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J. Biol. Chem., Vol. 281, Issue 39, 28615-28626, September 29, 2006
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112
334
From the
Cancer Biology Program, Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, the Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan, and the ¶Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10021
Received for publication, April 19, 2006 , and in revised form, June 21, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Kit belongs to a subfamily of receptor-tyrosine kinases (RTK) that include the receptors for colony-stimulating factor and platelet-derived growth factor. Ex vivo studies of mast cells or heterologous cells expressing Kit wild type (WT) or various mutants indicate that Kit activates multiple downstream pathways, which together are important for the proliferation, survival, migration, and adhesion of mast cells (3). Upon SCF binding, Kit becomes dimerized and activated. Activated Kit transphosphorylates tyrosine (Y) residues in the receptor cytoplasmic tail, creating docking sites for various SH2 domain-containing signaling molecules. Among the various tyrosine residues in the cytoplasmic tail of Kit, Tyr719, and Tyr567 are critical for transmitting Kit signals (4, 5). Recent studies of Kit "knock-in" mice indicate that Tyr719 and Tyr567 contribute to mast cell development. However, Tyr567 is more important than Tyr719 for the mast cell development in specific tissue such as peritoneal cavity (6). Tyr719, a site for recruiting the p85 regulatory subunit of PI3K (7), is required for SCF-evoked PI3K activation (8, 9). Tyr567 is reported to recruit various signaling molecules including Src family kinases (SFKs) (4, 10), Shp-2 (11), and Shc (12). Tyr567 is important for SCF-evoked SFK (4) (13, 14) and Ras activation (14, 15). However, the role of Shp-2 recruitment to Tyr567 is less clear. Importantly, activation of SFK from Tyr567 and activation of PI3K from Tyr719 both contribute to proliferation and survival of mast cells in vitro. SFK and PI3K can both activate the Rac/JNK pathway that promotes SCF-evoked mast cell proliferation (4). It is not well understood what signaling pathway activated by SFK and PI3K promotes SCF-evoked mast cell survival. One report suggested that Rac2 promotes survival by activating Akt and suppressing the expression of pro-apoptotic protein Bad (16). PI3K can activate Rac through Rac GEF (17). PI3K via activation of Rac also mediates SCF-evoked mast cell migration (18). However, it is not clear what molecule mediates the activation of Rac by SFK from Kit Tyr567.
Gab2 (Grb2-associated binder-2) is a member of Gab/Dos subfamily of scaffolding adapters that also include mammalian Gab1 and Gab3, Drosophila DOS (daughter of sevenless), and Caenorhabditis elegans Soc-1 (19). Like other Gab/DOS family members, Gab2 contains an N-terminal pleckstrin homology (PH) domain, several proline-rich motifs (PXXP), and multiple tyrosine phosphorylation sites (19). Gab2 PH domain preferentially binds PI 3,4,5-P3 (PIP3) (20). Two of the proline-rich motifs in Gab2 are Grb2-SH3 domain binding sites (21), and are important for coupling Gab2 to upstream receptors through the Shc·Grb2 complex (22). Gab2 plays an important role in transmitting signals downstream of receptors for several cytokines and growth factors as well as multichain immune receptors. Upon receptor activation, Gab2 becomes tyrosyl-phosphorylated and recruits SH2 domain containing signal relay molecules, including the tyrosine phosphatase Shp-2 and p85, the regulatory subunit of Class 1A PI3K. Gab2 association with Shp-2 is important for cytokine induced immediate early gene expression (23) and growth factor-induced Erk activation (24–27). Genetic and biochemical evidence indicate that Shp-2 via Gab/Dos is required for activation of the Ras/Erk pathway (28). However, the critical Shp-2 substrate in controlling Ras/Erk activation is still not clear. Gab2 association with p85 is critical for cytokine, Fc-receptor, and growth factor-evoked PI3K activation (19, 29).
The in vivo functions of Gab2 have been elucidated through the analysis of Gab2 knock-out (–/–) mice. Gab2 is essential for allergic responses and is reportedly important for RANK-mediated osteoclastogenesis (30, 31). In addition, we and others have found that Gab2–/– mice have selective loss of mast cells in certain tissues such as peritoneal cavity and stomach (30, 32). Because Kit is essential for mast development in vivo, these data strongly suggest that Gab2 mediates Kit regulated mast cell development. Consistent with this idea, SCF-evoked proliferation is reduced in Gab2–/– mast cells in vitro (32). However, the mechanism by which Gab2 participates in Kit-evoked mast cell proliferation is still not clear. In this study, we provide evidence that the scaffolding adapter Gab2 via Shp-2 mediates signal from Kit Tyr567 in an SFK-dependent manner to activate the Rac/JNK pathway that is important for the Kit-regulated proliferation. The KitY567-Gab2 pathway is likely to be critical for mast cell development in specific tissues in vivo.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Constructs—The HA-Gab2 WT and Gab2-
Shp-2 fragments, released by restriction enzyme digestion from pBluescript (23), were cloned into the retroviral vector pMXs-puro (a gift from T. Kitomura, Tokyo University). The GST-RBD (Rac Binding Domain of PAK1) plasmid was a kind gift from Dr. C. Carpenter (Beth Israel Deaconess Medical Center, BIDMC, Boston).
Mice and Cell Cultures—Gab2–/– mice (sv129/J x C57BL/6J background) (30) and KitY719F/Y719F knock-in mice (sv129 x C57BL/6J x BalbC background) (8) were described previously. To generate compound mutants, Gab2+/– mice were crossed with Kit +/Y719F mice to generate Kit +/Y719F + Gab2+/– double heterozygous mice, which were interbred to generate KitY719F/Y719F + Gab2–/– mice. Shp-2 floxed/floxed (fl/fl) mice (33) were mated with ERCreTM mice (34) to generate Shp-2 fl/fl-ERCreTM (C57BL/6J) mice. Bone marrow-derived mast cells (BMMC) were cultured as described previously (35). Briefly, BM from 2–4-month old WT mice and littermates with the indicated genotypes were incubated in Iscove's Modified Dulbecco's medium (IMDM) with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 2 mM L-glutamine, 0.1 mM nonessential amino acids (NEAA), 1 mM sodium pyruvate, 1000 units/ml penicillin, 1 mg/ml streptomycin, 50 µM 2-mercaptoethanol, 4 ng/ml of recombinant murine IL-3 (PeproTech, Rocky Hill, NJ). After 4 weeks, such cultures consist of 
95% mast cells, as indicated by surface expression of Fc
RI and Kit. 4–6 week-old BMMC cultures were used for all studies. For cell stimulation, BMMC were washed, starved in IMDM + 1% bovine serum albumin for 4–6 h at 37 °C, and resuspended in modified Tyrodes buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.4, 5.6 g/liter glucose, and 0.1% bovine serum albumin) before stimulation with the indicated concentrations of SCF (PeproTech). In some experiments, BMMC were pre-treated with 5 µM of SU6656 (Calbiochem), a specific inhibitor of SFK, for 1 h before SCF stimulation. BaF3 cells expressing similar levels of WT mouse Kit and Kit mutants (Y567F, Y569F, Y702F, and Y719F) (13) were cultured in RPMI with 10% heat-inactivated FBS, 2 mM L-glutamine, 1000 units/ml penicillin, 1 mg/ml streptomycin, and 1 ng/ml of recombinant murine IL-3. The MC/9 murine mast cells expressing HA-Gab2 WT or Grb2 (a mutant of Gab2 that cannot bind Grb2) (20) were cultured in RPMI supplemented with 10% FBS, 5% T-Stim supplement (BD Biosciences), 0.1 mM NEAA, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 1000 units/ml penicillin, 1 mg/ml streptomycin, 50 mM 2-mercaptoethanol.
Immunoprecipitation and Western Blots—Cells were lysed in 1% Nonidet P-40 lysis buffer as described previously (22). Total cell lysates or immunoprecipitates were resolved by SDS-PAGE, immunoblotted with the indicated primary antibodies, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham Biosciences), and developed by enhanced chemiluminesence (ECL) (Amersham Biosciences). The intensities of bands in Western blots were quantified by densitometry analysis using NIH Image 1.63F software.
Flow Cytometric Analysis BMMC—(2 x 105) were prebound with anti-DNP IgE, washed with phosphate-buffered saline containing 3% FBS, and stained with FITC-conjugated rat monoclonal anti-mouse IgE antibody R35–72 (IgG1) or FITC-rat monoclonal IgG1 as control. Stained cells were analyzed using a FACScan (Becton Dickinson). To measure surface Kit expression, BMMC were stained with FITC-rat monoclonal anti-CD117 or FITC-isotype control antibodies. All these FITC-conjugated antibodies were purchased from BD Pharmingen.
Proliferation and Apoptosis Assays—BMMC proliferation assays were performed as described (8). Briefly, 105 cells were starved without cytokine in IMDM + 10% FBS for 12–14 h, and plated in triplicate in 96-well plates in the presence of the indicated concentrations of growth factors for 24–30 h. [3H]thymidine (1 µCi) was added to each well for 4–6 h, cells were collected using a Cell Harvester (Skatron, Sterling, VA), and [3H]thymidine incorporation was determined using a Beta Plate Liquid Scintillation Counter (PerkinElmer Life Sciences). For quantifying apoptosis, cells were stained with FITC-Annexin V (Promega) according to the manufacturer's instructions, and analyzed by flow cytometry.
Retroviral Infection—of BMMC PMXs-puro retroviral plasmids were transfected into the ecotrophic packaging line Plate-E (36) using FuGENE (Roche Applied Science). Virus-containing culture supernatants were collected 2-days later. BM cells cultured in IL-3-containing IMDM medium for 12 days were spin-infected (2500 rpm, 90 min) with pMXs-puro virus supernatants in the presence of 4 µg/ml polybrene, and then incubated at 37 °C for 18–24 h. Infected cells were selected in the presence of 1 µg/ml of puromycin for 10–12 days, and then cultured in the absence of puromycin for 4 weeks.
Rac, Ras Activation, and JNK Kinase Assays—Rac-GTP pull-down assay was performed as described (37) with the following modifications. The GST-RBD (the Rac Binding Domain in PAK1) fusion protein was purified on the day of the assay. Cells (107) were lysed in 200 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 5 mM MgCl2, 10 mM Tris-Cl pH 7.5, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 2 µg/ml aprotinin, and incubated with GST-RBD (50 µg) at 4 °C for 40 min. Bound proteins were washed, resolved by SDS-PAGE, and immunoblotted with Rac-1 antibodies. JNK kinase assays were performed as described (8). Cells (107) were lysed in 1%Nonidet P-40 lysis buffer, and JNK1 was immunoprecipitated with anti-JNK1 antibodies. Immune complexes were subjected to in vitro kinase assay using GST-c-Jun-(1–135) (38) as the substrate in the presence of 25 µM ATP and 5 µCi [
-32P]ATP. The reactions were stopped, resolved by SDS-PAGE, and exposed to X-Omat film (PerkinElmer Life Sciences). Ras-GTP pull-down assay was performed as described (39).
Analysis of Tissue Mast Cells—Back skins and ears from WT, Kit Y719F/Y719F, Gab2–/–, KitY719F/Y719F+Gab2–/– mice (2–5-month old) were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 µm). Mast cells were visualized by staining tissue sections with toluidine blue (Histology core facility at Beth Israel Deaconess Medical Center), and counted under a microscope. Data were presented as the total number of mast cells per 1-cm tissue with similar thickness. Hypodermis of the back skin consists of the fat cells and smooth muscle cells underneath the dermis.
Statistical Analysis—Paired data were evaluated by two-tailed Student's t test. Comparisons of multiple groups were performed using two-way ANONA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Kit Tyr567 Signaling to Gab2 Depends on SFK Activity—To begin to address how Gab2 mediates SCF-evoked proliferation in mast cells, we first asked whether Gab2 forms a complex with Kit upon SCF stimulation. Gab2 was immunoprecipitated from starved and SCF-stimulated WT BMMC lysates, and subjected to immunoblotting with several antibodies (Fig. 2A). Consistent with a previous report (32), Gab2 became tyrosyl-phosphorylated in response to SCF stimulation. We also found that Gab2 associated with Kit, Shp-2, and p85 (Fig. 2A).
To examine how Kit signals to Gab2, we analyzed Gab2 tyrosyl phosphorylation in BaF3 cells (Kit-negative) reconstituted with WT Kit and the Kit mutants Y567F, Y569F, Y702F, and Y719F. These BaF3 cells express similar levels of WT and mutant Kit, as described previously (13). Upon SCF stimulation, Gab2 was robustly tyrosyl-phosphorylated (Fig. 2B) and associated with tyrosyl-phosphorylated Kit (data not shown) in Kit WT cells (Fig. 2B), as well as in cells expressing Kit Y569F, Y702F, and Y719F. In marked contrast, Gab2 tyrosyl phosphorylation (Fig. 2B) and its association with tyrosyl phosphorylated Kit (data not shown) were inhibited in Kit Y567F cells (Fig. 2B) although residual Gab2 tyrosyl phosphorylation could be seen in Kit Y567F cells after longer exposure (data not shown). Loss of SCF-evoked Gab2 tyrosyl phosphorylation is not because of the defective kinase activity of the Kit 567F mutant since we found that SCF-evoked Kit 567F autophosphorylation and proliferation of Baf3 Kit 567F cells were not impaired compared with Kit WT (13). This result indicates that Tyr567 is required for SCF-evoked Gab2 tyrosyl phosphorylation.
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Our previous data show that Shc via Grb2 recruits Gab2 to 
c of IL-3R/GM-CSFR (22). Therefore, our data support a model that Shc/Grb2 recruits Gab2 to Kit Tyr567. To test this hypothesis, we examined the biochemical behavior of Gab2 Grb2 mutant in MC/9 mast cell line. Previously, we established pool of MC/9 cells expressing HA-Gab2 WT and Gab2 Grb2 by retroviral transduction (20). Gab2 Grb2 is a Gab2 mutant that cannot bind Grb2 constitutively because its two Grb2 SH3 binding sites are mutated (21). Upon SCF stimulation, we found that HA-Gab2 WT became strongly tyrosyl phosphorylated and associated with Shc and Kit. In contrast, HA-Gab2 Grb2 was barely phosphorylated and lost its association with Shc and Kit (Fig. 2F). This data strongly support that the Grb2/Shc complex is required for Gab2 association with the activated Kit (Fig. 8).
To ask whether SFK activity is required for SCF-evoked Gab2 tyrosyl phosphorylation, we pretreated WT BMMC with the selective SFK inhibitor, SU6656, (40) before stimulating the cells with SCF. Notably, SU6656 strongly inhibited SCF-evoked Gab2 tyrosyl phosphorylation and its association with Shp-2 (Fig. 2G). Interestingly, Gab2-associated Shc tyrosyl phosphorylation is not affected by SU6656 treatment (Fig. 2G), suggesting that SFK activity is not required for Shc tyrosyl phosphorylation. Collectively, these data suggest that Kit Tyr567 via Shc is required for Gab2 recruitment, and Gab2 tyrosyl phosphorylation by SFK.
Kit-evoked Full Activation of Rac and JNK Requires Gab2—To investigate which Kit-activated downstream signaling pathway(s) is(are) regulated by Gab2, we first examined the activation of Erk and Akt in Gab2+/+ and Gab2–/– BMMC by performing immunoblotting using phosphospecific antibodies. Upon stimulation with SCF with doses that induce BMMC proliferation (Fig. 1B), Gab2–/– BMMC showed a 50% decrease in Erk phosphorylation at later time point (15 min) compared with Gab2+/+ BMMC. In contrast, Akt phosphorylation was similar in Gab2+/+ and Gab2–/– BMMC (Fig. 3A). We and others (26, 27) have shown that Gab2-mediated Erk activation is important for proliferation of the mammary epithelial cells. To ask whether decreased Erk activation is responsible for the impaired SCF-evoked proliferation of Gab2–/– BMMC, we examined the effects of the Mek inhibitor UO126. We found that pretreatment of BMMC with UO126, which inhibited Erk activation, did not inhibit SCF-evoked thymidine incorporation in Gab2+/+ BMMC (Fig. 3B), indicating that Gab2-activated Erk is not required for SCF-evoked mast cell proliferation.
Kit also activates the Rac/JNK pathway, which is known to be required for mast cell proliferation (4). We examined SCF-evoked Rac activation using the GST-RBD (Rac binding domain in PAK1) pull-down assay to measure the amount of GTP-bound Rac1. Whereas Gab2+/+ BMMC showed robust Rac1 activation, Rac1 activation was reduced (40–60%) in Gab2–/– BMMC (Fig. 3C). Like Rac1, we observed that SCF-evoked Rac2 activation was reduced in Gab2–/– BMMC (data not shown). We also analyzed JNK activation by immune complex kinase assay. Consistent with the decrease in Rac activation, Gab2–/– BMMC showed decreased SCF-evoked JNK activation compared with Gab2+/+ BMMC (Fig. 3D).
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65%) inhibited SCF-evoked Rac activation (Fig. 3E).
Gab2 Acts via Shp-2 to Activate Rac/JNK, Ras, and Promote SCF-evoked BMMC Proliferation—Besides recruiting and activating PI3K, Gab2 also binds and activates Shp-2. To test whether Gab2 association with Shp-2 is required for Rac/JNK activation and mast cell proliferation, we used retroviral gene transduction to reconstitute Gab2–/– BMMC with similar levels of Gab2 WT and Gab2-Shp-2, a Gab2 point mutant that cannot bind Shp-2. Compared with vector alone, Gab2 WT-expressing cells showed enhanced SCF-evoked Rac and JNK activation. However, Gab2-Shp-2 expressing cells displayed weak Rac and JNK activation, similar to vector-alone Gab2–/– BMMC (Fig. 4A). Furthermore, whereas Gab2 WT rescued the proliferation defect of Gab2–/– BMMC, expression of Gab2-Shp-2 failed to do so (Fig. 4B).
These data suggest that Gab2, via its association with Shp-2, is required for SCF-evoked activation of the Rac/JNK pathway and mast cell proliferation. However, the Shp-2 binding sites in Gab2 may also bind other SH2-containing signaling molecules such as SOCS proteins (42). To test whether Shp-2 itself is required for SCF-evoked Rac activation and mast cell proliferation, we acutely inhibited Shp2 expression in BMMC. To achieve this, we derived BMMC from mice bearing a homozygous "floxed" (fl/fl) allele of Shp-2 and the ERCreTM transgene (ERCreTM:fl/flShp2 mice). Addition of the estrogen analog, Tamoxifen, into the culture medium results in the acute activation of the ERCreTM fusion protein and excision of the floxed Shp-2 allele via Cre-loxp-mediated recombination.
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75% compared with control) occurred between day 3–4 after Tamoxifen addition to fl/fl Shp-2 ERCreTM BMMC (Fig. 5A). Shp-2 deletion resulted in reduced (50%) SCF-evoked cell proliferation, as revealed by reduced thymidine incorporation (Fig. 5B). SCF-evoked Rac activation and JNK phosphorylation also were decreased following Shp-2 deletion (Fig. 5C). In contrast, SCF-evoked Erk and Akt activation were minimally affected in the presence of Tamoxifen (Fig. 5D). As a negative control, Tamoxifen treatment had no effects on the proliferation of WT BMMC (data not shown).
Because previous biochemical and genetic evidence indicates that Shp-2 acts upstream of Ras in RTK signaling, we also examined the effect of Shp2 deletion on SCF-evoked Ras activation. Ras was activated robustly in control BMMC. In contrast, SCF-evoked Ras activation was reduced in Shp-2-deleted cells (Fig. 5E). Similarly, SCF-evoked Ras activation also was reduced (to a similar extent) in Gab2–/–, compared with Gab2+/+, BMMC (Fig. 5F). Thus, our result suggests that Shp-2 via interaction with Gab2 contributes to SCF-evoked Ras activation in mast cells. Consistent with this notion, we found that expression of Gab2 WT in Gab2–/– BMMC resulted in increased SCF-evoked Ras activation. However, expression of Gab2-Shp-2 in Gab2–/– BMMC failed to enhance SCF-evoked Ras compared with vector alone (Fig. 5G).
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35% decrease) in proliferation compared with WT BMMC. Notably, BMMC from compound mutant (i.e. Kit Y719F/Y719F+Gab2–/–) mice showed markedly impaired (80%) proliferative responses (Fig. 6A). Furthermore, although, SCF-evoked JNK (Fig. 6B) activation was partially impaired in Kit Y719F/Y719F (70%) or Gab2–/– BMMC (50%) (compared with WT BMMC), more pronounced inhibition (>90%) of JNK activation was observed in Gab2–/– Kit Y719F/719F BMMC (Fig. 6B). These data about Gab2 in Kit-evoked JNK activation and mast cell proliferation are very similar to the previous published report that Kit Tyr567 contributes to SCF-evoked Rac/JNK activation and mast cell proliferation (4), further supporting the model that Kit Y567->Gab2->Rac/JNK is critical for SCF-evoked mast cell proliferation (Fig. 8).
We also examined SCF-evoked PI3K activation in these cells by assessing the activation of Akt, a downstream effector of PI3K, using phospho-Akt antibodies. We found that SCF-evoked Akt phosphorylation was dramatically reduced by 90% in Kit Y719F/Y719F BMMC compared with WT BMMC (Fig. 6C), consistent with previous reports that Kit Tyr719 is the major route for SCF-evoked PI3K and Akt activation (8, 9). Also, as we observed in Fig. 3B, no change in Akt phosphorylation could be detected in Gab2–/– compared with WT BMMC (Fig. 4D). However, the residual level of Akt phosphorylation in Kit Y719F/Y719F BMMC was eliminated in Gab2–/– Kit Y719F/Y719F BMMC. This result indicates that whereas Gab2 is only responsible for a minor fraction (<10%) of SCF-evoked Akt (or PI3K) activation in BMMC, it nonetheless contributes to PI3K activation if the main route for PI3K activation through Kit719F is compromised.
The Gab2 Pathway Plays a Distinct Role in Mast Cell Development in Different Tissues—We also analyzed mast cells in various tissues in the aforementioned mice by toluidine blue staining (Fig. 7). Although there was no decrease in mast cell numbers in the dermis of the back skins of either Gab2–/– mice or Kit Y719F/Y719F mice, mast cell numbers were decreased (50%) (p < 0.05) significantly in compound Kit Y719F/Y719F+Gab2–/– mice compared with WT mice (Fig. 7A). Similarly, although there was an 30% (p < 0.05) or 40% decrease (p < 0.01) in mast cell numbers in the ears of Kit Y719F/Y719F and Gab2–/– mice, respectively, Gab2 deficiency combined with the Kit Y719F/Y719F mutation resulted in further decrease in mast cell numbers (80%) (p < 0.001) (Fig. 7C). These results indicate that Gab2 and Kit Tyr719 both contribute equally to mast cell development in the dermis and ears. In the hypodermis, Gab2 deficiency alone resulted in an 50% decrease (p < 0.01) in mast cell numbers (Fig. 7B), whereas the Kit Y719F/Y19F mutation alone did not affect mast cell numbers. Strikingly, Gab2 deficiency combined with the Kit Y719F/Y719F mutation almost eliminated (95% reduction) (p < 0.001) mast cells in this location (Fig. 7B). In agreement with previous reports (32) (6), we also found that Gab2 deficiency led to >95% decrease in peritoneal mast cells, whereas Kit Y719F/Y719F mutation resulted in an 75% decrease in these cells (data not shown). Furthermore, in double mutant mice, no mast cells were detected at this location (data not shown). These data indicate that the Gab2 pathway is more critical for mast cell development in specific tissues such as peritoneal cavity and hypodermis of back skins. However, both the Gab2 and the Kit Tyr719 pathways contribute to mast cell development in other tissues such as ears and the dermis of the back skins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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75% decrease in mast cell in the peritoneal cavity of Kit Y719F/Y19F mice. These genetic data further support the model that Kit Tyr567 signals to Gab2 (Fig. 8).
Our data strongly support a model that Tyr567 via Shc/Grb2 brings Gab2 to Kit, where Gab2 can be phosphorylated by SFK in trans in the Kit heterodimer (Fig. 8). This mode of Gab2 recruitment to Kit is analogous to our previous finding that Shc, when tyrosyl-phosphorylated, via Grb2 recruits Gab2 to cof IL-3/GM-CSFR (19). Consistent with this idea, we found that SCF-evoked Shc and Gab2 tyrosyl phosphorylation is dramatically reduced in Kit Y567F BaF3 cells (Fig. 2, B and E). Kit Tyr567 was reported to be important for Shc phosphorylation (15). In addition, a published report suggests that Shc can bind Tyr567 because a phosphorylated-Tyr567 containing peptide can precipitate Shc from cell lysates (12). Result from searching Scansite (44) suggests phosphorylated Tyr567 in Kit as a potential binding site for Shc SH2 domain. Importantly, the Gab2 Grb2 mutant lost its association with Shc and Kit in response to SCF stimulation (Fig. 2F). Consistent with the idea that Shc/Grb2-recruited Gab2 can be phosphorylated by SFK in trans in the Kit heterodimer, we found that SFK inhibitor only impairs SCF-evoked Gab2 tyrosyl phosphorylation whereas Gab2-associated Shc tyrosyl phosphorylation was not affected (Fig. 2G). This result suggests that Shc, upon being recruited to Kit Tyr567, is tyrosyl-phosphorylated mainly by kinase other than SFK.
At present, we cannot exclude the presence of other minor routes for recruiting Gab2 to Kit. For example, SFK activated through Kit Tyr567 phosphorylates another docking site for Shc or Grb2 in Kit cytoplasmic tail. However, we did observe normal SCF-evoked Gab2 tyrosyl phosphorylation in cells expressing a mutant of the Grb2 binding site (45), Kit Y702F (Fig. 2B). It is also possible that SFK directly binds Gab2. Consistent with this idea, a previous report showed that EGF-evoked Gab2 tyrosyl phosphorylation was correlated with the weak Gab2 association with Src through the interaction between the two proline-rich motifs in Gab2 and the Src SH3 domain (46).
Our results reveal a novel function of Gab2, via Shp-2, in activating the Rac/JNK pathway critical for mast cell proliferation (Fig. 3C). How Gab2/Shp-2 regulates Rac remains unclear. Ras can activate Rac in PI3K-dependent and -independent manners (17, 41). As SCF-evoked Akt is unaffected in Gab2–/– BMMC or BMMC with acute deletion of Shp-2 (Figs. 3A and 5D), PI3K activation probably is not affected in Gab2–/– or Shp-2 deleted BMMC. However, given that Gab2/Shp2 is important for SCF-evoked Ras activation (Fig. 5, E–G), it is possible that Gab2/Shp2 may regulate Rac activation via a Ras-dependent PI3K-independent pathway in mast cells. Consistent with this possibility, LY294002 pretreatment only partially inhibits SCF-evoked Rac activation (Fig. 3E). Notably, Ras interacts with the Rac exchange factor, which can activate Rac in a PI3K-independent manner (41). Alternatively, Gab2 via Shp-2 may inhibit the activity of a Rac GAP. In this regard, Shp-2 has been postulated to activate Rho via dephosphorylation of tyrosyl-phosphorylated p190RhoGAP, which has increased activity against Rho (47). Conceivably, Gab2/Shp2 may activate Rac by dephosphorylating a RacGAP expressed in mast cells. One attractive candidate for such a protein is GC/GAP, which is a Gab2-binding protein that has Rac GAP activity in vitro and ex vivo when overexpressed (48).
Previous genetic study in Drosophila and biochemical studies in mammalian cells indicate that Shp-2 acts at a step upstream of Ras (28). However, the Shp-2 substrate/target critical in Ras activation is still not unclear. Our data indicate that the Gab2·Shp-2 complex is involved in Kit-evoked Ras activation, which is consistent with the reported role of Kit Tyr567 in Kit-evoked Ras activation (14, 15). Together with our biochemical data that Kit Tyr567 signals to Gab2 (Fig. 2), these results strongly support a model that Kit Tyr567 via Gab2/Shp-2 regulates Ras activation. Furthermore, our result is also consistent with previous reports that Shp-2 via Gab1 activates Ras (49, 50) in EGF signaling by preventing the recruitment of Ras-GAP to Gab1 (50). However, we could not detect any increased Ras-GAP association with the Gab2-Shp-2, a Gab2 mutant that cannot bind Shp-2, compared with Gab2 WT in BMMC upon SCF stimulation.6 This suggests that Gab2/Shp-2 regulates SCF-evoked Ras activation via mechanism other than inhibiting recruitment of Ras-GAP to Gab2, which is a subject of continuing investigation in our laboratory.
Our biochemical data strongly suggest that the Gab2 and Kit Tyr719-PI3K activated pathways such as the Rac/JNK pathway contribute equally to mast cell development in some tissues, such as the dermis of back skin (Fig. 7A) and ears (Fig. 7C). However, analysis of mast cells in hypodermis (Fig. 7B), stomach (32), and peritoneal cavity (6, 30, 32, 43) (data not shown) suggest that the Kit Tyr567-Gab2 activated pathway is more important than the Kit Tyr719 pathway in mast cell development in these specific tissues. One possibility is that the signal from Kit Tyr719 might be transient or weak. In these tissues, the Kit Tyr567-Gab2 pathway becomes more critical for the mast cell maturation and development. In this scenario, it is also possible that the small of amount of PI3K activated by Gab2 (Fig. 6D) may contribute to mast cell growth/maturation in these tissues. Consistent with this idea, p85
(the regulatory subunit of PI3K) knock-out mice showed no detectable mast cells in stomach (51). The other possibility is that Gab2/Shp-2 via Ras may activate other downstream effector only expressed in mast cells in specific tissues. Known Ras effectors include Raf, PI3K, Rac, and Ral (52). Lastly, it is also possible that Gab2 may be involved in other signaling system important for mast cell development in specific tissue. It has been reported that 1 integrin cross-linking can activate Gab2 biochemically (53). Mac-1 (a 2 integrin) knock-out mice were reported to have defective mast cell development in the peritoneal cavity (54). 7 integrin is required for mast cell progenitor (MCP) homing to intestines (55). Therefore, Gab2 could mediate integrin-initiated signals that may be important for MCP homing to specific tissues.
Decreased mast cell proliferation in Gab2–/– BMMC because of impaired activation of the Rac/JNK pathway could explain decreased mast cell numbers in various tissues. Rac is also known to play important role in regulating actin cytoskeleton remodeling and cell migration (56). Therefore, it is also possible that Gab2–/– mast cells have impaired ability to migrate and adhere, which could also affect mast cell development in vivo. A recent study identified MCP from bone marrow as Kit+ as well as other cell surface makers 7+T1/ST2+ Lin–Sca-1–Ly6c–FcRIa–CD27– (57). Therefore, our data suggest the possibility that Gab2 may play a role in differentiation of MCP into mature mast cells. Future studies are required to clarify the role of Gab2 in these processes.
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FOOTNOTES |
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1 These authors contributed equally to this work.
2 Present address: Inst. of Molecular Medicine, Peking University, New Life Sciences Building, Room 238, Mail Box 135, 5 Yiheyuan Rd., Beijing 100871, China.
3 Supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and Japan Society for the Promotion of Science.
4 A recipient of the Junior Faculty Scholar Award from American Association of Hematology. To whom correspondence should be addressed: NRB 1030N, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-667-0908; Fax: 617-667-0610; E-mail: hgu{at}bidmc.harvard.edu.
5 The abbreviations used are: BM, bone marrow; Gab, Grb2-associated binder; SCF, stem cell factor; SFK, Src family kinase; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; RNAK, receptor activator of NF-
B; HA, hemagglutinin; BMMC, bone marrow-derived mast cells; PH, pleckstrin homology; GST, glutathione S-transferase; WT, wild type; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HA, hemagglutinin; IL, interleukin; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; ANOVA, analysis of variance.
6 M. Yu, Y. P. Wang, and H. Gu, unpublished observation.
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