Four Tyrosine Residues in Phospholipase C- 2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling
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.
- SourceAvailable from: Michael Alan Levine
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- "Targeted deletion of Plcγ2 but not Plcγ1 in mice results in an in vivo osteopetrotic phenotype , suggesting that PLCγ2 is the critical isoform for sRANKL-induced osteoclastogenesis. PLCγ2 has four tyrosine phosphorylation sites (Tyr753, Tyr759, Tyr1197, Tyr1217) [73-75]. In separate experiments the mutation of all four of these tyrosines had a dramatic effect on PLCγ2 activation as measured by intracellular calcium mobilization in B cells . "
ABSTRACT: Cherubism is a rare bone dysplasia that is characterized by symmetrical bone resorption limited to the jaws. Bone lesions are filled with soft fibrous giant cell-rich tissue that can expand and cause severe facial deformity. The disorder typically begins in children at ages of 2-5 years and the bone resorption and facial swelling continues until puberty; in most cases the lesions regress spontaneously thereafter. Most patients with cherubism have germline mutations in the gene encoding SH3BP2, an adapter protein involved in adaptive and innate immune response signaling. A mouse model carrying a Pro416Arg mutation in SH3BP2 develops osteopenia and expansile lytic lesions in bone and some soft tissue organs. In this review we discuss the genetics of cherubism, the biological functions of SH3BP2 and the analysis of the mouse model. The data suggest that the underlying cause for cherubism is a systemic autoinflammatory response to physiologic challenges despite the localized appearance of bone resorption and fibrous expansion to the jaws in humans.Orphanet Journal of Rare Diseases 05/2012; 7 Suppl 1(Suppl 1):S5. DOI:10.1186/1750-1172-7-S1-S5 · 3.96 Impact Factor
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- "Wild-type human SOCS-1 and mutant SOCS-1 cDNA were amplified by polymerase chain reaction (PCR) from genomic DNA, inserted into the pApuro2 vector, and confirmed by DNA sequencing. DNA transfection into 293T cells with Lipofectamine (Invitrogen Corp., Carlsbad, CA, USA) has been described elsewhere (Watanabe et al, 2001). cDNAs of mouse wild-type SOCS-1 or R105Q mutant, which was introduced into the Arg-105 point mutation of SOCS-1 to yield Gln, were co-transfected with pSVIINeo as described previously (Narazaki et al, 1998) and selected in the presence of 500 lg/ml of G418 (Nakarai Tesque, "
ABSTRACT: The aim of this study was to investigate whether the suppressor of cytokine signalling (SOCS)-1 can act as a tumour suppressor when functioning as a negative regulator of the Janus family tyrosine kinases (JAKs), which have been reported to play important roles in leukaemogenesis. For this purpose, we carried out molecular analysis of the SOCS-1 gene in human acute myeloid leukaemia (AML) and human haematopoietic cell lines. Sequencing alterations in the coding region were found in two of 90 primary AML samples and one of 17 cell lines. Hypermethylation of the SOCS-1 gene was also observed in 72% of primary cases and 52% of cell lines and aberrant methylation strongly correlated with reduced expression. Transfection of SOCS-1 into Jurkat cells harbouring the mutation and methylation suppressed cell growth at a low serum concentration. These findings indicate that SOCS-1 is frequently silenced in haematopoietic malignancies, mainly as a result of hypermethylation, and suggest that SOCS-1 may be able to function as a tumour suppressor.British Journal of Haematology 10/2004; 126(5):726-35. DOI:10.1111/j.1365-2141.2004.05107.x · 4.96 Impact Factor
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- "The reaction products were resolved on SDS-PAGE, dried, and subjected to quantification of radioactivity by using a Fuji FLA2000 bioimaging analyzer (Fuji Photo Film). Based on these analyses, the stimulation folds are shown in Figs. 3 E, 4 B, 5 C, 5 D, 9 B, 9 C, and 10 C. Phospholipase hydrolysis activity of PLC-γ2 was measured by quantitating inositol 1,4,5-trisphosphate (IP3) production as described previously (28). "
ABSTRACT: To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.Journal of Experimental Medicine 02/2002; 195(2):189-200. · 13.91 Impact Factor