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Department of Immunology and Signal Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Fuchu, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1) is
a cytoplasmic PTP containing two SH2 domains at the
NH2 terminus, followed by a catalytic domain,
which is preferentially expressed in hemopoietic cells
(11, 12, 13, 14). Point mutation in the gene encoding SHP-1 caused
a number of immunological abnormalities found in motheaten
(me) and motheaten viable (mev)
mice (15, 16), including expansion of
CD5+ B-1 B cells (17), reduced
number of B cell progenitors in the bone marrow and B-2 B cells in
periphery (18), and functional defects in T and NK cells
(19, 20). SHP-1 has been implicated in negative regulation
of the resting BCR complex (21) and in the threshold
determination for BCR signaling and negative selection
(22). Biochemical studies have demonstrated that SHP-1 is
recruited to tyrosine-phosphorylated immunoreceptor tyrosine-based
inhibitory motifs present in the cytoplasmic regions of CD22 (23, 24) and CD72 (25, 26), thus negatively regulating
BCR-initiating signals. In addition, we have previously demonstrated
that SHP-1 is constitutively associated with SLP-76 in the mouse
immature B cell line, WEHI-231 (27). SLP-76 is a
leukocyte-specific cytoplasmic protein comprised of acidic region at
the NH2 terminus containing three tyrosine
residues that can be phosphorylated by Syk family PTKs, followed by a
proline-rich region and a COOH-terminal SH2 domain (28).
This molecule is involved in the regulation of signaling events
initiated by TCR (28, 29, 30, 31, 32, 33) and Fc
RI (34).
Recently, it was demonstrated that SLP-76 is a direct substrate of
SHP-1 in T cells and NK cells, and that dephosphorylation of SLP-76 by
SHP-1 is a crucial mechanism for the negative regulation of lymphocyte
activation by inhibitory receptors (35).
To understand fully the molecular mechanisms of SHP-1 action in B cells, we identified physiological substrates of SHP-1 by using the substrate-trapping approach (36, 37). When cells were transfected with the catalytically inactive form of SHP-1, with the catalytic center cysteine (C453) being replaced with serine (SHP-1-C/S), BCR-induced tyrosine phosphorylation was strongly enhanced in a protein of about 70 kDa. Immunoprecipitation and Western blot analysis revealed that this protein is the B cell linker protein (BLNK) (38), also named SH2 domain leukocyte protein of 65 kDa (SLP-65) (39), preferentially expressed in B cells. Our results also demonstrated that in in vitro conditions, SHP-1-C/S protein binds tyrosine-phosphorylated BLNK and SHP-1 dephosphorylates BLNK. Furthermore, among members of mitogen-activated protein kinase (MAPK) family, only c-Jun NH2-terminal kinase (JNK) was more strongly induced upon BCR ligation in SHP-1-C/S-expressing cells than in control and wild-type SHP-1 (SHP-1-wt) transfectants. These results suggest that BLNK is a physiological substrate of SHP-1, and JNK is situated downstream of the signaling pathways initiated by SHP-1 and BLNK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WEHI-231 cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in RPMI 1640 (Sigma, St. Louis, MI) supplemented with 10% FBS (JRH Biosciences, Lenexa, KS), 50 mM 2-ME, 100 mg/ml streptomycin, and 100 U/ml penicillin (complete medium).
Antibodies
Goat anti-mouse IgM Ab was purchased from Cappel, Organon Teknika (Durham, NC). Polyclonal rabbit anti-human SHP-1 Ab, reacting with the mouse product, anti-Lyn Ab, and anti-Syk Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine (PY) mAb was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-flag epitope M2 mAb was purchased from Sigma. Rabbit anti-BLNK Ab was raised by immunizing a rabbit with mouse BLNK (aa 4–205) expressed in Escherichia coli as a GST fusion protein. Rabbit anti-mouse phospho-specific p38 Ab and phospho-specific JNK Ab were purchased from New England BioLabs (Beverly, MA), and rabbit anti-mouse phospho-specific ERK was purchased from Promega (Madison, WI). Rabbit anti-mouse ERK-2 Ab, anti-JNK Ab, and anti-p38 Ab were purchased from Santa Cruz Biotechnology. Alkaline phosphatase (AP)-conjugated goat anti-mouse IgG and AP-conjugated mouse anti-rabbit IgG were purchased from Bio-Rad Laboratories (Richmond, CA) and Jackson ImmunoResearch (West Grove, PA), respectively. HRP-conjugated anti-rabbit IgG was purchased from Santa Cruz Biotechnology.
Immunoprecipitation, protein preparation, and Western blot analysis
Immunoprecipitation and Western blot analysis were performed as described previously (27). Briefly, cells (2 x 107) were suspended in 5 ml of complete medium and stimulated for the indicated times with 25 mg/ml anti-IgM Ab. The reactions were stopped with 20 ml ice-cold PBS containing 1 mM Na3VO4 and 2 mM EDTA (PBS-VE). After washing twice with PBS-VE, cells were lysed in 2 ml of TNE buffer (1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 150 mM, NaCl, 2 mM Na3VO4, 2 mM EDTA) and centrifuged. The supernatants were immunoprecipitated with protein G-Sepharose coupled with anti-flag mAb or Abs against SHP-1 or BLNK. Immunoprecipitates were boiled in reducing Laemmli SDS sample buffer and subjected to 10% SDS-PAGE analysis, followed by transfer to a nitrocellulose membrane. The membranes were incubated with anti-PY mAb, anti-flag mAb, anti-SHP-1 Ab, or anti-BLNK Ab. The blots were visualized by incubating with AP-conjugated goat anti-mouse IgG or AP-conjugated mouse anti-rabbit IgG Ab, and then developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
In vitro kinase assay
In vitro kinase assay was performed as described previously
(40). The immunoprecipitates with Abs against Lyn and Syk
were washed four times with TNE buffer and then four times with kinase
buffer (20 mM HEPES, pH 8, 150 mM NaCl, 10 mM
MgCl2, 20 mM MnCl2). After
washing, each immunoprecipitate was suspended in 20 ml of kinase buffer
containing 10 µCi of [
-32P]ATP (6000
Ci/mM) and exogenous substrates, enolase for Lyn and myelin basic
protein for Syk, incubated for 3 min (Lyn) or 15 min (Syk) at 30°C.
The reaction was stopped by adding SDS-PAGE sample buffer. The samples
were resolved by 10% SDS-PAGE, and the resulting gels were treated
with 1 N KOH at 60°C for 90 min, dried, and subjected to
autoradiography. The intensities of bands were measured with a Bio-Rad
Imaging Densitometer, and the results were expressed as fold activation
with the intensity of unstimulated cells being 1.
Expression plasmid constructs and transfection
To generate transient expression constructs for SHP-1 and BLNK, PCR fragments containing entire open reading frames for SHP-1 and BLNK were cloned downstream of EF promoter in pEF-flag vector (30) (a gift of Dr. Gary Koretzky, University of Pennsylvania, Philadelphia, PA). The resulting expression vectors, pEF-flag-SHP-1-wt and pEF-flag-BLNK, encode full-length SHP-1 and BLNK, each containing 8-aa peptide tag at NH2 terminus, which is recognized by anti-flag mAb. To introduce a cysteine-to-serine substitution at the position 453 (C453S) in the PTP domain of SHP-1, in vitro mutagenesis was performed on a full-length SHP-1 cDNA cloned in pBluescript using GeneEditor in vitro site-directed mutagenesis system (Promega). Mutation was identified by the presence of an additional restriction site and then verified by sequencing. Mutated SHP-1 cDNA was then cloned into pEF-flag vector (pEF-flag-SHP-1-C/S). For transient transfection, WEHI-231 cells (2 x 107 cells/400 ml/cuvette) were electroporated with 40 µg of the indicated plasmid in the cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 (pH 7.6), 25 mM HEPES (pH 7.6), 2 mM EGTA, 5 mM MgCl2, 2 mM ATP, 5 mM glutathione), using Bio-Rad Gene Pulser II apparatus set at 270 V and 975 mF. After electroporation, cells were placed on ice for 10 min, transferred into 25 ml complete medium, and cultured for 16–18 h.
In vitro binding assay
To generate constructs for GST-SHP-1-wt and GST-SHP-1-C/S fusion proteins, cDNA fragments encoding PTP domain of SHP-1 (aa 274–595) were generated by PCR using pEF-flag-SHP-1-wt and pEF-flag-SHP-1-C/S expression plasmids as templates, respectively. The PCR products were ligated into pGEX-4T3 vector (Amersham Pharmacia Biotech, Uppsala, Sweden). The fusion proteins were generated in E. coli and affinity purified, as described previously (27). For in vitro binding assay, WEHI-231 cells expressing flag-tagged BLNK were either unstimulated or stimulated with pervanadate for 1 min, and lysed with TNE. The cell lysates were incubated overnight at 4°C with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) coupled to SHP-1-wt-GST, SHP-1-C/S-GST, or GST alone. Samples were washed five times with TNE and subjected to Western blot analysis with anti-flag and anti-PY mAbs.
In vitro dephosphorylation assay
WEHI-231 cells were transfected separately with pEF-flag-SHP-1-wt, pEF-flag-SHP-1-C/S, or pEF-flag-BLNK. Cells expressing BLNK were stimulated with pervanadate for 1 min, lysed with TNE. BLNK was immunoprecipitated with anti-flag mAb and released from protein G-Sepharose by heating in 0.5% SDS. The eluent was diluted into 100 mM citrate buffer, pH 5, containing 1 mM DTT (PTP buffer), and used as substrate for in vitro dephosphorylation assay. SHP-1-wt and SHP-1-C/S were also immunoprecipitated with anti-flag mAb, washed three times with PTP buffer, and mixed with phosphorylated BLNK. The mixture of SHP-1-wt:BLNK with or without Na3VO4 (1 mM) and of SHP-1-C/S:BLNK was incubated for 60 min on 37°C. After incubation, reactions were terminated by adding SDS-PAGE sample buffer, and the samples were subjected to Western blot analysis with anti-PY and anti-flag mAbs.
Assays for MAPKs
TNE-soluble supernatants from cells, unstimulated and anti-IgM stimulated, were separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Western blots were incubated with Abs against ERK, JNK, p38, and their phosphorylated forms, followed by incubation with HRP-conjugated anti-rabbit IgG. The blots were visualized by ECL Western blot detection kit (Amersham Pharmacia Biotech). The intensities of bands were measured with a Bio-Rad Imaging Densitometer, and the results were expressed as fold activation with the intensity of unstimulated cells being 1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first used the substrate-trapping method (36, 37)
to identify substrates for SHP-1. This method was developed based on
the fact that a mutant PTP with cysteine residue in the catalytic
center (C453) substituted by serine (PTP-C/S) loses its PTP activity,
but retains the ability to bind to substrate proteins. Expression of
PTP-C/S mutant allows cells to compete with endogenous PTP for
substrates, resulting in accumulation of PY on the substrate proteins
(36, 37). We first examined for proteins in WEHI-231 cells
whose tyrosine phosphorylation was altered when they transiently
expressed the mutant form of SHP-1, SHP-1-C/S. As shown in Fig. 1
, upon anti-IgM stimulation (25
mg/ml), the degree of tyrosine phosphorylation of a protein of about 70
kDa (p70), indicated by arrows, was enhanced in SHP-1-C/S transfectant
cells and slightly reduced in SHP-1-wt transfectants compared with
control cells. Tyrosine phosphorylation of a protein at a slightly
higher m.w., indicated by line, was not significantly different among
groups. Hyperphosphorylation of p70 was observed within 1 min after BCR
ligation and maintained for up to 1 h. We considered this
phosphoprotein of 70 kDa (pp70) as a candidate substrate for SHP-1 and
set out to characterize the nature of this protein by searching for
PY-containing signaling molecules of about 70 kDa. Immunoprecipitation
and Western blot analyses demonstrated that the phosphorylation state
of 75-kDa HS1 (41) and 72-kDa Syk in SHP-1-C/S-transfected
cells was not significantly different from that in SHP-1-wt
transfectants. These results suggested that pp70 is not HS1 nor Syk
(data not shown).
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It has recently been reported that a novel B cell-specific linker
protein, BLNK, is tyrosine phosphorylated after BCR stimulation
(38, 39). To test whether pp70 is BLNK, pEF-flag-BLNK
expression plasmid was introduced in WEHI-231 cells, together with
pEF-flag-SHP-1-wt, pEF-flag-SHP-1-C/S, or pEF-flag vector. Each
transfectant was either unstimulated or stimulated with 25 mg/ml
anti-IgM Ab for 5 min, and exogenously expressed BLNK was
immunoprecipitated with anti-flag mAb and immunoblotted with
anti-PY or anti-flag mAb. Results shown in Fig. 2A indicate that upon BCR
ligation, BLNK immunoprecipitated from SHP-1-C/S transfectants was
tyrosine phosphorylated more strongly than that from vector control
transfectants, and conversely, transfection of SHP-1-wt significantly
reduced tyrosine phosphorylation of BLNK as compared with vector
transfection. In this experiment, similar amounts of BLNK were
immunoprecipitated in SHP-1-C/S, SHP-1-wt, and control transfectants
(Fig. 2A, bottom panel). Furthermore, when total
cell lysates from transfectants of SHP-1-C/S or SHP-1-C/S and BLNK were
immunoblotted with anti-PY and anti-flag mAbs, exogenously
expressed BLNK was shown to migrate at a molecular mass similar to pp70
(Fig. 2B). These results suggested that pp70 might be
BLNK.
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, BLNK that was immunoprecipitated from
SHP-1-C/S transfectants was tyrosine phosphorylated upon BCR ligation
more robustly than that from control transfectants, and again the
degree of tyrosine phosphorylation of BLNK from SHP-1-wt transfectants
was lower than that from vector transfectants. Second, the binding of
BLNK and SHP-1 was examined in in vitro conditions. WEHI-231 cells that
were transfected with flag-tagged BLNK were either untreated or treated
with pervanadate for 1 min, and the total cell lysates were incubated
with Sepharose coupled to GST-SHP-1-wt, GST-SHP-1-C/S, or GST alone.
The precipitates were then immunoblotted with anti-flag and
anti-PY mAbs. The results in Fig. 4
demonstrated that GST-SHP-1-C/S, but not GST-SHP-1-wt nor GST, binds
tyrosine-phosphorylated BLNK. Taken collectively, it is reasonable to
conclude that BLNK is indeed a substrate for SHP-1.
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To test directly whether SHP-1 dephosphorylates
tyrosine-phosphorylated BLNK, we performed in vitro dephosphorylation
assay. BLNK was immunoprecipitated from pervanadate-stimulated WEHI-231
cells that had been transfected with flag-tagged BLNK. WEHI-231 cells
were also transfected with SHP-1, either wt or C/S mutant, and SHP-1
was immunoprecipitated with anti-flag mAb and added to
phosphorylated BLNK. As shown in Fig. 5,
pervanadate treatment induced tyrosine phosphorylation of BLNK and the
addition of SHP-1-wt resulted in almost complete dephosphorylation of
BLNK. However, SHP-1-wt with vanadate (a PTP active site competitor) or
SHP-1-C/S could not dephosphorylate BLNK, suggesting that SHP-1
directly dephosphorylates BLNK in vitro.
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It has been shown that upon Ag receptor stimulation, BLNK and
SLP-76 are rapidly tyrosine phosphorylated by Syk in B cells
(38) and by ZAP-70 (30, 33) in T cells,
respectively. Therefore, Syk or members of Src family PTKs might be
activated as a result of competition between SHP-1-C/S and endogenous
SHP-1, phosphorylating BLNK. To exclude this possibility, we examined
the phosphorylation state and kinase activity of major PTKs in WEHI-231
cells, Lyn and Syk. WEHI-231 cells transfected with SHP-1-wt or
SHP-1-C/S were stimulated with anti-IgM Ab for 3 min, and the
phosphorylation state and enzymatic activity of Lyn and Syk were
examined by immunoblotting with anti-PY mAb and in vitro kinase
assays, respectively. The results revealed that tyrosine
phosphorylation state as well as kinase activity (both
autophosphorylation and phosphorylation of exogenous substrates) of Lyn
and Syk were not significantly different between SHP-1-C/S- and
SHP-1-wt-expressing cells (Fig. 6). This
result indicates that enhanced tyrosine phosphorylation of BLNK in
SHP-1-C/S-expressing cells was not due to increased activity of Lyn or
Syk, but due to direct action of catalytic inactive mutant of
SHP-1.
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Finally, to assess the downstream signaling pathways initiated by
SHP-1 and BLNK, MAPK activation was examined in SHP-1-wt- and
SHP-1-C/S-expressing cells. WEHI-231 cells, transfected with SHP-1-wt,
SHP-1-C/S, or empty vector, were stimulated with anti-IgM Ab for 5,
15, and 30 min, and activation of MAPK family members, ERK, JNK, and
p38, was measured by immunoblotting with anti-phospho-ERK, -JNK,
and -p38 Abs. As shown in Fig. 7A, activation of ERK and p38
in SHP-1-C/S transfectants was not significantly different from that in
SHP-1-wt transfectants or control cells. However, activation of JNK was
strongly increased in SHP-1-C/S transfectants. Densitometric analysis
of three independent experiments further confirms that SHP-1 has a
selective, negative regulatory effect on JNK activation (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we try to elucidate the SHP-1 action by identifying its
substrate using the substrate-trapping approach (36, 37).
The results presented herein demonstrate that expression of SHP-1-C/S
in WEHI-231 cells leads to hyperphosphorylation of a 70-kDa protein
upon BCR stimulation (Fig. 1
), and that this protein turned out to be a
recently identified linker protein, BLNK (38, 39). This
conclusion is based on the findings that the m.w. of BLNK is identical
to a hyperphosphorylated protein of 70 kDa (Fig. 2B), that
both exogenously expressed and endogenous BLNK is hyperphosphorylated
upon BCR ligation in SHP-1-C/S-expressing cells and transfection of
SHP-wt decreases the degree of BLNK phosphorylation (Figs. 2A and 3), that upon tyrosine phosphorylation BLNK binds
SHP-1-C/S in vitro (Fig. 4), and that catalytically active SHP-1
dephosphorylates tyrosine-phosphorylated BLNK in vitro (Fig. 5). It is
thus concluded that BLNK is a physiological substrate for SHP-1 in
WEHI-231 cells. Physical association between BLNK and SHP-1-C/S in
vivo was not detected under our experimental conditions. Given that
SHP-1 acts on diverse substrates and BLNK binds many molecules and that
in cells, interaction between these two molecules is dynamic and each
interaction event may be transient, it may be difficult to constantly
observe interaction between PTPs and their substrates in vivo.
BLNK was identified as a phosphorylated adaptor protein that binds
multiple signaling molecules such as Grb2, Vav guanine nucleotide
exchange factor, PLC-1, and Nck adaptor protein (38, 39, 44). BLNK consists of an NH2-terminal
basic and acidic domain, a central proline-rich region, and a
COOH-terminal SH2 domain (38, 39). The binding of BLNK
with Vav, PLC-, and Nck is mediated by tyrosine
phosphorylation-dependent mechanisms involving SH2 domains of the
latter, whereas BLNK is constitutively associated with SH3 and SH2
domains of Grb2 (38, 39, 44). The functional significance
of tyrosine phosphorylation of BLNK by Syk was clearly illustrated by
the findings that overexpression of wild-type BLNK and mutant BLNK, in
which tyrosine-72, -84, -96, and -178 are substituted to phenylalanine,
respectively, enhances and reduces BCR-induced tyrosine phosphorylation
of PLC-1 and PLC-2 and BCR-mediated calcium mobilization and
transcriptional activation of the NF-AT (38). From these
results, the following scenario emerges. BCR ligation induces
activation of Lyn and subsequently Syk PTKs, which in turn
phosphorylate BLNK. Tyrosine-phosphorylated BLNK is then translocated
to the membrane and serves as a scaffold protein for SH2-containing
signaling molecules, including PLC-1. Membrane-located PLC-1 is
further tyrosine phosphorylated and activated by Syk, initiating the
phosphoinositide pathway.
The results presented in this study demonstrate that among MAPK family
members, JNK activation is selectively enhanced by transfection of
SHP-1-C/S (Fig. 7), suggesting that SHP-1 plays a negative regulatory
role in the JNK pathway. Preliminary results showed that calcium
responses were not significantly affected in SHP-1-C/S transfectants.
Although 20–25% of cells were successfully transfected as revealed by
control transfection with green fluorescence protein, the level of
phosphorylation of BLNK observed in SHP-1-C/S transfectants may not be
high enough to relay messages to downstream targets leading to calcium
mobilization. Indeed, phosphorylation of PLC-2 was not significantly
altered in SHP-C/S-expressing cells. One of the possibilities is that a
threshold for BLNK phosphorylation leading to PLC- activation and
calcium responses may be set higher than that leading to other
downstream signaling pathways, for example, MAPK. It is also possible
that the requirement for BLNK phosphorylation in calcium responses may
be more stringent in WEHI-231 cells than in other cells.
A recent study addressed the question of whether BLNK regulates
downstream targets selectively or exerts its effects nonspecifically,
because BLNK-binding Vav and Grb2 are involved in the regulation of two
small G proteins, Rac1 and Ras, respectively (45). As it
turns out, BLNK plays a critical role in coupling Syk activation to
activation of PLC2 and Rac1-JNK pathways, but not to Ras activation,
thus selecting downstream signaling pathways (45). Our
results suggest that SHP-1 directly regulates the phosphorylation state
of BLNK, thereby contributing to the fine tuning of which downstream
signaling pathways are activated. Whether SHP-1 regulates only JNK
pathway or controls multiple pathways involving Vav or Nck, for
example, remains to be seen.
A recent report demonstrated that expression of SHP-1-C/S in the
IgG2a-positive K46 B lymphoma line results in increased Syk kinase
activity, and thus Syk is a possible substrate for SHP-1
(46). Furthermore, it has been also reported that the
activity of Src family PTKs such as Lck and Fyn is enhanced in
thymocytes from SHP-1-deficient me/me mice, suggesting that
SHP-1 negatively regulates Src family PTKs (47). Thus, it
seems that SHP-1 is a common regulator for not only Src family but also
Syk PTKs. However, present study shows that PTKs such as Lyn and Syk
are not directly regulated by SHP-1 based on the findings that the
enzymatic activity of Lyn and Syk in SHP-1-C/S-expressing cells is not
significantly different from that in SHP-1-wt-expressing cells (Fig. 6). Definitive reasons for this discrepancy are not clear at present.
Given accumulating evidence suggesting that a PTP acts on different
substrates depending on the cellular milieu dictated by the cell type,
activation stage, or differentiation stage (48, 49), one
of the possible reasons for the discrepant phenomena may be due to
differences in the cell type assayed: for example, T cell
(47) vs B cell (present study, 46), or immature B cells
(present study) vs highly differentiated (class-switched) B cells
(46).
In summary, we demonstrate that an adaptor protein BLNK constitutes a physiological substrate for SHP-1 in WEHI-231 B cells. In vitro kinase assays reveal that the kinase activity of Lyn and Syk is not altered in SHP-1-C/S transfectant cells, suggesting that these PTKs are not substrates for SHP-1. Furthermore, SHP-1 selectively exerts its negative effect on BCR-induced activation of JNK.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Tokyo Women’s Medical University, School of Medicine, Tokyo 162-8666, Japan.
3 Address correspondence and reprint requests to Dr. Hidetaka Yakura, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.
4 Abbreviations used in this paper: BCR, B cell Ag receptor; AP, alkaline phosphatase; BLNK, B cell linker protein; ERK, extracellular signal-related kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; PY, phosphotyrosine; SH2, Src homology region 2; SHP-1, SH2 domain-containing phosphatase-1; SHP-1-C/S, SHP-1 in which the catalytic center cysteine (C453) was replaced with serine; SLP, SH2 domain leukocyte protein; wt, wild-type.
Received for publication September 10, 1999. Accepted for publication May 19, 2000.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CD3 induction of interleukin-2. Immunity 6:155.[Medline]
, Grb2, and Vav after B cell antigen receptor activation. J. Biol. Chem. 272:27362.2 and Rac1-JNK in B cells. Immunity 10:117.[Medline]
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K. Mizuno, Y. Tagawa, K. Mitomo, N. Watanabe, T. Katagiri, M. Ogimoto, and H. Yakura Src Homology Region 2 Domain-Containing Phosphatase 1 Positively Regulates B Cell Receptor-Induced Apoptosis by Modulating Association of B Cell Linker Protein with Nck and Activation of c-Jun NH2-Terminal Kinase J. Immunol., July 15, 2002; 169(2): 778 - 786. [Abstract] [Full Text] [PDF]
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H. Gary-Gouy, J. Harriague, A. Dalloul, E. Donnadieu, and G. Bismuth CD5-Negative Regulation of B Cell Receptor Signaling Pathways Originates from Tyrosine Residue Y429 Outside an Immunoreceptor Tyrosine-Based Inhibitory Motif J. Immunol., January 1, 2002; 168(1): 232 - 239. [Abstract] [Full Text] [PDF]
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 T. Adachi, J. Wienands, C. Wakabayashi, H. Yakura, M. Reth, and T. Tsubata SHP-1 Requires Inhibitory Co-receptors to Down-modulate B Cell Antigen Receptor-mediated Phosphorylation of Cellular Substrates J. Biol. Chem., July 6, 2001; 276(28): 26648 - 26655. [Abstract] [Full Text] [PDF]
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