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Cbl suppresses B cell receptor-mediated phospholipase C (PLC)-gamma2 activation by regulating B cell linker protein-PLC-gamma2 binding.

Yasuda T, Maeda A, Kurosaki M, Tezuka T, Hironaka K, Yamamoto T, Kurosaki T - J. Exp. Med. (2000)

Bottom Line: BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains.As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited.Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-0071, Japan.

ABSTRACT
Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.

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BCR-induced tyrosine phosphorylation in wild-type and Cbl-deficient DT40 cells. C8-10 was used for a Cbl-deficient clone. (A) Tyrosine phosphorylation in whole cell lysates from wild-type (wt) and Cbl-deficient (cbl−) DT40 cells. At the indicated time points after addition of M4 (4 μg/ml), whole cell lysates prepared from 2 × 106 cells were loaded onto an 8% SDS-PAGE gel. The blotted membrane was incubated with antiphosphotyrosine Ab. (B) Tyrosine phosphorylation and in vitro kinase activity of Syk. Wild-type and Cbl-deficient DT40 cells were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-Syk Ab. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top, anti-pY) or anti-Syk Ab (bottom). The remaining third was used for the in vitro kinase assay (middle). (C) Tyrosine phosphorylation and in vitro kinase activity of Btk. Wild-type and Cbl-deficient DT40 cells expressing T7-tagged Btk were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-T7 mAb. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top) or anti-T7 mAb (bottom). The remaining third was used for the in vitro kinase assay (middle).
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Figure 5: BCR-induced tyrosine phosphorylation in wild-type and Cbl-deficient DT40 cells. C8-10 was used for a Cbl-deficient clone. (A) Tyrosine phosphorylation in whole cell lysates from wild-type (wt) and Cbl-deficient (cbl−) DT40 cells. At the indicated time points after addition of M4 (4 μg/ml), whole cell lysates prepared from 2 × 106 cells were loaded onto an 8% SDS-PAGE gel. The blotted membrane was incubated with antiphosphotyrosine Ab. (B) Tyrosine phosphorylation and in vitro kinase activity of Syk. Wild-type and Cbl-deficient DT40 cells were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-Syk Ab. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top, anti-pY) or anti-Syk Ab (bottom). The remaining third was used for the in vitro kinase assay (middle). (C) Tyrosine phosphorylation and in vitro kinase activity of Btk. Wild-type and Cbl-deficient DT40 cells expressing T7-tagged Btk were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-T7 mAb. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top) or anti-T7 mAb (bottom). The remaining third was used for the in vitro kinase assay (middle).

Mentions: As Syk and Btk are the PTKs responsible for BCR-induced PLC-γ2 tyrosine phosphorylation 4350, one possible explanation for the enhanced PLC-γ2 phosphorylation in Cbl-deficient cells is that Syk and/or Btk are hyperactivated in Cbl-deficient DT40 cells, leading to hyperphosphorylation of PLC-γ2. To evaluate this possibility, the BCR-induced whole tyrosine phosphorylation pattern was compared in wild-type and mutant cells. As shown in Fig. 5 A, there were no significant changes between wild-type and Cbl-deficient cells, except that the band corresponding to Cbl itself was absent in the mutant cells. Consistent with the whole tyrosine phosphorylation data, both Syk and Btk were inducibly tyrosine phosphorylated to an almost similar extent in wild-type and Cbl-deficient DT40 cells. In addition, the BCR-induced in vitro kinase activities of Syk and Btk were almost the same between wild-type and mutant DT40 cells (Fig. 5B and Fig. C).


Cbl suppresses B cell receptor-mediated phospholipase C (PLC)-gamma2 activation by regulating B cell linker protein-PLC-gamma2 binding.

Yasuda T, Maeda A, Kurosaki M, Tezuka T, Hironaka K, Yamamoto T, Kurosaki T - J. Exp. Med. (2000)

BCR-induced tyrosine phosphorylation in wild-type and Cbl-deficient DT40 cells. C8-10 was used for a Cbl-deficient clone. (A) Tyrosine phosphorylation in whole cell lysates from wild-type (wt) and Cbl-deficient (cbl−) DT40 cells. At the indicated time points after addition of M4 (4 μg/ml), whole cell lysates prepared from 2 × 106 cells were loaded onto an 8% SDS-PAGE gel. The blotted membrane was incubated with antiphosphotyrosine Ab. (B) Tyrosine phosphorylation and in vitro kinase activity of Syk. Wild-type and Cbl-deficient DT40 cells were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-Syk Ab. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top, anti-pY) or anti-Syk Ab (bottom). The remaining third was used for the in vitro kinase assay (middle). (C) Tyrosine phosphorylation and in vitro kinase activity of Btk. Wild-type and Cbl-deficient DT40 cells expressing T7-tagged Btk were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-T7 mAb. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top) or anti-T7 mAb (bottom). The remaining third was used for the in vitro kinase assay (middle).
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Figure 5: BCR-induced tyrosine phosphorylation in wild-type and Cbl-deficient DT40 cells. C8-10 was used for a Cbl-deficient clone. (A) Tyrosine phosphorylation in whole cell lysates from wild-type (wt) and Cbl-deficient (cbl−) DT40 cells. At the indicated time points after addition of M4 (4 μg/ml), whole cell lysates prepared from 2 × 106 cells were loaded onto an 8% SDS-PAGE gel. The blotted membrane was incubated with antiphosphotyrosine Ab. (B) Tyrosine phosphorylation and in vitro kinase activity of Syk. Wild-type and Cbl-deficient DT40 cells were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-Syk Ab. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top, anti-pY) or anti-Syk Ab (bottom). The remaining third was used for the in vitro kinase assay (middle). (C) Tyrosine phosphorylation and in vitro kinase activity of Btk. Wild-type and Cbl-deficient DT40 cells expressing T7-tagged Btk were stimulated with M4 for the indicated amount of time, and were immunoprecipitated with anti-T7 mAb. Immunoprecipitates were divided, and one third was used for Western blotting with antiphosphotyrosine mAb (top) or anti-T7 mAb (bottom). The remaining third was used for the in vitro kinase assay (middle).
Mentions: As Syk and Btk are the PTKs responsible for BCR-induced PLC-γ2 tyrosine phosphorylation 4350, one possible explanation for the enhanced PLC-γ2 phosphorylation in Cbl-deficient cells is that Syk and/or Btk are hyperactivated in Cbl-deficient DT40 cells, leading to hyperphosphorylation of PLC-γ2. To evaluate this possibility, the BCR-induced whole tyrosine phosphorylation pattern was compared in wild-type and mutant cells. As shown in Fig. 5 A, there were no significant changes between wild-type and Cbl-deficient cells, except that the band corresponding to Cbl itself was absent in the mutant cells. Consistent with the whole tyrosine phosphorylation data, both Syk and Btk were inducibly tyrosine phosphorylated to an almost similar extent in wild-type and Cbl-deficient DT40 cells. In addition, the BCR-induced in vitro kinase activities of Syk and Btk were almost the same between wild-type and mutant DT40 cells (Fig. 5B and Fig. C).

Bottom Line: BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains.As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited.Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-0071, Japan.

ABSTRACT
Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.

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