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Originally published online as doi:10.1189/jlb.0705398 on December 5, 2005

Published online before print December 5, 2005
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(Journal of Leukocyte Biology. 2006;79:363-368.)
© 2006 by Society for Leukocyte Biology

Identification of tyrosine residues crucial for CD200R-mediated inhibition of mast cell activation

Shuli Zhang1 and Joseph H. Phillips2

DNAX Research Institute, Palo Alto, California

1 Correspondence at current address: Abgenix, Inc., 6701 Kaiser Dr., MS11, Fremont, CA 94555. E-mail: szhang2k{at}gmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD200 and its receptor CD200R are type-1 membrane glycoproteins, which contain two immunoglobulin-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R does not contain an immunoreceptor tyrosine-based inhibitory motif but contains three tyrosine residues (Y286, Y289, and Y297) in the cytoplasmic domain. Y297 is located in an NPxY motif. Previously, we have shown that engagement of CD200R in mouse mast cells induces its tyrosine phosphorylation and recruitment of inhibitory adaptor proteins Dok1 and Dok2, leading to the inhibition of Ras/mitogen-activated protein kinase activation. In the present study, we examined the roles of these three tyrosines in CD200R-mediated inhibition by site-directed mutagenesis in mouse mast cells. Our data show that Y286 and Y297 are the major phosphorylation sites and are critical for CD200R-mediated inhibition of mast cell activation, and Y289 is dispensable. Our data also suggest that the Src family kinase may mediate the phosphorylation of CD200R and Dok.

Key Words: mast cells • cell surface molecules • signal transduction • protein kinases/phosphatases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD200 (originally OX2) is a member of the immunoglobulin (Ig) superfamily, which is widely expressed on variety of cell types [1 ]. It is the ligand for a receptor (CD200R) whose expression is restricted to hematopoietc cells, particularly myeloid cells [1 2 3 4 ]. CD200 and its receptor are type I membrane glycoproteins and contain two extracellular Ig-like domains. Mouse CD200 has a short intracellular domain [19 amino acids (aa)], which lacks any known signaling motifs. Mouse CD200R, however, has a larger cytoplasmic domain (67 aa) containing three tyrosine residues (Y286, Y289, and Y297) [2 ].

Recent in vitro and in vivo studies have suggested that CD200R is a novel inhibitory receptor capable of regulating the activation threshold of myeloid cells such as mast cells and macrophages [5 6 7 ]. Engagement of CD200R by its ligand or agonist antibodies results in a potent inhibition of mast cell degranulation and cytokine production, and this inhibition does not require the coligation of CD200R to an activating receptor [6 , 7 ]. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase-recruiting, immunoreceptor tyrosine-based inhibitory motif (ITIM) [8 9 10 ] but contains an NPxY motif in its cytoplasmic domain. Recently, we have shown that engagement of CD200R induces its tyrosine phosphorylation and recruitment of inhibitory adaptor proteins Dok1 and Dok2, leading to the inhibition of Ras/mitogen-activated protein kinase (MAPK) activation [6 ]. In the present study, we examined the roles of three tyrosine residues in CD200R-mediated inhibition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant protein, cytokines, and antibodies
Recombinant mouse stem cell factor was purchased from PeproTech (Rocky Hill, NJ). Rabbit polyclonal anti-Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) and anti-phosphotyrosine monoclonal antibody (mAb; 4G10) antibody were obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-Dok1, Dok2, and anti-Myc (9E10) mAb were purchased from Santa Cruz Biotechnology (CA). Anti-dual-phosphorylated MAPK antibodies [extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kianse (JNK), and p38 MAPK] and control anti-MAPK were obtained from Cell Signaling Technology (Beverly, MA). Anti-Ras, Dok1, Dok2, and Ras-GTPase activating protein (RasGAP) mAb were purchased from PharMingen (San Diego, CA). Tyrosine kinase inhibitors genistein, herbimycin A, terreic acid [11 ], Syk inhibitor [12 ], PP2 [13 ], and PP3 were from Calbiochem (San Diego, CA).

A fusion protein consisting of the extracellular domain of mouse CD200 fused to the Fc domain of mouse IgG1 mutated in the CH2 domain (D265A) to inhibit binding to Fc receptors (FcRs; CD200-mIg), and control mIg was generated as described previously [4 ]. Anti-mouse CD200R antibody (DX109, rat IgG1) and anti-mouse CD200RLa (DX89, rat IgM) were generated as described previously [4 ].

Cell culture and flow cytometry
Mouse bone marrow-derived mast cells (BMMCs) were generated from BM of 2- to 3-week-old C57BL/6 mice as described previously [4 ]. They were cultured in RPMI-1640 (BioWittaker, Walkersville, MD) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.3 mg/ml L-glutamine, 20 mM Hepes, 50 uM 2-mercaptoethanol, and recombinant murine interleukin (rmIL)-3.

Surface expression of CD200R was analyzed by standard flow cytometry techniques. In brief, mast cells and transfectants were washed once with phosphate-buffered saline (PBS) and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD200R (DX109, rat IgG1) or FITC-conjugated anti-Myc antibody. After incubation at 4°C for 20 min, cells were washed twice in PBS with 0.5% bovine serum albumin and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Mutagenesis and retroviral transduction
The cDNA-encoding wild-type (WT) mouse CD200R with an N-terminal Myc tag in the pMXneo retroviral vector [14 ] was used as a template to generate tyrosine-to-phenylalanine point mutations with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). DNA fragments carrying point mutations of the cytoplasmic region of CD200R, Y286F, Y289F, Y297F, Y286/289F, Y286/297F, Y289/297F, and Y286/289/297F were cloned in the same vector. All mutants were confirmed by DNA sequencing. These constructs were transiently transfected into Pheonix ecotropic retrovirus packaging cells (a gift from Garry Nolan, Stanford University, CA) using Lipofectamine (Invitrogen, Carlsbad, CA). Viral supernatants were collected and used to infect BMMCs. After selection in G418 (Geneticin, Gibco-BRL, Grand Island, NY) at 1 mg/ml, mast cells were stained with FITC-anti-Myc antibody (9E10, Santa Cruz Biotechnology) and sorted for similar expression.

Degranulation and cytokine assays
Mast cell degranulation was determined using a ß-hexosaminidase release assay as described previously [6 , 7 ]. The degranulation was triggered by incubating mast cells with the DX89 antibody, which binds to the activating receptor CD200RLa and induces a strong degranulation response and cytokine production [6 ]. Briefly, 1 x 106 cells/ml mast cells were treated with DX89 antibody at 20 ng/ml in RPMI-1640 media in 96-well plates. Supernatants were assayed for ß-hexosaminidase activity after 1 h incubation and for cytokine secretion after 24 h incubation. For CD200 inhibition, the cells were pretreated with control or CD200-mIg at 2 ug/ml for 30 min before activation.

Cytokine production was measured with mouse inflammation cytometric bead array (CBA) kit from PharMingen, according to the manufacturer’s instruction. Briefly, the mouse inflammation CBA assay uses six bead populations with distinct fluorescence intensities and coated with capture antibodies specific for mIL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), interferon-{gamma}, or tumor necrosis factor {alpha} (TNF-{alpha}) and IL-12p70. The capture beads were mixed with phycoerythrin-conjugated detection antibodies and incubated with recombinant standards or test samples to form sandwich complexes. The six bead populations were mixed together and resolved in the FL3 channel of a BD FACScan flow cytometer (Becton Dickinson). Following acquisition of data, sample results were analyzed using the BD CBA analysis software (Becton Dickinson). Degranulation and cytokine production were expressed as an inhibition index, which is calculated by 100 xx [1–(control mIg-media)/(CD200-mIg-media)].

Immunoprecipitation and immunoblotting
Mast cells (1–2x107 cells/ml) were stimulated at 37°C with control or CD200-mIg (3 ug/ml) for various times as indicated. For CD200 inhibition, the cells were pretreated with control mIg or CD200-mIg (3 ug/ml) for 30 min at 37°C and then stimulated with DX89 antibody (20 ng/ml) for indicated times. Cells were then rinsed once with ice-cold PBS containing 1 mM Na3VO4 and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 5 mM EGTA, 50 mM NaF, 1 mM Na3VO4, plus protease inhibitor cocktails) for 20 min on ice. Lysates were clarified at 14,000 rpm for 10 min. The protein concentration of the supernatant was determined by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were analyzed by native polyacrylamide gel electrophoresis (Nu-PAGE; Invitrogen) and Western blotting. For Western blotting, primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (Pierce, Rockford, IL). For immunoprecipitations, antibodies were incubated with 0.5–1 mg cell lysate for 2 h at 4°C. The immune complexes were recovered by incubation with protein A-agarose beads or protein G Plus agarose beads (Santa Cruz Biotechnology) for 1 h at 4°C. After washing three times in lysis buffer and once in PBS containing 1 mM Na3VO4, the immune complexes were dissociated in sodium dodecyl sulfate sample buffer. The samples were analyzed by Nu-PAGE and Western blotting as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mutagenesis and overexpression of CD200R
Unlike the majority of myeloid inhibitory receptors, CD200R lacks an ITIM but contains three tyrosine residues Y286, Y289, and Y297 in the cytoplasmic domain, one of which is located in a polypyrimidine tract-binding protein (PTB) motif (NPxY297). Our recent studies have shown that engagement of CD200R by its ligand induced CD200R tyrosine phosphorylation and recruitment of inhibitory adaptor protein Dok1 and Dok2 in mouse mast cells overexpressing CD200R. Both Dok proteins are tyrosine-phosphorylated and bind to RasGAP, leading to the inhibition of Ras/MAPK activation [6 ]. To examine the role of each tyrosine residue in CD200R-mediated inhibition, we substituted these three tyrosine residues (Y286, Y289, and Y297) with phenylalanine (Fig. 1A ). These mutant constructs were transduced into BMMCs by retroviral-mediated gene transfer. After selection in G418, stable clones were sorted for similar CD200R expression by flow cytometry and used for subsequent analyses. As shown in Figure 1B , all the clones expressed a similar level of CD200R. Expression of the activating receptor CD200RLa was not affected by retroviral transduction, and all the clones expressed the same level of CD200RLa (data not shown).


Figure 1
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  		Figure 1. Surface expression of WT and mutant CD200R in mouse mast cells. (A) Schematic structure of WT and mutant forms of CD200R. Black boxes represent transmembrane domain (TM). EC, Extracellular; CP, cytoplasmic, Y, tyrosine; F, phenylalanine. (B) Surface expression of WT and mutant forms of CD200R on murine mast cells. Mouse mast cells were transduced with control vector, WT, or CD200R mutants. After selection in G418 (1 mg/ml), stable clones were sorted for similar CD200R expression and analyzed by flow cytometry using anti-Myc antibody.

 
Mast cell degranulation and cytokine production
Our recent studies have indicated that CD200R expression density may regulate the magnitude of the inhibitory response in mast cells [6 , 7 ]. In BMMCs, engagement of the endogenous CD200R by soluble CD200 ligand does not inhibit degranulation response and cytokine secretion induced by antigen cross-linking of Fc{epsilon}RI or an activating antibody DX89. However, overexpression of CD200R made these cells extremely sensitive to CD200-mediated inhibition.

To determine which tyrosine is important for the inhibition, we evaluated the degranulation and cytokine production in mast cells overexpressing WT and mutant forms of CD200R. Consistent with our previous findings, overexpressing WT CD200R resulted in potent inhibition of mast cell degranulation and cytokine production induced by DX89 mAb (Fig. 2 ). Y289F-expressing cells showed similar inhibition. However, there was much less inhibition in Y286F, Y297F, and three double mutant-expressing cells, and the inhibition was abolished in triple mutant-expressing cells. These data showed that Y286 and Y297 are critical for CD200R-mediated inhibition, and Y289 is dispensable.


Figure 2
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  		Figure 2. Inhibition of mast cell degranulation and cytokine production by CD200R engagement. Mast cells expressing WT and mutant CD200R were preincubated with control mIg or CD200-mIg at 2 ug/ml for 30 min and then stimulated with DX89 antibody at 20 ng/ml. The degranulation and cytokine production were measured as in Materials and Methods. Results are represented as the means of triplicate samples from one of three experiments. The extent of degranulation in the absence of CD200 inhibition was optical density 1.6–2.4, as measured by the release of ß-hexoaminidase. Cytokines produced by WT CD200R-overexpressing mast cells in the absence of CD200 inhibition were 22.9 ng TNF-{alpha}, 14.5 ng IL-6, and 6.1 ng MCP-1, respectively.

 
Tyrosine phosphorylation of CD200R and Dok
Our previous studies have shown that soluble CD200 ligand did not induce tyrosine phosphorylation of endogenous CD200R in BMMCs but could trigger the CD200R phosphorylation in mast cells overexpressing CD200R [6 ]. To determine which tyrosine in CD200R is required for its phosphorylation, we examined the CD200R phosphorylation in mast cells overexpressing WT and mutant forms of CD200R. As shown in Figure 3 , CD200 stimulation induced tyrosine phosphorylation of WT and Y289F CD200R. As expected, there was no phosphorylation of the Y286/289/297F mutant. It is surprising that mutants Y286F and Y297F were not phosphorylated, suggesting that these two tyrosines are the major sites of tyrosine phosphorylation and that their phosphorylation is interdependent, as mutation of one residue abolishes phosphorylation of the other. There was no detectable phosphorylation of three double Y-to-F mutants (data not shown). These results suggest that Y286 and Y297 are required for CD200R phosphorylation.


Figure 3
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  		Figure 3. Tyrosine phosphorylation of CD200R induced by CD200. Mast cells expressing WT and mutant CD200R were stimulated with control mIg (lane 1) or CD200-mIg (lane 2) at 3 ug/ml for 5 min. CD200R was immunoprecipitated (IP) from cell lysates using a rat anti-CD200R antibody and immunoblotted (IB) with antiphosphotyrosine antibody (pY). The same membrane was stripped and reblotted with anti-CD200R antibody. Control mIg had no effect on the tyrosine phosphorylation of WT and mutant CD200R.

 
CD200R cytoplasmic domain has a potential PTB motif, NPxY297. Our previous studies show that engagement of CD200R by its ligand induced tyrosine phosphorylation of Dok1 and Dok2 and their association with RasGAP and SHIP [6 ]. To test which tyrosine in the CD200R mediates this effect, we evaluated the phosphorylation of Dok1 and Dok2 in WT and mutant CD200R-expressing cells. After stimulation with CD200, Dok1 and Dok2 were immunoprecipitated from the cell lysates and separated by 8% Nu-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with antiphosphotyrosine antibody. As shown in Figure 4 , triggering CD200R resulted in similar tyrosine phosphorylation of Dok1 and Dok2 in WT and Y289F mutant CD200R-expressing cells. In Y286/289/297F-expressing cells, there was only background phosphorylation of Dok1 and Dok2. Tyrosine phosphorylation of Dok1 and Dok2 was greatly reduced in Y286F- and Y297F-expressing cells. Association of Dok with CD200R and RasGAP was only detected in WT and Y289F-expressing cells. It is interesting that in WT and Y289F mutant CD200R-expressing cells, CD200 stimulation also induced tyrosine phosphorylation of SHIP and its association with Dok1 and Dok2, which was not detected in Y286F-expressing cells. In Y286F- and Y286/289/297F-expressing cells, the phosphorylation of SHIP and its association with Dok1 and Dok2 were greatly reduced. These results suggest that Y286 and Y297 are required for the phosphorylation of Dok and association with CD200R and RasGAP as well as the phosphorylation of SHIP.


Figure 4
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  		Figure 4. Tyrosine phosphorylation of Dok1 and Dok2 in mast cells. Growth factor-starved mast cells expressing WT and mutant CD200R were stimulated with control mIg (lane 1) or CD200-mIg (Lane 2) at 3 ug/ml for 5 min. Dok1 and Dok2 were immunoprecipitated from cell lysates, and the immunoprecipitates were immunoblotted with the indicated antibodies. Control mIg had no effect on all mutant CD200R-expressing cells.

 
Inhibition of MAPK activation
We have shown previously that engagement of CD200R by its ligand inhibited the activation of ERK, p38 MAPK, and JNK [6 ]. To test which tyrosine in the CD200R mediates this inhibition, we examined the activation of these MAPKs in WT and mutant CD200R-expressing cells. Activation of mouse mast cells via the DAP12 pairing receptor, CD200RLa, caused a pronounced phosphorylation of ERK, p38, and JNK, which was directly associated with a potent degranulation response [6 ]. Preincubation of mast cells with CD200-mIg markedly reduced the activation-dependent phosphorylation of ERK in WT and Y289F-expressing cells (Fig. 5 ). There was a slight inhibition in Y286F-expressing cells and no inhibition in Y297F- and Y286/289/297F-expressing cells. The inhibition of p38 MAPK and JNK activation in Y286F-, Y297F-, and Y286/289/297F-expressing cells was also reduced greatly (data not shown). These results are consistent with the degranulation response, cytokine production, phosphorylation of CD200R, and Doks, confirming that Y286 and Y297 are critical for CD200R function, and Y289 is dispensable.


Figure 5
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  		Figure 5. Inhibition of MAPK activation by CD200. Mast cells expressing WT and mutant CD200R were pretreated with control mIg (lane 1) or CD200-mIg (lane 2) at 3 ug/ml at 37°C for 30 min and then stimulated with DX89 antibody (20 ng/ml) for 5 min. The activation of ERK was detected by immunoblotting with antiphospho-ERK (pErk)-specific antibody. The same membranes were stripped and reblotted with anti-SH2-containing tyrosine phosphatase-2 (SHP2) antibody for sample loading control.

 
Inhibition of tyrosine phosphorylation of CD200R and Dok by PP2
One of the earliest events in mast cell activation is the activation of the Src family kinase Lyn, followed by the activation of Syk. The activated kinases then in turn phosphorylate and activate other downstream targets including phospholipase C{gamma} (PLC{gamma}), Btk, phosphatidylinositol-3 kinase (PI-3K), protein kinase C (PKC), and others [15 , 16 ]. Previous studies have demonstrated that tyrosine kinase inhibitors specific for Src family kinases, Syk kinase, and Btk are capable of inhibiting Fc{epsilon}RI-mediated signaling in mast cells [11 , 12 , 17 ]. To determine which kinase may mediate the phosphorylation of CD200R and Dok proteins, we used PP2 (a Src family protein tyrosine kinase inhibitor), Syk inhibitor, and terreic acid (a specific Btk inhibitor) and analyzed their inhibitory activity on mast cells overexpressing WT CD200R. PP3 was used as a negative control for PP2. After preincubation of mast cells with these inhibitors, the phosphorylation of CD200R and Dok1 was determined by immunoprecipitation and immunoblotting. As shown in Figure 6 , PP2 inhibited the phosphorylation of CD200R and Dok1 completely, and terreic acid and Syk inhibitor had no effect. However, Syk inhibitor did inhibit the association of Dok1 with RasGAP, and the association of Dok1 with RasGAP was blocked completely by PP2. CD200-mediated phosphorylation of Dok2 was also inhibited by PP2 (data not shown). These data suggest that tyrosine phosphorylation of CD200R and Dok requires Src kinases, and Syk plays a role in promoting the association of RasGAP with Dok.


Figure 6
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  		Figure 6. PP2 inhibits tyrosine phosphorylation of CD200R and Dok1 induced by CD200. Mast cells overexpressing WT CD200R were pretreated with dimethyl sulfoxide (DM) or tyrosine kinase inhibitors terreic acid (TA; 20 uM), Syk inhibitor (SI; 0.5 uM), PP2 (5 uM), and PP3 (a negative control for PP2; 5 uM) at 37°C for 30 min and then stimulated with control mIg (C) or CD200-mIg (CD200) at 3 ug/ml at 37°C for 5 min. The phosphorylation of CD200R (A) and Dok1 (B) was detected by immunoprecipitation and immunoblotting as described in Figures 3 and 4 .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD200R is a novel inhibitory receptor expressed highly on myeloid cells and some T cells [4 ]. Most inhibitory receptors on immune cells contain a consensus amino acid sequence, ITIM, in the cytoplasmic domain [8 , 9 , 18 , 19 ]. The prototype ITIM consists of the sequence (I/V/L/S)-x-Y-x-x-(L/V), where x denotes any amino acid. Ligand-induced clustering of these ITIM-containing receptors results in tyrosine phosphorylation, often by a Src family kinase, which provides a docking site for the recruitment of tyrosine phosphatases SHP1 (and occasionally SHP2) and the inositol phosphatase SHIP. By recruiting phosphatase SHP1 and/or SHIP, ITIM-bearing inhibitory receptors suppress cell activation by promoting dephosphorylation reactions. Unlike other myeloid inhibitor receptors such as Fc{epsilon}RIIB [20 , 21 ], gp49B1 [22 , 23 ], paired Ig-like receptor-B [24 , 25 ], and mast cell function-associated antigen [26 , 27 ], CD200R lacks an ITIM but contains three tyrosine residues in the cytoplasmic domain. One tyrosine (Y297) is located in an NPxY motif, which may represent a potential PTB domain protein-binding motif. Previously, we have shown that engagement of CD200R induces its tyrosine phosphorylation and recruitment of inhibitory adaptor proteins Dok1 and Dok2 [6 ], containing an amino-terminal PH domain, a central PTB domain, and a carboxyl-terminal region with multiple potential tyrosine phosphorylation sites and proline-rich regions, which may serve as docking sites for SH3 domains. In the present study, we examined the roles of the three tyrosine residues in CD200R-mediated inhibition of mast cell activation.

Our mutagenesis studies show that among the three tyrosine residues (Y286, Y289, and Y297), Y286 and Y297 are required for the phosphorylation of CD200R and Doks as well as the CD200R-mediated mast cell activation, and Y289 is dispensable. Considering Y297 is located in the NPxY motif, which is believed to mediate the interaction of CD200R with Doks, it is expected that mutation of this Tyr would abolish the binding of Dok to CD200R and impede the function of CD200R. Our data support this hypothesis. It is surprising that mutation of Y286 also abolished the phosphorylation of CD200R and its association with Doks. This suggests that tyrosines work in concert to promote phosphorylation of each other.

One of the earliest events in mast cell activation is the activation of the Src family kinase Lyn, followed by the activation of Syk. The activated kinases then in turn phosphorylate and activate other downstream targets including PLC{gamma}, Btk, PI-3K, PKC, and others [15 , 16 ]. Another Src family kinase, Fyn, has also been shown to initiate complementary signals required for IgE-dependent mast cell degranulation [28 ]. Previous studies have demonstrated that tyrosine kinase inhibitors specific for Src family kinases (PP1/PP2), Syk kinase, and Btk are capable of inhibiting Fc{epsilon}RI-mediated signaling in mast cells [11 , 12 , 17 ]. To determine which tyrosine kinase may mediate the phosphorylation of CD200R and Dok proteins, we used different tyrosine kinase inhibitors to examine their effect on the phosphorylation of CD200R and Doks induced by CD200 stimulation. Our results suggest that Src family kinase Lyn or Fyn may mediate the phosphorylation of CD200R and Doks, as PP2 completely inhibited the phosphorylation of CD200R and Dok induced by CD200 (Fig. 6) . Our data also show that Syk plays a role in promoting the association of RasGAP with Dok. Using Lyn-, Fyn-, and Syk-deficient mast cells will help to further define the role of these kinases in CD200R-mediated inhibitory signaling.

Mast cell degranulation and cytokine secretion are dependent on ERK and p38 MAPK activation [29 30 31 ]. Our previous study has shown that CD200R engagement inhibits activation of ERK, p38 MAPK, and JNK [6 ]. The inhibition of these MAPKs by CD200 stimulation was reduced significantly in Y286F- and Y297F-expressing cells but was maintained in Y289F-expressing cells. In agreement with these results, CD200-mediated inhibition of mast cell degranulation and cytokine production was greatly reduced in Y286F- and Y297F-expressing cells but not in Y289F-expressing cells. These results confirmed that Y286 and Y297 are crucial for CD200R-mediated inhibition of mast cell activation, and Y289 is dispensable.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr. Paul Heyworth for his critical reading of the manuscript. We also thank Holly Cherwinski and Mike Bigler for outstanding technical assistance.


   FOOTNOTES
 
2 Correspondence: DNAX Research Institute, 901 California Ave., Palo Alto, CA 94304-1104. E-mail: joe.phillips{at}dnax.org Back

Received July 20, 2005; revised September 28, 2005; accepted October 4, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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