Tyrosine Residues in Phospholipase Cγ2 Essential for the Enzyme Function in B-cell Signaling

Institute of Cancer Research, Londinium, England, United Kingdom
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2002; 276(51):47982-92. DOI: 10.1074/jbc.M107577200
Source: PubMed


Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).

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    • "Activated BTK can interact with adapter protein BLNK/SLP65 through its SH2 domain. The complex can then activate phospholipase C (PLC)-γ2 [32], triggering a cascade of events that culminates in sustained intracellular calcium influx and indirect activation of downstream transcriptional signaling such as MEK/ERK, p38 MAPK, and JNK/SAPK pathways (Figure 2) [27,28,33-35]. Other downstream substrates of BTK include transcription factors BAP-135/TFII-I, NFκB, ARID3A, STAT3 and NFAT, where BTK plays a critical role in direct transcription regulation and the expression of hundreds of genes [29,31,34,36]. "
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    • "It has been proposed that the negatively charged XY-linkers of these PLCs prevent PtdIns(4,5)P2 gaining access to the active site, by a combination of steric exclusion and electrostatic repulsion of negatively charged membranes (Hicks et al., 2008). The PLCγ XY-linker possesses additional regulatory domains: a PH domain, two SH2 domains and an SH3 domain, and this enzyme is activated by tyrosine phosphorylation within the XY-linker region (Rodriguez et al., 2001; Ozdener et al., 2002; Sekiya et al., 2004). A recent study suggested that the general mechanism of PLC auto-inhibition mediated by the XY-linker region also applies to PLCγ isozymes and the crucial determinant for the auto-inhibition is the C-terminal SH2 domain (Gresset et al., 2010). "
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    • "tibody were greatly reduced . The interaction of GIT1 with PLCγ1 also appeared to be dependent upon integrin engagement . The data in Fig . 4A , B suggest that PLCγ1 is placed downstream of Src kinases . In addition , it has been reported that PLCγ isoforms can be phosphorylated directly by several members of the Src family ( Liao et al . , 1993 ; Rodriguez et al . , 2001 ) . In agreement with these previous observations , we found that purified Src , Fyn and Lck can phosphorylate PLCγ1 in vitro . Furthermore , unlike Syk tyrosine kinase , they were capable of phosphorylating the tyrosine residue critical for activation , Y783 ( Fig . 4D ) ; this residue was also phosphorylated in BE cells attached to Ma"
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