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*
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907; and
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
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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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and Igß)
components of the BCR complex become phosphorylated, most likely by Src
family kinases, on a pair of tyrosines located within their cytoplasmic
tails. This pair of tyrosines and the surrounding amino acids comprise
a functional motif known as an ITAM (immunoreceptor tyrosine-based
activation motif) that is conserved among multiple immune recognition
receptors (3, 4). Phosphorylation of the ITAM tyrosines
creates a docking site for the tandem pair of SH2 domains of the
protein-tyrosine kinase, Syk, leading to its recruitment from the
cytoplasm to the site of the aggregated receptor (5, 6).
This is a critical step in the signaling process because a multitude of
signaling pathways are attenuated in the absence of a functional Syk
kinase, including BCR-dependent phosphoinositide production, calcium
mobilization, and activation of mitogen-activated protein kinase, c-Jun
N-terminal kinase, and p90Rsk (7, 8).
The participation of Syk in these signaling cascades is a function of
its ability to bind to and remain bound to the Ag receptor (9, 10), its intrinsic catalytic activity (10), and its
ability to recruit downstream effector molecules to the site of the
aggregated BCR (1). These properties are, in turn,
dependent on the state of phosphorylation of key tyrosine residues.
Receptor-associated Syk becomes phosphorylated on multiple tyrosines by
a combination of autophosphorylation and phosphorylation in
trans by receptor-associated, Src-family kinases such as Lyn
(11, 12). Tyrosine phosphorylation in vivo occurs
predominantly on five residues: two on the activation loop within the
catalytic domain (Y519 and Y520 using the murine Syk numbering system)
and three within the linker region that separates the SH2 domains from
the catalytic domain (Y317, Y342, and Y346) (12). The
linker region phosphotyrosines lie within consensus sequences predicted
to mediate protein-protein interactions. Syk, in fact, has been
demonstrated to associate with a variety of signaling molecules
including phospholipase C-
(PLC-), Vav, and c-Cbl, which have SH2
or phosphotyrosine-binding (PTB) domains predicted to bind within this
region (13, 14, 15, 16).
The interaction of Syk with PLC- or Vav would reasonably be expected
to serve a positive role in the coupling of the BCR to intracellular
signaling pathways. However, the association of Syk with c-Cbl might
constitute an interaction that negatively regulates receptor-mediated
signaling. c-Cbl has structural features characteristic of an adaptor
protein including an amino-terminal PTB domain, a central RING finger
domain, an extensive proline-rich region suitable for binding SH3
domain-containing molecules, numerous phosphorylatable tyrosines
present within consensus SH2 domain docking sites, and a
carboxyl-terminal leucine zipper (17, 18). Consistent with
a role as an adaptor, c-Cbl binds numerous signaling molecules that are
known to be expressed in B cells including the protein-tyrosine kinases
Syk, Fyn, Lyn, Abl and Btk; the adaptor proteins Grb2, Shc, and Crk;
and the p85 subunit of phosphatidylinositol 3-kinase. However, rather
than serving a positive role as an adaptor in signaling, biochemical
and genetic evidence indicate that c-Cbl is a negative regulator of
signaling through Syk family kinases. The best evidence for a negative
role comes from studies in mast cells, where the overexpression of
c-Cbl inhibits the Fc
RI-induced phosphorylation and activation of
Syk (19) and in thymocytes from c-Cbl-deficient mice,
where the absence of c-Cbl accentuates the CD3-dependent activation of
the Syk homologue, ZAP-70 (20).
Our previous studies on the characterization of the in vivo sites of tyrosine phosphorylation on Syk identified Tyr317 as a negative regulatory site (12). Tyr317 is a likely candidate for an interaction site for c-Cbl because the amino acids surrounding this residue match a consensus sequence for a site recognized by the c-Cbl PTB domain (21). In addition, the mutation of this site diminishes interactions between Syk and Cbl when the two are coexpressed in the same cell (13, 22). To study the role of linker region tyrosines in the regulation of Syk function, we examined the structural requirements for functional interactions between c-Cbl and Syk in B cells. In this study, we report that the overexpression of c-Cbl inhibits BCR-induced activation of the NF-AT transcription factor in a manner dependent on the c-Cbl PTB domain and Syk Tyr317.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Syk-deficient DT40 chicken B cells (8) were obtained from Dr. Tomohiro Kurosaki. Stable cell lines expressing epitope-tagged forms of murine Syk (Syk(WT), Syk(Y130F), and Syk(Y130E)) were as described previously (9). Anti-Syk Abs were prepared as described (23). Goat anti-chicken IgM Abs were from Bethyl Laboratories (Montgomery, TX). Anti-Cbl Abs were purchased from Transduction Laboratories (Lexington, KY). The 9E10 anti-Myc hybridoma cell line was purchased from the American Type Culture Collection (Manassas, VA), and ascites fluid was prepared by the Purdue University Cancer Center Antibody Facility. The biotinylated ITAM and diphospho-ITAM (P2-ITAM) peptides were synthesized by the Purdue University Cancer Center Peptide Synthesis Facility. The sequence of the ITAM peptide is biotin-EDNLYEGLNLDDCSMYEDISRG. In the diphospho-form, the two tyrosines are phosphorylated. Streptavidin-Sepharose was obtained from Sigma (St. Louis, MO).
Syk constructs
The generation of cDNAs for murine Syk with a Myc-epitope tag at the carboxyl terminus have been described elsewhere (9). Site-directed mutagenesis was conducted using the Transformer mutagenesis kit (Clontech, Palo Alto, CA) and confirmed by DNA sequencing. The mutagenesis reactions were performed in the pBluescript KSII cloning vector (Stratagene, La Jolla, CA), and the products were subcloned into the pGEM/EPB expression vector (24). All Syk cDNA clones described here contain sequences encoding the Myc-epitope tag. Stable cell lines expressing Syk mutants were generated by transfection and antibiotic selection as described (9). The NF-AT-luciferase reporter construct was a gift of Dr. Anjana Rao (Harvard University, Boston, MA).
Cbl constructs
Human c-Cbl cDNA with a hemagglutinin (HA) tag at the N terminus (provided by Dr. Wallace Langdon, University of Western Australia) was subcloned into the XhoI site of a mammalian expression vector (pCAGGS) under the control of the chicken ß-actin promoter (25), creating the pCAGGS Cbl construct. The G306E Cbl point mutant was generated by PCR-directed mutagenesis using the Pfu polymerase (Stratagene). The PCR-amplified DNA was digested with SacII endonuclease, and the fragment containing the mutation was swapped into the SacII-digested wild-type pCAGGS Cbl construct. The nucleic acid sequence of the swapped region was determined and found to contain the appropriate mutation and is devoid of PCR-induced error.
Promoter-linked luciferase assays
Syk- DT40 cells (1 x 107) were transfected by electroporation (300 V, 330 µF) with vectors containing cDNAs for the various epitope-tagged Syk mutants (20 µg), the indicated amount of plasmids coding for c-Cbl or Cbl(G306E), and either the NF-AT-luciferase reporter plasmid (10 µg) or the Elk-1-GAL4 and GAL4-luciferase plasmids (10 µg each) supplied with the Pathfinder kit from Stratagene. Cells were harvested 48 h following transfection, plated at a density of 1 x 106 cells/ml, and activated with anti-IgM Abs (10 µg/ml unless indicated otherwise) or a mixture of PMA (50 ng/ml) and ionomycin (1.0 µM) for 6 h at 37°C. Luciferase activity was determined by using the luciferase assay system kit (Promega, Madison, WI) and was measured on a Lumat LB9501 luminometer (EG&G Wallac, Wellesly, MA). Luciferase activity is expressed as a fraction of that activity observed with activation by PMA plus ionomycin. Protein expression levels were determined by Western blotting with anti-Syk or anti-Cbl Abs.
Protein interaction assays
Syk-negative DT40 B cells (1 x 107)
were cotransfected with 20 µg each of plasmids encoding Cbl and
Syk(WT), Cbl and Syk(F317), or Cbl(G306E) and Syk(WT). Cells were
either unstimulated or treated with pervanadate (0.1 mM sodium
orthovanadate and 0.5 mM
H2O2) to inhibit
phosphotyrosine phosphatases and promote the phosphorylation of Syk.
Cells were treated with 0.3 mg/ml dithiobis(succinimidyl propionate)
(DSP) (Pierce, Rockford, IL) or DMSO carrier alone (final concentration
of 3%) for 1 h at room temperature with gentle rocking. The
cross-linking reaction was quenched by the addition of 50 mM Tris-HCl
(pH 7.4) and incubating for 15 min at room temperature. Cells were
lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Brij-97, 5 mM EDTA,
10 µg/ml leupeptin, 10 µg/ml aprotinin, and 100 µM sodium
orthovanadate. Anti-HA immune complexes were prepared and incubated in
a kinase reaction buffer containing 25 mM HEPES (pH 7.5), 10 mM
MnCl2, 5 mM p-nitrophenylphosphate,
and 25 µCi [-32P]ATP for 5 min at 30°C.
Proteins were separated by SDS-PAGE, transferred to polyvinylidene
difluoride membranes, incubated at 50°C for 1 h, and visualized
by autoradiography.
Syk-ITAM binding assays
Biotinylated ITAM or diphospho-ITAM peptides (50 µg) were
adsorbed to streptavidin-Sepharose (30 µl) by incubation for 1 h
at 4°C. The immobilized peptides were then washed with detergent
lysis buffer (1% Brij 96, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM
EDTA, 1 mM sodium orthovanadate, and 10 µg/ml each of leupeptin and
aprotinin). Stable cell lines expressing the various Syk mutants were
left untreated or were incubated with 10 mM
H2O2 for 5 min at 37°C
and then lysed in detergent lysis buffer. Lysates were centrifuged at
15,000 x g for 10 min at 4°C to remove nuclei and
unbroken cells. Supernatants were mixed with the immobilized peptides
and incubated for 1 h at 4°C. In some experiments, the unbound
supernatants were incubated with anti-Myc epitope Abs to isolate
the Myc-tagged Syk. The ITAM precipitates and immune complexes were
washed twice with lysis buffer and once with 25 mM HEPES (pH 7.5) and 1
mM sodium orthovanadate. Adsorbed proteins were separated by SDS-PAGE
and detected by Western blotting with anti-Syk Abs. In some
experiments, the P2-ITAM-associated proteins were
incubated in a kinase reaction buffer containing 25 mM HEPES (pH 7.4),
10 mM MnCl2, 10 mM
p-nitrophenylphosphate, and 25 µCi
[-32P]ATP for 5 min to allow
autophosphorylation of bound Syk. After separation by SDS-PAGE,
radiolabeled proteins were detected by autoradiography.
Phosphopeptide mapping
Syk(WT) was adsorbed to the immobilized
P2-ITAM peptide from lysates of
Syk(WT)-expressing cells as described above. The resin containing the
bound kinase was incubated in kinase reaction buffer containing 2 µM
[-32P]ATP for the times indicated. The
reactions were terminated by the addition of ice cold lysis buffer
containing 10 mM EDTA. The resin was collected by centrifugation.
Syk(WT) present in the supernatant (released from the resin) was
isolated by immunoprecipitation with anti-Myc epitope Abs. The
beads were washed two times with lysis buffer and then the
resin-associated proteins and the anti-Myc immune complexes were
separated by SDS-PAGE and transferred to nitrocellulose membranes.
Radiolabeled Syk proteins were detected by autoradiography, excised
from the membrane, and digested with trypsin for phosphopeptide mapping
as described (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used chicken DT40 cells as a model system to explore possible
functional interactions between Syk and c-Cbl in B lymphocytes. In DT40
cells that have been rendered Syk-deficient by gene disruption
(8), cross-linking of the Ag receptor is uncoupled from
activation of the NF-AT transcription factor as measured using an
NF-AT-driven luciferase reporter construct (Fig. 1
A). Receptor-mediated
activation of NF-AT activity could be restored by cotransfecting cells
with a cDNA directing the expression of Syk(WT) (Fig. 1A).
Thus, a form of Syk of murine origin can reconstitute Syk-dependent
signaling in a chicken B cell line as was demonstrated previously for
porcine Syk (8). Cells were then cotransfected with
increasing amounts of a c-Cbl expression plasmid to examine a possible
role for c-Cbl in modulating the ability of Syk to restore signaling to
the Syk- cells. The overexpression of human
c-Cbl led to a decrease in Syk- and BCR-dependent activation of NF-AT
(Fig. 1A). Cotransfection of the same plasmid lacking the
c-Cbl cDNA had no effect on Syk-dependent signaling. The overexpression
of c-Cbl had no significant effect on the level of expression of
Syk(WT) (Fig. 1B). These data indicate that the
overexpression of c-Cbl in B cells inhibits Syk-dependent signaling
through the Ag receptor.
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The linker region of Syk contains multiple tyrosines that could
serve as potential mediators of protein-protein interactions. Five of
these tyrosines can be phosphorylated in vitro in an
autophosphorylation reaction (11), and three of these five
are prominent in vivo sites of phosphorylation (12). To
explore the potential of linker region tyrosines to mediate the
Syk-c-Cbl interaction, we prepared a collection of Syk phosphorylation
site mutants (Fig. 2A). The
various Syk mutants were then tested for their abilities to restore
signaling to Syk-deficient cells and for c-Cbl to modulate this
signaling. These assays were conducted by the transient transfection of
DT40 cells with the NF-AT-luciferase construct and with plasmids
directing the expression of the mutant forms of Syk with or without
coexpression of wild-type c-Cbl. A form of Syk containing all five
tyrosines mutated to Phe (Syk(F5)) was able to support BCR-dependent
activation of NF-AT (Fig. 2B). However, coexpression of
c-Cbl had no significant effect on receptor-mediated stimulation of
NF-AT activity, suggesting that at least one of these sites was
important for mediating the negative effects of c-Cbl on Syk-dependent
signaling. To further identify the specific site involved, we prepared
Syk mutants in which only a single one of the tyrosine residues known
to be phosphorylated in vivo was present within the linker region
(Syk(Y317), Syk(Y342), and Syk(Y346)). As shown in Fig. 2B,
only signaling mediated by the Syk(Y317) mutant was strongly
susceptible to inhibition by c-Cbl.
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A, Syk(Y317)-dependent
signaling was inhibited by coexpression of increasing concentrations of
c-Cbl and appeared to be even more sensitive to c-Cbl expression than
Syk(WT)-dependent signaling. To further confirm the importance of
Tyr317, we expressed in Syk-deficient DT40 cells
a mutant form of Syk in which the only substitution was
Tyr317, which was replaced with Phe (Syk(F317)).
BCR-induced NF-AT activation was enhanced in the Syk(F317)-expressing
cells as compared with the cells expressing Syk(WT), consistent with
previous observations (12). The coexpression of increasing
amounts of c-Cbl with Syk(F317) was unable to inhibit BCR-dependent
activation of NF-AT activity (Fig. 3B). In fact, we observed
a small enhancement in the ability of Syk(F317) to couple the BCR to
the activation of NF-AT in the presence of c-Cbl.
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The primary sequence of amino acids surrounding Syk
Tyr317 matches the consensus sequence required
for high affinity interactions with the c-Cbl PTB domain
(21). We tested directly the importance of the PTB domain
for c-Cbl-dependent inhibition of signaling by coexpressing a mutant
form of c-Cbl in which an essential glycine residue in the PTB domain
was replaced by glutamate (Cbl(G306E)). We then measured BCR-stimulated
expression from the NF-AT-luciferase plasmid. As shown in Fig. 2
B, the expression of Cbl(G306E) had no significant effect
on signaling mediated by any of the mutant forms of Syk, even though
the expression levels of Cbl(G306E) were comparable to those of c-Cbl
(Fig. 2C). Furthermore, as shown in Fig. 4A, the transfection of cells
with increasing amounts of Cbl(G306E) cDNA did not inhibit
BCR-dependent activation of NF-AT in cells expressing Syk(WT). In fact,
we observed consistently a small increase in BCR-induced NF-AT
activation in cells expressing both Cbl(G306E) and Syk(WT). The
expression of Cbl(G306E) also did not inhibit the activation of NF-AT
in cells expressing Syk(Y317) (Fig. 4B), which is the form
of Syk that is particularly sensitive to inhibition by c-Cbl (Fig. 3A). These data indicate that the PTB domain of c-Cbl is
essential for the inhibition of Syk-dependent signaling. This region of
c-Cbl is highly conserved between the murine and human forms
(26), which likely accounts for the ability of the human
protein to interact with murine Syk.
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To explore the nature of the physical interactions between Syk and
c-Cbl in the DT40 B cell system, we attempted to coimmunoprecipitate
the two proteins from cells transiently transfected with cDNAs encoding
Syk and c-Cbl (with an HA epitope tag) as well as forms of Syk and
c-Cbl with mutations at Tyr317 or within the
c-Cbl PTB domain. Anti-HA immune complexes were isolated from cell
lysates and incubated with [-32P]ATP to
identify coimmunoprecipitating protein-tyrosine kinases. In transfected
but unstimulated cells, no Syk autophosphorylating activity could be
detected in the anti-HA immune complexes (Fig. 5, A and B,
lanes 1 and 3), regardless of the form of Syk or
c-Cbl expressed in the cells. Phosphoproteins of 53 and 56 kDa were
present in the phosphorylated immune complexes and corresponded to the
two splice variant forms of Lyn, which binds via its SH3 domain to the
proline-rich region of c-Cbl (19). This pattern was not
altered when anti-HA immune complexes from Syk-deficient cells were
analyzed. To help stabilize weak interactions that might be lost during
washing of the immune complexes, cells were preincubated with DSP, a
membrane-permeable, cleavable cross-linking reagent, before
immunoprecipitation. Interestingly, anti-HA immune complexes from
cells treated with the cross-linking reagent contained easily
detectable amounts of autophosphorylated Syk, tyrosine-phosphorylated
c-Cbl, and a third substrate of 54 kDa (Fig. 5, A and
B, lanes 5 and 6). This same protein
could be observed in anti-Syk immune complexes and has been
identified as the tubulin subunit by peptide mapping
(23). This interaction was completely independent of
either Syk Tyr317 or a functional c-Cbl PTB
domain. All three phosphoproteins were missing from immune complexes
prepared from Syk-deficient cells (data not shown).
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, A and B,
lanes 2 and 4). The association of Syk with c-Cbl
was not completely lost, but was decreased by greater than 2-fold when
anti-HA immune complexes were isolated from cells expressing either
Syk(NOREF>317) or c-Cbl(G306E). Mutation of Syk Tyr317 alters the dose-response curve for anti-IgM stimulation of NF-AT and Elk-1 activity
In mast cells, c-Cbl acts to inhibit the interactions of Syk with
the FcRI receptor (19). To examine whether a similar
mechanism might be operative in B cells, we first examined the response
of Syk-deficient DT40 cells transiently transfected with plasmids
expressing Syk(WT) or Syk(F317) along with the NF-AT-luciferase
reporter construct to increasing concentrations of anti-IgM Ab. As
shown in Fig. 6A,
Syk-deficient cells were nonresponsive to activating Ab at all
concentrations tested. Syk(F317)-expressing cells not only exhibited a
higher extent of luciferase production in response to activating Ab
than did Syk(WT) cells, but also responded at lower concentrations of
Ab. The altered dose response curves suggested that, in the absence of
the c-Cbl docking site, Syk(F317) exhibited an enhanced ability to
associate with the cross-linked Ag receptor.
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B). The
dose-response curve for the activation of Elk-1 by anti-IgM Abs was
shifted to lower concentrations in the Syk(F317)-expressing cells
comparable to that observed for the activation of NF-AT. Syk mutants lacking Tyr317 exhibit enhanced association with diphospho-ITAM peptides
To create a model system to explore the effect of
Tyr317 on Syk-receptor interactions, we
synthesized as a receptor mimic a diphosphopeptide corresponding in
sequence to the ITAM motif on CD79a. This peptide was also biotinylated
at the amino terminus. The diphosphopeptide
(P2-ITAM) and its nonphosphorylated counterpart
were linked to streptavidin-Sepharose beads and used as affinity
reagents to isolate Syk from detergent lysates of DT40 cells. As shown
in Fig. 7A, Syk(WT) could
readily be recovered from lysates prepared from the Syk(WT)-expressing
stable cell line by adsorption onto the immobilized
P2-ITAM, but not onto a resin containing the
immobilized, nonphosphorylated ITAM peptide. Syk(Y130E), a form of Syk
that does not bind to the Ag receptor (9), was used as a
control and failed to bind to either P2-ITAM or
the nonphosphorylated peptide as expected (Fig. 7A).
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B, Syk(WT) from
H2O2-treated cells, but not
from control cells, failed to bind to the phosphopeptide. To explore a
role for the phosphorylation of Tyr317 in the
Syk-P2-ITAM interaction, we also treated stable
cell lines expressing Syk(F317) with
H2O2 and applied lysates
from treated and control cells to the P2-ITAM
resin. Interestingly, a substantial fraction of Syk(F317) from the
H2O2-treated cells retained
the ability to bind to P2-ITAM (Fig. 7B). The amount bound was comparable to that observed for
Syk(Y130F), a mutant form of Syk with an enhanced ability to bind to
the Ag receptor in activated cells (9).
Tyrosine-phosphorylated Syk(F317) retained its specificity for binding
to the phosphorylated ITAM peptide and did not interact with the
nonphosphorylated peptide (Fig. 7C).
To determine whether the phosphorylation of
Tyr317 per se led to the dissociation of Syk from
P2-ITAM, we adsorbed Syk(WT) from control cell
lysates onto the phosphopeptide, washed the resin extensively, and then
incubated the bound protein with [-32P]ATP
for varying periods of time to allow it to autophosphorylate. At each
time point, the resin was washed thoroughly and the bound and released
Syk(WT) were separated by centrifugation and analyzed by SDS-PAGE (Fig. 8A). The phosphorylated
Syk(WT) proteins were transferred to nitrocellulose membranes, excised,
and digested with trypsin (12). Tryptic phosphopeptides
were separated by alkaline 40% PAGE and detected by autoradiography.
The identity of the major phosphopeptides was determined based on our
prior analysis of Syk phosphorylation sites (12). The bulk
of the tyrosine-phosphorylated Syk remained bound to the
P2-ITAM peptide even though it was extensively
phosphorylated on Tyr317 (Fig. 8B),
indicating that phosphorylation at this site does not preclude the
association of Syk with the phosphorylated ITAM. The only residue that
was exclusively phosphorylated in the released fraction of Syk was
Tyr130.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of c-Cbl to negatively regulate Syk-dependent signaling
through the BCR is dependent on the Syk linker region
Tyr317. This conclusion is based on analyses of
the ability of c-Cbl to inhibit BCR-stimulated activation of the NF-AT
transcription factor mediated by Syk and a collection of Syk mutants.
The activation of NF-AT following aggregation of the BCR is attenuated
in Syk-deficient DT40 cells, but restored upon the re-expression of
Syk(WT) or of forms of Syk with tyrosine to Phe substitutions within
the linker region. The coexpression of increasing amounts of c-Cbl
leads to a dose-dependent decrease in the BCR-stimulated activation of
NF-AT in cells expressing Syk(WT), which retains the full complement of
phosphorylation sites within the linker (Fig. 1A). A form of
Syk lacking all five potential linker region phosphorylation sites
(Syk(F5)) is still capable of coupling the BCR to NF-AT activation, but
is not subject to inhibition by c-Cbl (Fig. 2B). On the
other hand, a form of Syk with Tyr317 as the sole
phosphorylation site in the linker is inhibited by the expression of
c-Cbl. Other individual tyrosine residues within the linker region that
have been shown to be phosphorylated in vivo
(Tyr342 or Tyr346) are
unable to mediate an interaction with c-Cbl as demonstrated by an
inability of c-Cbl to inhibit signaling mediated by Syk(Y342) or
Syk(Y346) (Fig. 2B). These studies indicate that Syk is
subject to negative regulation by elevated levels of c-Cbl only as long
as Tyr317 is present within the linker region.
The ability of overexpressed c-Cbl to inhibit Syk(WT)-dependent NF-AT
activation by only 50% (Fig. 1A) may reflect the fact that
Syk(WT) is not quantitatively phosphorylated on
Tyr317 following receptor aggregation and
nonphosphorylated forms of Syk can still couple the BCR to NF-AT
activation or that c-Cbl bound to Syk can still act as an adaptor
protein to provide an alternative, but less efficient route to NF-AT
activation.
An important role for Tyr317 is further supported
by the observation that signaling through Syk(F317), which lacks only
this single site of tyrosine phosphorylation, is also insensitive to
inhibition by overexpressed c-Cbl (Fig. 3B). In the absence
of overexpressed c-Cbl, all Syk mutants tested that lack
Tyr317 (Syk(F5), Syk(Y342), Syk(Y346), Syk(F317))
demonstrate an enhanced signaling activity as compared with the
Tyr317-containing mutants (Syk(WT) or Syk(Y317)).
This finding suggests that endogenous c-Cbl acts to partially attenuate
signaling from any expressed form of Syk with a tyrosine at position
317. The substitution of Phe for Zap-70 Tyr292,
the Zap-70 c-Cbl binding site analogous to Syk
Tyr317, results also in a kinase with elevated
signaling activity, suggesting that Zap-70 is also negatively regulated
by endogenous c-Cbl in T cells (29). This is consistent
with the enhanced susceptibility to activation of Zap-70 in thymocytes
isolated from c-Cbl-deficient mice (20).
The interactions of c-Cbl with Tyr317 on Syk are
dependent on a functional PTB domain. The ability of c-Cbl to interact
with sites of tyrosine phosphorylation is abrogated if a single point
mutation is made within the c-Cbl PTB domain that changes
Gly306 to Glu. This mutation mimics that of a
loss-of-function mutation identified in the Caenorhabditis
elegans SLI-1 protein, a homologue of mammalian c-Cbl
(30). The G306E mutant of the c-Cbl PTB domain is no
longer able to bind to Zap-70 in T cells (31). Likewise,
the introduction of the G306E mutant of full-length c-Cbl into DT40
cells fails to inhibit Syk-mediated activation of NF-AT regardless of
the form of Syk that is expressed (Figs. 2B and
4A). Thus, the negative regulatory effect of c-Cbl on
Syk-mediated activation of NF-AT is dependent both on Syk
Tyr317 and an intact, functional, c-Cbl PTB
domain. These observations are consistent with the recently reported
requirement for the phosphorylation of Syk on
Tyr317 (Tyr323 in human
Syk) and for an intact c-Cbl PTB domain for the direct physical
interaction of Syk and c-Cbl in transfected COS-7 cells
(22). We also see a decrease in the interaction of
phosphorylated Syk with c-Cbl in B cell lysates when either Syk
Tyr317 or the c-Cbl PTB domain are mutated
(Fig. 5).
In RBL-2H3 mast cells, interactions mediated by the c-Cbl proline-rich
region and the amino-terminal region of Syk contribute to Syk-c-Cbl
interactions that can be observed in the absence of receptor engagement
(19). This interaction does not require either the Syk
linker region or the c-Cbl PTB domain. We have also been able to
identify Syk in association with c-Cbl in Cbl-containing immune
complexes if these are prepared from DSP cross-linked cells (Fig. 5).
Three prominent Syk substrates are present within these cross-linked
immune complexes: Syk itself, Cbl, and -tubulin. Both Syk and c-Cbl
have previously been identified as tubulin-binding proteins
(32), and it seems likely that tubulin, which is an
abundant protein in B cells, mediates this interaction. This
interaction is also unaffected by the loss of Syk
Tyr317 or by the inactivation of the c-Cbl PTB
domain. Based on the functional analyses described here, it does not
appear that this interaction leads to the negative regulation of
Syk-dependent signaling in DT40 cells. Because the coexpression of
Cbl(G306E) with Syk(WT) or of c-Cbl with Syk(F317) does lead to small
enhancements in signaling (Figs. 3A and 4A),
interactions that occur between Syk and c-Cbl that are not mediated by
the binding of the PTB domain to Tyr317 and do
not lead to inhibition may also be important for some signaling
events.
The coexpression of c-Cbl with Syk in COS cells results in a reduction in the level of Syk expression, presumably by targeting Syk for degradation (22). We have not observed a comparable effect of c-Cbl expression on the level of Syk protein present in transfected DT40 B cells, suggesting that this may be either a cell type-specific phenomenon or a consequence of the relative levels at which Syk is expressed. Therefore, we sought to examine other potential mechanisms by which c-Cbl interacting with Syk Tyr317 might lead to a decrease in receptor-mediated signaling.
Previous studies have shown that a mutant form of Syk (Syk(Y130F)) with
an enhanced affinity for the BCR exhibits enhanced activity, whereas a
mutant with reduced affinity (Syk(Y130E)) exhibits reduced activity
(9). Thus, one way to regulate signaling strength is to
modulate the interaction of Syk with the BCR. The dose response for
BCR-stimulation of NF-AT or Elk-1 activity shifts considerably to lower
concentrations of anti-IgM in cells expressing Syk(F317) as
compared with cells expressing Syk(WT), suggesting that a major effect
of the Tyr317 mutation might be to promote the
interaction of Syk with the BCR (Fig. 6). This would be consistent with
previous observations in mast cells where the overexpression of c-Cbl
was shown to inhibit the association of Syk with the FcRI receptor
(19).
The altered binding of Syk Tyr130 mutants to the
receptor is reflected also in their binding to a biotinylated peptide
corresponding in sequence to the phosphorylated ITAM region of CD79a.
Interestingly, Syk(F317) behaved in these assays similarly to
Syk(Y130F) in retaining an ability to bind to the
P2-ITAM even when isolated in a heavily
tyrosine-phosphorylated form from
H2O2-treated cells (Fig. 7). The inability of Syk phosphorylated on Tyr130
to bind to the P2-ITAM is due to the positioning
of a negative charge in the inter-SH2 domain region (9).
However, the reduced ability of Syk phosphorylated on
Tyr317 to bind appears to require an interacting
protein because it cannot be mimicked in an in vitro system containing
only P2-ITAM and associated Syk (Fig. 8). The
binding of endogenous c-Cbl is a likely candidate because it is, so
far, the only protein known to interact with Syk at this site.
The data reported in this paper provide added insights into the molecular mechanisms that regulate the activity of Syk in B lymphocytes. These data implicate c-Cbl as an important component of a regulatory apparatus that negatively controls the coupling of Syk to intracellular pathways leading to the activation of the transcription factors NF-AT and Elk-1. A component of this regulation appears to be an altered association of Syk with the Ag receptor.
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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. E-mail address:
3 Abbreviations used in this paper: BCR, B cell Ag receptor; ITAM, immunoreceptor tyrosine-based activation motif; PTB, phosphotyrosine binding; DSP, dithiobis(succinimidyl propionate; Sky(WT), wild-type murine Syk; HA, hemagglutinin; P2-ITAM, diphospho-ITAM.
Received for publication January 27, 1999. Accepted for publication September 13, 1999.
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References |
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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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J. J. Hong, T. M. Yankee, M. L. Harrison, and R. L. Geahlen Regulation of Signaling in B Cells through the Phosphory |
) or Cbl(G306E) (
). Transfected cells either remained
unactivated (
) or were activated by treatment with anti-IgM Abs
(
, 
), or no additional plasmid (
). Transfected cells were
activated by treatment with the indicated concentrations of
anti-IgM Abs. The level of expression of luciferase relative to the
PMA plus ionomycin control is indicated. B,
Syk- DT40 B cells were cotransfected with the Elk-1-GAL4
and GAL4-luciferase reporter plasmids, 10 µg of plasmid coding for
Syk(WT) (
