Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7865-7870, July 1996 Immunology

Evidence for a preformed transducer complex organized by the B cell antigen receptor (tyrosine phosphatase/tyrosine kinase/signal transduction) JORGEN WIENANDS*, OLIVER LARBOLETITE, AND MICHAEL RETH Max-Planck-Institut fur Immunbiologie, Stubeweg 51, D-79108 Freiburg, Germany Communicated by Klaus Rajewsky, Universitdt zu Koln, Cologne, Germany, April 23, 1996 (received for review March 21, 1996)

ABSTRACT The B cell antigen receptor (BCR) consists of one or more PTKs and which PTK is the first to become the membrane-bound immunoglobulin (mlg) molecule and activated. A direct structural analysis of the resting and/or the Ig-a/Ig-18 heterodimer, which functions as signaling sub- activated BCR is hampered by the fact that lysis of cells by unit of the receptor. Stimulation of the BCR activates protein detergent can disrupt important interactions between the tyrosine kinases (PTKs) that phosphorylate a number of receptor and intracellular proteins. This makes it difficult to substrate proteins, including the Ig-c/Ig-I8 heterodimer ofthe know whether the small number ofBCR molecules (<5%) that BCR itself. How the PTKs become activated after BCR are associated with PTKs before receptor stimulation (17) engagement is not known at present. Here, we show that reflects a low stoichiometry of association or whether this BCR-negative J558L cells treated with the protein tyrosine small number is a consequence of the experimental proce- phosphatase inhibitor pervanadate/H202 display only a weak dures. substrate phosphorylation. However, in BCR-positive trans- The myeloma cell line J558L was used previously to identify fectants of J558L, treatment with pervanadate/H202 induces the signaling components of the BCR (22-24), to functionally a strong phosphorylation of several substrate proteins. Treat- characterize the ITAM (5), and to analyze the role of CD45 ment with pervanadate/H202 does not result in receptor (25). Like other myelomas, the J558L line has switched off crosslinking, yet the pattern of protein phosphorylation is many genes for surface markers on B cells, including CD19, similar to that observed after BCR stimulation by antigen. The CD20, CD21, CD22, CD40, and CD45. Crosslinking of the response requires cellular integrity because tyrosine phos- BCR on mlg/mb-1 transfectants of this line, however, still phorylation of most substrates is not visible in cell lysates. results in a rapid PTK activation and phosphorylation of Cells that express a BCR containing an Ig-a subunit with a substrate proteins, many ofwhich become also phosphorylated mutated immunoreceptor tyrosine-based activation motif dis- in B lymphoma cell lines and normal B cells (26). play a delayed response. The data suggest that, once expressed Treatment of living cells with pervanadate/H202 inhibits on the surface, the BCR organizes protein tyrosine phos- protein tyrosine phosphatases (PTPs), thereby modifying the phatases, PTKs, and their substrates into a transducer com- intracellular equilibrium between dephosphorylation and plex that can be activated by pervanadate/H202 in the absence phosphorylation (27-31). In the present study, we have used of BCR crosslinking. Assembly of this preformed complex pervanadate/H202 to activate protein tyrosine phosphoryla- seems to be a prerequisite for BCR-mediated signal trans- tion in J558L and different transfectants of J558L. We describe duction. here that only BCR-positive transfectants of this line re- sponded to the drug with a rapid and efficient protein tyrosine The B cell antigen receptor (BCR) is composed of multiple phosphorylation. subunits. Antigen bound by transmembrane immunoglobulins (mIg) of different classes transmits an activation signal to the cell interior through the mlg-associated Ig-a/Ig-f3 heterodimer MATERIALS AND METHODS (1, 2). The immunoreceptor tyrosine-based activation motif Cell Culture. The J558L myeloma cells and its transfectants (ITAM) (3) plays a critical role in this process and is respon- were maintained in RPMI 1640 medium supplemented with sible for the communication of the BCR with cytoplasmic 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml peni- protein tyrosine kinases (PTKs) (4-8). The amino acid se- cillin, 50 mg/ml streptomycin, and 200 mM 2-mercaptoetha- quence of ITAM is DEx6D/ExxYxxLx7YXXI/L and a single nol. Surface IgD-positive transfectants of J558L are copy of the motif is present in the cytoplasmic part of both Ig-a 558Lam7.1, expressing wild-type IgD-BCR (32), and and Ig-13. The current model of BCR signaling predicts that J558L8m2.6, expressing a glycosylphosphatidylinositol-linked crosslinking of the BCR results in activation of the Src-related form of IgD (33). The J558L,um15-25 cells express the mIgM PTKs, Lyn, Blk, Fyn, and/or Lck (9-11), which first phos- molecule intracellularly but not on the cell surface (34). phorylate one or two tyrosine residues of the ITAMs of both Surface IgM-positive transfectants are J558L,um3, which ex- Ig-a and Ig-3 (5). This in turn leads to the SH2-mediated presses a wild-type IgM-BCR (34), and J558LM1, J558LM4, recruitment of the PTK Syk from the cytosol to the receptor and J558LM6, which produce an IgM-BCR with a mutated and its subsequent activation (6-8). Thereafter, a number of Ig-a subunit (5). The IgM molecule of J558L,uTM cells additional effector molecules and PTK substrates like HS1 contains the transmembrane part of the major histocompati- (12-14) and SHC (13, 15) are recruited into the complex and bilty complex class I protein and is transported onto the cell become activated. Indeed, the use of PTK inhibitors, coim- surface independently of Ig-a and Ig-13 (35). The J558Ltks cells munoprecipitation studies, and gene targeting experiments secrete the IgM molecule (34). The transfectants J558L-y2am/ have shown that PTK activity is essential to BCR signaling for mbl and produce mIgG2a that is expressed on the both early and late events (16-21). However, it is a matter of J558Ly2am debate whether the unligated BCR is already associated with Abbreviations: BCR, B cell antigen receptor; ITAM, immunoreceptor tyrosine-based activation motif; mlg, membrane-bound form of im- The publication costs of this article were defrayed in part by page charge munoglobulins; PTK, protein tyrosine kinase; PTP, protein tyrosine payment. This article must therefore be hereby marked "advertisement" in phosphatase; NIP, hapten 4-hydroxy-5-jodo-3-nitrophenylacetyl. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

7865 Downloaded by guest on October 2, 2021 7866 Immunology: Wienands et al. Proc. Natl. Acad. Sci. USA 93 (1996) cell surface either as wild-type IgG2a-BCR or independent of previously shown (27-31), treatment of cells with pervana- Ig-a/Ig-,3 (36). All mlg molecules of the different cell lines date/H202 inhibits PTPs and results in an increased tyrosine carry the antigen-binding site of the B1-8 idiotype and are thus phosphorylation of PTK substrate proteins. When BCR- specific for the hapten 4-hydroxy-5-jodo-3-nitrophenylacetyl negative (-) J558L myeloma cells are incubated for 2 min with (NIP). increasing doses of pervanadate/H202 (Fig. la) only two Stimulation of Cells. For stimulation of the cells with proteins, plOO and p120, become tyrosine phosphorylated. pervanadate/H202, 1 ml of a 20 mM sodium orthovanadate This phosphorylation event is first detected at a concentration stock solution was mixed with 330 gl of 30% H202 and of 25 ,M pervanadate/H202 (Fig. la, lane 7) and increases incubated for 5 min at room temperature, yielding a solution slightly with higher doses of pervanadate/H202 (lanes 9, 11, of 6 mM pervanadate plus remaining H202. To stimulate cells and 13). In the IgD-BCR positive (+) transfectant with 25 ,uM pervanadate/H202, the prepared solution was J558L8m7.1, even low doses of pervanadate/H202 activate diluted 1:5 with H20 and 5 ,lI was added to 1-5 x 107 cells tyrosine phosphorylation and additional substrates were de- suspended in 1 ml RPMI 1640 medium without supplements. tected. Apart from plOO and p120, substrate proteins of 80, 72, As pervanadate/H202 is unstable, a fresh solution was pre- 65, 55, and 35 kDa are phosphorylated only in the transfectant. pared each time. The sodium orthovanadate stock solution, Using PTK specific antibodies, p72 and p55 were identified as which is also unstable, was titrated from time to time to ensure Syk and Lyn, respectively (data not shown). The p35 phos- optimal results. BCR crosslinking was induced by incubating phoprotein was recognized by the tandem SH2 domains of Syk the cells with 10 ,tg/ml NIP-coupled bovine serum albumin (NIP7-BSA). All stimulations were carried out at 37°C. Ap- (data not shown), which are known to bind specifically to proximately 20 sec before the end of the desired stimulation tyrosine-phosphorylated Ig-a (7). Note that a dose as low as 5 time, cells were pelleted and lysed for 25 min on ice in 1 ml lysis ,uM pervanadate/H202 induces weak phosphorylation of p65 buffer consisting of 137 mM NaCl, 50 mM Tris-HCl (pH 7.8), (lane 4), whereas phosphorylation of plOO, p120, and p80 10% vol/vol glycerol, 1 mM sodium orthovanadate, 2 mM requires 10 ,uM (lane 6) and that of Syk, Lyn, and Ig-a requires EDTA (pH 8.0), 1% vol/vol Nonidet P-40, plus protease 25-50 ,uM of pervanadate/H202 (lanes 8 and 10). Phosphor- inhibitors leupeptin and aprotinin (10 ,ug/ml each), and 1 mM ylation of all of the substrates was further enhanced at higher phenylmethylsulfonyl fluoride. Cell debris were spun down for doses of pervanadate/H202 (lanes 12 and 14). This shows that 20 min at 16,500 x g at 4°C and cleared cellular lysates were substrates in the BCR-positive J558LSm7.1 transfectants are analyzed by anti-phosphotyrosine immunoblotting using the more sensitive to PTK phosphorylation induced by pervana- 4G10 antibody (Upstate Biotechnology, Lake Placid, NY) as date/H202 than the BCR-negative J558L parental cells. described (13). A time course experiment reveals further differences be- In Vitro Kinase Assay. To stimulate protein tyrosine phos- tween the two cell lines studied (Fig. lb). In J558L cells phorylation in cellular lysates, 1-5 x 107 cells were lysed in 1 incubated with 25 ,M pervanadate/H202, the phosphoryla- ml 50 mM Tris HCl (pH 7.4), 5 mM MnCl2, 0.1 mM NaVO3, tion of plOO and p120 requires an incubation time of at least and protease inhibitors, and the lysates were cleared as de- 2 min (lane 9), whereas in J558L6m7.1 cells, phosphorylation scribed above. ATP was added to a final concentration of 250 ofplOO, p120, and p65 is already visible 10 sec after stimulation AM and the samples were incubated at 37°C for 15 min in the (lane 4). Phosphorylation of the other PTK substrates in presence of either recombinant PTKs obtained from baculo- J558L8m7.1 such as p80, Syk, Lyn, and Ig-a requires a longer virus-infected insect cells or with various concentrations of incubation with pervanadate/H202 (lanes 6, 8, and 10). Max- pervanadate/H202. The reactions were stopped by adding imal phosphoprotein content is reached 3 min poststimulation Laemmli's sample buffer and boiling for 4 min at 95°C. (lane 12) and gradually declines thereafter (lanes 14, 16, and 18). Together, these results demonstrate that in pervanadate/ H202-treated J558L cells, PTK substrate proteins are phos- RESULTS phorylated either very inefficiently (in the case of plOO and The Pervanadate/H202-Induced Phosphorylation of Most p120) or not at all (in the case of p65, p80, Syk, Lyn, and Ig-a) PTK Substrates in J558L Requires BCR Expression. As unless these cells express a BCR on their cell surface. Thus,

0 5 10 25 50 75 ioo [PM] 0 10 30' 1' 2' 3' 4' 5' 10' [time] -+ -+ -+-- -+ - + - + BCR -+-+-+ +--+-+- + - + BCR

112 :za as a Ali ..A. - 'I" .0i, - i -I pl 00/1 20 112 _ - 20 - pl 00/1 lmw- I..,.Ir I. p80 p80 84 = dEft 40* 84 - , - Syk - Syk on* 4m Sb qj. 2 " p65 x p65 53 - - Lyn 53 - - Lyn

- Ig-a 35 - .-all A* .i, as - Ig-a 35 -

1 2 3 4 5 6 7 8 910 1112 13 14 1 2 3 4 5 6 7 8 9 1011 1213 14 15 16 1718 ANTI - YP ANTI - YP FIG. 1. Pervanadate/H202 induces a strong and rapid PTK substrate phosphorylation only in BCR-expressing J558L cells. (a) Dose response of pervanadate/H202-induced protein tyrosine phosphorylation in the BCR-negative (-) J558L parental cells (lanes 1, 3, 5, 7, 9, 11, and 13) and in the IgD-BCR positive (+) transfectant J558L6m7.1 (lanes 2, 4, 6, 8, 10, 12, and 14). Approximately 1 x 107 cells per ml in RPMI 1640 medium without supplements were stimulated for 2 min at 37°C with the indicated amounts ofpervanadate/H202 and than lysed in 1% Nonidet P-40. Cleared cellular lysates were analyzed by anti-phosphotyrosine immunoblotting. Each lane contains proteins obtained from 5 x 105 cells. Numbers on the left indicate the relative molecular mass in kDa. Prominent PTK substrate proteins are indicated on the right. (b) Time course of pervanadate/ H202-induced protein tyrosine phosphorylation in J558L (lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17) and J558L8m7.1 (lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18). The cells (1 X 107 cells per ml) were stimulated for the indicated times with 25 ,uM pervanadate/H202 at 37°C. Downloaded by guest on October 2, 2021 Immunology: Wienands et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7867

expression of a BCR in J558L seems to result into a reorga- to the cell surface due to the lack of Ig-a, and J558L,uTM (lane nization of PTKs and their substrate proteins. This would 7), which expresses a cell surface chimeric mIgM protein mean that there is a transducer complex organized by the BCR, containing the transmembrane part of the H2-K protein. The which can be rapidly activated by pervanadate/H202 only in mIgG2a molecule can be expressed either alone or as an BCR-positive J558L cells. It is worth noting that we did not find IgG2a-BCR in association with the Ig-a/Ig-,3 heterodimer on any biochemical evidence for a direct effect of pervanadate/ the cell surface of J558L (24, 36). The two forms of IgG2a are H202 on the BCR itself. First, incubation of BCR-positive B expressed on the transfectants J558Ly 2am and J558Lmbl/,y cells with different doses of pervanadate/H202 did not result 2am, respectively. After treatment of these transfectants with in patching or capping of the BCR, which was easily detected pervanadate/H202, only the latter shows phosphorylation of after crosslinking of the BCR with anti-g antibodies (data not p80, p65, and Ig-a (lane 13). This confirms that the Ig-a/Ig-f3 shown). Second, upon incubation of soluble IgM or IgG with heterodimer is required for the pervanadate/H202-induced doses of pervanadate/H202 up to 100-fold higher than those phosphorylation of these substrate proteins. used in our stimulation protocol, no covalent linkage between Ig-a and Ig-13 both carry an ITAM sequence that mediates the antibody molecules could be detected by a subsequent the interaction between the BCR and intracellular signaling Western blot analysis (data not shown). Thus, pervanadate/ molecules and that may thus be involved in the organization of H202 does not lead to a linkage of BCR molecules that could the BCR transducer complex. We therefore analyzed the account for receptor crosslinking and aggregation. transfectants J558LM1, J558LM4, and J558LM6, which pro- The Efficient Response to Pervanadate/H202 Depends on duce a mutated Ig-ca subunit. J558LM1 and J558LM4 express Ig-a/Ig-13 Expression and the ITAM Sequence. The phenom- an Ig-a protein in which either both or only the first of the two enon of BCR-dependent substrate phosphorylation was ana- ITAM tyrosines were changed to phenylalanine, respectively. lyzed in different Ig-transfectants of J558L (Table 1) stimu- In J558LM6, the Ig-a protein has an almost complete deletion lated for 2 min with 100 ,uM pervanadate/H202 (Fig. 2). The of its cytoplasmic part. The mutated Ig-a proteins of these cells same set of BCR-dependent substrate proteins efficiently are expressed together with wild-type Ig-f3 as part of an phosphorylated in the IgD-BCR-positive J558L8m7.1 (lane 4) IgM-BCR that is transported to the cell surface in amounts are also seen in the IgM-BCR-positive J558L,um3 cells (lane similar to the wild-type IgM-BCR in J558L,um3 (data not 8). However, other Ig transfectants of J558L that lack Ig-ar shown). In response to pervanadate/H202, all BCR- expression are similarly to the J558L parental cells (lane 2) in dependent substrate proteins (apart from Ig-a) are still phos- that only plOO and p120 become phosphorylated. These trans- phorylated in the mutant cell lines (lanes 9-11). In J558LM1 fectants are J558LSm2.6 (lane 3), which express a glyco- cells, however, phosphorylation seems to be less efficient than sylphosphatidylinositol-linked IgD molecule, J558L,us (lane in J558L,um3 cells (compare lanes 8 and 9). To analyze the 5), expressing the secreted form of IgM, J558L,uml5-25 (lane difference between the wild-type (w) and Mi-mutated (m) 6), which produces mIgM molecules that are not transported IgM-BCR in more detail, we compared the kinetics of per- Table 1. Phenotype of different J558L transfectants J558L cell Response to line BCR components Surface expression pervanadate/H202 Parental Al-light chain, Ig-j3, Sm2.6 S-heavy chain, Glycosylphosphatidylinositol-linked IgD Al-light chain, Ig-13 8m7.1 Bm-heavy chain, Wild-type IgD-BCR + Al-light chain, Ig-a, Ig-13 ,uLs secreted ,u-heavy chain, Al-light chain, Ig-,B ,uml5-25 ,um-heavy chain, Al-light chain, Ig-,B ,uTM ,u-heavy chain with IgM without Ig-a/Ig-3 transmembrane part of MHC-I, Al-light chain, Ig-,3 ,um3 ,um-heavy chain, Wild-type IgM-BCR + Al-light chain, Ig-a, Ig-,B Ml ,um-heavy chain, IgM-BCR Delayed Al-light chain, Ig-aMl,* Ig-,B M4 ,um-heavy chain, IgM-BCR +§ Al-light chain, Ig-aM4,t Ig-0 M6 ,um-heavy chain, IgM-BCR +§ Al-light chain, Ig-aM6,* Ig-,d y2am y2am-heavy chain, IgG2a without Ig-a/Ig-P3 Al-light chain, Ig-f3 mb-l/-y2am y2am-heavy chain, Wild-type IgG2a-BCR + Al-light chain, Ig-,B *Both tyrosine residues of the Ig-a ITAM are mutated to phenylalanine. tFirst tyrosine residue of the Ig-a ITAM is mutated to phenylalanine. tIg-a mutant, lacking 52 amino acids of its cytoplasmic part including the ITAM. §Kinetic of tyrosine phosphorylation not tested. Downloaded by guest on October 2, 2021 7868 Immunology: Wienands et al. Proc. Natl. Acad. Sci. USA 93 (1996)

s phosphorylated after 30 sec (lane 5) and 1 min (lane 7). The ( 0 to substrate phosphorylation peaks at 2.5 to 5 min (lane 9 and 11)

______- ______(V and declines with longer incubation periods (lane 13). This '..Z C -T'MsacoEv` VtEI E$t.vco gtr s o h time course is very similar to that observed in J558L,m7.1 cells Q Qy o6-tz 4-t Z , (see Fig. lb). The response of the J558LM1 mutant is signif- icantly delayed in that plOO/pl2O is first phosphorylated after 30 sec (lane 6) and the phosphorylation ofp65 and p80 requires 112- 4 p100/120 1 min (lane 8). These data show that an efficient pervanadate/ H202-induced phosphorylation of BCR-dependent substrates thus 84 - 4 p80 require the wild-type sequence of the Ig-a ITAM. We conclude that the tyrosine residues of the Ig-a ITAM (and 4 p65 probably also those of the Ig-13 ITAM) not only serve as PTK 4 Lyn substrates after BCR triggering, but also have a function in the 53 - organization of the BCR transducer complex. The Induction of BCR-Regulated Substrate Phosphoryla- tion Requires Cellular Integrity. We next asked whether the 4 Ig- P phosphorylation difference between J558L and BCR-positive J558L8m7.1 cells could also be observed when detergent 35 - 4 Ig-ta lysates of these cells were incubated with pervanadate/H202. After incubation of intact cells (Fig. 4, lanes 3 and 8) and 29 - Nonidet P-40 lysates of these two cell lines (Fig. 4, lanes 4 and 1 234 5678910111213 9) with 100 ALM of pervanadate/H202 for 10 min, most BCR-dependent PTK-substrates did not become phosphory- ---- ANTI - YP lated in the cell lysates. In particular, phosphorylation of p120, FIG. 2. The pervanadate/H202-induced phosphorylation of the plOO, p65, and Ig-a is induced by pervanadate/H202 treatment BCR-dependent PTK substrates in J558L requires the expression of in living J558LSm7.1 cells but not in cell lysates (Fig. 4, the Ig-a/Ig-,B heterodimer. Substrate phosphorylation was induced compare lanes 8 and 9). However, one of the substrates (p80) with 100 ,uM pervanadate/H202 for 2 min at 37°C in J558L parental was more strongly phosphorylated by postlysis pervanadate/ cells (lane 2) and different mb-i and/or mIg transfectants of J558L H202 treatment of J558L and J558LSm7.1 cell lysates than by (lanes 3-13). The transfectants express a wild-type or mutant BCR of treatment of the intact cells. Total cellular lysates of both cell different classes. Lanes: 3, glycosylphosphatidylinositol-linked mIgD; in kinase assay using a 4, wild-type IgD-BCR; 5, secreted form of IgM; 6, cytoplasmic mIgM lines were also subjected to an vitro without Ig-a; 7, mutated IgM with the extracellular part of IgM fused to the transmembrane part of MHC class I [H2-Kk]; 8, wild-type J558L J558L8mZ1 IgM-BCR; 9, IgM-BCR containing an Ig-a subunit with two Y>F mutations; 10, IgM-BCR containing an Ig-a subunit with one Y>F 11, containing a deletion mutant ofIg-a that lacks mutation; IgM-BCR ______bC 9 Z.Z 9 Q. t the cytoplasmic part; 12, surface IgG2a without Ig-a3; 13, wild-type -0 -.0 Pff!QA IgG-BCR. For methods, see Fig. 1. vanadate/H202-induced protein phosphorylation in J558Lttm3 and J558LM1 cells (Fig. 3). The substrate p65 is 112 = 4 p1 tyrosine phosphorylated within 10 sec of stimulation of 00 J558L,m3 cells (lane 3). Other substrate proteins become 84 - 4 p80 - 4*0 mm 4 p65 O 1i0 30' 1' 2.5' 5' 10' [time] I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ wm wm wmwmwmwm wm BCR 53 _ 4 p56

112 _ - pl 00/1 20 a.hi. . 4 Ig-a p80 - 84 - - Syk 35 -4 Ig-cz rn4j&* x p65 29 - - Lyn 53 - 1 2 3 4 5 6 7 8 9 10 ANTI yp FIG. 4. Lysis of the cells by detergent prevents the pervanadate/ 35 - - Ig-a H202 induced phosphorylation of most PTK substrates in J558L. J558L parental cells (lanes 1-5) and IgD-BCR expressing J558L8m7.1 cells (lanes 6-10) were left untreated (lanes 1 and 6) or were stimulated 1 2 3 4 5 6 7 8 9 1011121314 for 2 min at 37°C with either 10 ,ug/ml NIP7-BSA (lanes 2 and 7) or ANTI YP with 100 ,uM pervanadate/H202 (lanes 3 and 8). Alternatively, cleared cellular lysates from J558L cells (lanes 4 and 5) or from J558LSm7.1 FIG. 3. A Y>F mutation of the two tyrosines of the Ig-a ITAM cells (lanes 9 and 10) were incubated for 2 min at 37°C with 100 ,uM sequence delays the pervanadate/H202-induced substrate phosphor- pervanadate/H202 (lanes 4 and 9) or in an in vitro kinase assay for 10 ylation. J558L,m3 cells expressing the wild-type IgM-BCR (w) (lanes min at 37°C in the presence of 100 ,uM ATP with a mixture of the 1, 3, 5, 7, 9, 11, and 13) and J558L,umM1 cells expressing an IgM-BCR baculovirus-expressed PTKs: Syk, Lyn, and Blk (lanes 5 and 10). The with a mutated Ig-a (m) (lanes 2,4,6,8, 10, 12, and 14) were stimulated phosphoproteins from all samples were affinity-purified by Glutathion with 25 ,uM pervanadate/H202 at 37°C for the indicated times. For beads loaded with 10 jig/ml GST-SH2 fusion protein containing the methods, see Fig. 1. SH2 domain of Src. For methods, see Fig. 1. Downloaded by guest on October 2, 2021 Immunology: Wienands et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7869 mixture of the baculovirus produced PTKs Syk, Lyn, and Blk. lines showing that the pervanadate/H202-induced tyrosine In this assay, nearly the same pattern of phosphoproteins was phosphorylation does not require surface expression of a T cell found as upon postlysis stimulation with pervanadate/H202 receptor TCR (28, 29). We have been able to reproduce these (Fig. 4, compare lanes 5 and 10 with lanes 4 and 9). In published results in our laboratory using both a TCR-positive summary, using two different assays, we did not detect a and a TCR-negative variant of the human T cell line Jurkat difference in phosphorylation between the cell lysates ofJ558L (data not shown). Beside the TCR, however, the T cells studied and J558L8m7.1. These experiments indicate that PTKs, and may have several receptors wvhose transducer complex may most of their substrates, are separated from each other in the become activated by pervanadate/H202. In contrast, the lysate suggesting that the BCR-organized transducer complex J558L myeloma line seems to lack many such receptors. Many is disrupted upon exposure to detergents. This may explain B cell surface markers, such as CD19, CD20, CD22, CD23, and why such a complex has not been detected by a commonly used CD45, are not expressed on these cells and while all of the T method, i.e., immunoprecipitation. Fig. 4 also shows that upon cell lines as well as a number of different B cell lines respond treatment of J558L8m7.1 cells either with the antigen NIP- to fetal calf serum treatment with the induction of protein BSA (Fig. 4, lane 7) or with pervanadate/H202 (Fig. 4, lane 8), tyrosine phosphorylation, no such response is observed in the same substrate proteins become phosphorylated, although J558L cells (data not shown). The BCR-positive transfectants the antigen-induced phosphorylation is much weaker than that of J558L are thus useful cells to study the signaling function of induced by pervanadate/H202. the BCR without interference from other receptors. The rapid onset of tyrosine-phosphorylation observed in DISCUSSION activated B cells cannot easily be explained by diffusion and/or stimulation-dependent recruitment of signaling proteins to the Using pervanadate/H202 to change the PTP-PTK balance BCR. If, however, most or all BCR-regulated signaling ele- inside living cells, we have found a striking difference between ments are already connected with the unligated BCR, their J558L parental cells and BCR-positive transfectants. Only activation should be more rapid and more specific. Our BCR-positive cells respond to the drug with a rapid and concept of a preformed BCR transducer complex would thus efficient protein tyrosine phosphorylation. Thus, surface ex- fit with the observed kinetic of BCR signaling. Other receptors pression of the BCR may cause a redistribution of relevant on the B cell surface might not be able to organize a transducer PTKs and their substrates, presumably by organizing these complex by themselves, but could use the BCR transducer proteins into a BCR transducer complex. In the absence of a complex once it is assembled by the BCR. For example, BCR, these proteins seem to be separated from each other, signaling through CD38 on B cells requires surface expression thereby preventing a successful activation of the phosphory- of a BCR together with Ig-a/Ig-,3 (37). Furthermore, in lation cascade. Because pervanadate and H202 act inside the immature B cell lines, anti-CD38 antibodies induce tyrosine cells without crosslinking the BCR, our results suggest that phosphorylation and activation of a subset of proteins similar some of these signaling components are already associated to that induced with anti-IgM antibodies (38). Conversely, in with the resting (i.e., the unligated) BCR and constitute a some cases CD2, Thy-1 and Ly-6 require expression of the preformed BCR-associated transducer complex. TCR on the T cell surface to transduce an activation signal A good candidate for a BCR transducer element is p65. It (39). is the first PTK substrate to become phosphorylated after The PTP1C tyrosine phosphatase has been implicated in ligation of the BCR by antigen (26) as well as after treatment negative feedback control to terminate signaling from the of BCR-positive J558L cells with pervanadate/H202. Its phos- antigen-ligated BCR (40-42). Our finding that the PTP in- phorylation requires the presence of the Ig-a/Ig-13 signaling hibitor pervanadate/H202 activates the transducer complex of subunits of the BCR. Furthermore, in cells expressing a BCR is also under with a mutated Ig-a ITAM, the phosphorylation of p65 is the unligated BCR suggests that this complex delayed and less efficient. The residual phosphorylation of p65 negative control of PTPs. This would imply that inhibition of in these cells may be due to the presence of endogenous Ig-f3 PTPs is critically involved in the onset of BCR signaling while with its own ITAM sequence. Another putative BCR trans- turning off the signal involves the activation of PTPs. Indeed, ducer element is p80 that, like p65, becomes tyrosine phos- one or more PTP may be an integral component of the phorylated earlier than the PTKs Lyn and Syk. The late transducer complex as we could coprecipitate a PTP activity phosphorylation of Lyn and Syk in our assay suggests that these with the BCR from unstimulated J558L,um3 cells (data not enzymes are secondary response elements rather than the shown). primary transducer of the BCR. This is also supported by data In summary, our studies suggest that our understanding of from Lyn- or Syk-deficient cell lines (18) or knockout animals the initial events of BCR signaling is still fragmentary and that (19-21) showing that signal transduction from the BCR is not the role of PTKs and PTPs has to be critically reevaluated. completely abolished by deletion of either of these molecules. We thank Dr. Heinrich Flaswinkel for recombinant PTKs, Dr. Peter The finding that pervanadate/H202 induces BCR-depen- Weiser for IgG2a transfectants, and Drs. Lise Leclercq, Peter J. dent substrate phosphorylation only in living cells, but not in Nielsen, and Peter Shaw for critically reading the manuscript and for cell lysates, indicates that association between the BCR and its helpful discussions. This work was supported by the Deutsche For- presumed transducer complex is not resistant to detergents. schungsgemeinschaft (Grant SFB 388). We have tested many different detergents, including digitonin, and none of them preserved the BCR-dependent substrate 1. Reth, M. (1992) Annu. Rev. Immunol. 10, 97-121. phosphorylation. Most strikingly, only a weak tyrosine phos- 2. Cambier, J. C., Pleiman, C. & Clark, M. C. (1994) Annu. Rev. phorylation of Ig-a and Ig-f3 could be detected, even in the Immunol. 12, 457-486. presence of recombinant Lyn, Blk, and Syk. This may be the 3. Reth, M. (1989) Nature (London) 338, 383. reason why a BCR-transducer complex could so far not be 4. Law, D. A., Chan, V. W. F., Datta, S. K. & DeFranco, A. 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