(Received for publication, October 5, 1995; and in revised form, January 12, 1996)
From the
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.
B lymphocytes can recognize a myriad of foreign antigens by
virtue of the diversity of their B cell antigen receptors (BCR). ()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-
/Ig-
heterodimer(1) . The
signaling cascade initiated by Ig-
and Ig-
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- and Ig-
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
p56
co-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-
, and Ig-
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-
and
Ig-
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-
and Ig-
, 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.
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.
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.
To examine the association between Syk and the BCR, lysates
were immunoprecipitated with anti-Ig- 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-
antiserum to ensure equal
loading of lanes.
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.
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.
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.
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- 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-
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).
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- antiserum to examine loading integrity (lower panel). B, cell lysates (400 µg of
protein) used in A were immunoprecipitated with anti-Ig-
antiserum, and the precipitates were separated on a 10% SDS-PAGE gel
and immunoblotted with 4G10 (upper panel). The positions of
Ig-
and Ig-
are indicated. The blot was then stripped and
reprobed with anti-Ig-
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- and Ig-
were more highly
phosphorylated in the Syk-expressing transfectants than in the parental
100-33 cells. To confirm this interpretation, the Ig-
/Ig-
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-
and Ig-
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.
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.
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).
Figure 9:
Failure of Syk to reconstitute BCR-induced
inositol phosphate production. The inositol-containing phospholipids of
AtT20 transfectants were labeled overnight with
[H]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
H present in the
inositol phosphates divided by the
H 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).
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-/Ig-
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- and Ig-
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-
and Ig-
in vivo, even before BCR
cross-linking(13) , and can phosphorylate a glutathione S-transferase Ig-
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-
/Ig-
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.