©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of B Cell Antigen Receptor-induced Signaling Events in a Nonlymphoid Cell Line by Expressing the Syk Protein-tyrosine Kinase (*)

(Received for publication, October 5, 1995; and in revised form, January 12, 1996)

James D. Richards (1) Michael R. Gold (2) Sharon L. Hourihane (3) Anthony L. DeFranco (1) Linda Matsuuchi (3)(§)

From the  (1)Department of Microbiology and Immunology, G. W. Hooper Foundation, University of California, San Francisco, San Francisco, California 94143 and the Departments of (2)Microbiology and Immunology and (3)Zoology, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

B cell antigen receptor (BCR) cross-linking activates both Src family and Syk tyrosine kinases, resulting in increased cellular protein-tyrosine phosphorylation and activation of several downstream signaling enzymes. To define the role of Syk in these events, we expressed the BCR in the AtT20 mouse pituitary cell line. These nonlymphoid cells endogenously expressed the Src family kinase Fyn but not Syk. Anti-IgM stimulation of these cells failed to induce most of the signaling events that occur in B cells. BCR-expressing AtT20 transfectants were generated that also expressed Syk. Syk expression reconstituted several signaling events upon anti-IgM stimulation, including Syk phosphorylation and association with the BCR, tyrosine phosphorylation of numerous proteins including Shc, and activation of mitogen-activated protein kinase. In contrast, Syk expression did not reconstitute anti-IgM-induced inositol phosphate production. A catalytically inactive Syk mutant could associate with the BCR and become tyrosine phosphorylated but could not reconstitute downstream signaling events. Expression of the Src family kinase Lck instead of Syk also did not reconstitute signaling. Thus, wild type Syk was required to reconstitute several BCR-induced signaling events but was not sufficient to couple the BCR to the phosphoinositide signaling pathway.


INTRODUCTION

B lymphocytes can recognize a myriad of foreign antigens by virtue of the diversity of their B cell antigen receptors (BCR). (^1)The antigen recognition and binding functions of the BCR are performed by membrane immunoglobulin (Ig), whereas the ability to transduce signals across the plasma membrane is fulfilled by the membrane Ig-associated Ig-alpha/Ig-beta heterodimer(1) . The signaling cascade initiated by Ig-alpha and Ig-beta upon BCR cross-linking can promote different biological outcomes, depending on the differentiation state of the B cell and the nature of additional signals received by the cell. Immature B cells become anergized or undergo apoptosis upon antigen binding, whereas mature B cells enter the cell cycle and can be induced to differentiate into antibody-secreting plasma cells(1) .

The earliest signaling event initiated by BCR cross-linking is an increase in the tyrosine phosphorylation of numerous proteins(2, 3, 4, 5) . Upon stimulation, the cytoplasmic tails of Ig-alpha and Ig-beta become tyrosine phosphorylated. This promotes the binding and activation of intracellular tyrosine kinases(6) . This tyrosine kinase activity is essential for BCR-mediated responses. B cells that are treated with tyrosine kinase inhibitors (7, 8, 9) or that fail to express the correct tyrosine kinases (10) do not activate downstream signaling pathways or exhibit biological responses upon stimulation.

Two types of intracellular tyrosine kinases that have been implicated in BCR signaling are Syk and members of the Src family of tyrosine kinases. Both Syk and the Src family kinases p59, p53/56, p55, and p56co-immunoprecipitate with the BCR and become activated upon BCR cross-linking(1, 11, 12, 13, 14) . Evidence for the significance of these two classes of kinases in BCR signaling has been provided by experiments with a chicken B cell line rendered deficient for Lyn or Syk expression by homologous recombination(10) . Mutant cell lines lacking either Syk or Lyn exhibited markedly decreased anti-Ig-induced protein-tyrosine phosphorylation and activation of phospholipase C. In addition, the Lyn cells failed to tyrosine phosphorylate and activate Syk as effectively as did the wild type cells, implying that Lyn may act upstream of Syk(15) .

To identify lymphoid-specific proteins that functionally link the BCR to the activation of downstream signaling pathways, we have attempted to reconstitute BCR signaling in a nonlymphoid cell line. Previously, we transfected the genes encoding the BCR proteins µ heavy chain, light chain, Ig-alpha, and Ig-beta into the AtT20 mouse pituitary cell line(16) . Although the BCR was expressed on the surface of these cells, cross-linking it with anti-Ig antibodies failed to elicit most of the signaling responses normally associated with the BCR, with the exception that the cytoplasmic tails of Ig-alpha and Ig-beta became tyrosine phosphorylated. Notably, a general increase in protein-tyrosine phosphorylation did not occur upon anti-Ig treatment, suggesting that one or more tyrosine kinases required for BCR function may have been absent. Of the five tyrosine kinases known to be activated by the BCR, the AtT20 cells expressed only Fyn. Because Syk may be essential for BCR signaling, we isolated a cDNA encoding the murine Syk kinase, transfected it into the BCR AtT20 cells, and selected clones that expressed Syk at levels comparable to Syk expression in B cells. Syk expression was sufficient to reconstitute in a stimulation-dependent manner several BCR-induced signaling events in the BCR AtT20 cells, including tyrosine phosphorylation of Shc and activation of MAP kinase. Consistent with a requirement for both a Src family kinase and Syk, BCR signaling in AtT20 cells was not reconstituted by expression of Lck instead of Syk. These results are compatible with a ``sequential kinase'' model of BCR signaling (13, 17) in which Src family kinases are important for the initial tyrosine phosphorylation of Ig-alpha and Ig-beta, whereas subsequent recruitment of Syk to the BCR and Syk activation are required for additional, downstream signaling events to occur. However, Syk expression did not reconstitute activation of the phosphoinositide signaling pathway. Thus, additional lymphoid-specific proteins besides Syk may be required to mediate BCR-induced activation of phospholipase C.


EXPERIMENTAL PROCEDURES

Cell Lines

The mouse endocrine cell line AtT20/D16V-WT#6 (AtT20) (18) was grown at 37 °C in Dulbecco's modified Eagle's medium (Stem Cell Technologies, Vancouver, BC) containing 4.5 g/liter glucose, 10% fetal calf serum (Canadian Life Technologies, Burlington, ON), and penicillin/streptomycin and kept in an atmosphere of 10% CO(2). AtT20 cells expressing the 5HT serotonin receptor, SR1, have been described previously(16) . WEHI-231, Bal17, and A20 B cells were grown as described previously (19) except that the medium was supplemented with 5% FCS. Jurkat T cells were grown in the same conditions as the B cell lines.

Antibodies

To generate anti-Syk antiserum, rabbits were immunized with a synthetic peptide, PYEPTGGPWGPDRGLQREAL (single-letter code), corresponding to amino acids 316-335 of murine Syk, coupled to keyhole limpet hemocyanin. The peptide was synthesized by Dr. Ian Clark-Lewis (Biomedical Research Centre, University of British Columbia), and the antiserum was generated with the assistance of the University of British Columbia Animal Care Facility. In addition, a glutathione S-transferase fusion protein containing amino acids 259-333 of murine Syk was produced in Escherichia coli and purified by glutathione-agarose affinity chromatography. This fusion protein was used to immunize rabbits at Caltag Laboratory, Inc. (Healdsburg, CA). Rabbit anti-porcine Syk antiserum was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit anti-Lck antiserum was a gift from Dr. Pauline Johnson (University of British Columbia). Rabbit anti-Fyn antiserum was a gift from Dr. Roger Perlmutter (University of Washington). Affinity purified rabbit anti-Blk antibodies and rabbit anti-Lyn antibodies were previously described(13) . Rabbit anti-murine Ig-alpha antiserum and mouse anti-phosphotyrosine monoclonal antibody, 4G10, have been described previously(20) . An additional rabbit anti-Ig-alpha antiserum was a gift from Drs. Jason Cyster and Chris Goodnow (Stanford University). Rabbit anti- light chain antibodies were purchased from ICN Biomedicals Canada, Ltd. (St. Laurent, Quebec). Rabbit anti-Shc antibody was purchased from Upstate Biotechnology Inc. Anti-MAP kinase antibody anti-ERK2 (sc-154, Santa Cruz Biotechnology, Santa Cruz, CA) was used for p42 MAP kinase activity assays. For immunoblotting MAP kinase in electrophoretic mobility shift assays, antibody sc-94 was used (Santa Cruz Biotechnology). Affinity purified Goat anti-IgM µ chain specific antibodies used in cell stimulations were purchased from Jackson ImmunoResearch (West Grove, PA) or Bio-Can (Mississauga, ON).

Library Construction and Screening and Sequence Analysis

WEHI-231 cells were grown to log phase, and total RNA was purified(21) . Poly(A) RNA was isolated as described(22) . cDNA was synthesized with reverse transcriptase as recommended by Promega (Madison, WI), except that for first strand synthesis 1000 units of Moloney murine leukemia virus RNase H RT (Promega) and 31 units of avian myeloblastosis virus RT XL (Life Sciences, St. Petersburg, FL) were used. cDNA ends were made blunt by incubating at 37 °C for 30 min with dNTPs and 10 units of T4 DNA Polymerase (NEB, Beverly, MA). Reaction products were extracted with phenol:chloroform and precipitated with ethanol. The cDNAs were ligated to adapters, and fragments less than 1 kilobase were removed following electrophoretic separation. The cDNAs were then ligated into the ZAPII vector (Stratagene, La Jolla, CA) in the XhoI site. The ligation was then packaged with Gigapack II Gold Packaging Extract (Stratagene).

Approximately 220,000 phage were plated on the XL-1 Blue bacterial strain. Phage plaques were transferred in duplicate to Hybond-N filters (Amersham Corp.), and the DNA was cross-linked onto the filters by UV irradiation with a Stratalinker 1800 (Stratagene). The filters were screened with either of two P-labeled DNA probes. The first was a murine syk DNA probe that was generated by polymerase chain reaction using the 5` oligonucleotide primer 5`-GACTACCTGGTCCAGGGGGGC-3` and the 3` primer 5`-GTCTGCCTGCTCAAGAACCCT-3`, which were chosen based on the porcine syk sequence. The second probe was a 1.1-kilobase fragment of the porcine syk gene (gift of K. Chu and D.R. Littman, University of California, San Francisco). Filters were hybridized overnight at 42 °C. Hybridization and washing conditions were as described(23) , except that the filters probed with porcine syk were hybridized with 30% formamide and washed at 37 °C. Four plaques that hybridized with both probes were picked and purified by two additional rounds of screening and excised into pBluescript SK. The longest cDNA, U4.1, was sequenced in both directions using the dideoxy chain termination procedure with Sequenase (U. S. Biochemical Corp.) and found to contain an open reading frame encoding the full-length Syk kinase. The other three clones were partially sequenced and found to be fragments of the same gene. Sequence alignments were performed by the GAP program of the Wisconsin Package (Genetics Computer Group, Madison, WI), which also yielded the percent identity scores.

Site-directed Mutagenesis and Subcloning

To generate the catalytically inactive mutated form of Syk, the lysine at position 396 was mutated to an alanine by site-directed mutagenesis with the oligonucleotide 5`-CTTCAGGATTGCCACAGCCACG-3`, using standard techniques (22) . The open reading frames encoding both wild type and catalytically inactive Syk were subcloned into the HindIII and XbaI sites of the pRc/CMV expression vector (InVitrogen, San Diego, CA).

DNA Transfection and Preparation of AtT20 Cell Lines

A murine B cell antigen receptor-expressing AtT20 cell line was made by co-transfecting cells using the calcium phosphate technique (18) with 20 µg of a plasmid containing the hygromycin resistance gene expressed from a TK promoter, pHS-53, and 25 µg each of the following plasmids: pRSV µ membrane cDNA, pRSV 1 genomic clone, pCMV mb-1, and pLpA B29. These plasmids encoding the four chains of the BCR have been previously described(16) . Transfected cells were selected in complete growth medium containing 175 µg/ml hygromycin (Calbiochem and Boehringer Mannheim), and drug-resistant clones were recovered and screened by immunofluorescence for the expression of surface µ chain using goat anti-mouse µ-fluorescein isothiocyanate (ICN Biochemicals, Mississauga, ON). All four BCR chains must be expressed in order for the BCR to be expressed at the cell surface(16) . The original clone with the highest level of surface µ chain was subcloned three successive times, yielding the recipient cell line for future transfections, 100-33. The 100-33 cell line expresses high levels of µ chain on its surface and is homogeneous. In addition to the immunofluorescence experiments, surface expression of chain, µ chain, and Ig-alpha was confirmed by surface biotinylation of 100-33 cells, selective immunoprecipitation with anti-µ, anti-, and anti-Ig-alpha antibodies, analysis on SDS-PAGE, transfer to nitrocellulose, and identification of biotinylated proteins using streptavidin-HRP and enhanced chemiluminescence detection (ECL, Amersham Corp.).

The 100-33 cell line was transfected as described above, with 20 µg of pSV2neo and 100 µg/transfection of the pRc/CMV expression vector containing either the syk or lck cDNA. A cDNA clone encoding Lck was obtained from Dr. Jamey Marth (Biomedical Research Centre, University of British Columbia). Alternatively, 100 µg of the expression vector encoding Syk or catalytically inactive Syk was introduced into 100-33 cells alone in the pRc/CMV vector. Transfected cells were selected in growth medium containing 0.4 mg/ml geneticin (Life Technologies, Inc.) with individual clones recovered and screened for expression of Lck or Syk protein using specific antibodies.

Cell Stimulation and Preparation of Cell Lysates

Cells grown in 10-cm dishes were washed three times with phosphate-buffered saline containing 1 mg/ml glucose and the medium was replaced with buffer A (25 mM NaHepes, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl(2), 1 mM Na(2)HPO(4), 0.5 mM MgSO(4), 1 mg/ml glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, and 1 mg/ml bovine serum albumin). After 15 min at 37 °C, the cells were washed three times with phosphate-buffered saline/glucose, and fresh 37 °C buffer A was added back. Cells were stimulated for 5 min with 20 µg/ml goat anti-mouse IgM. Reactions were terminated by aspirating the buffer and washing the cells three times with ice-cold phosphate-buffered saline/glucose containing 1 mM Na(3)VO(4). Cells were lysed by adding 1 ml of buffer B (20 mM Tris-HCl, pH 8, 137 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin) to each dish. For MAP kinase enzyme assays, the cells were lysed in 1 ml of buffer C (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40, 1 mM dithiothreitol, 1 mM Na(2)MoO(4), 0.2 mM Na(3)VO(4), 10 µg/ml aprotinin, 2 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 0.23 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor). After rocking for 20 min in the cold, detergent-insoluble material was removed by centrifugation, and the lysates were stored at -80 °C until assayed. Protein concentrations were determined using the bicinchoninic acid assay (Pierce).

Immunoprecipitations

Unless otherwise indicated, immunoprecipitations were performed as follows. Cell lysates were precleared for 1 h with protein A-Sepharose (Sigma), with or without precoating with normal rabbit serum(24) . The appropriate antiserum was added to the precleared lysates with new protein A-Sepharose and incubated with rocking at 4 °C for 1-3 h. Beads were then washed once with buffer B plus 0.5 M NaCl and twice with buffer B, except anti-Shc immunoprecipitates were washed 3 times with buffer B and 1 time with 20 mM Tris, pH 7.5/50 µM Na(3)VO(4). Beads were then pelleted, and bound proteins were eluted with SDS-PAGE sample buffer containing 100 mM dithiothreitol and separated by SDS-PAGE gels.

Immunoblotting

Cell lysates were separated by SDS-PAGE and transferred to BA85 nitrocellulose (Schleicher & Schuell) or Immobilon (Amersham Corp.) membranes. For MAP kinase electrophoretic mobility shift assays, whole cell lysates were separated on 12.5% low bis-acrylamide (12.36% acrylamide, 0.1% bis-acrylamide, final concentrations) SDS-PAGE gels. Filters were blocked for 1 h to overnight with 5% (w/v) bovine serum albumin or nonfat dried milk in TBS (10 mM Tris, pH 7.5, 150 mM NaCl). The filters were washed for 10 min in TBS/0.05% Tween 20 (TBST) or TBST/0.5% Nonidet P-40 (TBST-Nonidet P-40) and then incubated with the primary antibody for 2-3 h at room temperature at the indicated dilution in TBST or TBST-Nonidet P-40. Antibodies used were the 4G10 anti-phosphotyrosine mAb (60 ng/ml), rabbit anti-murine Syk antiserum (diluted 1:2000), rabbit anti-Ig-alpha (diluted 1:1000), or affinity purified rabbit anti-ERK1/ERK2 antibodies (1:1000; Santa Cruz Biotechnology). The 4G10 mAb was detected with HRP-conjugated sheep anti-mouse IgG (1:10,000; Amersham Corp.), whereas rabbit antibodies were detected with HRP-conjugated goat anti-rabbit IgG (1:20,000; Bio-Rad, Mississauga, ON) or protein A-HRP (1:10,000; Amersham Corp.). The filters were washed extensively with TBS/0.1% Tween 20 or TBST-Nonidet P-40, and immunoreactive bands were visualized by enhanced chemiluminescence detection (ECL, Amersham Corp.). Blots to be reprobed were stripped of bound antibodies by incubating at 50 °C for 30 min in 100 mM 2-mercaptoethanol/2% SDS/62.5 mM Tris, pH 6.7.

To examine the association between Syk and the BCR, lysates were immunoprecipitated with anti-Ig-alpha antiserum as described above. The beads were split into 2 fractions, and the immunoprecipitates were separated on 7.5% gels by SDS-PAGE and transferred to nitrocellulose. Membranes were immunoblotted with 4G10 or anti-Syk antiserum as described above. Membranes were then stripped as above and reprobed with anti-Ig-alpha antiserum to ensure equal loading of lanes.

In Vitro Kinase Assay

Lysates were precleared with protein A-Sepharose beads that had been coated with normal rabbit serum. Lysates were then immunoprecipitated with the appropriate antiserum and fresh protein A-Sepharose. Beads were then washed twice with buffer B with 0.5 M LiCl and once with kinase assay buffer(24) . Autophosphorylation reactions were performed as described(24) , and reaction products were separated by SDS-PAGE and visualized by autoradiography. The in vitro kinase activity of Syk was determined as above, except lysates were precleared with protein A-Sepharose alone, beads were washed in buffer B plus 0.5 M NaCl instead of LiCl, and reaction products were separated on 8% gels.

p42 MAP Kinase (ERK2) Activity

Cell lysates (0.5 mg of protein) in buffer C were incubated for 1.5 h at 4 °C with 15 µl of agarose beads to which affinity purified rabbit anti-ERK 2 antibodies had previously been covalently coupled. The beads were washed three times with buffer C and once with MAP kinase assay buffer (20 mM NaHepes, pH 7.2, 5 mM MgCl(2), 1 mM EGTA, 5 mM 2-mercaptoethanol, 2 mM Na(3)VO(4), 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Reactions were initiated by adding 30 µl of kinase assay buffer containing 1 mg/ml myelin basic protein (MBP; Sigma) and 5 µCi of [P]ATP. Reactions were carried out for 15 min at 30 °C and terminated by addition of 35 µl of 2 times SDS-PAGE sample buffer. After boiling, portions of the sample (15-20 µl) were separated on 15% SDS-PAGE gels and then transferred to nitrocellulose. The MBP bands were detected by Ponceau S staining of the filter. After autoradiography, the stained MBP bands were excised, and P incorporation was determined by liquid scintillation counting. The upper portion of the blot was analyzed by anti-MAP kinase immunoblotting to confirm that equal amounts of p42 MAP kinase had been precipitated in all lanes.

Phosphoinositide Breakdown

Inositol phosphate production was measured essentially as described(16) . Cells were grown overnight in medium containing 10 µCi of [^3H]inositol and washed and stimulated as described above, except that they were stimulated for 30 min to allow for the accumulation of inositol phosphates. Inositol phosphate production is expressed as the percentage of inositol phosphate release, which was calculated as the amount of ^3H-labeled inositol phosphates divided by the sum of [^3H]inositol phosphates plus the [^3H]inositol-labeled phospholipids present in the trichloroacetic acid-insoluble fraction. The trichloroacetic acid-insoluble fraction was washed once in ice-cold 10% trichloroacetic acid before it was sonicated, resuspended in methanol, and quantitated by scintillation counting.

Measurement of Intracellular Free Calcium

Cells were grown in 2-well coverglass chambers (Nunc, Naperville, IL) and incubated with 2-4 µM indo-1 acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) for 90 min at 37 °C. Cells were washed three times with buffer D (buffer A minus glutamine, sodium pyruvate, and 2-mercaptoethanol) and then stimulated with buffer D alone or buffer D plus either 20 µg/ml goat anti-mouse IgM, 10% dialyzed FCS, or 1 µM serotonin (5-hydroxytryptamine, Sigma). Fluorescence intensity of intracellular indo-1 was monitored by image analysis on a laser-based fluorescence image cytometer (ACAS 470, Meridian Instruments, Inc.) as described(25) .


RESULTS

Tyrosine Kinase Expression in AtT20 Cells

In vitro kinase assays were performed to determine which of the tyrosine kinases that associate with the BCR in B cells were expressed in the BCR AtT20 cells. AtT20 lysates were incubated with antibodies specific to these kinases, and the immunoprecipitates were incubated with [-P]ATP and allowed to autophosphorylate in vitro (Fig. 1). Lysates from the Jurkat T lymphoma cell line and the B lymphoma cell lines WEHI-231 and Bal17 also were used as controls. In the anti-Fyn immunoprecipitates from Jurkat, Bal17, and AtT20 lysates, a band of approximately 59 kDa became labeled. No labeled band corresponding to Fyn was observed after anti-Fyn immunoprecipitation from WEHI-231 cell lysates, in agreement with a previous report that WEHI-231 cells have little or no fyn mRNA(19) . Although they expressed Fyn, AtT20 cells did not express Lyn, Blk, or Lck. Moreover, the AtT20 cells did not express Syk. In the anti-Syk immunoprecipitates from WEHI-231 and Bal17 lysates, but not from AtT20 lysates, a P-labeled doublet of approximately 72 kDa was observed. Both bands in the doublet are Syk; purified baculovirus-expressed Syk also autophosphorylates in vitro to form two or more bands, which represent differentially phosphorylated forms of Syk. (^2)Subsequent immunoblotting experiments (see below) confirmed that Syk protein was not expressed in AtT20 cells. Thus, AtT20 cells expressed at least one Src family tyrosine kinase implicated in BCR signaling, but they did not express Syk.


Figure 1: Tyrosine kinase expression in BCR AtT20 transfectants. The indicated cell lines were lysed in 1% Triton X-100 lysis buffer. Cell lysates were incubated with antiserum specific for p59, p53/56, p56, p55, or Syk (for Src family kinases: 150 µg of protein for Jurkat, WEHI-231, and Bal17 lanes; 300 µg for AtT20 lane; for Syk: 250 µg protein for each lane). Kinase expression and activity were assessed by autophosphorylation as described under ``Experimental Procedures,'' and reaction products were visualized by autoradiography. The positions of these kinases are indicated.



Isolation of a cDNA Clone Encoding the Murine Syk Protein-tyrosine Kinase

In order to clone the murine syk cDNA, we constructed a murine B cell cDNA library and screened it with both a polymerase chain reaction-generated fragment of murine syk and a fragment of the porcine syk cDNA. Four plaques that hybridized positively with each probe were obtained. DNA sequencing of the longest cDNA clone revealed a full-length open reading frame encoding the entire murine Syk kinase, as determined by its homology with porcine Syk(26) . Partial sequencing of the other cDNAs revealed that they were fragments of the same gene. The cDNA nucleotide sequence and deduced amino acid sequence of the full-length clone are shown in Fig. 2. An open reading frame is present beginning at nucleotide 220 and terminating at nucleotide 2106. It codes for a 629-amino acid polypeptide with a deduced molecular mass of 71,371 daltons. At the amino acid level, murine Syk is 91.5 and 92.9% identical to porcine Syk (26) and human Syk(27) , respectively. It is 56.3% identical to murine ZAP-70(28) , and 55.2% identical to human ZAP-70(29) .


Figure 2: Nucleotide and deduced amino acid sequence of murine syk. The two SH2 domains of Syk are underlined with solid lines. The kinase domain is underlined with a dashed line. The lysine that was mutated to alanine to create mutant Syk is boxed. @ represents the stop codon. These sequence data are available from EMBL/GenBank/DDBJ under accession number U36776.



Syk Expression and Activity in AtT20 Transfectants

The BCR AtT20 clonal cell line 100-33 was generated, as described under ``Experimental Procedures.'' In turn, 100-33 cells were used to generate BCR AtT20 transfectants that expressed either wild type Syk, catalytically inactive Syk (lysine 396 changed to alanine), or wild type Lck. Individual AtT20 clonal transfectants and the B cell lines WEHI-231, Bal17, and A20 were screened by immunoblotting with anti-Syk antiserum (Fig. 3A). The 100-33 and lck10 cell lines were both negative for Syk. Syk was expressed in the syk13, syk38, and syk41 clonal transfectants, and the mutated form of Syk was expressed in the kd17, kd16, and kd21 clonal transfectants. Syk expression in the syk38, syk41, and kd17 clones was similar to that in the WEHI-231 B cell line, whereas it was higher in the syk13 clone and lower in the kd16 and kd21 clones. Lck was expressed in lck10 cells (data not shown).


Figure 3: Syk expression and activity in AtT20 transfectants and B cell lines. A, relative levels of Syk expression were determined from the 100-33 parental BCR-expressing AtT20 cells and from 100-33 cells transfected with lck (lck10), with wild type syk (syk13, syk38, and syk41), or with mutated syk (kd17, kd16, and kd21), as well as from the B cell lines WEHI-231, Bal17, and A20. Cell lysates (15 µg of protein/lane) were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with anti-Syk antiserum, as described under ``Experimental Procedures.'' The position of Syk is indicated. B, Syk activity was determined from the indicated cells. Cell lysates (250 µg) were immunoprecipitated with anti-Syk antiserum, and the ability of Syk to autophosphorylate was determined as described under ``Experimental Procedures.'' The position of the labeled Syk doublet is indicated. The positions of molecular mass markers are indicated on both panels.



The catalytic activity of Syk was analyzed in several AtT20 clones expressing either wild type or mutated Syk. Syk was immunoprecipitated, and in vitro autophosphorylation reactions were performed. The syk13 and syk38 transfectants expressed catalytically active Syk, as shown by the labeled doublet at 72 kDa (Fig. 3B). In contrast, no Syk activity was immunoprecipitated from kd16, kd17, or kd21, even though the mutated Syk protein was expressed (Fig. 3A). These results confirmed that the mutated Syk was catalytically inactive.

Association of Both Wild Type and Kinase Inactive Syk with the BCR

Syk associates with the BCR in B cells, and upon BCR stimulation the extent of this association increases and Syk becomes tyrosine phosphorylated(12, 13, 14) . We determined whether Syk was regulated similarly in the AtT20 transfectants. Cell lysates from unstimulated and anti-IgM-stimulated cells were immunoprecipitated with an anti-Ig-alpha antiserum. The immunoprecipitates were split and examined by immunoblotting with either an anti-Syk antiserum or with the anti-phosphotyrosine monoclonal antibody 4G10 (Fig. 4, A and B, respectively). In the Syk-expressing AtT20 transfectants, as well as in WEHI-231 cells, there was often a low basal level of association between Ig-alpha/Ig-beta and Syk (Fig. 4A). However, the amount of BCR-associated Syk clearly increased upon anti-IgM stimulation. The syk38 transfectant was atypical in that it generally had only a very modest level of Syk associated with the BCR. Anti-phosphotyrosine immunoblotting revealed that both wild type and kinase inactive BCR-associated Syk from stimulated AtT20 cells were tyrosine phosphorylated and that there was no consistent difference in the extent of tyrosine phosphorylation between these two populations of Syk. As with autophosphorylated Syk ( Fig. 1and Fig. 3B), both bands in the doublet are Syk; the upper band is a more highly phosphorylated form of Syk and is recognized by our anti-Syk antiserum, although inefficiently (see Fig. 5, especially the WEHI-231 lanes). Whereas WEHI-231 cells appeared to have a similar level of Syk/BCR association as that seen in the AtT20 transfectants, the WEHI-231 BCR-associated Syk was tyrosine phosphorylated to a much greater degree. The reasons for this difference are not known. However, these experiments demonstrate that Syk/BCR association and Syk tyrosine phosphorylation were regulated in AtT20 cells as they are in B cells. The observation that kinase inactive Syk also associated with Ig-alpha/Ig-beta and became tyrosine phosphorylated upon stimulation demonstrates that Syk kinase activity was not required for association with the BCR and that Syk must have been phosphorylated by a separate tyrosine kinase.


Figure 4: Anti-IgM-induced Syk/BCR association and tyrosine phosphorylation of BCR-associated Syk. The indicated cells were incubated for 5 min with or without 20 µg/ml anti-IgM and lysed in 1% Triton X-100 lysis buffer. Lysates (2.5 mg for AtT20 transfectants, 1.5 mg for WEHI-231 cells) were immunoprecipitated with an anti-Ig-alpha antiserum. The immunoprecipitates were split into two fractions, each of which was separated on a 7.5% gel by SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes and immunoblotted with either anti-Syk antiserum (A) or the anti-phosphotyrosine mAb 4G10 (B). The WEHI-231 lanes in B were exposed for substantially less time than the rest of the lanes on that membrane (25 s versus 15 min). The membranes were then stripped of bound antibody and reprobed with anti-Ig-alpha antiserum to verify equal loading had occurred (not shown).




Figure 5: BCR-induced tyrosine phosphorylation of total cellular Syk. The indicated cells were incubated for 5 min with or without 20 µg/ml anti-IgM and lysed in 1% Triton X-100 lysis buffer. For each lane, 1 mg of lysate was immunoprecipitated with an anti-Syk antiserum. Immunoprecipitates were separated by SDS-PAGE (8% gel) and immunoblotted with the anti-phosphotyrosine mAb 4G10 (upper panel). The lack of visible IgH in the WEHI-231 lanes reflects a shorter exposure time for these lanes than for the AtT20 lanes (1 versus 20 min). The membrane was then stripped of bound antibody and reprobed with anti-Syk antiserum, and all lanes were exposed for the same amount of time (lower panel).



Differential Tyrosine Phosphorylation of Wild Type and Kinase Inactive Syk upon BCR Cross-linking

Only a small fraction of total cellular Syk could be immunoprecipitated with Ig-alpha/Ig-beta. In order to examine the effect of BCR cross-linking on the tyrosine phosphorylation of total cellular Syk, Syk was immunoprecipitated directly from lysates of unstimulated or anti-IgM-stimulated cells. Its tyrosine phosphorylation state was then examined by anti-phosphotyrosine immunoblotting (Fig. 5). The syk13 and syk38 clones exhibited a strong induction of Syk tyrosine phosphorylation upon BCR cross-linking, although this phosphorylation was less robust than in WEHI-231 cells. The kd16 and kd17 transfectants also showed a BCR-induced tyrosine phosphorylation of kinase inactive Syk but consistently to a lesser extent than seen with wild type Syk. This difference was not due to a smaller amount of catalytically inactive Syk being immunoprecipitated, as shown by reprobing this blot with anti-Syk antiserum (Fig. 5). Thus, BCR cross-linking induced a more extensive tyrosine phosphorylation of wild type Syk than catalytically inactive Syk, although this difference was not seen in the population of Syk associated with the BCR (Fig. 4B). One possible explanation for these results is that in addition to being phosphorylated by a separate tyrosine kinase, wild type Syk was able to autophosphorylate and dissociate from the BCR.

Syk Reconstitution of BCR-induced Tyrosine Phosphorylation of Cellular Proteins

A direct consequence of the rapid activation of tyrosine kinases upon BCR stimulation in B cells is the tyrosine phosphorylation of a variety of cellular proteins(2, 3, 4, 5) . These effects were largely absent in the BCR-expressing AtT20 cells, which lacked Syk (16) . In order to test whether Syk expression would reconstitute these events, we examined lysates from unstimulated and anti-IgM-stimulated AtT20 transfectants by immunoblotting with the anti-phosphotyrosine mAb, 4G10. In the 100-33 cells, BCR cross-linking weakly induced the tyrosine phosphorylation of 30-40-kDa proteins that included Ig-alpha and Ig-beta (Fig. 6A), as previously reported (16) . However, a general increase in the number and intensity of tyrosine phosphorylated bands was not seen. In contrast, the wild type Syk-expressing clones exhibited a dramatic increase in both the number and intensity of tyrosine phosphorylated bands upon anti-IgM stimulation. The pattern of induced bands was similar in the different clones and included the same 30-40-kDa bands as were seen in the 100-33 cells. The anti-IgM-induced bands also included a prominent band just above the 69-kDa marker that was probably Syk (see above). Unlike the Syk-expressing transfectants, the Lck-expressing transfectant lck10 did not exhibit a BCR-induced increase in tyrosine phosphorylated proteins. In fact, compared with the 100-33 cells, Lck expression often led to a decrease in the extent of tyrosine phosphorylation. Thus, Syk fulfills a unique role in BCR signaling that cannot be performed by Lck.


Figure 6: Anti-IgM-induced increase in protein-tyrosine phosphorylation in AtT20 transfectants expressing wild type but not catalytically inactive Syk. A, the indicated cell lines were left unstimulated or stimulated for 5 min with 20 µg/ml anti-IgM. Cell lysates (10 µg of protein) were separated on a 7.5% SDS-PAGE gel and immunoblotted with the anti-phosphotyrosine mAb, 4G10 (upper panel). The blot was stripped of bound antibodies and reprobed with anti-Ig-alpha antiserum to examine loading integrity (lower panel). B, cell lysates (400 µg of protein) used in A were immunoprecipitated with anti-Ig-alpha antiserum, and the precipitates were separated on a 10% SDS-PAGE gel and immunoblotted with 4G10 (upper panel). The positions of Ig-alpha and Ig-beta are indicated. The blot was then stripped and reprobed with anti-Ig-alpha as in A (lower panel). C, cell lysates were prepared and analyzed as in A, except that 18 µg of protein were loaded in each of the first 4 lanes, and 15 µg were loaded in the last 6 lanes.



The increased tyrosine phosphorylation of the 30-40-kDa proteins suggested that upon anti-IgM stimulation, Ig-alpha and Ig-beta were more highly phosphorylated in the Syk-expressing transfectants than in the parental 100-33 cells. To confirm this interpretation, the Ig-alpha/Ig-beta heterodimer was immunoprecipitated from these cells, and their level of tyrosine phosphorylation was determined by immunoblotting (Fig. 6B). This experiment demonstrated that the tyrosine phosphorylation of Ig-alpha and Ig-beta was increased by BCR cross-linking and that this response was enhanced in the Syk-expressing clones.

Although tyrosine kinases generally require their kinase activity to promote signaling, their ability to interact with other proteins may allow them to perform distinct signaling functions that do not require a functional kinase domain. For example, a kinase-independent activity of Lck in coreceptor-assisted T cell activation has been observed(30) . Therefore, we tested whether Syk kinase activity was required for the tyrosine phosphorylation events seen in the AtT20 cells. AtT20 transfectants expressing the BCR and catalytically inactive Syk were stimulated with anti-IgM antibodies and examined for protein-tyrosine phosphorylation (Fig. 6C). To better visualize the anti-IgM-induced increase in tyrosine phosphorylation of the 30-40-kDa proteins seen in the 100-33 cells, more protein was loaded in each lane than in the experiment shown in Fig. 6A. In contrast to the syk13 transfectant, the kd16, kd17, and kd21 cells did not exhibit a general anti-IgM-induced increase in protein-tyrosine phosphorylation. Transfectants expressing catalytically inactive Syk did show an increase in tyrosine phosphorylation of the 30-40-kDa proteins. However, unlike with the wild type Syk-expressing clones, this increase was similar in magnitude to the increase seen in the 100-33 cells. BCR stimulation of the catalytically inactive Syk-expressing transfectants also induced the tyrosine phosphorylation of another band just above the 69-kDa marker, which was not seen in the 100-33 cells. This upper band was probably the catalytically inactive Syk protein (see above). Thus, Syk expression reconstituted the tyrosine phosphorylation of a variety of cellular proteins upon BCR stimulation, and the kinase activity of Syk was required for this reconstitution.

BCR-induced Tyrosine Phosphorylation of Shc

We next wanted to determine whether Syk expression would also reconstitute the activation of BCR-associated signaling pathways. Stimulation by a variety of receptors, including the BCR, leads to extensive tyrosine phosphorylation of the adapter protein Shc(31, 32) , which allows Shc to participate in the activation of Ras(33) . We therefore examined the ability of BCR stimulation to induce the tyrosine phosphorylation of Shc in the AtT20 transfectants. Shc immunoprecipitates from lysates of unstimulated and anti-IgM-stimulated AtT20 cells were examined by immunoblotting with anti-phosphotyrosine antibodies (Fig. 7). As was seen in B cells, anti-IgM treatment induced Shc tyrosine phosphorylation in the transfectants expressing wild type Syk. In contrast, BCR cross-linking did not induce Shc phosphorylation in the 100-33 cells or in the transfectants expressing catalytically inactive Syk. The lck10 cells also failed to tyrosine phosphorylate Shc upon stimulation (data not shown). Thus, Syk kinase activity was required to reconstitute the BCR-induced tyrosine phosphorylation of Shc.


Figure 7: BCR-induced tyrosine phosphorylation of Shc. The indicated cells were incubated with or without 20 µg/ml anti-IgM for 5 min. The Shc in cell lysates (1 mg of protein) was immunoprecipitated with anti-Shc antibodies, resolved on 10.5% SDS-PAGE gels, and immunoblotted with the anti-phosphotyrosine mAb, 4G10. The position of Shc is indicated.



Reconstitution of BCR-induced MAP Kinase Activation by Wild Type Syk

The MAP kinases are a family of serine/threonine kinases that are activated through multiple signaling pathways by a variety of receptors(34) , including the BCR(35, 36) . These kinases phosphorylate a number of transcription factors, including p62(37) and NF-IL6(38) , and may therefore mediate anti-Ig-induced changes in gene expression. To examine whether Syk expression would reconstitute MAP kinase activation, the p42 ERK2 form of MAP kinase was immunoprecipitated from lysates of unstimulated or anti-IgM-stimulated cells and was subjected to an in vitro kinase assay using MBP as an exogenous substrate. MBP phosphorylation was visualized by SDS-PAGE followed by autoradiography (Fig. 8A) and quantified by scintillation counting (Fig. 8B). The 100-33 cell line did not exhibit a significant increase in p42 MAP kinase activity upon anti-IgM stimulation. In contrast, anti-IgM treatment increased p42 MAP kinase activity by 6.9-fold in syk13 cells and by 3.4-fold in syk38 cells. Higher Syk expression and/or BCR association in the syk13 cells than in the syk38 cells may explain the difference in the magnitude of MAP kinase activation between these two transfectants. The activity of p42 MAP kinase in the anti-IgM-stimulated syk13 and syk38 clones was similar in magnitude to that in anti-IgM-stimulated WEHI-231 cells. In contrast to the wild type Syk-expressing transfectants, anti-IgM caused little or no increase in p42 MAP kinase activity in the cells expressing catalytically inactive Syk. The lck10 clone also failed to activate MAP kinase upon stimulation. Additionally, anti-IgM stimulation of wild type Syk-expressing transfectants resulted in decreased electrophoretic mobility of both the p42 and p44 forms of MAP kinase, which is a characteristic of MAP kinase phosphorylation. This change in mobility was not observed in the 100-33 cells, in the lck10 cells, or in the transfectants expressing kinase inactive Syk (data not shown).


Figure 8: BCR-induced activation of p42 MAP kinase in AtT20 transfectants expressing wild type Syk. A, the indicated cell lines were incubated with or without 20 µg/ml anti-IgM for 5 min. The p42 ERK2 isoform of MAP kinase was immunoprecipitated from cell lysates (0.5 mg of protein). The activity of the immunoprecipitated p42 MAP kinase was assessed by in vitro kinase assay using MBP as a substrate. Reaction products were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. Autoradiographs from three representative experiments are shown. Immunoblotting with anti-MAP kinase antibodies indicated that similar amounts of MAP kinase were precipitated from each sample (not shown). B, to quantify the effects of BCR cross-linking on p42 MAP kinase activity, the MBP bands were excised and counted. The anti-IgM-induced increases in MAP kinase activity for the various cell lines are shown (mean ± S.E., n = number of independent cell stimulations assayed in separate experiments).



Failure of Syk to Reconstitute Inositol Phosphate Production or [Ca] Elevation in AtT20 Cells

The activation of the phosphoinositide signaling pathway is another important BCR-mediated event in B cells. Inositol-containing phospholipids are hydrolyzed by phospholipase C to generate inositol phosphates and diacylglycerol, which lead to an elevation in intracellular free Ca and the activation of protein kinase C, respectively. These signaling pathways are important for BCR-dependent gene induction (1, 23) and apoptosis in an immature B cell line(39) . We previously reported that BCR Syk AtT20 clones do not generate inositol phosphates upon anti-IgM stimulation (16) . Therefore, we tested whether Syk expression would reconstitute this pathway in response to anti-IgM. As previously reported(16) , FCS stimulation of all the AtT20 cell lines, as well as serotonin treatment of the serotonin receptor-expressing transfectant, SR1, induced increases in inositol phosphate production over the level observed in unstimulated cells (Fig. 9). In contrast, neither the 100-33 cells nor the syk13 or syk38 transfectants exhibited an increase in inositol phosphate production in response to anti-IgM stimulation.


Figure 9: Failure of Syk to reconstitute BCR-induced inositol phosphate production. The inositol-containing phospholipids of AtT20 transfectants were labeled overnight with [^3H]inositol. Cells were left unstimulated (dotted bars) or stimulated for 30 min with 10% dialyzed FCS (shaded bars), 20 µg/ml anti-IgM (black bars), or 1 µM serotonin (hatched bar) as indicated. Cells were lysed and total inositol phosphate generation was measured, as described under ``Experimental Procedures.'' Inositol phosphate production is calculated as the ^3H present in the inositol phosphates divided by the ^3H present in inositol phosphates plus phosholipids and is expressed as a percentage. The values shown are the mean and S.E. of triplicate samples. The experiment shown is representative of between three and six experiments, depending on the cell line.



We also tested these cell lines for increases in intracellular free Ca in response to FCS, serotonin, and anti-IgM treatment. The AtT20 cells were loaded with the calcium-sensitive dye indo-1, and stimulation-induced changes in intracellular free calcium were monitored at the single cell level by image cytometry(25) . Small Ca increases were observed after stimulation of all the AtT20 cell lines with FCS or after stimulation of the SR1 cells with serotonin. In contrast, we did not observe a Ca increase in the 100-33, syk13 or syk38 lines in response to anti-IgM antibodies (data not shown).


DISCUSSION

As a strategy for determining the requirements for signal transduction by the BCR, we expressed this receptor in the nonlymphoid AtT20 cell line. Signaling events observed in B cells, such as rapid tyrosine phosphorylation of many cellular proteins, did not occur in the BCR AtT20 transfectants. We found that AtT20 cells expressed a Src family tyrosine kinase implicated in BCR signaling, Fyn, but did not express the Syk tyrosine kinase. Therefore, we isolated a full-length murine syk cDNA and used it to express Syk in the BCR AtT20 cells. Syk expression was sufficient to reconstitute numerous BCR-induced signaling events in a stimulation-dependent manner, including the association of Syk with the BCR, the tyrosine phosphorylation of numerous cellular proteins including Syk and Shc, and the activation of MAP kinase. In contrast, BCR cross-linking of these cells failed to activate the phosphoinositide signaling pathway. The kinase activity of Syk was required for the reconstitution of downstream signaling events, although it was not required to recruit Syk to the BCR or for Syk tyrosine phosphorylation. Unlike Syk expression, Lck expression could not reconstitute any of these signaling events. These results demonstrate that Syk is a key component in BCR signaling and suggest that other lymphoid-specific components are not required for many signaling events triggered by BCR ligation in B cells. In addition, these results are complementary to data from a chicken B cell line that was rendered deficient for Syk expression, in that these Syk B cells exhibited profound defects in BCR signaling (10) .

Several features of our results are consistent with a model of how antigen receptors activate tyrosine kinases and thus initiate signaling (13, 17) . Clustering of antigen receptors leads to phosphorylation of the cytoplasmic domains of receptor subunits on tyrosines present in a conserved sequence now called the immunoreceptor tyrosine-based activation motif (ITAM)(40) . According to the model, this initial phosphorylation is mediated by one or more Src family kinases. In B cells, Syk then binds via its SH2 domains to phosphorylated Ig-alpha/Ig-beta ITAMs, whereas in T cells the Syk homologue ZAP-70 binds to phosphorylated T cell receptor subunit ITAMs. Syk or ZAP-70 becomes activated by this ITAM binding and/or by subsequent tyrosine phosphorylation (41, 42) and phosphorylates downstream signaling targets.

Consistent with this model, we have found substantial Ig-alpha and Ig-beta phosphorylation upon BCR cross-linking in both the Syk and Syk AtT20 cells. The AtT20 cells were found to express Fyn, so it is possible that Fyn is responsible for this phosphorylation. This possibility is supported by observations that Fyn can associate with Ig-alpha and Ig-beta in vivo, even before BCR cross-linking(13) , and can phosphorylate a glutathione S-transferase Ig-alpha fusion protein in vitro(43) . Also in agreement with the model were the observations that only a small amount of Syk was associated with Ig-alpha/Ig-beta in unstimulated cells, whereas the amount of Syk bound to the BCR clearly increased following anti-IgM treatment. This increase occurred with both wild type Syk and catalytically inactive Syk, and both forms of BCR-associated Syk also became tyrosine phosphorylated upon stimulation. This last observation suggests that the initial tyrosine phosphorylation of Syk was mediated by a separate kinase, possibly Fyn. This interpretation is also supported by the observation that Src family kinases can phosphorylate Syk and ZAP-70 in COS cells(15, 44) and in vitro(45) . In addition, Syk has been shown to interact directly with the Src family kinase Lyn in vivo(46) . In our experiments, BCR-associated wild type and kinase inactive Syk were tyrosine phosphorylated to a similar extent following BCR cross-linking. In contrast, when total cellular Syk was examined, it was found that wild type Syk was tyrosine phosphorylated much more extensively upon stimulation than was catalytically inactive Syk. The simplest explanation for this difference is that wild type Syk can undergo autophosphorylation either immediately before dissociating from the BCR or after dissociation occurs. Alternatively, the increased tyrosine phosphorylation of total cellular Syk may have been an indirect result of a Syk-mediated event, such as the inhibition of a protein-tyrosine phosphatase.

A key feature of the model described above is that Syk phosphorylates downstream signaling targets. Consistent with this prediction, anti-IgM stimulation led to the increased tyrosine phosphorylation of numerous cellular proteins, including Shc, in transfectants expressing wild type but not catalytically inactive Syk. These results are consistent with recently reported experiments that showed that Syk kinase activity greatly enhanced Shc phosphorylation over what was seen in Syk chicken B cells(47) . In addition, our experiments demonstrated that no additional lymphoid-specific components were required for BCR-induced tyrosine phosphorylation of cellular proteins.

One consequence of the BCR-induced tyrosine phosphorylation of cellular target proteins is the activation of multiple signaling pathways. One important signaling pathway activated by BCR cross-linking in B cells is the MAP kinase pathway(35, 36) . MAP kinase can phosphorylate a number of transcription factors and may thus tie receptor-mediated signaling events to changes in gene expression and biological activity. Indeed, in many cell types MAP kinase activation has been associated with the regulation of cellular proliferation, differentiation, secretion, and metabolic activity(34) . Expression of wild type but not catalytically inactive Syk enabled the BCR to activate p42 MAP kinase in AtT20 cells. The level of p42 MAP kinase activity in anti-IgM-stimulated syk13 and syk38 cells was similar to that in anti-IgM-stimulated WEHI-231 B cells. The mechanism by which the BCR activates MAP kinase in B cells is not known at this time, although there is evidence for both protein kinase C-dependent and -independent mechanisms of activation(36) . BCR signaling leads to a protein kinase C-independent increase in the active form of Ras in B cells(48, 49) , and Ras can initiate a kinase cascade that activates MAP kinase. Therefore, it seems likely that BCR-induced activation of MAP kinase in B cells occurs at least in part via Ras. Although we have not determined how BCR stimulation activates MAP kinase in AtT20 cells, we have shown that BCR stimulation of these cells leads to tyrosine phosphorylation of Shc. Shc can form a phosphorylation-dependent complex with both the adapter protein Grb2 and the Ras guanine nucleotide exchange factor mSOS and can thereby participate in the activation of Ras(33) . Thus, it seems likely that MAP kinase was activated through the Ras pathway in the AtT20 cells as well. In contrast, it seems unlikely that MAP kinase was activated in the AtT20 cells in a protein kinase C-dependent manner, because BCR cross-linking of these cells apparently did not activate phospholipase C (see below), which is upstream of protein kinase C.

Despite its ability to lead to the activation of MAP kinase, Syk expression in AtT20 cells was not sufficient to allow BCR-induced activation of the phosphoinositide signaling pathway. In B cells, phospholipase C-1 and/or phospholipase C-2 is activated by tyrosine phosphorylation to hydrolyze inositol-containing phospholipids to generate inositol phosphates and diacylglycerol(1) . These two second messengers then mediate the release of intracellular calcium stores and the activation of protein kinase C, respectively. Although this pathway was not activated in the AtT20 cells, phospholipase C-1 was tyrosine phosphorylated upon anti-IgM treatment in the wild type Syk-expressing transfectants (data not shown). It is possible that the AtT20 phospholipase C-1 was not phosphorylated extensively enough or on the proper tyrosine residues to become activated or that it failed to localize to the cell membrane where its substrate resides. It has been reported previously that chicken B cells lacking Syk activity do not activate phospholipase C-2 in response to BCR cross-linking, demonstrating that Syk is necessary for this activation to occur(10) . Our experiments indicate that Syk activity, although perhaps necessary, is not sufficient to link the BCR to the activation of phospholipase C. Other B cell proteins in addition to Syk may be needed to couple the BCR to phospholipase C-1, perhaps by localizing phospholipase C-1 to the cell membrane.

The conclusion that Syk plays a unique role in BCR signaling events was underscored by the observation that Lck did not functionally substitute for Syk to restore BCR-induced signaling. Presumably, the inability of Lck to substitute for Syk reflects important structural and functional differences between Src family kinases and Syk. However, because AtT20 cells do not express CD45 (data not shown), a protein-tyrosine phosphatase that can dephosphorylate the negative regulatory tyrosines of Src family kinases(50) , it is also possible that Lck could not be fully activated in these cells.

We have generated a heterologous system that allows us to identify, via a gain-of-function strategy, the lymphoid-specific proteins that are sufficient to link the BCR to the activation of downstream signaling events. Thus far, we have focused on the Syk tyrosine kinase. In previous reconstitution experiments performed in COS cells(15, 29, 44, 51) , Src family kinases and either Syk or ZAP-70 were expressed at very high levels and could interact and become activated even in the absence of receptor expression or stimulation. In contrast, in the system employed here, both Syk and the BCR were expressed at levels comparable with what is seen in B cells. Moreover, Syk/BCR association, Syk activation, and BCR signaling all occurred in a stimulation-dependent manner, indicating that physiologically relevant interactions between Syk, Fyn, and the BCR were likely occurring as they would in B cells. Together with gene knockout experiments(10) , the experiments described here establish that Syk is necessary for BCR signaling. We have also demonstrated that in the absence of other added lymphoid-specific components, the activation of Syk is sufficient to couple the BCR to downstream signaling events such as tyrosine phosphorylation of Shc and activation of MAP kinase but is not sufficient for activation of the phosphoinositide signaling pathway.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R01AI20038 (to J. D. R. and A. L. D.) and T32AI07334-07 (to J. D. R.), British Columbia Health Research Foundation Grant 99(94-1) (to L. M. and M. R. G.), Medical Research Council of Canada Grant MT11528 (to L. M.), National Science and Engineering Research Council of Canada Grant OGP0121457 (to L. M.), and funds from the University of British Columbia (to L. M. and M. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada. Tel.: 604-822-4881; Fax: 604-822-2416.

(^1)
The abbreviations used are: BCR, B cell antigen receptor; FCS, fetal calf serum; MAP, mitogen-activated protein; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; MBP, myelin basic protein; ITAM, immunoreceptor tyrosine-based activation motif.

(^2)
S. Harmer and A. L. DeFranco, unpublished observations.


ACKNOWLEDGEMENTS

We thank Andy Chan and Art Weiss for polymerase chain reaction primers, Vivien Chan for the affinity purified anti-Lyn and anti-Blk antibodies, Bill Hyun for assistance with the ACAS 470, and Graham Redgrave for assistance with sequence analysis. We thank Inge van Oostveen and Helen Ting for technical assistance. We also thank Vivien Chan, Mary Crowley, Sandip Datta, Julie Hambleton, Stacey Harmer, Janice Kim, Debbie Law, Trish Roth, and Steve Weinstein for critical reading of the manuscript.


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