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Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, Fc
RI,
FcRII, and FcRIII (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The importance of Syk to
receptor-mediated signaling in hemopoietic cells is underscored by the
signaling defects that adversely affect B cell differentiation, mast
cell activation, and platelet activation in mice that lack Syk (5, 14, 15). In B cells, the pathway leading to the activation of Syk is
initiated by the phosphorylation of a pair of tyrosines residing within
a conserved motif, the ITAM3
(immunoreceptor tyrosine-based activation motif) (16), located on the
cytoplasmic domains of the receptor components Ig-
and Ig-ß (17, 18). ITAM phosphorylation is initiated by the aggregation of surface Ig
and is thought to be catalyzed by members of the Src family of protein
tyrosine kinases. When phosphorylated, this domain functions as a
docking site for the recruitment of Syk to the receptor: the
interaction occurring between the ITAM phosphotyrosines and the tandem
pair of SH2 domains located in the amino-terminal half of Syk (19). The
binding of Syk to phospho-ITAMs results in both a relocalization of the
kinase to the site of the aggregated receptor and an increase in its
intrinsic kinase activity (20).
Much less is known of how Syk participates in the propagation of
downstream signals following its activation. One important clue has
been the observation that Syk becomes phosphorylated on tyrosine
subsequent to receptor cross-linking (21, 22). These phosphorylations
are characterized by an increase in the intrinsic activity of Syk (1, 22), a shift in its electrophoretic mobility when analyzed by SDS-PAGE,
a change in receptor affinity (23), and the creation of docking sites
for the binding of Syk-interacting proteins such as Lyn, Lck, PLC-,
and Vav (24, 25, 26, 27). While the importance of many individual tyrosines on
Syk has been suggested through the use of site-directed mutants, no
studies have fully addressed the more complicated issue of which
tyrosines are actually phosphorylated under conditions that lead to the
activation of Syk in intact B cells and which kinases are responsible
for catalyzing these phosphorylations. The primary candidates for the
kinases responsible for Syk phosphorylation are Syk itself and one or
more members of the Src family.
Previously, we demonstrated that the incubation of purified Syk in vitro with ATP leads to the covalent modification of multiple tyrosines (28). In the present study, we have combined metabolic labeling and peptide mapping approaches to identify the tyrosine residues on Syk that become phosphorylated in response to the activation of B cells. Our results indicate that Syk is phosphorylated on multiple tyrosines in response to either receptor engagement or inducers of oxidative stress. The phosphorylation of specific sites on Syk is dependent on the catalytic activities of both Syk and Lyn. These tyrosines include residues in the inter-SH2 domain region and within the catalytic loop, which are phosphorylated primarily by Syk itself, and residues within the linker region that connects the tandem SH2 domains to the catalytic domain, which are the preferred Lyn-dependent phosphorylation sites. This includes a site (Tyr317) that negatively regulates Syk function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type, Syk-, and Lyn- chicken DT40 B cells (29) were generously provided by Dr. Tomohiro Kurosaki (Kansai Medical University, Moriguchi, Japan). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% chicken serum, 50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin G, and 100 µg/ml streptomycin. Preparation of a cDNA for the expression of Myc epitope-tagged Syk using the pGEM/EPB vector (30) was described previously (31). cDNAs for the expression of site-directed mutants were constructed using the Transformer mutagenesis kit (Clontech, Palo Alto, CA). All mutations were confirmed by sequencing. Syk- or Lyn- DT40 B cells were transfected with 25 µg of the various linearized Syk-Myc DNA-containing plasmids and 2.5 µg of p3'SS (Stratagene, La Jolla, CA) by electroporation using a Cell-Porator (Life Technologies, Gaithersburg, MD) at 300 V, 330 µF. Stable transfected cell lines were selected in hygromycin (2 mg/ml). The 9E10 anti-Myc hybridoma cell line was obtained from American Type Culture Collection (Manassas, VA). Rabbit anti-phosphotyrosine (1) and anti-Syk (23) antisera were prepared by Purdue University Cancer Center Antibody Production Facility (West Lafayette, IN). Goat anti-chicken IgM Abs were obtained from Bethyl Laboratories (Montgomery, TX). The synthetic peptide ADENYYK was prepared by Purdue Cancer Center Peptide Synthesis Facility. Cloning, expression, and isolation of the GSTp42.5 form of Syk were as described (28). The NF-AT-luciferase reporter construct was a gift of Anjana Rao, Harvard University (Boston, MA).
Metabolic labeling
A total of 2.5 x 106 DT40 cells was preincubated in 15 ml of phosphate-free RPMI 1640 for 1 h and then incubated for an additional 2 h in the presence of 5 mCi of [32P]orthophosphate (DuPont/NEN, Boston, MA). Cells were collected, washed, and resuspended in PBS. Where indicated, cells were activated for 15 min at 4°C by the addition of affinity-purified goat anti-chicken IgM (50 µg/ml). In some experiments, cells were activated instead by treatment for either 20 min with pervanadate (final concentration of 0.1 mM sodium orthovanadate and 0.5 mM H2O2) at 37°C or 10 min with 10 mM H2O2 at 37°C. Cells were lysed in buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Brij 96, 5 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were centrifuged at 15,000 x g for 10 min at 4°C. The supernatants were precleared by incubation with protein A-Sepharose for 1 h at 4°C. Syk was immunoprecipitated from the unbound fraction by the addition of 20 µl of protein A-Sepharose that had been previously incubated with 5 µl of mouse ascites fluid containing the anti-Myc epitope mAb. The immune complexes were washed eight times with RIPA buffer (150 mM NaCl, 10 mM Tris/HCl, pH 7.2, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS), dissociated in SDS-sample buffer, and separated by SDS-PAGE. The separated proteins were then electrophoretically transferred to nitrocellulose membranes and detected by autoradiography.
To obtain in vitro autophosphorylated Syk, the epitope-tagged protein
was immunoprecipitated from lysates of Syk-expressing DT40 cells with
anti-Myc epitope Abs, as described above. The immune complexes were
washed twice with lysis buffer, followed by two washes with 25 mM
HEPES, pH 7.5, and 1 mM sodium orthovanadate, and then incubated in
vitro for 30 min (or for the times indicated) at 30°C in 25 mM HEPES,
pH 7.4, 10 mM MnCl2, 5 mM
p-nitrophenylphosphate, 5 mM ATP, and 100 µCi of
[-32P]ATP. Phosphoproteins were separated by SDS-PAGE,
transferred to nitrocellulose membranes, and detected by
autoradiography.
Phosphopeptide mapping
Phosphoproteins were excised from nitrocellulose membranes and digested with trypsin essentially as described (32). In brief, membrane pieces were first incubated in 0.5% polyvinylpyrrolidone (PVP-10; Sigma, St. Louis, MO), 100 mM acetic acid for 30 min at 37°C. After extensive washing with H2O, membranes were incubated for 2 h at 37°C with 10 µg L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) in 50 mM NH4HCO3 and then for an additional 2 h with freshly added 10 µg of trypsin. Samples were lyophilized and resuspended in alkaline PAGE sample buffer containing 0.125 M Tris/HCl, pH 6.8, 6 M urea, and a small amount of bromphenol blue (33). The tryptic phosphopeptides were resolved by electrophoresis on an alkaline 40% polyacrylamide gel, as described (33). Samples were electrophoresed at 180 V until the blue tracking dye had migrated to Rf = 0.5. Phosphopeptides were detected by autoradiography.
The synthetic peptide ADENYYK was phosphorylated in vitro using
GSTp42.5 immobilized on glutathione-agarose (28) at a peptide
concentration of 1 mM for 2.5 min at 30°C in buffer containing 10 mM
MnCl2, 5 mM p-nitrophenylphosphate, 25 mM HEPES,
pH 7.4, 1 µM ATP, and 5 µCi of [-32P]ATP. The
reaction mixture containing the phosphopeptide was resolved by alkaline
PAGE. The generation of tryptic phosphopeptides from in vitro
phosphorylated GSTp42.5 and their separation by HPLC was as described
previously (28).
Measurement of NF-AT promoter activity
Syk-DT40 cells (5 x 106) were transfected with vectors containing the various Syk mutants (20 µg) along with NF-AT-luciferase (10 µg) by electroporation, as described above. Cells were harvested 40 h following transfection, plated at a density of 1 x 106/ml, and activated with anti-IgM Abs or with a combination of PMA (50 ng/ml) and ionomycin (1.0 µM) for 6 h at 37°C. Luciferase activity was determined using the luciferase assay system kit (Promega, Madison, WI) and measured on a Lumat LB 9501 luminometer (EG&G Wallac, Wellesley, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To develop a convenient procedure for the analysis of sites of
tyrosine phosphorylation on Syk, we investigated the use of alkaline
40% polyacrylamide gels for the separation of tryptic phosphopeptides
(33, 34). We first examined phosphopeptides derived from Syk(WT), a
form of murine Syk with a Myc epitope tag at the extreme carboxyl
terminus expressed in a Syk-DT40 B cell (31). Syk(WT) was
recovered from cell lysates with anti-Myc epitope Abs, incubated
with [-32P]ATP to allow autophosphorylation to occur,
and then digested with trypsin. The tryptic phosphopeptides were
separated electrophoretically to generate the phosphopeptide map shown
in Fig. 1
, lane 4. A complete
digest of autophosphorylated Syk(WT) yielded a series of
phosphopeptides that were well resolved by one-dimensional alkaline
PAGE.
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-32P]ATP, and digested
with trypsin. The resulting phosphopeptides were separated by alkaline
PAGE and detected by autoradiography. Examples of these analyses are
shown in Fig. 1
(lanes 1, 2, 3,
5, and 6). These studies positively identified
peptides containing phosphotyrosines at positions 130
(lane 1), 342 (lane 2), 346
(lane 3), 290 (lane 5), and 317
(lane 6).
The migration position of phosphopeptides containing Tyr519
and Tyr520 was confirmed using a synthetic peptide
corresponding in sequence to the tryptic fragment containing
these residues (ADENYYK). This peptide was phosphorylated in
vitro, using as a catalyst a truncated form of Syk expressed in insect
cells as a fusion protein coupled to glutathione
S-transferase (GSTp42.5) (28). The resulting phosphopeptides
were analyzed by alkaline gel electrophoresis and are shown in Fig. 2. This reaction resulted primarily in
the phosphorylation of ADENYYK on a single tyrosine with only small
amounts of the doubly phosphorylated peptide being observed. These
peptides comigrated with HPLC-purified and sequenced phosphopeptides
corresponding to singly (lane 4) and doubly
(lane 5) phosphorylated ADENYYK derived from in vitro
autophosphorylated GSTp42.5 (28).
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A. The identity of each
of the phosphopeptides on the map that were analyzed as described above
is indicated. The location of each of these sites on the Syk molecule
(Fig. 3B) and the sequence of each of these tryptic peptides
(Fig. 3C) are also indicated.
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A stable line of Syk-DT40 B cells transfected to
express Syk(WT) was used for the initial characterization of Syk
phosphorylation in intact cells. Cells were preincubated in the
presence of [32P]orthophosphate, and then either remained
untreated or were activated by treatment with anti-IgM Abs,
pervanadate, or H2O2. Syk(WT) was then
immunoprecipitated with anti-Myc epitope Abs. The resulting immune
complexes were washed extensively, separated by SDS-PAGE, and
transferred to nitrocellulose membranes. As shown in Fig. 4A, Syk(WT) from both
untreated and activated cells was a phosphoprotein. The phosphate
content of Syk(WT) increased following activation. However, as shown
previously, Syk(WT) contains little or no phosphotyrosine in the
absence of activation (31).
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B, proteolysis of Syk(WT) recovered from unstimulated
cells generated four prominent phosphopeptides, indicating that Syk is
phosphorylated on multiple serine and/or threonine residues in the
absence of activation. Additional phosphopeptides were obtained from a
tryptic digest of Syk(WT) recovered from B cells activated by treatment
with anti-IgM Abs (Fig. 4B), pervanadate, or
H2O2 (Fig. 4C). These represented
sites of tyrosine phosphorylation previously identified from the study
of in vitro autophosphorylated Syk. Phosphorylation was observed on
peptides corresponding to Tyr130, Tyr317,
Tyr342, Tyr346, Tyr519, and
Tyr520. Linker region tyrosines are phosphorylated in the absence of Syk catalytic activity
The phosphorylation of Syk on tyrosine in intact cells occurs most
likely as a combination of autophosphorylation and phosphorylation by
receptor-associated, Src family kinases. To determine which site or
sites on Syk could be phosphorylated in the absence of Syk
autophosphorylating activity (and in the absence of endogenous Syk), a
catalytically inactive Syk mutant (Syk(K396R)) was prepared in which
Arg replaced an essential Lys in the active site. Syk-DT40
B cells were established that expressed the epitope-tagged Syk(K396R)
protein. These were incubated in the presence of
[32P]orthophosphate with or without stimulation with
anti-IgM Abs. The metabolically labeled proteins were
immunoprecipitated with anti-Myc epitope Abs (Fig. 5A) and subjected to tryptic
phosphopeptide mapping (Fig. 5B). In the absence of
activation, Syk(K396R) was extensively phosphorylated. Immunoblotting
experiments using Abs directed against phosphotyrosine indicated that
the protein was not phosphorylated on tyrosine (data not shown). Upon
receptor engagement, however, Syk(K396R) did become phosphorylated on
tyrosine. As shown in Fig. 5B, the prominent sites of
tyrosine phosphorylation were Tyr317, Tyr342,
and Tyr346.
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-32P]ATP. The resulting phosphoproteins were
separated by SDS-PAGE and transferred to nitrocellulose. Interestingly,
Syk(K396R) became phosphorylated in the immune complex, and this
phosphorylation was enhanced by receptor engagement (Fig. 5A). To determine the sites that were labeled, the band
corresponding to Syk(K396R) was digested with trypsin to generate a
phosphopeptide map. As shown in Fig. 5C, Syk(K396R) was
phosphorylated predominantly on Tyr317, Tyr342,
and Tyr346. These results indicated that an active protein
tyrosine kinase coimmunoprecipitated with the catalytically inactive
Syk(K396R) that could catalyze its phosphorylation upon the addition of
[-32P]ATP. Lyn, which is the only Src family kinase
expressed in DT40 B cells, was a likely candidate. To explore this,
Syk(K396R) was expressed in a Lyn-DT40 B cell line that
lacks any detectable Src family kinase activity (29). Anti-Myc
epitope immune complexes prepared from this cell line were devoid of
any significant tyrosine kinase activity (Fig. 5A). Thus,
Lyn appeared to preferentially phosphorylate Syk on hinge region
Tyr317, Tyr342, and Tyr346 both in
vitro and in vivo.
To explore an absolute requirement for Lyn for Syk phosphorylation,
Syk(WT) was expressed in Lyn-DT40 B cells, which were then
treated with or without H2O2. Peptide maps of
metabolically labeled Syk(WT) recovered from treated cells revealed a
low level of tyrosine phosphorylation occurring only at sites
corresponding to Tyr317, Tyr342, and
Tyr346 (Fig. 5D).
Phosphorylation of Syk on Tyr317 retards its electrophoretic mobility on SDS-PAGE
The tyrosine phosphorylation of Syk in vitro or in vivo typically
results in a shift in its electrophoretic mobility on SDS-PAGE. To
identify the site responsible for this mobility shift, Syk(WT) was
immunoprecipitated from Syk(WT)-expressing DT40 B cells with
anti-Myc epitope Abs and autophosphorylated in vitro with
[-32P]ATP. The resulting phosphoprotein migrated as a
doublet on SDS-PAGE (Fig. 6A).
The upper and lower phosphoprotein bands were excised separately and
subjected to tryptic peptide mapping, as described above. As shown in
Fig. 6B, only the peptide map generated from the upper band
exhibited the phosphopeptide containing Tyr317.
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A) or H2O2 (not
shown) exhibited a reduced electrophoretic mobility. In contrast,
Syk(Y317F) showed little change in mobility. Phosphopeptide mapping of
metabolically labeled Syk(Y317F) from peroxide-treated cells revealed
the expected loss of the Tyr317-containing phosphopeptide
(Fig. 6C). However, the remaining sites of tyrosine
phosphorylation (Tyr130, Tyr342,
Tyr346, Tyr519, and Tyr520) were
unaffected by the mutation. Mutation of Tyr317 to Phe enhances Ag receptor signaling
While the linker region Tyr342 has been reported
previously as a binding site for the SH2 domains of Vav and PLC-
(15, 31), a role for Tyr317 has yet to be explored. To
examine this question, Syk-DT40 cell lines were
transiently transfected with cDNAs expressing Syk(WT) or Syk(Y317F)
along with a NF-AT reporter construct. Cells lacking Syk failed to
stimulate NF-AT activity following receptor cross-linking (Fig. 7). Expression of Syk(WT) restored
signaling. Syk(Y317F), expressed at a level comparable with that of
Syk(WT) (Fig. 7), exhibited a greatly enhanced ability to stimulate the
B cell receptor-dependent induction of NF-AT activity (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP. These phosphopeptides,
which varied in size from 4 to 30 amino acids, could not be completely
resolved in a single HPLC run (28) and, in our hands, were not well
resolved by conventional two-dimensional thin-layer
electrophoresis/thin-layer chromatography. To separate the
phosphopeptides, we adopted an alternative procedure, alkaline PAGE,
which separates acidic peptides on the basis of charge and size (33, 34). This technique is particularly well suited to the analysis of
tryptic phosphopeptides, which generally contain only a single
positively charged amino acid and carry a net negative charge at pH 9
(34). In the specific case of Syk, the tryptic phosphopeptides
representing the major sites of tyrosine phosphorylation varied
sufficiently in mass and charge that they could be completely resolved
in a single one-dimensional separation. By comparing the migration
positions of the phosphopeptides derived from autophosphorylated Syk
with those of purified and sequenced phosphopeptides (28), and through
the use of phosphorylated synthetic peptides and site-directed mutants,
we could develop a standard peptide map useful for the analysis and
identification of Syk phosphorylation sites that are modified in vitro
or in vivo in response to a variety of stimuli (
Figs. 1–3). Using this one-dimensional mapping approach, we mapped the major tyrosine residues on Syk that are phosphorylated in intact cells in response to external stimuli such as anti-IgM Abs or oxidizing agents (pervanadate or H2O2). These stimuli produce comparable patterns of protein tyrosine phosphorylation of cellular proteins that are all dependent on the expression of cell surface Ag receptors with intact cytoplasmic ITAMs (35). However, the extent of phosphorylation resulting from the treatment of B cells with pervanadate or H2O2 is generally greater than that seen with anti-IgM Abs. We found that the tyrosines on Syk that are phosphorylated in activated B cells are the tyrosines that also are phosphorylated in an in vitro autophosphorylation reaction. The ability of Syk to autophosphorylate on these presumably physiologically relevant sites most likely explains why the aggregation of a chimeric CD16-Syk molecule alone can signal in T cells (36). However, not all of the autophosphorylation sites that can be identified in vitro also become modified in vivo. The sites of autophosphorylation that we have been unable to detect in vivo, which include Tyr358 or any of the carboxyl-terminal tyrosines (623, 624 or 625), are sites that are among the slowest to be modified during autophosphorylation (31) and seem unlikely to play an important role in the receptor-mediated activation of Syk.
The tyrosines that are phosphorylated in vivo are present in multiple
locations throughout the Syk molecule (Fig. 3) at sites that have been
implicated as important mediators of Syk function in hemopoietic cells.
Tyr130 is located between the tandem SH2 domains at a site
implicated in the regulation of the Syk-receptor interaction (31). The
ability of Syk to bind the Ag receptor and to participate in
receptor-mediated signaling is enhanced by the replacement of
Tyr130 with Phe and abrogated by its replacement with Glu
(31). Phosphorylation at this site would be expected to lead to the
dissociation of Syk from the receptor ITAMs. Tyr130 does
not appear to be a major site of phosphorylation modified in response
to receptor cross-linking (Fig. 4B), but is phosphorylated
in cells treated with protein tyrosine phosphatase inhibitors such as
pervanadate and H2O2 (Fig. 4C).
Thus, it is possible that Tyr130 is phosphorylated, but
only transiently, following receptor engagement. Alternatively,
Tyr130 might be phosphorylated primarily in response to
agents such as pervanadate and H2O2 that can
activate Syk in the absence of B cell receptor aggregation (35). Since
the placement of a negatively charged amino acid such as Glu at this
site results in an enzyme with an elevated basal catalytic activity
(31), the phosphorylation of Tyr130 could provide an
intriguing alternative mechanism for the activation of Syk in the
absence of receptor cross-linking.
Tyr342 and Tyr346 are located in the linker
region that separates the tandem SH2 domains from the catalytic domain.
This region of Syk and the corresponding Tyr315/319 region
of ZAP-70 have been implicated as docking sites for SH2
domain-containing molecules based on the location of the sites, the
sequence of amino acids surrounding the phosphotyrosines, and studies
with site-directed mutants. However, these sites had not been
demonstrated previously to be phosphorylated in vivo. The importance of
these residues as docking sites is indicated by studies showing that
the substitution of both Tyr342 and Tyr346 with
Phe blocks the ability of a CD8-Syk chimera to bind
PLC-1 (37), and the substitution of Syk Tyr342 or ZAP-70
Tyr315 with Phe blocks their ability to bind to Vav (25, 38). In the case of ZAP-70, this severely disrupts signaling (38).
Therefore, these sites are likely to be important, positive regulators
of Syk function.
Phosphorylation of the linker region Tyr317 is responsible
for the mobility shift of tyrosine-phosphorylated Syk frequently
observed on SDS-polyacrylamide gels. This suggests that phosphorylation
at this site may cause a conformational change in the protein. Since
the sequence of the tetrapeptide surrounding Tyr317 (NPYE)
strongly predicts a ß-turn (39), it is possible that the disruption
of this turn through phosphorylation of the tyrosine could account for
this altered mobility. The substitution of Tyr317 with Phe
also results in a form of the kinase with an enhanced ability to induce
NF-AT activity following receptor cross-linking (Fig. 7). Thus, it
would appear that the phosphorylation of Tyr317 serves as a
negative regulatory event, perhaps acting as a feedback signal to
down-regulate Syk activity. This Tyr is located in a region of Syk
roughly equivalent to that of Tyr292 of ZAP-70, which is
also phosphorylated in activated T cells (40). In ZAP-70,
Tyr292 serves also as a negative regulatory site, and its
replacement with Phe results in a form of the kinase with an enhanced
ability to participate in receptor-mediated signaling when expressed in
either T or B cells (41, 42). Tyr292 has recently been
shown, when phosphorylated, to be the site of interaction between
Zap-70 and the PTB domain of Cbl (43), a negative regulator of Syk
(44). Although the amino acid sequences in these regions of Syk
(VSFNPpYEPTGG) and ZAP-70 (LNSDGpYTPEPE) have diverged, Syk
Tyr317 still fits the determined consensus for a Cbl
interaction site (43).
Tyr519 and Tyr520 are located in the activation loop within the Syk catalytic domain and are homologous to ZAP-70 Tyr492 and Tyr493. These are sites whose phosphorylation is important for the participation of each of these kinases in receptor-mediated signaling pathways. For ZAP-70, the critical residue for phosphorylation is Tyr493, since its replacement with Phe results in a loss of function, while the replacement of Tyr492 with Phe enhances signaling (41, 42, 45, 46, 47). For Syk, the replacement of both Tyr519 and Tyr520 with Phe also reduces the receptor-mediated stimulation of protein tyrosine phosphorylation (19, 48). A reduction in protein tyrosine phosphorylation in transfected COS cells is also observed when either Tyr519 or Tyr510 is replaced with Phe, suggesting that the phosphorylation of both sites might be required for maximal activity (49). The phosphorylation of Tyr519 and Tyr520 also creates a docking site on Syk for the SH2 domain of Lck, and this interaction is thought to be important for signaling (24, 49).
The phosphorylation sites on Syk can be divided into two primary classes: 1) those that are preferentially phosphorylated by Lyn and do not require the catalytic activity of Syk; and 2) those that require the activities of both Syk and Lyn, but are not directly phosphorylated by Lyn. The linker region tyrosines, 317, 342, and 346, are the principal sites on Syk phosphorylated by the Lyn kinase both in vivo and in vitro. The finding of a preferential phosphorylation of Syk by Lyn at sites located within the linker region rather than within the catalytic domain was somewhat unexpected since the Lck kinase preferentially phosphorylates ZAP-70 at Tyr493, which is equivalent to Syk Tyr520 and is located within the activation loop (41). It is consistent, however, with other observations in both B cells and mast cells that have indicated that Syk is phosphorylated in trans by other tyrosine kinases primarily on sites outside the catalytic domain (48, 50). However, it is well established that Syk and ZAP-70 show fundamental differences in their intrinsic activities when expressed in B and T cells, with Syk being much less dependent on associated Src family kinase activity (51, 52, 53). This difference may be reflected, in part, by how they interact with Src family members and how important the phosphorylation of activation loop tyrosines is to changes in catalytic activity. It is also formally possible that a tyrosine kinase other than Lyn, but one that is dependent for activity on the expression of Lyn, is responsible for catalyzing the phosphorylation of the linker region sites. It is important to note that the contributions of Lyn-dependent phosphorylations on the coupling of Syk to downstream signaling pathways such as NF-AT induction are greatly dependent on the sites that are being modified. Some phosphorylations are likely to be stimulatory (Tyr342 and/or Tyr346), while others are inhibitory (Tyr317). Thus, the consequences of coclustering Lyn-associated receptors with the Ag receptor on Syk-mediated signaling may well be dependent on the exact location of the sites that are being phosphorylated.
The phosphorylation of Tyr130, Tyr519, and
Tyr520 only occurs to a discernible extent when a
catalytically active form of Syk is expressed in cells containing
endogenous Lyn. These sites are not appreciably phosphorylated by Lyn,
but are refractory to phosphorylation when Syk(WT) is expressed in
Lyn- cells (Fig. 5D). This may reflect a
requirement for a Lyn-catalyzed phosphorylation of receptor ITAMs to
create docking sites for the recruitment of Syk to the Ag receptor. The
recruitment and binding of Syk to the Ag receptor ITAMs could then
stimulate Syk-catalyzed autophosphorylation reactions that lead to the
modification of Tyr130, Tyr519, and
Tyr520. The phosphorylated Ag receptor would act as a
scaffold for the recruitment and proper positioning of Syk molecules to
allow enhanced intermolecular phosphorylations to occur, as has been
proposed previously for ZAP-70 (54). A recent model for Syk activation
in RBL-2H3 mast cells proposed an initial Lyn-catalyzed phosphorylation
of activation loop tyrosines, which would then initiate a cascade of
Syk-catalyzed intermolecular phosphorylations that would account for
the bulk of Tyr519 and Tyr520 phosphorylation
(50). Our results cannot rule out a direct contribution of Lyn to a
low-level phosphorylation of Tyr519 and Tyr520.
Finally, the presence of phosphoserine or phosphothreonine on Syk isolated from metabolically labeled cells raises the possibility that Syk might also be regulated by protein-serine/threonine kinases. The kinase or kinases responsible for this phosphorylation have not yet been identified. However, since Syk has been reported to coimmunoprecipitate with protein kinase C µ (55), this enzyme is a potential candidate. Studies to identify the sites of serine/threonine phosphorylation on Syk are currently under way.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Geahlen, Department of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Building, Purdue University, West Lafayette, IN 47907.
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; GST, glutathione S-transferase; NF-AT, nuclear factor of activated T cells; PLC, phospholipase C.
Received for publication April 29, 1998. Accepted for publication July 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 16:2333.[Medline]
R1-mediated tyrosine phosphorylation and activation of a 72 kDa protein tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107. chain after receptor aggregation. J. Biol. Chem. 268:23318.Iibß3. J. Biol. Chem. 269:28859. receptor signaling. J. Biol. Chem. 268:15900.RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein. J. Immunol. 152:5429.[Abstract]
receptor I (FcRI) and receptor II (FcRII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J. Biol. Chem. 268:24442. protein during signal transduction via the B cell antigen receptor. EMBO J. 13:83.[Medline]
/Igß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.-tubulin on tyrosine. J. Biol. Chem. 271:4755.1 in B lymphocytes. J. Biol. Chem. 270:11806. interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk. Mol. Cell. Biol. 16:1305.[Abstract]
and 
enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins. Mol. Cell. Biol. 15:3171.[Abstract]
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