Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling.
Activation of phospholipase C-gamma2 (PLCgamma2) 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 PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 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 PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 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 PLCgamma2.
Watanabe
D
D
Osaka University Medical School, Department of Molecular Medicine, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan.
Hashimoto
S
S
Ishiai
M
M
Matsushita
M
M
Baba
Y
Y
Kishimoto
T
T
Kurosaki
T
T
Tsukada
S
S
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium