Article

Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling

Osaka University, Suika, Ōsaka, Japan
Journal of Biological Chemistry (Impact Factor: 4.6). 11/2001; 276(42):38595-601. DOI: 10.1074/jbc.M103675200
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
95 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cherubism (OMIM#118400) is a genetic disorder with excessive jawbone resorption caused by mutations in the signaling adaptor protein SH3BP2. Studies on the mouse model for cherubism carrying a P416R knock-in mutation have revealed that mutant SH3BP2 enhances TNF-α production and RANKL-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of TRAP-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2(KI/KI) ). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2(KI/+) ) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves SYK and PLCγ2 phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders. © 2014 American Society for Bone and Mineral Research.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2014; 29(12). DOI:10.1002/jbmr.2295 · 6.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bruton's tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function, however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC sealing zone formation. Because CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not impact OC sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.Leukemia accepted article preview online, 12 February 2014; doi:10.1038/leu.2014.69.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 02/2014; 28(9). DOI:10.1038/leu.2014.69 · 9.38 Impact Factor
  • Source