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INTRODUCTION |
Tyrosine phosphorylation is a critical control mechanism for
growth, differentiation, cell cycle control, and cytoskeletal function.
The signal transduction pathways involving tyrosine phosphorylation are
regulated by the concerted action of protein-tyrosine kinases and
protein-tyrosine phosphatases. Although much of the work has centered
on the actions of the multitude of protein-tyrosine kinases, it is now
apparent that the protein-tyrosine phosphatases play an equally diverse
and important role.
SHP-1 is a member of the SH21
domain-containing family of nonmembrane protein-tyrosine phosphatases
that is predominately expressed in hematopoietic cells. SHP-1 and the
related phosphatases SHP-2 and csw in Drosophila
all share a similar structure of two tandem SH2 domains in the N
terminus followed by the catalytic domain and a C-terminal tail (1). In
resting cells, the SHP-1 SH2 domains sterically inhibit the catalytic
activity of SHP-1 through interactions with the catalytic domain (2,
3). Following lymphocyte activation, the SH2 domains allow SHP-1 to
bind to tyrosine-phosphorylated immunoreceptor tyrosine-based
inhibitory motifs (4, 5). This binding serves to recruit SHP-1
to the membrane and relieve the steric inhibition, activating the
phosphatase (2).
A naturally occurring SHP-1 deficiency exists in the motheaten mouse.
These mice do not express any detectable SHP-1 protein and exhibit a
panoply of hematopoietic defects, ranging from the overproliferation of
macrophages and neutrophils to abnormal B- and T-cell development and
hyper-responsiveness (6). This suggests that SHP-1 plays an inhibitory
role in hematopoietic cells. Consistent with this idea, SHP-1 has been
shown to negatively regulate signaling downstream of the erythropoietin
receptor, c-Kit, the granulocyte/macrophage colony-stimulating
factor receptor, and the B- and T-cell antigen receptor (5, 7-9).
The identification of physiological substrates of SHP-1 has been of
considerable interest. In B cells, CD72 has been identified as an
in vivo substrate of SHP-1 (10). Additionally,
SIRP-
and p62DOK have been identified
as SHP-1 substrates in macrophages (11, 12). In T cells, the adapter
protein SLP-76 and the cytosolic tyrosine kinase ZAP-70 have been shown
to be substrates of SHP-1 (13, 14). We were interested in identifying additional substrates of SHP-1 in T cells. We initially focused our
efforts on Lck because of the prolonged Lck catalytic activity observed
in motheaten thymocytes (15) and because Lck is an upstream activator
of ZAP-70 (16).
Lck is a lymphoid cell-specific member of the Src family of nonreceptor
tyrosine kinases that is essential for both the development of T cells
in the thymus and the response of mature T cells to signals arising
from the T-cell antigen receptor (17, 18). Like all Src family kinases,
Lck is activated and inhibited by tyrosine phosphorylation. Tyr-394 is
the site of stimulatory phosphorylation, whereas Tyr-505 is the site of
inhibitory phosphorylation. Here we have examined whether SHP-1
directly regulates Lck phosphorylation.
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EXPERIMENTAL PROCEDURES |
DNA Constructs and Antibodies--
The cDNAs encoding
wild-type murine Lck (WT), F505 Lck, F394 Lck, and
SH2 Lck have been
described previously (19-21). For the transient transfections used in
this study, the Lck cDNAs were subcloned into the expression vector
pCEP4 (Invitrogen). The SHP-1 cDNA was a kind gift from Dr. Matt
Thomas (Washington University, St. Louis, MO). The catalytically
inactive SHP-1 cDNAs (D419A and C453S) were constructed by
polymerase chain reaction-mediated mutagenesis. For transient
transfections, the SHP-1 cDNAs were subcloned in frame into the
vector CS3+MT (a kind gift from Dr. Jon Cooper, Fred Hutchinson Cancer
Research Center, Seattle, WA), which appends six copies of the Myc
epitope tag to the N terminus. SHP-1 constructs lacking both SH2
domains (
SH2 and 
SH2 D419A) were constructed by subcloning
a restriction fragment encoding amino acids 203-595 in frame into the
vector CS3+MT. The CD4 cDNA was a kind gift from Dr. Andrey Shaw
(Washington University) and was subcloned into the vector pCMX (22).
The GST-SHP-1 fusion protein constructs were created by the polymerase
chain reaction amplification of the nucleotides encoding amino acids
214-595 from either wild-type SHP-1 or catalytically inactive SHP-1
(C453S). The polymerase chain reaction product was subcloned in frame
into the vector pGEX-2TK (Amersham Pharmacia Biotech). The fidelity of
all polymerase chain reaction products was confirmed by automated DNA sequencing.
Polyclonal rabbit anti-Lck antibodies and rabbit anti-phosphotyrosine
antibodies have been described previously (23, 24). The mouse
monoclonal anti-Myc antibody 9E10 (25) and the mouse monoclonal
anti-phosphotyrosine antibody 4G10 (26) were a kind gift from Jill
Meisenhelder and Dr. Tony Hunter (The Salk Institute, La Jolla, CA).
The mouse monoclonal anti-CD4 antibody OKT4 (27) was a kind gift from
Dr. Bob Hyman (The Salk Institute).
Cell Culture and Transfections--
293, a human embryonic
kidney cell line (28), was grown in Dulbecco's modified Eagle's
medium (Cellgro, Mediatech) supplemented with 10% calf serum
(Hyclone). 293 cells were seeded onto 5-cm gelatin-coated Petri dishes
and were transfected using a calcium phosphate transfection system
(Life Technologies, Inc.) as per the manufacturer's protocol.
Cell Lysis and Immunoprecipitations--
Cells were washed once
with Tris-buffered saline and lysed in ice-cold radioimmune
precipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1%
sodium deoxycholate, 0.1% SDS, 100 kallikrein-inactivating units/ml
aprotinin, and 1 mM Na3VO4).
Lysates were clarified by centrifugation at 35,000 × g
for 30 min. Lysates were subjected to immunoprecipitation using either
a rabbit anti-Lck antibody or a monoclonal anti-CD4 antibody (OKT4).
Immune complexes were collected on Pansorbin cells (Calbiochem), washed
three times in radioimmune precipitation buffer, and used for
subsequent analysis.
Immunoblotting--
Immunoprecipitated Lck and total cellular
lysates were resolved by SDS-polyacrylamide gel electrophoresis and
transferred to an Immobilon-P membrane (Millipore). Western blotting
was carried out with rabbit anti-Lck antibodies, anti-phosphotyrosine
antibodies, or anti-Myc antibodies as described previously (23, 24).
Bound antibodies were detected either by enhanced chemiluminescence or
by 125I-protein A (ICN) and a PhosphorImager (Molecular
Dynamics) as indicated.
In Vivo Labeling and Two-dimensional Tryptic Peptide
Mapping--
293 cells were transiently transfected with the DNA
constructs as indicated. At 24-48 h post-transfection, cells were
washed twice in Tris-buffered saline. Cells were biosynthetically
labeled in 2 ml of phosphate-free Dulbecco's modified Eagle's
medium supplemented with 10% dialyzed fetal calf serum and
32Pi
(H332PO4, ICN, 0.5 mCi/ml) for
2 h at 37 °C. Cells were washed with Tris-buffered saline and
lysed in radioimmune precipitation buffer, and 32P-labeled
Lck was isolated by immunoprecipitation as described. 32P-labeled Lck was resolved by SDS-polyacrylamide gel
electrophoresis, transferred to a nitrocellulose membrane, and excised
from the membrane following identification by autoradiography. The
excised membrane containing the labeled Lck was then digested with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin as
described previously (29). Two-dimensional tryptic peptide mapping was
carried out on cellulose thin layer chromatography plates (EM Science)
by electrophoresis at pH 8.9 in the first dimension followed by
ascending chromatography in phosphochromatography buffer in the second
dimension as described previously (30). The labeled peptides were
visualized using a PhosphorImager.
In Vitro Dephosphorylation Reactions--
293 cells were
transiently co-transfected with cDNAs encoding WT Lck or F505 Lck
and CD4. At 24 h post-transfection, the CD4-Lck complex was
immunoprecipitated as described above with OKT4. The CD4-Lck
immunoprecipitates were washed once in TN (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and once in phosphatase assay
buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM dithiothreitol, and 5 mM EDTA).
Immunoprecipitated CD4-Lck was resuspended in phosphatase assay buffer
and incubated with 5 µg of soluble, purified GST-SHP-1 fusion protein
in a final reaction volume of 20 µl for 1 h at 4 °C unless
otherwise indicated. To terminate the reaction, the immunoprecipitates
were washed three times with radioimmune precipitation buffer and
resuspended in SDS-polyacrylamide gel electrophoresis sample buffer.
For the two-dimensional tryptic peptide analysis of the in
vitro dephosphorylated products, the above procedure was employed except that the transfected 293 cells were labeled biosynthetically with 32Pi for 2 h prior to immunoprecipitation.
Peptide dephosphorylation reactions were carried out using
purified GST-SHP-1 and a Lck-derived phosphopeptide consisting of the
sequence CIEDNEpYTAREGA in which pY represents phosphorylated Tyr-394.
The Lck-derived phosphopeptide was synthesized by Jill Meisenhelder
using an ABI 432A peptide synthesizer (Applied Biosystems Inc.) and
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) synthesis methods. The peptide was resuspended in SHP-1 assay buffer (50 mM NaOAc-HOAc, pH 5.0, 2 mM EDTA, and 2 mM dithiothreitol). Reactions were carried out in a volume
of 20 µl in 96-well microtiter plates, and phosphate release was
measured by malachite green assay (31). The amount of phosphate
released during the reaction was calculated from a standard curve
generated using sodium phosphate buffer, pH 7.0.
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RESULTS |
Tyrosine Phosphorylation of Lck Is Reduced by Co-expression of
SHP-1--
To examine whether the phosphorylation of Lck on tyrosine
was affected by SHP-1, we co-expressed Lck and SHP-1 by transient transfection in 293 cells. We immunoprecipitated Lck from these cells
and analyzed the level of Lck tyrosine phosphorylation by Western
blotting using anti-phosphotyrosine antibodies (Fig.
1). In all of our experiments, we used a
mutant of SHP-1 lacking both SH2 domains to augment SHP-1 activity
(
SH2). Deletion of the SH2 domains has been shown to increase
catalytic activity by relieving the steric hindrance of the catalytic
site by the SH2 domain (2, 3). We confirmed the increased catalytic
activity of 
SH2 SHP-1 and the absence of the activity of

SH2 D419A SHP-1 in vitro (data not shown). Lck is
tyrosine-phosphorylated in 293 cells when expressed alone (Fig.
1A, lane 1). Multiple forms of Lck are observed
when the protein is overexpressed in 293 cells. This heterogeneity is
attributable to differing degrees of serine phosphorylation,
which is catalyzed in part by activated mitogen-activated protein
kinases (32). When wild-type SHP-1 was co-expressed with Lck, the
tyrosine phosphorylation of Lck was reduced ~1.8-fold as quantified
using a PhosphorImager (Fig. 1A, lane 2).
Co-expression of catalytically inactive SHP-1 (D419A) did not reduce
the tyrosine phosphorylation of Lck (Fig. 1A, lane
3), indicating that the catalytic activity of SHP-1 was required
for the reduction of Lck phosphotyrosine levels. The level of Lck
protein was slightly higher in the immunoprecipitates from cells
co-expressing Lck and SHP-1 compared with immunoprecipitates from cells
expressing Lck alone (Fig. 1B, lanes 2 and
3 compared with lane 1). Therefore, the
quantitation of the Lck phosphotyrosine levels was normalized for the
Lck protein level present in the immunoprecipitates. The expression of
wild-type and catalytically inactive SHP-1 was shown to be equivalent
by Western blotting of the cell lysates using anti-Myc antibodies
(Fig. 1C, lanes 2 and 3).

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Fig. 1.
Effect of SHP-1 co-expression on wild-type
Lck tyrosine phosphorylation. 293 cells were transiently
co-transfected with wild-type Lck and either Myc-tagged, catalytically
activated SHP-1 ( SH2) or catalytically inactive SHP-1 ( SH2
D419A). Lck was immunoprecipitated using anti-Lck antibodies and
divided into equal fractions. A, Lck immunoprecipitates were
analyzed by Western blotting using anti-phosphotyrosine antibodies and
125I-protein A. B, Lck immunoprecipitates were
analyzed by Western blotting using anti-Lck antibodies and
125I-protein A. C, total cell lysates were
analyzed by Western blotting using anti-Myc antibodies (9E10) and
125I-protein A. Lane 1, wild-type Lck alone;
lane 2, wild-type Lck and  SH2 SHP-1; lane
3, wild-type Lck and  SH2 D419A SHP-1. Data shown are
representative of five independent experiments
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SHP-1-induced Dephosphorylation Occurs Predominately at
Tyr-394--
When Lck is overexpressed in 293 cells, it is
tyrosine-phosphorylated at both Tyr-394 and Tyr-505 (33). Therefore,
the reduction of total Lck tyrosine phosphorylation induced by SHP-1
could be a result of the reduced phosphorylation of Tyr-394, Tyr-505,
or both. To distinguish between these possibilities, we examined the
effect of SHP-1 on the phosphorylation of two mutants of Lck, F505 Lck
and F394 Lck. These mutants are tyrosine-phosphorylated only at Tyr-394
or Tyr-505, respectively. We co-expressed these mutants of Lck in 293 cells with either catalytically active 
SH2 SHP-1 or catalytically
inactive 
SH2 D419A SHP-1 by transient transfection and analyzed
Lck tyrosine phosphorylation by Western blotting using
anti-phosphotyrosine antibodies (Fig. 2).
As with wild-type Lck, both F505 Lck and F394 Lck were
tyrosine-phosphorylated in 293 cells (Fig. 2A, lanes
1 and 4). The tyrosine phosphorylation of F505 Lck was
reduced by co-expression of catalytically active SHP-1 (Fig.
2A, lane 2). Catalytically inactive SHP-1 did not affect the tyrosine phosphorylation of F505 Lck (Fig. 2A,
lane 3). In contrast, the tyrosine phosphorylation of F394
Lck was not affected by co-expression with catalytically active SHP-1 (Fig. 2A, lane 5), suggesting that expression of
SHP-1 does not alter the phosphorylation of Tyr-505.

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Fig. 2.
Effect of SHP-1 co-expression on Lck
phosphorylation site mutants. 293 cells were transiently
transfected with either F505 Lck or F394 Lck alone or these kinases
together with Myc-tagged  SH2 SHP-1 or  SH2 D419A SHP-1. Lck
was immunoprecipitated using anti-Lck antibodies and divided into equal
fractions. A, Lck immunoprecipitates were analyzed by
Western blotting using the anti-phosphotyrosine antibody 4G10 and
enhanced chemiluminescence. B, Lck immunoprecipitates were
analyzed by Western blotting using anti-Lck antibodies and enhanced
chemiluminescence. Lane 1, F505 Lck alone; lane
2, F505 Lck and  SH2 SHP-1; lane 3, F505 Lck and
 SH2 D419A SHP-1; lane 4, F394 Lck alone; lane
5, F394 Lck and  SH2 SHP-1; lane 6, F394 Lck and
 SH2 D419A SHP-1. Data shown are representative of seven
independent experiments.
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To confirm that the dephosphorylation of wild-type Lck by SHP-1
occurred at Tyr-394, we analyzed Lck phosphorylation by two-dimensional tryptic peptide analysis. To do this, we co-expressed wild-type Lck
with either catalytically active SHP-1 or catalytically inactive SHP-1
and labeled the co-transfected cells biosynthetically with 32Pi. Lck was isolated by immunoprecipitation,
and its phosphorylation was analyzed by two-dimensional tryptic peptide
analysis (Fig. 3). In cells expressing
Lck alone, both Tyr-505 and Tyr-394 were phosphorylated with 1.4-fold
more phosphate present at Tyr-505 than at Tyr-394 (Fig.
3A). The expression of catalytically active SHP-1 reduced
the amount of phosphate at Tyr-394, increasing the ratio of phosphate
in the Tyr-505-containing peptide to that of the Tyr-394-containing
peptide to 5.6-fold (Fig. 3B). Co-expression of
catalytically inactive SHP-1 did not change the relative
phosphorylation state of Tyr-394 or Tyr-505 (Fig. 3C). These
results demonstrate that SHP-1 causes the dephosphorylation of Lck at
Tyr-394.

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Fig. 3.
Analysis of Lck phosphorylation in 293 cells
co-expressing SHP-1. Lck was immunoprecipitated from
32P-labeled 293 cells co-expressing Lck and SHP-1 and
analyzed by two-dimensional tryptic peptide mapping on thin layer
cellulose plates. A, Lck from cells expressing wild-type Lck
alone; B, Lck from cells co-expressing wild-type Lck and
 SH2 SHP-1; C, Lck from cells co-expressing Lck and
 SH2 D419A SHP-1. Origins are marked with arrowheads.
Arrows indicate the directions of electrophoresis and
chromatography. Y505, peptide containing phosphorylated
Tyr-505; Y394, peptide containing phosphorylated Tyr-394.
Values in the lower left-hand corner of each
panel are the ratio of the PhosphorImager units present in
Tyr-505 to that of Tyr-394 (set as 1). Data shown are representative of
three independent experiments.
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The Lck SH2 Domain Protects Tyr-505 from
Dephosphorylation--
The crystal structure of the inactive forms of
Hck and c-Src, two other Src family kinases, shows that the SH2 domain
binds intramolecularly to a conserved phosphorylated tyrosine residue in the extreme C terminus of the molecule, keeping the kinase in an
inactive conformation (34, 35). Because Lck is activated either by
mutation of this conserved tyrosine (Tyr-505) or by mutation of the SH2
domain (20, 36), it is reasonable to infer that the SH2 domain of Lck
interacts intramolecularly with the C-terminal tyrosine residue in the
same manner as Hck or c-Src. It is a possibility that SHP-1 can
dephosphorylate either Tyr-394 or Tyr-505, but the Lck SH2 domain
interferes with the dephosphorylation of Tyr-505 by SHP-1, thus giving
rise to the apparent preference of SHP-1 to dephosphorylate Tyr-394.
Alternatively, the apparent preferential dephosphorylation of Tyr-394
could be specified by the amino acid sequences surrounding Tyr-394 and
Tyr-505. To examine whether the Lck SH2 domain protected against
dephosphorylation of Tyr-505, we co-expressed a mutant of Lck that
lacks the SH2 domain (
SH2 Lck) with catalytically activated SHP-1 by
transient transfection and analyzed Lck phosphorylation by
two-dimensional tryptic peptide analysis (Fig.
4). In cells expressing
SH2 Lck alone,
both Tyr-505 and Tyr-394 were phosphorylated, although there was
0.57-fold less phosphate present at Tyr-505 than at Tyr-394 (Fig.
4A), the opposite of what we observed with wild-type Lck.
The change in the ratio of phosphate between Tyr-394 and Tyr-505 in
SH2 Lck could be attributable to increased dephosphorylation of
Tyr-505 by endogenous tyrosine phosphatases because this site is no
longer protected by the SH2 domain, or it could be attributable to
increased Lck phosphorylation at Tyr-394. Co-expression of catalytically active SHP-1 with
SH2 Lck reduced the amount of phosphate at Tyr-505, further decreasing the ratio of phosphate in the Tyr-505-containing peptide to that of the
Tyr-394-containing peptide to 0.26-fold (Fig. 4B).
Co-expression of catalytically inactive SHP-1 with
SH2 Lck did not
change the relative phosphorylation state of Tyr-394 or Tyr-505 (Fig.
4C). These results suggest that the specificity of SHP-1 for
Tyr-394 is attributable in part to the Lck SH2 domain sterically
hindering the access of SHP-1 to Tyr-505.

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Fig. 4.
Effect of SHP-1 co-expression on the
phosphorylation of SH2 Lck. Lck was
immunoprecipitated from 32P-labeled 293 cells co-expressing
SH2 Lck and SHP-1 and analyzed by two-dimensional tryptic peptide
mapping on thin layer cellulose plates. A, Lck from cells
expressing SH2 Lck alone; B, Lck from cells transiently
co-expressing SH2 Lck and  SH2 SHP-1; C, Lck from
cells co-expressing SH2 Lck and  SH2 D419A SHP-1. Origins are
marked with arrowheads. Arrows indicate the directions of
electrophoresis and chromatography. Y505, peptide containing
phosphorylated Tyr-505; Y394, peptide containing
phosphorylated Tyr-394. Values in the lower left-hand corner
of each panel are the ratio of the PhosphorImager units
present in Tyr-505 to that of Tyr-394 (set as 1). Data shown are
representative of three independent experiments.
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SHP-1 Can Dephosphorylate Lck in Vitro--
The results of the
co-expression experiments demonstrated that the expression of SHP-1
reduced the phosphorylation of Lck specifically at Tyr-394 in
vivo. However, these experiments did not distinguish between
direct dephosphorylation of Lck by SHP-1 or indirect induction of Lck
dephosphorylation. To determine whether SHP-1 could dephosphorylate Lck
directly, we expressed and purified recombinant GST fusion proteins
consisting of the catalytic domain and the C terminus of either
wild-type SHP-1 or a catalytically inactive SHP-1 (C453S). These
purified GST-SHP-1 fusion proteins did not contain the tandem SH2
domains and are therefore fully active. To obtain a suitable substrate,
we co-expressed CD4 and either F505 Lck or wild-type Lck in 293 cells
and immunoprecipitated the CD4-Lck complex with anti-CD4 antibodies. We
then incubated the immunoprecipitates with the recombinant GST-SHP-1
fusion proteins and analyzed Lck phosphorylation by Western blotting
with anti-phosphotyrosine antibodies (Fig.
5). We isolated the CD4-Lck complex by
immunoprecipitation of CD4 because our Lck antiserum contains anti-GST
reactivity, and we wanted to avoid binding the GST-SHP-1 fusion
protein to the immunoprecipitate during the in vitro
dephosphorylation reaction. In addition, immunoprecipitation of the
CD4-Lck complex by anti-CD4 antibodies eliminates the potential of
interference because of the antibody directly bound to Lck. Wild-type
GST-SHP-1 dephosphorylated both F505 Lck (Fig. 5A, compare
lane 2 with lane 1) and wild-type Lck (Fig.
5A, compare lane 5 with lane 4). The
extent of dephosphorylation was considerably greater with F505 Lck.
This suggested that SHP-1 directly dephosphorylated Lck at Tyr-394
in vitro. In contrast, the catalytically inactive GST-SHP-1
did not dephosphorylate either F505 Lck or wild-type Lck (Fig.
5A, lanes 3 and 6).

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Fig. 5.
In vitro dephosphorylation of Lck
by GST-SHP-1. 293 cells were transiently co-transfected with CD4
and either F505 Lck or wild-type Lck. The CD4-Lck complex was
immunoprecipitated and incubated in vitro with purified
GST-SHP-1 fusion proteins. A, CD4-Lck immunoprecipitates
were analyzed by Western blotting using anti-phosphotyrosine antibodies
and enhanced chemiluminescence. B, CD4-Lck
immunoprecipitates were analyzed by Western blotting using anti-Lck
antibodies and enhanced chemiluminescence. Lane 1, F505 Lck
alone; lane 2, F505 Lck incubated with wild-type GST-SHP-1;
lane 3, F505 Lck incubated with catalytically inactive
GST-SHP-1 (C453S); lane 4, WT Lck alone; lane 5,
WT Lck incubated with GST-SHP-1; lane 6, WT Lck incubated
with GST-SHP-1 (C453S). Data shown are representative of two
independent experiments. C, CD4-F505 Lck immunoprecipitates
were incubated with decreasing amounts of GST-SHP-1. Following the
incubation, immunoprecipitates were analyzed by Western blotting using
anti-phosphotyrosine antibodies and 125I-protein A. Lane 1, no GST-SHP-1; lane 2, 3.5 µM GST-SHP-1; lane 3, 700 nM
GST-SHP-1; lane 4, 350 nM GST-SHP-1; lane
5, 70 nM GST-SHP-1; lane 6, 35 nM GST-SHP-1; lane 7, 7 nM
GST-SHP-1. Data shown are representative of three independent
experiments.
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We used SHP-1 at a concentration of 3.5 µM in the above
experiment. To determine how much SHP-1 was required for efficient dephosphorylation of Lck in vitro, we incubated
CD4-F505 Lck immunoprecipitates with varying amounts of purified
GST-SHP-1 and analyzed Lck phosphorylation by Western blotting with
anti-phosphotyrosine antibodies. Greater than 75% dephosphorylation
could be achieved using GST-SHP-1 at a concentration of 70 nM (Fig. 5C, lane 5), and 50%
dephosphorylation was observed using a GST-SHP-1 at a concentration of
7 nM (Fig. 5C, lane 7). We also
determined the kinetics of dephosphorylation of a Lck-derived peptide
containing phosphorylated Tyr-394 (data not shown). After establishing
reaction conditions that allowed linear reaction rates as a function of
both incubation time and phosphatase concentration, we measured
dephosphorylation of the Lck peptide as a function of peptide
concentration. The Lck peptide exhibited an average
Km of 118 ± 40 µM in four
independent experiments.
The Catalytic Domain of SHP-1 Exhibits Site-specific
Dephosphorylation of Lck in Vitro--
To examine the apparent
specificity for Tyr-394 in vitro in another way, we
co-expressed CD4 and wild-type Lck in 293 cells, labeled the cells
biosynthetically with 32Pi, and isolated the
CD4-Lck complex by immunoprecipitation. We then incubated the
immunoprecipitates with active and inactive GST-SHP-1 catalytic domain
fusion proteins in vitro and analyzed dephosphorylation of
Lck by two-dimensional tryptic peptide analysis (Fig.
6). The amount of phosphate at Tyr-505
and Tyr-394 in the substrate alone was approximately equal (Fig.
6A). Wild-type GST-SHP-1 dephosphorylated Tyr-394
preferentially, yielding a product that contained 10-fold more
phosphate at Tyr-505 than at Tyr-394 (Fig. 6B). As expected,
catalytically inactive GST-SHP-1 or GST did not affect either Tyr-394
or Tyr-505 phosphorylation (Fig. 6, C and D). In
this experiment we chose to analyze Lck phosphorylation at the reaction
end point instead of analyzing initial rates of dephosphorylation.
Therefore, it is important to note that the difference in
dephosphorylation of Tyr-394 versus Tyr-505 could actually
be greater than the difference that we observed.

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Fig. 6.
Site-specific dephosphorylation of Lck by
GST-SHP-1. 293 cells were transiently transfected with CD4 and
wild-type Lck. After biosynthetically labeling the cells with
32Pi, the CD4-Lck complex was
immunoprecipitated and incubated in vitro with GST-SHP-1
fusion proteins. Lck phosphorylation was analyzed by two-dimensional
tryptic peptide mapping on thin layer cellulose plates. A,
wild-type Lck alone; B, wild-type Lck incubated with
wild-type GST-SHP-1; C, wild-type Lck incubated with GST;
D, wild-type Lck incubated with catalytically inactive
GST-SHP-1 (C453S). Origins are marked with arrowheads.
Arrows indicate the directions of electrophoresis and
chromatography. Y505, peptide containing phosphorylated
Tyr-505; Y394, peptide containing phosphorylated Tyr-394.
Values in the lower left-hand corner of each
panel are the ratio of the PhosphorImager units present in
Tyr-505 to that of Tyr-394 (set as 1). Data shown are representative of
two independent experiments.
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DISCUSSION |
We examined here whether the Src family kinase Lck was a substrate
of the SHP-1 protein-tyrosine phosphatase. Our results demonstrate that
Lck is dephosphorylated directly and specifically by SHP-1 at Tyr-394,
a conserved tyrosine in the activation loop (37, 38). Because
phosphorylation of Tyr-394 activates Lck (21, 39), SHP-1 is therefore
an inhibitor of Lck activity.
In contrast to the phosphorylation of Tyr-394, phosphorylation of
Tyr-505 in Lck by the ubiquitous tyrosine protein kinase Csk (40, 41)
induces formation of a biologically inactive conformation by allowing
intramolecular binding of the SH2 domain to the phosphorylated C
terminus (34, 42, 43). We did not observe any dephosphorylation of
Tyr-505 in wild-type Lck or in F394 Lck by SHP-1. Both of these
proteins contain an intact SH2 domain capable of intramolecular binding
to phosphorylated Tyr-505, and this could protect Tyr-505 from
dephosphorylation. Our results in Fig. 4 demonstrate that deletion of
the SH2 domain renders Tyr-505 susceptible to dephosphorylation by
SHP-1. Some of the specificity of SHP-1 for phosphorylated Tyr-394
therefore comes from the protection of phosphorylated Tyr-505 by the
Lck SH2 domain. The tyrosine phosphatase CD45 has been shown to
dephosphorylate either Tyr-394 or Tyr-505 in Lck (44, 45). Our results
demonstrate that unlike CD45, SHP-1 is unable to displace the
intramolecular interaction between the Lck SH2 domain and
phosphorylated Tyr-505. This would suggest that in a physiological
context, SHP-1 would only act on Lck phosphorylated at Tyr-394.
However, it still is a possibility that SHP-1 could act on Lck
phosphorylated at Tyr-505, although it presumably would require the
prior displacement of the Lck SH2 domain by another molecule.
Because we demonstrated dephosphorylation of Lck in vivo at
Tyr-394 when SHP-1 was overexpressed or when Lck was incubated with a
high concentration of SHP-1 in vitro (3.5 µM),
it could be argued that our data do not provide evidence that
Tyr-394 is a high affinity substrate of SHP-1. However, we were able to
demonstrate significant dephosphorylation of Lck when it was incubated
with as low a concentration as 7 nM SHP-1. In
addition, the Km value of 118 ± 40 µM that we obtained for the Tyr-394-containing phosphopeptide is not dissimilar to the Km values
(72 and 80 µM) of two peptides corresponding to two sites
of SHP-1 dephosphorylation in the physiological substrate
SIRP-
that have been confirmed by structural studies (46).
This is consistent with the hypothesis that the Tyr-394 of Lck is a
physiological substrate of SHP-1.
Loss of SHP-1 is known to cause hyperactivity of T cells and lower
T-cell selection thresholds (15, 47-51). This suggests that the
dephosphorylation of tyrosine-phosphorylated substrates by SHP-1 plays
a role in the regulation of T cell activation and selection in
wild-type mice. Plas et al. (14) have provided evidence that
ZAP-70 may be a target of SHP-1. Because ZAP-70 phosphorylation and
activity play an important role in T cell activation and maturation
(52, 53), a loss of ZAP-70 dephosphorylation attributable to SHP-1
deficiency could well contribute to the hyperactivation of T cells in
motheaten mice. Our data demonstrate that Lck is also a direct target
of SHP-1 and is inhibited by SHP-1. Because Lck activates ZAP-70 by
phosphorylation at Tyr-493 in ZAP-70 (16), our results could suggest
that the effects of SHP-1 on ZAP-70 phosphorylation and activity are
indirect. However, it is equally possible that SHP-1 could act on both
Lck and ZAP-70 to inhibit signaling. Nevertheless, our results suggest
that the failure of SHP-1 to dephosphorylate Lck at Tyr-394 may
contribute to the phenotype of motheaten mice. Indeed, our data are
consistent with the prolonged activity of Lck in observed in motheaten
thymocytes (15) and suggest strongly that SHP-1 directly inhibits Lck
activity in vivo.