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Departments of
*
Pathology and Molecular Microbiology and
Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and
Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, MO 63104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
Igß, are phosphorylated at tyrosine residues within their
immunoreceptor tyrosine-based activation motifs (ITAMs) 2 . These
serve as a docking site for the Src-homology domain 2 (SH2) domains of
p72syk (Syk), thereby localizing and
activating the Syk tyrosine kinase 4, 5 . Tyrosine phosphorylation, by
one or more of these kinases, may regulate downstream signaling by
phosphatidylinositol-3 kinase, phospholipase C-
1 and 2, and the
Ras-Raf-MAP kinase pathway 2 . The balance of tyrosine phosphorylation
by protein tyrosine kinases and protein tyrosine phosphatases (PTPases)
is essential throughout B cell development. Mice lacking one or more
hematopoietic tyrosine kinase or PTPase are severely impaired in B cell
differentiation and function 3, 6, 7 The SH2-containing protein tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), is a cytoplasmic PTPase with two amino-terminal SH2 domains and is expressed predominantly in cells of hematopoietic origin 8 . SHP-1-deficient mice (motheaten or viable motheaten (mev)) suffer from hematological, immunological, and inflammatory abnormalities 9, 10, 11, 12, 13 . Various levels of SHP-1 have been observed in the B cell-rich areas of the germinal center, suggesting a role for SHP-1 in the critical proliferative, differentiation, and selective events that occur there 14 . However, the B cell defect in SHP-1-deficient mice is first evident much earlier in B cell differentiation and may be at least partially due to selective pressures imposed by inflammatory bone marrow macrophages or other SHP-1-deficient cell types 7 . The use of bone marrow chimeric animals demonstrates that SHP-1-deficient (mev) B cells are altered in both development and activation, with skewing toward the development of B-1 B cells, down-regulation of the BCR, and increased Ca2+ mobilization after BCR engagement 7 .
SHP-1 associates with the BCR in resting B cells and dissociates
rapidly after BCR stimulation 15 . SHP-1 may also regulate B cell
activation by inducible associations with other transmembrane molecules
such as CD22 16, 17, 18 and, possibly, FcRIIB1 19 . Furthermore,
SHP-1 is reported to associate with cytoplasmic signaling molecules
including Vav, Grb2, mSos, and SLP-76 20, 21 . Tyrosine-phosphorylated
peptide sequences, termed immunoreceptor tyrosine-based inhibitory
motifs (ITIMs), can bind to the SH2 domains of SHP-1 and activate SHP-1
catalytic activity 22 . ITIMs with a consensus sequence of
(I/V)X(p)YXXL have been identified in FcRIIB1, CD22, the NK cell
inhibitory receptor, and the erythropoietin (EPO) and IL-3 receptors
22, 23 . ITIMs may target SHP-1 catalytic activity to nearby
phosphotyrosine residues. SHP-1 associated with the
tyrosine-phosphorylated EPO receptor may directly dephosphorylate and
inactivate the tyrosine kinase Jak2 24, 25 Similarly, constitutive
association of SHP-1 with the ß IFN receptor may permit SHP-1 to
regulate the activity of Jak1 and/or Stat1 26 . It has recently
been demonstrated that SHP-1 can dephosphorylate and inactivate the
tyrosine kinase ZAP-70 27 . These data demonstrate that SHP-1 may
regulate a variety of responses in T cells, B cells, NK cells, and
erythroid precursors by dephosphorylating signaling molecules
associated with membrane receptors. However, other enzymes including
the related PTPase, SHP-2, and the polyphosphate inositol phosphatase,
SHIP (SH2-containing inositol phosphatase), can also bind to ITIM
sequences on inhibitory receptors 28, 29, 30 . The presence of an ITIM
sequence does not by itself indicate that a receptor’s function is
mediated by SHP-1.
To better define the role of SHP-1 in the activation of mature B cells, we have expressed a catalytically inactive form of SHP-1 in K46, a murine B cell line with a mature (membrane IgG) phenotype 31 . Our results indicate that SHP-1 affects proximal and late events in BCR signaling and identify several molecules in which the tyrosine phosphorylation state is affected by SHP-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine K46 B lymphoma cell line (IgG2a, 
) 31 was a gift
of Dr. L. Justement (University of Alabama, Birmingham, AL) and was
maintained in Iscove’s modified DMEM, supplemented with 10%
heat-inactivated FCS (HyClone, Logan, UT), 2 mM
L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,
50 µg/ml gentamicin, and 1 mM sodium pyruvate. Transfected clones
were selected and grown continuously in 1.25 mg/ml G418 (Life
Technologies, Grand Island, NY). Splenocytes were prepared from C57BL/6
mice.
Construct and expression
A cysteine to serine substitution (C453S) in the active site of
SHP-1 that ablates catalytic activity was previously described 32 . A
c-myc epitope was appended by PCR to the C terminus to
distinguish overexpressed SHP-1 (C453S) from endogenous SHP-1. The
construct was cloned into the expression vector BSREN 33 .
SHP-1 (C453S) BSREN or control vector was electroporated into K46
cells 31 . Expression of transfected SHP-1 (C453S) was confirmed by
immunoblotting for the overexpressed SHP-1 and for the c-myc
epitope (Fig. 1
A). Clones with
high levels of SHP-1 (C453S) expression and unchanged levels of
membrane Ig expression (Fig. 1B) were chosen for further
analysis.
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Rabbit anti-mouse SHP-1 antisera were developed by immunization with either the purified SH2 or catalytic domains of murine SHP-1. Rabbit anti-Syk, specific for residues 260–370, was a gift of Dr. J. Bolen (DNAX, Palo Alto, CA; 34 ; anti-Syk antiserum recognizing the 28 C-terminal residues of Syk was a gift of Dr. R. Geahlen (Purdue University, West Lafayette, IN; 35 . Rabbit antisera for mouse Syk and horseradish peroxidase (HRP)-conjugated 4G10 antiphosphotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-active MAP kinase rabbit antiserum was purchased from Promega (Madison, WI). FITC-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West Grove, PA).
B cell stimulation
K46 cells were resuspended at 5 x 106 to 2 x 107/ml in PBS for experiments. Stimulation conditions were as reported by others 35, 36 . Cells were stimulated at 37°C with 0.1–30 µg/ml goat anti-mouse IgG (Jackson ImmunoResearch) for 0.5–30 min, as indicated. The standard stimulation conditions were 10 µg/ml Ab for 5 min. For assays of extracellular signal-regulated kinase (ERK) activation, cells were stimulated for 2 min with 20 µg/ml goat anti-mouse IgG as described 37, 38 . After stimulation, cells were rapidly pelleted at 4°C and lysed in ice-cold Nonidet P-40 lysis buffer unless otherwise indicated. Nonidet P-40 lysis buffer contained 150 mM NaCl, 1% Nonidet P-40, and 50 mM Tris-HCl (pH 8.0). RIPA lysis buffer contained 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris-HCl (pH 8.0). Lysis buffers were supplemented with 21 µg/ml aprotinin, 2 mM leupeptin, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml soybean trypsin inhibitor, 5 mM iodoacetamide, 0.4 mM sodium orthovanadate, and 10 mM sodium fluoride (all inhibitors purchased from Sigma, St. Louis, MO). SHP-1 and Syk immunoprecipitations were performed in the presence of 1 mg/ml chicken OVA to reduce background. Normal murine C57BL/6 splenocytes were brought to 108/ml in PBS and stimulated with 5 mM pervanadate as described 28 .
Immunoprecipitation and immunoblotting
Lysates were precleared on ice with Pansorbin cells (Calbiochem, San Diego, CA). Equal amounts of protein as determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) were analyzed by SDS-PAGE and immunoblotting. Rabbit antisera were immunoprecipitated with protein A-conjugated Sepharose beads (Sigma) and detected with HRP-conjugated protein A (Boehringer Mannheim, Indianapolis, IN). mAbs were precipitated with protein G-conjugated Sepharose beads (Boehringer Mannheim) and detected with HRP-conjugated goat anti-mouse IgG (Caltag, South San Francisco, CA).
Syk kinase assays
Prewarmed cells (2 x 107/ml) were stimulated
with 1 µg/ml avidin with or without 10 µg/ml biotinylated goat
anti-mouse IgG (Jackson ImmunoResearch) for 2 min at 37°C. Cells
were pelleted and lysed with ice-cold Nonidet P-40 lysis buffer
containing 5 mM EDTA, 1 mg/ml OVA, 4 mM leupeptin, 1 µM pepstatin A,
10 µg/ml soybean trypsin inhibitor, 10 mM sodium fluoride, 10 mM
sodium molybdate, and 200 µM sodium vanadate. After washing Syk
immunoprecipitations three times in lysis buffer, the
immunoprecipitations were split: one half was used for an immunoblot to
control for loading and the other half was used in a Syk kinase assay.
The Syk kinase assay was performed as previously described 39 .
Briefly, immunoprecipitates were washed once in 10 mM Tris (pH 7.4),
0.5 M LiCl, and twice in kinase buffer (10 mM Tris (pH 7.4), 10 mM
MgCl2). Kinase assays were then performed in 25 µl of
kinase buffer supplemented with 10 µCi [-32P]ATP and
1 µg glutathione S-transferase (GST)-Band 3 (produced as
described in 40 . Kinase assays were incubated at room temperature
for 5 min and stopped by the addition of Laemmli loading buffer and
boiling for 5 min. After separation by SDS-PAGE gel, proteins were
transferred to nitrocellulose and exposed for autoradiography and
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Analysis of Syk phosphorylation in vivo
A fusion protein of GST-Syk was expressed in Sf9 insect cells in the presence or absence of SHP-1. Sf9 cells were lysed in lysis buffer, and cell lysates were tumbled with glutathione agarose for 1 h. Agarose beads were washed three times in lysis buffer, and GST-Syk was eluted by boiling in SDS-PAGE sample buffer. After SDS-PAGE, samples were immunoblotted sequentially with anti-phosphotyrosine and anti-Syk.
Calcium mobilization
Calcium mobilization was studied with the calcium-sensitive dyes, Fluo-3 AM and Fura Red AM (Molecular Probes, Eugene, OR), as described 41 . Briefly, cells (5 x 106/ml) were loaded with 3 µM Fluo 3 AM and 6 µM Fura Red AM for 30 min at 30°C. Labeled cells were washed and resuspended at 1–2 x 106 cells/ml in Iscove’s modified DMEM supplemented with 10% FCS. Cells were prewarmed to 37°C before analysis on a FACSCaliber (Becton Dickinson Immunocytometry Systems, San Jose, CA). Fluo-3 and Fura Red fluorescence data were collected over time from viable cells, selected on forward and orthogonal scatter profile. Cells were stimulated with 3, 10, or 30 µg/ml goat anti-mouse IgG, as indicated. Ratiometric data were analyzed and graphed using FlowJo software (Tree Star, San Carlos, CA).
Homotypic adhesion
K46 or K46 SHP-1 (C453S) were cultured at 104 cells/well in a final volume of 200 µl/well in Corning/Costar (Corning, NY) tissue culture-treated 96-well flat-bottom trays. Cells were unstimulated or stimulated with goat anti-mouse IgG (0.1–10 µg/ml) at 37°C for 18–20 h. Images of undisturbed cultures were acquired on an inverted microscope with a cooled charged-coupled device (CCD) camera (Photometrics, Tucson, AZ) using IP Lab software (Signal Analytics, Vienna, VA).
Analysis
The public domain image analysis program, NIH Image, was used for densitometric analysis of immunoblots and for measurement of B cell clusters. NIH Image was developed at the National Institutes of Health and is available on the Internet (http://rsb.info.nih.gov/nih-image/).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To define the role of SHP-1 in the activation of mature B cells,
we overexpressed SHP-1 (C453S) in K46 B lymphoma cells (Fig. 1
). A
single amino acid substitution in the active site of SHP-1 (C453S)
ablates phosphatase activity 32 . This enzymatically dead form of
SHP-1 is incapable of dephosphorylating ITIM sequences and binds to
ITIM sequences for significantly longer periods of time, preventing the
endogenous phosphatase from inhibiting signal transduction (Julie
Blasioli and M.L.T., unpublished data). Therefore, it is likely that
this mutation functions as an efficient dominant-negative mutation.
SHP-1 (C453S) was modified with a C-terminal c-myc epitope.
An expression construct encoding the c-myc epitope tagged
SHP-1 (C453S) was electroporated into K46 cells. Expression of
transfected SHP-1 (C453S) was confirmed by immunoblotting for the
overexpressed SHP-1 and for the c-myc epitope (Fig. 1A). Clones with SHP-1 (C453S) expression 2- to 3-fold over
endogenous SHP-1 levels plus unchanged levels of membrane Ig
expression (Fig. 1B) were chosen for further analysis.
Dominant-negative SHP-1 increases BCR-stimulated tyrosine phosphorylation
We tested the effects of SHP-1 (C453S) overexpression on protein
tyrosine phosphorylation in B cells stimulated by anti-BCR Ab. K46
B cells (5 x 106–107/ml) were activated
with intact goat anti-mouse IgG for varying times at 37°C, and
equal amounts of total cell lysate protein were analyzed for tyrosine
phosphorylation. When compared with K46 cells, K46 (C453S) cells
demonstrated increased protein tyrosine phosphorylation, evident at
many doses of BCR stimulation (Fig. 2A). This increase in
phosphotyrosine content was observed within 30–60 s after stimulation
and persisted for at least 10 min (Fig. 2B). Some proteins
remained hyperphosphorylated for at least 30 min. Some proteins were
hyperphosphorylated even in unstimulated cells expressing SHP-1 (C453S)
(Fig. 2, A and B). The degree of
hyperphosphorylation correlated with the level of expression of SHP-1
(C453S) (Fig. 2B and data not shown). Tyrosine
phosphorylation of 55- to 60-kDa, 70-kDa, and 110-kDa proteins was
increased at all doses of stimulus and at all time points. SHP-1
(C453S) expression also resulted in increased tyrosine phosphorylation
of a similar set of protein bands in K46 cells stimulated with
pervanadate (data not shown). These proteins may show increased
tyrosine phosphorylation due to increased kinase activity or decreased
dephosphorylation by SHP-1. Alternatively, these proteins may be
protected from other phosphatases by binding the SH2 domains of
SHP-1 (C453S). To examine this issue, we analyzed the association of
Syk with SHP-1.
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Although SHP-1 is reported to interact with a number of
cell-surface and cytoplasmic signaling proteins, few have been directly
shown to be targets for dephosphorylation by SHP-1. SHP-1 has been
shown to regulate ZAP-70 and certain JAK tyrosine kinases 24, 26, 27 .
Therefore, we examined the phosphorylation state of the ZAP-70-related
tyrosine kinase, Syk, in two K46 clones overexpressing SHP-1 (C453S).
Syk immunoprecipitates were resolved by SDS-PAGE and probed with
anti-phosphotyrosine (Fig. 3A, top) and
anti-Syk (Fig. 3A, bottom). Syk,
immunoprecipitated from SHP-1 (C453S) mutant cells, demonstrated
increased tyrosine phosphorylation compared with that from control
K46 cells. When corrected for the amount of Syk protein in the
anti-Syk immunoblot (Fig. 3A, bottom),
tyrosine phosphorylation was 4.5- to 12-fold higher in Syk
immunoprecipitates from activated SHP-1 (C453S) mutant cells than from
control cells (Fig. 3A). Immunoprecipitation kinases assays
were performed to determine whether the increase in Syk tyrosine
phosphorylation resulted in altered catalytic activity. Syk tyrosine
kinase activity was increased in both resting cells and cells
stimulated with anti-IgG (Fig. 3B).
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SHP-1 associates with and regulates ZAP-70 in activated T cells
27 . To examine whether SHP-1 associates with Syk in B cells, Syk was
immunoprecipitated from either K46 or K46 SHP-1 (C453S) cells and
immunoblot analysis was performed (Fig. 4, A and B). SHP-1
was detected in Syk immunoprecipitates from resting K46 cells as well
as from activated cells (Fig. 4A). This result was observed
with two different anti-Syk antisera, directed against the unique
interdomain region of Syk 34 or the C-terminal 28 amino acids of Syk
35 . Surprisingly, SHP-1 coimmunoprecipitated with Syk from either
resting or activated K46 cells (Fig. 4B). This result was
observed both with an anti-SHP-1 antiserum directed against the two
SH2 domains of SHP-1 and with one directed against the catalytic domain
of SHP-1. SHP-1 association with Syk was not substantially altered in
the lysates of cells expressing SHP-1 (C453S). In resting K46 cells,
Syk does not appear to be tyrosine phosphorylated. Therefore, this
result suggests that the interaction between Syk and SHP-1 may not be
phosphotyrosine-dependent in this instance. Because the constitutive
association of Syk and SHP-1 was unexpected, we decided to examine
whether these two enzymes were constitutively associated in primary B
cells. In contrast, coimmunoprecipitation of SHP-1 with Syk
immunoprecipitation from murine splenic B cells requires stimulation
(Fig. 4C), demonstrating that the association is inducible.
The molecular basis for the constitutive association in K46 cells and
inducible association in splenic B cells is unknown. However, it is
possible that the transformed state of K46 results in increased basal
activation, potentially affecting the steady-state association among
Syk, SHP-1, and other proteins. One potential mechanism by which
Syk and SHP-1 could associate is through the formation of a
tri-molecular complex with CD22. However, coexpression of Syk, SHP-1,
and CD22 in HeLa cells did not result in the formation of a
tri-molecular complex despite appropriate phosphorylation of CD22 and
the association with SHP-1 (D.R.P., Silke Paust, and M.L.T.,
unpublished data). We also could not demonstrate a direct association
of SHP-1 and Syk in vitro or by coexpression. Thus, the kinase and
phosphatase may associate through a novel mechanism.
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That SHP-1 and Syk associate in B cells suggests that as reported
for SHP-1 and ZAP-70 27 , these enzymes regulate each other. To
confirm that the association SHP-1 and Syk is physiologically
significant, we examined whether SHP-1 could dephosphorylate Syk. A
GST-Syk fusion protein was expressed in Sf9 cells in the presence or
absence of SHP-1. GST-Syk purified from cells expressing SHP-1 was
decreased in phosphorylation when compared with GST-Syk purified from
cells that did not express SHP-1 (Fig. 4D). Furthermore,
phosphorylated recombinant Syk protein stimulated SHP-1 phosphatase
activity in vitro (data not shown).
Downstream alterations in BCR signal transduction in cells expressing SHP-1 (C453S)
BCR-stimulated Ca2+ mobilization and ERK
phosphorylation were measured to assess later changes in signal
transduction. Compared with parental cells, K46 cells expressing SHP-1
(C453S) demonstrated more pronounced increases in cytoplasmic
Ca2+ after stimulation with all concentrations of
anti-BCR Abs examined (Fig. 5). The
kinetics of Ca2+ mobilization were accelerated and the
fraction of responding cells was increased among K46 cells expressing
SHP-1 (C453S). Similarly, BCR cross-linking resulted in greater MAP
kinase activation as measured by ERK phosphorylation in cells
expressing SHP-1 (C453S) (Fig. 6). Active
ERK was not detected in either resting wild-type K46 cells or resting
SHP-1 (C453S) transfectants. However, when cells were treated with goat
anti-mouse IgG, active ERK levels were an average
(n = 3) of 2.8-fold higher in K46 cells expressing
SHP-1 (C453S). These results support the idea that SHP-1 (C453S)
affects early events in the BCR signal transduction cascade such as Syk
activation.
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The experiments detailed above show that SHP-1 (C453S) alters
protein tyrosine phosphorylation in K46 B cells during the first 30 min
after stimulation of the BCR. Because the transformed cell lines used
in this assay proliferate regardless of stimulation, it is not
practical to measure the effects of dominant-negative SHP-1 on
BCR-stimulated proliferation. Adhesion was measure as a parameter of
late changes in activation. SHP-1 (C453S) permitted a dramatic increase
in homotypic adhesion of BCR-stimulated cells after overnight
stimulation with anti-BCR Abs (Fig. 7). Although control K46 cells formed
small aggregates after overnight stimulation (Fig. 7B),
cells expressing SHP-1 (C453S) formed aggregates 3.5–7 times larger
(Fig. 7D). In both control and mutant cells, aggregation was
increased as the dose of goat anti-mouse IgG was increased from 0.1
to 10 µg/ml. Mutant cells showed enhanced aggregation compared with
control cells at all doses of stimulus tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1 19 and may contribute to the regulation of B cell
activation by these membrane receptors. B cell differentiation is
severely impaired in the absence of SHP-1 activity 7 . Motheaten B
cells demonstrate increased B cell activation, as measured by
Ca2+ mobilization 7 and proliferation 15 . It is
possible that the B-1 lymphocytes that preferentially survive in the
motheaten mouse have a different activation program. To separate the
role of SHP-1 in B cell activation from its role in B lymphocyte
differentiation, it is essential to interrupt its function specifically
in mature B cells without subjecting these cells to the developmental
abnormalities of the motheaten mouse.
We examined the effects of dominant-negative SHP-1 on activation of the
B lymphoma line, K46, by anti-BCR Abs. The catalytically inactive
SHP-1 (C453S) can compete with endogenous wild-type SHP-1 for
association with other signaling molecules and for access to substrates
and thus can serve as a dominant-negative mutation. Our results
demonstrate that expression of dominant-negative SHP-1 in mature B
cells causes increased tyrosine phosphorylation of a number of proteins
after BCR engagement (Fig. 2). These proteins include the tyrosine
kinase Syk (Fig. 3), which is a substrate for dephosphorylation by
SHP-1 (Fig. 4D). We also observed an association of SHP-1
with Syk in splenic B cells and in wild-type or SHP-1 (C453S) K46 cells
(Fig. 4). That SHP-1 (C453S) altered early tyrosine kinase activity is
supported by the increased changes in early downstream events in BCR
signal transduction, such as Ca2+ mobilization (Fig. 5) and
ERK phosphorylation (Fig. 6). Longer-term events are also affected by
SHP-1. A full day after stimulation by goat anti-mouse IgG, K46
cells expressing SHP-1 (C453S) show increased homotypic adhesion (Fig. 7). Recent data demonstrate that CD45 regulates integrin-mediated
adhesion in lymphocytes 43, 44 and macrophages 45 . Together, these
results point to an emerging role for protein tyrosine phosphatases in
the regulation of lymphocyte adhesion.
These results support the hypothesis that Syk is negatively regulated
by SHP-1 in B cells. K46 B cells expressing SHP-1 (C453S) showed
increased Syk tyrosine phosphorylation and tyrosine kinase activity
(Fig. 3). In addition, Syk was dephosphorylated when coexpressed with
SHP-1 in insect cells (Fig. 4D). This finding adds Syk to
the growing list of receptor-associated tyrosine kinases that are
regulated by SHP-1 24, 25, 27, 46 . Furthermore, we found that SHP-1
and Syk associate physically both in wild-type K46 B cells and in K46
expressing SHP-1 (C453S) (Fig. 4). A complex containing both SHP-1 and
Syk could be immunoprecipitated from K46 cells with various antisera
specific for either enzyme. This complex likely contained a relatively
small fraction of each enzyme in K46 B cells, and was most clearly
identified when SHP-1 or Syk was immunoprecipitated from limiting
numbers of cells. SHP-1 may interact with Syk by a mechanism
independent of the binding of the SH2 domains of SHP-1 to
phosphorylated tyrosine residues in Syk. Thus, BCR engagement and
subsequent Syk tyrosine phosphorylation did not alter Syk and SHP-1
association in K46 B cells. This finding is consistent with the
observation that a functional interaction between SHP-1 and Jak2
requires neither functional SHP-1 SH2 domains, nor tyrosine
phosphorylation of Jak2 25 . In contrast, the association was
increased by pervanadate stimulation in splenic B cells. The
association may be mediated by an adapter protein expressed in B cells,
but not other cell types (Fig. 4; D.R.P. and M.L.T., unpublished data).
The SH2 domains of SHP-1 may bind to regulatory ITIMs while other
interactions mediate a direct association between SHP-1 and its
substrates.
These results support a model in which SHP-1 is recruited to
tyrosine-phosphorylated transmembrane receptors, then acts to regulate
the tyrosine phosphorylation of signal transduction molecules
associated with or downstream of the transmembrane receptors. If SHP-1
dephosphorylates and inactivates signaling molecules associated with
CD19 or CD22, this could reduce the tyrosine phosphorylation of the
transmembrane receptors indirectly by inactivating those tyrosine
kinases that are responsible for phosphorylating them. Evidence for
such an indirect mechanism has been reported in SHP-1 regulation of
signal transduction through the EPO receptor 25 . Similarly, SHP-1
recruited to the phosphorylated NK cell inhibitory may directly
dephosphorylate downstream molecules including ZAP-70 and phospholipase
C-2 47
These results do not rule out SHP-1 in the regulation of other tyrosine
kinases. Src-family tyrosine kinases including Lyn are differentially
phosphorylated in cells expressing SHP-1 C453S (Fig. 2; L.B.D., Y.T.H.,
and M.L.T., unpublished data). We have not seen evidence for changes in
phosphorylation of the tyrosine kinase Btk in SHP-1 (C453S) expressing
cells, because the 77-kDa Btk does not comigrate with any of the
differentially phosphorylated bands observed in Fig. 2 (data not
shown). The possibility that SHP-1 and Btk may not act on the same
pathways is suggested by the fact that Btk mutant mice lack B-1 B cells
3 , a subset that is selectively retained in mice with the SHP-1
mutations motheaten and viable motheaten
7 .
At least four different mechanisms could account for the increased tyrosine phosphorylation of specific cellular proteins in B cells expressing catalytically inactive SHP-1. Some of these proteins may be targets for dephosphorylation by SHP-1. Syk is a candidate for such direct regulation by virtue of its association with SHP-1 in vivo, its dephosphorylation in cells coexpressing SHP-1, and because SHP-1 dephosphorylates the related kinase, ZAP-70 27 . Second, the kinases that phosphorylate these proteins may be targets for regulation by SHP-1. In this regard, SHP-1 selectively regulates the level of EPO receptor tyrosine phosphorylation by Jak2, an enzyme regulated by SHP-1, but not by c-Fes, an enzyme not known to be regulated by SHP-1 25 . Third, catalytically inactive SHP-1, expressed in excess, could bind to phosphotyrosine via its SH2 domains, denying access to phosphorylated substrates by endogenous SHP-1 as well as other endogenous phosphatases. Finally, the active site of catalytically inactive SHP-1 may stably bind to substrates and prevent endogenous, functional phosphatases from dephosphorylating these substrates. In these studies, we allowed SHP-1 and other phosphatases to dephosphorylate their substrates in intact cells before lysis. This maintains normal cellular structures and protein-protein interactions, thereby permitting SHP-1 to act on its normal substrates. SHP-1 may exert its regulatory effects on B cell development and activation by changing the threshold for activation through these receptors and nonreceptor tyrosine kinases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew L. Thomas, Howard Hughes Medical Institute, Department of Pathology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110. E-mail address:
3 Abbreviations used in this paper: BCR, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; EPO, erythropoietin; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-base inhibitory motif; mev, viable motheaten, PTPase, protein tyrosine phosphatase; SH2, Src-homology domain 2; SHP-1, SH2-containing protein tyrosine phosphatase-1; Syk, p72syk; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein.
Received for publication June 15, 1998. Accepted for publication November 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Igß immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.RIIB1. Science 268:293./ß interferon-stimulated Jak/Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol. Cell. Biol. 15:7050.[Abstract]
RIIb1 require distinct phosphatases. J. Exp. Med. 186:473. and Ig-ß components of the B cell antigen receptor. Eur. J. Immunol. 23:911.[Medline]
1 upon B cell activation. J. Exp. Med. 183:547.This article has been cited by other articles:
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  N. R. D. Paling and M. J. Welham Tyrosine phosphatase SHP-1 acts at different stages of development to regulate hematopoiesis Blood, June 1, 2005; 105(11): 4290 - 4297. [Abstract] [Full Text] [PDF]
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 C. L. Kepley, S. Taghavi, G. Mackay, D. Zhu, P. A. Morel, K. Zhang, J. J. Ryan, L. S. Satin, M. Zhang, P. P. Pandolfi, et al. Co-aggregation of Fc{gamma}RII with Fc{epsilon}RI on Human Mast Cells Inhibits Antigen-induced Secretion and Involves SHIP-Grb2-Dok Complexes J. Biol. Chem., August 20, 2004; 279(34): 35139 - 35149. [Abstract] [Full Text] [PDF]
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 Z. Zhang, E. Jimi, and A. L. M. Bothwell Receptor Activator of NF-{kappa}B Ligand Stimulates Recruitment of SHP-1 to the Complex Containing TNFR-Associated Factor 6 That Regulates Osteoclastogenesis J. Immunol., October 1, 2003; 171(7): 3620 - 3626. [Abstract] [Full Text] [PDF]
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L. P. Ganesan, H. Fang, C. B. Marsh, and S. Tridandapani The Protein-tyrosine Phosphatase SHP-1 Associates with the Phosphorylated Immunoreceptor Tyrosine-based Activation Motif of Fc{gamma}RIIa to Modulate Signaling Events in Myeloid Cells J. Biol. Chem., September 12, 2003; 278(37): 35710 - 35717. [Abstract] [Full Text] [PDF]
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Y. Hokazono, T. Adachi, M. Wabl, N. Tada, T. Amagasa, and T. Tsubata Inhibitory Coreceptors Activated by Antigens But Not by Anti-Ig Heavy Chain Antibodies Install Requirement of Costimulation Through CD40 for Survival and Proliferation of B Cells J. Immunol., August 15, 2003; 171(4): 1835 - 1843. [Abstract] [Full Text] [PDF]
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J. Kabat, F. Borrego, A. Brooks, and J. E. Coligan Role That Each NKG2A Immunoreceptor Tyrosine-Based Inhibitory Motif Plays in Mediating the Human CD94/NKG2A Inhibitory Signal J. Immunol., August 15, 2002; 169(4): 1948 - 1958. [Abstract] [Full Text] [PDF]
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 C. H. Nielsen and R. G. Q. Leslie Complement's participation in acquired immunity J. Leukoc. Biol., August 1, 2002; 72(2): 249 - 261. [Abstract] [Full Text] [PDF]
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 K. Yoshida and D. Kufe Negative Regulation of the SHPTP1 Protein Tyrosine Phosphatase by Protein Kinase C delta in Response to DNA Damage Mol. Pharmacol., December 1, 2001; 60(6): 1431 - 1438. [Abstract] [Full Text] [PDF]
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P. Merciris, M.-D. Hardy-Dessources, and F. Giraud Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase Blood, November 15, 2001; 98(10): 3121 - 3127. [Abstract] [Full Text] [PDF]
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 T. Oka, T. Yoshino, K. Hayashi, N. Ohara, T. Nakanishi, Y. Yamaai, A. Hiraki, C. A. Sogawa, E. Kondo, N. Teramoto, et al. Reduction of Hematopoietic Cell-Specific Tyrosine Phosphatase SHP-1 Gene Expression in Natural Killer Cell Lymphoma and Various Types of Lymphomas/Leukemias : Combination Analysis with cDNA Expression Array and Tissue Microarray Am. J. Pathol., October 1, 2001; 159(4): 1495 - 1505. [Abstract] [Full Text]
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H.-C. Hsu, L. D. Shultz, X. Su, J. Shi, P.-A. Yang, M. J. Relyea, H.-G. Zhang, and J. D. Mountz Mutation of the Hematopoietic Cell Phosphatase (Hcph) Gene Is Associated with Resistance to {{gamma}}-Irradiation-Induced Apoptosis in Src Homology Protein Tyrosine Phosphatase (SHP)-1-Deficient ""Motheaten"" Mutant Mice J. Immunol., January 15, 2001; 166(2): 772 - 780. [Abstract] [Full Text] [PDF]
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K. Mizuno, Y. Tagawa, K. Mitomo, Y. Arimura, N. Hatano, T. Katagiri, M. Ogimoto, and H. Yakura Src Homology Region 2 (SH2) Domain-Containing Phosphatase-1 Dephosphorylates B Cell Linker Protein/SH2 Domain Leukocyte Protein of 65 kDa and Selectively Regulates c-Jun NH2-Terminal Kinase Activation in B Cells J. Immunol., August 1, 2000; 165(3): 1344 - 1351. [Abstract] [Full Text] [PDF]
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Z.-H. Xie, J. Zhang, and R. P. Siraganian Positive Regulation of c-Jun N-Terminal Kinase and TNF-{alpha} Production But Not Histamine Release by SHP-1 in RBL-2H3 Mast Cells J. Immunol., February 1, 2000; 164(3): 1521 - 1528. [Abstract] [Full Text] [PDF]
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K. L. Berg, K. A. Siminovitch, and E. R. Stanley SHP-1 Regulation of p62DOK Tyrosine Phosphorylation in Macrophages J. Biol. Chem., December 10, 1999; 274(50): 35855 - 35865. [Abstract] [Full Text] [PDF]
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D. R. Plas, C. B. Williams, G. J. Kersh, L. S. White, J. M. White, S. Paust, T. Ulyanova, P. M. Allen, and M. L. Thomas Cutting Edge: The Tyrosine Phosphatase SHP-1 Regulates Thymocyte Positive Selection J. Immunol., May 15, 1999; 162(10): 5680 - 5684. [Abstract] [Full Text] [PDF]
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R. K. Ganju, S. A. Brubaker, R. D. Chernock, S. Avraham, and J. E. Groopman beta -Chemokine Receptor CCR5 Signals through SHP1, SHP2, and Syk J. Biol. Chem., June 2, 2000; 275(23): 17263 - 17268. [Abstract] [Full Text] [PDF]
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A.-K. Somani, K. Yuen, F. Xu, J. Zhang, D. R. Branch, and K. A. Siminovitch The SH2 Domain Containing Tyrosine Phosphatase-1 Down-regulates Activation of Lyn and Lyn-induced Tyrosine Phosphorylation of the CD19 Receptor in B Cells J. Biol. Chem., January 12, 2001; 276(3): 1938 - 1944. [Abstract] [Full Text] [PDF]
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T. Adachi, J. Wienands, C. Wakabayashi, H. Yakura, M. Reth, and T. Tsubata SHP-1 Requires Inhibitory Co-receptors to Down-modulate B Cell Antigen Receptor-mediated Phosphorylation of Cellular Substrates J. Biol. Chem., July 6, 2001; 276(28): 26648 - 26655. [Abstract] [Full Text] [PDF]
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