinchoninic
acid assay (Pierce).
Antibody Coupling to Protein A-Sepharose--
Anti-Lyn or
anti-IgG (isotype control) antibody was incubated at 4 °C overnight
under rocking conditions with protein A-Sepharose 4B (Amersham
Pharmacia Biotech) in PBS (about 10 µg of antibody per 10 µl of
beads). Beads were then washed two times in 0.1 M sodium
borate (pH 8.6) and two times in 0.2 M tri-ethanolamine (pH
8.2). Beads were then resuspended in 0.2 M tri-ethanolamine solution containing 40 mM dimethyl pimelimidate
dihydrochloride (Pierce) and incubated for 1 h at room temperature
with continual rocking. The antibody-coupled beads were washed two
times in 200 mM ethanolamine (pH 8.2), two times in 0.1 M sodium borate (pH 8.0), two times in PBS, and resuspended
in PBS supplemented with 0.2% NaN3.
Immunoprecipitation and Immunoblotting--
Lysates were
precleared before immunoprecipitation by incubating 1 mg of cell lysate
with protein A-Sepharose beads (Amersham Pharmacia Biotech) at 4 °C
for 1 h and for an additional 1 h with 40 µl of rabbit
preimmune serum. Lysates were then incubated for 2 h at 4 °C
with the appropriate antibody (anti-Lyn, anti-IgG isotype control) and
25 µl of protein A- or protein G-Sepharose beads. Immune complexes
were then collected by centrifugation, washed four times in lysis
buffer, and boiled for 5 min in reducing SDS-gel sample buffer. Samples
were then electrophoresed through SDS-polyacrylamide and transferred to
nitrocellulose (Bio-Rad Laboratories, Mississauga, Ontario). After 1-h
incubation in 3% gelatin, the filters were incubated for 1 h at
room temperature with anti-CD19, anti-Lyn, or anti-phosphotyrosine 4G10
antibodies followed by horseradish peroxidase-labeled secondary
antibody (Amersham Pharmacia Biotech, Arlington Heights, ICN) or, for
analysis of biotinylated cells, with horseradish peroxidase-avidin
(Pierce). Immune complexes were detected using an enhanced
chemiluminescence system (Amersham Pharmacia Biotech). Stripping and
reprobing of the blots were performed according to Amersham Pharmacia
Biotech's recommended protocol.
Assay of Phosphatase Activity--
For analysis of
CD19-associated phosphatase activity, anti-CD19 immunoprecipitates were
prepared (as described above) from 1 mg of lysates of unstimulated or
anti-IgM antibody-stimulated motheaten and wild-type B cells.
Phosphatase assays were also performed on anti-CD19 and anti-IgG
(control) immunoprecipitates prepared from lysates of wild-type splenic
B cells immunodepleted of SHP-1 by overnight incubation with an excess
of anti-SHP-1 antibody followed by addition of 100 µl of protein
A-Sepharose. For this experiment, the complete immunodepletion of SHP-1
protein was confirmed by Western immunoblotting analysis (data not
shown). The amount of SHP-1 antibody utilized to completely
immunodeplete SHP-1 protein from lysates was predetermines by titration
and Western immunoblotting analysis (data not shown).
Immunoprecipitates were washed twice in phosphatase buffer (10 mM Tris-HCl, 1.0 mM EDTA, 1 mg/ml bovine
serum albumin, 0.1% 2- In Vitro Kinase Assay--
Lyn kinase activity was evaluated
using immunoprecipitates prepared as described above from unstimulated
and stimulated splenic B cells. The immunoprecipitates were washed in
kinase buffer (20 mM HEPES, pH 7.6, 150 mM
NaCl, 5 mM MnCl2, 0.25 mM
Na3VO4, 0.5% Nonidet P-40, 0.1 mM
2- In Vitro Binding Assays--
Glutathione
S-transferase (GST)-SHP-1 fusion proteins were generated by
subcloning the following murine cDNA or polymerase chain
reaction-amplified fragments into pGEX2T: the full-length SHP-1
cDNA (GST-SHP-1), a full-length SHP-1 cDNA containing a Cys-453
Cyanogen Bromide Cleavage Analysis--
In vitro
[ BCR-evoked CD19 Tyrosine Phosphorylation Is Enhanced, but
CD19-associated Phosphatase Activity Is Only Marginally Altered, in
SHP-1-deficient B Cells--
Previous data from our laboratory and
others have revealed modulation of inhibitory coreceptors to represent
a major mechanism whereby SHP-1 mediates its down-regulatory effects on
BCR signaling (5, 18-20). However, data garnered from the analysis of
mice deficient for both the CD45 and SHP-1 PTPs (31) raised the
possibility that SHP-1 also influences the tyrosine phosphorylation
and, by extension, signaling functions of the positive regulatory
coreceptor, CD19. To begin addressing this issue, SHP-1-deficient B
cells from me/me and
mev/mev mice were
evaluated with regards to the kinetics of CD19 phosphorylation following BCR ligation. As illustrated in Fig.
1, anti-phosphotyrosine immunoblotting
analysis of CD19 immunoprecipitates from the SHP-1-deficient cells
revealed tyrosine phosphorylation of the 115- to 120-kDa species
representing CD19 to be markedly increased both constitutively and
inducibly in the mev/mev
and to a lesser extent in the me/me cells compared with
wild-type cells. The increased phosphorylation of CD19 detected in the
motheaten cells cannot be attributed to expansion of the
CD5+ B-1 cell population in these mice, because
CD5+ CH12 cells exhibited normal levels of CD19
phosphorylation both before and after BCR cross-linking (data not
shown). These data support the contention that SHP-1 modulates CD19
tyrosine phosphorylation and are consistent with previous data
revealing BCR-evoked CD19 phosphorylation to be augmented in a B cell
line derived from me/me mice (41).
To more directly investigate the possibility that CD19 represents a
SHP-1 substrate, the SHP-1-deficient B cells were next compared with
wild-type B cells in terms of the levels of tyrosine phosphatase
activity coimmunoprecipitated with CD19 from these respective cell
populations. The results of this analysis demonstrated levels of
CD19-associated phosphatase activity to be markedly increased following
BCR ligation in both wild-type and me/me B cells (Fig.
2). Although levels of phosphatase
activity coprecipitated with CD19 were lower in me/me
compared with wild-type B cells and were also relatively reduced in B
cells immunodepleted for SHP-1, the reduction in coprecipitated
phosphatase activity observed in the SHP-1-deficient cells was only
modest. These findings indicate that SHP-1 is not the major
CD19-associated tyrosine phosphatase and suggest that SHP-1
contribution to the direct dephosphorylation of CD19 may be small. This
interpretation of the data is consistent with the data shown in Fig.
1A, indicating that tyrosine dephosphorylation of CD19
proceeds normally in motheaten B cells and is not diminished in the
SHP-1-deficient compared with wild-type B cells at the 10-min time
point following BCR stimulation. This conclusion is also supported by
our observation of minimal amounts of SHP-1 in anti-CD19
immunoprecipitates from unstimulated and stimulated B cells (data not
shown), a finding also consistent with the lack of ITIMs in the CD19
cytosolic region. Along similar lines, SHP-1 deficiency has been shown
to have negligible impact on the enhanced dephosphorylation of CD19,
which occurs in conjunction with Fc BCR-induced Activation of the Lyn PTK Is Enhanced in
SHP-1-deficient Cells--
Although the precise profile of effectors
that mediate induction of CD19 tyrosine phosphorylation following BCR
ligation is unclear, an important role for the Lyn PTK has been
suggested by the detection of Lyn kinase activity in anti-CD19
immunoprecipitates and by data implicating the CD19-Lyn complex in
regulation of B cell survival (25, 26, 34). In view of these
observations, the kinase activity contained in immunoprecipitates from
me/me and
mev/mev B cells was next
investigated using an in vitro assay of kinase activity. The
results of this analysis revealed Lyn-mediated autophosphorylation and
phosphorylation of exogenous substrate (GST-Ig Lyn Associates with the SHP-1 N-terminal SH2 Domain--
In view
of the apparent role for SHP-1 in modulating Lyn kinase activity, the
possibility that these enzymes associate with one another in resting
and stimulated B cells was next investigated. As shown in Fig.
4A (upper panel),
anti-SHP-1 immunoblotting analysis of Lyn immunoprecipitates prepared
from resting and BCR-ligated WEHI-231 B cells revealed SHP-1 to be
present in Lyn immunoprecipitates from stimulated but not unstimulated
cells. Association of SHP-1 with Lyn has also been previously detected
in U937 myeloid leukemia cells, although, in the latter study the
association of these enzymes appeared to occur constitutively (46). By
contrast, in the current study, SHP-1 binding to Lyn was up-regulated
by cell stimulation, a result that suggests the interaction may be phosphotyrosine-dependent and involve binding of the SHP-1
SH2 domains with phosphorylated tyrosine residues on Lyn. To
investigate this possibility, GST fusion proteins containing
full-length SHP-1, a catalytically inert form of SHP-1 (SHP-1 C453S),
and one or both of the SHP-1 SH2 domains were evaluated for their
capacities to interact with in vitro phosphorylated Lyn. The
results of this analysis revealed the interaction of phosphorylated Lyn
with the N-terminal, but not the C-terminal, SHP-1 SH2 domain (Fig.
4B). As shown in Fig. 4B, the fusion protein
containing the C- and the N-terminal SHP-1 SH2 domain precipitated more
Lyn protein than did the GST-N-terminal SHP-1 fusion protein. This
observation is consistent with previous data indicating that a single
SH2 domain may be sufficient for SHP-1 interaction with target
substrate, but may be less efficient than the combined SH2 domains in
promoting such interactions (47). In addition, these data do not
preclude the possibility that SHP-1 association with Lyn involves other sites within these respective proteins. The Lyn SH3 domain has, for
example, been implicated in the Lyn-SHP-1 interaction detected in
myeloid cell lines (46), and the association of SHP-1 with the related
Src PTK appears to involve interactions between not only the SHP-1 SH2
domains and phosphorylated Src, but also the Src SH2 domain and
phosphorylated SHP-1 (48, 49). In addition, both SHP-1 and Lyn
associate with other signaling effectors, such as PI3K (50, 51), that
are recruited to the BCR following its stimulation and that may create
a structural framework at the membrane that enhances SHP-1 association
with Lyn. Although the relative importance of these various
interactions to SHP-1-Lyn binding remains to be determined, the current
data are consistent with the conclusion that SHP-1 physically
associates with Lyn in B cells and is therefore appropriately
positioned to induce dephosphorylation and deactivation of the
kinase.
The data shown in Fig. 4B also reveal the signal intensity
of the Lyn species precipitated with the SHP-1 C453S fusion protein to
be greater than that of the species precipitated with the wild-type SHP-1 fusion protein. This observation most likely reflects the substrate trapping properties of SHP-1 C453S and, by extension, indirectly supports the contention that Lyn represents a SHP-1 substrate. Conversely, the association between SHP-1 and Lyn may also
provide a framework for Lyn to phosphorylate SHP-1. SHP-1 has in fact
been shown to represent a substrate for Lyn in an exogenous expression
system (46), and several Src-family kinases (Src, Lck, and Lyn) can
phosphorylate SHP-1 in vitro (46, 49, 52). Moreover, in the
BCR-stimulated splenic B cells studied here, the tyrosine
phosphorylation of Lyn-associated SHP-1 was found to be increased
compared with that detected in the total pool of SHP-1 (data not
shown). These findings suggest that SHP-1 is phosphorylated by Lyn
in vivo and raise the possibility that SHP-1 and Lyn engage
in a reciprocal functional relationship wherein Lyn phosphorylates and
potentially activates SHP-1 and SHP-1 then dephosphorylates and
deactivates Lyn. This functional paradigm has been suggested with
respect to SHP-1 modulation of Lyn-dependent apoptotic
responses to DNA damage (46) and also in relation to SHP-1 modulation
of ZAP-70-dependent proliferative response to T cell
antigen receptor engagement (53). However, at present the relationship
between SHP-1 tyrosine phosphorylation status and its catalytic
activity is unclear (52, 54), and accordingly, the relevance of Lyn to
SHP-1 activation remains to be determined. By contrast, the available
data provide compelling evidence that SHP-1 negatively regulates Lyn
activity and thus suggest that SHP-1 modulation of Lyn plays a role in
SHP-1-mediated inhibition of BCR signaling.
SHP-1 Catalyzes the Dephosphorylation of Tyr-397 within the Lyn
Kinase Domain--
As for other Src-family PTKs, Lyn contains two
major sites of tyrosine phosphorylation, these being the
autophosphorylation site (Tyr-397) within the protein kinase domain and
the negative regulating tyrosine phosphorylation site (Tyr-508) within
the C-terminal tail (55). To determine whether SHP-1 targets the autophosphorylation site on Lyn, as is predicted by the detection of
increased Lyn activity in SHP-1-deficient cells, the effects of SHP-1
on Lyn tyrosine phosphorylation were examined using CNBr cleavage
analysis. Prior to this analysis, the capacity of recombinant SHP-1 to
induce Lyn dephosphorylation in vitro was initially assessed by evaluating the effects of GST-SHP-1 and catalytically inert GST-SHP-1 C453S fusion proteins on 32P-labeled Lyn
immunoprecipitates prepared from WEHI-231 and CH12 cells. As
illustrated in Fig. 5A, signal
intensity and, by extension, the phosphorylation status of the
radiolabeled Lyn was markedly reduced in the SHP-1-treated compared
with the SHP-1 C453S-treated samples. Based on these findings, the
sites on Lyn, which are dephosphorylated by SHP-1, were next examined
by subjecting SHP-1 and SHP-1 C453S-treated 32P-labeled Lyn
to CNBr cleavage. As illustrated in Fig. 5B, CNBr treatment
of Lyn is predicted to generate multiple cleavage fragments, most
notably including an 8-kDa fragment containing the autophosphorylation site, Tyr-397, and a 4-kDa fragment encompassing the inhibitory tyrosine phosphorylation site, Tyr-508. In the current study, these
latter two fragments, which have been previously confirmed to be
phosphotyrosine-containing regions of Lyn (55), were both easily
detected following CNBr hydrolysis of SHP-1 C453S-treated Lyn (Fig.
5B). However, although phosphorylation of the Lyn regions represented by both the 8.0- and the 4.0-kDa Lyn CNBr cleavage fragments was diminished following treatment with wild-type SHP-1, the
reduction in phosphorylation of the 8-kDa cleavage fragment was much
more dramatic than that of the 4-kDa fragment, particularly in the
context of Lyn pretreatment with 1.0 µg of SHP-1. These data suggest
that both Tyr-397 contained within the 8-kDa fragment and Tyr-508
contained within the 4-kDa fragment can be dephosphorylated by SHP-1,
but also indicate that SHP-1 preferentially dephosphorylates Tyr-397.
Thus, these findings reveal the capacity of SHP-1 to directly
dephosphorylate Lyn at the autophosphorylation site as is consistent
with a role for SHP-1 in negatively regulating Lyn activity. The
current data also suggest that the C-terminal inhibitory phosphotyrosine on Lyn is not regulated by SHP-1, a conclusion supported by previous data revealing the phosphorylated C-terminal tyrosine on Lyn to be targeted by the CD45 PTP (55). Thus
phosphorylation and activation of Lyn appears to be regulated in a
fashion highly similar to that of the related Lck PTK, the latter of
which is also tyrosine-dephosphorylated at its autophosphorylation site by SHP-1 (52) and at its inhibitory C-terminal site by CD45 (56). A
role for CD45 in activating Lyn is also suggested indirectly by the
diminution of BCR-induced CD19 tyrosine phosphorylation detected in
CD45-deficient mice (32). Thus CD45 and SHP-1 appear to exert opposing
effects on Lyn activity and thereby engender a biochemical
counterbalance critical to the modulation of CD19 and potentially to
the regulation of other signaling effectors involved in BCR signal
delivery.
The data reported in this study establish the capacity of SHP-1 to
modulate CD19 tyrosine phosphorylation but link this effect to
SHP-1-mediated dephosphorylation of Lyn rather than to
dephosphorylation of CD19 per se. Importantly, although Lyn
interactions with CD19 appear to exert a positive effect on BCR signal
delivery, recent data derived largely from the analysis of
Lyn-deficient mice, have revealed a pivotal role for this PTK in the
negative regulation of BCR signaling (57, 58). This inhibitory effect
of Lyn appears to be realized at least in part through its
phosphorylation of the inhibitory coreceptor CD22, an event that evokes
SHP-1 binding to CD22 and that is required for CD22 to negatively
modulate BCR signaling (59, 60). Interestingly, SHP-1 association with CD22 also appears relevant to the modulation of CD19 tyrosine phosphorylation, as BCR-evoked phosphorylation of CD19 is increased in
the context of CD22 deficiency (61). These data suggest the involvement
of SHP-1 and Lyn in a complex BCR inhibitory signaling axis wherein
CD19-positive effects on BCR signal are antagonized by SHP-1-mediated
down-regulation of Lyn activation and by extension, CD19, whereas
CD22-negative effects on BCR signal delivery are enhanced by
Lyn-mediated phosphorylation of CD22 and the consequent recruitment of
SHP-1 and dephosphorylation of Lyn and/or other effectors. SHP-1
interactions with Lyn may also influence the activities of several
other BCR-activated signaling effectors, such as Syk and PI3-K, which
appear to be substrates for both these enzymes (50, 51, 62, 63). Thus
the capacity of SHP-1 to dephosphorylate and deactivate Lyn may impact
upon many of the intracellular biochemical events evoked by BCR
ligation and, in this context, is likely to represent a very
significant mechanism whereby SHP-1 realizes its inhibitory effects on
BCR signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
and
chains (1, 2). The signals transmitted consequent to
antigen engagement drive B lymphocyte activation via a complex
signaling network, which biochemically links the receptor complex to
cellular responses such as to proliferation, differentiation, and
antibody secretion. Transmission of BCR signals via this intracellular
circuitry is further regulated by the integration of accessory signals
from BCR comodulators (3) and is highly dependent on reversible
protein-tyrosine phosphorylation mediated by the balanced activities of
protein-tyrosine kinases (PTKs) and phosphatases (PTPs) (4, 5).
(6-12). These initial interactions induce Ras
activation, phosphoinositide turnover, increases in intracellular free
calcium, and other intermediary events, which ultimately transduce the
BCR-evoked signal to the nucleus and consequent proliferation,
apoptosis, maturation, or other physiological responses. The mechanisms
whereby BCR ligation can induce such a wide diversity of biological
outcomes are not well understood but are likely to involve modulation
of the BCR signaling pathway by a spectrum of transmembrane and
cytosolic signaling effectors that qualitatively and/or quantitatively
alter the relay and downstream interpretation of BCR signal (13).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
mercaptoethanol, and 100 µg/ml penicillin/streptomycin.
-mercaptoethanol, 0.01%
NaN3, pH 7.34) and then incubated for 12 h at 37 °C
overnight in phosphatase buffer containing 2 mM
p-nitrophenyl phosphate (Sigma). Under these conditions,
SHP-1 activity for the substrate has been shown previously to increase
linearly with the amount of SHP-1 used in the reaction (38, 39).
Reactions were terminated by addition of 0.2 N NaOH, and
absorbance was measured at 410 nm by spectrophotometry.
-mercaptoethanol) and then incubated for 30 min at 30 °C in 20 µl of kinase buffer containing 10 µCi of [
-32P]ATP
(ICN) with or without 10 µg of GST-Ig
/
fusion protein (provided
by Dr. Y. Wu, Toronto, Ontario). Samples were resuspended in SDS-gel
sample buffer, boiled, and centrifuged at 14,000 × g
for 10 min and resolved over 10% SDS-PAGE gels. The
32P-labeled proteins were electrophoretically transferred
to Immobilon-P membranes (Millipore Corp., Bedford, MA) and then
visualized by autoradiography. Lyn quantification was performed by
anti-Lyn immunoblotting of the membranes using ECL.
Ser mutation (GST-SHP-1 (C453S)), the SHP-1 N-terminal SH2 domain
(amino acids 1-95), the SHP-1 C-terminal SH2 domain (amino acids
110-205), and the SHP-1 N- and C-terminal SH2 domains (amino acids
1-221). These expression plasmids were transfected into
Escherichia coli JM101, and the fusion proteins were
purified from isopropyl
-D-thiogalactopyranoside-induced bacteria using glutathione-conjugated Sepharose beads (Amersham Pharmacia Biotech). Equimolar amounts of each GST-SHP-1 fusion protein and GST-beads were
then incubated at 4 °C for 1 h with 0.9 µg of in
vitro 32P-labeled purified Lyn protein. Beads were
washed seven times, and the complexes were resuspended in SDS-sample
buffer, boiled, analyzed by SDS-PAGE, and transferred to
nitrocellulose, and the Lyn protein was visualized by autoradiography.
-32P]ATP-labeled Lyn was immunoprecipitated using
anti-Lyn antibody and incubated at 37 °C with equal amounts of either GST-SHP-1 or GST-SHP-1 (C453S) protein in 200 µl of
phosphatase buffer (10 mM Tris-HC1, 1.0 mM
EDTA, 1 mg/ml bovine serum albumin, 0.1% 2-
-mercaptoethanol, 0.01%
NaN3, pH 7.34). The immune complexes were then resolved
over SDS-PAGE and transferred to nitrocellulose. The 56-kDa
Lyn-containing bands were then excised from the membranes and subjected
to CNBr cleavage as described previously (40). The excised Lyn protein
was incubated with 60 mg/ml CNBr in 70% formic acid for at least
2 h at room temperature. Samples were then washed and dried, and
the CNBr-generated peptide fragments were resuspended in tricine SDS
sample buffer, resolved by separation on 10-20% gradient Tricine
SDS-PAGE (Novex, San Diego, CA), transferred to an Immobilon-P
membrane, and visualized by autoradiography. Complete digestion of each
sample was confirmed by the absence of higher molecular weight
32P-labeled bands, and the CNBr fragments were quantitated
by phosphorimaging (Molecular Dynamics).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
CD19 tyrosine phosphorylation is increased in
SHP-1-deficient motheaten B cells. Purified splenic B cells
isolated from C57BL/6J-wild-type (+/+) or viable motheaten
(Mev) mice (A) or from C3HeBFeJ-wild-type
(+/+) or motheaten (Me) mice (B) were stimulated
with 40 µg/ml goat F(ab')2 anti-mouse IgM antibody for
the indicated times. Lysate proteins (1 mg) were then
immunoprecipitated with anti-CD19 antibody (1D3), resolved on 10%
SDS-PAGE, and transferred to nitrocellulose membranes, and the
membranes were then immunoblotted with anti-phosphotyrosine antibody.
Loading of equivalent amounts of CD19 protein was confirmed by
reblotting with either horseradish peroxidase-avidin (A) or
anti-CD19 (B) antibodies (lower left and
right panels, respectively). Arrows indicate the
position of CD19. Mobilities of molecular mass standards are shown on
the left.
RIIB-mediated inhibition of BCR
signaling (42, 43). Taken together, these data suggest a minimal role
for SHP-1 in the direct dephosphorylation of CD19.
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Fig. 2.
BCR-induced increases in CD19-associated PTP
activity are only modestly influenced by SHP-1 deficiency. Splenic
B cells purified from C3HeBFeJ wild-type (C3H+/+) or
motheaten (Me) mice were stimulated with goat
F(ab')2 anti-mouse IgM antibody (40 µg/ml) for 5 min.
Cells were lysed in 1% CHAPS lysis buffer, and lysate proteins (1 mg)
were then immunoprecipitated with anti-CD19 or isotype IgG control
antibodies. CD19 was also immunoprecipitated from normal anti-IgM
antibody-stimulated splenic B cell lysates pretreated with an excess of
anti-SHP-1 antibody (SHP-1-depleted). The precipitated
proteins were incubated for 12 h at 37 °C in phosphatase buffer
containing 1 mM p-nitrophenyl phosphate. After
addition of 0.2 M NaOH, absorbance was measured at 410 nm
using an enzyme-linked immunosorbent assay plate reader. Phosphatase
activity was quantified in millimoles based on a standardized
concentration curve of the product p-nitrophenol
(PNP). Bars indicate the standard deviations
obtained from three independent experiments.
/
) to be both constitutively and inducibly higher in the me/me and
mev/mev B cells than in
wild-type B cells (Fig. 3, A
and B). Similarly, analysis of the Lyn kinase activity
contained in CD19 immunoprecipitates from resting and BCR-stimulated
cells demonstrated CD19-associated Lyn activity to be both
constitutively and inducibly enhanced in SHP-1-deficient compared with
wild-type cells (Fig. 3C). Lyn association with CD19 was
also increased in the context of SHP-1 deficiency, an observation
consistent with previous data linking increases in Lyn activity to
enhanced phosphorylation of the CD19 tyrosine residues mediating Lyn
binding to CD19 (44). As shown in Fig. 3D, tyrosine
phosphorylation status of Lyn was also evaluated by
anti-phosphotyrosine immunoblotting analysis of Lyn immunoprecipitates from the cells under study. The results of this analysis revealed tyrosine phosphorylation of Lyn both before and after BCR ligation to
be increased in the motheaten cells. These results, therefore, indicate
SHP-1 deficiency to be associated with heightened Lyn activity and
strongly suggest that Lyn is a SHP-1 substrate. Interestingly, increases in Lyn activity have also been detected in pre-B cell lines
derived from motheaten bone marrow (45). However, in this latter
system, the augmentation in Lyn activity was ascribed to the expression
of increased Lyn protein in these mutant cells. By contrast in the
current analysis, levels of Lyn protein were found to be comparable in
splenic B cells isolated from either wild-type or motheaten mice. Thus,
these data suggest that SHP-1 induces the dephosphorylation and
deactivation of Lyn and that the SHP-1 effect on CD19 phosphorylation
may be mediated via the down-regulation of Lyn activity.
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Fig. 3.
Lyn and CD19-associated Lyn activities are
enhanced in SHP-1-deficient B cells. Splenic B cells (2 × 107) purified from C57BL/6J wild-type (+/+) and viable
motheaten (Mev) mice (A) and from
C3HeBFeJ wild-type (+/+) and motheaten (Me) mice
(B) were stimulated with goat F(ab')2 anti-mouse
IgM antibody (40 µg/ml) for the indicated times. Lysate proteins were
then immunoprecipitated with either anti-Lyn (A,
B, and D) or anti-CD19 (C) antibody.
Immunoprecipitates were then subjected to an in vitro kinase
assay in the presence of [ -32P]ATP with (A
and B) or without (C) exogenous substrate
GST-Ig
/
. Samples were resolved on SDS-PAGE, transferred to an
Immobilon-P membrane, and visualized by autoradiography. Alternatively,
the Lyn immunoprecipitates were subjected to immunoblotting with
anti-phosphotyrosine antibody (D). Levels of Lyn protein
were determined by reprobing the blots with anti-Lyn antibody
(lower segment of each panel), the positions of Lyn and GST-
Ig
/
are shown on the right.
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Fig. 4.
SHP-1 associates with Lyn. A,
lysates were prepared from unstimulated or goat F(ab')2
anti-mouse IgM (40 µg/ml)-stimulated cells and the lysate proteins
(500 µg) immunoprecipitated with bead-coupled anti-Lyn or anti-IgG
isotype control antibodies. The immunoprecipitated as well as total
cell lysate proteins were then immunoblotted with anti-SHP-1
(upper panel) or anti-Lyn (lower panel)
antibodies. B, anti-Lyn antibody (first lane on
the left) or glutathione-Sepharose-bound GST fusion proteins
containing either GST alone (GST), the SHP-1 N- and
C-terminal (GST-SH2(N+C)) domains, the single N
(GST-SH2(N)) or the single C
(GST-SH2(C))-terminal SH2 domains, the full-length wild-type
SHP-1 (GST-SHP-1) or the catalytically inert SHP-1
(GST-SHP-1(C453S)) proteins were used to precipitate
in vitro 32P-labled purified Lyn proteins (0.9 µg/lane) at 4 °C for 1 h, and the precipitates were subjected
to SDS-PAGE, transferred to Immobilon-P, and visualized by
autoradiography.
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Fig. 5.
SHP-1 dephosphorylates the
autophosphorylation site of Lyn. A, WEHI-231 or CH12
cells were stimulated with goat F(ab')2 anti-mouse IgM
antibody for 5 min. Lysate proteins were then immunoprecipitated with
bead-coupled anti-Lyn antibody and subjected to an in vitro
Lyn kinase assay in the presence of [ -32P]ATP. The
32P-labeled Lyn was then incubated for 2 h at 37 °C
with 20 µg of GST fusion proteins containing 20 µg of catalytically
inert (C453S) or active SHP-1. Lyn immunoprecipitates were then
resolved on SDS-PAGE, transferred to membrane, and visualized by
autoradiography (upper panel). The level of Lyn protein was
determined by immunoblotting the filters with anti-Lyn antibody
(lower panel). B, CNBr analysis of SHP-1-treated
Lyn. The diagram shows the predicted CNBr cleavage sites within p56 Lyn
(hatched lines) and the tyrosine residues representing the
major sites for autophosphorylation (Y397) and inhibitory
regulation (Y508). The 8- and 4-kDa cleavage fragments
encompassing Tyr-397 and Tyr-508, respectively, are shown under the
cleavage map. Following 32P-labeling in vitro,
purified Lyn was incubated for 5 min at 37 °C with GST fusion
proteins containing catalytically inert SHP-1 (C453S) (4.5 µg) or
active SHP-1 (0.5 and 1 µg). The labeled Lyn was then purified and
subjected to CNBr cleavage, and the fragments were analyzed by
electrophoresis over a 10-20% gradient of Tricine SDS-PAGE, followed
by transfer to membrane, and autoradiography analysis. This result is a
representative example of three independent experiments.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council (MRC) of Canada and the Arthritis Society of Canada.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.
§ A recipient of a National Cancer Institute of Canada Studentship Award.
¶ A recipient of an Arthritis Society/MRC fellowship.
** A Senior Scientist of the Medical Research Council of Canada and the Arthritis Society of Canada. To whom correspondence should be addressed: Mount Sinai Hospital, Rm. 656A, 600 University Ave., Toronto, Ontario M5G 1X5, Canada. Tel.: 416-586-8723; Fax: 416-586-8731; E-mail: ksimin@mshri.on.ca.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M006820200
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ABBREVIATIONS |
---|
The abbreviations used are:
BCR, B cell
antigen receptor;
FcRIIB, B cell receptor for IgG Fc region;
GST, glutathione S-transferase;
me/me, motheaten;
mev/mev, viable
motheaten;
PAGE, polyacrylamide gel electrophoresis;
PI3K, phosphatidylinositol 3-kinase;
PTK, protein-tyrosine kinase;
PTP, protein-tyrosine phosphatase;
SH, Src homology domain;
Tricine, N-tris-[hydroxymethyl]methylglycine;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PBS, phosphate-buffered saline;
ITIM, immunoreceptor tyrosine-based
inhibitory motif.
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