Limits...
Cbl-b positively regulates Btk-mediated activation of phospholipase C-gamma2 in B cells.

Yasuda T, Tezuka T, Maeda A, Inazu T, Yamanashi Y, Gu H, Kurosaki T, Yamamoto T - J. Exp. Med. (2002)

Bottom Line: Cbl-b interacted with PLC-gamma2 and helped the association of PLC-gamma2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK).Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+.These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-gamma2 complex formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Genetic studies have revealed that Cbl-b plays a negative role in the antigen receptor-mediated proliferation of lymphocytes. However, we show that Cbl-b-deficient DT40 B cells display reduced phospholipase C (PLC)-gamma2 activation and Ca2+ mobilization upon B cell receptor (BCR) stimulation. In addition, the overexpression of Cbl-b in WEHI-231 mouse B cells resulted in the augmentation of BCR-induced Ca2+ mobilization. Cbl-b interacted with PLC-gamma2 and helped the association of PLC-gamma2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK). Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+. Both NH(2)-terminal tyrosine kinase-binding domain and COOH-terminal half region of Cbl-b were essential for its association with PLC-gamma2 and the regulation of Ca2+ mobilization. These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-gamma2 complex formation.

Show MeSH

Related in: MedlinePlus

Role of Cbl-b on Btk-dependent Ca2+ mobilization, IP3 production, and tyrosine phosphorylation of PLC-γ2. Wild-type cells, Cbl-b–deficient cells, wild-type cells expressing T7-tagged Btk (C38-9 and C38-11), and Cbl-b–deficient cells expressing T7-tagged Btk (C37-4 and C37-10) were analyzed for (A) BCR-induced Ca2+ mobilization, (B) IP3 production, and (C) tyrosine phosphorylation of PLC-γ2, as described in Fig. 2. Similar results were obtained using two other Cbl-b–deficient clones. All experiments were performed more than three times.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2194016&req=5

fig5: Role of Cbl-b on Btk-dependent Ca2+ mobilization, IP3 production, and tyrosine phosphorylation of PLC-γ2. Wild-type cells, Cbl-b–deficient cells, wild-type cells expressing T7-tagged Btk (C38-9 and C38-11), and Cbl-b–deficient cells expressing T7-tagged Btk (C37-4 and C37-10) were analyzed for (A) BCR-induced Ca2+ mobilization, (B) IP3 production, and (C) tyrosine phosphorylation of PLC-γ2, as described in Fig. 2. Similar results were obtained using two other Cbl-b–deficient clones. All experiments were performed more than three times.

Mentions: To further confirm that Cbl-b positively regulates Btk-mediated PLC-γ2 activation, we examined the role of Cbl-b in Btk-dependent intracellular Ca2+ ([Ca2+]i) increase upon BCR stimulation. Consistent with the findings of Takata and Kurosaki (11) and Fluckiger et al. (46), the overexpression of Btk in wild-type DT40 cells resulted in an enhanced and sustained increase in [Ca2+]i after BCR stimulation. In contrast, the overexpression of Btk in Cbl-b–deficient cells induced an initial but not sustained increase in [Ca2+]i. Within 4 min after BCR stimulation, [Ca2+]i of these cells decreased to a level comparable with that in parental Cbl-b–deficient cells (Fig. 5 A). The production of IP3 and the tyrosine phosphorylation of PLC-γ2 were enhanced and sustained in wild-type DT40 cells overexpressing Btk, but those in Cbl-b–deficient cells overexpressing Btk were transient (Fig. 5, B and C). Thus, we concluded that Cbl-b is a positive regulator of Btk-dependent activation of PLC-γ2/Ca2+ signaling.


Cbl-b positively regulates Btk-mediated activation of phospholipase C-gamma2 in B cells.

Yasuda T, Tezuka T, Maeda A, Inazu T, Yamanashi Y, Gu H, Kurosaki T, Yamamoto T - J. Exp. Med. (2002)

Role of Cbl-b on Btk-dependent Ca2+ mobilization, IP3 production, and tyrosine phosphorylation of PLC-γ2. Wild-type cells, Cbl-b–deficient cells, wild-type cells expressing T7-tagged Btk (C38-9 and C38-11), and Cbl-b–deficient cells expressing T7-tagged Btk (C37-4 and C37-10) were analyzed for (A) BCR-induced Ca2+ mobilization, (B) IP3 production, and (C) tyrosine phosphorylation of PLC-γ2, as described in Fig. 2. Similar results were obtained using two other Cbl-b–deficient clones. All experiments were performed more than three times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194016&req=5

fig5: Role of Cbl-b on Btk-dependent Ca2+ mobilization, IP3 production, and tyrosine phosphorylation of PLC-γ2. Wild-type cells, Cbl-b–deficient cells, wild-type cells expressing T7-tagged Btk (C38-9 and C38-11), and Cbl-b–deficient cells expressing T7-tagged Btk (C37-4 and C37-10) were analyzed for (A) BCR-induced Ca2+ mobilization, (B) IP3 production, and (C) tyrosine phosphorylation of PLC-γ2, as described in Fig. 2. Similar results were obtained using two other Cbl-b–deficient clones. All experiments were performed more than three times.
Mentions: To further confirm that Cbl-b positively regulates Btk-mediated PLC-γ2 activation, we examined the role of Cbl-b in Btk-dependent intracellular Ca2+ ([Ca2+]i) increase upon BCR stimulation. Consistent with the findings of Takata and Kurosaki (11) and Fluckiger et al. (46), the overexpression of Btk in wild-type DT40 cells resulted in an enhanced and sustained increase in [Ca2+]i after BCR stimulation. In contrast, the overexpression of Btk in Cbl-b–deficient cells induced an initial but not sustained increase in [Ca2+]i. Within 4 min after BCR stimulation, [Ca2+]i of these cells decreased to a level comparable with that in parental Cbl-b–deficient cells (Fig. 5 A). The production of IP3 and the tyrosine phosphorylation of PLC-γ2 were enhanced and sustained in wild-type DT40 cells overexpressing Btk, but those in Cbl-b–deficient cells overexpressing Btk were transient (Fig. 5, B and C). Thus, we concluded that Cbl-b is a positive regulator of Btk-dependent activation of PLC-γ2/Ca2+ signaling.

Bottom Line: Cbl-b interacted with PLC-gamma2 and helped the association of PLC-gamma2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK).Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+.These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-gamma2 complex formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Genetic studies have revealed that Cbl-b plays a negative role in the antigen receptor-mediated proliferation of lymphocytes. However, we show that Cbl-b-deficient DT40 B cells display reduced phospholipase C (PLC)-gamma2 activation and Ca2+ mobilization upon B cell receptor (BCR) stimulation. In addition, the overexpression of Cbl-b in WEHI-231 mouse B cells resulted in the augmentation of BCR-induced Ca2+ mobilization. Cbl-b interacted with PLC-gamma2 and helped the association of PLC-gamma2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK). Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+. Both NH(2)-terminal tyrosine kinase-binding domain and COOH-terminal half region of Cbl-b were essential for its association with PLC-gamma2 and the regulation of Ca2+ mobilization. These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-gamma2 complex formation.

Show MeSH
Related in: MedlinePlus