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Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI)-induced degranulation of mast
cells. To examine the mechanism of Syk regulation, the two tyrosine
residues at 519 and 520 in the putative activation loop of rat
Syk were mutated to phenylalanine either singly or in combination. The
various mutants were expressed in a Syk-negative variant of the RBL-2H3
(rat basophilic leukemia 2H3) mast cell line. In these transfected cell
lines, mutant Syk did show increased tyrosine phosphorylation in vivo
and increased enzymatic activity in vitro after FcRI aggregation.
There were conformational changes detected by an Ab when the wild-type
and mutant Syk were either tyrosine phosphorylated or bound to
tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif
peptides. However, these mutant Syk were incapable of transducing
FcRI signaling. In cells in which the expression level of mutant Syk
was similar to that of the wild-type Syk, FcRI cross-linking induced
no increase in cellular protein tyrosine phosphorylation, no increase
in tyrosine phosphorylation of phospholipase C-
2 and
mitogen-activated protein kinase, and no histamine release.
Overexpression of Y519F or Y520F Syk mutants partially reconstituted
the signaling pathways. These results indicate that these tyrosines in
the putative activation loop are not essential for the enzymatic
activity of Syk or for the conformational changes induced by binding of
tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif
peptides. However, these tyrosines are necessary for Syk-mediated
propagation of FcRI signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI)2 on mast cells,
like the T and B cell receptors, is a multimeric protein complex
consisting of ligand-binding domain(s) and signal-transducing subunits.
These receptors lack intrinsic enzymatic activity, but they all contain
the immunoreceptor tyrosine-based activation motif (ITAM), which is
critical for cell activation (1, 2, 3, 4). Ag binding to these receptors
results in phosphorylation of the tyrosines in the ITAM. Syk/ZAP-70
protein tyrosine kinases then associate with the
tyrosine-phosphorylated ITAM, which results in tyrosine phosphorylation
of Syk/ZAP-70 and an increase in their enzymatic activity (5, 6, 7, 8).
The importance of Syk/ZAP-70 activation in signaling through Ag and Fc
receptors has been demonstrated by a number of experiments. The
peripheral CD4+ T cells from ZAP-70-deficient patients are
incapable of transducing the normal TCR-mediated intracellular signals
(9). In Syk-deficient avian B cells, B cell receptor (BCR) engagement
fails to induce intracellular calcium mobilization and does not result
in the full spectrum of intracellular protein tyrosine phosphorylations
(10). Similarly, in Syk-deficient mast cells, FcRI aggregation
induces no detectable increase in total cellular protein tyrosine
phosphorylation and no degranulation (11, 12), defects that are
reconstituted by the expression of wild-type Syk (11).
Because of the critical role of Syk/ZAP-70 in signaling, there is much
interest in understanding the mechanism of the regulation of these
enzymes. Most protein tyrosine kinases are substrates for
phosphorylation on tyrosine residues. Phosphopeptide mapping has
identified 6 tyrosine phosphorylation sites in ZAP-70 and 10 in Syk
(13, 14). The tyrosine phosphorylation of Syk/ZAP-70 at some of these
sites may be important in regulating the catalytic activity of these
molecules and may also provide docking sites for binding of substrates
and other molecules. There are two adjacent tyrosine residues in the
catalytic domain of Syk (Y518, Y519) and ZAP-70 (Y492, Y493). These two
tyrosines located in the "activation segment or loop" are probably
important for regulating the function of kinases (15). In ZAP-70, the
phosphorylation of the two tyrosines requires an Src family kinase, but
they are autophosphorylated in Syk (13). In Syk-deficient B cells, the
expression of Syk with both of these tyrosines mutated to
phenylalanines abrogates the function of Syk in Ag signaling but not
binding to ITAM (16). In 3T3E cells (NIH3T3 cells transfected with the
, ß, and subunits of the FcRI), Y518F- and
Y519F-mutated Syk expressed by recombinant vaccinia viruses has
basal activity nearly identical to that of wild-type Syk but reduced
tyrosine phosphorylation and no increase in activity after FcRI
aggregation (17). Mutation of the two tyrosine residues separately in
ZAP-70 has interesting effects. When the first tyrosine is mutated
(Y492F), there is an increase in the intrinsic kinase activity in COS-7
cells (18) and also an increase in its capacity to reconstitute
BCR-induced signaling in Syk-deficient B cells (19). When the second
tyrosine is mutated (Y493F), there is no change in enzymatic activity
(18), but it cannot be activated by Lck in COS-7 and Sf9 insect
cells (18, 19) and fails to reconstitute BCR-mediated signaling in
Syk-negative B cells (19).
Structural studies have suggested a mechanism for the regulation of protein tyrosine kinases by the activation loop tyrosines (20, 21, 22, 23). In the insulin receptor kinase there are three tyrosines (Y1158, Y1162 and Y1163) in the activation loop; Y1162 but not Y1163 lies within the catalytic center such that it prevents both ATP and substrate binding. In the activated form, phosphorylated Y1163 is the key residue that stabilizes the molecule in a different conformation and allows kinase activity. These studies suggest that activation could occur optimally only by transphosphorylation. The structure of the fibroblast growth factor receptor kinase domain suggests an alternative model in which Y653 in the activation loop interferes with substrate binding but not with ATP binding. Phosphorylation of this tyrosine is thought to induce a conformational change that would allow substrate access to the catalytic center.
The purpose of the present studies was to characterize the role of the
activation loop tyrosines in the function of Syk in FcRI signaling.
Therefore the tyrosines were mutated either singly or in combination,
and these mutant forms were expressed by stable transfection in a
Syk-deficient variant of the RBL-2H3 (rat basophilic leukemia 2H3)
cells. The use of these cells allowed the function of Syk to be
examined in a background completely deficient of this kinase and
without any modification such as tags. Our results suggested that
phosphorylation of Y519 or Y520 (the double tyrosines in rat Syk that
are equivalent to Y518, Y519 in chicken Syk) are not critical
for the in vitro kinase activity of Syk but are essential for
propagating the FcRI signaling in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Triton X-100, Nonidet P-40, ATP, protein A agarose beads, and
protease inhibitors were obtained from Sigma (St. Louis, MO). The
materials for electrophoresis were purchased from Novex (San Diego,
CA), and polyvinylidene difluoride transfer membrane was purchased from
Millipore (Bedford, MA). The plasmid expressing the human cytoplasmic
domain of erythrocyte band 3 protein (cdb3) was kindly provided by Dr.
P. S. Low (Purdue University, West Lafayette, IN). The cdb3
protein was purified as described previously (24). The rabbit
anti-rat Syk Abs have been described previously; the anti-SykI
is to a sequence between the second Syk Src homology region 2 (SH2) and
kinase domains, and the anti-SykC is to a peptide corresponding to
the carboxyl-terminal amino acids (6). Mouse monoclonal
anti-FcRI (mAb BC4) and the anti-trinitrophenol-specific
IgE have been described previously (25, 26). The horseradish
peroxidase-conjugated anti-phosphotyrosine Ab PY-20 was from ICN
Immunobiologics (Lisle, IL). Phosphoplus p44/42 mitogen-activated
protein kinase (MAPK) (Tyr204) Ab kit was from New
England BioLabs (Beverly, MA). The sources of other materials not
indicated were as described previously (6).
Construction of plasmids and stable transfections
Y519 and/or Y520 of rat Syk were mutated to phenylalanine using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s directions. Following is the nomenclature used to refer to the different mutations in Syk: Y519F (FY), Y520F (YF), and FY plus YF (double mutation; FF). The identities of all mutations were verified by standard DNA sequencing. Mutant Syk cDNAs were cloned into the SacI site of the pSVL expression vector (Pharmacia LKB, Piscataway, NJ). The expression constructs were cotransfected with pSV2-neo vector into Syk-negative TB1A2 cells by electroporation, and stable transfected clones were selected with 400 µg/ml of active G418 (Life Technologies, Grand Island, NY) as described previously (11). The cell lines were screened for the expression of mutated Syk by immunoblotting with anti-Syk Abs, and four clones with each mutation were selected for further study.
Cell culture and activation
TB1A2 cells and their transfectants were cultured as monolayers
in Eagle’s MEM supplemented with 15% heat-inactivated FBS,
penicillin, streptomycin, and amphotericin (27). For activation, cell
monolayers after overnight culture were stimulated as described
previously (11). When cells were cultured with Ag-specific IgE,
activation was with the Ag DNP coupled to human serum albumin at
concentrations from 0.01 to 1.0 µg/ml or the calcium ionophore A23187
at 0.25 to 2 µM. Cells were also stimulated with 0.3 µg/ml
anti-FcRI Abs (mAb BC4) or 40 nM PMA. After the indicated
times, the supernatants were removed for histamine analysis.
Immunoprecipitation and immunoblotting
After stimulation for the indicated times, the cell monolayers were rinsed with ice-cold PBS containing 1 mM Na3VO4 and protease inhibitors (2 mM PMSF, 90 mU/ml aprotinin, 50 µg/ml leupeptin, and 50 µg/ml pepstatin) and solubilized in Brij lysis buffer (3% Brij-96, 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM Na3VO4, and 10 mM 2-ME plus protease inhibitors) or with modified RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, in addition to protease inhibitors and Na3VO4). The postnuclear supernatants were immunoprecipitated with Abs bound to protein A-agarose beads. After rotation at 4°C for 1 h, the beads were washed four times with ice-cold lysis buffer, and the proteins were eluted by boiling for 5 min with sample buffer as described previously (7). Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. The blots were probed with 40 ng/ml anti-phosphotyrosine mAb PY-20 conjugated to horseradish peroxidase and, after stripping, were reblotted with other Abs. In all blots, proteins were visualized by enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL).
In vitro kinase assay
Syk immunoprecipitated with rabbit anti-SykI Abs as
described above was further washed with kinase buffer (30 mM HEPES, pH
7.5, 10 mM MgCl2, and 2 mM MnCl2) and
resuspended in 50 µl of kinase buffer. The reactions were started by
the addition of 3 µCi of [-32P]ATP and 4 µM ATP
with or without cdb3 as substrate. After the indicated incubation time
at room temperature, the reactions were stopped by the addition of 50
µl of 2x Laemmli sample buffer and boiling for 10 min. The eluted
proteins were separated under reducing conditions by SDS-PAGE (10%
gels), electrotransferred to membranes, and visualized by
autoradiography. The same membranes were immunoblotted with
anti-Syk Abs as described above.
Studies of the conformation changes in Syk induced by ITAM peptides
These studies were essentially as described previously (28). The
phosphorylated synthetic peptide used was based on the subunit of
FcRI and immunoprecipitation was from lysates of the different
transfected cell lines with anti-SykC in the presence or absence of
this peptide. For the effect of the ITAM on autophosphorylation, Syk
was immunoprecipitated with anti-SykI Abs, and the reaction was as
described previously (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the potential role of the activation loop tyrosines in
Syk on FcRI signaling, Y519 and Y520 of Syk were singly or in
combination changed to phenylalanines by site-directed mutagenesis. The
mutated Syk cDNA was transfected into a Syk-negative variant of RBL-2H3
cells and clones were selected with G418 containing media. Expression
of Syk was assessed by anti-Syk immunoblotting. Clones were
selected with the level of expression of Syk comparable with or higher
than that expressed in cells transfected with wild-type Syk (Fig. 1
). In general, for every mutant at least
two clones with Syk expressed at levels similar to wild-type and two
with levels that were higher were used for further characterization.
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RI induced tyrosine phosphorylation of the different Syk
mutants
FcRI aggregation results in tyrosine phosphorylation and
increased kinase activity of Syk (6, 29). To test whether receptor
stimulation still induced tyrosine phosphorylation of the different
mutant Syk, cells were activated with anti-FcRI mAb. Repeated
experiments showed that FcRI aggregation induced tyrosine
phosphorylation of the different mutant Syk (Fig. 2). There were, however, differences in
the extent of this phosphorylation. The tyrosine phosphorylation of FY
mutant was comparable with that of wild-type Syk, whereas the level of
the tyrosine phosphorylation of the YF mutant was weaker than that of
wild-type Syk, although the difference was not dramatic. Unlike the
single mutants, the FF double mutant was tyrosine phosphorylated less
than the wild-type Syk after receptor stimulation. This decreased
phosphorylation of the FF mutated Syk was also apparent in time course
experiments (data not shown). The level of Syk expression in cells had
no effect on the extent of the receptor-induced tyrosine
phosphorylations. Therefore, FcRI aggregation results in tyrosine
phosphorylation of Syk even when both of the activation loop tyrosines
are mutated. This phosphorylation must be on sites other than the
activation loop.
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The functional activity of the mutant Syk was tested in an
immune-complex kinase assay using the cdb3 band protein as exogenous
substrate. Surprisingly, cdb3 band was phosphorylated by all of these
mutated Syk (Fig. 3), and none of the
different Syk showed decreased enzymatic activity compared with
wild-type Syk, even though the in vivo tyrosine phosphorylation of the
double mutant (FF) was clearly weaker than that of wild-type Syk (see
above). The extent of the autophosphorylation of the different mutants
of Syk was also similar. Since no receptor-induced increase in Syk
kinase activity was detected in these experiments, shorter incubation
times were used for the in vitro kinase reaction. Under these
conditions, there was no phosphorylation of the exogenous substrate by
wild-type or the different Syk mutants, but all showed FcRI
aggregation-induced increase in autophosphorylation (Fig. 4). These results indicate that mutation
of the activation loop tyrosines does not affect the in vitro kinase
activity of Syk and that FcRI aggregation still induces an increase
in the autophosphorylation activity of Syk.
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RI signaling
Receptor-induced tyrosine phosphorylation of cellular proteins is
one of the earliest events after FcRI aggregation (30). We therefore
examined the FcRI-induced tyrosine phosphorylation of total cellular
proteins in the different transfected cell lines (Fig. 5). Transfection of wild-type Syk
restored receptor-mediated cellular protein tyrosine phosphorylations
in the Syk-negative TB1A2 cells. In the cells expressing mutant Syk at
levels comparable with those of cells transfected with the wild type,
receptor aggregation failed to induce an obvious increase in cellular
protein tyrosine phosphorylations. FcRI-induced protein tyrosine
phosphorylation was partially reconstituted in cells that were
overexpressing either the FY or the YF mutant forms of Syk. However,
these phosphorylations were weaker in the cells with the YF compared
with the FY mutant Syk. There were minimal receptor-mediated protein
tyrosine phosphorylations in the cells expressing double-mutated (FF)
Syk. Therefore, although the different Syk mutants have in vitro kinase
activity, they are defective in propagating intracellular signals.
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RI activation results in degranulation by a pathway
in which Syk is essential (11, 12). Therefore, we investigated the
effects of the mutation of these tyrosines on FcRI-stimulated
histamine release. Syk-negative cells and cells transfected with
wild-type or mutated Syk were activated by either FcRI aggregation
or by the calcium ionophore A23187 (Fig. 6). The histamine release stimulated by
the calcium ionophore A23187 was similar among all of these clones and
was therefore used as an internal control. As we have observed
previously, the transfection of wild-type Syk reconstituted histamine
release. In cells with the mutated forms of Syk, there was no
FcRI-induced histamine release when the expression level was the
same as in the cells with wild-type Syk. However, overexpression of
either the FY or the YF mutant form of Syk partially reconstituted
secretion. In cells expressing the double mutant form of Syk (FF),
there was no FcRI-induced secretion even in the lines in which there
was overexpression of the protein. Therefore, Y519 and Y520 in Syk are
essential for propagation of the intracellular signals that result in
degranulation.
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and MAPK activation
Previous studies have shown that FcRI-induced tyrosine
phosphorylation of PLC-2 is dependent on Syk (11). In cells in which
the expression of mutant forms of Syk were comparable with the level of
wild-type Syk, there was no detectable tyrosine phosphorylation of
PLC-2 (data not shown). Therefore, we examined whether
overexpression of FY, YF, or the double mutant (FF) forms of Syk could
support the FcRI-mediated tyrosine phosphorylation of PLC-2. In
cells overexpressing FY Syk, aggregation of FcRI induced some
tyrosine phosphorylation of PLC-2, although this increase was much
weaker than that of wild-type Syk (Fig. 7). Overexpression of the YF Syk resulted
in only a very slight increase in PLC-2 tyrosine phosphorylation,
while the double mutant Syk failed to reconstitute the FcRI-mediated
PLC-2 tyrosine phosphorylation.
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RI-induced activation of MAPK is also downstream of Syk (31).
Therefore, we investigated the role of the activation loop tyrosines on
regulating activation of MAPK in these transfected cells (Fig. 8). FcRI aggregation did not induce
activation of MAPK in the Syk-negative cells. Transfection of wild-type
Syk reconstituted FcRI-induced tyrosine phosphorylation. In the
cells expressing the different mutant Syk at levels comparable with
those of wild-type, receptor stimulation did not result in
phosphorylation of MAPK. The overexpression of Syk FY or of YF
partially reconstituted this MAPK activation, which was greater with
the FY than with the YF mutation. In contrast, FcRI aggregation did
not induce any tyrosine phosphorylation of MAPK in cells overexpressing
the double mutant (FF) Syk. As an internal control, MAPK became
phosphorylated to the same extent when all of the different cell lines
were stimulated with PMA to directly activate protein kinase C.
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The interaction of Syk with tyrosine-phosphorylated ITAM
peptides or the tyrosine phosphorylation of Syk results in a
conformational change recognized by a polyclonal anti-peptide Ab
(anti-SykC) raised to the carboxyl-terminal sequence of Syk (28).
Anti-SykC also immunoprecipitates Syk that is tyrosine phosphorylated
in vivo after cell activation (28). For example, addition of synthetic
diphosphorylated ITAM peptide corresponding to the subunits of
FcRI results in a conformational change that allows the
immunoprecipitation of nonphosphorylated Syk by anti-SykC (28).
Therefore, anti-SykC was used to detect conformational changes.
There was enhanced precipitation of Syk with the anti-SykC Abs when
diphosphorylated ITAM peptide was added to the lysates of cells
expressing the different mutant forms of Syk (Fig. 9). There was no detectable difference in
the extent of this precipitation in mutants that had either one or both
of the tyrosines mutated. The amount of Syk immunoprecipitated with
anti-SykC increased when lysates were from stimulated cells, and
this correlated with the extent of its in vivo tyrosine phosphorylation
(data not shown). FcRI was also coprecipitated with Syk in amounts
that correlated with the extent of Syk tyrosine phosphorylation (data
not shown). Therefore, the mutation of the tyrosines in the activation
loop does not interfere in the conformational changes induced in Syk by
either tyrosine phosphorylation or by interaction with diphosphorylated
ITAM peptides.
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ITAM peptide induced any change in
the kinase activity of the Syk mutants. Syk was immunoprecipitated
with anti-SykI Abs and subjected to immune complex kinase assays
with or without added ITAM peptide (Fig. 10). The slow autophosphorylation of
the different Syk mutants was increased by the addition of the
diphosphorylated ITAM peptides. This effect was less when both Y519 and
Y520 were mutated. These results indicate that the conformational
changes induced by the ITAM peptides still result in enhanced kinase
activity when these tyrosines are mutated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-induced signaling in
mast cells, the mechanism of its regulation is not fully understood.
One mechanism for regulation is by binding to tyrosine-phosphorylated
ITAM peptides, which results in a conformational change and an increase
in the kinase activity of Syk (28). Here we demonstrate another
mechanism for regulating the in vivo function of Syk that requires the
activation loop tyrosines. When Syk mutated in these tyrosines was
transfected into Syk-negative TB1A2 cells, FcRI-aggregation failed
to induce cellular protein tyrosine phosphorylation and phosphorylation
of PLC-2 or MAPK and did not result in histamine release. However,
receptor cross-linking still induced increased tyrosine phosphorylation
and increased enzymatic activity of the mutant Syk. In vitro binding of
a phosphorylated peptide based on the ITAM of FcRI induced
increased kinase activity of the mutant Syk. Furthermore, the
mutated Syk also showed similar conformational changes as wild-type Syk
after cell stimulation. These results suggest that these activation
loop tyrosines are not essential for the in vitro enzymatic activity of
Syk, but they are necessary for in vivo propagation of FcRI
signaling. Expression level
The expression level of the mutant Syk was an important parameter for determining the functional impairment in signal transduction. There was no reconstitution of signal transduction when the FY or the YF mutants were expressed at the same level as the wild-type Syk. However, overexpression of these two mutant forms exhibited some of the wild-type responses in activation of signaling pathways and in degranulation. Therefore, overexpression of these mutant forms could mask defects induced by these mutations and/or induce alternative signaling pathways that are not active in the native state. In contrast, the overexpression of the doubly mutated form of Syk still did not activate any of the intracellular signaling pathways.
Syk tyrosine phosphorylation
Mutation of either of the two tyrosines had effects on the in vivo tyrosine phosphorylation of Syk. There was minimal if any change when the first tyrosine was mutated but some decrease after mutation of the second tyrosine. However, when both tyrosines were mutated, there was a more dramatic decrease, although there was still in vivo tyrosine phosphorylation of Syk. However, all of the different mutants autophosphorylated to an equal extent in an in vitro kinase reaction, suggesting that the in vivo differences are not due to changes in autophosphorylating activity but to interactions of Syk with other molecules. Such interactions of Syk could be with other kinases that can potentially phosphorylate Syk and other downstream molecules. For example, several Src family kinases interact by their SH2 domain with tyrosine-phosphorylated Syk (7, 34, 35, 36, 37, 38), and the in vitro binding of Lck requires the phosphorylation of the two activation loop tyrosines (36). The activation loop tyrosines of Syk are autophosphorylated in vitro probably by transphosphorylation, which then can bind other molecules including kinases (such as Lyn) that are critical for downstream signal transduction (14).
The strong tyrosine phosphorylation of Syk, which has the two tyrosines in the activation loop mutated suggests that other tyrosines in the molecule are potential sites both for autophosphorylation (transphosphorylation) and for phosphorylation in vivo by other kinases. This is not surprising, as Syk has multiple tyrosines that are phosphorylated in vitro. The difference between in vitro and in vivo results could suggest that these alternative sites are not as good for transphosphorylation in vivo as they are in vitro.
Effect of activation loop mutations on substrate tyrosine phosphorylation
The surprising observation was that the mutation of either or both
of the activation loop tyrosines in Syk did not significantly reduce
its in vitro kinase activity. Thus, by both autophosphorylation and by
the ability to tyrosine phosphorylate a substrate in vitro, there was
no change in the activity of Syk. However, there was a dramatic
difference in the capacity of Syk to phosphorylate in vivo substrates,
such as PLC-2, when these tyrosines were mutated. Therefore, the
effect of these mutations of Syk on signaling could be due either to
subtle changes in the catalytic activity of Syk or to effects on the
interaction of Syk with substrates.
Function of activation loop tyrosines in Syk activation and function
Structural studies indicate that activation loop tyrosines are important in regulating the function of protein kinases. In protein tyrosine kinases that are receptors, the binding of ligand results in a conformational change, which allows transphosphorylation of the activation loop tyrosines. Phosphorylation with the resulting change in the charge induces a conformational change in the activation loop and allows the binding of substrates. This results in further transphosphorylation of the molecule and the phosphorylation of substrates. Comparison of the crystal structure of insulin and of the fibroblast growth factor receptor suggests that there are different mechanisms by which the activation loop inhibits transphosphorylation of protein tyrosine kinases. The inhibitory effect in the insulin receptor is present because the activation loop inhibits binding of both ATP and of the substrate, whereas in the case of the fibroblast growth factor receptor there is inhibition of only the substrate. These differences would suggest that inhibition by the activation loop is stronger in insulin than in the fibroblast growth factor receptor. Furthermore, depending on the concentration of substrate, it is possible that there could be activation of the kinase in the absence of phosphorylation of the activation loop residues in the case of fibroblast growth factor. The capacity of Syk to phosphorylate substrate even when it is not tyrosine phosphorylated suggests that there is weak inhibition by the activation loop.
There are several studies of the effect of mutations in the activation
loop tyrosines on the function of Syk and the related ZAP-70. Although
they are structurally similar, there are differences between these two
kinases in their signaling capacity (39). For example, activation of
ZAP-70 but not of Syk requires an Src family kinase, and the enzymatic
activity of Syk is at least 100-fold greater than that of ZAP-70
(40, 41, 42, 43). In vitro the activation loop tyrosines of ZAP-70 but not of
Syk require Lck to become tyrosine phosphorylated (13, 14). With
ZAP-70, mutation of the first activation loop tyrosine increases,
whereas mutation of the second or both tyrosines has minimally or no
effect on enzymatic activity. When such ZAP-70 mutants are transfected
into a Syk-negative avian B cell line, the ZAP-70 mutated at the first
activation loop tyrosine (FY) results in increased activation signals
compared with the wild-type ZAP-70, while the kinase with
mutation of the second (YF) or both tyrosines (FF) does not function
(19, 44, 45). In transient transfection in COS cells, the FY mutation
results in a fourfold increase in ZAP-70 basal kinase activity,
while the YF mutant has the same basal kinase activity as wild-type
ZAP-70. Incubation with pervanadate, a tyrosine phosphatase inhibitor,
results in an increase in the tyrosine phosphorylation in cells
transfected with wild-type and FY mutant, but no obvious change with
the YF mutant (18). Also in transfected COS cells, a chimeric Syk with
both of the activation loop tyrosines mutated does not induce tyrosine
phosphorylation of endogenous PLC-1, and it is only minimally
precipitated by the SH2 domain of PLC-1, even though the mutated Syk
protein is tyrosine phosphorylated (46). In Jurkat T cells,
overexpression of ZAP-70 with both of these mutated tyrosines inhibits
TCR-induced activation of NFAT (a nuclear factor enhancing IL-2 gene
transcription), intracellular calcium increase, and activation of
extracellular signal-regulated kinases. However, this FF mutant
is tyrosine phosphorylated; binds to the TCR 
subunit; associates
with Lck; and, by in vitro kinase reaction, has increased basal kinase
activity compared with wild-type ZAP-70 (47). These results suggest
that there are differences in the regulation of Syk compared with
ZAP-70 by the two tyrosines in the activation loop. However, altogether
the data strongly indicate that the activation loop tyrosines are
critical in signaling by both of these kinases.
The loss of signaling by Syk mutated at these tyrosines could also imply that the activation loop tyrosines once phosphorylated are potential binding sites for both intramolecular and intermolecular interactions. The change in the charge of these tyrosines once phosphorylated could allow intramolecular interactions that stabilize a conformation that results in interactions with potential substrates. Alternatively, the phosphorylated tyrosines in the activation loop could be potential binding sites for interaction with other molecules. The structure of insulin crystallized in the active form suggests that the two activation loop tyrosines do not interact with other residues in the kinase and therefore could potentially be docking sites for downstream signaling proteins (22) such as insulin receptor substrate 2 (48, 49), Grb-2 (50, 51), or SHP-1 (52). For Syk there is evidence that the two tyrosines once phosphorylated are involved in interaction with Lck (36). Therefore, further work is required to define the interactions of Syk with other molecules that may be disrupted by mutation of the activation loop tyrosines that would explain the in vivo dramatic changes in downstream signaling.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: FcRI, receptor with high affinity for IgE; ITAM, immunoreceptor tyrosine-based activation motif; BCR, B cell receptor; SH2, Src homology region 2; MAPK, mitogen-activated protein kinase; FY, Syk Y519F mutation; YF, Syk Y520F mutation; FF, Syk FY plus YF mutation; PLC, phospholipase C.
Received for publication May 7, 1998. Accepted for publication June 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107. chain. J. Biol. Chem. 268:23318. and ß subunits of the high-affinity receptor for immunoglobulin E, FcRI. Mol. Cell. Biol. 15:272.[Abstract]
RI aggregation in rat basophilic leukemia cells: p72syk is a minor component but the major protein tyrosine kinase of pp72. J. Biol. Chem. 269:16902.RI signaling. Immunol. Today 13:195.[Medline]
RI is not shared by a G protein-coupled receptor. J. Biol. Chem. 270:10960./Igß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590. complex. J. Exp. Med. 181:1997.1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk. Mol. Cell. Biol. 16:1305.[Abstract]
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M. Simon, L. Vanes, R. L. Geahlen, and V. L. J. Tybulewicz Distinct Roles for the Linker Region Tyrosines of Syk in Fc{epsilon}RI Signaling in Primary Mast Cells J. Biol. Chem., February 11, 2005; 280(6): 4510 - 4517. [Abstract] [Full Text] [PDF]
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S. Atwell, J. M. Adams, J. Badger, M. D. Buchanan, I. K. Feil, K. J. Froning, X. Gao, J. Hendle, K. Keegan, B. C. Leon, et al. A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase J. Biol. Chem., December 31, 2004; 279(53): 55827 - 55832. [Abstract] [Full Text] [PDF]
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 J. R. Faeder, W. S. Hlavacek, I. Reischl, M. L. Blinov, H. Metzger, A. Redondo, C. Wofsy, and B. Goldstein Investigation of Early Events in Fc{epsilon}RI-Mediated Signaling Using a Detailed Mathematical Model J. Immunol., April 1, 2003; 170(7): 3769 - 3781. [Abstract] [Full Text] [PDF]
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 J. Zhang, E. Berenstein, and R. P. Siraganian Phosphorylation of Tyr342 in the Linker Region of Syk Is Critical for Fc{varepsilon}RI Signaling in Mast Cells Mol. Cell. Biol., December 1, 2002; 22(23): 8144 - 8154. [Abstract] [Full Text] [PDF]
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Z.-H. Xie, I. Ambudkar, and R. P. Siraganian The Adapter Molecule Gab2 Regulates Fc{epsilon}RI-Mediated Signal Transduction in Mast Cells J. Immunol., May 1, 2002; 168(9): 4682 - 4691. [Abstract] [Full Text] [PDF]
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E. Bulanova, V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus The IL-15R{alpha} Chain Signals Through Association with Syk in Human B Cells J. Immunol., December 1, 2001; 167(11): 6292 - 6302. [Abstract] [Full Text] [PDF]
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 K. Sada, J. Zhang, and R. P. Siraganian SH2 domain-mediated targeting, but not localization, of Syk in the plasma membrane is critical for Fc{epsilon}RI signaling Blood, March 1, 2001; 97(5): 1352 - 1359. [Abstract] [Full Text] [PDF]
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K. Sada, J. Zhang, and R. P. Siraganian Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function J. Immunol., January 1, 2000; 164(1): 338 - 344. [Abstract] [Full Text] [PDF]
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J. Zhang and R. P. Siraganian CD45 Is Essential for Fc{epsilon}RI Signaling by ZAP70, But Not Syk, in Syk-Negative Mast Cells J. Immunol., September 1, 1999; 163(5): 2508 - 2516. [Abstract] [Full Text] [PDF]
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D. Vial, H. Okazaki, and R. P. Siraganian The NH2-terminal Region of Focal Adhesion Kinase Reconstitutes High Affinity IgE Receptor-induced Secretion in Mast Cells J. Biol. Chem., September 1, 2000; 275(36): 28269 - 28275. [Abstract] [Full Text] [PDF]
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J. Zhang, M. L. Billingsley, R. L. Kincaid, and R. P. Siraganian Phosphorylation of Syk Activation Loop Tyrosines Is Essential for Syk Function. AN IN VIVO STUDY USING A SPECIFIC ANTI-Syk ACTIVATION LOOP PHOSPHOTYROSINE ANTIBODY J. Biol. Chem., November 3, 2000; 275(45): 35442 - 35447. [Abstract] [Full Text] [PDF]
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