Introduction

The mechanisms by which receptor activation translates into cellular functions has been an area of intense investigation over the past three decades. A paradigm has evolved in which ligand binding to receptors activates multiple signaling pathways that coordinately generate second messengers important in cellular functions (reviewed in Jordan et al., 2000). In biological systems involving receptor tyrosine kinases, the receptor or its associated signaling subunits is typically phosphorylated on multiple tyrosine residues that, in turn, recruit effector molecules. The concept of molecular scaffolding suggests that signaling components are required to organize in a specific manner at a macromolecular level to mediate receptor functions (reviewed in Leo et al., 2002; Smith and Scott, 2002). However, the mechanism by which such a scaffold coordinates activation of downstream pathways is less well defined. More recently, the concept of cytoplasmic scaffolds has emerged. Molecules, such as IRS-1, Grb2 and Gab1, which typically reside in the cytosol but are recruited to the plasma membrane following receptor engagement, are also capable of binding multiple effector proteins. While a large body of biochemical data demonstrates the existence of such large molecular complexes, the concept of how these macromolecular complexes coordinate the generation of second messengers has been less well elucidated.

In B cells, binding of foreign antigens to the B cell antigen receptor (BCR) induces activation of three distinct families of cytoplasmic protein tyrosine kinases (PTKs), Src, Syk, and Tec, which are required for B cell proliferation, differentiation and apoptosis (reviewed in DeFranco, 1997; Benschop and Cambier, 1999; Kurosaki, 1999). All three PTK families phosphorylate adapter proteins, which, in turn, coordinate enzymes that generate second messengers including phosphoinositides and GTPases (Takata et al., 1994; Cox et al., 1996; Law et al., 1996; Fu et al., 1998; Okada et al., 2000). The requirement for all three PTK families in B cell function has been demonstrated by the B cell developmental and functional defects revealed in human and mice with mutations or deletions in these PTKs (Tsukada et al., 1993; Cheng et al., 1995; Hibbs et al., 1995; Kerner et al., 1995; Khan et al., 1995; Nishizumi et al., 1995; Turner et al., 1995; Hendricks et al., 1996).

The generation of second messengers requires the tyrosine phosphorylation of a number of effector molecules, which include phospholipase C (PLC) , the Vav Rho-GTPase guanine nucleotide exchange factor (GEF), the Shc adapter protein that regulates the Grb2/Son of Sevenless (SoS) Ras-GTPase GEF complex, and phosphoinositide 3 kinase (PI3K). Studies over the past four years have identified two B cell adapter molecules, BLNK for B cell linker protein (also known as SLP-65, BASH and BCA) and BCAP (for B cell adapter for PI3K), which link the cytoplasmic PTKs with the phosphorylation of downstream effector molecules (Fu et al., 1998; Gangi-Peterson et al., 1998; Goitsuka et al., 1998; Wienands et al., 1998; Okada et al., 2000). B cells lacking BLNK fail to elicit [Ca²⁺]_i flux following BCR crosslinking and exhibit attenuated activation of all three families of MAPKs (Ishiai et al., 1999). DT40 B cells lacking BCAP demonstrate decreased activation of the PI3K pathway, and bcap^-/- mice demonstrate defects in B cell development and function (Okada et al., 2000; Yamazaki et al., 2002).

As BLNK encodes no intrinsic enzymatic activity, its function is to serve as a scaffold by assembling macromolecular complexes that include enzymes (PLC, Vav and Btk) and additional linker proteins (Grb2 and Nck) (Fu and Chan, 1997; Fu et al., 1998; Wienands et al., 1998; Hashimoto et al., 1999; Jumaa et al., 2001). The functional significance for tyrosine phosphorylation of BLNK has been suggested by the inducible association of BLNK with a number of effector signaling molecules and the ability of a mutant BLNK molecule with decreased tyrosine phosphorylation to attenuate BCR-induced calcium-dependent responses (Fu et al., 1998). In this study, we analyzed roles for BLNK tyrosine phosphorylation and demonstrate that BLNK tyrosine phosphorylation is important for organizing 'cis'-mediated interactions of specific signaling components.

Results

In vivo phosphorylation of BLNK in B cells

To understand how tyrosine phosphorylation of BLNK regulates BCR function, we analyzed the in vivo phosphorylation sites within human BLNK (hBLNK) in resting and activated B cells. Phosphorylated hBLNK from resting and BCR-activated Daudi B cells were digested with trypsin and peptides resolved by 2-dimensional TLC. Four major peptides, designated as A, B, D and E, containing both phosphoserine (pS) and phosphothreonine (pT), but not phosphotyrosine (pY) residues were isolated from resting cells (Figure 1A, left; data not shown). Following BCR activation, pY was detected in peptides A and B and an additional peptide (designated C) containing pY was also detected in BCR-activated cells (Figure 1A, right; data not shown). No pY residues were detected in peptides D and E isolated from resting or activated B cells. Hence, BCR activation results in tyrosine phosphorylation of peptides A, B and C.

Identification of tyrosine phosphorylation sites within BLNK by peptide analysis

To facilitate the identification of the phosphorylated tyrosine residues, we analyzed the pattern of peptides derived from purified hBLNK, which was phosphorylated in vitro by its upstream PTK-Syk (Fu et al., 1998). The in vitro phosphorylated products demonstrated the presence of three major species that co-migrate with peptides A, B and C from the in vivo labeling experiments (Figure 1B, left; Supplementary figure 1A−F, available at The EMBO Journal Online).

Manual Edman sequencing of peptide A failed to release ³²P within the first 13 cycles and suggested that this peptide may represent the tryptic peptide encoding amino acids (aas) 51−106 (Figure 1C). This peptide encompasses Y52 (cycle 2), Y72 (cycle 22), Y84 (cycle 34) and Y96 (cycle 46). Digestion with additional enzymes failed to cleave this peptide into smaller fragments and prohibited sequencing of smaller derivative peptides (data not shown). Hence, we examined the ability of Syk to phosphorylate a mutant hBLNK molecule, designated as hBLNK(3F) in which Ys 72, 84 and 96 (but not Y52 since cycle 2 was not phosphorylated) were mutated to phenylalanine (F). While hBLNK(3F) was phosphorylated on peptides B and C, no ³²P was incorporated into peptide A (Figure 1B, right) and this is consistent with one or more of the three Y residues (Ys 72, 84 and 96) being phosphorylated by Syk (see below).

Edman sequencing of in vitro phosphorylated tryptic peptides B and C both released counts in cycle 9 (Supplementary figure 2A and B). Only Y178 of the 33 aa tryptic peptide encoding aas 170−202 would have released counts in this cycle (Figure 1C). Since this peptide also contains Y189 (cycle 20), which could not have been detected using manual Edman sequencing, we subjected peptides B and C to additional enzymatic digestion with endoproteinase-Glu C (endo-gluC). Manual sequencing revealed ³²P in cycles 2 and 3 for both peptides B and C, which correspond to phosphorylation of Y178 (cycle 3) and Y189 (cycle 2) (Supplementary figure 2C and D). Consistent with this biochemical analysis, in vitro phosphorylation by Syk of a mutant hBLNK in which Y178 and Y189 were both mutated to Phe resulted in the loss of both peptides B and C without any effect on peptide A (data not shown). Together, these data are consistent with peptides B and C being derived from phosphorylation of Y178 and Y189, though the exact biochemical difference between peptides B and C remains unclear.

Confirmation of BLNK phosphorylation by immunologic analysis

To confirm the identity of these phosphorylation sites, we generated a panel of phosphopeptide-specific antibodies specific for each site. Immunoblotting with each phosphospecific antibody demonstrated the absence of phosphorylation in resting Daudi cells and the inducible phosphorylation of all five Ys on both alternatively spliced hBLNK and hBLNK-s (short) molecules following BCR crosslinking (Figure 2A). No significant differences in the kinetics of phosphorylation were observed among the five Y sites (Supplementary figure 3).

To evaluate the roles of BLNK phosphorylation, we expressed the chicken BLNK (cBLNK) ortholog mutant in which the five conserved cBLNK Ys were mutated to F (Ys 91, 103, 115, 194 and 205 in cBLNK) in blnk^-/- DT40 B cells (Figure 2B). However, this mutant cBLNK molecule [designated cBLNK(Y5F)] still demonstrated detectable tyrosine phosphorylation following BCR crosslinking (data not shown). A search of the cBLNK sequence revealed an additional tyrosine residue (Y248) that contains the preferred phosphorylation sequence (YE/DXP) for the Syk PTK. Additional mutation of Y248 to F [designated cBLNK(Pan F)] demonstrated no residual tyrosine phosphorylation following BCR crosslinking (Figure 2C). Since cBLNK(Pan F) still retained its ability to bind to Grb2, the overall structural integrity of cBLNK(Pan F) appears to be preserved as this interaction is mediated through SH3−proline interactions (data not shown).

To determine if BLNK phosphorylation sites occurred in a coordinated fashion, each of the six cBLNK Ys were re-introduced onto the cBLNK(Pan F) background. While no tyrosine phosphorylation was detected in the cBLNK(Pan F) mutant, each of the single Y reconstituted mutants was phosphorylated following BCR crosslinking (Figure 2D). Together, the biochemical and immunological studies indicate that Ys 72, 84, 96, 178 and 189 within hBLNK, and Ys 91, 103, 115, 194, 205 and 248 within cBLNK are phosphorylated following BCR activation.

Tyrosine phosphorylation of BLNK is required for [Ca²⁺]_i and MAPK activation

To understand the biological implications for tyrosine phosphorylation of BLNK, we analyzed the effects of the cBLNK(Pan F) mutation. Since BLNK associates with PLC following BCR crosslinking (Fu et al., 1998), we first analyzed the ability of cells expressing cBLNK(Pan F) to regulate the phosphoinositide-signaling pathway. No tyrosine phosphorylation of PLC2 was observed in cells expressing cBLNK(Pan F) (Figure 3A). In addition, cBLNK(Pan F) did not co-immunoprecipitate with PLC2. Correspondingly, cells expressing cBLNK(Pan F) failed to induce increases in [Ca²⁺]_i or NF-AT transcriptional activation following BCR crosslinking (Figure 3B and C).

Since BLNK is also required for the efficient activation of the three families of MAPKs (Ishiai et al., 1999), we analyzed the requirements for the tyrosine phosphorylation of BLNK in MAPK activation. Expression of cBLNK(Pan F) failed to restore activation of Erk2, JNK, or p38 phosphorylation following BCR crosslinking (Figure 3D−F). Hence, tyrosine phosphorylation of BLNK is required for both [Ca²⁺]_i and MAPK signaling pathways activated by the BCR.

Binding specificity of effector molecules to BLNK tyrosine phosphorylation sites

To assess binding specificity of the six phosphorylated tyrosine sites, we generated a panel of phosphorylated cBLNK peptides and assessed their binding to BLNK interacting proteins. Immunoblotting indicated that Ys 103, 194 and 205 of cBLNK preferentially bound PLC2, Y115 preferentially bound Btk, and Y91 preferentially bound to both Vav and Nck (Figure 4B).

To evaluate the functional significance of these binding specificities, we generated a panel of mutants in which the three predicted PLC2 binding sites were mutated in various combinations (Y194F, Y103F/Y194F and Y103F/Y194F/Y205F, respectively). These cDNAs were expressed in blnk^-/- DT40 cells and a minimum of two individual clones expressing comparable BLNK and BCR levels were analyzed (data not shown). While mutation of a single PLC2 binding site (Y194F) caused a significant reduction in [Ca²⁺]_i, mutation of two PLC2 sites (Y103F/Y194F) resulted in a further reduction, and mutation of all three PLC2 binding sites abrogated [Ca²⁺]_i mobilization (Figure 5A). Correspondingly, these mutant cells also demonstrated a graded reduction in transcriptional activation of an NF-AT responsive element, tyrosine phosphorylation of PLC2, and ability of PLC2 to bind BLNK (Figure 5B−D).

Reciprocally, we examined the effects of restoring the PLC2 binding sites in the cBLNK(Pan F) mutant molecule. Restoration of one (Y194 only) or two (Y103/Y194 only) of the PLC2 sites failed to induce any significant increases in [Ca²⁺]_i or NF-AT transcriptional activation following BCR crosslinking (Figure 6A and B). Restoration of all three PLC2 binding sites (Y103/Y194/Y205 only) resulted in minimal BCR-induced [Ca²⁺]_i and no significant NF-AT transcriptional activation. Despite the significant decrease in [Ca²⁺]_i and NF-AT activation, the binding of PLC2 to cBLNK and the tyrosine phosphorylation of PLC2 was recapitulated in cells expressing cBLNK(Y103/194/205 only) and cBLNK (Y103/194 only) (Figure 6C). These data indicate that recruitment of PLC to cBLNK as well as tyrosine phosphorylation of PLC2 was insufficient to restore BLNK function, and an additional level of organization of signaling proteins on BLNK was required to mediate NF-AT transcriptional activation.

Because Btk is implicated in activating PLC, we next analyzed the functional significance of the Btk binding site by solely mutating Y115 to F. As compared with cells expressing wild-type cBLNK, cells expressing cBLNK(Y115F) demonstrated reduced [Ca²⁺]_i and NF-AT transcriptional activation following BCR crosslinking (Figure 7A and B). In contrast to the PLC binding mutant, the overall tyrosine phosphorylation of PLC2 was not decreased and the association of cBLNK(Y115F) was comparable to cells expressing wild-type cBLNK (Figure 7C).

Phosphorylation of PLC and Btk binding sites within a single BLNK molecule is required for normal BCR function

Finally, we analyzed the potential for cooperativity between distinct BLNK-binding effectors in regulating NF-AT transcriptional activation. We first examined whether PLC2 and non-PLC2 binding mutants of BLNK could complement each other in trans (Figure 8A). Consistent with the analysis in Figures 5 and 6, expression of either cBLNK molecules alone resulted in no BCR-inducible NF-AT transcriptional activation (Figure 8B). Moreover, co-expression of equal molar amounts of both mutant molecules in blnk^-/- cells also failed to complement each other to restore NF-AT transcriptional activation. Hence, NF-AT transcriptional activation cannot be mediated through 'trans'-crosstalk between PLC2 and non-PLC2 binding BLNK molecules.

Conversely, we analyzed the ability of a cBLNK molecule containing both PLC2 and Btk binding sites in mediating BCR-activated signaling pathways in cis (Figure 9A). While expression of the PLC binding mutant, cBLNK(Y103/194/205 only), only minimally restored BCR induced [Ca²⁺]_i mobilization (Figures 6A and 9B), the additional reconstitution of the Btk binding site, cBLNK(Y103/115/194/205 only), fully restored the initial and partially the latter phase of BCR-induced Ca²⁺ mobilization (Figure 9B). Additionally, while the PLC2 binding cBLNK (Y103/Y194/Y205 only) mutant was devoid of transcriptional activation of NF-AT, expression of cBLNK(Y103/115/194/205 only) restored 50% of the BCR-activated NF-AT transcriptional activity (Figure 9C) and tyrosine phosphorylation of PLC2 (Figure 9D). Together, these data suggest that phosphorylation of Ys on a single scaffold to bind PLC2 and other effectors (e.g. Btk) is required for normal BCR function.

Discussion

Assembly of macromolecular signaling complexes to coordinate the propagation of signaling pathways has gained great acceptance in a number of biological systems. Studies of yeast have demonstrated the critical importance of the Ste5p and Pbs2p scaffolding proteins in dictating the in vivo substrate specificities of the MAPK pathways and, in turn, specific biological responses of yeast mating and glycerol production reactions, respectively (reviewed in van Drogen and Peter, 2002). In lymphocytes, the specificity and sensitivity of signaling pathways is paramount to cellular fate. Lymphocytes must discern the fine differences between foreign and self-ligands that result in markedly distinct biological responses of cellular proliferation of pathogen-specific lymphocytes and apoptosis of self-reactive lymphocytes. One mechanism by which these ligands affect cellular fates is through different kinetics and dynamics of signaling pathways activated through the antigen receptors (Healy et al., 1997; Hippen et al., 2000; Kimura et al., 2000). Alterations in the generation of second messengers following receptor engagement, such as [Ca²⁺]_i and small GTPases, are associated with the induction of lymphocyte unresponsiveness, a state known as anergy (Dolmetsch et al., 1997; Macian et al., 2002). Hence, the coordinated generation of second messengers in lymphocytes is required for normal cellular function.

In B cells, we and others have previously demonstrated the requirement of the BLNK adapter protein in B cell development and in BCR-signal transduction (Ishiai et al., 1999; Jumaa et al., 1999; Minegishi et al., 1999; Pappu et al., 1999; Xu et al., 2000; Yamazaki et al., 2002). Mice and humans deficient in BLNK demonstrate developmental blocks at the pro- to pre-B cell transition, and additionally, in mice, the immature to mature B cell transition and underscores the requirements for BLNK in both pre-BCR and IgM BCR signaling pathways, respectively. In the chicken DT40 B cell system, blnk^-/- DT40 B cells exhibit absent [Ca²⁺]_i and MAPK responses (Ishiai et al., 1999). In this study, we demonstrate the importance for the phosphorylation of multiple tyrosine residues within a single BLNK molecule to generate a single molecular scaffold. Fine mapping of BLNK phosphorylation sites permitted us to identify subsets of tyrosine residues that bind distinct effector molecules. Phosphorylation of Ys 103, 194 and 205 within cBLNK (and correspondingly Ys 84, 178 and 189 within hBLNK) facilitates PLC2 binding; Y115 within cBLNK (and correspondingly Y96 in hBLNK) facilitates Btk binding; Y91 within cBLNK (and correspondingly Y72 in hBLNK) facilitates Nck and Vav binding.

Mutation of the multiple PLC2 binding sites within BLNK reduces the association of PLC2 with BLNK and activation of PLC2-mediated signaling pathways. The correlative nature of the number of PLC2 sites within BLNK and the magnitude of PLC2 function supports an amplification role for the multiple PLC2 binding sites within BLNK. Furthermore, the inability of a mutant BLNK molecule that contains solely PLC2 binding sites [cBLNK(103/194/205 only)] to restore wild-type [Ca²⁺]_i or NF-AT transcriptional activation hinted that other binding partners might be required for normal [Ca²⁺]_i signaling. As Btk is also required for normal [Ca²⁺]_i and in view of the reported association of BLNK with Btk (Hashimoto et al., 1999; Jumaa et al., 2001), we tested the hypothesis that the dual binding of Btk and PLC2 with BLNK may be required for normal [Ca²⁺]_i. Indeed, while cBLNK(Y115F) bound PLC2 with similar stoichiometry as wild-type BLNK, cells expressing cBLNK(Y115F) still exhibited an attenuated [Ca²⁺]_i response. The ability of BLNK, Btk and PLC2 to co-migrate as a large molecular weight complex (>600K M_r) in size fractionation studies as opposed to the 70K M_r BLNK in resting B cells also supports the notion that BLNK serves as a scaffold to nucleate a multi-component signaling (data not shown). Finally, the inability of two distinct PLC-binding site mutants to complement each other in NF-AT transcriptional activation (Figure 8B), and the partial restoration of NF-AT transcriptional activation by a BLNK molecule capable of binding both PLC2 and Btk (Figure 9C), is likewise consistent with the notion that BLNK provides a molecular platform on which specific signaling components are spatially organized in cis for appropriate activation (Figure 9A). The ability of cBLNK(Y103/115/194/205 only) to partially restore [Ca²⁺]_i mobilization and NF-AT transcriptional activation (as compared with wild-type cBLNK) suggests that other signaling effector proteins through Ys 91 and 248 in cBLNK (and Y72 within hBLNK) are also required for normal levels of NF-AT activation. Candidates for Y91 include Vav and Nck, both of which can bind a phosphopeptide encompassing Y91 (Figure 4B) and have been demonstrated to play important roles in the [Ca²⁺]_i and NF-AT transcriptional responses (Costello et al., 1999).

While others have reported a stable association of Btk with BLNK (Hashimoto et al., 1999), we have been unable to demonstrate a high stoichiometry of association of Btk with wild-type BLNK, although some degree of association has been intermittently observed (data not shown). This may reflect the transient nature of this interaction and/or the inability to co-immunoprecipitate complexes that are less accessible to detergent solubilization. As a result, we were unable to interpret the inability of cBLNK(Y115F) mutant to interact with Btk. A model has recently been proposed in which Btk tyrosine phosphorylation and activation occur in a BLNK-dependent fashion (Baba et al., 2001). However, we were unable to demonstrate any differences in Btk phosphorylation or kinase activity in BLNK-expressing or -deficient DT40 cells (data not shown). In addition, no differences in Btk phosphorylation and activation were observed in pro-B cell cultures derived from the bone marrows of blnk^+/- and blnk^-/- mice (our unpublished data). These data do not favor a model in which Btk activation is dependent upon BLNK, but rather a model in which Btk is activated in a BLNK-independent fashion. Additionally, the tyrosine phosphorylation of BLNK and association of PLC2 with BLNK occurs in a Btk-independent fashion (Fu et al., 1998; our unpublished data). While btk^-/- DT40 B cells are unable to mobilize [Ca²⁺]_i following BCR crosslinking (Ishiai et al., 1999), cells expressing cBLNK(Y115F) are still capable of mediating a moderate degree of BCR-induced [Ca²⁺]_i. As the latter still express wild-type Btk, the binding of Btk to Tyr 115 of cBLNK may only partially contribute to PLC2 activation. Given the low affinities of most single domain mediated interactions, it is likely that the multi-modular nature of signaling proteins all contribute to the stable assembly and localization of signaling complexes. Hence, normal [Ca²⁺]_i signaling requires not only both BLNK-dependent PLC2−BLNK interaction and BLNK-independent Btk activation, but also the assembly of Btk−BLNK−PLC2 macromolecular complexes.

The presence of three PLC2 binding sites within BLNK represents an atypical example of scaffolding. In growth factor receptors, a single predominant tyrosine (Y1021 in the PDGF receptor and Y992 in the EGF receptor) binds PLC1 (Rotin et al., 1992; Kashishian and Cooper, 1993; Larose et al., 1993; Valius et al., 1993). In T cells, phosphorylation of Y192 within the transmembrane 'linker for activation of T cells' (LAT) adapter protein binds PLC1 (Zhang et al., 2000; Paz et al., 2001). Our combinatorial analysis of the three PLC2 binding sites within BLNK supports an amplification role for PLC-mediated signaling. As [Ca²⁺]_i represents a major regulator of biological function, differential BLNK phosphorylation may alter the dynamics and kinetics of second messenger generation. Finally, as tyrosine phosphorylation of BLNK regulates many signaling pathways, differential BLNK phosphorylation may also result in the activation of subsets of signaling functions that alter the cellular fate of a given B cell response. Additional studies are ongoing to determine if tyrosine phosphorylation of BLNK may represent such a key regulatory point in determining the outcome of B cell function.

Materials and methods

Cells, antibodies and plasmids

Parental DT40 B cells and their derivatives were cultured in RPMI-1640 supplemented with 10% FCS, 1% chicken serum, 50 M 2-mercaptoethanol, 2 mM L-glutamine and antibiotics. Anti-BLNK antiserum was generated by immunizing rabbits with a bacterially expressed GST fusion protein containing human BLNK (aas 4−205). This antiserum reacts with both human and chicken BLNK (data not shown). Phosphospecific antibodies were generated at Biosource International (Hopkinton, MA) by immunizing rabbits with coupled peptides corresponding to each of the phosphorylated tyrosine residues of murine BLNK (Y72, aas 67−79; Y84, aas 79−90; Y96, aas 93−104; Y178, aas 172−184; and Y189, aas 184−196). Sera were negatively depleted by affinity chromatography using unphosphorylated peptide and the flow-through purified with phosphorylated peptide. These antibodies react with both human and murine BLNK (data not shown).

Additional antibodies used in this study include: anti-hBLNK mAb (2B11 or 2C9), anti-phosphotyrosine mAb (4G10, UBI and PY20; Transduction Labs), anti-chicken IgM mAb (M4; courtesy of Dr Max Cooper), anti-myc mAb (9E10), anti--actin (Sigma), anti-GST (Sigma), anti-pErk2 (Promega), anti-Erk2 (Santa Cruz Biotech), anti-pJNK (Promega), anti-PLC2 (Santa Cruz Biotech), anti-Btk (Pharmingen), anti-Vav (Santa Cruz Biotech), anti-Nck (Santa Cruz Biotech), anti-JNK (Pharmingen), anti-phospho p38 (New England Biolabs) and anti-p38 (Santa Cruz Biotech) antibodies.

GST fusion proteins were produced using the pGEX-KT vector (Hakes and Dixon, 1992). Mutants of BLNK were produced by a PCR-directed mutagenesis strategy. All junctions and PCR products were confirmed by standard dideoxy DNA sequencing.

Transfection of cells and biochemical analysis of cells

Stable transfectants were generated by electroporating 10⁷ blnk^-/- DT40 cells with 20 g of pApuro containing wild-type BLNK or BLNK mutations as described previously (Kong et al., 1995). Analysis of NF-AT transcriptional activation was performed as described previously (Kong et al., 1995).

Cells were resuspended at 10⁸ cells/ml in PBS for 15 min at 37°C. DT40 cells and their derivatives were crosslinked with an anti-BCR M4 mAb (4 g/ml) for the indicated time periods described in the figures at 37°C. Cells were sedimented at 5000 g and lysed in an equal volume of 10 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40 supplemented with protease and phosphatase inhibitors (lysis buffer) for 15 min at 4°C. Cellular debris was sedimented at 15 000 g for 10 min at 4°C and the supernatant harvested for studies.

For immunoprecipitations, cellular lysates were incubated with antiserum for 2 h and captured with protein A−Sepharose (Pharmacia) for 1 h at 4°C. Immune complexes were washed three times with lysis buffer (1 ml each) and prepared for analysis. Protocols for SDS−PAGE and immunoblotting have been published previously (Chan et al., 1995).

For the phosphorylated peptide precipitation assays, resting Daudi cell lysates were incubated with each of the peptides for 2 h and captured with streptavidin agarose (Sigma) for 20 min at 4°C. The associated complexes were washed three times with lysis buffer (1 ml each) and prepared for analysis.

Retroviral infections were performed utilizing the Phoenix amphotrophic packaging cell line using standard protocols (Swift et al., 1999). In brief, the Phoenix cells were transfected with a plasmid expressing human PLC2 and a GFP marker using calcium phosphate precipitation. The resulting supernatant was placed onto the target DT40 derivatives with 10 g/ml protamine sulfate and spun at 2 000 r.p.m. for 1 h. The infected cells were subsequently sorted based on fluorescence.

Fluorimetry

For analysis of intracellular calcium levels, cells were loaded with Fura-2 (Molecular Probes) and calcium-sensitive fluorescence was monitored using a Hitachi F2000 Fluorescence spectrophotometer at wavelengths 340 nm and 540 nm. Cells were stimulated with soluble anti-BCR M4 mAb (4 g/ml). Maximal fluorescence was determined following lysis of cells with Triton X-100 while minimum fluorescence was determined following chelation with EGTA.

Analysis of ³²P-labeled BLNK peptides

Daudi B cells expressing a myc-epitope tagged hBLNK were labeled with [³²P]orthophosphate for 4 h in phosphate-deficient media and analyzed under resting or BCR-activating conditions. BCR stimulation was performed by addition of anti-IgM F(ab')₂ for 2 min and terminated by the addition of cold lysis buffer. BLNK was immunoprecipitated from ³²P-labeled cells with an anti-myc 9E10 mAb, resolved by SDS−PAGE, transferred to nitrocellulose and visualized by autoradiography. Tryptic peptide mapping was performed as described previously (Luo et al., 1991). Tryptic peptides were separated by TLC utilizing HTLE-7000 electrophoretic apparatus (CBS, Del Mar, CA) as described previously (Chan et al., 1995).

For sequencing analysis, labeled peptides were eluted in 0.1 ml pH 1.9 buffer and sonicated as described previously (Hellman et al., 1995). Eluted peptides were cleared by centrifugation at 15 000 g for 10 min at 4°C. Subsequent digestion with endo-gluC (Boehringer Mannheim) was accomplished by washing the peptide three times with H₂O and resuspending it in 0.1 ml of 25 mM ammonium carbonate. Eluted peptides were then digested with 5 g of endo-gluC overnight at room temperature. The sample was heated to 85°C for 30 min to inactivate the remaining endo-gluC, washed three times with H₂O and resuspended in 0.02 ml of 30% acetonitrile for manual Edman sequencing. Manual Edman sequencing was performed using the Sequelon AA Reagent Kit (Millipore) according to manufacturer's instructions.

Supplementary data

Supplementary data are available at The EMBO Journal Online.

Acknowledgements

The authors thank Alec Cheng and members of the Chan laboratory for critical review of the manuscript.

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