Tyrosine Residues in Phospholipase Cγ2 Essential for the Enzyme Function in B-cell Signaling
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m107577200
ISSN1083-351X
AutoresRosie Rodriguez, Miho Matsuda, Olga Perišić, Jerónimo Bravo, Angela Paul, Neil P. Jones, Yvonne Light, Karl Swann, Roger Williams, Matilda Katan,
Tópico(s)T-cell and B-cell Immunology
ResumoPhospholipase Cγ (PLCγ) 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 PLCγ1; however, despite the critical importance of PLCγ2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCγ2 nor the sites in PLCγ2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCγ2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCγ2, we have demonstrated that two tyrosine residues, Tyr753 and Tyr759, were important for the PLCγ2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCγ2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCγ2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCγ2 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 PLCγ2. Further analysis of PLCγ2 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 Tyr753 and Tyr759, shown preferential phosphorylation of Tyr753. Phospholipase Cγ (PLCγ) 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 PLCγ1; however, despite the critical importance of PLCγ2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCγ2 nor the sites in PLCγ2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCγ2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCγ2, we have demonstrated that two tyrosine residues, Tyr753 and Tyr759, were important for the PLCγ2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCγ2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCγ2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCγ2 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 PLCγ2. Further analysis of PLCγ2 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 Tyr753 and Tyr759, shown preferential phosphorylation of Tyr753. phospholipase C B-cell receptor phosphate-buffered saline green fluorescent protein epidermal growth factor glutathioneS-transferase Src homology 2 and 3, respectively pleckstrin homology The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C occurs in response to a large number of extracellular signals (reviewed in Refs. 1Williams R.L. Katan M. Structure. 1996; 4: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2Katan M. Biochim. Biophys. Acta. 1998; 1436: 5-17Crossref PubMed Scopus (191) Google Scholar, 3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar). Four families of mammalian phosphoinositide-specific phospholipase C (PLC),1 PLCβ (β1–β4), PLCγ (γ1, γ2), PLCδ (δ1-δ4), and PLCε, have been described. Each family is characterized by the distinct domain organization and type of signaling pathways that regulate enzyme activity.PLCγ isoforms are mainly regulated through receptors with intrinsic tyrosine kinase activity (e.g. growth factor receptors) or receptors (such as B- and T-cell antigen receptors) that are linked to the activation of nonreceptor tyrosine kinases through a complex signaling network (3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 5Carpenter G. Ji Q.-S. Exp. Cell. Res. 1999; 253: 15-24Crossref PubMed Scopus (207) Google Scholar). The two isoforms of PLCγ have distinct tissue distributions; whereas PLCγ1 is expressed ubiquitously, the pattern of expression of PLCγ2 is characterized by high levels in cells of hematopoietic origin. Transgenic studies suggested that the biological function of these isoforms is reflected in their cellular distribution. Thus, a deficiency in PLCγ1 is embryonic lethal in mice (6Ji Q.-S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2999-3003Crossref PubMed Scopus (217) Google Scholar), whereas homozygous disruption of PLCγ2 allowed normal development but resulted in functional and signaling disorders in a subset of cell types including B-cells, platelets, and mast cells (7Wang D. Feng J. Wen R. Marine J.-C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar).The importance of PLCγ2 in signaling in B-cells has not only been documented in experiments using transgenic animals deficient in PLCγ2 (7Wang D. Feng J. Wen R. Marine J.-C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar) but also by studies of a chicken B-cell lymphoma cell line (DT40) (reviewed in Refs. 8Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (137) Google Scholar and 9Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar) with the property of extraordinarily high frequency of homologous recombination when DNA constructs are introduced into the cells. Generation of a number of targeted mutations in specific genes in DT40 cells provided valuable information about signaling components linking the activation of the B-cell receptor (BCR) to an increase in intracellular calcium concentrations. Using this system, it has been found that protein-tyrosine kinases from Src, Tec (e.g. Btk), and Syk/ZAP70 families are essential signaling components of the BCR pathway (10Takata T. Sabe H. Hata A. Inazu T. Homma Y. Nukuda T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar, 11Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar). In addition, an adapter BLNK (B-cell linker protein), inositol 1,4,5-trisphosphate receptors, and PLCγ2 itself were required for calcium responses triggered by the BCR (12Miyaka T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Lino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (338) Google Scholar, 13Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 14Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 15Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907Crossref PubMed Scopus (183) Google Scholar). Although each of these components may have more than one function and could be integrated in different pathways in B-cells, the current model (8Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (137) Google Scholar, 9Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar) suggests that the Src family kinase Lyn interacts with BCR and becomes activated upon the receptor aggregation. Activation of Syk kinase results in phosphorylation of BLNK that could provide binding sites for PLCγ2 and a number of other proteins. Syk, together with Btk, has also been implicated in phosphorylation of PLCγ2, which, through inositol 1,4,5-trisphosphate production, results in calcium mobilization. A similar pathway seems to be involved in calcium responses to oxidative stress after exposure of B-cells to hydrogen peroxide (16Tomlinson M.G. Woods D.B. McMahon M. Wahl M.I. Witte O.N. Kurosaki T. Bolen J.B. Johnston J.A. BMC Immunol. 2001; 2: 4-15Crossref PubMed Scopus (52) Google Scholar, 17Qin S. Stadtman E.R. Chook P.-B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7118-7123Crossref PubMed Scopus (50) Google Scholar, 18Qin S. Inazu T. Takata M. Kurosaki T. Homma Y. Yamamura H. Eur. J. Biochem. 1996; 236: 443-449Crossref PubMed Scopus (62) Google Scholar). It has been reported that the BCR complex and tyrosine kinases Syk, Lyn, and Btk, are components required for calcium responses. In addition, phosphorylation of several protein components, including BLNK and PLCγ2, has been described.Despite extensive genetic dissection of B-cell signal transduction, it has not been shown which tyrosine kinase(s) directly phosphorylate PLCγ2 or which sites in PLCγ2 become phosphorylated in response to BCR activation or oxidative stress. Similarly, the relative importance of specific tyrosine residues for signaling function of PLCγ2 has not been clarified. More generally, the molecular mechanism of activation of PLCγ and the role of phosphorylation in this process is not well understood. Previous studies of PLCγ phosphorylation have been mainly restricted to PLCγ1 in signaling through growth factor receptors (19Wahl M.I. Nishibe S. Kim J.-W. Kim H. Rhee S.-G. Carpenter G. J. Biol. Chem. 1990; 265: 3944-3948Abstract Full Text PDF PubMed Google Scholar, 20Kim J.-W. Sim S.-S. Kim U.-H. Nishibe S. Wahl M.I. Carpenter G. Rhee S.-G. J. Biol. Chem. 1990; 265: 3940-3943Abstract Full Text PDF PubMed Google Scholar, 21Kim H.-K. Kim J.-W. Zilberstein A. Margolis B. Kim J.-G. Schlessinger J. Rhee S.-G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar). These studies revealed multiple phosphorylation sites, not all of which appear to be functionally critical at least in the context of a specific signaling pathway.To analyze phosphorylation and importance of specific tyrosine residues in PLCγ2, we used DT40 cell lines stimulated by BCR cross-linking or by oxidative stress. In experiments where the human wild-type and mutated PLCγ2 constructs were tested for reconstitution of calcium responses in DT40 PLCγ2− cells, two tyrosine residues have been identified as important for PLCγ2 phosphorylation and activation in B-cells. Further experiments, using purified protein components, implicated tyrosine kinase Btk and possibly some kinases from the Src family in direct phosphorylation of PLCγ2 and suggested that the requirement for Syk kinase in PLCγ2 activation mainly involves phosphorylation of the adapter protein BLNK.DISCUSSIONPhosphorylation of both PLCγ1 and PLCγ2 has been well documented for the majority of cellular systems where the activation of PLCγ isoforms takes place (3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 5Carpenter G. Ji Q.-S. Exp. Cell. Res. 1999; 253: 15-24Crossref PubMed Scopus (207) Google Scholar). However, phosphorylation sites and the importance of specific tyrosine residues that become phosphorylated have been analyzed only for PLCγ1 in cells stimulated through growth factor receptors. Within a complex profile of PLCγ1-phosphorylated peptides, obtained after EGF stimulation, two main tyrosine-phosphorylated residues have been mapped as Tyr771 and Tyr1254, and one minor site has been found to correspond to Tyr783 (19Wahl M.I. Nishibe S. Kim J.-W. Kim H. Rhee S.-G. Carpenter G. J. Biol. Chem. 1990; 265: 3944-3948Abstract Full Text PDF PubMed Google Scholar). More recently, the use of phosphospecific antibodies to Tyr(P)783confirmed phosphorylation of this site in stimulated cells (23Matsuda M. Paterson H.F. Rodriguez R. Fensome A.C. Ellis M.V. Swann K. Katan M. J. Cell Biol. 2001; 153: 599-612Crossref PubMed Scopus (70) Google Scholar, 31Yu H. Fukami K. Itoh T. Takenawa T. Exp. Cell Res. 1998; 243: 113-122Crossref PubMed Scopus (36) Google Scholar). Similar patterns of phosphorylation have been seen after stimulation of fibroblasts with platelet-derived growth factor and in several other systems (3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 5Carpenter G. Ji Q.-S. Exp. Cell. Res. 1999; 253: 15-24Crossref PubMed Scopus (207) Google Scholar, 21Kim H.-K. Kim J.-W. Zilberstein A. Margolis B. Kim J.-G. Schlessinger J. Rhee S.-G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar). These (Tyr771, Tyr783, and Tyr1254) and some additional sites have been identified after in vitro phosphorylation of purified PLCγ1 by EGF receptor kinase (20Kim J.-W. Sim S.-S. Kim U.-H. Nishibe S. Wahl M.I. Carpenter G. Rhee S.-G. J. Biol. Chem. 1990; 265: 3940-3943Abstract Full Text PDF PubMed Google Scholar). Interestingly, mutational studies have revealed that only Tyr783 was critical, while other residues had less impact on PLCγ1 function when tested in platelet-derived growth factor signaling (21Kim H.-K. Kim J.-W. Zilberstein A. Margolis B. Kim J.-G. Schlessinger J. Rhee S.-G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar), demonstrating that not all phosphorylation sites may be functionally important. Taking into account the complexity of the phosphorylation pattern and possible functional redundancy, the studies of PLCγ2 described here focused on a mutagenesis approach based on information obtained for the PLCγ1 isoform. Comparison of PLCγ1 and PLCγ2 sequences has revealed that of three tyrosine residues in the loop region between the C-SH2 and the SH3 domain, only two are conserved (Tyr753 and Tyr759 in PLCγ2, the latter corresponding to phosphorylation site Tyr783 in PLCγ1), while there is no conservation of sequences in the C-terminal region, including residue Tyr1254 in PLCγ1 (Fig. 1). Our mutagenesis analysis of tyrosines in PLCγ2 within the C-SH2/SH3 loop region demonstrated that both Tyr753 and Tyr759 are required to restore calcium signaling in DT40 cells deficient in PLCγ (Fig.2 B). Thus, the conserved residue corresponding to Tyr759 in PLCγ2 and Tyr783 in PLCγ1 is important for the function of both isoforms. The other conserved residue (753 in PLCγ2/775 in PLCγ1) has not been mutated in PLCγ1 and has not been identified as one of the major phosphotyrosine sites in response to EGF stimulation. Further studies are required to establish whether or not this site is functionally important in any of a number of different signaling pathways leading to phosphorylation of PLCγ1.Comparison between properties of a double mutant within the C-SH2/SH3 loop region in PLCγ1 (Y771F/Y783F, where Tyr771 is unique for PLCγ1) observed in previous studies (21Kim H.-K. Kim J.-W. Zilberstein A. Margolis B. Kim J.-G. Schlessinger J. Rhee S.-G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (444) Google Scholar) with the PLCγ2 Y753F/Y759F double mutant in the same region described here (Figs. 2and 3) reveals several similarities. For example, both proteins (PLCγ1 Y771F/Y783F and PLCγ2 Y753F/Y759F) retained full in vitro catalytic activity. Also, when the function of these proteins has been analyzed in the context of platelet-derived growth factor signaling for PLCγ1 and in B-cell signaling for PLCγ2, these mutations not only inhibited generation of inositol 1,4,5-trisphosphate and calcium mobilization but also abolished phosphorylation of the PLCγ protein. In the case of PLCγ1, it has been shown that the Y771F/Y783F mutation resulted in a loss of not only phosphorylation in the C-SH2/SH3 loop region but also phosphorylation of Tyr1254 at the C terminus. Since the phosphorylation profile of PLCγ2 in stimulated B-cells also appears to be complex, it is possible that the double mutation Y753F/Y759F in PLCγ2 could have a similar effect on other potential phosphorylation sites. It has been speculated that the main impact of tyrosine phosphorylation on the function of PLCγ isoforms could be to, through conformational changes, increase the access of the enzyme to phosphatidylinositol 4,5-bisphosphate present in the plasma membrane and in this way result in a higher rate of substrate hydrolysis (3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 5Carpenter G. Ji Q.-S. Exp. Cell. Res. 1999; 253: 15-24Crossref PubMed Scopus (207) Google Scholar). However, these conformational changes in the C-SH2/SH3 loop region may also be required to expose additional phosphorylation sites.Genetic analysis of DT40 cells has suggested the importance of several nonreceptor tyrosine kinases for PLCγ2-mediated calcium signaling (8Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (137) Google Scholar,9Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar). However, it has not been established which of these enzymes could phosphorylate PLCγ2 directly. This was examined here (Figs. 5, 6, and8) using purified preparations of PLCγ2 constructs and various tyrosine kinases with an emphasis on Btk and Syk, both essential for PLCγ2 signaling.The role of Btk in B-cell signaling has been extensively studied. B-cells deficient in Btk and stable cell lines where the wild-type or different Btk mutants have been transfected into these deficient cells have been assessed for calcium signaling and PLCγ2 phosphorylation (11Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar, 16Tomlinson M.G. Woods D.B. McMahon M. Wahl M.I. Witte O.N. Kurosaki T. Bolen J.B. Johnston J.A. BMC Immunol. 2001; 2: 4-15Crossref PubMed Scopus (52) Google Scholar, 27Hashimoto S. Iwamatsu A. Ishiai M. Okawa K. Yamadori T. Matsushita M. Baba Y. Kishimoto T. Kurosaki T. Tsukada S. Immunology. 1999; 94: 2357-2364Google Scholar, 35Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (357) Google Scholar, 36Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). While the calcium responses in Btk−cells were abolished, in most reports only reduction in PLCγ2 phosphorylation has been observed, suggesting the involvement of additional tyrosine kinases in phosphorylation of this PLC. Thein vitro phosphorylation study using purified components described here demonstrated that Btk could directly phosphorylate PLCγ2, including an important residue, Tyr753 (Figs. 5,6, and 8). Our studies have also shown that additional sites are phosphorylated by Btk in vitro. However, the identity of all sites remains to be established, together with their physiological relevance. Furthermore, studies of Btk have also suggested that the role of this protein in calcium signaling could be more complex than a requirement for PLCγ2 tyrosine phosphorylation. Mutations in the Btk PH and SH2 domains as well as a mutation affecting the catalytic activity resulted in a loss of signaling function, as measured by restoration of calcium responses in DT40 Btk− cells (11Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar). While the Btk PH domain could be involved in critical membrane binding interactions, it is possible that the Btk SH2 and/or SH3 domains provide important sites for a formation of a signaling complex. It has been reported recently that a tyrosine kinase-inactivating mutation (in the active site and different from the nonactive site mutation affecting the catalytic activity in a preceding study) did not abolish the function of Btk in calcium signaling (16Tomlinson M.G. Woods D.B. McMahon M. Wahl M.I. Witte O.N. Kurosaki T. Bolen J.B. Johnston J.A. BMC Immunol. 2001; 2: 4-15Crossref PubMed Scopus (52) Google Scholar). This further emphasizes the potential scaffolding role of Btk and the possibility that the important tyrosine residues phosphorylated by Btk, and possibly other critical residues in PLCγ2, could also be phosphorylated by another kinase. Surprisingly, the studies using a panel of different tyrosine kinases (Fig. 8) have identified Lck, an Src family kinase where a link to B-cell signaling was not confirmed in all studies (10Takata T. Sabe H. Hata A. Inazu T. Homma Y. Nukuda T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar, 32Corey S.J. Anderson S.M. Blood. 1999; 93: 1-14Crossref PubMed Google Scholar, 33Sattethwaite A. Witte O.N. Annu. Rev. Immunol. 1996; 14: 131-154Crossref PubMed Scopus (33) Google Scholar, 34Campbell M.A. Sefton B.M. Mol. Cell. Biol. 1992; 12: 2315-2321Crossref PubMed Scopus (157) Google Scholar), as a tyrosine kinase that can efficiently phosphorylate a peptide incorporating Tyr753 and Tyr759 residues of PLCγ2.Protein-tyrosine kinase Syk has also been implicated in B-cell signaling and shown to be required for both PLCγ2 phosphorylation and calcium responses (10Takata T. Sabe H. Hata A. Inazu T. Homma Y. Nukuda T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar). It has been shown that the essential adapter protein BLNK, forming complexes with a number of signaling components including PLCγ2, needs to be phosphorylated by Syk in order to bind other proteins (13Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 14Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Therefore, the role of Syk in calcium responses could be to phosphorylate both PLCγ2 and BLNK or to phosphorylate only BLNK, thereby enabling formation of signaling complexes. The data presented here (Fig. 5) show that Syk does not efficiently phosphorylate PLCγ2, but it does phosphorylate BLNK. Furthermore, phosphorylation of BLNK by Syk, in the absence of additional components, could be sufficient to provide docking sites for direct binding of PLCγ2 (Fig. 7).In summary, we identified tyrosine residues Tyr753 and Tyr759 as important for activation and tyrosine phosphorylation of PLCγ2 in B-cells. Based on this observation, the roles of various tyrosine kinases that genetic analysis has implicated in regulation of PLCγ2 were further assessed. Direct phosphorylation of PLCγ2 by Btk is observed; however, the role of Syk may not be to phosphorylate PLCγ2 directly but to provide docking phosphotyrosine sites on the adapter protein BLNK, essential in B-cell signaling. The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C occurs in response to a large number of extracellular signals (reviewed in Refs. 1Williams R.L. Katan M. Structure. 1996; 4: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2Katan M. Biochim. Biophys. Acta. 1998; 1436: 5-17Crossref PubMed Scopus (191) Google Scholar, 3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar). Four families of mammalian phosphoinositide-specific phospholipase C (PLC),1 PLCβ (β1–β4), PLCγ (γ1, γ2), PLCδ (δ1-δ4), and PLCε, have been described. Each family is characterized by the distinct domain organization and type of signaling pathways that regulate enzyme activity. PLCγ isoforms are mainly regulated through receptors with intrinsic tyrosine kinase activity (e.g. growth factor receptors) or receptors (such as B- and T-cell antigen receptors) that are linked to the activation of nonreceptor tyrosine kinases through a complex signaling network (3Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (818) Google Scholar, 4Rhee S.-G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 5Carpenter G. Ji Q.-S. Exp. Cell. Res. 1999; 253: 15-24Crossref PubMed Scopus (207) Google Scholar). The two isoforms of PLCγ have distinct tissue distributions; whereas PLCγ1 is expressed ubiquitously, the pattern of expression of PLCγ2 is characterized by high levels in cells of hematopoietic origin. Transgenic studies suggested that the biological function of these isoforms is reflected in their cellular distribution. Thus, a deficiency in PLCγ1 is embryonic lethal in mice (6Ji Q.-S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2999-3003Crossref PubMed Scopus (217) Google Scholar), whereas homozygous disruption of PLCγ2 allowed normal development but resulted in functional and signaling disorders in a subset of cell types including B-cells, platelets, and mast cells (7Wang D. Feng J. Wen R. Marine J.-C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The importance of PLCγ2 in signaling in B-cells has not only been documented in experiments using transgenic animals deficient in PLCγ2 (7Wang D. Feng J. Wen R. Marine J.-C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar) but also by studies of a chicken B-cell lymphoma cell line (DT40) (reviewed in Refs. 8Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (137) Google Scholar and 9Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar) with the property of extraordinarily high frequency of homologous recombination when DNA constructs are introduced into the cells. Generation of a number of targeted mutations in specific genes in DT40 cells provided valuable information about signaling components linking the activation of the B-cell receptor (BCR) to an increase in intracellular calcium concentrations. Using this system, it has been found that protein-tyrosine kinases from Src, Tec (e.g. Btk), and Syk/ZAP70 families are essential signaling components of the BCR pathway (10Takata T. Sabe H. Hata A. Inazu T. Homma Y. Nukuda T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar, 11Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar). In addition, an adapter BLNK (B-cell linker protein), inositol 1,4,5-trisphosphate receptors, and PLCγ2 itself were required for calcium responses triggered by the BCR (12Miyaka T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Lino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (338) Google Scholar, 13Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 14Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 15Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907Crossref PubMed Scopus (183) Google Scholar). Although each of these components may have more than one function and could be integrated in different pathways in B-cells, the current model (8Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (137) Google Scholar, 9Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar) suggests that the Src family kinase Lyn interacts with BCR and becomes activated upon the receptor aggregation. Activation of Syk kinase results in phosphorylation of BLNK that could provide binding sites for PLCγ2 and a number of other proteins. Syk, together with Btk, has also been implicated in phosphorylation of PLCγ2, which, through inositol 1,4,5-trisphosphate production, results in calcium mobilization. A similar pathway seems to be involved in calcium responses to oxidative stress after exposure of B-cells to hydrogen peroxide (16Tomlinson M.G. Woods D.B. McMahon M. Wahl M.I. Witte O.N. Kurosaki T. Bolen J.B. Johnston J.A. BMC Immunol. 2001; 2: 4-15Crossref PubMed Scopus (52) Google Scholar, 17Qin S. Stadtman E.R. Chook P.-B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7118-7123Crossref PubMed Scopus (50) Google Scholar, 18Qin S. Inazu T. Takata M. Kurosaki T. Homma Y. Yamamura H. Eur. J. Biochem. 1996; 236: 443-449Crossref PubMed Scopus (62) Google Scholar). It has been reported that the BCR complex and t
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