Four Tyrosine Residues in Phospholipase C-γ2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling

Dai Watanabe, Shoji Hashimoto, Masamichi Ishiai, Masato Matsushita, Yoshihiro Baba, Tadamitsu Kishimoto, Tomohiro Kurosaki, Satoshi Tsukada

Research output: Contribution to journalArticle

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Abstract

Activation of phospholipase C-γ2 (PLCγ2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCγ2, the exact activation mechanism of PLCγ2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197 and 1217 in rat PLCγ2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCγ2, PLCγ2-deficient DT40 cells were reconstituted with a series of mutant PLCγ2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCγ2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCγ2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCγ2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCγ2.

Original languageEnglish
Pages (from-to)38595-38601
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number42
DOIs
Publication statusPublished - Oct 19 2001
Externally publishedYes

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B-Cell Antigen Receptors
Phosphorylation
Calcium Signaling
Type C Phospholipases
Tyrosine
Calcium
Chemical activation
Substitution reactions
Phosphotransferases
Cells
Agammaglobulinaemia tyrosine kinase
Dissection
Phenylalanine
Rats
Assays
B-Lymphocytes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Four Tyrosine Residues in Phospholipase C-γ2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling. / Watanabe, Dai; Hashimoto, Shoji; Ishiai, Masamichi; Matsushita, Masato; Baba, Yoshihiro; Kishimoto, Tadamitsu; Kurosaki, Tomohiro; Tsukada, Satoshi.

In: Journal of Biological Chemistry, Vol. 276, No. 42, 19.10.2001, p. 38595-38601.

Research output: Contribution to journalArticle

Watanabe, Dai ; Hashimoto, Shoji ; Ishiai, Masamichi ; Matsushita, Masato ; Baba, Yoshihiro ; Kishimoto, Tadamitsu ; Kurosaki, Tomohiro ; Tsukada, Satoshi. / Four Tyrosine Residues in Phospholipase C-γ2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 42. pp. 38595-38601.
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