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Blood, 1 March 2004, Vol. 103, No. 5, pp. 1779-1786. Prepublished online as a Blood First Edition Paper on November 6, 2003; DOI 10.1182/blood-2003-07-2260. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-07-2260v1 103/5/1779    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Qu, X. Articles by Yamamura, H. Search for Related Content PubMed PubMed Citation Articles by Qu, X. Articles by Yamamura, H. Related Collections Immunobiology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY Negative regulation of FcRI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b Xiujuan Qu, Kiyonao Sada, Shinkou Kyo, Koichiro Maeno, S. M. Shahjahan Miah, and Hirohei Yamamura From the Division of Proteomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcRI) on mast cells induces a number of biochemical events, including protein-tyrosine phosphorylation leading to degranulation and multiple cytokine gene transcription. Here, we have demonstrated that a second member of the Cbl family of ubiquitin-protein ligase Cbl-b translocates into the lipid raft after FcRI engagement. Overexpression of Cbl-b in the lipid raft inhibits FcRI-mediated degranulation and cytokine gene transcription through the distinct mechanism. A point mutation of Cys373 in the RING finger domain of Cbl-b abrogates the suppression of FcRI-mediated degranulation but not cytokine gene transcription. The antigen-induced tyrosine phosphorylation of FcRI, Syk, phospholipase C- (PLC-), activation of c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), inhibitor of nuclear factor B kinase (IKK), and Ca++ influx were all suppressed in the cells overexpressing Cbl-b in the lipid raft. In particular, the expression amount of Gab2 protein and thereby its FcRI-mediated tyrosine phosphorylation were dramatically down-regulated by ubiquitin-protein ligase activity of Cbl-b. These results suggest that Cbl-b is a negative regulator of both Lyn-Syk-LAT and Gab2mediated complementary signaling pathways in FcRI-mediated mast cell activation.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Mast cells play a central role in inflammatory and immediate allergic reactions. The aggregation of FcRI triggers the activation of Lyn protein-tyrosine kinase (PTK), resulting in the rapid tyrosine phosphorylation of its and subunits.1 We isolated nonreceptor type PTK Syk.2,3 Tyrosine phosphorylated subunit of FcRI then recruits and activates Syk, the essential PTK for antigen-induced Ca++ influx, degranulation, and cytokine production in mast cells.3-9 Syk phosphorylates an adaptor protein linker for activation of T cells (LAT) which is located in the lipid raft (also referred to as the glycolipid-enriched microdomains [GEMs]) to accumulate the signaling molecules and is essential for FcRImediated mast cell activation.6,7,10 In contrast to this Lyn-Syk-LAT pathway, recent findings by using the genetic approach have revealed the existence of another complementary signaling pathway. Expression of the adaptor/scaffold protein Gab2 is necessary for FcRI-mediated activation of phosphatidylinositol 3-kinase (PI3-kinase) and downstream mast cell activation.11 Src family PTK Fyn is required for tyrosine phosphorylation of Gab2, independent of Lyn and LAT.12 Both of these 2 pathways are important for the FcRI-induced degranulation and cytokine production. Cbl-b, a second member of the Cbl-family of E3 ubiquitin-protein ligase, was originally isolated from the human breast cancer cells and is expressed in a variety of healthy tissues and cancer cells.13 It consists of an amino-terminal variant Src homology domain 2 (SH2) domain, a RING finger domain, a proline-rich region, a carboxyl-terminal leucine-zipper domain, and potential tyrosine phosphorylation sites. Genetic studies revealed that c-Cbl and Cbl-b have the distinct and overlapped functions in T-cell regulations.14 Although c-Cbl regulates thymocyte development and cell surface T-cell receptor (TCR) expression, Cbl-b–/– mice display a normal development of thymocytes and impaired peripheral T-cell activation mediated by CD28.15,16 Cbl-b targets PI3-kinase by ubiquitination in T cells.17,18 In addition, lack of Cbl-b results in the enhanced tyrosine phosphorylation and guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchanging activity of Vav1.15,16 Therefore, Cbl-b might indirectly regulate the activation of Vav1 through phosphatidylinositol 3-phosphate, the product of PI3-kinase.19 Analysis of B cells from Cbl-b–deficient mice showed that lack of Cbl-b results in the sustained phosphorylation of Ig, Syk, phospholipase C-2 (PLC-2), and prolonged Ca++ mobilization.20 However, Cbl-b–deficient avian pro-B cells display reduced PLC-2 activation and Ca++ mobilization, suggesting that Cbl-b may play a different role in immature and mature B cells.21 In mast cells, c-Cbl catalyzes FcRI-mediated ubiquitination of FcRI and Syk, resulting in an inhibition of the downstream inflammatory mediator release.22-24 However, the function of Cbl-b in mast cells has not been demonstrated yet. The lipid raft constitutes a membrane domain that concentrates sphingolipids, cholesterol, glycophosphatidylinositol-linked proteins, and numerous signaling molecules.25 Engagement of the immune receptors induces the accumulation of the signaling molecules into the lipid raft for both the effective receptor signals to the downstream and the degradation of the activated/tyrosine phosphorylated signaling molecules by ubiquitination.26 Engagement of FcRI induces the translocation of a ubiquitin-protein ligase c-Cbl into the lipid raft.27 Our present experiment shows that Cbl-b translocates into the lipid raft after the FcRI engagement. Targeting of the ubiquitin-protein ligase Cbl-b by the localization signal to the lipid raft might emphasize the activity of Cbl-b to have a dominant-active function. The present study demonstrated that the overexpression of Cbl-b in the lipid raft inhibits FcRI-mediated tyrosine phosphorylation of FcRI, Syk, Gab2, and, therefore, the downstream degranulation and cytokine gene transcription, suggesting that Cbl-b functions as a negative regulator of both Lyn-Syk-LAT and complementary signaling pathways from FcRI. In particular, overexpression of Cbl-b in the lipid raft dramatically down-regulates the protein amount of Gab2. This result gives us a new insight to develop gene therapy methods to treat immune and allergic diseases by using Cbl-b.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Materials and antibodies Protein A-agarose beads, mouse monoclonal anti-dinitrophenyl IgE monoclonal antibody (mAb; anti-DNP IgE, clone SPE-7), anti-Flag mAb (M2), Ca++ ionophore A23187 [GenBank] , thapsigargin, and PMA (phorbol 12-myristate 13-acetate) were purchased from Sigma (St Louis, MO). Protein-G–Sepharose beads were from Amersham (Piscataway, NJ). Antiphosphotyrosine (pTyr) mAb (4G10), anti-JNK/SAPK1 (c-Jun N-terminal kinase/stress-activated protein kinase 1) antibody, anti–PLC-1 mAb, and anti-Gab2 antibody were purchased from Upstate Biotechnology (Lake Placid, NY). Anti–Cbl-b, anti-Lyn, anti–PLC-2, anti-Syk, anti-p38, and anti-IKK (inhibitor of nuclear factor B kinase) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Antihemagglutinin epitope (HA) mAb was from Covance (Princeton, NJ). Antiphospho-p44/42 ERK (extracellular signal regulated kinase) (Thr202/Tyr204), anti-p44/42 ERK, antiphospho-Akt (Ser473), and anti-Akt antibodies were from New England Biolabs (Beverly, MA). Antiphospho-SAPK/JNK (Thr183/Tyr185), antiphospho-specific p38 MAP (mitogen-activated protein) kinase (Thr180/Tyr182), and antiphospho-IKK (Ser180)/IKK (Ser181) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Antigen 2, 4-dinitrophenylated bovine serum albumin (DNP-BSA; 30 mol DNP/1 mol BSA) was from LSL (Tokyo, Japan). Anti-FcRI mAbs were kindly provided by Dr Juan Rivera28 and Dr Reuben P. Siraganian (National Institutes of Health, Bethesda, MD). Construction of DNA The human Cbl-b cDNA was kindly provided by Dr Stanley Lipkowitz (National Naval Medical Center, Bethesda, MD). The cDNA encoding Cbl-b with the membrane localization signal was constructed by the polymerase chain reaction (PCR)–based method.9,29 We generated a chimeric molecule of Cbl-b with the myristoylation and palmitoylation signals. The 16 amino acids of c-Src N-terminus region with a Ser3 to Cys mutation [GEM-tag, MGCNKSKPKDASQRRR] was fused to the second amino acid in the N-terminal of human Cbl-b (GEM–Cbl-b). The cDNA of GEM–Cbl-b was then used to generate its mutant form which could not function as ubiquitin-protein ligase by a point mutation with a Cys373 to Ala (C373A) using the Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).30 All mutations were confirmed by DNA sequencing. The cDNA of Flag-tagged ubiquitin (Flag-Ub) was a gift from Dr Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science, Japan). All cDNAs were then subcloned into the pSVL expression vector (Amersham). The schematic diagram of Cbl-b mutants used in this study was shown in Figure 2A. View larger version (18K): [in this window] [in a new window]   Figure 2.. Generation of the stable cell lines expressing the lipid raft–anchored forms of Cbl-b. (A) Schematic diagram of the mutant forms of Cbl-b used in the current experiments. Both GEM-Cbl-b and GEM-Cbl-b (C373A) are chimeric molecules with the myristoylation and palmitoylation signals from c-Src (AA 1-16), in which Ser3 was substituted to Cys (GEM-tag). These cDNAs were subcloned into the pSVL expression vector. 4H indicates 4-helix; EF, EF-hand Ca++ binding domain; RING, RING finger; Pro rich, Pro-rich region; LZ, leucine zipper domain. (B) RBL-2H3 cells were stably cotransfected with pSVL-GEM-Cbl-b or pSVL-GEM-Cbl-b (C373A) together with pSV2-neo vector and selected with G418. Two or 3 positive cloned lines overexpressing each of the different kinds of chimeric proteins were selected for further analysis. Total cell lysates from the parental RBL-2H3 and selected cloned lines were analyzed by the immunoblotting with anti-Cbl-b, anti-HA, and anti-FcRI antibodies as an internal control. (C) GEM-Cbl-b mainly localizes in GEMs. Cell homogenates from control RBL-2H3 cells and cells overexpressing GEM-Cbl-b were fractionated by the sucrose density gradient centrifugation, and fractions 1 to 3, 4 to 6, and 7 to 10 were each collected and reacted with anti-Cbl-b antibody prebound to protein-G-Sepharose, respectively. The immunoprecipitates from collected fractions were analyzed by the immunoblotting with anti-HA mAb. Similar results were obtained when the other cloned lines were examined. The results were representative of 4 experiments.   Cell culture and transfection Rat basophilic leukemia RBL-2H3 cells were maintained as monolayer cultures in Dulbecco modified Eagle medium (DMEM) (Sigma), 100 U/mL penicillin, and 10% heat-inactivated fetal calf serum. For stable transfection, each 20 µg linearized expression construct and 2 µg pSV2-neo vector were cotransfected into 5 x 106 RBL-2H3 cells by electroporation (950 µF, 310 V).31 Stably transfected cell lines were selected by 0.4 mg/mL active G418 (Invitrogen, Carlsbad, CA). Cell lines were screened by the level of protein expression by the immunoblotting of total lysates with anti-HA and anti–Cbl-b antibodies. The immunoblotting with anti-FcRI mAb was used as an internal control. RBL-2H3 cells stably expressing Flag-Ub were used for immunoprecipitation study to demonstrate ubiquitination of Gab2. Cell activation, immunoprecipitation, and immunoblotting The cell monolayers cultured overnight with anti-DNP IgE (1:5000) were washed once with Tyrode-HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid) buffer (10 mM HEPES, pH 7.4, 127 mM NaCl, 4 mM KCl, 0.5 mM KH2PO4, 1 mM CaCl2, 0.6 mM MgCl2, 10 mM LiCl2, 5.6 mM glucose, and 0.1% BSA) and then stimulated with 30 ng/mL antigen DNP-BSA in the same buffer for the indicated times.32 For the immunoprecipitation studies, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and solubilized in 1% Triton lysis buffer (1% Triton X-100, 50 mM Tris (tris(hydroxymethyl)aminomethane), pH 7.4, 150 mM NaCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 100 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 90 mU/mL aprotinin) on ice. For immunoprecipitation with anti–Cbl-b, anti-pTyr or anti-Gab2 antibody, cells were solubilized in the denature buffer (1% Triton X-100 lysis buffer containing 0.1% sodium dodecylsulfate [SDS] and 0.5% deoxycholic acid) to dissociate protein complexes. Cell lysates were precleared by centrifugation, and then resultant supernatants were incubated with the indicated antibodies prebound to protein A-agarose or protein G-Sepharose beads. After rotation for 1 hour at 4°C, the beads were washed 4 times with lysis buffer, and the immunoprecipitated proteins were eluted by heat treatment at 100°C for 5 minutes with 2 x sampling buffer. For the preparation of total cell lysates, monolayers were rinsed as described earlier with PBS and lysed by the direct addition of 2 x sampling buffer. Total cell lysates, immunoprecipitated proteins, and subcellular fractions were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electronically transferred to polyvinylidene diflouride transfer membrane (Millipore, Bedford, MA). After blocking with 5% skim milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween20), the blots were probed with the indicated primary antibodies for 1 hour at 4°C. After washing with TBST, the membrane was reacted with the appropriate horseradish peroxidase–conjugated secondary antibodies for 30 minutes at room temperature. After extensive washing with TBST, proteins were visualized by the enhanced chemiluminescence reagent (Western Lightning; PerkinElmer Life Sciences, Boston, MA). Densitometric analysis of immunoblotting was performed by using National Institutes of Health (NIH) Image software. Relative amounts of protein-tyrosine phosphorylation which compared to the maximum phosphorylation in the control cells were normalized by protein expression and indicated at the bottom of the figures. In some experiments, relative amounts of protein expression were analyzed. Subcellular fractionation The low-density detergent-insoluble fractions were prepared by sucrose density gradient centrifugation essentially as described.33,34 The proteins were concentrated with 0.02% deoxycholic acid and 10% trichloroacetic acid. For the immunoprecipitation study, 1 to 3 fractions, 4 to 6 fractions, and 7 to 10 fractions were collected together. Analysis of -hexosaminidase release Degranulation of different cell lines was determined by the measurement of -hexosaminidase release. Cells (105) were seeded in 24-well plates and cultured overnight with or without anti-DNP IgE. The cell monolayers were washed once with Tyrode-HEPES buffer and then stimulated with different concentrations of the antigen DNP-BSA or Ca++ ionophore A23187 [GenBank] in the same buffer. After incubation for 1 hour at 37°C, the medium was recovered for the analysis of -hexosaminidase activity. Total cell lysates were obtained by the addition of 1% NP-40 in the same medium. The medium and total cell lysates were incubated with 1.3 mg/mL P-nitrophenyl-N-acetyl--d-glucopyranoside (Nacalai, Osaka, Japan) in 0.1 M sodium citrate buffer (pH 4.5) for 30 minutes at 37°C. The reaction was terminated by the addition of 0.2 M glycine buffer (pH 10.7). The release of the product 4-P-nitrophenol was monitored by the absorbance at 405 nm by using a Microplate reader (Model 550; Bio-Rad, Hercules, CA). The antigen-induced -hexosaminidase release was expressed as a percentage of the maximal release induced by A23187 [GenBank] .31 Measurement of [Ca++]i Intracellular free Ca++ concentration ([Ca++]i) was measured by means of fluorescent indicator fura-2 (Dijindo, Kumamoto, Japan) as described previously.31 The cell monolayers of the different cell lines sensitized with anti-DNP IgE were detached from the tissue-culture dish and then loaded with 3 µM fura-2 for 30 minutes at 37°C. Cells were stimulated by either 30 ng/mL antigen DNP-BSA or 1 µM thapsigargin. Fura-2 fluorescence was monitored with the fluorescence spectrophotometer (Hitachi F-4500, Tokyo, Japan) by the excitation wavelength at 340, 380 nm and the emission wavelength at 510 nm. RNase protection assay The total RNA was purified by RNeasy Mini Kit (Qiagen, Hilden, Germany). The production of the cytokine mRNA was quantitatively measured by using Multi-probe RNase Protection Assay Kit (BD Biosciences, San Jose, CA).35 Briefly, by Riboquant In Vitro Transcription Kit (BD Biosciences), 32P-labeled RNA probes were synthesized by using Rat Cytokine Multi-Probe Template Sets (BD Biosciences). The synthesized probes were purified by using the Spin+STE 10 Column (BD Biosciences) and then hybridized overnight at 56°C with 20 µg total RNA purified from cells with or without either the antigen or PMA plus calcium ionophore stimulation. After the RNase treatment, the protected double-stranded RNAs were separated by the urea gel and visualized by autoradiography. In vitro protein kinase assay Either unstimulated or antigen-stimulated RBL-2H3 cells or cells transfected with the different cDNAs were solubilized in Triton lysis buffer. Anti-Lyn immunoprecipitates were resuspended in the kinase buffer without adenosine triphosphate (ATP), then incubated with 40 µL kinase buffer (40 mM HEPES, pH 7.5, 10 mM MgCl2, 4 µM ATP, 4 µCi [0.148 MBq] -32P] ATP) and 2.5 µg acid-treated enolase (Sigma) at 30°C for 5 minutes. Reactions were terminated by heat treatment at 100°C for 5 minutes in the sample buffer, and eluted proteins were separated by 10% SDS-PAGE. The gels were incubated with 1 N KOH for 1 hour at 56°C to remove phosphoserine and most of the phosphothreonine. After the gel fixing and drying, radiolabeled proteins were visualized by autoradiography. Immunoprecipitation of Lyn was confirmed by immunoblotting.36,37    Results Top Abstract Introduction Materials and methods Results Discussion References   Aggregation of FcRI induces the translocation of Cbl-b into the lipid raft Engagement of FcRI results in the translocation and concentration of many signaling molecules, including c-Cbl, into the lipid raft.27 In RBL-2H3 mast cell line, 2 kinds of Cbl family of ubiquitin ligase, c-Cbl and Cbl-b, are expressed.37 Therefore, we first examined whether there is a similar change in Cbl-b association with the lipid raft or not (Figure 1). The result shows that the majority of Cbl-b localized in detergent-soluble fractions (Figure 1, upper panels). After FcRI engagement, Cbl-b protein was shifted into the lower-density fractions and was detectable in the lipid raft fractions (Figure 1, fractions 2 and 3). Densitometric analysis revealed that 3% of total Cbl-b was translocated into the lipid raft (Figure 1A). Recently, we have demonstrated that antigen stimulation causes the inducible association of Cbl-b with Lyn, which is known to be localized in the lipid raft.37 Both Lyn and FcRI were colocalized in the lipid raft fraction prior to antigen stimulation (Figure 1, middle and bottom panels). These results demonstrated that antigen stimulation induces the translocation of Cbl-b into the lipid raft in RBL-2H3 mast cells (Figure 1). View larger version (72K): [in this window] [in a new window]   Figure 1.. Aggregation of FcRI induces the translocation of Cbl-b into the lipid raft. The cell homogenates of either nonstimulated or antigen (Ag)–stimulated RBL-2H3 cells were fractionated by sucrose density gradient centrifugation. The proteins were concentrated with 0.02% deoxycholic acid and 10% trichloroacetic acid, separated by SDS-PAGE, and analyzed by immunoblotting with anti-Cbl-b (upper), anti-Lyn (middle), and anti-FcRI antibodies (bottom). Molecular size markers are indicated at the left in kilodaltons. The results were representative of 3 experiments.   To investigate the role of Cbl-b in the lipid raft during mast cell activation, we generated stable cell lines overexpressing the mutant forms of Cbl-b which constitutively localize in the lipid raft. The myristoylation and palmitoylation of cytoplasmic proteins by addition of 16 amino acids from the N-terminus of c-Src in which Ser3 is substituted to Cys (GEM-tag) can direct molecules into the lipid raft.9,29 This GEM-tag was fused to the N-terminus of wild-type Cbl-b (Figure 2A).30 Expression constructs of GEM-Cbl-b or GEM-Cbl-b (C373A) which loses the ability as a ubiquitin-protein ligase were stably transfected into RBL-2H3 cells and cloned lines were screened by immunoblotting (Figure 2B). For further analysis, 2 or 3 cloned lines transfected with each kind of cDNA were selected in which the level of expression of the mutant form of Cbl-b was highest among the clones. The molecular weight of GEM-Cbl-b (C373A) seems to be higher than that of GEM-Cbl-b (Figure 2B). The intracellular localization of chimeric protein was further analyzed by the subcellular fractionation study. The GEM-tagged Cbl-b was mainly localized in the lipid raft (Figure 2C). Densitometric analysis demonstrated that 55% of GEM-Cbl-b was constitutively localized in the fraction of the lipid raft (Figure 2C). Overexpression of Cbl-b in the lipid raft suppresses FcRI-mediated Ca++ mobilization and degranulation To examine the function of Cbl-b in the lipid raft, we examined the FcRI-induced -hexosaminidase release by using the stable cloned lines overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) (Figure 2B). The cell lines were stimulated by antigen or Ca++ ionophore A23187 [GenBank] . Antigen-induced -hexosaminidase release was suppressed in GEM-Cbl-b–overexpressing cells compared with that in the control cells (Figure 3A). However, overexpression of GEM-Cbl-b (C373A) relatively enhanced the antigen-induced -hexosaminidase release, suggesting that GEM-Cbl-b (C373A) has a dominant-negative function for the endogenous Cbl-b (Figure 3A). The A23187 [GenBank] -induced release as a percentage of the total -hexosaminidase activity was comparable among all these cell lines (Figure 3A, legend). In addition, thapsigargin-induced release was similar among these clones (data not shown). Similar results were obtained when the other cloned lines overexpressing the mutant form of Cbl-b were tested. These results demonstrate that overexpression of Cbl-b in the lipid raft results in a suppression of FcRI-mediated degranulation, depending on its ubiquitin-protein ligase activity. View larger version (49K): [in this window] [in a new window]   Figure 3.. Overexpression of GEM-Cbl-b suppresses FcRI-mediated Ca++ mobilization and degranulation in RBL-2H3 cells. (A) Analysis of FcRI-mediated -hexosaminidase release. Control cells, GEM-Cbl-b or GEM-Cbl-b (C373A) overexpressing cells were cultured overnight with anti-DNP IgE and then stimulated with the indicated concentrations of antigen DNP-BSA (ng/mL) or Ca++ ionophore A23187 [GenBank] . The antigen-induced -hexosaminidase release was presented as a percentage of the A23187 [GenBank] -induced maximal -hexosaminidase release. The A23187 [GenBank] -induced average releases ± SD as a percentage of total activity were 42% ± 2% (ctrl.), 37% ± 1% (GEM-Cbl-b), and 34% ± 1% (GEM-Cbl-b [C373A]), respectively. The results are the mean values ± SD from 3 independent experiments. The results were representative of 10 experiments. (B) FcRI-mediated Ca++ mobilization is suppressed by the overexpression of GEM-Cbl-b. Intracellular free Ca++ concentration ([Ca++]i) was measured by means of fluorescent indicator fura-2. IgE-sensitized control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were loaded with fura-2 and then activated with either 30 ng/mL antigen (top row) or 1 µM thapsigargin (bottom row). The traces show the 340:380 fluorescence ratios. (C-D) Overexpression of GEM-Cbl-b suppresses tyrosine phosphorylation of PLC-. Control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (107) were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti–PLC-1 mAb (C) or anti–PLC-2 antibody (D). The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb along with anti–PLC-1 mAb (C) or anti–PLC-2 antibody (D). The numbers at the bottom of the figures are the normalized densitometric analysis of PLC-1 and PLC-2 tyrosine phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of 3 experiments (B-D).   Because antigen-induced increase in [Ca++]i is critical for mast cell degranulation, we compared Ca++ mobilization. The parental control cells and cells overexpressing GEM-Cbl-b were stimulated by either antigen or thapsigargin (Figure 3B). As shown, the response to the antigen stimulation was suppressed in the cells overexpressing GEM-Cbl-b compared with that in control cells. Overexpression of GEM-Cbl-b (C373A) resulted in an increase in Ca2+ mobilization (Figure 3B). In contrast, there was similar response to thapsigargin among the clones, indicating that overexpression of GEM-Cbl-b or GEM-Cbl-b (C373A) has no effects on Ca++ release from the intracellular storage. Thus, the overexpression of Cbl-b in the lipid raft suppresses the signals upstream of the elevation of [Ca++]i. Then, we focused on PLC- which hydrolyzes phosphatidylinositol to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP3) to release Ca++ ion from the internal stores, resulting in a Ca++ influx. Mast cells express 2 kinds of PLC-, PLC-1 and PLC-2. Compared with the control cells, FcRI-induced tyrosine phosphorylation of PLC-1 and PLC-2 was reduced in the cells overexpressing GEM-Cbl-b (Figure 3C-D). Overexpression of GEM-Cbl-b (C373A) lightly enhanced the antigen-induced tyrosine phosphorylation of PLC-1 and PLC-2 (Figure 3C-D). Therefore, these results indicate that overexpression of Cbl-b in the lipid raft down-regulates FcRI-induced activation of PLC-1 and PLC-2, and thereby the subsequent Ca++ mobilization and degranulation. The ubiquitin-protein ligase activity is necessary to regulate this pathway. Overexpression of Cbl-b in the lipid raft suppresses the activation of MAP kinases and cytokine gene transcription Because overexpression of Cbl-b in the lipid raft suppresses the antigen-induced immediate degranulation, we next examined the transcription of cytokine genes by the RNase protection assay (Figure 4A). Compared with the control cells, there was significant suppression of the antigen-induced increase in the mRNA of interleukin-3 (IL-3), IL-4, IL-6, IL-10, and tumor necrosis factor (TNF) in the cells overexpressing GEM-Cbl-b (Figure 4A). Unlike degranulation, expression of GEM-Cbl-b (C373A) could not restore the defect of cytokine gene transcription. The production of control housekeeping genes (L32 and GAPDH) was comparable among the control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) (Figure 4A). Moreover, PMA plus calcium ionophore–induced production of cytokine genes (0 minute versus 60 minutes induced by PMA plus calcium ionophore) were identical among the clones (Figure 4A). These results suggest that the endogenous Cbl-b plays a negative regulatory role in FcRI-mediated transcriptional activation of cytokine genes, independent of its ubiquitin-protein ligase activity. Perhaps the other domains in Cbl-b contribute to this suppression through the passive adaptor function of Cbl-b. View larger version (35K): [in this window] [in a new window]   Figure 4.. Overexpression of GEM-Cbl-b suppresses FcRI-mediated activation of MAP kinases and cytokine gene transcription in RBL-2H3 cells. (A) Analysis of FcRI-mediated cytokine gene transcription. Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were cultured overnight with anti-DNP IgE and then stimulated with 30 ng/mL antigen DNP-BSA or 10 ng/mL PMA plus 0.5 µM A23187 [GenBank] (P/I). After the indicated times, total RNA was extracted and hybridized with the 32P-labeled RNA probes. The protected double-stranded RNA was separated by the urea gel and analyzed by the autoradiography. Similar results were obtained when the other cloned lines were examined. The results were representative of 10 experiments. (B-E) Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Total cell lysates or detergent-soluble cell lysates (1-3 x 105 cell equivalents per lane) were separated by SDS-PAGE and analyzed by the immunoblotting with antiphospho-SAPK/JNK (pJNK) and anti-JNK (B), antiphospho-p44/42 ERK (pERK) and anti-p44/42 ERK (C), and antiphospho-p38 MAP kinase (p-p38) and anti-p38 MAP kinase antibodies (D), antiphospho-IKK/ (pIKK/) and anti-IKK antibodies (E), respectively. The numbers at the bottom of the figures are the normalized densitometric analysis of JNK, ERK, p38, and IKK phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of at least 3 experiments (B-E).   Then we tested the effects on the activation of the upstream MAP kinases. Overexpression of Cbl-b in the lipid raft suppressed the antigen-induced phosphorylation of JNK (Figure 4B) and extracellular signal regulated kinase (ERK) (Figure 4C). Suppression of JNK was more remarkable than ERK. However, overexpression of GEM-Cbl-b (C373A) could result in the suppression of neither JNK nor ERK, suggesting that Cbl-b–mediated negative regulation of JNK and ERK is RING finger domain dependent. In contrast, phosphorylation of p38 MAP kinase displayed similar patterns among the different cell lines (Figure 4D). In addition to MAK kinases, we tested IKK which is known to regulate nuclear factor B (NF-B) (Figure 4E). Similar to MAP kinases, activation of IKK was down-regulated by the overexpression of GEM-Cbl-b but not by GEM-Cbl-b (C373A). These results suggest that the suppression of multiple cytokine gene transcriptions by Cbl-b is regulated by both RING finger–dependent and –independent specific mechanisms. Overexpression of Cbl-b in the lipid raft suppresses tyrosine phosphorylation of FcRI and Syk To date, several proximal molecules have been identified which possess a critical function in the activation of mast cells induced by the antigen stimulation.38 The sequential activation of 2 types of protein-tyrosine kinases Lyn and Syk is critical for mast cell activation. Therefore, we tested the effect of the overexpression of Cbl-b in the lipid raft on the antigen-induced activation of Lyn and Syk. Antigen-induced tyrosine phosphorylation of FcRI and subunits was dramatically suppressed in the cells overexpressing GEM-Cbl-b, compared with that in the control cells (Figure 5A). In parallel to this result, the enzymatic activity of Lyn was suppressed by the overexpression of GEM-Cbl-b (Figure 5B). Similar results were obtained for tyrosine phosphorylation of Syk (Figure 5C). The inhibition of the activation of Lyn and Syk were abrogated by a point mutation in the RING domain of Cbl-b (GEM-Cbl-b [C373A]). The expressing protein amounts of FcRI, Lyn, and Syk were not affected by the overexpression of Cbl-b in the lipid raft (Figure 5, lower panels). Therefore, overexpression of Cbl-b down-regulates the kinase activity of Lyn, tyrosine phosphorylation of FcRI and subunits which cause the suppression of FcRI signals. View larger version (26K): [in this window] [in a new window]   Figure 5.. Overexpression of GEM-Cbl-b suppresses tyrosine phosphorylation of FcRI and Syk. (A) Control cells and cells overexpressing GEM-Cbl-b were primed with anti-DNP IgE and then stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (3 x 106 cells) were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti-FcRI mAb. The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb, anti-FcRI mAb. (B) Cells were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti-Lyn antibody.Anti-Lyn immunoprecipitates were subjected to the in vitro protein kinase assay by using enolase as an external substrate. Radiolabeled proteins were separated by SDS-PAGE and visualized by autoradiography. Immunoprecipitated Lyn was analyzed by immunoblotting with anti-Lyn antibody. (C) Cells were solubilized in the denature buffer and immunoprecipitated with anti-pTyr mAb. The immunoprecipitates and detergent-soluble cell lysates were analyzed by the immunoblotting with anti-pTyr mAb and anti-Syk antibody. The numbers at the bottom of the figures are the normalized densitometric analysis of FcRI,FcRI, and Syk phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of 5 experiments.   Overexpression of Cbl-b in the lipid raft down-regulates Gab2 Earlier findings revealed that the adaptor/scaffold protein Gab2 is critical for the complementary signaling pathway by regulating PI3-kinase in mast cells.11 FcRI-mediated tyrosine phosphorylation of Gab2 was dramatically suppressed, and the expressing amount of Gab2 protein was also markedly decreased in the cells overexpressing GEM-Cbl-b (Figure 6A). Overexpression of GEM-Cbl-b (C373A) resulted in an increase in tyrosine phosphorylation of Gab2 (Figure 6A). The mobility shift of Gab2 is lightly enhanced by the expression of GEM-Cbl-b (C373A). This result suggests that overexpression of Cbl-b in the lipid raft may affect the stability of Gab2, depending on the activity of ubiquitin-protein ligase. In fact, antigen stimulation induces the multi-ubiquitination of Gab2 in RBL-2H3 mast cells (Figure 6B). The protein amount of Gab2 was decreased after the antigen stimulation. Therefore, overexpression of Cbl-b in the lipid raft might directly affect the protein amount of Gab2 by ubiquitin-protein ligation and degradation of Gab2. View larger version (40K): [in this window] [in a new window]   Figure 6.. Overexpression of GEM-Cbl-b dramatically inhibits the FcRI-induced tyrosine phosphorylation of Gab2 and stimulates the degradation of Gab2. (A) Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (107) were solubilized in the denature buffer and immunoprecipitated with anti-Gab2 antibody. The immunoprecipitates and detergent-soluble cell lysates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb and anti-Gab2 antibody. The numbers at the bottom of the figures were the normalized densitometric analysis of Gab2 phosphorylation (Gab2 tyrosine phosphorylation/Gab2 protein). The levels of Gab2 expression were 20% (GEM-Cbl-b overexpressing cells) and 90% (GEM-Cbl-b [C373A] overexpressing cells) compared with the control cells. (B) Cells stably expressing Flag-Ub were primed with anti-DNP IgE and stimulated with 1 µg/mL antigen DNP-BSA for the indicated times. Either nonstimulated or antigen-stimulated cell lysates (3 x 106) were solubilized in the denature buffer and immunoprecipitated with anti-Gab2 antibody. The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-Flag mAb and anti-Gab2 antibody. In the immunoblotting with anti-Gab2 antibody, the membrane was exposed for 1 minute (short exposure) and 20 minutes (long exposure). Ubiquitination of Gab2 (Gab2-Ub) was indicated. Similar results were obtained when the other cloned lines were examined. The levels of Gab2 expression were 60% (1 minute) and 50% (3 or 10 minutes after the antigen stimulation) compared with the unstimulated cells. The results were representative of 4 experiments.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   We have demonstrated that overexpression of Cbl-b in the lipid raft suppresses antigen-induced degranulation and cytokine gene transcription (Figures 3A and 4A). Among the signaling molecules, overexpression of Cbl-b in the lipid raft dramatically down-regulates the protein amount of Gab2 and, therefore, the antigen-induced tyrosine phosphorylation of Gab2 (Figure 6A). Although Cbl-b possesses multiple domains and tyrosine phosphorylation sites, the effect on Gab2 is mediated by the RING finger domain because overexpression of GEM-Cbl-b (C373A) could not suppress the expression level of Gab2, relatively enhanced its antigen-induced tyrosine phosphorylation and downstream degranulation (Figures 3A and 6A). Our results suggest that Cbl-b has a function to down-regulate the adaptor/scaffold protein Gab2 by ubiquitination in the lipid raft (Figure 6). Genetic analysis revealed that Gab2 is essential for FcRI-mediated activation of PI3-kinase.11 Therefore, Cbl-b may negatively regulate the complementary signaling pathway from FcRI in mast cells.12 Furthermore, overexpression of Cbl-b in the lipid raft strongly suppresses tyrosine phosphorylation of FcRI, subunits, and Syk (Figure 5). Previous results have demonstrated that c-Cbl catalyzes ubiquitination of TCR and down-regulates TCR-CD3 complex in thymocytes, whereas Cbl-b promotes ubiquitination of p85 of PI3-kinase and inhibits the signals by CD28 costimulation in peripheral T cells.14 In B cells, it is reported that Cbl-b negatively regulates B-cell antigen receptor–mediated signaling through ubiquitination of Syk.20 All together, these findings suggest that endogenous Cbl-b could inhibit both Lyn-Syk-LAT and Gab2-mediated complementary signaling pathways from FcRI in mast cells. Here we proposed that Gab2 is a possible direct target of Cbl-b in FcRI signaling. Although tyrosine phosphorylation of Gab2 was completely abolished, phosphorylation of Akt, which is also the downstream of PI3-kinase is not affected (data not shown).11 This defect might be explained by the fact that RBL-2H3 cells are factor independent because of a point mutation in c-Kit (Asp817 to Tyr).39 A positive regulatory signal from the constitutive active form of c-Kit may be involved in the normal activation of Akt.40 Mast cells express 2 isoforms of PLC-, PLC-1 and PLC-2. Antigen stimulation induces the translocation of PLC-1 into the lipid raft, whereas PLC-2 distributes in the plasma membrane and Golgi region and does not redistribute appreciably after FcRI engagement.41 Antigen-induced translocation and activation of PLC-1, but not PLC-2, is sensitive to the PI3-kinase inhibitor, wortmannin.41 Moreover, antigen-induced tyrosine phosphorylation of PLC-2, not PLC-1, requires the activation of Vav1.42 Overexpression of Cbl-b in the lipid raft suppresses tyrosine phosphorylation of Vav1 (data not shown). A genetic study revealed that both Vav1 and Slp-76, which associates with Vav1, play critical roles in FcRI-mediated mast cell activation.42,43 Our results indicate that overexpression of Cbl-b in the lipid raft affects FcRI-mediated activation of Vav1. Therefore, although the antigen-induced activation of PLC-1 and PLC-2 is mediated by the distinct pathways, endogenous Cbl-b could negatively regulate the activation of both PLC-1 and PLC-2 (Figure 3C-D). In immune receptor signaling, adaptor and molecular scaffold proteins lack the enzymatic and transcriptional domains, but they have multiple motifs and domains to create protein complexes to integrate signals from cell surface receptors.44 In addition to the passive adaptor function, these molecules possess the active regulatory function in immune receptor signaling. We have reported that an adaptor protein 3BP2 [PDB] is a putative ligand of SH2/SH3 domains of Src family PTK Lyn to unclamp the closed-inactive conformation and stimulate the enzymatic activation of Lyn.36,45 In this model, an adaptor protein 3BP2 [PDB] acts as an active regulator of nonreceptor type of PTK. Moreover, point mutations in the 3bp2 gene are correlated with human inherited disease cherubism, suggesting that 3BP2 [PDB] may have a specific role in the transcriptional regulation in osteoclasts.46 In the current study, overexpression of Cbl-b in the lipid raft results in the suppression of the protein amount of Gab2 prior to stimulation, suggesting that Gab2 might be synthesized and degraded in the rapid turnover in resting cells (Figure 6A). This finding leads to the idea that Cbl-b has a potential active function in nonstimulated mast cells. Our result also demonstrates that RING finger–independent, passive adaptor function may be involved in the regulation of antigen-induced cytokine gene transcription (Figure 4A). Because FcRI-mediated activation of JNK, ERK, and IKK depends on the function of RING-finger of Cbl-b, the additional pathways for activating the transcriptional factor might be involved in the regulation of cytokine gene transcription (Figure 4). In T cells, it is expected that Ser/Thr kinases might be involved in controlling TCR-mediated transcriptional activation of the NF-B p65 subunit.47,48 A possible role of p65 phosphorylation is to serve as a binding site for transcriptional co-activator such as cyclic adenosine monophosphate (cAMP) response element-binding protein/p300.47 It is possible that an adaptor function of Cbl-b down-regulates the transcription of cytokine genes through the co-activator of transcriptional factor in mast cells. This present study suggests that endogenous Cbl-b negatively regulates FcRI, Lyn, PLC-1, PLC-2, Gab2, and subsequent mast cell activation. Overexpression of the novel chimeric protein GEM-Cbl-b has a strong inhibitory role on allergic reaction. Here, we propose that a ubiquitin-protein ligase Cbl-b is a target molecule to develop novel anti-allergic drugs and gene therapy for the immune diseases.    Acknowledgements   We thank Dr Reuben P. Siraganian, Dr Stanley Lipkowitz, Dr Keiji Tanaka, and Dr Juan Rivera for providing the reagents. We also thank Dr Keiko Kawauchi-Kamata and Dr Zhi-Hui Xie for the instructions.    Footnotes   Submitted July 7, 2003; accepted October 31, 2003. Prepublished online as Blood First Edition Paper, November 6, 2003; DOI 10.1182/blood-2003-07-2260. Supported in part by research funding from Hyogo Science and Technology Association (K.S.); the 21st Century COE Program and the Grad-in-Aids for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. 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