(Received for publication, February 13, 1997)
From the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907
Syk (p72syk) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor.
Syk (p72syk) is a 72-kDa intracellular
protein-tyrosine kinase expressed in a variety of hematopoietic cells
including B-cells, mast cells, macrophages, platelets, and thymocytes.
Although it has been implicated in signaling through various receptor
types, the role of Syk in receptor-mediated signaling has been best
characterized for immune-recognition receptors such as the B-cell
antigen receptor (BCR)1 (1-4). Aggregation
of these receptors results in an increase in the tyrosine
phosphorylation of cellular proteins that are commonly involved in
signaling, increased inositol 1,4,5-trisphosphate production, calcium
mobilization, and activation of the mitogen-activated protein kinase
pathway (5). In syk/
mice, B-cell and
pre-B-cell antigen receptors fail to deliver signals for cellular
survival and expansion leading to a failure of B-cell maturation,
indicating a critical role for Syk in B-cell signaling (6, 7).
Signaling through the BCR requires the phosphorylation of a pair of
uniformly spaced tyrosines in the cytoplasmic domains of the Ig-
(CD79
) and Ig-
(CD79
) subunits. These tyrosines are present
within an immunoreceptor tyrosine-based activation motif, which
consists of two YXX(L/I) cassettes separated by 6-8 amino
acids (4, 8). These phosphorylated immunoreceptor tyrosine-based
activation motif tyrosines serve as a docking site for the two tandem
Src homology 2 (SH2) domains of Syk (9, 10). The recruitment of Syk to
the receptor results in its activation as characterized by an increase
in its enzymatic activity and an increase in its phosphotyrosine
content resulting from autophosphorylation and phosphorylation by a Src
family protein-tyrosine kinase (11, 12).
We have shown recently (13) that the majority of activated, tyrosine-phosphorylated Syk in an anti-IgM-treated B-cell has dissociated from the receptor and is found in the soluble, cytosolic fraction. To begin to explore the role of phosphorylation in modulating the properties of Syk, we have focused on Tyr-130, an in vitro site of Syk autophosphorylation that lies within the inter-SH2 domain region (14). In this study, we provide evidence that phosphorylation of Syk at Tyr-130 modulates not only its ability to interact with the antigen receptor but also its intrinsic activity.
Syk DT40 chicken B-cells
(15) were obtained from Dr. Tomohiro Kurosaki. Unconjugated and
fluorescein isothiocyanate-conjugated goat anti-chicken IgM were
purchased from Bethyl Laboratories, Inc. Anti-phosphotyrosine and
anti-Syk antisera have been described previously (9, 16). The 9E10
anti-Myc hybridoma cell line was purchased from ATCC, and ascites fluid
was prepared by the Purdue University Cancer Center Antibody Production
Facility. The cytoplasmic fragment of human erythrocyte band 3 (cfb3)
was obtained from Dr. Philip Low, Purdue University.
The cloning of murine
syk cDNA has been described elsewhere (14). To generate
Myc epitope-tagged Syk, oligonucleotides containing the sense and
antisense sequence of the Myc epitope along with a stop codon were
annealed and then ligated to the syk cDNA. This cDNA
was subcloned into the XhoI site of the pGEM/EPB expression vector, which contains a heavy chain enhancer/promoter cassette for
B-cell-specific expression (17). Site-directed mutagenesis was carried
out using the Transformer mutagenesis kit (CLONTECH) and confirmed by
DNA sequencing. Syk DT40 cells were electroporated with
25 µg of the various Syk-Myc DNA-containing plasmids and 2.5 µg of
p3'SS (Stratagene), which contains a hygromycin resistance gene. Cells
were selected in hygromycin (2 mg/ml) and were screened for Syk
expression by both immune complex kinase assays and Western blotting.
Clones expressing comparable levels of Myc-tagged wild-type Syk
(Syk(WT)), Myc-tagged Syk with Tyr-130 mutated to Glu (Syk(Y130E)), and
Myc-tagged Syk with Tyr-130 mutated to Phe (Syk(Y130F)) were
selected.
DT40 cells were activated on
ice with 25 µg/ml goat anti-chicken IgM for 15 min at 4 °C.
Activation was terminated by the addition of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Brij 96, 5 mM EDTA, 1 mM sodium orthovanadate, and 10 µg/ml each leupeptin and aprotinin). After incubation on ice for 15 min, nuclei and unbroken cells were removed by centrifugation at
15,000 × g for 5 min at 4 °C. Procedures for
immunoprecipitations and immune complex kinase assays have been
previously described in detail (9). Briefly, the Myc epitope-tagged Syk
proteins were immunoprecipitated with anti-Myc epitope monoclonal
antibodies coupled to protein A-Sepharose (Sigma). Syk activity was
detected in the immune complexes by Western blotting with anti-Syk,
anti-phosphotyrosine antibodies, or by autophosphorylation in the
presence of [-32P]ATP (5 µM). Where
indicated, cfb3 (3 µg) was included as an exogenous substrate.
Phosphoproteins were separated by SDS-PAGE, transferred to
polyvinylidene difluoride membranes, and treated with 1 N
KOH at 55 °C for 2 h. Phosphotyrosine-containing proteins were detected by autoradiography.
Anti-Myc epitope immune complexes obtained
from lysates of Syk(WT)-expressing DT40 cells were phosphorylated
in vitro with [-32P]ATP (5 µM) for the times indicated. The phosphorylated kinase was isolated by SDS-PAGE, transferred to nitrocellulose, detected by
autoradiography, excised from the membrane, and incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin as described previously (14). Tryptic phosphopeptides were
compared by 40% polyacrylamide alkaline gel electrophoresis to a set
of standard phosphopeptides derived from in vitro
autophosphorylated glutathione S-transferase-Syk (14). These
standard phosphopeptides were isolated by high performance liquid
chromatography and identified by a combination of Edman sequencing and
mass spectrometry as described previously (14). Relative levels of each
phosphopeptide were determined by densitometric analysis of
autoradiograms of the dried alkaline gels.
Prolonged
incubation (30 min) of Syk in vitro with ATP leads to its
autophosphorylation on multiple sites including Tyr-130, which lies
between the amino-terminal tandem pair of SH2 domains (14). To
determine if Tyr-130 represented a major early site of
autophosphorylation, the sites on Syk that become phosphorylated at
shorter times of incubation with ATP were examined. For these studies,
Syk bearing a Myc epitope tag at the extreme carboxyl terminus,
Syk(WT), was expressed in Syk DT40 B-cells. Syk(WT) was
immunoprecipitated and autophosphorylated in vitro with
[
-32P]ATP for varying periods of time. Tryptic
phosphopeptides generated by complete digestion of phospho-Syk(WT) were
separated by electrophoresis on 40% polyacrylamide alkaline gels and
identified by comparison with a series of known phosphopeptides (14).
The relative extents of phosphorylation of the various sites as a
function of time are illustrated in Fig. 1. The most
rapidly phosphorylated residues were Tyr-317 and -130.
Role of Tyr-130 in Syk Kinase Activity
To explore the role of Tyr-130 in Syk-receptor interactions, additional DT40 cell lines were prepared that lacked endogenous Syk but expressed mutant forms of epitope-tagged Syk in which Tyr-130 was replaced by either Phe, to prevent phosphorylation at this site, or Glu, to position a negatively charged amino acid at this site. Phosphopeptide mapping studies of in vitro autophosphorylated Syk(Y130F) and Syk(Y130E) mutants confirmed the absence of phosphate at Tyr-130. Lack of phosphate at this site did not, however, preclude autophosphorylation at the other sites (data not shown).
To compare the basal activity of Syk(Y130F) and Syk(Y130E) to that of
Syk(WT), the three kinases were immunoprecipitated individually from
transfected DT40 cells and assayed in the resulting immune complexes
for phosphorylation of the Syk substrate, cfb3. As shown in Fig.
2A, the intrinsic activity of Syk(Y130F) was
lower than that of Syk(WT) (1.3-fold). In contrast, the basal activity
of Syk(Y130E) was substantially higher (2-fold) than that of Syk(WT). Furthermore, receptor cross-linking resulted in an increase (2.2-fold) in the intrinsic kinase activity of the recovered Syk(WT), but either
had no effect or decreased the activity of the mutant kinases (Fig.
2B). Western blotting analyses with anti-Syk antibodies confirmed that equivalent levels of each kinase were present in the
anti-Myc epitope immune complexes prepared from the untreated or
anti-IgM-treated Syk-expressing DT40 cell lines (Fig.
2C).
Association of Syk with the BCR Complex
To explore the effect
of Tyr-130 mutations on Syk-receptor interactions, intact BCR complexes
were immunoprecipitated from Brij 96 lysates of the DT40-derived cell
lines expressing wild type or mutant forms of Syk. The presence of
BCR-associated kinases was detected by immune complex kinase assays. As
shown in Fig. 3A, a low level of
receptor-associated Syk(WT) autophosphorylating activity could be
observed co-immunoprecipitating with the clustered antigen receptor.
The level of receptor-associated Syk(Y130F) activity was significantly
higher than that of Syk(WT). In contrast, little or no
receptor-associated Syk(Y130E) could be detected. Similar amounts of
the 53- and 56-kDa forms of Lyn (as determined by Lyn
autophosphorylating activity) were recruited to the clustered antigen
receptors regardless of the nature of the Syk kinase expressed in the
cell. This level of Lyn was similar to that observed associating with
the cross-linked receptor from Syk-negative DT40 cells (data not
shown). The third, and smallest, of the major proteins phosphorylated in these complexes has not been identified but, by analogy to anti-IgM
immune complexes from human or murine cells, likely represents chicken
Ig-.
To determine if the different levels of receptor-associated Syk activity were due to differences in the level of Syk protein bound to the receptor, anti-IgM immune complexes were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-Syk antibodies. As shown in Fig. 3B, Syk(WT) protein was found in the anti-IgM immune complexes prepared from anti-IgM-treated cells. The level of Syk(Y130F) associated with the receptor following receptor cross-linking was consistently higher than that of Syk(WT). No Syk(Y130E) protein could be detected in anti-IgM immunoprecipitates isolated from Syk(Y130E)-expressing cells.
Effect of Tyr-130 on Receptor-mediated Protein PhosphorylationDT40 cells expressing Syk(WT), Syk(Y130F), or
Syk(Y130E) were compared for their abilities to support
receptor-mediated protein tyrosine phosphorylation. Flow cytometric
analyses of BCR expression using fluorescein isothiocyanate-conjugated
anti-IgM antibodies did not reveal any significant differences in the
surface receptor expression between the three cell lines (data not
shown). Lysates from resting or anti-IgM-treated cells were analyzed by
immunoblotting with anti-phosphotyrosine antibodies (Fig.
4A). Receptor engagement in
Syk(WT)-expressing cells resulted in the increased tyrosine phosphorylation of multiple intracellular proteins. Phosphorylation of
these proteins was enhanced in cells expressing the Syk(Y130F) mutant.
In contrast, there was a marked decrease in phosphotyrosine-containing proteins in activated cells expressing the Syk(Y130E) mutant. A closer
examination of the differences between two additional clones of cells
expressing Syk(WT) and Syk(Y130F) indicated that both the rate and
extent of receptor-mediated tyrosine phosphorylation was increased in
the Syk(Y130F)-expressing cells (Fig. 4C).
To characterize the state of Syk tyrosine phosphorylation in response to BCR activation, the three DT40 clones were treated with increasing concentrations of the activating anti-IgM antibody. Anti-Myc epitope immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine antibody. In the absence of receptor cross-linking, only Syk(Y130E) contained phosphotyrosine at detectable levels (Fig. 4B). Following receptor engagement, both Syk(WT) and Syk(Y130F) were extensively phosphorylated while Syk(Y130E) exhibited only a modest increase in phosphotyrosine content (Fig. 4B).
The activation of Syk in B-cells takes place at the site of clustered antigen receptors and is accompanied by a substantial increase in the phosphotyrosine content of the kinase (11, 16, 18). The activated tyrosine-phosphorylated kinase, however, dissociates from the antigen receptor, since it is found primarily in the soluble fraction of cells that have been lysed by Dounce homogenization (13). Data presented in this study indicate that the phosphorylation of Syk on Tyr-130 is likely to enhance this dissociation and is also involved in the receptor-mediated activation of the kinase.
Syk has multiple tyrosine residues that are potential sites of
phosphorylation. Tyrosine residues 130, 290, 317, 342, 346, 358, 519, 520, 623, and 624 are all phosphorylated when Syk is incubated in
vitro with ATP (14). Based on previous studies of Syk and by
analogy to the Syk homolog, ZAP-70, some phosphorylation sites are
likely to regulate the catalytic activity of Syk (Tyr-519 and -520)
(10, 19, 20) while others form docking sites for SH2 domain-containing
proteins such as phospholipase C-1 (Tyr-342 and/or -346) (21), Lck
(Tyr-519 and -520) (22), and others (23). None of the previously
characterized sites, however, have been implicated in mediating
kinase-receptor interactions. We focused our attention on Tyr-130 since
it is a prominent and early site of Syk autophosphorylation (Fig. 1)
and is localized within the inter-SH2 domain region.
Several observations are consistent with a role for Tyr-130 phosphorylation in modulating the interactions of Syk with the antigen receptor. First, if the phosphorylation of Tyr-130 is prevented by its replacement with Phe, receptor binding is enhanced. Low levels of Syk(WT) activity or protein can be detected binding to the aggregated antigen receptor (Fig. 3), consistent with previous observations (9, 10). It is likely that this interaction of Syk with the antigen receptor is transient in nature. Functional coupling of Syk(WT) to the antigen receptor is also indicated since anti-IgM antibodies induce Syk(WT) phosphorylation (Fig. 4B), Syk(WT) activation (Fig. 2B), and the phosphorylation of intracellular proteins (Fig. 4, A and C). When Tyr-130 is replaced by Phe to prevent phosphorylation at this site, the level of receptor-associated kinase that can be recovered increases significantly (Fig. 3). This increased association of Syk(Y130F) with the receptor also enhances the receptor-mediated phosphorylation of intracellular targets (Fig. 4, A and C). On the other hand, when Tyr-130 is replaced with Glu, no receptor-associated kinase can be detected in immunoprecipitated BCR complexes from anti-IgM-activated cells (Fig. 3). As a result, the receptor-mediated phosphorylation of intracellular proteins on tyrosine is greatly diminished in the Syk(Y130E)-expressing cells (Fig. 4A). The receptor-mediated increases in tyrosine phosphorylation that do remain in Syk(Y130E)-expressing cells likely result either from a transient or low affinity interaction of Syk(Y130E) with the receptor or from an interaction of Syk(Y130E) with Lyn, which is still activated by receptor engagement in these cells.
Several observations also indicate that the phosphorylation of Tyr-130 may be an important step in the activation of Syk. When Tyr-130 is replaced by Phe, the intrinsic activity of the kinase is reduced compared with that of the wild-type enzyme (Fig. 2A). More significantly, we have been unable to observe a receptor-coupled activation of Syk(Y130F) (Fig. 2B) despite the enhanced ability of the enzyme to bind aggregated receptors (Fig. 3A). Either the Syk(Y130F) kinase is not activated by receptor engagement or the percentage of the expressed enzyme that is activated is below our detection limits. If Tyr-130 is replaced by Glu to permanently position a negatively charged residue at this site, the kinase now exhibits a basal activity twice that of Syk(WT) and nearly three times that of Syk(Y130F) (Fig. 2A). It is interesting to note that the phosphorylation of downstream target proteins on tyrosine in response to receptor aggregation appears more dependent on the length of time Syk remains associated with the receptor than on increases in its intrinsic activity (Fig. 4, A and C).
The mechanisms by which the phosphorylation of Tyr-130 alters Syk
activity and receptor binding have yet to be determined. By analogy to
Tyr-126 of the Syk-family kinase, ZAP-70, Tyr-130 would be expected to
lie near the apex of a coiled coil of -helices located within the
inter-SH2 domain region (24). It has been hypothesized that this region
functions to properly position the SH2 domains in an orientation
appropriate for the inter-SH2 domain interactions required for binding
a dually phosphorylated immunoreceptor tyrosine-based activation motif
(24). This region may also be in a position to mediate intramolecular
interactions between the coiled coil domain and the kinase active site
(24). The amino acids surrounding Tyr-130 share the sequence of
LXX(E/D)Y with Syk phosphorylation sites on
-tubulin and
erythrocyte band 3, proteins that physically associate with Syk (13,
25). On band 3, this site forms a loop that surrounds the tyrosine and
serves as a site involved in protein/protein associations (26).
Phosphorylation of this tyrosine destabilizes the loop (26) and
disrupts these interactions (27). By analogy, phosphorylation of
Tyr-130 might reasonably be expected to cause changes in the local
conformation of the inter-SH2 domain region that alters both the
SH2-SH2 interface interactions and the interactions between the
inter-SH2 region and the kinase active site. Thus phosphorylation of
Tyr-130 could negatively influence the binding of Syk to the antigen
receptor and at the same time allow increased access of the catalytic
site to protein substrates. Further experiments on the structure and function of Syk will be required to formally validate this model.
We thank Dr. Tomohiro Kurosaki for the
generous gift of the Syk DT40 cell line and Dr. Frederick
W. Alt for the pGEM/EPB expression vector.