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International Immunology Advance Access originally published online on August 3, 2007
International Immunology 2007 19(9):1049-1061; doi:10.1093/intimm/dxm070
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Negative autoregulation of Src homology region 2-domain-containing phosphatase-1 in rat basophilic leukemia-2H3 cells

Tomoko Ozawa1, Kazuko Nakata1, Kazuya Mizuno1 and Hidetaka Yakura1,2

1 Department of Immunology and Signal Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Science, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan
2 Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan

Correspondence to: K. Mizuno; E-mail: kzmizuno{at}tmin.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
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 Discussion
 Funding
 References
 
Src homology region 2-domain-containing phosphatase-1 (SHP-1) plays an important role in the regulation of signaling from various receptors in hematopoietic cells. In mast cells, SHP-1 has been shown to negatively regulate the initial signaling triggered by high-affinity receptor for IgE (Fc{epsilon}RI) and positively regulate downstream outputs. To clarify the molecular mechanisms of SHP-1 in mast cells, we determined substrates for SHP-1 by using the substrate-trapping approach. When phosphatase-inactive SHP-1 was over-expressed in rat basophilic leukemia (RBL)-2H3 cells, tyrosine phosphorylation of a 68-kDa protein was enhanced before and after Fc{epsilon}RI aggregation. Immunoprecipitation and western blot analyses revealed that this protein is SHP-1, either endogenous or ectopically expressed. Fc{epsilon}RI-induced activation of Lyn and Syk was comparable between cells expressing wild-type (wt) and phosphatase-inactive SHP-1. In vitro phosphatase assay and combined transfection, immunoprecipitation and immunoblot analyses showed that tyrosine 536 of SHP-1 was potent phosphorylation site and that SHP-1 could dephosphorylate this site that had been phosphorylated by Lyn. Furthermore, the phosphatase activity of SHP-1 immunoprecipitated from cells expressing a phosphatase-inactive SHP-1 was increased compared with that from vector-transfected or wt SHP-1-expressing cells. Finally, expression of phosphatase-inactive SHP-1 resulted in decreased activation of mitogen-activated protein kinases and suppressed transcription of cytokine genes, whereas wt SHP-1 enhanced these processes. Taken collectively, these results suggest that SHP-1 may be a physiological substrate of SHP-1 in RBL-2H3 cells and that dephosphorylation of SHP-1 leads to a decrease in its catalytic activity and an enhancement of downstream signaling. A negative autoregulatory circuit of SHP-1 may contribute to mast cell regulation.

Keywords: Fc receptors, mast cells, SHP-1, signal transduction


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
 Funding
 References
 
Mast cells play a central role in the induction of IgE-mediated immediate hypersensitivity and allergic diseases. Cross-linking of the high-affinity receptor for IgE (Fc{epsilon}RI) with IgE and multivalent antigen leads to mast cell activation that results in degranulation, release of granules containing preformed chemical mediators and the synthesis and secretion of cytokines and chemokines (1, 2). Fc{epsilon}RI belongs to the multichain immune receptor superfamily, consisting of one IgE-binding {alpha} chain, one ß chain and a homodimer of disulfide-linked {gamma} chains (3). Among them, ß and {gamma} chains that contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic regions are essential for mediating downstream signaling events (4). Upon cross-linking of Fc{epsilon}RI, the Src family protein tyrosine kinase (PTK), Lyn, constitutively associated with the ß chain, is activated and phosphorylates tyrosine residues in the ITAMs of the ß and {gamma} chains. Syk PTK is then recruited to the phosphorylated ITAMs of {gamma} chain via its Src homology region 2 (SH2) domains and becomes tyrosine phosphorylated and activated by Lyn (5–7). The activated PTKs further phosphorylate a number of signaling molecules, including phospholipase C (PLC){gamma} (8, 9), Vav (10), SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) (11), linker for activation of T cells (LAT) (12) and linker for activation of B cells (LAB)/non-T cell activation linker (NTAL) (13). Phosphorylation of these proteins induces Ca2+ mobilization and activation of mitogen-activated protein kinases (MAPKs) and is ultimately converged into degranulation and production of cytokines and chemokines. Thus, tyrosine phosphorylation process is critical for Fc{epsilon}RI-mediated mast cell activation.

Because the extent of tyrosine phosphorylation of cellular proteins is strictly balanced by the opposite action of PTKs and protein tyrosine phosphatases (PTPs), much attention has recently been focused on a role of PTPs in immune receptor-mediated signaling (14). The cytosolic Src homology region 2-domain-containing phosphatase-1 (SHP-1) has two SH2 domains in N-terminus, one catalytic domain and the other C-terminal regulatory domain containing two tyrosine residues that are phosphorylated by Src family PTKs (15). SHP-1 is implicated as a negative regulator of receptor-mediated signaling in lymphocytes because B and T cells derived from SHP-1-deficient motheaten (me) mice are hyperresponsive to antigen receptor stimulation (16, 17). In mast cells, following Fc{epsilon}RI engagement, SHP-1 is recruited to the phosphorylated tyrosine residues in immunoreceptor tyrosine-based inhibitory motifs (ITIMs) found in the cytoplasmic regions of inhibitory receptors such as gp49B1 (18, 19), PIR-B (20), leukocyte mono-Ig-like receptor 1 (LMIR1) (21) and myeloid-associated Ig-like receptor-1 (MAIR-I) (22). As a result, SHP-1 is activated and is predicted to dephosphorylate various signaling molecules, mediating negative regulation of Fc{epsilon}RI-initiated downstream signal transduction. However, with one exception (23), precise molecular mechanisms of the negative regulation by SHP-1 have been largely unknown.

To understand the physiological function of PTPs in cellular signaling, it is necessary to identify their substrates and define the regulation of their activities. To this end, the substrate-trapping approach has been widely used (24, 25). This procedure is based on the following findings. In the PTP catalytic domain, highly conserved two amino acids, cysteine and aspartic acid, are required for dephosphorylation reaction (26). The PTP possessing a mutation of cysteine to serine (C/S mutant) or aspartic acid to alanine (D/A mutant) loses its catalytic activity but still retains its ability to bind phosphorylated tyrosine residues of the substrates, thus competing with endogenous PTPs. It is, therefore, expected that tyrosine phosphorylation of substrate proteins is increased in the cells expressing substrate-trapping mutants.

In the present study, to elucidate the role of SHP-1 in Fc{epsilon}RI-mediated signaling events, we determined substrates of SHP-1 in mast cells by introducing a wild-type (SHP-1-wt) or a substrate-trapping mutant of SHP-1 (SHP-1-C/S) into rat basophilic leukemia (RBL)-2H3 cells. The results demonstrated that the transfection of SHP-1-C/S enhanced tyrosine phosphorylation of endogenous or exogenously introduced SHP-1. The activity of Lyn, responsible for phosphorylating and activating SHP-1 (15, 27), was comparable among vector-transfected cells, cells expressing SHP-1-wt and SHP-1-C/S. Significantly, SHP-1 could dephosphorylate phosphorylated tyrosine 536 in the C-terminal regulatory domain of SHP-1 in vitro and enhanced tyrosine phosphorylation was detected in the exogenously introduced SHP-1-C/S in which tyrosine 564 was substituted to phenylalanine but not in that containing a tyrosine to phenylalanine substitution at tyrosine 536 in vivo, suggesting that tyrosine 536 is a potent phosphorylation site both in vitro and in vivo and that SHP-1 itself is a substrate of SHP-1. Furthermore, increased phosphorylation of SHP-1 augmented its catalytic activity, inhibiting MAPK activation and cytokine responses. Taken collectively, our results might provide a novel, autoregulatory mechanism for SHP-1 activity.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Antibodies and reagents
Anti-dinitrophenol (DNP) IgE mAb (SPE-7), dinitrophenol-conjugated human serum albumin (DNP-HSA) and anti-flag epitope-M2 mAb were purchased from Sigma-Aldrich (St Louis, MO, USA). HRP-conjugated anti-phosphotyrosine (PY) mAb (PY20) and antibodies against SHP-1, SHP-2, ERK, c-myc, Lyn, Syk, extracellular signal-regulated kinase (ERK)2, c-Jun N-terminal kinase (JNK)2, p38, PLC{gamma}1, PLC{gamma}2 and AKT were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-specific JNK, anti-phospho-specific p38 and anti-phospho-specific AKT (serine 473) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-specific ERK antibody was from Promega (Madison, WI, USA). Anti-glutathione S-transferase (GST) antibody was raised in rabbits by immunizing with GST protein. All HRP-conjugated secondary antibodies were described (28). Enhanced chemiluminescence western blotting detection kit and glutathione Sepharose 4B were obtained from Amersham Bioscience (Buckinghamshire, UK).

Cell culture and stimulation
The RBL cell line, RBL-2H3, was obtained from the Human Science Research Resource Bank (Osaka, Japan). The cells were maintained as monolayers in Eagle's MEM (EMEM: Sigma-Aldrich) supplemented with 10% heat-inactivated FCS, 100 µg ml–1 streptomycin and 100 Uml–1 penicillin (complete EMEM). For cell stimulation, cells were incubated with 100 ng ml–1 of SPE-7 for 5 h. After washing monolayers twice with PBS to remove excess SPE-7, the cells were incubated in complete EMEM for 1 h and, then, stimulated with 50 ng ml–1 of DNP-HSA for various periods of times.

Immunoprecipitation and western blot analysis
After simulation, the reaction was stopped with ice-cold PBS containing 1 mM Na3VO4 and 2 mM EDTA (PBS-VE). Monolayer was washed twice with PBS-VE and lysed in TNE buffer (1% Nonidet P-40, 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM Na3VO4 and 2 mM EDTA). The total cell lysates (TCLs) thus prepared were subjected to immunoprecipitation and western blot analysis as described (28).

Expression constructs and transfection
Expression constructs for flag-tagged SHP-1-wt (pEF-flag-SHP-1-wt) and SHP-1-C/S (pEF-flag-SHP-1-C/S), in which cysteine 453 in the PTP domain of SHP-1 was substituted to serine, have been described (28). cDNA encoding SHP-1-wt tagged with c-myc epitope at N-terminus was generated by PCR using pEF-flag-SHP-1-wt as a template and sub-cloned into pEFIII vector (a gift from Dr G. Koretzky, University of Pennsylvania, Philadelphia, PA, USA), yielding pEF-myc-SHP-1-wt. To introduce a tyrosine to phenylalanine substitution at positions 536 and 564 of SHP-1-C/S, PCR-directed mutagenesis was performed using pEF-flag-SHP-1-C/S as a template and mutations were confirmed by DNA sequencing, thus yielding pEF-flag-SHP-1-C/S-Y536F and pEF-flag-SHP-1-C/S-Y564F, respectively. For transient transfection, 40 µg of the indicated plasmid was transfected into 2 x 107 single-cell suspension of RBL-2H3 cells in the cytomix buffer (28) at 290 V, 975 µF by using Gene Pulser II Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA). After electroporation, cells were placed on ice for 10 min and transferred to complete EMEM and cultured for 16–18 h. The expression level of transfected gene was 30–35% as determined by flow cytometry with pEF-green fluorescent protein (GFP) transfectant. To increase the amount of transduced protein expression, 80 µg of plasmid was used for the transfection. Transfected cells were incubated overnight and the electroporation with 80 µg of plasmid was repeated on the next day. Following overnight incubation, cells were used for the experiments. In this case, the expression level of transfected gene was 55–65%.

Expression of GST fusion protein
cDNA encoding the C-terminal domain of SHP-1 (amino acids 516–595) was generated by PCR using pEF-flag-SHP-1-wt as template. After confirming the sequence, PCR product was sub-cloned into pGEX-4T3 vector (Amersham Bioscience), thus yielding pGEX-SHP-1-C-wt. The fusion protein, GST-SHP-1-C-wt, was generated in Escherichia coli and affinity purified by eluting from glutathione Sepharose 4B beads with 5 mM glutathione in 50 mM Tris–HCl, pH 8.0, as recommended by the manufacturer. To introduce a tyrosine to phenylalanine substitution at positions 536 and 564 of SHP-1, PCR-directed mutagenesis was performed using pGEX-SHP-1-C-wt as a template, yielding pGEX-SHP-1-C-Y536F and pGEX-SHP-1-C-Y564F, respectively. After mutations were confirmed by DNA sequencing, the fusion proteins, GST-SHP-1-C-Y536 and GST-SHP-1-C-Y564, were generated as described above.

In vitro kinase and dephosphorylation assay
In vitro kinase assay for Lyn was performed as described previously (28). In vitro dephosphorylation assay was performed by using either p-nitrophenyl phosphate (pNPP) or GST-SHP-1-C as a substrate. Affinity-purified GST-SHP-1-C-wt, -Y536F or -Y564F was incubated with 1 mM ATP and active Lyn that were immunoprecipitated from RBL-2H3 cells stimulated for 1 min. The phosphorylated GST-SHP-1-C or 5 mM pNPP in PTP buffer (28) was incubated with SHP-1, SHP-1-C/S or SHP-2 that was immunoprecipitated from RBL-2H3 cells or SHP-1-C/S-expressing cells in the case of SHP-1-C/S stimulated for 2 min, in the absence or presence of Na3VO4 (2 mM) for 15 min at 37°C. For the experiments using pNPP as a substrate, the reaction was terminated by adding 1 N NaOH, and the amount of p-nitrophenol released was measured at 405 nm by spectrophotometer. The reaction using GST-SHP-1-C was stopped by addition of SDS-PAGE sample buffer and the samples were subjected to western blot analysis with anti-PY mAb.

Real-time PCR
Total RNA was isolated using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA, USA) and reverse transcribed using a cDNA ExScript RT Reagent Kit (Takara Bio, Otsu, Japan) with random hexamer primers. Real-time PCR was performed using Smart Cycler II System (Takara Bio) with SYBR Premix Ex Taq (Takara Bio) and commercially available primer sets specific for tumor necrosis factor {alpha} (TNF{alpha}), IL-6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Takara Bio). Levels of TNF{alpha} and IL-6 were first normalized with GAPDH levels and determined relative to the unstimulated vector-transfected group, which was arbitrarily designated a value of 1.0.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Tyrosine phosphorylation of a 68-kDa protein is increased in SHP-1-C/S-expressing cells
To identify substrates of SHP-1 in RBL-2H3 cells, we over-expressed a trapping mutant SHP-1-C/S, in which a critical cysteine 453 in the catalytic center was substituted to serine. Since cysteine 453 is required for the interaction with phosphate group in the substrate to form a thiophosphate intermediate (29), this mutation decreases catalytic activity but still retains the ability to bind to substrate proteins, competing with endogenous PTP for the substrate proteins. Thus, the expression of SHP-1-C/S in RBL-2H3 cells should cause accumulation of phosphorylated tyrosine residues in potential substrates. We selected this mutant because it was effective to identify B cell substrates such as B cell linker protein (BLNK) (28) and SLP-76 (30). RBL-2H3 cells transfected with vector alone, pEF-flag-SHP-1-wt or pEF-flag-SHP-1-C/S were incubated with DNP-specific IgE mAb, SPE-7 (100 ng ml–1), for 5 h and stimulated with DNP-HSA (50 ng ml–1) for the indicated length of time. TCLs from these cells were subjected to SDS-PAGE and anti-PY western blotting. As shown in Fig. 1(A), increased tyrosine phosphorylation was observed in proteins migrating at 75, 68 (p68) and 55 kDa following Fc{epsilon}RI cross-linking. Almost equal protein loading was verified by anti-ERK immunoblotting (Fig. 1A, bottom panel). Among them, hyperphosphorylation of p68 was more evident even before stimulation and almost unchanged up to 15 min following Fc{epsilon}RI cross-linking. Tyrosine phosphorylation of p68 was induced in RBL-2H3 cells transfected with vector alone or SHP-1-wt but to a lesser extent. Since previous report (23) demonstrated that over-expression of another trapping mutant, SHP-1-D/A, enhanced phosphorylation of Syk and ß- and {gamma}-chain of Fc{epsilon}RI, we examined the effects of over-expression of SHP-1-C/S on cellular tyrosine phosphorylation. Although repeated transfection with increased amount of plasmid resulted in at least 4-fold over-expression of SHP-1 compared with control transfectants (Fig. 1D), the pattern of the total cellular tyrosine phosphorylation induced by Fc{epsilon}RI cross-linking was almost identical to that shown in Fig. 1(A and C). These results suggest that p68 is a candidate substrate for SHP-1 in RBL-2H3 cells.


Figure 1
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  		Fig. 1. Tyrosine phosphorylation of a 68-kDa protein is increased in SHP-1-C/S-expressing cells. (A) RBL-2H3 cells, transfected with vector alone, pEF-flag-SHP-1-wt or pEF-flag-SHP-1-C/S, were cultured with 100 ng ml–1 of SPE-7 for 5 h and then stimulated with 50 ng ml–1 of DNP-HSA for the indicated periods of time. TCLs were size fractionated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-PY mAb or anti-ERK antibody. A band migrating at 68 kDa (p68), indicated by an arrowhead, showed enhanced phosphorylation in SHP-1-C/S-expressing cells. The molecular weight markers used are shown in kilodaltons on the left. (B) Expression of SHP-1-wt and SHP-1-C/S was confirmed by immunoblotting the same membrane with anti-flag mAb. (C and D) Cells after repeated transfection were cultured with 100 ng ml–1 of SPE-7 for 5 h and then stimulated with 50 ng ml–1 of DNP-HSA for 5 min. TCLs were subjected to SDS-PAGE and immunoblotted with anti-PY mAb (C) or anti-SHP-1 antibody and anti-flag mAb (D). In (D), the intensity of SHP-1 was determined relative to the unstimulated vector-transfected cells, which was arbitrarily designated a value of 1.0.

 
SHP-1 is hyperphosphorylated in SHP-1-C/S-expressing cells
Given the similarity in electrophoretic mobility between p68 (Fig. 1A) and exogenously introduced flag-tagged SHP-1 (Fig. 1B), we performed experiments to determine whether p68 is SHP-1 by using combined immunoprecipitation and western blot analysis. RBL-2H3 cells transfected with vector, SHP-1-wt or SHP-1-C/S were either left unstimulated or stimulated for 5 and 15 min, and ectopically expressed SHP-1 was immunoprecipitated with anti-flag mAb, divided into two parts, and immunoblotted one with anti-PY mAb and another with anti-flag mAb. The results shown in Fig. 2(A) clearly demonstrated that basal as well as stimulation-induced tyrosine phosphorylation was higher in cells expressing SHP-1-C/S than in those expressing SHP-1-wt.


Figure 2
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  		Fig. 2. Tyrosine phosphorylation of exogenously introduced and endogenous SHP-1 is increased in SHP-1-C/S-expressing cells. (A) RBL-2H3 cells were transfected with vector alone, SHP-1-wt or SHP-1-C/S. Following stimulation for 5 and 15 min with DNP-HSA, exogenously introduced SHP-1 was immunoprecipitated with anti-flag mAb and the samples were divided into two parts, one for anti-PY immunoblot and another for anti-flag immunoblot. (B) After depleting flag-tagged SHP-1 by three rounds of immunoprecipitation with anti-flag mAb from the TCLs used in (A), endogenous SHP-1 was immunoprecipitated with anti-SHP-1 antibody and the samples were divided into two parts, one for anti-PY immunoblot and another for anti-SHP-1 immunoblot. Complete removal of exogenously expressed SHP-1 was confirmed by anti-flag immunoblot of the TCLs after anti-flag immunoprecipitation. (C) SHP-2 was immunoprecipitated from the TCL prepared as in (A) and immunoblotted with anti-PY mAb and anti-SHP-2 antibody.

 
We further examined the effect of the expression of SHP-1-wt or SHP-1-C/S on the tyrosine phosphorylation of endogenous SHP-1. Each transfectant was either left unstimulated or stimulated for 5 and 15 min, and exogenously expressed SHP-1 was precleared from lysates by immunoprecipitation with anti-flag mAb. Having confirmed that supernatants did not contain flag-tagged SHP-1 by anti-flag immunoblot (Fig. 2B, bottom panel), endogenous SHP-1 was immunoprecipitated with anti-SHP-1 antibody, divided into two parts, and immunoblotted one with anti-PY mAb and another with anti-SHP-1 antibody. As shown in Fig. 2(B), phosphorylation of endogenous SHP-1 in vector- (lane 1) and SHP-1-wt-transfected cells (lane 4) was weak before stimulation and induced ~2-fold following Fc{epsilon}RI cross-linking. The level of SHP-1 phosphorylation was slightly reduced in SHP-1-wt-expressing cells. In contrast, the expression of SHP-1-C/S strongly increased tyrosine phosphorylation in endogenous SHP-1 before and after stimulation (lanes 7–9). As a control, we could detect little difference in tyrosine phosphorylation of SHP-2 among three transfectants (Fig. 2C). Thus, we conclude that p68 is SHP-1 itself and that the tyrosine phosphorylation of endogenous SHP-1 is negatively regulated by SHP-1.

Activity of Lyn and phosphorylation of Syk are not affected in SHP-1-C/S-expressing cells
SHP-1 contains in its C-terminal regulatory region two tyrosine residues that become phosphorylated by the action of Src family PTKs (15). Thus, to prove that SHP-1 is a substrate of SHP-1, we have to rule out the possibility that SHP-1 regulates the catalytic activity of PTKs responsible for SHP-1 phosphorylation. To this end, we determined the phosphorylation state of Lyn and Syk and the kinase activity of Lyn, a major Src family PTK in RBL-2H3 cells. RBL-2H3 cells transfected with SHP-1-wt or SHP-1-C/S were cultured in the presence of SPE-7 for 5 h and stimulated with DNP-HSA for 2 and 5 min, and immunoprecipitated Lyn and Syk were subjected to anti-PY immunoblotting. As shown in Fig. 3(A), the phosphorylation state of both PTKs was comparable among cells transfected with vector alone, SHP-1-wt- and SHP-1-C/S-expressing cells. Furthermore, in vitro kinase assay revealed that the kinase activity of Lyn was not significantly affected by the expression of SHP-1-C/S (Fig. 3B), indicating that enhanced phosphorylation of SHP-1 in SHP-1-C/S-expressing cells was not due to the increased activity of Lyn or Syk.


Figure 3
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  		Fig. 3. Activity of Lyn and phosphorylation of Syk are comparable among cells transfected with vector alone, SHP-1-wt- and SHP-1-C/S-expressing cells. RBL-2H3 cells transfected with either vector alone, SHP-1-wt or SHP-1-C/S were sensitized with SPE-7 and stimulated with 50 ng ml–1 of DNP-HSA for the times indicated. (A) Lyn (left) and Syk (right) immunoprecipitated from each transfectant were subjected to immunoblotting with anti-PY mAb. The amount of Lyn and Syk immunoprecipitated was estimated by immunoblotting using anti-Lyn and anti-Syk antibodies, respectively. (B) The activity of Lyn was determined by in vitro kinase assay using enolase as an exogenous substrate. Kinase activity was expressed as fold activation; the density of the sample from unstimulated SHP-1-wt-expressing cells was arbitrarily assigned to 1.0. The results were confirmed to be representative of three independent experiments.

 
SHP-1 dephosphorylates the C-terminal domain of SHP-1
To directly examine whether SHP-1 can dephosphorylate SHP-1, we performed in vitro dephosphorylation assay using tyrosine-phosphorylated GST fusion protein containing C-terminal domain of SHP-1 (GST-SHP-1-C) as a substrate (see Methods). As shown in Fig. 4(A), tyrosine phosphorylation of GST-SHP-1-C was reduced by the addition of SHP-1 in a dose-dependent manner (upper panel, lanes 3–5). This dephosphorylation activity was almost completely diminished in the presence of 2 mM of Na3VO4 (a PTP-active site competitor: upper panel, lane 6), implying direct dephosphorylation of SHP-1 by SHP-1 in vitro. In contrast, SHP-2 that was immunoprecipitated from RBL-2H3 cells failed to dephosphorylate GST-SHP-1-C (lane 7), supporting the specificity of this assay. Since two tyrosine residues in C-terminal regulatory domain, tyrosine 536 and 564, have been reported to be phosphorylated by Src family PTKs (15), we next tried to identify the dephosphorylation sites by SHP-1. To this end, we introduced a tyrosine to phenylalnine substitution at a position of 536 or 564 of GST-SHP-1-C and performed in vitro dephosphorylation assay. As shown in Fig. 4(B), tyrosine phosphorylation could not be observed in GST-SHP-1-C-Y536F (lane 3). In contrast, GST-SHP-1-Y564F was effectively phosphorylated by Lyn and this tyrosine phosphorylation was reduced by the addition of SHP-1 (Fig. 4B, lanes 5 and 6). Thus, tyrosine 536 is a potent phosphorylation site for Lyn and a dephosphorylation site by SHP-1 at least in vitro. In addition, SHP-1-C/S immunoprecipitated from flag-SHP-1-C/S-expressing cells stimulated for 2 min failed to dephosphorylate GST-SHP-1-C-Y536F (Fig. 4C, lane 3), confirming the lack of PTP activity in this trapping mutant. Finally, we addressed the question whether tyrosine 536 becomes phosphorylated in vivo after Fc{epsilon}RI cross-linking and whether phsophosrylation of this site is enhanced in the presence of C/S background. RBL-2H3 cells were transfected with SHP-1-wt, SHP-1-C/S or two SHP-1-C/S mutants, in which a tyrosine to phenylalanine substitution was introduced at tyrosine 536 (SHP-1-C/S-Y536F) or tyrosine 564 (SHP-1-C/S-Y564F), and were either unstimulated or stimulated for 2 and 5 min. Ectopically expressed SHP-1 was immunoprecipitated with anti-flag mAb and then endogenous SHP-1 was immunoprecipitated with anti-SHP-1 antibody. Ectopically expressed and endogenous SHP-1 were subjected to anti-PY and anti-flag immunoblots and anti-PY and anti-SHP-1 immunoblots, respectively. The results shown in Fig. 4(D) demonstrated that enhanced tyrosine phosphorylation was detected in SHP-1-C/S and SHP-1-C/S-Y564F before and after stimulation but not in SHP-1-C/S-Y536F (Fig. 4D, top two panels, lanes 4–6 and 10–12). Similarly, tyrosine phosphorylation was markedly increased in endogenous SHP-1 immunoprecipitated from SHP-1-C/S- and SHP-1-C/S-Y564-expressing cells (Fig. 4D, bottom two panels, lanes 4–6 and 10–12) but not in that from SHP-1-C/S-Y536-expressing cells (Fig. 4D, bottom two panels, lanes 7–9). In addition, tyrosine phosphorylation of endogenous SHP-1 in SHP-1-C/S-Y536-expressing cells was slightly enhanced compared with that in SHP-1-wt-expressing cells (Fig. 4D, bottom two panels, lanes 1–3 versus lanes 7–9). Taken collectively, it is likely that tyrosine 536 of SHP-1 is a potent phosphorylation site both in vitro and in vivo, that SHP-1 dephosphorylates this tyrosine and that phosphorylation of tyrosine 536 is required for the dominant negative effect of SHP-1-C/S.


Figure 4
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  		Fig. 4. SHP-1 dephosphorylates tyrosine 536 in the C-terminal domain of SHP-1 in vitro. (A) GST-SHP-1-C-wt was tyrosine phosphorylated by Lyn and used as a substrate for the assay. SHP-1 and SHP-2 (control) were immunoprecipitated from RBL-2H3 cells stimulated for 2 min. Phosphatase samples and phosphorylated GST-SHP-1-C-wt were incubated in PTP buffer in the presence or absence of Na3VO4 (2 mM) for 15 min at 37°C. The reactions were terminated by the addition of SDS-PAGE sample buffer and subjected to immunoblotting with anti-PY mAb to determine the degree of dephosphorylation of the substrate. (B) GST-SHP-1-C-wt, GST-SHP-1-C-Y536F and GST-SHP-1-C-Y536F were tyrosine phosphorylated by Lyn and used as substrates for in vitro dephosphorylation assay by SHP-1 as described in (A). (C) GST-SHP-1-C-Y564F was tyrosine phosphorylated by Lyn and used as a substrate for the assay by SHP-1 or SHP-1-C/S that was immunoprecipitated from flag-SHP-1-C/S-expressing cells stimulated for 2 min. (D) RBL-2H3 cells, transfected with SHP-1-wt, SHP-1-C/S, SHP-1-C/S-Y536F or SHP-1-C/S-Y564F, were either left unstimulated or stimulated for 2 and 5 min. Ectopically expressed SHP-1 was immunoprecipitated with anti-flag mAb and immunoblotted with anti-PY and anti-flag mAbs (top two panels). Following the immunoprecipitation with anti-flag mAb, endogenous SHP-1 was immunoprecipitated with anti-SHP-1 antibody and immunoblotted with anti-PY mAb and anti-SHP-1 antibody (bottom two panels).

 
SHP-1 associates with SHP-1 in vivo
We next examined whether SHP-1 interacts with SHP-1 in the cells. RBL-2H3 cells transfected with pEF-flag-SHP-1-wt and pEF-myc-SHP-1-wt were either unstimulated or stimulated for 2 and 5 min. Having confirmed that both exogenously introduced proteins were equally expressed in the cells (Fig. 5, lower panels), myc-tagged SHP-1 was immunoprecipitated and immunoblotted with anti-flag mAb. As shown in Fig. 5 (top panel), flag-tagged SHP-1 was coimmunoprecipitated with myc-tagged SHP-1 in the absence of Fc{epsilon}RI cross-linking and the SHP-1:SHP-1 association was maintained even after stimulation. This association appeared to be specific because we could not detect flag-tagged SHP-1 in immunoprecipitates with control IgG (Fig. 5, upper panel, lanes 4–6) and coimmunoprecipitation between flag-tagged SHP-1 and myc-tagged SHP-2 (data not shown). Regarding the molecular mechanisms of SHP-1 dephosphorylation, there are at least two possibilities, dephosphorylating by autocatalytic mechanisms and trans-dephosphorylation by SHP-1 in the vicinity. These results suggest that SHP-1 can be dephosphorylated by trans-dephosphorylation mechanism.


Figure 5
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  		Fig. 5. SHP-1 associates with SHP-1 in vitro. RBL-2H3 cells, transfected with pEF-flag-SHP-1-wt and pEF-myc-SHP-1-wt, were either left unstimulated or stimulated for 2 and 5 min. Myc-tagged SHP-1 was immunoprecipitated with either anti-Myc antibody or normal rabbit IgG, and immunoblotted with anti-flag mAb or anti-c-myc antibody. The expression of flag-tagged and myc-tagged SHP-1 was confirmed by immunoblotting of the TCL with anti-flag mAb and anti-c-myc antibody, respectively.

 
Negative regulation of SHP-1 activity by SHP-1
The phosphorylation of C-terminal tyrosine residues 536 and 564 is reportedly involved in the activation of SHP-1 catalytic activity (27). It might be therefore predicted that hyperphosphorylated endogenous SHP-1 in SHP-1-C/S-expressing cells exhibits enhanced catalytic activity. We addressed this possibility by in vitro dephosphorylation assay using pNPP as a substrate. First, we determined the time course of tyrosine phosphorylation of the SHP-1 following Fc{epsilon}RI cross-linking. RBL-2H3 cells were stimulated for the times indicated. The results demonstrated that phosphorylation of SHP-1 peaked within 1 min of Fc{epsilon}RI cross-linking, and then gradually declined to the basal level within 10 min (Fig. 6A). Based on these results, we compared the catalytic activity of total SHP-1, both endogenous and exogenously expressed, and endogenous SHP-1 from cells transfected with vector alone, SHP-1-wt- or SHP-1-C/S-expressing cells either unstimulated or stimulated for 1 min. In this study, we used the transfectants that expressed higher amounts of SHP-1 by repeated transfection. SHP-1 immunoprecipitated from each cell was divided into three parts, each for anti-PY or anti-SHP-1 immunoblotting or for in vitro dephosphorylation assay. As shown in Fig. 6(B, middle panel), the amount of total SHP-1 immunoprecipitated from SHP-1-wt- or SHP-1-C/S-expressing cells was higher than that from vector-transfected cells, as expected by the fact of higher SHP-1 expression in SHP-1-wt- and SHP-1-C/S-expressing cells (Fig. 1D). Tyrosine phosphorylation of total SHP-1 was induced in control cells after Fc{epsilon}RI cross-linking (Fig. 6B, upper panel, lane 2), which was increased by the expression of SHP-1-C/S (Fig. 6B, upper panel, lane 6). In contrast, the expression of SHP-1-wt decreased phosphorylation of total SHP-1 compared with vector-transfected cells (Fig. 6B, upper panel, lane 4). Consistent with tyrosine phosphorylation states, the increase in the catalytic activity of total SHP-1 after stimulation was most prominent in SHP-1-C/S-expressing cells (160% increase compared with that in unstimulated vector-transfected cells) and was marginal in SHP-1-wt-expressing cells (115% increase) although higher amount of SHP-1 was present in the immunoprecipitates from SHP-1-wt-expressing cells than in those from vector-transfected cells. Next, we determined tyrosine phosphorylation and the catalytic activity of endogenous SHP-1 that was immunoprecipitated after depleting exogenously expressed SHP-1 by anti-flag immunoprecipitation. As shown in Fig. 6(C), tyrosine phosphorylation of endogenous SHP-1 was consistently decreased in SHP-1-wt-expressing cells, whereas the introduction of SHP-1-C/S increased tyrosine phosphorylation of endogenous SHP-1 before and after Fc{epsilon}RI cross-linking. In agreement with these phosphorylation states, the catalytic activity of endogenous SHP-1 was up-regulated either before (132% increase) or after (193% increase) stimulation in SHP-1-C/S-expressing cells as compared with that in unstimulated vector-transfected cells (Fig. 6C). In contrast, endogenous SHP-1 from SHP-1-wt-expressing cells showed decreased activity as compared with that from control cells. Furthermore, the tyrosine phosphorylation and the PTP activity of the endogenous SHP-1 in SHP-1-C/S-Y536F- and SHP-1-C/S-Y564F-expressing cells were comparable to those in vector-transfected cells and SHP-1-C/S-expressing cells, respectively (data not shown). These results suggest that the regulation of SHP-1 activiry was mediated, at least in part, by tyrosine phosphorylation of SHP-1 and that SHP-1 negatively regulates its own catalytic activity.


Figure 6
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  		Fig. 6. Dephosphorylation of SHP-1 decreases SHP-1 catalytic activity. (A) Kinetics of tyrosine phosphorylation of SHP-1 following Fc{epsilon}RI cross-linking. RBL-2H3 cells were sensitized with SPE-7 and stimulated with DNP-HSA for 0.5–10 min. SHP-1 was immunoprecipitated and immunoblotted with anti-PY antibody and anti-SHP-1 antibody. (B) Tyrosine phosphorylation and phosphatase activity of total SHP-1 immunoprecipitated from each transfectant. Cells transfected with vector alone, SHP-1-wt (wt) or SHP-1-C/S (C/S) were either unstimulated or stimulated for 1 min. Total SHP-1, both endogenous and exogenously expressed, was immunoprecipitated from each transfectant with anti-SHP-1 antibody, and divided into three parts, each for anti-PY blot, anti-SHP-1 blot or measuring phosphatase activity. For phosphatase activity, SHP-1 immunoprecipitated from cells transfected with vector alone (open bar), SHP-1-wt-expressing cells (hatched bar) or SHP-1-C/S-expressing cells (solid bar), either unstimulated or stimulated for 1 min, was subjected to the assay using pNPP as a substrate. The results are expressed as mean catalytic activity ± SEM (error bars) from five independent experiments, with the activity of SHP-1 from unstimulated vector-transfected cells being arbitrarily assigned to 1.0. *P < 0.02. (C) Tyrosine phosphorylation and phosphatase activity of endogenous SHP-1 immunoprecipitated from each transfectant. Cells transfected with vector alone, SHP-1-wt (wt) or SHP-1-C/S (C/S) were either unstimulated or stimulated for 1 min. After depleting exogenously expressed SHP-1 with anti-flag mAb, endogenous SHP-1 was immunoprecipitated from each transfectant and subjected to the assays as described in (B).

 
Fc{epsilon}RI-mediated MAPK activation and cytokine gene transcription are increased in SHP-1-wt-expressing cells
Finally, we examined whether the expression of either SHP-1-wt or SHP-1-C/S would influence the downstream signaling events and the final outputs, degranulation and cytokine production. RBL-2H3 cells were transfected with vector, SHP-1-wt or SHP-1-C/S and stimulated for the times indicated. These transfectants did not show significant differences in tyrosine phosporylation of PLC{gamma}1 (data not shown) and PLC{gamma}2 (Fig. 7A), phosphorylation and activation of AKT (Fig. 7A), intracellular Ca2+ mobilization and degranulation upon Fc{epsilon}RI cross-linking (data not shown). In contrast, the expression of SHP-1-wt increased the Fc{epsilon}RI-mediated phosphorylation of ERK, JNK and p38, whereas the expression of SHP-1-C/S decreased the phosphorylation of JNK and p38 (Fig. 7A). Since Fc{epsilon}RI-mediated MAPK activation has been demonstrated to lead to gene transcription of several cytokines (23), we next compared the induction of mRNA for TNF{alpha} and IL-6 that are the major cytokines induced by Fc{epsilon}RI cross-linking in RBL-2H3 cells (23). In accordance with MAPK activation (Fig. 7A), real-time PCR analysis revealed that TNF{alpha} mRNA production was strongly increased in the cells expressing SHP-1-wt, whereas it was decreased in SHP-1-C/S-expressing cells (Fig. 7B, left panel). The change in mRNA level for IL-6 was basically similar to that in TNF{alpha} mRNA (Fig. 7B, right panel). Although we examined downstream signaling events and final outputs by using the transfectants with higher amount of transduced SHP-1 expression, the results obtained were comparable to those shown in Fig. 7 (data not shown). In accordance with the level of tyrosine phosphorylation of endogenous SHP-1, SHP-1-C/S-Y536F- and SHP-1-C/S-Y564F-expressing cells showed almost identical phenotypes on downstream signaling and final outputs to cells transfected with vector alone and SHP-1-C/S-expressing cells, respectively (data not shown). Combined with the results shown in Fig. 6(B and C), these results suggest that the increased SHP-1 activity in SHP-1-C/S-expressing cells may lead to negative regulation of Fc{epsilon}RI-mediated MAPK activation and cytokine gene transcription.


Figure 7
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  		Fig. 7. Fc{epsilon}RI-induced MAPK activation and cytokine gene transcription are increased in SHP-1-wt-expressing cells. (A) RBL-2H3 cells transfected with vector, SHP-1-wt or SHP-1-C/S were stimulated for the times indicated. TCL was immunoblotted with antibodies against AKT, ERK, JNK and p38 and their phosphorylated forms (pAKT, pERK, pJNK and pp38). To estimate tyrosine phosphorylation of PLC{gamma}2, PLC{gamma}2 was immunoprecipitated and immunoblotted with either anti-PY mAb (pPLC{gamma}2) or anti-PLC{gamma}2 antibody (PLC{gamma}2). This result is representative of three independent experiments. (B) Real-time PCR analysis to quantify TNF{alpha} and IL-6 mRNA production. The transfectants used in (A) were either unstimulated or stimulated for indicated times. Total RNA was prepared, and first-strand cDNA was synthesized with random hexamer primers. Values obtained were normalized with an internal GAPDH control and fold increases were determined relative to the unstimulated vector-transfected group, which was arbitrarily designated a value of 1.0. *P < 0.01, **P < 0.05. Data are shown as the mean ± SEM (error bars) from three independent RNA samples.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Because a point mutation of SHP-1 gene causes a wide variety of immunological disorders observed in me mice (31, 32), SHP-1 is implicated as an important regulatory molecule in lymphocyte signaling. In mast cells, SHP-1 is recruited to phosphorylate tyrosine residues in ITIMs of inhibitory receptors such as gp49B1 (18, 19), PIR-B (20), LMIR1 (21) and MAIR-I (22), and negatively regulates Fc{epsilon}RI-initiated signal transduction. However, precise molecular mechanisms whereby SHP-1 mediates negative regulation are largely unknown. In the present study, we approached this problem by determining substrates of SHP-1 with the substrate-trapping approach (24, 25). Over-expression of catalytically inactive SHP-1-C/S mutant in RBL-2H3 cells resulted in an increased tyrosine phosphorylation of p68 in the absence or presence of Fc{epsilon}RI cross-linking. Immunoprecipitation and immunoblotting studies revealed this protein to be SHP-1 itself. This conclusion is based on the following findings. First, p68 migrates at a position almost identical to SHP-1 (Fig. 1). Secondly, tyrosine phosphorylation of both endogenous and exogenously introduced SHP-1 was enhanced in SHP-1-C/S-expressing cells (Fig. 2A and B). Thirdly, the activity of Lyn, an upstream activator of SHP-1, in SHP-1-C/S transfectant was not significantly different from that in SHP-1-wt-expressing cells (Fig. 3). Finally, in vitro PTP assay demonstrated that SHP-1 could dephosphorylate GST-SHP-1-C fusion protein that had been phosphorylated by Lyn (Fig. 4).

A previous study addressed this question by over-expressing another substrate-trapping mutant SHP-1-D/A in RBL-2H3 cells (23). They showed that over-expression of SHP-1-D/A led to an increased tyrosine phosphorylation not only of the ß and {gamma} subunits of Fc{epsilon}RI but also of Syk. In addition, they found two other candidate substrate proteins, pp25 and pp30, that stably formed complex with SHP-1-D/A both in vitro and in vivo. The precise reason why we could not detect alterations in tyrosine phosphorylation of Syk (Fig. 3A), proteins of ~25 and 30 kDa (Fig. 1) or ß or {gamma} subunits of Fc{epsilon}RI (data not shown) is not known. However, given differences in affinity to substrates and in sensitivity to a PTP inhibitor, Na3VO4 (24) between SHP-1-C/S and -D/A, it is very likely that these two mutants trap distinct members of substrates. In addition, this hypothesis might be further supported by the previous observation that over-expression of SHP-1-D/A failed to induce enhanced tyrosine phosphorylation of ~68-kDa protein (23).

The present study also demonstrates that the expression of SHP-1-C/S in RBL-2H3 cells causes enhancement of tyrosine phosphorylation (Fig. 2B) and activity (Fig. 6C) of endogenous SHP-1, thus negatively regulating Fc{epsilon}RI-mediated MAPK activation and the cytokine mRNA transcription. These results might provide a novel autoinhibitory regulation of SHP-1 activity as follows: upon Fc{epsilon}RI cross-linking, Lyn phosphorylates and activates SHP-1 that is in turn dephosphorylated and inactivated by SHP-1 itself. The activity of SHP-1 is regulated by at least three mechanisms. The interaction of SH2 domains of SHP-1 with tyrosine-phosphorylated peptides (33) or tyrosine phosphorylation in C-terminal regulatory region (27) leads to activation of SHP-1, whereas serine phosphorylation in C-terminal tail (34) results in inhibition of its activity. The first mode was revealed by the observations that truncation of the SH2 domains increased the phosphatase activity by ~30-fold relative to wt enzyme and that the increase in PTP activity was induced by the addition of tyrosine-phosphorylated peptides capable of specifically binding to the SH2 domains of SHP-1 (33). This was further supported by crystal structure of the C-terminally truncated form of SHP-1, maintaining the inactive conformation by the protrusion of the N-SH2 domain to the active site of catalytic domain (35). Binding of N-SH2 domain with phosphopeptides would disrupt the autoinhibiting interaction on the interface between the N-SH2 and the catalytic domain, leading to exposure of the active site to the substrates. The second mode of regulation was proposed by a study using site-specific PY mimetics (27). Phosphorylated tyrosine 536 in the C-terminal tail causes the interaction with the N-SH2, thus relieving autoinhibiting conformation as in the case of the first mechanism. Thirdly, in platelets where SHP-1 is constitutively associated with PKC{alpha} and Vav, upon activation of the thrombin receptor, SHP-1 becomes phosphorylated on serine 591 by protein kinase C (PKC){alpha}, leading to inhibition of SHP-1 activity by unknown mechanisms (34).

The results presented herein suggest that one of the PTPs dephosphorylating critical C-terminal regulatory residues of SHP-1 is SHP-1 itself. We could not rule out the possibility that SHP-1 is also involved in dephosphorylation of proteins interacting with the SH2 domain of SHP-1. In fact, we detected several PY-containing proteins upon stimulation in the SHP-1 immunoprecipitates, although there was little difference in tyrosine phosphorylation of SHP-1-interacting proteins in two transfectants, either before or after stimulation. The association of SHP-1 with several proteins might explain the discrepancy in the degree of SHP-1 activation between in vitro study using recombinant SHP-1 (33) and our present study. In the cellular milieu, the activity of SHP-1 might be intricately determined by SH2-domain-dependent association with positive regulatory tyrosine-phosphorylated proteins and SH2-domain-independent binding with unknown modifying proteins. At this stage, however, direct dephosphorylation of the C-terminal tail of SHP-1 by SHP-1 might be a major mechanism for determining SHP-1 activity in RBL-2H3 cells. To further support the importance of tyrosine phosphorylation in the regulation of SHP-1 activity, we found that the expression of SHP-1-C/S-Y536F fails to induce the increased phosphorylation of endogenous SHP-1 (Fig. 4D, bottom two panels, lanes 7–9), indicating the requirement of phosphorylated tyrosine 536 for the dominant negative effect of SHP-1-C/S as proposed previously (27).

The expression of SHP-1-wt in RBL-2H3 cells enhances Fc{epsilon}RI-induced MAPK activation and cytokine gene transcription, whereas the expression of SHP-1-C/S negatively regulates both phenotypes (Fig. 7). These findings are almost consistent with the previous study (23). One could assume that the expression of catalytically inactive forms of SHP-1 would function as dominant negative mutants and positively regulate downstream signaling pathways. As discussed previously (23), however, the positive effects of SHP-1 may be due to modulations in some inhibitory or activating molecules upstream of MAPK by SHP-1. Thus, it is important to identify pathways that are specifically regulated by catalytically altered SHP-1. The experiments to this end are currently under study.

Despite structural similarity between SHP-1 and SHP-2, their roles in cell signaling are quite different. In contrast to negative regulatory SHP-1, SHP-2 plays a positive role in signal transduction in most cases (36). The underlying reason for these functional differences remains to be determined. However, one possibility is that the function of SHP-1 is mediated mainly through its PTP activity, whereas SHP-2 functions as an adaptor molecule linking cell surface receptors to downstream signaling events. Thus, phosphorylated tyrosine residues in SHP-2 provide docking sites for other adaptor molecules, facilitating receptor-mediated signal transduction. Interestingly, in this context, there are reports suggesting a positive role for SHP-1. For example, as presented in this study, over-expression of substrate-trapping mutant of SHP-1 suppressed MAPK pathways, reducing signal transduction and activation of transcription in non-hematopoietic cells (37, 38). SHP-1 can also serve as a positive regulator by preferentially dephosphorylating the C-terminal inhibitory tyrosine residue of Src family PTKs in platelets and T cells (39). Thus, SHP-1 may influence signaling events in both negative and positive ways depending on the cellular milieu and the type of stimulation. Given that Lyn activity was comparable between SHP-1-wt- and SHP-1-C/S-expressing cells, it is important to determine the physiological significance of rapid activation and subsequent autoinactivation of SHP-1 after Fc{epsilon}RI engagement.

In summary, to elucidate the regulatory mechanisms of SHP-1, we identified substrates for SHP-1 by expressing a substrate-trapping mutant of SHP-1 in RBL-2H3 cells. In the event, SHP-1 turned out to be hyperphosphorylated in trapping mutant-expressing cells. Given that the activity of two receptor-proximal PTKs, Lyn and Syk, was not significantly changed in cells transfected with vector alone, wt- and trapping mutant-expressing cells and that SHP-1 dephosphorylates tyrosine 536 in the C-terminal domain of SHP-1 in vitro, we concluded that SHP-1 is one of its substrates. Furthermore, the activity of SHP-1 was inhibited as a result of its own enzymatic reaction, leading to enhancement of MAPK activation and cytokine gene expression. Thus, an autoregulatory mechanism for SHP-1 activity may play a decisive role in mast cell function.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Japanese Ministry of Education, Culture, Sports, Science and Technology (to KM 15590446, to HY 16616010).


   Abbreviations
 
BLNK, B cell linker protein
DNP, dinitrophenol
ERK, extracellular signal-regulated kinase
Fc{epsilon}RI, high-affinity receptor for IgE
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
GFP, green fluorescent protein
GST, glutathione S-transferase
ITAM, immunoreceptor tyrosine-based activation motif
ITIM, immunoreceptor tyrosine-based inhibitory motif
JNK, c-Jun N-terminal kinase
LAB, linker for activation of B cells
LAT, linker for activation of T cells
LMIR1, leukocyte mono-Ig-like receptor 1
MAIR-I, myeloid-associated Ig-like receptor-1
MAPK, mitogen-activated protein kinase
me, motheaten
PLC, phospholipase C
pNPP, p-nitrophenyl phosphate
PTK, protein tyrosine kinase
PTP, protein tyrosine phosphatase
Py, phosphotyrosine
RBL, rat basophilic leukemia
SH2, Src homology region 2
SHP-1, Src homology region 2-domain-containing phosphatase-1
SLP-76, SH2 domain-containing leukocyte phosphoprotein of 76 kDa
TCL, total cell lysates
TNF{alpha}, tumor necrosis factor {alpha}
Wt, wild type

   Notes
 
Transmitting editor: T. Watanabe

Received 26 September 2006, accepted 11 June 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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K. Nakata, T. Yoshimaru, Y. Suzuki, T. Inoue, C. Ra, H. Yakura, and K. Mizuno
Positive and Negative Regulation of High Affinity IgE Receptor Signaling by Src Homology Region 2 Domain-Containing Phosphatase 1
J. Immunol., October 15, 2008; 181(8): 5414 - 5424.
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