Regulation of FcεRI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells by Kiyonao Sada, S. M. Shahjahan Miah, Koichiro Maeno, Shinkou Kyo, Xiujuan Qu, and Hirohei Yamamura Blood Volume 100(6):2138-2144 September 15, 2002 ©2002 by American Society of Hematology

Aggregation of FcεRI induces tyrosine phosphorylation of 3BP2 in RBL-2H3 cells.(A) Generation of cell lines expressing HA-tagged 3BP2 wild type. Aggregation of FcεRI induces tyrosine phosphorylation of 3BP2 in RBL-2H3 cells.(A) Generation of cell lines expressing HA-tagged 3BP2 wild type. The RBL-2H3 cells were stably transfected with either empty pMT3 vector or pMT3-HA-3BP2, together with pSV2-neo, by electroporation (950 μF, 310 V). Clones resistant to G418 were selected and screened by level of protein expression. Two positive cloned lines with the highest expression were chosen for further analysis. Total cell lysates of the parental RBL-2H3 and transfected cells were analyzed by immunoblotting (IB) with anti-3BP2, anti-HA, and anti-FcεRIβ antibodies used as an internal control. (B) Analysis of tyrosine phosphorylation of 3BP2. Cell lines expressing HA-3BP2 and vector-transfected control cells (pMT3) were cultured overnight with anti-DNP IgE and then stimulated with 10 ng/mL of antigen (Ag) DNP-BSA for the indicated times or incubated without antigen for 1 minute. Tyrosine phosphorylation of 3BP2 was analyzed by immunoprecipitation (IP). Cells were lysed in 1% Triton lysis buffer and cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr mAb. The membrane was stripped and reprobed with anti-HA antibody. Similar results were obtained when the other lines were examined. (C) Tyrosine phosphorylation of endogenous 3BP2. RBL-2H3 cells sensitized with anti-DNP IgE were either unstimulated or stimulated with antigen for 1 minute. Cells were lysed in the denature buffer and cell lysates were immunoprecipitated with anti-pTyr mAb or control IgG. Immunoprecipitates were analyzed by immunoblotting with anti-3BP2 antibody. Molecular size markers are indicated at the left in kilodaltons. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

FcεRI-mediated tyrosine phosphorylation of 3BP2 does not depend on calcium influx from external sources.Cell lines expressing HA-3BP2 sensitized with anti-DNP IgE were stimulated with 10 ng/mL of antigen DNP-BSA for the indicated times without or with the p... FcεRI-mediated tyrosine phosphorylation of 3BP2 does not depend on calcium influx from external sources.Cell lines expressing HA-3BP2 sensitized with anti-DNP IgE were stimulated with 10 ng/mL of antigen DNP-BSA for the indicated times without or with the presence of 0.5 mM EDTA in medium. Cell lysates were immunoprecipitated with anti-HA antibody, and then immunoprecipitates were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr and anti-HA antibodies. Similar results were obtained when the other lines were examined. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

Overexpression of 3BP2-SH2 domain suppresses FcεRI-mediated degranulation in RBL-2H3 cells.(A) Generation of cell lines expressing the HA-tagged SH2 domain of 3BP2. Overexpression of 3BP2-SH2 domain suppresses FcεRI-mediated degranulation in RBL-2H3 cells.(A) Generation of cell lines expressing the HA-tagged SH2 domain of 3BP2. The RBL-2H3 cells were stably transfected with pMT3-HA-3BP2-SH2 (454-559) by electroporation. G418-resistant clones were screened by immunoblotting and 2 positive cloned lines with highest expression were selected for further analysis. Total cell lysates of the transfected cell lines were analyzed with anti-HA and anti-FcεRIβ antibodies used as an internal control. (B) Analysis of FcεRI-mediated β-hexosaminidase release. Control cells transfected with pMT3 vector alone and cells overexpressing 3BP2-SH2 domain were cultured overnight with anti-DNP IgE and then stimulated with the indicated concentrations of the antigen DNP-BSA (ng/mL) or with the calcium ionophore A23187 (μM). The antigen- or A23187-induced releases are normalized by expression as a percentage of the total β-hexosaminidase activity. (C) Analysis of thapsigargin-induced β-hexosaminidase release. Cells were stimulated with the indicated concentrations of thapsigargin (μM); releases are normalized by expression as a percentage of β-hexosaminidase activity induced by 1 μM A23187. The results are the mean values ± SE from 3 independent experiments. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

FcεRI-induced tyrosine phosphorylation of cellular proteins is not affected by the overexpression of 3BP2-SH2 domain.Control cells (pMT3) and cells overexpressing SH2 domain of 3BP2 were primed with anti-DNP IgE and stimulated with 10 ng/mL antigen DNP-BSA ... FcεRI-induced tyrosine phosphorylation of cellular proteins is not affected by the overexpression of 3BP2-SH2 domain.Control cells (pMT3) and cells overexpressing SH2 domain of 3BP2 were primed with anti-DNP IgE and stimulated with 10 ng/mL antigen DNP-BSA for the indicated times. Total cell lysates (105 cell equivalents per lane) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr mAb. Similar results were obtained when the other lines were examined. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

Overexpression of 3BP2-SH2 domain suppresses FcεRI-mediated tyrosine phosphorylation of PLC-γ and calcium mobilization.(A and B) Cells transfected with vector alone (pMT3) and cells overexpressing 3BP2-SH2 domain were stimulated with antigen for the indicat... Overexpression of 3BP2-SH2 domain suppresses FcεRI-mediated tyrosine phosphorylation of PLC-γ and calcium mobilization.(A and B) Cells transfected with vector alone (pMT3) and cells overexpressing 3BP2-SH2 domain were stimulated with antigen for the indicated times; then cell lysates were immunoprecipitated with either anti–PLC-γ2 (A) or anti–PLC-γ1 (B) antibody. The immunoprecipitated proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr mAb. The membrane was then stripped and reprobed with anti–PLC-γ2 or anti–PLC-γ1 antibody. Similar results were obtained when the other lines were examined. (C) Intracellular Ca++ concentration ([Ca++]i) was measured by means of fluorescent indicator fura-2. Sensitized cells were loaded with fura-2 and then activated with either 10 ng/mL antigen (top) or 1 μM thapsigargin (bottom). The traces show the 340:380 fluorescence ratio. Similar results were obtained when the other lines were examined. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

Aggregation of FcεRI induces the association of 3BP2-SH2 domain with LAT.Cells transfected with vector alone (pMT3) and cells overexpressing 3BP2-SH2 domain were stimulated with antigen for the indicated times and cell lysates were then immunoprecipitated w... Aggregation of FcεRI induces the association of 3BP2-SH2 domain with LAT.Cells transfected with vector alone (pMT3) and cells overexpressing 3BP2-SH2 domain were stimulated with antigen for the indicated times and cell lysates were then immunoprecipitated with anti-HA antibody. The immunoprecipitated proteins were separated by 10% and 14% SDS-PAGE and analyzed by immunoblotting with anti-pTyr, anti-LAT, and anti-HA antibodies. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology

The mutant form of 3BP2-SH2 domain does not suppress FcεRI-mediated degranulation.Control cells and cells expressing 3BP2-SH2 domain or the mutant form of 3BP2-SH2 domain (R486K) were stimulated with 10 ng/mL antigen. The mutant form of 3BP2-SH2 domain does not suppress FcεRI-mediated degranulation.Control cells and cells expressing 3BP2-SH2 domain or the mutant form of 3BP2-SH2 domain (R486K) were stimulated with 10 ng/mL antigen. (A) Cell lysates were immunoprecipitated with anti–PLC-γ2 antibody. The immunoprecipitated proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr mAb and anti–PLC-γ2. Similar results were obtained when the other lines were examined. (B) Analysis of β-hexosaminidase release. The antigen-induced releases are normalized by expression as a percentage of the total β-hexosaminidase activity. The results are the mean values ± SE from 3 independent experiments. Kiyonao Sada et al. Blood 2002;100:2138-2144 ©2002 by American Society of Hematology