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

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.

PLC␥ 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 (3)(4)(5). The two isoforms of PLC␥ have distinct tissue distributions; whereas PLC␥1 is expressed ubiquitously, the pattern of expression of PLC␥2 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 PLC␥1 is embryonic lethal in mice (6), whereas homozygous disruption of PLC␥2 allowed normal development but resulted in functional and signaling disorders in a subset of cell types including B-cells, platelets, and mast cells (7).
The importance of PLC␥2 in signaling in B-cells has not only been documented in experiments using transgenic animals deficient in PLC␥2 (7) but also by studies of a chicken B-cell lymphoma cell line (DT40) (reviewed in Refs. 8 and 9) 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 (10,11). In addition, an adapter BLNK (B-cell linker protein), inositol 1,4,5-trisphosphate receptors, and PLC␥2 itself were required for calcium responses triggered by the BCR (12)(13)(14)(15). Although each of these components may have more than one function and could be integrated in different pathways in B-cells, the current model (8,9) 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 PLC␥2 and a number of other proteins. Syk, together with Btk, has also been implicated in phosphorylation of PLC␥2, 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 (16 -18). 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 PLC␥2, has been described.
Despite extensive genetic dissection of B-cell signal transduction, it has not been shown which tyrosine kinase(s) directly phosphorylate PLC␥2 or which sites in PLC␥2 become phosphorylated in response to BCR activation or oxidative stress. Similarly, the relative importance of specific tyrosine residues for signaling function of PLC␥2 has not been clarified. More generally, the molecular mechanism of activation of PLC␥ and the role of phosphorylation in this process is not well understood. Previous studies of PLC␥ phosphorylation have been mainly restricted to PLC␥1 in signaling through growth factor receptors (19 -21). 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 PLC␥2, we used DT40 cell lines stimulated by BCR cross-linking or by oxidative stress. In experiments where the human wild-type and mutated PLC␥2 constructs were tested for reconstitution of calcium responses in DT40 PLC␥2 Ϫ cells, two tyrosine residues have been identified as important for PLC␥2 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 PLC␥2 and suggested that the requirement for Syk kinase in PLC␥2 activation mainly involves phosphorylation of the adapter protein BLNK.

EXPERIMENTAL PROCEDURES
Generation of DT40 Cell Lines Expressing Human PLC␥2-For the expression of human PLC␥2 in DT40 cells, the full-length cDNA (22) was subcloned into the pApuro vector as described previously (23). The original sequencing data contained a sequencing error close to the C terminus; the corrected frame of the amino acid sequence shows good alignment with the rat PLC␥2 sequence in this region (Fig. 1B).
The mutations of tyrosine residues, Y753F, Y759F, and Y753F/ Y759F, were generated using a two-stage PCR-based overlap extension method and introduced into pApuro/PLC␥2 construct. The plasmids were used for stable transfection of DT40/PLC␥2 Ϫ cells (15) as described previously for the wild type PLC␥2 (23). Briefly, the linearized constructs of PLC␥2 were introduced into the cells by electroporation (950 V, 25 microfarads, ϱ ⍀), and puromycin (0.35 g/ml) was added to the medium. 10 -12 days after the selection, colonies were picked, and the puromycin selection was repeated for 5-8 days. Subsequently, the puromycin-resistant colonies were grown in normal medium (RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.) and 1% (v/v) chicken serum (Life Technologies)), and the expression of PLC␥2 was confirmed by Western blotting.
Analysis of PLC␥2 Phosphorylation and Calcium Responses in DT40 Cell Lines after Stimulation-DT40 cell lines (DT40 PLC␥2-deficient cells and these cells stably transfected with either the wild type or Y753F, Y759F, and Y753F/Y759F mutants of human PLC␥2) were stimulated by the addition of either anti-chicken IgM or H 2 O 2 . Typically, a 6 ϫ 10 6 -cell aliquot of these cell lines was stimulated with 10 g/ml goat anti-chicken IgM (M4) (Universal Biologicals) or 2 mM H 2 O 2 at 37°C for up to 5 min in 200 l of PBS. The cell pellet was resuspended in 200 l of lysis buffer (1% Triton, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na 3 VO 4 , 0.5% Nonidet P-40, protease inhibitor mixture (Roche), and phosphatase inhibitor mixture (Sigma)), and the cells were lysed by incubation for 30 min at 4°C. The supernatant was removed and added to anti-PLC␥2 antibody-protein G complexes (prepared by mixing 2 g of anti-PLC␥2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with 9 l of protein G (Roche)) and incubated at 4°C for 1.5 h. After the incubation, immunocomplexes were washed, resuspended in SDS gel loading buffer, and subjected to SDS-PAGE (7.5% polyacrylamide gels) and subsequent Western blotting. For detection of PLC␥2 in immunocomplexes or in cell extracts and membrane fractions (prepared as described in Ref. 14), the anti-PLC␥2 antibody (1:4000; Santa Cruz Biotechnology) was used, while the detection of PLC␥2 phosphorylation present in immunocomplexes was performed using anti-phosphotyrosine antibody (1:1000; Transduction Laboratories). After incubation with the secondary antibody (anti-rabbit or anti-mouse Ig horseradish peroxidase-linked whole antibody from Amersham Biosciences, Inc., diluted 1:3000), the visualization was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Total protein transferred to polyvinylidene difluoride membrane was stained using Amido Black solution. For detection of BLNK in PLC␥2 immunoprecipitates, mouse anti-chicken antibody (described in Ref. 14) was used for Western blotting; the same protein was detected using anti-phosphotyrosine antibody.
For measurements of intracellular calcium concentrations in DT40 cells, a cell suspension containing 5 ϫ 10 6 cells was loaded with 2 M Fluo-3 AM (Molecular Probes, Inc., Eugene, OR) in RPMI medium for 1 h at room temperature. The cells were washed with PBS, resuspended in RPMI medium, and stimulated with 10 g/ml M4 antibody or 2 mM H 2 O 2 , and their calcium mobilization was simultaneously measured at 40°C, with constant stirring in a LS-50B fluorimeter (PerkinElmer Life Sciences). The excitation wavelength was 490 nm, and emission was monitored at a wavelength of 535 nm.
Immunofluorescence Confocal Microscopy-Localization of PLC␥2 constructs containing a GFP tag was analyzed after transfection of A431 cells, exactly as described previously (23), using EGF for stimulation. Similar experiments were performed with A20 B-cells, microinjected with the PLC␥2 plasmids. The images were recorded before and after stimulation of A20 cells with anti-IgG2a (30 g/ml).
Constructs for Expression of Recombinant Proteins-For expression of the full-length PLC␥2 protein containing the His 6 tag at the C terminus, the DNA fragment encoding the PLC␥2 sequence (with the PCR-generated tag) was subcloned into baculovirus vector pVL1393 (Pharmingen) using the XbaI site. The fragment of PLC␥2 with the Y753/Y759F mutations was also subcloned into pVL1393 using EcoRI and XbaI sites. The PLC␥2 construct for baculovirus expression, encoding a deletion of 1187-1265 amino acid residues, was also made as a His 6 tag protein at the C terminus. A region encoding a PLC␥-specific array of domains of PLC␥2 (␥2SA) (amino acids 468 -919) was subcloned from the pEGFP/␥2SA construct described previously (23) into bacterial expression vector pGEX-2T (Amersham Biosciences) using the BglII and EcoRI site. After generation of Y753F/Y759F mutations by PCR in the pEGFP/␥2SA construct, the same strategy was used to subclone the fragment into pGEX-2T vector. Both ␥2SA constructs contained a GST tag at the N terminus. Expression of pEGFP/␥2SA constructs incorporating either the wild-type sequence or Y753F/Y759F mutations was analyzed in A431 cells, and translocation of encoded GFP fusion proteins was monitored as described previously (23).
The constructs for expression of different protein-tyrosine kinases (Btk, Syk, Lck, Fyn, and Src) and the adapter protein BLNK, using baculovirus expression, incorporated PCR-generated His 6 tag in frame with the N terminus of the protein. The full-length cDNA encoding human Btk was subcloned into pVL1393 using the BglII and BamHI sites. To generate a truncated Btk (⌬213-Btk), a fragment encoding residues 214 -659 was produced by PCR and cloned as a BamHI/NotI fragment into pVL1393 cut with the same enzymes. The construct encoding GST-Syk fusion protein described previously (13) was used to make a truncated catalytically active version of Syk (⌬318-Syk). The region encoding amino acid residues 319 -635 was amplified using PCR with a forward primer encoding a BamHI site upstream of the initiation codon and a reverse primer encoding a NotI site downstream from the stop codon. Following digestion with BamHI and NotI, the PCR product was cloned into the pVL1393 baculovirus transfer vector cut with the same enzymes. The His 6 -BLNK construct, containing an N-terminal His 6 tag (Met-Asp-His 6 ) attached to residue 2 of human BLNK, was cloned in pVL1393 using the previously described BLNK plasmid (13). Constructs for baculovirus expression of Src family kinases were made by subcloning the cDNAs (24) encoding activated Src (Y527F Src), activated Fyn (Y531F Fyn), and activated Lck (Y525F Lck) into the baculovirus transfer vector pVLplink.2. 2 All viruses were constructed using Baculogold genomic DNA (Pharmingen), according to the manufacturer's instructions.
Expression and Purification of Recombinant Proteins-Insect (Sf9) cells, grown at 27°C in shaker flasks in TNM-FH medium (supplemented with 10% (v/v) fetal calf serum, 1ϫ lipid mixture (Life Technologies), 5 mM glutamine, penicillin (50 IU/ml), streptomycin (50 g/ml), and fungizone (0.25 g/ml)), were infected with baculoviruses for 48 -56 h. For purification of His 6 -tagged PLC␥2, BLNK, ⌬318-Syk, and the fulllength Btk and ⌬213-Btk, cell pellets were sonicated in PBS supplemented with 5% glycerol and 2 mM imidazole (40 ml of buffer/liter of original Sf9 culture) and centrifuged at 100,000 ϫ g. The supernatant was loaded onto a 5-ml nickel column equilibrated in 20 mM Tris-HCl, pH 8.0, 50 mM KH 2 PO 4 , pH 8.0, 0.4 M NaCl, 5% glycerol, and 15 mM imidazole. The column was washed first with the same buffer and then with a buffer containing 50 mM KH 2 PO 4 , pH 8.0, 0.1 M (NH 4 ) 2 SO 4 , 5% glycerol, and 20 mM imidazole, followed by elution in a buffer containing 10 mM HEPES, pH 7.5, 5% glycerol, 100 mM (NH 4 ) 2 SO 4 , 100 mM EDTA, pH 8.0, and increasing concentrations of imidazole. For purification of Src family kinases Fyn, Src, and Lck, a buffer containing 25 mM Tris, pH 7.5, 0.1% (v/v) Triton X-100, 1 mM dithiothreitol, and complete protease inhibitors (Roche Molecular Biochemicals) was used. The cells were lysed by sonication and subjected to centrifugation at 100,000 ϫ g for 1.5 h at 4°C. The supernatant was added to Probond nickel resin (Invitrogen) equilibrated in 20 mM HEPES, pH 8.0, 400 mM NaCl, 5% (v/v) glycerol, 1 mM 2-mercaptoethanol, and 5 mM imidazole, and incubation was carried out for 1.5 h at 4°C. The resin was washed with the equilibration buffer, and bound proteins were eluted with the same buffer containing increasing concentrations of imidazole. The His 6 -Src family kinases were eluted with 100 mM (Fyn), 60 mM (Src), or 30 mM FIG. 1. Domain organization and sequence similarity between PLC␥1 and PLC␥2. A, PLC␥1 and PLC␥2 share the same domain organization including the N-terminal PH domain, EF-hand domain, catalytic domain, and the C2 domain. In addition, and specific to the PLC␥ family, they have a specific array of domains (␥SA) inserted through a loop in the catalytic domain comprising the "split PH domain," two SH2 domains and one SH3 domain. Residues at the boundaries for all domains are indicated for PLC␥2, for two regions located between the second SH2 (C-SH2) domain and the SH3 domain (region I), and for the C terminus (region II). B, alignment of mammalian PLC␥1 and PLC␥2 sequences (ClustalW 1.81) is shown for regions I and II, where the main tyrosine phosphorylation sites in PLC␥1 have been mapped. These tyrosine residues in PLC␥1 (771, 783, and 1254) are boxed. Conserved tyrosine residues between PLC␥1 and PLC␥2 within the C-SH2/SH3 linker are indicated by the arrows. Accession numbers are as follows: human PLC␥1, P19174; rat PLC␥1, P10686; bovine PLC␥1, P08487; human PLC␥2, P16885; rat PLC␥2, P24135.
(Lck) imidazole in 20 mM Hepes, pH 8.0. Proteins were either bufferexchanged into the appropriate assay buffer using a NAP-5 (Sephadex G-25) column (Amersham Biosciences) or subjected to further purification. PLC␥2 was further purified on a heparin-Sepharose column (Amersham Biosciences) followed by gel filtration on a Superdex 200 16/60 column (Amersham Biosciences). His 6 -BLNK and ⌬213-Btk were further purified on a 5-ml HiTrapQ column (Amersham Biosciences) followed by gel filtration on a Superdex 200 16/60 column, while ⌬318-Syk was purified by gel filtration on a Superdex 75 16/60. For purification of GST-Syk, the supernatant was incubated with glutathione-Sepharose (Amersham Biosciences) equilibrated in PBS, and bound protein was isolated by centrifugation at 4,000 ϫ g for 5 min.
The ␥2SA domains were expressed as GST fusion proteins in Escherichia coli. After induction with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside (Calbiochem), cells were grown for 18 h at 20°C. Cell pellets were resuspended in (8 ml/liter of original culture) PBS supplemented with 2 mM dithiothreitol, 1 mM EDTA, 1% (v/v) Triton X-100 with complete protease (Roche) and phosphatase inhibitors (Sigma), lysed by sonication, and subjected to centrifugation (10,000 ϫ g for 10 min). The supernatant was added to glutathione-Sepharose and incubated at 4°C for 45 min. After extensive washing with PBS, the fusion protein was eluted with 50 mM Tris, pH 8.0, supplemented with 10 mM reduced glutathione (Sigma) and 0.1% (v/v) Triton X-100.
In Vitro Assays for Analysis of Protein Phosphorylation, Formation of Protein Complexes, and PLC Activity-For phosphorylation reaction in vitro, purified preparations of PLC␥2 (5 g), ␥2SA proteins (1-5 g), or synthetic peptides (10 -30 g) (Genosphere Biotechnologies) were used as a substrate for the purified protein kinases (0.1-0.5 g). The reaction mixture contained 50 mM Tris, pH 8.0, 2 mM MnCl 2 , 2 mM MgCl 2 , 1 mM Na 3 VO 4 , 50 M ATP, 2 mM dithiothreitol and, when specified, also included 1-5 Ci of [␥-32 P]ATP. Reactions were carried at 30°C for 20 -30 min (or longer, when indicated) and terminated by the addition of 4ϫ SDS loading buffer, and protein was subjected to SDS-PAGE (7.5 and 10% polyacrylamide for PLC␥2 and ␥2SA proteins, respectively) or a 10-20% gradient of polyacrylamide (Invitrogen) for the peptides). Further analysis was by Western blotting using anti-phosphosphotyrosine antibody as described above or, when [␥- 32  The activity of the wild-type (middle panel) and PLC␥2 Y753F/Y759F mutant (right panel) proteins was measured over the range of calcium concentrations using an in vitro assay for phosphatidylinositol 4,5-bisphosphate hydrolysis.
NaCl, 1 mM EDTA, and 1 mM dithiothreitol and subjected to gel filtration on a Superose 12 PC 3.2/3.0 column (Amersham Biosciences) using the same buffer. The conditions used to phosphorylate BLNK were as described for PLC␥2, except that GST-Syk bound to glutathione-Sepharose was used; after incubation, BLNK was separated from the enzyme by centrifugation.
Analysis of interaction between PLC␥2 and nonphosphorylated or phosphorylated BLNK was also performed by band shift on 12.5% polyacrylamide native PHAST gels (Amersham Biosciences) after incubation of protein components at 25°C for 15 min. To ensure full phosphorylation of BLNK for this analysis, BLNK was prepared from Sf9 cells co-infected with Syk, further phosphorylated by Syk in vitro, and purified using chromatography steps described above.
Phospholipase C activity was measured using detergent-mixed micelles containing sodium cholate and [ 3 H]phosphatidylinositol 4,5bisphosphate at different concentrations of free calcium, as previously described (25).
All mass spectra were acquired in reflector mode using a Voyager-DE TM STR BioSpectrometry TM work station fitted with a 337-nm nitrogen laser. All samples were prepared using the dried droplet method with freshly prepared ␣-cyano-4-hydroxycinamic acid at 10 mg/ml in 50% MeCN, 0.1% trifluoroacetic acid.

Calcium Responses in DT40 Cell Lines after B-cell Receptor and Hydrogen Peroxide Stimulation-PLC␥2
is an essential component in calcium signaling triggered either by the stimulation of BCR (8,9) or, as described below (Fig. 2B), by stress responses to hydrogen peroxide in DT40 cells. The stimulation of the PLC␥2 activity in these cells by both agonists is accompanied by phosphorylation of the enzyme at tyrosine residues (15,17). To analyze which tyrosine residues may be involved in activation of PLC␥2 in these systems, the sequences of PLC␥1 and PLC␥2 from two regions were compared. The first region corresponds to linker between the C-terminal SH2 (C-SH2) domain and the SH3 domain (within the "specific array of domains" unique to the PLC␥ family (␥SA)), and the second region is located at the C terminus of PLC␥ (Fig. 1). In PLC␥1, two phosphorylated residues have been mapped to region I (Tyr 771 and Tyr 783 ) and one residue (Tyr 1254 ) within region II. However, only one of these residues, Tyr 783 , appears to be critical for the enzyme signaling function after platelet-derived growth factor stimulation (21). The amino acid sequence alignment of mammalian PLC␥1 and PLC␥2 enzymes shows conservation of Tyr 783 , which corresponds to Tyr 759 in PLC␥2 (Fig.  1B). The sequence around this residue, however, is not strictly conserved. Residues Tyr 771 and Tyr 1254 seem to be unique for PLC␥1. Analysis of sequence similarity between PLC␥1 and PLC␥2 has also revealed that another tyrosine residue within the C-SH2/SH3 linker, Tyr 775 in PLC␥1 and Tyr 753 in PLC␥2, is conserved.
To analyze the role of conserved tyrosine residues within the region I for PLC␥2 signaling in B-cells, stable cell lines were generated by transfection of human PLC␥2 into PLC␥2-deficient DT40 cells. As shown in Fig. 2A, human PLC␥2 containing the wild-type sequences restored calcium responses to BCR  2 and 3). The phosphorylation was also analyzed before (lane 4) and 2 and 5 min after stimulation by hydrogen peroxide (lanes 5 and 6). PLC␥2 from various cell extracts was isolated by immunoprecipitation and analyzed by Western blotting using either anti-phosphotyrosine antibody (PY, top panels) or antibody to PLC␥2 (PLC␥2, bottom panels). B, the PLC␥2-deficient DT40 cells (␥2 Ϫ ) (lane 1) and these cells stably transfected either with the wild-type human PLC␥2 (wt) (lanes 2 and 3) or PLC␥2 mutants Y753F/Y759F (lanes 4 and 5), Y753F (lanes 6 and 7), and Y759F (lanes 8 and 9) were analyzed for PLC␥2 phosphorylation. After immunoprecipitation using anti-PLC␥2 antibody, Western blotting was performed using anti-phosphotyrosine antibody (PY, top panel). The PLC␥2 protein was visualized on the same nitrocellulose membrane by Amido Black staining (PLC␥2, bottom panel). C, immunoprecipitation of the wild-type PLC␥2 (lanes 1 and 2) and PLC␥2 Y753F/Y759F mutant (lanes 3 and 4) from the stable DT40 cell lines was performed as described for A and B. The presence of BLNK in the immunoprecipitates from stimulated (lanes 1 and 3) and unstimulated cells (lanes 2 and 4) was analyzed by Western blotting. stimulation (bottom panel) to levels similar as measured in the wild-type DT40 cells (top panel) but lacking in PLC␥2-deficient cells (middle panel). In addition to the wild-type PLC␥2, the constructs incorporating mutations Y753F, Y759F, and Y753F/ Y759F were also used to generate stable DT40 cell lines (PLC␥2 Ϫ /wtPLC␥2, PLC␥2 Ϫ /PLC␥2 Y753F, PLC␥2 Ϫ /PLC␥2 Y759F, and PLC␥2 Ϫ /PLC␥2 Y753F/Y759F). Expression of PLC␥2 in all cell lines was initially analyzed by Western blotting, and clones expressing similar amounts of PLC␥2 were selected for further study. Immunoprecipitation confirmed that these cell lines expressed similar amounts of human wild-type or mutant PLC␥2 (see Fig. 3B, bottom panel). The selected cell lines were analyzed for calcium responses to stimulation by either the M4 antibody, which binds to BCR, or to hydrogen peroxide (Fig. 2B). In all DT40 cell lines, the M4 antibody and hydrogen peroxide had a similar effect on calcium responses. Both agonists stimulated calcium responses in the wild-type DT40 cells and PLC␥2 Ϫ /wtPLC␥2. In contrast, DT40 cell lines PLC␥2 Ϫ /PLC␥2 Y753F, PLC␥2 Ϫ /PLC␥2 Y759F, and PLC␥2 Ϫ / PLC␥2 Y753F/Y759F showed no calcium responses (in addition to those seen in PLC␥2-deficient cells). These results indicate that Tyr 753 and Tyr 759 are essential for PLC␥2 function in B-cells.
To confirm the possibility that the mutation of tyrosine residues had an effect on PLC␥2 signaling function, rather than by causing more general changes in the catalytic properties of this PLC, the wild-type and PLC␥2 Y753F/Y759F mutant were expressed using a baculovirus system, and the purification based on the presence of His 6 tag was carried out. Preparations of pure proteins (Fig. 2C, left panel) were analyzed for PLC activity in vitro using conditions measuring basal catalytic activity. Under similar conditions, measurements of PLC␥1 activity gave the same values for the enzyme isolated from nonstimulated and stimulated cells (26). In this assay, the wild-type and PLC␥2 Y753F/Y759F mutant had similar specific activities (in the range of 120 -180 mol/mg). Measurements of PLC activity over the range of calcium concentrations (Fig. 2C, middle and right panels) also demonstrated similar calcium dependence with the highest activity at 5-10 M. These data demonstrated an intact function of the PLC␥2 Y753F/Y759F catalytic domain and suggested the importance of Tyr 753 and Tyr 759 residues in the context of the BCR signal transduction and stress responses. Further evidence ruling out gross changes in protein structure and correct folding is presented in Figs. 3C and 4, demonstrating that Y753F/Y759F mutation did not affect interaction with BLNK or the ability of PLC␥2 to translocate to the plasma membrane, previously shown to require functional SH2 domains (23).
Phosphorylation of PLC␥2 in DT40 Cell Lines-The tyrosine phosphorylation of PLC␥2, endogenously present in the wildtype DT40 cell, has previously been observed after stimulation by the M4 antibody or hydrogen peroxide (15,17). In the experiments shown in Fig. 3A, the phosphorylation of human PLC␥2 in the DT40 PLC␥2 Ϫ /wtPLC␥2 cell line could also be detected 2 and 5 min following stimulation with either M4 (right panel) or hydrogen peroxide (left panel). The stimulation in the presence of hydrogen peroxide appeared to be more potent and particularly prominent after 5 min of stimulation.
An attempt was made to map tyrosine residues in PLC␥2 that become phosphorylated after stimulation of DT40 cells. Analysis of tryptic peptides from tyrosine-phosphorylated PLC␥2 demonstrated that several peaks (resolved by reversephase chromatography) contained tyrosine-phosphorylated peptides. Further analysis of the peak fractions by mass spectroscopy revealed that masses corresponding to phosphorylated peptides containing Tyr 753 and Tyr 759 were present under two of these peaks. However, the fractions contained a mixture of peptides, and phosphorylation of Tyr 753 and Tyr 759 was not confirmed by sequencing due to limiting amounts (data not shown).
Further analysis of PLC␥2 Y753F/Y759F mutant in DT40 cell has suggested that, as previously shown for the wild type PLC␥2 (13)(14)(15), it could also interact with BLNK (Fig. 3C) and the plasma membrane (Fig. 4). Whether or not levels of the mutant in glycolipid-enriched microdomains were comparable with that of the wild-type was not demonstrated conclusively due to the background presence of PLC␥2 in the absence of stimulation (data not shown). Nonetheless, the translocation of a PLC␥2 construct incorporating the Y753F/Y759F mutation in a system previously used to demonstrate a requirement for functional SH2 domains (23) was clearly demonstrated (Fig.  4B). Using the same approach, translocation of the Y753F/ Y759F mutant was also confirmed in A20 B-cells (data not shown). The data presented in Fig. 3 have demonstrated that the residues Tyr 753 and Tyr 759 are not only important for calcium signaling function in B-cells (Fig. 2B) but also for phosphorylation of the entire PLC␥2 protein. Although the mapping of tyrosines phosphorylated in vivo has not shown this conclusively, the loss of phosphorylation observed for the Y753F/ Y759F mutant suggests that these are the tyrosine residues that become phosphorylated in response to stimulation. Furthermore, this phosphorylation could be a requirement for phosphorylation of other tyrosine residues, which may be present in other regions of PLC␥2.

Role of Btk and Syk in Phosphorylation and Complex Formation in Vitro-Genetic studies of B-cell signal transduction
have demonstrated that nonreceptor tyrosine kinases from at least three families, Src, ZAP-70/Syk, and Tec, were important for an increase in intracellular calcium (8,9). The tyrosine kinases Syk and Btk, present in DT40 and other B-cells, have been considered to directly phosphorylate PLC␥2. Previous analysis of PLC␥2 phosphorylation in cells after overexpression of a particular tyrosine kinase (27,28), however, has not been conclusive, since the possibility that this tyrosine kinase could contribute to PLC␥2 phosphorylation through activation of another tyrosine kinase(s) endogenously present in the cell could not be ruled out. Furthermore, concentrations of tyrosine kinases or the PLC␥2 substrate could not be controlled in those experiments. To circumvent these problems, purified proteins (prepared either as His 6 or GST fusion proteins) were used in a phosphorylation assay in vitro (Fig. 5). While Btk was able to use purified PLC␥2 protein as a substrate, phosphorylation of this PLC by Syk kinase was much lower (Fig. 5, B and C).
Using the same preparation of Syk kinase, autophosphorylation (Fig. 8A) and phosphorylation of purified BLNK protein (Fig. 4B, bottom panel) could be demonstrated clearly. Essentially the same results were obtained using a purified GST fusion protein of the full-length Syk as with a truncated, catalytically active His 6 -tagged protein. Syk kinase could also phosphorylate ␥SA of PLC␥1 at tyrosine residue 783, as demonstrated using a specific antibody to this phosphorylation site (data not shown). When longer incubation times and increased concentrations of PLC␥2 were used (Fig. 5C, bottom panel) or when greater amounts of purified kinases were included in the reaction (data not shown), phosphorylation of PLC␥2 by Syk could also be measured. Detailed kinetic analysis, directly comparing phosphorylation of PLC␥2 by Syk and Btk, is illustrated in Fig. 5D and has allowed calculation of apparent K m values and relative values for V max . The difference between K m values was about 2-fold (50.0 M for Btk and 83.3 M for Syk), and the difference between values for V max (expressed as units/min/mg) was about 7-fold (6.6 for Btk and 1.1 for Syk). The kinetic analysis was extended to phosphorylation of BLNK by Syk (Fig. 5E), demonstrating even greater differences between phosphorylation of BLNK and PLC␥2 by Syk than when the two kinases were compared for PLC␥2 phosphorylation. ( Figs. 1B and 6A). This peptide was phosphorylated, separated from nonphosphorylated peptide, and analyzed by mass spectrometry, demonstrating an increase in mass (by 80, from 2420.00 to 2499.98) corresponding to the phosphorylation of one tyrosine residue. When two additional peptides incorporating either the Y753F (peptide 2) or Y759F (peptide 3) mutation were used, it was shown that Tyr 753 was phosphorylated in preference to Tyr 759 (Fig. 6B). Further phosphorylation studies in vitro using the wild type and Y753F/Y759F mutant in the context of the full-length PLC␥2 demonstrated phosphorylation of both proteins (data not shown). Thus, additional Btk phosphorylation sites, outside the region represented by the peptide, are present in PLC␥2. Some of the additional sites could be within the ␥2SA protein (containing 24 tyrosine residues), as suggested in Fig. 8D.
Interaction of purified PLC␥2 and BLNK has been analyzed by gel filtration (Fig. 7A) and band shift on native gels (Fig. 7B) and demonstrated that phosphorylation of BLNK by Syk resulted in incorporation of PLC␥2 in high molecular weight complexes. These data are consistent with previous observations of co-immunoprecipitation of these proteins after B-cell stimulation (13,14). When purified preparation of PLC␥2 Y753F/Y759F mutant protein was tested, it was also incorporated into a complex with phosphorylated BLNK in this in vitro assay (Fig. 7B, right panel).
In Vitro Phosphorylation of PLC␥2 by Various Tyrosine Kinases-In addition to Syk and Btk, several other nonreceptor tyrosine kinases from the Src family were tested for their ability to phosphorylate PLC␥2 (Fig. 8, A and B). It has been previously reported that partially purified preparations of several of these kinases could phosphorylate PLC␥2 in vitro (29). The Src family kinases used in our study included Src, Lck, and Fyn, and all contained a mutation (corresponding to Y527F in Src) known to prevent phosphorylation and inhibition by other tyrosine kinases in cells (30). The proteins were expressed using a baculovirus system and contained a His 6 tag for purification. Like Syk and Btk, all Src kinases were autophosphorylated in vitro (Fig. 8A). Also, all Src kinases, like Btk, phosphorylated PLC␥2 (Fig. 8B). Thus, among the tyrosine kinases tested at the specific conditions, only Syk kinase was unable to efficiently phosphorylate the full-length PLC␥2.
Since the mutagenesis of tyrosine residues identified Tyr 753 and Tyr 759 as important for PLC␥2 signaling function and tyrosine phosphorylation in stimulated DT40 cells, ␥2SA pro-tein (which includes these tyrosine residues) was also used as a substrate. ␥2SA encoding the wild-type sequences and the protein incorporating Y753F/Y759F mutations were expressed as GST fusion proteins (Fig. 8C). When the panel of proteintyrosine kinases (Syk, Btk, Src, Lck, and Fyn) was used with the wild-type ␥2SA as a substrate, Btk and Lck phosphorylated this protein better than other kinases. Further comparison of these kinases using both the wild-type and Y753F/Y759F ␥2SA demonstrated that the mutation abolished phosphorylation by Lck but not with Btk (Fig. 8D). This demonstrates that Lck can phosphorylate one or both of these tyrosine residues in PLC␥2.
The studies of phosphorylation of Tyr 753 and Tyr 759 were also performed in the context of a synthetic peptide corresponding to residues 745-764 in PLC␥2 (peptide 1) (Fig. 8E). Phosphorylation of the peptide by Syk, Btk, Lck, Fyn, and Src was analyzed in a reaction mixture containing [␥-32 P]ATP, and the peptide was separated from other components by SDS-PAGE. When low concentrations of the enzymes (0.1 g) and short incubation times (20 min) were used, Lck was clearly the most efficient tyrosine kinase from the panel (Fig. 8E). Purified preparations of Lyn, prepared as a GST fusion protein, could phosphorylate the peptide to levels comparable with Btk and Fyn but not Lck (data not shown). Analyses of the peptide phosphorylated by Lck by mass spectrometry revealed phosphorylation of only one tyrosine residue in the peptide (an increase of the peptide mass by 80, from 2420.00 to 2500.14). Further analysis using peptides with either Tyr 753 or Tyr 759 replaced by phenylalanine identified Tyr 753 as the main site phosphorylated by Lck (data not shown).

DISCUSSION
Phosphorylation of both PLC␥1 and PLC␥2 has been well documented for the majority of cellular systems where the activation of PLC␥ isoforms takes place (3)(4)(5). However, phosphorylation sites and the importance of specific tyrosine residues that become phosphorylated have been analyzed only for PLC␥1 in cells stimulated through growth factor receptors. Within a complex profile of PLC␥1-phosphorylated peptides, obtained after EGF stimulation, two main tyrosine-phosphorylated residues have been mapped as Tyr 771 and Tyr 1254 , and one minor site has been found to correspond to Tyr 783 (19). More recently, the use of phosphospecific antibodies to Tyr(P) 783 confirmed phosphorylation of this site in stimulated cells (23,31). Similar patterns of phosphorylation have been seen after stimulation of fibroblasts with platelet-derived growth factor and in several other systems (3)(4)(5)21). These (Tyr 771 , Tyr 783 , and Tyr 1254 ) and some additional sites have been identified after in vitro phosphorylation of purified PLC␥1 by EGF receptor kinase (20). Interestingly, mutational studies have revealed that only Tyr 783 was critical, while other residues had less impact on PLC␥1 function when tested in platelet-derived growth factor signaling (21), demonstrating that not all phos-phorylation sites may be functionally important. Taking into account the complexity of the phosphorylation pattern and possible functional redundancy, the studies of PLC␥2 described here focused on a mutagenesis approach based on information obtained for the PLC␥1 isoform. Comparison of PLC␥1 and PLC␥2 sequences has revealed that of three tyrosine residues in the loop region between the C-SH2 and the SH3 domain, only two are conserved (Tyr 753 and Tyr 759 in PLC␥2, the latter corresponding to phosphorylation site Tyr 783 in PLC␥1), while there is no conservation of sequences in the C-terminal region, including residue Tyr 1254 in PLC␥1 (Fig. 1). Our mutagenesis analysis of tyrosines in PLC␥2 within the C-SH2/SH3 loop region demonstrated that both Tyr 753 and Tyr 759 are required to restore calcium signaling in DT40 cells deficient in PLC␥ (Fig. 2B). Thus, the conserved residue corresponding to Tyr 759 in PLC␥2 and Tyr 783 in PLC␥1 is important for the function of both isoforms. The other conserved residue (753 in PLC␥2/775 in PLC␥1) has not been mutated in PLC␥1 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 PLC␥1.
Comparison between properties of a double mutant within the C-SH2/SH3 loop region in PLC␥1 (Y771F/Y783F, where Tyr 771 is unique for PLC␥1) observed in previous studies (21) with the PLC␥2 Y753F/Y759F double mutant in the same region described here (Figs. 2 and 3) reveals several similarities. For example, both proteins (PLC␥1 Y771F/Y783F and PLC␥2 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 PLC␥1 and in B-cell signaling for PLC␥2, these mutations not only inhibited generation of inositol 1,4,5-trisphosphate and calcium mobilization but also abolished phosphorylation of the PLC␥ protein. In the case of PLC␥1, 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 Tyr 1254 at the C terminus. Since the phosphorylation profile of PLC␥2 in stimulated B-cells also appears to be complex, it is possible that the double mutation Y753F/Y759F in PLC␥2 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 PLC␥ 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 (3)(4)(5). 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 PLC␥2-mediated calcium signaling (8,9). However, it has not been established which of these enzymes could phosphorylate PLC␥2 directly. This was examined here (Figs. 5, 6, and 8) using purified preparations of PLC␥2 constructs and various tyrosine kinases with an emphasis on Btk and Syk, both essential for PLC␥2 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 PLC␥2 phosphorylation (11,16,27,35,36). While the calcium responses in Btk Ϫ cells were abolished, in most reports only reduction in PLC␥2 phosphorylation has been observed, suggesting the involvement of additional tyrosine kinases in phosphorylation of this PLC. The in vitro phosphorylation study using purified components described here demonstrated that Btk could directly phosphorylate PLC␥2, including an important residue, Tyr 753 (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 PLC␥2 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 Ϫ cells (11). 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 (16). 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 PLC␥2, 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 (10,32,33,34), as a tyrosine kinase that can efficiently phosphorylate a peptide incorporating Tyr 753 and Tyr 759 residues of PLC␥2.
Protein-tyrosine kinase Syk has also been implicated in Bcell signaling and shown to be required for both PLC␥2 phosphorylation and calcium responses (10). It has been shown that the essential adapter protein BLNK, forming complexes with a number of signaling components including PLC␥2, needs to be phosphorylated by Syk in order to bind other proteins (13,14). Therefore, the role of Syk in calcium responses could be to phosphorylate both PLC␥2 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 PLC␥2, 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 PLC␥2 (Fig. 7).
In summary, we identified tyrosine residues Tyr 753 and Tyr 759 as important for activation and tyrosine phosphorylation of PLC␥2 in B-cells. Based on this observation, the roles of various tyrosine kinases that genetic analysis has implicated in regulation of PLC␥2 were further assessed. Direct phosphorylation of PLC␥2 by Btk is observed; however, the role of Syk may not be to phosphorylate PLC␥2 directly but to provide docking phosphotyrosine sites on the adapter protein BLNK, essential in B-cell signaling.