From the Howard Hughes Medical Institute, Department of Pathology and Molecular Microbiology, Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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CD22 phosphorylation is an early event of B cell
antigen receptor engagement and results in the recruitment of the
negative regulatory tyrosine phosphatase, SHP-1. Peptides representing the potential phosphorylation sites within the cytoplasmic domain of
CD22 have been used to stimulate SHP-1 catalytic activity and to
inhibit the binding of SHP-1 to CD22 (Doody, G., Justement, L.,
Delibrias, C., Matthews, R., Lin, J., Thomas, M., and Fearon, D. (1995) Science 269, 242-244). However, the sites of
phosphorylation within the cytoplasmic domain of CD22 and the
importance of each for the recruitment and activation of SHP-1 remain
unknown. Here we demonstrate that there are multiple sites within the
cytoplasmic domain of CD22 that interact with the Src homology 2 domains of SHP-1. Nevertheless, a minimum of two tyrosines in CD22 is
required for the association with SHP-1. Furthermore, both Src homology 2 domains of SHP-1 are necessary for efficient binding to CD22.
Engagement of the B cell antigen receptor
(BCR)1 results in an increase
in tyrosine phosphorylation. CD22, a B cell-specific transmembrane
lectin, is one of the initial substrates phosphorylated upon BCR
engagement. The cytoplasmic domain of CD22 contains six tyrosines and
has the potential of being a docking site for various Src homology 2 (SH2) domain-containing proteins. Indeed, it has been shown that CD22
binds to multiple enzymes including Syk, p53/56lyn (Lyn),
phosphatidylinositol 3-kinase, phospholipase C The importance of CD22 in BCR signaling is demonstrated by the
development of mice ablated in CD22 gene expression (5-8). These mice
exhibit a spontaneous decrease in IgM expression and elevated calcium
mobilization in response to BCR cross-linking. This suggests that CD22
is important in the negative regulation of BCR signaling.
Interestingly, an elevated calcium response is also observed in mice
deficient in either the Src family kinase, Lyn, or the tyrosine
phosphatase, SHP-1 (9, 10), suggesting that these enzymes also
contribute to the negative regulation of the BCR. Indeed, by examining
mice that are heterozygous for a genetic defect for two or more of
these genes, it has been demonstrated that CD22, Lyn, and SHP-1
interact to control BCR negative regulation (10). BCR activation
results in Lyn phosphorylation of the cytoplasmic domain of CD22, thus
allowing for the recruitment of SHP-1 (10). Various studies have shown
that SHP-1 is essential for B cell development and BCR negative
regulation (9-13). Furthermore, SHP-1 functions to regulate Syk kinase
activity.2 Therefore, the
interaction of CD22 and SHP-1 is central to BCR regulation.
SHP-1 is a cytosolic enzyme containing two SH2 domains at the amino
terminus. In its native form, SHP-1 has low basal catalytic activity.
However, truncation of the SH2 domains or binding to the SH2 domains
substantially increases its catalytic activity, suggesting that the SH2
domains regulate the catalytic activity by an allosteric mechanism (3,
14-16). Further supportive evidence for this model comes from the
recently solved crystal structure for a highly related phosphatase,
SHP-2 (17). The resolution of the crystal structure shows that the
first SH2 domain of SHP-2 interacts with the active site of the
phosphatase domain to preclude substrate binding. Binding to the first
SH2 domain may result in a conformational change allowing access to the
catalytic site. Thus, it is likely that the SH2 domains of SHP-1 have
two functions, to recruit the enzyme to appropriate locations and to
regulate enzymatic activity.
The tyrosine-phosphorylated sequence recognized by the SH2 domains of
SHP-1 has been termed an immunoreceptor
tyrosine-based inhibitory motif
(ITIM) with the consensus sequence
(V/L/I)X(p)YXXL. The cytoplasmic domain of CD22
has five potential ITIMs (Table I). Tyrosine-phosphorylated peptides
representing three of these sequences have been shown to increase SHP-1
activity and inhibit binding to CD22 (3). To determine which of these
potential ITIMs are necessary for SHP-1 binding, we co-expressed SHP-1
with a chimeric protein containing mutations of the ITIM sequences. Here we demonstrate that the ITIMs have redundant functions, and furthermore, two intact ITIMs are required for optimal binding to
SHP-1. In addition, both SH2 domains of SHP-1 are required for the
interaction with the cytoplasmic domain of CD22.
Cell Lines and Antibodies--
HeLa cells were obtained from the
ATCC (Rockville, MD) and maintained in Dulbecco's modified Eagle's
medium supplemented with 10% bovine calf serum and
L-glutamine. Transfection of HeLa cells was carried out as
described previously (18). An antiphosphotyrosine monoclonal antibody
4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY),
and rabbit antivesicular stomatitis virus serum was purchased from
Access Biomedical (San Diego, CA). Rabbit anti-SHP-1 antiserum was made
as described previously (19). Horseradish peroxidase-conjugated
protein A was purchased from Boehringer Mannheim, and
peroxidase-conjugated goat anti-mouse IgG was purchased from Cappel
Organon Teknika Corp. (West Chester, PA).
cDNA Reagents--
Mouse SHP-1(C453S) cDNA has been
described previously (19). Mutations were introduced into the SH2
domains of SHP-1 by polymerase chain reaction (PCR) site overlap
extension as described.3 To
construct SHP-1( Immunoprecipitation and Immunoblot Analysis--
Following
transfection/infection and overnight expression, HeLa cells were washed
with phosphate-buffered saline (PBS) and lysed in lysis buffer (1%
Nonidet P-40, 50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 5 mM EDTA, 10 mM sodium fluoride, 10 mM sodium molybdate, 1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM
phenylmethylsulfonyl fluoride, 50 µM phenylarsine oxide, and 5 mM iodoacetamide) on ice for 10 min. The lysates were
precleared with a 10% solution of Pansorbin cells (Calbiochem) and
centrifuged at 10,000 rpm for 10 min at 4 °C. An aliquot was removed
as the crude lysate sample; the remaining lysate was immunoprecipitated with the appropriate antibodies. Immunoprecipitates and crude lysates
were resolved on a 7.5% SDS-polyacrylamide gel under reducing conditions. The gel was transferred onto nitrocellulose and blocked in
PBS containing 0.05% Tween 20 and 3% bovine serum albumin for 1 h at room temperature or overnight at 4 °C. Antibodies (anti-VSVG, 1:2500; anti-SHP-1, 1:1000; 4G10, 1:3000) were diluted in PBS containing 0.05% Tween 20 and incubated with the membranes for 1 h at room temperature. Following the primary antibody, membranes were
washed twice in PBS containing 1% Nonidet P-40 and once with PBS
containing 0.05% Tween 20 for 5 min, developed with appropriate secondary antibodies in PBS containing 0.05% Tween 20 for 30 min, and
washed as described above. Proteins were visualized using the enhanced
chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom).
Phosphorylated CD22 Associates with SHP-1--
To define the
site(s) of interaction between CD22 and SHP-1, a chimeric protein of
the intact cytoplasmic domain of CD22, and the extracellular and
transmembrane domains of the VSVG protein (G/22) and SHP-1 were
expressed in HeLa cells. The G/22 chimeric protein is expressed on the
membrane surface as determined by flow cytometric analysis (data not
shown). Co-expression of G/22 with catalytically inactive SHP-1
(SHP-1(C453S)) results in tyrosine phosphorylation of G/22 and
association with SHP-1 (Fig. 1). In contrast, under these conditions co-expression of G/22 with wild type
SHP-1 results in no detectable tyrosine phosphorylation of G/22 and no
association of the two proteins. These data suggest that G/22 is a
substrate of SHP-1. In addition, expression of G/22 alone in HeLa cells
results in less tyrosine phosphorylation of G/22 compared with that
seen when co-expressed with SHP-1(C453S). This indicates that binding
of SHP-1 protects G/22 from dephosphorylation by endogenous
phosphatases. Of note is the presence of a doublet detected by VSVG
immunoblotting; the slower migrating band corresponds to
tyrosine-phosphorylated G/22. To define the sites of interaction between SHP-1 and G/22, SHP-1(C453S) was used in our subsequent studies.
Two Tyrosines Are Required for Phosphorylation of CD22 and
Association with SHP-1--
The cytoplasmic domain of CD22 contains
six tyrosine residues; tyrosine 795 does not fit the consensus ITIM
sequence for SHP-1 and therefore was left unchanged. The remaining five
tyrosines were all mutated to phenylalanine (Table
I). To examine whether any single ITIM
tyrosine was sufficient for SHP-1 binding, mutations were created such
that only a single ITIM was left intact. Co-expression of single ITIM
G/22 with SHP-1(C453S) revealed that only tyrosines 765 and 825 were
consistently phosphorylated (data not shown). Neither pervanadate
treatment nor co-expression with Lyn enhanced the tyrosine
phosphorylation levels of the other single ITIM G/22 proteins.
Co-expression of the single ITIM G/22 with SHP-1(C453S) resulted in
poor and inconsistent association of the two proteins as measured by
co-immunoprecipitation (data not shown). These findings suggest that
more than one tyrosine is required for efficient tyrosine
phosphorylation and binding to SHP-1.
Previously, it was demonstrated that phosphotyrosine peptides
representing the ITIM sequences of tyrosines 765, 825, or 845 but not
799 or 810 were capable of increasing SHP-1 activity and inhibiting
binding of SHP-1 to CD22 (3). When all ITIM tyrosines were mutated to
phenylalanines, the G/22 protein was not tyrosine-phosphorylated or
associated with SHP-1 (Fig.
2A). This indicates that
phosphorylated tyrosines are required for the interaction between CD22
and SHP-1. To determine which phosphorylated tyrosines in CD22 were
required for efficient binding to SHP-1, chimeric proteins were
expressed containing two or three intact ITIMs (Fig. 2). When
co-expressed in HeLa cells with SHP-1(C453S), the double ITIM G/22
chimeric protein containing tyrosines 765 and 825 was sufficient to
mediate SHP-1 binding and tyrosine phosphorylation at a level
comparable with that of wild type G/22, whereas that containing
tyrosines 825 and 845 was slightly reduced compared with wild type G/22 (Fig. 2A). Interestingly, the double ITIM G/22 protein that
comprised tyrosines 799 and 810 was neither tyrosine-phosphorylated nor associated with SHP-1 (Fig. 2A). The double ITIM G/22
protein containing tyrosines 765 and 845 was less
tyrosine-phosphorylated and associated with less SHP-1(C453S) than wild
type G/22. Based on these findings, one can speculate that tyrosine 825 is the primary SHP-1 binding site whereas tyrosines 765 and 845 are
secondary SHP-1 binding sites, of which one is potentially the target
for the phosphatase domain of SHP-1.
The triple ITIM G/22 protein containing tyrosines 765, 825, and 845 confirmed the results from the double ITIM G/22 proteins. Compared with
wild type G/22, this triple ITIM G/22 protein was tyrosine-phosphorylated and bound SHP-1 at an equivalent amount (Fig.
2B). However, the triple ITIM G/22 protein containing
tyrosines 765, 799, and 825 was equally tyrosine-phosphorylated as wild type G/22 but associated with less SHP-1. One possible explanation is
that in the absence of tyrosine 845, tyrosine 799 destabilizes the
complex and thus does not allow for efficient binding of SHP-1.
Both SH2 Domains of SHP-1 Associate with CD22--
To determine
whether both SH2 domains of SHP-1 were required for binding to CD22,
mutations that eliminate phosphotyrosine binding (21) were introduced
either into the individual SH2 domains or into both SH2 domains. The
mutations were constructed using the cDNA encoding catalytically
inactive SHP-1(C453S). When co-expressed with wild type G/22 protein or
the double ITIM G/22 containing tyrosines 765 and 825, mutations that
disrupted phosphotyrosine binding to both SH2 domains completely
prevented the association of SHP-1 with G/22 (Fig.
3). Results were the same for all
combinations of double and triple ITIM G/22 proteins containing
tyrosines 765, 825, and 845. SHP-1 with a single functional SH2 domain
was capable of binding the CD22 cytoplasmic domain; however, the
binding was dramatically decreased. Thus, both SH2 domains of SHP-1 are
required for efficient binding to the cytoplasmic domain of CD22. The
absence of tyrosine phosphorylation and association with SHP-1 to the G/22 protein in which all five tyrosines were mutated to phenylalanines confirmed that phosphorylated tyrosines interact with the SH2 domains
of SHP-1.
CD22 Is a Substrate of SHP-1--
It is widely recognized that
SHP-1(C453S) is a substrate-trapping mutant, whereas SHP-1( CD22 is required for the negative regulation of the BCR. This
effect is mediated by the recruitment and activation of SHP-1 to the
cytoplasmic domain of CD22. Here, we demonstrate that at least two
functional ITIMs and both SH2 domains of SHP-1 are required for
efficient binding to the cytoplasmic domain of CD22. The requirement for both SHP-1 SH2 domains likely reflects the increased affinity achieved by binding tandem SH2 domains (23). Thus, it is conceivable that the tandem SH2 domains of SHP-1 allow for SHP-1 to preferentially bind to the cytoplasmic domain of CD22 because of the increased binding
affinity achieved by tandem SH2 domains compared with proteins with
single SH2 domains. This phenomenon may explain why the two triple ITIM
G/22 proteins exhibited a similar level of tyrosine phosphorylation but
bound different amounts of SHP-1. Interestingly, CD22 is a potential
substrate for SHP-1, and thus, it appears that there is an inherent
feedback mechanism allowing for the disengagement of SHP-1 from CD22.
This is supported by our finding that under the conditions we used,
SHP-1(C453S) co-immunoprecipitates with G/22, which is
tyrosine-phosphorylated. However, wild type SHP-1 does not
co-immunoprecipitate, and G/22 is not tyrosine-phosphorylated. Thus,
dephosphorylation of G/22 causes disassociation of the two proteins.
Therefore, the requirement for both SHP-1 SH2 domains and at least two
phosphorylated ITIM tyrosines may also reflect the necessity of
maintaining SHP-1 binding to CD22 for a period of time sufficient to
allow dephosphorylation of substrates involved in BCR signal
transduction. In addition, the requirement for two ITIM sequences adds
to the biological specificity, localizing SHP-1 activity to appropriate
sites to control BCR signal transduction.
The cytoplasmic domain of CD22 contains six tyrosines, of which five
are potential ITIM sequences. Lyn has recently been shown to be
required for the phosphorylation of CD22 cytoplasmic domain (10-13)
and the subsequent recruitment of SHP-1. CD22 also binds Syk,
phospholipase C That CD22 is a substrate of SHP-1 was confirmed by co-expression of
SHP-1( Interestingly, not all the single ITIM G/22 mutants were consistently
tyrosine-phosphorylated; this could be because of a specific order
required for phosphorylation. Recently this phenomenon has been
described for the T cell receptor Several recent studies have shown that SHP-1 binds to several
potentially biphosphorylated proteins, such as signaling inhibitory receptor proteins, paired immunoglobulin-like receptor B, and killer
inhibitory receptors (22, 26-31). Thus, the requirement for two ITIM
sequences may be a general mechanism directing biological specificity
and duration of SHP-1 activity. The recently solved crystal structure
for SHP-2 has provided important insights as to how the tandem SH2
domains may work in unison to regulate the catalytic activity of the
enzyme (17). The amino SH2 domain interacts with the phosphatase domain
preventing access to the catalytic site and preventing optimal
phosphotyrosine binding. The carboxyl SH2 domain, in contrast, is
freely available. Thus, as previously proposed (21), it is possible
that the carboxyl SH2 domain acts to recruit SHP-1 to appropriate
locations placing the amino SH2 domain in close proximity to a second
ITIM sequence, which under these conditions engages, allowing for the
conformation change needed for high affinity binding and enzymatic
activity. Our data support this model for the recruitment and
activation of SHP-1, by showing that for efficient binding, both SHP-1
SH2 domains are required. However, we were not able to determine
whether there is any difference in the specificity of each SH2 domain, that is whether there are ITIM preferences for each SH2 domain. This
may be important in spatially orienting SHP-1 correctly to dephosphorylate appropriate substrates.
INTRODUCTION
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Abstract
Introduction
References
, and SHP-1 (1-4). However, neither the sequence nor the mechanism of these interactions has been delineated.
EXPERIMENTAL PROCEDURES
P), PCR site overlap extension was used to generate
a BglII-BalI fragment with a deletion of the
phosphatase catalytic domain signature motif
(451VHCSAG456); the PCR primers used were
5'-CACAGCAGAATACAAACTGC-3' (forward, outer),
5'-TCATGCAGGGCCCATCATTACGGGTACC-3' (forward, inner),
5'-TATCCAATGACGATGATGGTACCCGTAATGATGGG-3' (reverse, inner), and
5'-GGTCGTTTCGATGAACTGG-3' (reverse, outer). This
BglII-BalI PCR fragment was cloned into a
modified Myc-tagged SHP-1 construct.2 CD22 constructs were
made as a fusion protein comprising the extracellular and transmembrane
domains of vesicular stomatitis virus G protein (VSVG) and the
cytoplasmic domain of mouse CD22 (amino acids 717-850). Substitutions
of the tyrosines into phenylalanines were introduced by PCR site
overlap extension (20). All mutations were confirmed by automated DNA
sequencing. SHP-1 cDNAs were cloned into pBluescript (Stratagene)
behind the T7 promoter; CD22 cDNAs were cloned into pGEM-3Z
(Promega) behind the T7 promoter.
RESULTS
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Fig. 1.
SHP-1(C453S) associates with G/22 chimeric
protein when co-expressed in HeLa cells. A fusion protein of VSVG
and the cytoplasmic domain of CD22 (G/22) was co-expressed in HeLa
cells with either SHP-1 or SHP-1 in which the active site cysteine was
mutated to serine, SHP-1(C453S). Cell lysates were immunoprecipitated
with anti-VSVG. Immunoprecipitates and crude lysates were resolved on a
7.5% SDS-polyacrylamide gel. Immunoblots were sequentially performed
for SHP-1, VSVG, and phosphotyrosine (PTyr).
CD22 potential ITIM sequences
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Fig. 2.
Two phosphotyrosines within the cytoplasmic
domain of CD22 are required for SHP-1 binding. Double
(A) or triple (B) ITIM G/22 chimeric proteins
were co-expressed with SHP-1(C453S) in HeLa cells. The nonmutated
tyrosines were: A, tyrosines 765 and 825 (lane
3), 825 and 845 (lane 4), 765 and 845 (lane
5), 799 and 810 (lane 6); or B, tyrosines
765, 799, and 825 (lane 3) and 765, 825, and 845 (lane
4). The relative position of the intact ITIM sequence is indicated
by Y, and the position of the mutated ITIM is indicated by
F. The wild type G/22 protein (YYYYY) (lane
1) and the non-ITIM-containing G/22 protein (FFFFF)
(lane 2) were also expressed in HeLa cells with
SHP-1(C453S). Lysates were immunoprecipitated with anti-VSVG.
Immunoprecipitates and crude lysates were resolved on a 7.5%
SDS-polyacrylamide gel. Immunoblots were sequentially performed for
SHP-1, VSVG, and phosphotyrosine (PTyr).
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Fig. 3.
Both SH2 domains are required for binding to
the cytoplasmic domain of CD22. SHP-1(C453S), SHP-1(R30K, R33E,
C453S), SHP-1(R136K, C453S), or SHP-1(R30K, R33E, R136K, C453S) was
co-expressed with either wild type G/22 (YYYYY) (lanes
1-5), G/22 in which tyrosines 765 and 825 were mutated to
phenylalanine (YFFYF) (lanes 6-10), or G/22 in
which all five tyrosines were mutated to phenylalanine
(FFFFF) (lanes 11-15) in HeLa cells. Lysates
were immunoprecipitated with anti-VSVG. Immunoprecipitates and crude
lysates were resolved on a 7.5% SDS-polyacrylamide gel. Immunoblots
were performed for SHP-1, VSVG, and phosphotyrosine
(PTyr).
P), which
lacks the phosphatase catalytic signature motif, is a nontrapping
mutant (22). Using these two mutants, one can discriminate between
SHP-1 substrates and binding proteins. Substrates bind to the
SHP-1(C453S) phosphatase domain, and the phosphorylated tyrosine is
protected from dephosphorylation by other endogenous phosphatases.
However, this does not occur with SHP-1(
P); thus the substrate
should exhibit greater tyrosine phosphorylation in the presence of
SHP-1(C453S) compared with SHP-1(
P) or wild type SHP-1. To this end,
the wild type G/22 protein was co-expressed with wild type SHP-1,
SHP-1(C453S), and SHP-1(
P). The level of tyrosine phosphorylation of
G/22 when co-expressed with SHP-1(
P) was greatly reduced compared
with that co-expressed with SHP-1(C453S) (Fig.
4). In addition, less SHP-1
co-immunoprecipitated with G/22 in the presence of SHP-1(
P) compared
with SHP-1(C453S). These results indicate that SHP-1 binds to G/22 via
its phosphatase domain as well as the SH2 domains, thus confirming the
earlier results that CD22 is a substrate of SHP-1. Nonetheless, the
lack of SHP-1(C453S) binding to CD22 when both SH2 domains are mutated
demonstrates that the SH2 domains are required for substrate trapping
to occur (Fig. 3).
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Fig. 4.
CD22 is a substrate of SHP-1. Wild type
SHP-1 (lane 3), SHP-1(C453S) (lane 2), or
SHP-1( P) (lane 4) was co-expressed with wild type G/22 in
HeLa cells. Lysates were immunoprecipitated with anti-VSVG.
Immunoprecipitates and crude lysates were resolved on a 7.5%
SDS-polyacrylamide gel. Immunoblots were performed for SHP-1, VSVG, and
phosphotyrosine (PTyr).
DISCUSSION
, and phosphatidylinositol 3-kinase (1, 4). It is not
known, however, which tyrosines are phosphorylated by Lyn and to which
tyrosines these other molecules may bind. However, we have been unable
to demonstrate binding of Syk or phosphatidylinositol 3-kinase to G/22
under similar conditions.4 In
this study, we focused on mapping the interaction between CD22 and
SHP-1. We have shown that three tyrosines, 765, 825, and 845, are
capable of associating with SHP-1. Moreover, our results implicate
tyrosine 825 as the primary SHP-1 binding site because the amount of
SHP-1 associated with wild type G/22 was equal to that containing only
tyrosines 765 and 825 or tyrosines 825 and 845. In addition, our
results implicate tyrosine 765 as the target dephosphorylation site of
SHP-1 because G/22 was maximally tyrosine-phosphorylated in the
presence of tyrosines 765 and 825.
P) with wild type G/22. SHP-1(C453S) acts as a substrate-trapping mutant but needs the SH2 domains to bring CD22 to
within close proximity prior to dephosphorylating (or binding via its
catalytically inactive phosphatase domain). In contrast, SHP-1(
P)
does not act as a substrate-trapping mutant as it does not have a
functional phosphatase domain, and thus it only interacts with CD22 via
its SH2 domains. SHP-1 substrates should exhibit greater tyrosine
phosphorylation in the presence of SHP-1(C453S) compared with
SHP-1(
P). Indeed, this was observed when wild type G/22 was
co-expressed with SHP-1(C453S) and SHP-1(
P) (Fig. 4).
chain (24). Our results show that
tyrosines 765 and 825 are tyrosine-phosphorylated as single ITIMs,
whereas tyrosine 845 is not and requires a second tyrosine as
demonstrated by the double ITIM G/22 protein containing tyrosines 825 and 845. It may be likely that tyrosines 765 and 825 also act to
recruit a tyrosine kinase that then phosphorylates tyrosine 845 enabling complete binding and activation of SHP-1. Our results also
show that there is specificity to which tyrosine SHP-1 binds, because
the double ITIM G/22 protein containing tyrosines 799 and 810 did not
bind to SHP-1. It remains to be determined whether the spacing between
the tyrosines is critical for SHP-1 binding, as is the case for the
immunoreceptor tyrosine-based activation motif and tyrosine kinases
(25).
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ACKNOWLEDGEMENTS |
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We thank David Plas, Tanya Ulyanova, and Andy Chan for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grant GM56455 and the Human Frontiers Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Division of Biology and Biomedical Sciences,
Washington University, School of Medicine.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should addressed: Howard Hughes Medical Inst., Dept. of Pathology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110. Tel.: 314-362-8722; Fax: 314-362-8888; E-mail: mthomas{at}immunology.wustl.edu.
The abbreviations used are: BCR, B cell antigen receptor; ITIM, immunoreceptor tyrosine-based inhibitory motif; SH2, Src homology 2; VSVG, vesicular stomatitis virus G protein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.
2 L. Dustin, D. Plas, T. Hu, and M. L. Thomas, submitted for publication.
3 Wang, L., Blasioli, J., Plas, D., Thomas, M., L., and Yokoyama, W., (1999) J. Immunol., in press.
4 X. He, D. Plas, and M. L. Thomas, unpublished data.
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REFERENCES |
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