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INTRODUCTION |
The XLP1 syndrome is an
immune deficiency condition marked by an individual's inability to
control acute Epstein-Barr virus infections (1, 2). It manifests itself
in various phenotypes, which include fulminant infectious
mononucleosis, dysgammaglobulinemia, and malignant B cell lymphomas
(1-3). Patients of XLP exhibit specific immune defects such as
abnormal natural killer (NK) and T cell-mediated cytotoxicity (4-7).
However, the basis for such defects is not yet fully understood.
Cloning of the gene SH2D1A, whose mutation or deletion is
responsible for the onset of the XLP syndrome (8-10), and subsequent studies on its encoded protein, termed SH2D1A or SAP, have shed much
light on the pathogenesis of the disease. SAP is a protein of 128 amino
acids consisting of an N-terminal SH2 domain and a 26-residue
C-terminal tail. Its small size has led to the assumption that it may
act as a natural inhibitor of SH2 domain-dependent interactions (3, 8). Evidence in support of such a notion comes from
the observation that SAP interacts physically with the signaling
lymphocyte activation molecule, SLAM, through a Tyr-containing motif
that is conserved among members of the SLAM family of lymphocyte
surface receptors (3) and that this interaction serves to block a
competing interaction between SLAM and SHP-2, a phosphotyrosine
phosphatase that plays important roles in various cellular signaling
events (11, 12). The structural basis for the specific recognition of
SLAM by SAP has recently been unraveled by both x-ray crystallographic
and NMR methods (13, 14). We and others (13-15) have shown that the
SAP SH2 domain binds to a Tyr-containing motif in SLAM using a unique,
"three-pronged" mechanism, rather than a "two-pronged" one
employed by most conventional SH2 domains. These studies also
identified a consensus motif recognized by the SAP SH2 domain
represented by the sequence
(Thr/Ser)-X-Tyr(P)-X-X-(Val/Ile), where
X denotes any amino acid. The "three-prongs" in the
motif correspond to residue Thr or Ser at the N terminus, Tyr(P) in the
middle, and Val or Ile at the C terminus (14). Significantly, the presence of any two (of the three) prongs in a given ligand was
shown to be sufficient for its binding to SAP (14, 15).
Protein complexes analogous to the one between SAP and SLAM have also
been documented between SAP and other members of the SLAM family of
receptors (also called the CD2 receptor family) (3), which now consists
of nine related members, including the recently identified SF2000 and
SF2001 (16). These receptors are expressed in various subsets of immune
cells, such as T, B, and NK cells, and share a similar overall
structure characterized by two extracellular Ig-like domains, a single
transmembrane segment, and, with the exception of CD48 and BCM1-L, a
short cytoplasmic tail (3). Of note, four members of the family, namely
SLAM (also called CD150), 2B4 (or CD244), CD84, and Ly-9 (or CD229), contain sequences in their cytoplasmic regions that conform to the
consensus motif recognized by SAP (3, 5, 17, 18, 20). Not surprisingly,
these receptors have all been shown to interact with SAP in cells (3,
17). However, SAP binds to SLAM constitutively, whereas it binds to the
other three receptors in a phosphorylation-dependent manner
(5, 17, 18, 20).
The physiological significance of SAP interaction with the SLAM family
of receptors was underscored in findings that disease-causing missense
SAP mutants displayed markedly reduced affinities for SLAM, both
in vitro and in vivo, and were deficient in
blocking the interaction between SHP-2 and SLAM (14, 21). Moreover, the
binding of SAP to 2B4 appeared to play a pivotal role in regulating target-killing activities of NK cells (5, 18, 20). Thus, NK cells
isolated from XLP patients exhibited defects in 2B4-mediated cytotoxicity due to the absence of an active SAP-2B4 complex (5, 22).
These observations suggest that SAP plays important roles in signaling
through the SLAM family of receptors and that disruption of one or more
of these signaling pathways underlies the complex phenotypes of
XLP (17).
Notwithstanding the role of SAP as an inhibitor of SH2 domain-mediated
interactions, it was recently shown by Veillette and co-workers (23)
that SAP promotes a specific association of the T cell-specific
tyrosine kinase, FynT, with SLAM in T cells. In addition,
SAP-dependent interaction of FynT with SLAM was found to be
critical for the tyrosine phosphorylation of SLAM and for the
subsequent recruitment of downstream signaling molecules such as SHIP
and p62Dok to the receptor. These findings led to the proposal that SAP
functions as an adaptor molecule in SLAM signaling rather than as an
inhibitor of SH2 domain interaction (23).
To clarify the role of SAP in signaling through the SLAM family of
receptors, we systematically mapped the binding sites in these
receptors for a series of SH2 domain-containing signaling proteins that
include SAP and its homologue EAT-2, SHP-2, SHP-1, SHIP, and FynT,
using peptide arrays synthesized on a functionalized cellulose
membrane. The notion of SAP as an adaptor in SLAM signaling was
explored by examining a direct physical interaction between SAP and
FynT in vitro, and the functional significance of SAP-FynT interaction in SLAM phosphorylation and signaling was investigated in
HEK293 cells that were made to stably express SLAM. Our results are
consistent with a model in which SAP plays a dual functional role, both
as an adaptor to bridge an interaction between FynT and SLAM and as an
inhibitor to modulate the association of SH2 domain-containing proteins
with phosphorylated SLAM.
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EXPERIMENTAL PROCEDURES |
Subcloning, Expression, and Purification of SH2 Domains--
DNA
sequences encoding the SH2 domains of SHIP, FynT, SHP-1, and SHP-2 were
amplified by PCR, subcloned into the pGEX4T2 vector, and confirmed by
DNA sequencing. Proteins were expressed in Escherichia coli
and affinity-purified on glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's recommendations. Bound
proteins were eluted using 20 mM glutathione in 50 mM Tris and 100 mM NaCl, pH 8.0. Proteins were
concentrated, and buffer was changed to phosphate buffered saline
(PBS), pH 7.4, prior to use in binding studies.
Synthesis of Peptide Arrays on Derivatized Cellulose Membranes
and Binding Studies--
An array of undecamer peptides modeled after
Tyr-containing sites in the cytoplasmic domains of the SLAM family of
receptors were assembled on a derivatized cellulose membrane using
Auto-Spot Robot ASP 222 (Abimed) and standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) solid phase
peptide chemistry. The peptide membrane was moistened sequentially with
ethanol and water prior to screening using purified GST fusion proteins
or GST alone. Specifically, the membrane was washed three times with
TBS-T buffer containing 20 mM Tris-HCl, 140 mM
NaCl, and 0.1% (v/v) Triton X-100, pH 7.6, and blocked with 5% bovine
serum albumin in TBS-T for one h at room temperature. Approximately 1.0 µM of a GST fusion protein was added directly into the
blocking solution and incubated with the peptide membrane at room
temperature for an additional hour. The membrane was then washed three
times with TBS-T and once with TBS prior to addition of anti-GST
antibodies. After incubation for 30 min at room temperature, the
cellulose sheet was washed three times with TBS and developed with the
ECF Western blotting kit (Amersham Biosciences) following the
manufacturer's protocols and documented using a Fluor-S Multi-Imager (Bio-Rad). For reprobing, the peptide sheet was stripped by treating it
sequentially with buffer A, containing 8.0 M urea, 1%
(w/v) SDS, and 0.5% (v/v)
-mercaptoethanol, and buffer B,
containing 10% (v/v) acetic acid and 50% (v/v) ethanol, followed by
several washes of deionized water. To avoid possible background
artifacts resulting from incomplete stripping, binding studies for each SH2 domain were initially conducted using a fresh strip of peptide array generated under identical conditions.
Cell Culture, Transfection, and Pull-down Studies--
Human
embryonic kidney 293 cells (American Type Culture Collection) were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
(v/v) fetal bovine serum (Sigma), 10 units/ml penicillin, and 10 µg/ml streptomycin. For transient protein expression, cells were
grown to 70% confluency and transfected with the expression (pMES)
vectors for FynT or mutants (5 µg) by means of LipofectAMINE (15 µl) (Invitrogen). Deletion mutants of FynT were generated by two
consecutive rounds of PCR, and the Y417F mutant was prepared by
site-directed mutagenesis. A Myc tag sequence was inserted at the C
terminus of the SH2 and SH3 domain deletion mutants by PCR to
facilitate their detection. Cells were generally harvested between
60-72 h post transfection and lysed in PLC buffer (50 mM
Hepes pH7.5, 150 mM NaCl, 10% (v/v) glycerol, 1% Triton
X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM Na2P2O7, 100 mM NaF,
and 1 mM Na3VO4) supplemented with
protease inhibitors (1 µM pepstatin A, 2 µM E64, 1 µM bestatin, 10 µM leupeptin, 2 µg/ml aprotinin and 100 µM phenylmethylsulfonyl
fluoride) (Sigma).
For pull-down studies, aliquots of cleared HEK 293 lysate containing
50-200 µg of total proteins were mixed with 25 µg of biotin-labeled or GST-fused SAP or SAP mutants in the presence or
absence of a SLAM or a control peptide. The generation and production
of SAP mutants were reported previously (14). Protein complexes were
pelleted using either 30 µl of streptavidin or glutathione-Sepharose
beads (Amersham Biosciences), washed five times with PLC buffer,
resuspended in 1× SDS loading buffer, and resolved on 10-15%
SDS-PAGE. Protein bands were transferred to polyvinylidene difluoride
Western blotting membranes (Roche Diagnostics) and incubated in a
blocking solution of 5% bovine serum albumin (Sigma) for 1 h at
room temperature. The membrane was then washed three times in TBS-T and
probed with mouse anti-Fyn monoclonal antibody (Santa Cruz
Biotechnology) for 1 h at room temperature. A goat anti-mouse
IgG-HRP conjugate (Bio-Rad) was then applied to the membrane for 40 min
at room temperature before it was developed using enhanced
chemiluminescence (Pierce) on Kodak film according to the
manufacturer's instructions.
Immunoprecipitation and Western Blot--
The DNA sequence
encoding human SLAM was amplified by PCR from an expressed sequence tag
clone (Invitrogen), and subcloned into the pRc/CMV2 vector. The
pRc/CMV2/SLAM construct was stably transfected in HEK 293 cells by
LipofectAMINE followed by G418 restriction. The identity of SLAM was
verified by immunoblotting using anti CD150 monoclonal antibodies
(Santa Cruz Biotechnology).
The HEK 293/SLAM cells transfected with constructs for FynT or FynT
mutants and/or SAP were biotinylated using Sulfo-NHS-LC-Biotin (Pierce)
and lysed in a buffer containing 1% Nonidet P-40, 150 mM
NaCl, 10 mM Tris-HCl, pH 7.7, 1 mM sodium
pervanadate, and 10 mM NaF. After precleaning the lysate
with preimmune serum absorbed on protein A- or G-Sepharose beads, SLAM
was precipitated with anti-SLAM monoclonal antibodies (anti CD150, 4 µg per reaction; lysate containing 800 µg of total protein in 500 µl). The precipitated proteins were separated by SDS-PAGE and
transferred under semi-dry conditions to polyvinylidene difluoride
membranes (Millipore). Membranes were probed sequentially with
anti-Tyr(P) mouse monoclonal antibody (Cell Signaling Tech), rabbit
anti-SHP2, mouse anti-SAP (a generous gift from Dr. Cox Terhorst,
Harvard) or anti-FLAG monoclonal antibody, anti-Fyn (mouse), or
anti-c-Myc (mouse). Biotinylated SLAM was probed with
streptavidin-horseradish peroxidase (Sigma). The blotted membranes were
developed using enhanced chemiluminescence (Pierce).
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RESULTS |
Binding of SAP and Related SH2 Domains to Tyr-containing Motifs in
the SLAM Family of Receptors--
The cytoplasmic regions of the SLAM
family of receptors contain multiple conserved Tyr phosphorylation
sites that completely or partially conform to the
(Thr/Ser)-X-Tyr(P)-X-X-(Val/Ile) motif recognized
by the SAP SH2 domain (3, 24-26). To date, four of the SLAM family
members, namely SLAM, 2B4, Ly-9, and CD84, have been shown to interact
with SAP via one or more of these Tyr phosphorylation sites (5, 17,
18). To determine systematically the preferred binding sites for SAP
and related proteins in these receptors, an array of undecamer peptides
modeled after these sites was generated on a derivatized cellulose
membrane using the SPOT method of multiple peptide synthesis (27). To
assess the role of phosphorylation on binding, both phosphorylated and
unphosphorylated versions of the same peptides were synthesized.
Sequences of the array of peptides and their origins are listed in
Table I.
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Table I
Amino acid sequences of the peptides represented in the SPOT array
Note that both phosphorylated (Tyr(P)-containing) and unphosphorylated
(Tyr-containing) versions of the same peptides were included in the
array. Each peptide is 11 amino acids in length, with a 5-amino acid
extension in both the N and C terminus of the central Tyr(P) or Tyr
residue. Peptide spots in the array are defined by the number followed
by Tyr(P) or Tyr, which indicate phosphorylated and unphosphorylated
peptides, respectively. For instance, peptide 1-Tyr(P) respresents a
phosphorylated peptide derived from the SLAM Tyr-307 site.
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The peptide array was subsequently screened for binding to purified SAP
and EAT-2 as well as the SH2 domains of SHIP, SHP-2, SHP-1, and FynT.
SHIP and SHP-1 are phosphoinositide and phosphotyrosine phosphatases, respectively, that play important roles in inhibitory signaling through immune receptors (28), whereas the function of SHP-2
in lymphocyte signaling and activation is less well defined. EAT-2 has
the same overall structure as SAP and shares extensive sequence
identity to the latter (29). Interestingly, EAT-2 is expressed in B
cells and macrophages, whereas SAP is mainly found in T and NK cells,
suggesting a likely complimentary role for these two homologous
proteins in the immune system (3, 29). Because their C-terminal tails
are not involved in peptide recognition (14, 29), intact SAP and EAT-2
were used in the binding studies together with the other SH2 domains.
As seen in Fig. 1, with the exception of
peptide 10-Tyr(P) (phosphotyrosine-containing peptide number 10)
derived from the Tyr-581 site in Ly-9 (see Table I), all phosphorylated
(Tyr(P)) peptides in the array displayed strong binding to SAP,
implying that SAP can be recruited to each of the four SLAM family
members via multiple sites. Interestingly, only two unphosphorylated
(Tyr) peptides, namely peptides 3-Tyr (Tyr-containing peptide number 3)
from the SLAM Tyr-281 site and 8-Tyr (Tyr-containing peptide number 8)
from CD84 Tyr-262, were capable of binding to SAP. This result is
consistent with those reported in the literature showing that SAP could
associate with SLAM and CD84 in a constitutive manner (8, 17), whereas
it could associate with 2B4 and Ly-9 only following their tyrosine
phosphorylation (17, 18). In comparison, the homologous protein EAT-2
exhibited an almost identical binding pattern to the array of
Tyr(P)-containing peptides as SAP (Fig. 1). However, EAT-2 displayed
only weak and, most likely, nonspecific binding to the array of
Tyr-containing peptides. It can thus be predicted from this result that
EAT-2 can only be recruited to the SLAM receptors in a
phosphorylation-dependent manner, as shown in a recent
study by Morra et al. (29) to be indeed the case.

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Fig. 1.
Comparable binding patterns for SAP, SHP2,
and SHIP to SLAM receptor family members. Binding patterns for
SAP, EAT-2, and SH2 domains from SHP-2, SHIP, FynT, and SHP-1 to an
array of peptides derived from the SLAM family of receptors (see Table
I for sequence information) are shown. The N-terminal SH2 domain of
SHP-2 was used because no significant binding was detected for the
C-terminal SH2 domain (not shown). All proteins were used as
GST-fusion. Bright (fluorescent) spots indicate positive binding. See
"Experimental Procedures" for details of experimentation.
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The N-terminal SH2 domain of SHP-2 and the SHIP SH2 domain also
displayed binding profiles highly similar to those of SAP and EAT-2,
particularly in respect to the array of Tyr(P) peptides (Fig. 1). All
four proteins employed the same sites in SLAM, CD84 and Ly-9, although
they appeared to bind to the SLAM Tyr(P)-281 (column
3, pY rows in Fig. 1) and Ly-9 Tyr(P)-602
(column 12, pY rows) sites more
strongly than to other sites in the same receptors. A notable
difference was detected in 2B4-derived peptides. Whereas SAP and EAT-2
were apparently capable of binding to all four sites (corresponding to
the peptides depicted in columns 4-7, pY
rows in Fig. 1) in 2B4, SHP-2 and SHIP bound only to two of them
with high affinity. On the contrary, SH2 domains from SHP-1 and FynT displayed negative to weak binding to both the Tyr(P)- and
Tyr-containing peptides. Collectively, these results indicate that SAP,
EAT-2, SHP-2 and SHIP may be bona fide ligands for the SLAM
family of receptors, whereas SHP-1 and FynT are not.
To quantitate these interactions, we measured the affinities of the
above SH2 domains for two peptides derived from the Tyr-281 site in
SLAM by fluorescence polarization (15). In agreement with results
obtained using peptide arrays, the SH2 domains of SHIP and SHP-2 (N- or
N, C-tandem) were found to bind strongly to the SLAM Tyr(P)-281
peptide, with Kd values for the corresponding
peptide-protein complexes ranging from
sub to low micromolar (Fig. 2, and Table
II), which are comparable with those of
the SAP- or EAT-2-peptide complexes (Table II). It was also observed
that the C-terminal SH2 domain of SHP-2 was essentially inactive in
binding and that the tandem SH2 domains displayed a higher affinity
than the N-terminal SH2 domain alone. This was likely due to the fact
that the former was more stable than the latter (data not shown) rather
than because of a cooperative effect of the two SH2 domains in tandem.
In contrast, the SH2 domains of FynT and SHP-1 showed significantly
reduced binding to peptide SLAM Tyr(P)-281. As seen also in the peptide
array studies, none of the SH2 domains, except SAP, exhibited
significant binding to the SLAM Tyr-281 peptide.

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Fig. 2.
Binding of SH2 domains to a SLAM
peptide. Binding curves for the tandem SH2 domains of SHP-2 and
the SH2 domains of SHIP and FynT to fluorescein-labeled SLAM Tyr(P)-281
peptide. Data points shown represent relative fluorescence polarization
values (Y) at various protein concentrations (X) from a single
measurement. They were fitted to the equation Y = Bmax × X/(Kd + X), where
Bmax represents maximal levels of fluorescence
polarization, to yield the corresponding Kd values
using the software Prism 3.0 (Graphpad Software Inc.). Average
Kd values from two independent experiments are
reported in Table II.
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Table II
Relative affinities of various SH2 domains for fluorescein-labeled
peptides SLAM Tyr(P)-281 and Tyr-281
Sequences of the SLAM Tyr(P)-281 and Tyr-281 peptides corresponded to
those of spots 3-Tyr(P) and 3-Tyr in the peptide array (see also Table
I). The peptides were labeled with fluorescein using an N-terminal
anchoring sequence containing KGG to avoid end effects. See legend to
Fig. 2 for the derivation of Kd values. ND, not
determined due to weak binding.
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SAP Interacts with FynT in Vitro as Well as in Vivo, an
Interaction Augmented by Concomitant Binding of SLAM Peptides--
The
finding that FynT associates with SLAM in a SAP-dependent
manner (21) suggests that SAP may either indirectly or directly interact with SLAM. To explore the latter possibility, we employed a
purified SAP protein, either as GST-fusion or labeled with biotin, in
an attempt to pull down FynT from the lysate of HEK 293 cells that
transiently overexpress FynT. As seen in Fig.
3A, both GST-SAP and
biotin-SAP could pull down FynT, whereas GST and glutathione- (lane 1) or streptavidin-Sepharose beads (lane 4)
failed to do so, indicating that SAP interacts specifically with FynT
in vitro. To find out whether SAP and FynT can form a
complex in vivo, we transiently expressed the two proteins
in HEK 293 cells. As shown in Fig. 3B, FynT is detected in
anti-SAP immunoprecipitates from cells simultaneously expressing the
two proteins but not in cells expressing SAP alone.

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Fig. 3.
SAP interacts simultaneously with FynT and
SLAM. A, binding of SAP to FynT assayed in a pull-down
study using GST-SAP and biotinylated SAP (Bt-SAP). GST,
glutathione- (lane 1) and streptavidin-Sepharose beads
(lane 4) were included as controls. The same amount of
lysate (150 µg of total protein), protein (25 µg), and beads (30 µl) were used for all lanes. Protein bands corresponding to those of
FynT were indicated. WCL, whole cell lysate. B,
detection of FynT in anti-SAP immunoprecipitates (IP) from
lysates of HEK 293 cells either tranfected with SAP alone or with both
SAP and FynT constructs. The blot was reprobed with anti-SAP antibody
to show comparable amounts of SAP precipitated. Lysates were
pre-cleared using normal serum prior to IP. WB, Western
blot. C, binding of biotinylated SAP to FynT in the absence
or presence of 10-100 µM of SLAM Tyr(P)-281
(SLAM-pY281) or SLAM Tyr-281 (SLAM-Y281) peptides
(corresponding to peptide 3-Tyr(P) and 3-Tyr in Table I, respectively).
Experimental conditions were the same as for panel A.
Similar results were obtained using GST-SAP (data not shown).
D, binding of biotinylated SAP to FynT in the presence or
absence of a control peptide from the Numb-associated protein Nak
containing the sequence GFSNMSFEDFP (30).
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Because SAP also binds to SLAM through the Tyr-281 site, we were
interested in finding out whether SAP functions as an adaptor for FynT
and SLAM or whether the two interactions compete and are thereby
mutually exclusive. To this end, we examined the effects of two SLAM
peptides, namely SLAM Tyr(P)-281 and SLAM Tyr-281, in SAP-FynT
interaction. As shown in Fig. 3C, neither the phosphorylated nor the unphosphorylated SLAM peptide inhibited the interaction of SAP
with FynT. On the contrary, these two peptides appeared to enhance SAP
binding to FynT. This "enhancing" effect was particularly prominent
for the SLAM Tyr-281 peptide, which was found to increase the amount of
SAP-associated FynT significantly even when applied at 10 µM. By comparison, an unrelated peptide derived from the Numb-associated kinase (Nak) had no such effect (Fig.
3D).
A Direct Physical Association of FynT and SAP Requires the
Full-length SAP and the FynT SH3 Domain--
To determine the specific
regions in the two proteins that mediate their interaction, we
generated various truncation mutants of SAP and FynT. Because SAP
contains a 26-amino acid tail C-terminal to its SH2 domain, we were
interested in its role in binding. As shown in Fig.
4A, although the SAP SH2
domain alone is capable of binding to FynT, its affinity is
significantly reduced compared with that for full-length SAP,
suggesting that the tail of SAP is either directly involved in binding
FynT or indirectly modifying the SAP-FynT interaction. To map out the
domain(s) in FynT responsible for SAP-binding, two mutants were
constructed with either the SH2 or the SH3 domain deleted (mutants
SH2 and
SH3, Fig. 4B). The role of the kinase domain
was explored using a kinase-dead mutant bearing a mutation of residue
Tyr-417 (Y417F) in the activation loop (23). The binding of these
mutants to SAP was examined in a GST pull-down study. As shown in Fig.
4B, the
SH3 mutant of FynT failed completely to bind SAP,
whereas the
SH2 mutant displayed significantly reduced binding
compared with the wild-type FynT. In contrast, the kinase domain mutant
Y417F appeared to retain full binding capacity to SAP. These results
indicate that the FynT SH3 domain is essentially for binding to SAP,
whereas the SH2 domain may also contribute to the FynT-SAP
interaction.

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Fig. 4.
Molecular characterization of SAP-FynT
interaction. A, differential binding of SAP and the SAP
SH2 domain to FynT. Equal amounts of GST or GST fusion proteins were
used to pull down FynT from HEK 293 cell lysate. B, mapping
SAP-binding domain(s) in FynT. GST or GST-SAP was used to pull down
FynT or FynT mutants from HEK 293 cell lysate. Blots were probed with
either anti-FynT (for FynT and mutant Y417F) or anti-Myc (for mutants
FynT- SH2 and FynT- SH3). MW, molecular mass;
WB, Western blot; WCL, whole cell lysate.
C, binding of purified SAP to isolated FynT SH3 domain
labeled with fluorescein. The curve shown is generated from a typical
binding experiment using fluorescence polarization.
Kd = 2.74 ± 0.54 µM.
D, binding of purified FynT SH3 domain to
fluorescence-labeled SAP protein. Kd = 2.53 ± 0.35 µM.
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To further explore the molecular basis of FynT-SAP interaction, we
produced purified SAP and a FynT SH3 domain and labeled them one at a
time with fluorescein to determine their affinities for each other.
Fluorescence polarization measurements using the labeled FynT SH3
domain and an unlabeled SAP protein yielded a saturation binding curve
with a Kd value of 2.75 µM. A reverse
binding experiment using labeled SAP and unlabeled FynT SH3 domain
produced a similar Kd value of 2.5 µM.
In contrast, the FynT SH2 domain does not bind strongly to SAP in parallel experiments (Kd = 97 µM, data
not shown). Collectively these results demonstrate that the SH3 domain
of FynT is directly involved in binding SAP.
Regulation of SLAM Signaling by the Formation of a FynT-SAP-SLAM
Complex--
To validate the role of SAP as an adaptor for SLAM and
FynT in vivo, we derived a human embryonic HEK 293 kidney
cell line that stably expresses the SLAM receptor by means of G418
restriction. FynT or a FynT mutant was then transiently expressed in
these cells with or without co-expression of SAP. Receptor-associated SAP, FynT (or mutants), and SHP-2 in anti-SLAM immunoprecipitates were
assayed respectively using specific antibodies against these proteins.
As shown in Fig. 5A, SAP
associated with SLAM constitutively in cells transfected with an empty
vector (panel ii, lane 4). However,
the level of receptor-bound SAP was dramatically increased in cells
bearing FynT (Fig. 5A, lane 2), an observation
that is in agreement with previous reports (8, 23). In contrast, basal
levels of SLAM-associated SAP were detected in cells expressing FynT
mutant
SH3 or Y417F, suggesting that the SH3 and kinase domains of
FynT are important for SAP-SLAM interaction. Interestingly, the FynT
SH2 mutant was also largely ineffective in promoting the SAP-SLAM
complex, as the amount of the SLAM-bound SAP in cells expressing this
mutant was only moderately higher than the basal level (Fig.
5A, panel ii, lane 6). Binding of SAP
to SLAM appeared to be correlated with the extent of tyrosine
phosphorylation of the receptor. Although SLAM phosphorylation was
detectable in the cells containing FynT but not SAP (Fig.
5A, panel iv, lane 1), the presence of
SAP drastically increased the extent of SLAM phosphorylation (Fig.
5A, panel iv, lane 2). In comparison,
a complete absence or significant decrease in SLAM phosphorylation was
observed in cells expressing any of the FynT mutants, regardless of the
presence or absence of SAP. The deficiency of the FynT
SH2 and
SH3 mutants in phosphorylating SLAM cannot simply be attributed to
their relatively low levels of expression compared with wild-type FynT
(Fig. 5B), but rather their incompetence in SLAM
phosphorylation reflects the importance of the SH2 and SH3 domains of
FynT, especially the latter, in proper signaling of SLAM.

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Fig. 5.
Functional characterization of SAP in SLAM
recruitment of FynT and in SLAM phosphorylation and binding to
SHP-2. A, SLAM-bearing HEK 293 cells were transfected
with FynT or FynT mutant constructs with or without SAP co-expressing.
Cell lysates were subjected to precipitation using anti-SLAM
( -SLAM) monoclonal antibodies, and the resulting protein
complexes were resolved in SDS-PAGE. Proteins associated with SLAM,
such as SAP, FynT (mutants), and SHP-2, were probed using appropriate
antibodies as indicated. SLAM phosphorylation was monitored using
anti-Tyr(P)-100 ( -pTyr) mouse monoclonal antibody.
IP, immunoprecipitate; WB, Western blot.
B, expression of various proteins in HEK 293 cells as probed
using specific antibodies on the corresponding cell lysates. Note that
the levels of expression for the FynT mutants SH2 and SH3 (not
shown) were generally lower than those of wild type FynT and the kinase
mutant Y417F (not shown). In the bottom panel,
biotinylated SLAM (Biotin-SLAM) proteins
precipitated from HEK 293 cell lysates using anti-SLAM antibody were
probed with streptavidin-conjugated horseradish peroxidase
(SA-HRP) to reveal the pattern of a broad band resulting
from extensive glycosylation (8). MW, molecular mass.
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Elevated levels of SLAM phosphorylation also correlated with the
increased binding of FynT to the receptor (Fig. 5A,
panels iii and iv, lanes 1 and
2). Although FynT appeared to associate weakly with SLAM in
the absence of SAP, possibly mediated by a weak binding of its SH2
domain to phosphorylated SLAM (Fig. 2; note that no SLAM
phosphorylation or receptor binding was detected on the FynT
kinase-dead mutant Y417F), SAP was seen to significantly enhance FynT
binding to the receptor (Fig. 5A, panels ii and
iii, lanes 1 and 2). It should be
noted that the amount of SLAM-associated FynT
SH2 and
SH3 mutants
were difficult to assess because they comigrated with the
immunoglobulin heavy chain. These results demonstrate that SAP plays a
role of an adaptor in promoting the recruitment of FynT to SLAM.
Interestingly, the binding of SAP to SLAM was seen to also block the
interaction of SHP-2 with the receptor (Fig. 5A, panel
v, lanes 1 and 2). Thus, SAP is capable of
not only bridging the interaction of FynT with SLAM but also inhibiting
the binding of SHP-2 to phosphorylated SLAM.
Disease-causing SAP Mutants Exhibit Defects in Binding to
FynT--
A number of missense point mutations have been identified in
the SH2D1A gene isolated from XLP patients. These mutations
generally result in single amino acid changes within the protein's SH2
domain, except that, in one case, the stop codon is mutated to an Arg, which leads to the addition of 11 extra amino acids to the C terminus of SAP (the "Tail" mutant). We previously reported that a group of
ten SAP mutants associated with XLP displayed reduced affinities for
SLAM and SLAM-derived peptides and that these mutants were incompetent
in blocking the interaction between SHP-2 and SLAM compared with
wild-type SAP (14, 21). To assess the relevance of the SAP-FynT
interaction to the pathogenesis of the XLP syndrome, we examined the
binding of the same group of mutants to FynT in a GST pull-down assay.
As shown in Fig. 6, with the exception of
the Tail mutant, binding of each of the remaining nine mutants to FynT was either significantly compromised or completely abolished, compared with SAP. It should be noted that the Tail mutant bound to
FynT as strongly as wild-type SAP, implying that this mutant is
defective in other characteristics than just its ability to interact
with FynT.

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Fig. 6.
SAP mutants are deficient in binding to
FynT. A, binding of GST-fused SAP and ten
disease-causing mutants to FynT. ~15 µg of GST fusion protein was
used to pull down FynT from 150 µl (200 µg total protein) of HEK
293 lysates for each lane. B, Coomassie blue staining of
SDS-PAGE showing equal application of proteins in each lane
(lanes 2-12) of panel A.
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DISCUSSION |
A central question regarding the role of SAP in the pathogenesis
of the XLP syndrome is how SAP regulates the signaling and, thereby,
the activities of immune cells such as the T and NK cells. The small
size of the SAP protein favors the notion that it may act as a natural
inhibitor to block or modulate the binding of other SH2 domain-mediated
interactions. A number of studies suggest that this mechanism may be
physiologically relevant because SAP is indeed capable of inhibiting
the binding of SHP-2 to SLAM in COS-7 cells and to the homologous
receptor 2B4 in NK cells (5, 8). Nonetheless, this mechanism of SAP
action seems to contradict the "adaptor" model put forward by
Latour et al. (23).
Our observation that the SH2 domains of SHIP and SHP-2 bind
to the same sites in the SLAM family of receptors as does SAP strongly
suggests that these three proteins are binding partners competing for
the same receptors. In the case of SLAM, SAP binds with the highest
affinity among the three proteins to the Tyr-281 site, and it therefore
should compete favorably against SHP-2 in binding to the same site as
shown previously (8, 21) as well as in the present study. However,
because all four members of the SLAM family of receptors examined in
this study contain multiple tyrosine phosphorylation sites, the
efficiency of these SH2 domain-containing proteins in competing against
each other would depend on their relative affinities for these sites
and their local concentrations.
Because of its small size and single domain structure, the notion of
SAP acting as an adaptor in SLAM receptor signaling is not readily
comprehended, because adaptor proteins often contain multiple
protein-interacting domains and motifs (19). Our finding that SAP
simultaneously binds to tyrosine sites in the SLAM receptor and to the
SH3 domain of the FynT kinase suggests that it can indeed serve a role
as an adaptor for the recruitment of FynT to the receptor. Although the
structural basis for such a unique interaction is not yet clear, the
functional consequence is that more kinase is recruited to the receptor
by SAP, resulting in enhanced receptor phosphorylation. It should be
pointed out that basal levels of receptor-associated FynT and receptor
phosphorylation are detectable in cells containing no SAP. Although our
results agree with those reported by Sayos et al. (8), they
differ from those by Veillette and co-workers (23). This discrepancy may be due to higher kinase concentrations in the systems of Sayos et al. (8) and ours as a result of transient overexpression of FynT than those in the latter study. Nonetheless, our observation that a small amount of FynT can be recruited to the receptor
independent of SAP may be functionally significant. It has been
previously shown, and confirmed in the present study, that SAP
interacts with the NK cell-activating receptor 2B4 in a
phosphorylation-dependent manner (5, 17, 18, 20).
Apparently, a basal level association of a kinase with the receptor
would facilitate its initial phosphorylation and hence the subsequent
recruitment of SAP.
Based on findings reported here and by others (8, 23), we propose a
unified model for SAP signaling through SLAM, where it plays a dual
functional role both as an adaptor for FynT and an inhibitor of SH2
domain-mediated interactions. In this model, the initial interaction
takes place between SAP and SLAM, which can occur constitutively. The
binding of SAP to SLAM promotes its association with FynT through an
adaptor function of SAP. Subsequently, receptor-associated FynT can
phosphorylate Tyr residues in the cytoplasmic region of the receptor or
of a neighboring receptor molecule following receptor cross-linking or
activation. This, in turn, creates docking sites for SH2
domain-containing proteins such as SHP-2 and SHIP. Because SAP is also
capable of binding to the same phosphorylated sites, the amount of
receptor-associated SHP-2 or SHIP and, thereby, the outcome of
downstream signaling, would depend on the relative affinities and
abundance of these proteins in the cell at a given time.
A similar mechanism could also be employed in principle by other
members of the SLAM family of receptors. CD84 contains a site (Tyr-262)
that can bind SAP constitutively. The receptors 2B4 and Ly-9 lack such
a site, and their association with SAP could only concur with tyrosine
phosphorylation. A basal level phosphorylation of these receptors may
contribute to their initial recruitment of SAP and, subsequently, of
FynT (or another kinase), ultimately leading to full-fledged
phosphorylation of the receptors. Although further studies are needed
to substantiate this model, it is of particular interest to find out
that disease-causing SAP mutants display defects in binding to both
SLAM and FynT.