Báo cáo khoa học: SHP-1 dephosphorylates 3BP2 and potentially downregulates 3BP2-mediated T cell antigen receptor signaling ppt

SHP-1 dephosphorylates 3BP2 and potentially
downregulates 3BP2-mediated T cell antigen receptor
signaling
Zhenbao Yu
1
, Meryem Maoui
1
, Zhizhuang J. Zhao
2
, Yang Li
1
and Shi-Hsiang Shen
1,3
1 Health Sector, Biotechnology Research Institute, National Research Council of Canada, Montre
´
al, Canada
2 Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
3 Department of Medicine, McGill University, Montre
´
al, Canada
Protein tyrosine phosphorylation plays a critical role
in various signal-transduction pathways in T lympho-
cytes [1]. For example, ligation of the T cell antigen
receptor (TCR) activates Src family protein tyrosine
kinases (PTKs) such as Lck and Fyn, which in turn
phosphorylate TCR n chain and CD3 e, d and c sub-
units within the immunoreceptor tyrosine-based activa-
tion motif (ITAM), resulting in the recruitment and
activation of the ZAP70 and Syk PTKs [2,3]. These
activated PTKs further induce the tyrosine phosphory-
lation of multiple intracellular proteins, including the

adapter proteins LAT [4] and SLP-76 [5]. Phosphoryla-
tion of these adapter proteins creates docking sites
for various Src-homology 2 (SH2) domain-containing
proteins such as PLCc, Grb2, Grap, Gads, Nck, Vav,
c-CBL and Tec family tyrosine kinase Itk, leading to
stimulation of downstream signaling pathways, and
ultimately to T cell activation [6,7].
Dephosphorylation of these tyrosine-phosphorylated
proteins is a necessary counterpart for maintaining a
balance between activation and quiescence of TCR
signaling [8]. SHP-1 is one such enzyme which
can counterbalance PTK effects and terminate recep-
tor-initiated signaling [9]. SHP-1 is expressed primarily
in hematopoietic cells and plays a critical role in the
negative regulation of TCR signaling and T cell
development. Accordingly, thymocytes derived from
motheaten (me) mice, which lack the expression of
functional SHP-1, hyperproliferate in response to TCR
stimulation [10–18]. SHP-1 displays its negative func-
tion at diverse stages of TCR signaling. For instance,
SHP-1 constitutively associates with TCR and appears
Keywords
3BP2; protein phosphatases; protein–protein
interaction; SHP-1; T cell-receptor
Correspondence
Zhenbao Yu, Health Sector, Biotechnology
Research Institute, National Research
Council of Canada, Montre
´
al, Que

´
bec
H4P 2R2, Canada
Fax: +1 514 496 6319
Tel: +1 514 496 6377
E-mail:
(Received 14 December 2005, revised 28
February 2006, accepted 16 March 2006)
doi:10.1111/j.1742-4658.2006.05233.x
Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1
(SHP-1) is a critical inhibitory regulator in T cell-receptor (TCR) signaling.
However, the exact molecular mechanism underlying this is poorly defined,
largely because the physiological substrates for SHP-1 in T cells remain
elusive. In this study, we showed that adaptor protein 3BP2 serves as a
binding protein and a physiological substrate of SHP-1. 3BP2 is phosphor-
ylated on tyrosyl residue 448 in response to TCR activation, and the phos-
phorylation is required for T cell signalling, as indicated by transcriptional
activation of nuclear factor activated in T cells (NFAT). Concurrently,
phosphorylation of Tyr566 at the C-terminus of SHP-1 causes specific
recruitment of 3BP2 to the phosphatase through the SH2 domain of the
adaptor protein. This leads to efficient dephosphorylation of 3BP2 and
thereby termination of T cell signaling. The study thus defines a novel
function of the C-terminal segment of SHP-1 and reveals a new mechanism
by which T cell signaling is regulated.
Abbreviations
GST, glutathione S-transferase; IP, immunoprecipitation; ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine
kinase; PTPase, protein tyrosine phosphatase; pTyr, phosphotyrosine; SH2, src homology 2; SHP, SH2 domain-containing PTPase; TCR,
T cell antigen receptor.
FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2195
to dephosphorylate the TCR CD3e subunit and more

distal signaling effectors following TCR activation [11].
It has been also reported that SHP-1 is phosphorylated
by activated Src family kinase Lck [19] in T cells and
it, in turn, dephosphorylates and inactivates Lck and
Fyn kinases [12,18,20]. SHP-1 is also thought to regu-
late the activity of Syk family kinase ZAP70 [21,22].
Moreover, SHP-1 is known to interact with the adapter
proteins Grb2 and SLP-76, although the physiological
meaning is unclear [11,23,24]. In this study, we identi-
fied 3BP2 as a novel SHP-1 substrate and binding
protein.
3BP2 was originally described as a PTK c-Abl SH3
domain-binding protein [25]. It contains an N-terminal
PH domain, a central proline-rich region that interacts
with c-Abl, and a C-terminal SH2 domain. Recently,
the SH2 domain of 3BP2 has been shown to bind to
the PTKs Syk and ZAP70 [26]. As a result, overexpres-
sion of 3BP2 in T cells leads to increased nuclear fac-
tor activated T cell (NFAT-) and AP-1-dependent
transcription [26,27]. It has also been shown that 3BP2
plays a positive regulatory role in NK-cell-mediated
cytotoxicity [28] and participates in the regulation of
FccR1-mediated degranulation in basophilic cells [29].
However, the mechanism by which 3BP2 exerts its pos-
itive effect on downstream signaling molecules remains
elusive.
In this study, we demonstrate that the interplay of
3BP2 and SHP-1 has an important role in T cell signa-
ling. On the one hand, 3BP2 is phosphorylated on tyr-
osyl residue 448, and the tyrosine phosphorylation is

critical for TCR signaling. On the other hand, SHP-1
is phosphorylated on Tyr566 at the C-terminus and
thereby recruits 3BP2 through SH2 domain inter-
action. This leads to dephosphorylation of 3BP2 and
termination of T cell signaling. This study thus pro-
vides a novel mechanism by which 3BP2 and SHP-1
regulate T cell signaling.
Results
3BP2 interacts with SHP-1 in a yeast two-hybrid
screen
To demonstrate the molecular mechanism of SHP-1-
mediated regulation of TCR signaling, we searched
for SHP-1-interacting proteins from human T cells
using a modified yeast two-hybrid screen [30]. The
full-length SHP-1 with mutation of Cys455 to Ser
(SHP-1 ⁄ C455S), which abolishes the protein tyrosine
phosphatase (PTPase) catalytic activity but retains the
binding ability to its substrates, was cloned into
plasmid pBTM-116-Src [31] for two-hybrid screening.
Transformation of the plasmid in yeast results in the
expression of Lex DNA-binding domain ⁄ SHP-1–
C455S fusion protein and c-Src kinase. Expression of
c-Src allows the identification of tyrosine phosphoryla-
tion-dependent SHP-1-interacting proteins. From
1.1 · 10
7
transformants with a human Jurkat T cell
cDNA library, 124 were positive for both HIS3 and
LacZ expression. Sequence analyses of the 124 positive
clones revealed that, among others, 11 independent

clones of different lengths represented overlapping
cDNAs of the SH3 domain-binding protein 2 (3BP2).
3BP2 was originally characterized as an Abl
SH3-interacting protein [25]. It is composed of an
N-terminal PH domain, a proline-rich region and a
C-terminal SH2 domain. Interestingly, all of the 3BP2
clones isolated in our two-hybrid screening contained
at least the sequence encoding the entire SH2 domain,
suggesting that the SH2 domain of 3BP2 is involved
in mediating the SHP)1 ⁄ 3BP2 interaction. Our addi-
tional studies demonstrated that the catalytic inactive
Cys-to-Ser mutant of SHP-2, an enzyme structurally
similar to SHP-1, was incapable of interacting with
3BP2 in the system (data not shown). This indicates a
high specificity of the interaction between 3BP2 and
SHP-1.
3BP2 associates with SHP-1 in 293T cells when
coexpressed with Lck
To determine whether 3BP2 associates with SHP-1 in
mammalian cells, we carried out a coimmunoprecipitat-
ion assay. Because the interaction of 3BP2 with SHP-1
was first identified from a T cell cDNA library in a
modified yeast two-hybrid system in which a Src family
kinase c-Src was expressed, we cotransfected 293T cells
with C-terminal myc-tagged 3BP2 (3BP2–myc), catalyt-
ically inactive SHP-1 (SHP-1 ⁄ C455S), and Src family
kinases, Fyn and Lck, which are known to be involved
in TCR signaling. 3BP2–myc was immunoprecipitated
with an anti-myc IgG. As shown in Fig. 1A, SHP-
1 ⁄ C455S was detected by western blot with the anti-

(SHP-1) IgG in the anti-myc immunoprecipitant from
293T cells cotransfected with wild-type Lck or catalyti-
cally activated Lck (Lck ⁄ Y505F). However, under the
same conditions, SHP-1 ⁄ C455S could not be coimmu-
noprecipitated with 3BP2–myc from 293T cells cotrans-
fected with catalytically activated Fyn (Fyn ⁄ Y531F) or
the plain control vector (Fig. 1A). In reciprocal experi-
ments involving immunoprecipitating SHP-1 ⁄ C445S
with anti-(SHP-1) IgG, 3BP2–myc was detected in
the immunoprecipitant from cells transfected with cata-
lytically activated Lck but not in the immunoprecipi-
tant from the cells transfected with wild-type Lck
SHP-1 and 3BP2 in T cell receptor signaling Z. Yu et al.
2196 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC)
(Fig. 1A, right) although SHP-1 could be coimmuno-
precipitated with 3BP2–myc by anti-myc IgG from the
wild-type Lck-transfected cell. This indicates that low
amounts of associated proteins could not be detected
in some coimminoprecipitation experiments.
3BP2, SHP-1, and Lck all contain SH2 domains and
potential tyrosine phosphorylation sites that can medi-
ate protein–protein interactions. To examine whether
the association of 3BP2 with SHP-1 could be mediated
by Lck, we cotransfected these three proteins into
293T cells and carried out a two-step immunoprecipi-
tation experiment. Whole-cell lysates were first subjec-
ted to immunoprecipitation with anti-Lck IgG or
control antibody (mouse IgG or protein A–Seph-
arose 4B beads alone) and the unbound proteins were
then subjected to immunoprecipitation with anti-myc

IgG. If the interaction of 3BP2 with SHP-1 is mediated
by Lck, removal of Lck from whole-cell lysates by
anti-Lck IgG immunoprecipitation should reduce the
amount of SHP-1 coprecipitated with 3BP2–myc.
However, as shown in Fig. 1B, although Lck was
essentially depleted from whole-cell lysates by anti-Lck
IgG (Fig. 1B, upper, lane 4), coimmunoprecipitation
of SHP-1 with 3BP2–myc was not affected (Fig. 1B,
lower, lanes 8–10). Moreover, neither 3BP2–myc
(Fig. 1B, middle, lane 7) nor SHP-1 (Fig. 1B, lower,
lane 7) was detected in the anti-Lck immunoprecipi-
tates. Parallel reciprocal experiments showed that Lck
was not coimmunoprecipitated with 3BP2–myc by
anti-myc IgG either (Fig. 1B, upper, lanes 8 and 9).
Taken together, these results indicate that the associ-
ation of 3BP2 with SHP-1 is not mediated by Lck,
although its kinase activity is required for the interac-
tion in 293 cells.
3BP2-myc
SHP-1
++++
++++
kinase
-
F135
Y-
n
yF
k
c

L
F505
Y
-
kc
L
IP: anti-myc IP: anti-SHP-1
Western blot:
anti-SHP-1
82
62
47
82
62
47
3BP2-myc
SHP-1
++++
++++
-
F135
Y-n
y
F
k
c
L
F
50
5Y-k

c
L
Western blot:
anti-myc
A
IP: anti-myc IP: anti-SHP-1Whole cell lysates
3BP2-myc
SHP-1
++++
++++
kinase
-
F135
Y
-
n
yF
k
c
L
F505Y-kcL
++++
++++
-
F
1
3
5Y
-
ny

F
kcL
F505Y
-k
c
L
++++
++++
-
F13
5Y-
n
yF
kcL
F505Y
-k
cL
Western blot:
anti-pTyr
3BP2-myc
SHP-1
82
62
47
160
LCW
sbAon
GgI
kcL-
i

t
n
a
s
bA
on
GgI
kcL-itna
sbAon
GgI
kcL-
i
t
n
a
first IP
Lck
unbound precipitant second IP: anti-myc
Western blot:
anti-SHP-1
3BP2
SHP-1
82
62
47
82
62
47
82
62

47
1234 5 6 7 8 910
1234 5678910
1234 5678910
Western blot:
anti-myc
Western blot:
anti-Lck
B
Fig. 1. 3BP2 associates with SHP-1 when coexpressed with Lck in 293T cells. (A) 293T cells were transfected with 3BP2–myc, SHP-
1 ⁄ C445S and Src family of kinases Fyn or Lck as indicated. The cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal
bovine IgG for 48 h after transfection and then lysed without any treatment. Whole-cell lysates were subjected to immunoprecipitation and
western blot analysis with anti-myc IgG, anti-SHP-1 IgG or anti-phosphotyrosine IgG. Molecular mass (kDa) is indicated to the left of the gel.
(B) 293T cells were transfected with 3BP2–myc, SHP-1 ⁄ C445S and autoactivated Lck (Lck ⁄ Y505F). Forty-eight hours after transfection the
cells were lysed and the whole-cell lysates were subjected to immunoprecipitation with anti-Lck IgG, IgG or without antibody as control. The
unbound proteins after the first immunoprecipitation were subjected to immunoprecipitation with anti-myc IgG. The proteins collected in
each step were analyzed by western blot as indicated.
Z. Yu et al. SHP-1 and 3BP2 in T cell receptor signaling
FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2197
3BP2 interacts with SHP-1 through the SH2
domain of 3BP2
Because both 3BP2 and SHP-1 contain SH2 domains,
the interaction of 3BP2 with SHP-1 might be through
either binding of the SH2 domains of SHP-1 to tyro-
sine-phosphorylated 3BP2 or that of the SH2 domain
of 3BP2 to phosphorylated SHP-1. We thus construc-
ted a glutathione S-transferase (GST) fusion protein of
the SHP-1 SH2 domains (GST–SHP-1–2SH2) and also
of the 3BP2 SH2 domain (GST)3BP2–SH2), and
carried out GST pull-down experiments to determine

which of these two possibilities accounts for the
observed association. As shown in Fig. 2A, SHP-
1 ⁄ C455S was precipitated by GST)3BP2–SH2. In the
same condition, SHP-2 could not be pulled down by
GST)3BP2–SH2. Western blot with anti-phophotyro-
sine IgG indicates that both SHP-1 and SHP-2 were
phosphorylated. These results suggest that 3BP2 specif-
ically interacts with SHP-1 but not SHP-2. In contrast,
3BP2–myc could not be pulled down by GST–SHP-1–
2SH2 (Fig. 2B). Note that, under the same conditions,
GST–SHP-1–2SH2 was able to pull down S2V, a siglec
family receptor previously identified as an SHP-1-bind-
ing protein [32], suggesting that the GST–SHP-1–2SH2
fusion protein was properly folded. These results sug-
gest that 3BP2 interacts with SHP-1 through the SH2
domain of 3BP2 and, presumably, tyrosine-phosphor-
ylated SHP)1.
Interaction of the SH2 domain of 3BP2 with
SHP-1 is mediated by phosphorylation
of SHP-1 at Tyr566
We next determined which tyrosine residue(s) of SHP-
1 is (are) involved in the interaction using tyrosine-to-
phenylalanine mutants. SHP-1 contains a C-terminal
noncatalytic tail that bears three potential phosphoryl-
ated tyrosine residues (Tyr538, Tyr543 and Tyr566).
We mutated each of them and cotransfected the result-
ing mutants with 3BP2–myc and Lck into 293T cells.
As shown in Fig. 3B, although both SHP-1 ⁄ Y538F
and SHP-1 ⁄ Y543F were detected in the anti-myc
immunoprecipitants, SHP-1 ⁄ Y566 was not detectable,

suggesting that 3BP2 binds to the phosphorylated
Tyr566 of SHP-1. Interestingly, we found that both
wild-type and catalytically inactive SHP-1 (SHP-
1 ⁄ C455S) could be coprecipitated by 3BP2–myc with
anti-myc IgG (Fig. 2B and data not shown), indicating
that Tyr566, the 3BP2-binding site of SHP-1 was not
dephosphorylated by SHP-1 in this condition. How-
ever, western blot analyses of the anti-SHP-1
immunoprecipitants with anti-phosphotyrosine IgG
showed that the tyrosine phosphorylation level of
SHP-1 ⁄ Y566F mutant is much lower than that of
wild-type SHP-1, SHP-1 ⁄ Y538F, SHP-1 ⁄ Y543F and
SHP-1 ⁄ C455S ⁄ Y566F (Fig. 3F), suggesting that
Tyr566 is the major phosphorylation site of SHP-1 in
this condition. The anti-phosphotyrosine western blot
Western blot:
anti-SHP-1
Western blot:
anti-SHP-2
LCW
TSG
2HS-
2
PB3
-
TSG
SHP-1
SHP-2
IP :anti-SHP-2
Western blot:

anti-pTyr
Western blot:
anti-pTyr
IgG
IgG
SHP-1
SHP-2
pervanadate
- +
Western blot:
anti-myc
c
y
m-2PB3
cy
m
-
V2
S
cym-V2S+cy
m-
2P
B3
cym-2PB
3
cy
m-V2S
c
ym-V2S+cy
m-

2PB3
3BP2-myc
S2V-myc
WCL GST-SHP-1-2SH2
A
B
IP :anti-SHP-1
Fig. 2. SHP-1 associates with 3BP2 through
the SH2 domain of 3BP2. 293T cells were
transfected with SHP-1 ⁄ C455S, SHP-
2 ⁄ C459S, 3BP2–myc and 3BP2–myc plus
S2V-myc, respectively. Forty-eight hours
after transfection, the cells were treated
with 0.5 m
M pervanadate for 30 min. The
whole-cell lysates were incubated with GST,
GST)3BP2–SH2 or GST–SHP-1–2SH2
bound on glutathione Sepharose and subjec-
ted to immunoprecipitation as indicated. The
proteins precipitated were analyzed by
western blot with anti-SHP-1, anti-SHP-2,
anti-phosphotyrosine or anti-myc IgG.
SHP-1 and 3BP2 in T cell receptor signaling Z. Yu et al.
2198 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC)
analysis of the anti-myc immunoprecipitants showed
that 3BP2 was phosphorylated and dephosphorylated
by wild-type SHP-1, and the SHP-1 ⁄ Y538F and SHP-
1 ⁄ Y543F mutants (Fig. 3E), but not by the catalyti-
cally inactivated mutants (C455S and C455S ⁄ Y566F).
3BP2 was also partially dephosphorylated by SHP-

1 ⁄ Y566F mutant (Fig. 3E,D, lane 7) although it did
not associate with this mutant (Fig. 3B, lane 7), sug-
gesting that SHP-1 may be also able to directly de-
phospharylate 3BP2 without association of the two
proteins through the SH2 domain–phosphotyrosine
interaction in the condition with the overexpression of
the two proteins.
To exclude the possibility that the major tyrosine-
phosphorylated protein in the anti-myc precipitants is
not 3BP2–myc but another protein of similar mole-
cular mass that might be comimmunoprecipitated with
3BP2–myc, we treated the whole-cell lysates by adding
SDS to 1% and heating the samples at 100 °C for
10 min before immunoprecipitation. This should dis-
rupt protein–protein interactions. Treated samples
were then diluted 10 times with lysis buffer and
subjected to immunoprecipitation. Such treatment is
expected to eliminate the coimmunoprecipitation of
any 3BP2-binding proteins from 3BP2 with anti-myc
IgG. As shown in Fig. 3G, the tyrosine-phosphoryl-
ated protein with the same molecular mass as 3BP2–
myc was detected in the anti-myc precipitants from
the SDS-treated samples as well as in those from non-
treated samples. This result further confirms that
3BP2 was tyrosine phosphorylated. More significantly,
the phosphorylated 3BP2 was nearly completely de-
phosphorylated by wild-type SHP-1, but not by its
catalytically inactive mutant SHP-1 ⁄ C455S (Fig. 3E),
suggesting that 3BP2 is a potential substrate for
SHP-1.

A
whole cell lysates IP: anti-myc IP: anti-SHP-1
Western blot: anti-pTyr
3BP2-myc
+++++++
Lck +
+
++++++-
SHP-1
TW
S
554C
F
8
35Y
F
3
45Y
F
6
65
Y
F
6
6
5Y
/S
554C
TW
-

+++++++
+
+
++++++
-
T
W
S
5
54C
F
8
35Y
F345Y
F
665Y
F
6
65Y
/S
5
54
C
TW
-
+++++++
+
+
++++++
-

T
W
S55
4C
F835Y
F345Y
F66
5Y
F6
6
5Y/S554
C
TW
-
120
60
40
80
3BP2-myc
SHP-1
3BP2-myc +++++++
Lck +
+
++++++-
SHP-1
T
W
S5
5
4C

F8
3
5Y
F3
4
5Y
F
665Y
F
6
65Y/S
5
54
C
T
W
-
+++++++
+
+
++++++
-
T
W
S554
C
F
8
35Y
F

34
5Y
F
66
5Y
F66
5
Y
/S
5
5
4
C
TW
-
120
60
40
80
3BP2-myc
SHP-1
IP: anti-myc IP: anti-myc
WB: anti-myc WB: anti-SHP-1
+++++++
+
+
++++++
-
T
W

S5
5
4C
F83
5Y
F
34
5
Y
F665
Y
F6
6
5
Y
/S5
5
4
C
TW
-
IP: anti-SHP-1
WB: anti-SHP-1
BC
E
D
F
37685421
37685421
37685421

3768542137685421
37685421
3BP2-myc +++
Lck +
+
++-
SHP-1
S
5
54
C
TW

+++
+
+
++
-
S
554C
T
W

+++
+
+
++
-
S5
5

4
C
T
W

SDS treatment - +
WCL IP: anti-myc
WB:
anti-pTy
r
3BP2-myc +++
Lck +
+
++-
SHP-1
S5
54C
T
W

+++
+
+
++
-
S5
54C
TW

WCL

WCL
WB: anti-Lck WB: anti-SHP-1
82
62
47
82
62
47
WB:
anti-myc
82
62
47
G
Fig. 3. SHP-1 associates with 3BP2 through the phosphorylated tyrosine residue 566 of SHP-1 (A–F) 293T cells were cotransfected with
3BP2–myc, Lck ⁄ Y505F and SHP-1 or its mutants. Forty-eight hours after transfection, the cells were lysed and whole-cell lysates were sub-
jected to immunoprecipitation and western blot with the indicated antibodies. (G) 293T cells were cotransfected with 3BP2 myc, Lck ⁄ Y505F
and SHP-1 or its catalytically inactive mutant (SHP-1 ⁄ C455S). Forty-eight hours after transfection, the cells were lysed. The whole-cell
lysates were equally divided into two portions. One portion of the lysates was treated with 1% SDS at 100 °C for 10 min. The other portion
was left untreated. The lysates were then diluted 10 times with lysis buffer and subjected to immunoprecipitation and western blot as des-
cribed.
Z. Yu et al. SHP-1 and 3BP2 in T cell receptor signaling
FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2199
Tyr448 is the major phosphorylated residue of
3BP2 in response to TCR engagement and is
critical for 3BP2 function in TCR signaling
3BP2 is a positive regulator of TCR signaling and is
phosphorylated on tyrosine residues in response to
TCR engagement [26]. To further study the function
of 3BP2 phosphorylation in the regulation of TCR

signaling, we determined which tyrosine residue(s) of
3BP2 can be phosphorylated. Four potential phos-
phorylation sites, namely Tyr174, Tyr183, Tyr448
and Tyr485, were predicted (.
nctu.edu.tw). We mutated these tyrosine residues to
phenylalanine and transfected these mutants into Jur-
kat cells. The tyrosine-phosphorylation status of these
mutants was examined following stimulation with anti-
CD3 IgG OKT3. As shown in Fig. 4, although none
of the mutations Y174F, Y183F and Y485F exerted
any evident effect on the tyrosine phosphorylation of
3BP2, mutation Y448F almost completely abolished
tyrosine phosphorylation, suggesting that Tyr448 is the
major tyrosine-phosphorylated residue in response to
TCR activation.
We next determined the effects of these 3BP2
mutants on NFAT activation. To do so, we
cotransfected 3BP2 or its mutants with NFAT-lucif-
erase (firefly) reporter into Jurkat cells and stimulated
the cells with anti-CD3 IgG, and PMA plus ionomy-
cin, respectively. To determine whether the expression
of 3BP2 and its mutants affects stimulation of the
expression of NFAT-driven luciferase by PMA ⁄ iono-
mycin, we transfected the Jurkat cells with NFAT-
luciferase vector, pRL-TK vector which expresses TK
promoter-driven Renilla luciferase and 3BP2 or its
mutants, and then stimulated the cells with PMA plus
ionomycin. Firefly luciferase activity was normalized
by Renilla luciferase activity. As the results show that
3BP2 and its mutants did not affect the T cell response

to the PMA ⁄ ionomycin stimulation (data not shown),
we normalized the transfection efficiencies determined
by the stimulation with PMA plus ionomycin. As
shown in Fig. 5, although mutation on other tyrosine
residues of 3BP2 did not exert appreciable effects on
3BP2-mediated NFAT activation, the Y448F mutation
reduced the effect of 3BP2 on NFAT activation. These
results suggest that phosphorylation of 3BP2 on
Tyr448 plays an important role for its function in
TCR signaling.
SHP-1 dephosphorylates 3BP2 in TCR signaling
and negatively regulates 3BP2-induced NFAT
activation
Because tyrosine phosphorylations of SHP-1 at the
C-terminal residues can activate its phosphatase
activity [33] and 3BP2 interacts with phosphorylated
SHP-1, it is expected that the tyrosine-phosphorylated
3BP2 is a potential substrate for activated SHP-1
during their interaction. The substrate characteristic
of 3BP2 for SHP-1 was primarily demonstrated in
293T cells where the tyrosine-phosphorylated 3BP2
was largely dephosphorylated by wild-type SHP-1, but
not by catalytically inactive mutant SHP-1 ⁄ C455S
(Fig. 3E). To further investigate the substrate nature
of 3BP2 for SHP-1 during T cell signaling, we cotrans-
fected 3BP2–myc with wild-type SHP-1, its catalyti-
3BP2-myc
3BP2-myc
0 2' 10' 0 2' 10' 0 2' 10' 0 2 10' 0 2' 10'
3BP2 WT 3BP2/Y174F 3BP2/Y183F 3BP2/Y448F 3BP2/Y485F

62
82
62
82
IP: anti-myc
WB: anti-pTyr
IP: anti-myc
WB: anti-myc
OKT3
Fig. 4. Identification of Tyr448 as the major phosphorylated residue of 3BP2 in response to TCR engagement. Jurkat T cells were transfect-
ed with 3BP2 or its mutants as indicated. Forty-eight hours after transfection, the cells (5 · 10
7
) were stimulated with anti-CD3 IgG (OKT3)
at 37 °C for 0, 2 or 10 min as described in Experimental procedures. Whole-cell lysates were immunoprecipitated with anti-myc IgG and the
precipitated proteins were subjected to western blot analysis with anti-phosphotyrosine (pTyr) and anti-Myc IgG, respectively.
SHP-1 and 3BP2 in T cell receptor signaling Z. Yu et al.
2200 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC)
cally inactive mutant SHP-1 ⁄ C455S, and mutant SHP-
1 ⁄ Y566F, which is not capable of associating with
3BP2, into human Jurkat T cells, respectively. The
tyrosine-phosphorylation status of 3BP2 was examined
following anti-CD3 IgG stimulation in the transfected
cells. As shown in Fig. 6, the tyrosine-phosphorylation
level of 3BP2 was dramatically reduced when cotrans-
fected with wild-type SHP-1. In contrast, neither the
catalytically inactive SHP-1 (SHP-1 ⁄ C455S) nor
mutant SHP-1 ⁄ Y566F exerted any detectable effect on
the tyrosine phosphorylation of the cotransfected 3BP2
in Jurkat cells. Because Tyr448 is the major phosphor-
ylated site of 3BP2, SHP-1-mediated dephosphoryla-

tion of 3BP2 is expected to take place mainly on this
tyrosine residue. These results suggest that SHP-1 via
Tyr566 recruits 3BP2 as its potential substrate for dep-
hosphorylation during TCR signaling.
To further determine if the SHP-1-mediated de-
phosphorylation of 3BP2 affects its function in TCR,
we cotransfected Jurkat cells with NFAT-luciferase
reporter, 3BP2 and SHP-1 or SHP-1 mutants. As
shown in Fig. 7, expression of 3BP2 resulted in both
constitutive and anti-CD3 IgG-induced NFAT activa-
tion. Expression of SHP-1, however, inhibited anti-
CD3-induced NFAT activation. SHP-1 also nearly
completely inhibited 3BP2-mediated NFAT activation
in response to anti-CD3 stimulation in 3BP2-transfect-
ed cells. Furthermore, SHP-1 also inhibited the consti-
tutive NFAT activation in 3BP2-transfected cells but
not the basal NFAT activity in the cells without 3BP2
transfection. In contrast, the catalytically inactive
mutant SHP-1 ⁄ C455S or mutant SHP-1 ⁄ Y566F, which
abolished its interaction with 3BP2, was incapable of
suppressing the 3BP2-mediated NFAT activation in
3BP2 transfected cells. Taken together, these results
suggest that SHP-1 negatively regulates the function of
3BP2 in TCR signaling through dephosphorylation of
3BP2 on its Tyr448 residue.
Discussion
It has been reported that SHP-1 plays a negative role
in TCR signaling. However, the precise mechanism by
which SHP-1 regulates TCR signaling is largely
unknown. In this study, we reported the identification

of a novel SHP-1-interacting adapter protein 3BP2.
3BP2 is composed of an N-terminal PH domain, an
SH3-binding proline-rich region, and a C-terminal
SH2 domain. In addition to SHP-1 reported here, the
SH2 domain of 3BP2 has been shown to bind to sev-
eral phosphorylated proteins including ZAP70, PLCc,
LAT, Grb2 and Cbl26. 3BP2 was initially identified as
an Abl SH3 domain-binding protein of unknown func-
tion [25]. Recently, 3BP2 has been shown to interact
with the Syk and ZAP70 proteins of the Syk family of
tyrosine kinases. In addition, 3BP2 plays a positive
adapter function on basal and TCR-mediated NFAT
and AP-1 transcriptional activation in human Jurkat
Fig. 5. Effect of the mutation of tyrosine residues on 3BP2-induced
NFAT activation. Jurkat T cells were cotransfected with NFAT-lucif-
erase reporter and 3BP2–myc or its mutants as indicated. Twenty
hours after transfection, the cells were incubated with either no
addition, anti-CD3 IgG (OKT3) or PMA plus ionomycin at 37 °C for
6 h as described in Experimental procedures. Luciferase activity in
cell extracts was assayed and the data were normalized by the
maximal response obtained in the presence of PMA plus ionomy-
cin. The results shown are means ± SE from three independent
assays performed in two separate experiments.
S5
54C
/1
-PHS
F665Y
/1
-

PHS
TW/1-PHS
OKT3 - + - + - + - +
rotceV
82
62
82
62
3BP2-myc
3BP2-myc
WB:
anti-myc
WB:
anti-pTyr
Fig. 6. 3BP2 is dephosphorylated by SHP-1 in activated Jurkat
T cells. Jurkat T cells were transfected with 3BP2 and SHP-1 or its
mutants as indicated. Forty-eight hours after transfection, the cells
(5 · 10
7
cell equivalents) were stimulated with anti-CD3 antibody
(OKT3) at 37 °C for 2 min as described in Experimental procedures.
Whole-cell lysates were immunoprecipitated with anti-myc IgG and
the precipitated proteins were subjected to western blot analysis
with anti-phosphotyrosine (pTyr) and anti-myc IgG.
Z. Yu et al. SHP-1 and 3BP2 in T cell receptor signaling
FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2201
T cells [26]. However, the molecular mechanism by
which 3BP2 regulates TCR signal transduction remains
unclear. We found that 3BP2 is a potential substrate
of SHP-1 and SHP-1 is likely to negatively regulate

3BP2-mediated NFAT activation in TCR signaling. In
addition, we identified the major tyrosine phosphoryla-
tion site of 3BP2, Tyr448. Mutation of this tyrosine
residue reduced 3BP2-mediated NFAT activation.
Thus, tyrosine phosphorylation is crucial for 3BP2
function in TCR signaling and dephosphorylation of
the phosphorylated 3BP2 by SHP-1 negatively regu-
lates 3BP2 activity. Tyrosine phosphorylation of 3BP2
has been also demonstrated in mast cells in response
to aggregation of high affinity IgE receptor [29,34], in
NK cells upon stimulation with anti-FcR IgG [28] and
recently in T cells upon TCR activation [35]. In RBL-
2H3 mast cells, phosphorylation of Tyr448 of 3BP2
creates a binding site for the SH2 domain of Lyn, a
Src family protein tyrosine kinase, and interaction of
Lyn with 3BP2 positively regulates the kinase activity
of Lyn [34]. In NK cells, Tyr183 of 3BP2 is phosphor-
ylated and binds Vav and PLCc during activation of
NK cells through natural cytotoxicity receptors and
this phosphorylation is necessary for the enhancement
of natural cytotoxicity by 3BP2 [28]. Qu et al. [35]
recently found that both Tyr183 and Tyr448 could be
phosphorylated in response to TCR activation by anti-
CD3 IgG together with PMA. However, in our study,
mutation of Tyr183 to phenylalanine did not have
obvious effect on 3BP2 phosphorylation in response to
anti-CD3 IgG-induced TCR activation in the absence
of PMA. This suggests that both cross-linking of TCR
and direct activation of protein kinase C are required
for the phosphorylation of Tyr183 of 3BP2. Thus, it is

likely that 3BP2 is selectively activated in response to
various upstream signalings.
Usually, the SH2 domain of SHP-1 associates with
tyrosine-phosphorylated proteins during its interac-
tions with other signal molecules. Interestingly, the
association of 3BP2 with SHP-1 is through the SH2
domain of 3BP2 and the tyrosine-phosphorylated
phosphatase. SHP-1 contains three tyrosine residues
(Tyr538, Tyr543 and Tyr566) in its C-terminal tail. It
has been reported that at least two of these tyrosine
residues could be phosphorylated in response to the
stimulation of T cell-receptor [19], CSF receptor and
c-Kit [36]. However, the biochemical consequence and
physiological significance of tyrosine-phosphorylation
on SHP-1 remain elusive. It has been suspected that
tyrosine phosphorylation of SHP-1 may regulate its
phosphatase activity as observed in other phosphatases
[33]. In this study, however, we found that phosphory-
lation of SHP-1 at tyrosine residues on its C-terminal
tail confers to the phosphatase an ability to recruit
adapter protein 3BP2 and thereby affects signaling.
Site-directed mutation experiments further revealed
that 3BP2 interacts with SHP-1 through its phosphor-
ylated Tyr566 residue. The sequence surrounding
Tyr566 (Tyr566 ⁄ Glu567 ⁄ Asn568) is strikingly similar
to the optimal 3BP2 SH2 domain-binding motif (Tyr ⁄
Glu ⁄ Asn) [37].
In this study, we demonstrated that SHP-1 interacts
with 3BP2 through the tyrosine-phosphorylated C-ter-
minal segment of the former and the SH2 domain of the

latter. This interaction allows 3BP2 to be dephosphoryl-
ated more efficiently by the catalytic domain of SHP-1.
We thus defined a novel function for the C-terminal seg-
ment of SHP-1. It has been known that tyrosine phos-
phorylation of SHP-1 at its C-terminal segment also
initiates interaction with adapter protein Grb2 and
mSOS [23]. However, this does not seem involve the cat-
alytic activity of the enzyme and thus the physiological
meaning remains unclear. Furthermore, like SHP-1,
SHP-2 is also known to be phosphorylated at its C-ter-
minal segment. However, because these two enzymes
share minimum sequence identity at their C-termini, in
contrast to high sequence homologies in their SH2 and
catalytic domains, we believe this may allow the
enzymes interact with distinct proteins. This may
explain the often-opposite functions of the two enzymes.
rotcev
2
P
B
3
1-PHS
+
2P
B3
1
-
PHS
+
2P

B
3
S/
C1-
P
H
S
+2P
B3
F
6
65
Y1-P
H
S
S/
C1-
P
HS
F66
5Y1
-PHS
Fig. 7. SHP-1 negatively regulates 3BP2-induced NFAT activation.
Jurkat T cells were cotransfected with the NFAT-luciferase reporter
gene and empty vector, 3BP2–myc and SHP-1 or its mutants as
indicated. Twenty hours after transfection, the cells were incubated
with either no addition, anti-CD3 IgG (OKT3) or PMA plus iono-
mycin at 37 °C for 6 h as described in Experimental procedures.
Luciferase activity in cell extracts was assayed and the data were
normalized by the maximal response obtained in the presence of

PMA plus ionomycin. The results shown are means ± SE from
three independent assays performed in two separate experiments.
SHP-1 and 3BP2 in T cell receptor signaling Z. Yu et al.
2202 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC)
Experimental procedures
Reagents and antibodies
Rabbit anti-SHP-1 polyclonal IgG was generated as des-
cribed previously [38]. Mouse anti-SHP-1 and anti-SHP-2
monoclonal IgG were obtained from Transduction Labor-
atories (Lexington, KY). An anti-(human CD3-a) (OKT3)
monoclonal IgG was purified from the culture medium
of OKT3 hybridomas by protein A–Sepharose affinity
chromatography. Rabbit anti-(mouse IgG) was obtained
from BD Biosciences Pharmingen (San Diego, CA). Anti-
phosphotyrosine (4G10) and anti-myc (9E10) monoclonal
IgG were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Anti-hemagglutinin (anti-HA) monoclonal IgG
(clone 12CA5) was prepared from the culture medium of
hybridomas (ATCC, Manassas, VA). Anti-(mouse IgG-
horseradish peroxidase) and anti-(rabbit IgG-horseradish
peroxidase) were from Bio-Rad Laboratories (Hercules,
CA). Nitrocellulose membrane Hybond-ECL was from
Amersham Pharmacia Biotech (Little Chalfont, UK). West-
ern Lightning Chemiluminescence Reagent kit was pur-
chased from Perkin–Elmer Life Sciences Inc. (Boston, MA).
Protease inhibitor cocktail tablets were from Roche Diag-
nostics (Mannheim, Germany).
Plasmids
Plasmids expressing SHP-1 and its mutants were construc-
ted as described previously [31,38]. Myc-tagged 3BP2 plas-

mid and HA-tagged 3BP2 plasmid were constructed by
amplifying the full-length 3BP2 encoding region using total
RNA from Jurkat cells and inserting the amplified PCR
product into the HindIII site of pcDNA3.1 ⁄ myc-His (–) C
vector (Invitrogen, Carlsbad, CA) and pACTAG-2 vector
(kindly provided by M. Tremblay, McGill University).
3BP2 mutants were generated by PCR-based mutagenesis.
Fyn kinase in pRK5 vector was a kind gift from S Stamm
(Max Planck Institute of Biochemistry, Germany). Lck and
its activated mutant (Lck ⁄ Y505F) constructs were kindly
provided by B Sefton and G Chiang (The Salk Institute for
Biological Studies). NFAT-luciferase reporter was kindly
provided by G Crabtree (Stanford University School of
Medicine).
Cell culture and transfection
293T cells and Jurkat T cells were maintained as described
previously [31,39]. 293T cells were transfected with different
sets of plasmid DNAs using standard calcium phosphate pre-
cipitation methods. In some experiments, the transfected cells
were treated with 0.5 mm sodium pervanadate in regular
medium for 30 min. Sodium pervanadate was prepared by
mixing 100 mm sodium orthovanadate (Sigma, St. Louis,
MO) and 50 mm H
2
O
2
(Sigma) and incubating the mixture
at room temperature for at least 30 min. Jurkat T cells (10
7
in 400 lL of medium) were transfected with 20–25 lg DNA

by electroporation using a gene pulser (BTX Corp., San
Diego, CA) at 260 V for 50 ms. Empty vector was added to
some samples to make an equal amount of DNA in each
transfection. Forty-eight hours after transfection, Jurkat
T cells were washed and suspended in NaCl ⁄ P
i
. For stimula-
tion, cells were incubated with 2 lgÆmL
)1
of OKT3 on ice for
5 min and then with 10 lgÆmL
)1
of rabbit anti-(mouse IgG)
for an additional 5 min. The samples were then incubated at
37 °C for the indicated times.
Yeast two-hybrid screen
The cDNA encoding the full-length of SHP-1 with Cys455
to Ser mutation (SHP-1-C455S) was PCR-amplified from the
corresponding plasmid [40] and cloned in-frame downstream
of the DNA binding domain of Lex A in pBTM-116-src vec-
tor [30] to form the bait construct (Lex A–SHP-1–C455S)
[31]. The human Jurkat cDNA library expressed as fusion
proteins with the activation domain of GAL4 in the pACT2
vector was obtained from Clontech Laboratories (Palo Alto,
CA). The bait DNA and library DNA were sequentially
transformed into yeast strain L40a and 1.1 · 10
7
primary
transformants were screened for growth on medium lacking
leucine, tryptophan and histidine. The positive colonies were

further screened for the expression of b-galactosidase. The
plasmid DNA was recovered from His
+
⁄ LacZ
+
colonies
and identified by DNA sequencing.
Immunoprecipitation and immunoblot analysis
Immunoprecipitation and western blot experiments were
carried out as described previously [31]. Briefly, cells were
washed with cold NaCl ⁄ P
i
once and lysed in a lysis buffer
containing 50 mm Hepes (pH 7.4), 150 mm NaCl, 1% Tri-
ton X-100, 5 mm b-mercaptoethanol, 0.5 mm vanadate and
an EDTA-free mixture of protease inhibitors. The samples
were centrifuged at 20 000 g for 10 min at 4 °C. An aliquot
of this whole-cell lysate was removed and the remaining
lysate was subjected to immunoprecipitation. For immuno-
precipitations, cell lysates were incubated with optimal con-
centrations of antibodies for 2 h at 4 °C, followed by
incubation with 50 lL of 50% suspension of Protein A–
Sepharose CL-4B beads for 1 h. The Sepharose CL-4B
beads were washed at 4 °C with lysis buffer four times. The
proteins were resolved on a SDS ⁄ PAGE gel and transferred
to nitrocellulose membranes (Hybond-ECL). The mem-
branes were blocked with 5% milk in Tris-buffered saline
(TBS) (pH 7.6) overnight and then incubated with the first
antibodies for 2 h. After washing four times with TBS con-
taining 0.05% Tween-20 (TBS-T), the membranes were

incubated with the second antibody conjugated to horserad-
ish peroxidase for 1 h and then washed four times
with TBS-T. The blots were developed using the western
Z. Yu et al. SHP-1 and 3BP2 in T cell receptor signaling
FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2203
Lightning Chemiluminescence Reagent kit (Roche) accord-
ing to the manufacturer’s instruction.
Expression, purification of GST fusion proteins,
and GST pull-down
For the construction of a plasmid expressing GST)3BP2–
SH2 domain fusion protein, the cDNA fragment encoding
amino acid residues 452–561 of 3BP2 was amplified by
PCR and inserted into pGEX-5X1 vector (Amersham Phar-
macia Biotech) Construction of GST–SHP-1–2SH2 has
been described previously [41]. Fusion proteins were
expressed in Escherichia coli strain DH5a by induction with
25 lm isopropyl-d-thiogalactopyranoside at 25 °C for 16 h
and purified as described previously [42]. For binding
assays, glutathione–Sepharose beads with  1 lg of bound
GST or GST fusion protein were incubated at 4 °C for 2 h
with 1 mL of cell lysates. The beads were washed four
times with the lysis buffer and the bound proteins were
analyzed by SDS ⁄ PAGE and western blot.
NFAT reporter assay
Jurkat T cells (2 · 10
7
) were transiently transfected with
5 lg of pNFAT-luciferase and 20 lg of indicated plasmids
by electroporation. Twenty hours after transfection, cells
were aliquoted into a 12-well plate in 1 mL of culture med-

ium and triplicate samples were either left unstimulated,
stimulated with OKT3 (2 lgÆmL
)1
) or with PMA
(50 ngÆmL
)1
) plus 1 lm ionomycin for 6 h. Cells were then
harvested and washed with 1 mL of NaCl ⁄ P
i
. Harvested
cells were lysed and assayed for luciferase activity as previ-
ously described [40]. Luciferase activity was determined in
triplicate for each experimental condition and normalized
by the transfection efficiencies determined by the maximum
stimulation with PMA plus ionomycin.
Acknowledgements
This study was supported in part by the National Sci-
ence and Engineering Research Council of Canada
Grant 0GP0183691. We thank Dr J.A. Cooper for
kindly providing the pBTM-116-src vector, Dr S.
Stamm for Fyn kinase vector, Dr B.M. Sefton and Dr
G.G. Chiang for Lck constructs, Dr G.R. Crabtree for
NFAT-luciferase reporter and Dr M. Tremblay for
pACTAG-2 vector.
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FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2205