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,
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Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, Academy of Science of Ukraine, Kiev, Ukraine; and
Department of Microbiology and
Regional Primate Research Center, University of Washington, Seattle, WA 98195
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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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CDw150 is a cell surface molecule recognized by two mAb, IPO-3 and A12
anti-signaling lymphocytic activation molecule (SLAM) (5, 6, 7).
CDw150 is expressed on CD45RO+ CD45RA-
peripheral blood T cells, at low levels on blood and tonsillar B cells,
and on immature thymocytes and dendritic cells. Activating T cells, B
cells, or dendritic cells increases the expression of CDw150 (8, 9, 10).
In lymph nodes, CDw150 is localized in the cytoplasm of germinal center
cells and on the surface of the mantle zone B cells, and also is found
on a subpopulation of endothelial cells (11, 12). Ligation of CDw150 on
B lymphocytes with mAb IPO-3 induces a rapid elevation of intracellular
free calcium ([Ca2+]i) and augments
proliferation induced by CD40 mAb and IL-4 (8). Engaging CDw150 with
mAb results in IL-2- and CD28-independent but cyclosporin A-sensitive
proliferation (13). Ligation of CDw150 also induces IFN-
production
by CD4+ T cell clones and Ig production by activated B
cells (9, 13, 14). Thus, CDw150 may be involved in expanding Th0/Th1
immune responses (15).
Just how CDw150 exerts these effects on lymphocytes is not known. CDw150 is a sialylated phosphoglycoprotein ranging from 70 to 95 kDa in size (8, 9) and belongs to the CD2 subset of the Ig superfamily of type I transmembrane glycoproteins (9). It has a 77-residue intracytoplasmic tail containing several tyrosines within motifs for Src-homology 2 (SH2)-domain binding sites. Recently, Sayos et al. (16) used CDw150’s cytoplasmic tail as bait in a yeast two-hybrid screen to isolate clones encoding a 15-kDa polypeptide with a single SH2 domain. This molecule, SAP (for SLAM-associated protein), is expressed in T cells and some B cells and physically associates with CDw150 in vivo. Two groups (16, 17) have found that the X-linked gene encoding SAP or DSHP is mutated in patients with X-linked lymphoproliferative disease (XLP). The fact that patients with XLP have uncontrolled B cell proliferation following infection with EBV (18) suggests that DSHP/SAP via CDw150 normally controls B cell proliferation directly or indirectly via T cells (16). However, Nichols et al. (17) found that DSHP/SAP may be expressed in activated germinal center B cells, implying that it may also normally function directly to regulate B cell growth.
Using human B cells, we found that CDw150 associates with both tyrosine and serine/threonine kinase activities (8). Here we report that in human B cells CDw150 can be tyrosine-phosphorylated after BCR ligation. It associates with the Src-family kinase Fgr, an inositol polyphosphate 5'-phosphatase, SH2-containing inositol phosphatase (SHIP), and also with the protein tyrosine phosphatase (PTPase) CD45. Ligation of CDw150 on B cells induced rapid tyrosine dephosphorylation of the SHIP phosphatase. Our results also demonstrate that CDw150 is a signaling molecule that can regulate CD95-mediated apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit antisera against Syk, Lyn, Fyn, Lck, Fgr, SHIP, and p120cbl were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse biotinylated anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) was used for Western blotting. The PhosphoPlus Akt (ser473) Ab kit was purchased from New England Biolabs (Beverly, MA), and ApoAlert annexin V apoptosis kit was obtained from Clontech (Palo Alto, CA). Glutathione-agarose was purchased from Sigma (St. Louis, MO), and protein A- and protein G-Sepharose were purchased from Pharmacia (Piscataway, NJ). For cross-linking of surface receptors and biochemical experiments we used the following mAb: IPO-3 (IgG1) anti-CDw150 (6, 12), IPO-4 (IgM) anti-CD95 (12, 19), and G28–5 (IgG1) anti-CD40 (20), mAb 9.4 anti-CD45 (21), and G28–8 anti-Bgp95 (22). F(ab')2 of goat anti-human IgM (Jackson ImmunoResearch, West Grove, PA) were used for IgM cross-linking on human B cell lines. F(ab')2 of mouse mAb against CDw150 (IPO-3) were prepared using immobilized pepsin (Pierce, Rockford, IL), and F(ab')2 of goat anti-mouse IgG (Jackson ImmunoResearch) were used as secondary cross-linking reagent.
Plasmid constructs
A GST-fusion protein construct of the cytoplasmic tail of CDw150 (GST-CDw150ct) was prepared for this study. Forward and reverse primers with the appropriate restriction sites for in-frame cloning into the pGEX-2T plasmid were used to amplify a cDNA fragment using pfu polymerase (Stratagene, La Jolla, CA). The template used for amplifying the cytoplasmic domain of CDw150 was pSurSLAM cDNA (9), kindly provided by Dr. G. Aversa (DNAX, Palo Alto, CA). Plasmids with the correct nucleotide sequence were transformed into the bacterial strain XLI-BlueMRF' (Stratagene) for fusion protein production. Plasmids containing GST-CDw150ct were also transformed into Escherichia coli strain TKX1 (Stratagene) for production of tyrosine-phosphorylated GST-CDw150ct (GST-CDw150ct-PY). Tyrosine phosphorylation of these fusion proteins apparently was restricted only to the corresponding cytoplasmic tails, as GST was not tyrosine-phosphorylated when expressed alone in the same bacterial strain (data not shown). Expression and purification of GST fusion proteins were performed as described (23, 24).
Cell stimulation
The Burkitt’s lymphoma cell lines Namalwa and Raji, the lymphoblastoid cell lines CESS, MP-1, and RPMI-1788, and the HPB-ALL T cell line were maintained as described (25). Cells were stimulated by ligation of receptors with Abs as described (23, 24). Cross-linking of CDw150 was performed either with mAb IPO-3 or in two steps using F(ab')2 of IPO-3 followed by F(ab')2 of goat anti-mouse IgG. For induction of apoptosis, cells were incubated in triplicate in 24-well plates at 5 x 105/ml in the presence of one or more of the following mAbs: MOPC 21, IPO-3, IPO-4, and G28–5 (0.1–10 µg/ml). After culture for 1–72 h, cells were harvested, washed once with PBS and twice with HEPES binding buffer (26), and resuspended in annexin V-FITC at a final concentration of 0.5 µg/ml. Samples were incubated in the dark at room temperature for 15 min, washed once with HEPES binding buffer, and fixed in 1% paraformaldehyde in HEPES binding buffer before visualization. Then samples were analyzed using a fluorescent microscope Axiolab (Carl Zeiss, Jena, Germany), and the percentage of annexin V-binding cells was calculated based on analysis of >500 cells per sample.
Biochemical methods
Cell lysis, immunoprecipitation, SDS-PAGE, in vitro kinase
assays, and phosphoamino acid analysis were performed as described (8, 25, 27). Western blotting was performed with an enhanced
chemiluminescence kit (Amersham Life Science, Arlington Heights, IL).
For evaluation of kinase activity, immunoprecipitates were washed with
Nonidet P-40 lysis buffer or with Chicago high salt buffers and were
subjected to in vitro kinase assays with myelin basic protein, enolase,
or fusion proteins as potential substrates. The concentration of
substrates was 50 µg/ml. Immunoprecipitates were incubated for 15 min
at 30°C in 20 µl of kinase buffer (40 mM PIPES, 10 mM
MgCl2, 10 mM MnCl2, pH 7.0) with 10 mCi of
[-32P] ATP (300 Ci/mM, DuPont-NEN, Burbank, CA). The
reaction was stopped by boiling for 5 min with 20 µl 2x sample
buffer containing 30 mM EDTA. Proteins were resolved by SDS-PAGE
and phosphoamino acid analysis was performed. Sequential
immunoprecipitation was performed as described previously with some
modifications (28). Briefly, after primary immunoprecipitations with
rabbit or mouse Abs and in vitro kinase assays, 32P-labeled
bands of interest were excised from dried gels and proteins were eluted
in 50 mM Tris buffer, pH 8.0, containing 0.1 mM EDTA, 150 mM NaCl, and
0.1% SDS. Before secondary immunoprecipitations, inhibitors of
proteases and phosphatases were added. To reduce nonspecific binding,
Nonidet P-40 and bovine serum protein were added to final
concentrations of 0.5% and 0.1%, respectively. After secondary
immunoprecipitations, proteins were resolved by SDS-PAGE.
For biotinylation of cell surface molecules, 107 cells were washed three times with PBS, incubated 15 min at room temperature with 0.5 mg/ml Sulfo-NHS-biotin (Pierce), washed twice with RPMI 1640 culture media and once with PBS, and lysed in Nonidet P-40 lysis buffer. These lysates were used for standard immunoprecipitations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To define the signal transduction pathways linked to CDw150 in B cells, we looked for surface and intracellular molecules associating with CDw150. First, we partially purified CDw150 from a B lymphoblastoid cell line using affinity column chromatography with immobilized mAb IPO-3. Silver staining revealed several proteins associated with CDw150 and particularly proteins that were about 200, 140, and 59 kDa in size (data not shown). The CDw150 cytoplasmic tail (CDw150ct) has several tyrosines such as Y254 and Y300 (with YxxV motifs) that after phosphorylation may form potential binding sites for SH2 domains. To test whether tyrosine-phosphorylated CDw150 could be bound by other proteins, we prepared GST-fusion proteins of unphosphorylated CDw150ct (GST-CDw150ct) and its tyrosine-phosphorylated form (GST-CDw150ct-PY). These fusion proteins were used for precipitations followed by in vitro kinase assays and Western blotting. To define the molecules associated with CDw150 in vivo, we also examined CDw150 immunoprecipitates using anti-CDw150 mAb.
In vitro kinase assays showed that only GST-CDw150ct-PY and not
GST-CDw150ct and GST alone specifically precipitated from B cell
lysates kinase activities that phosphorylated intracellular substrates
(Fig. 1
A, lanes
3–5). Several phosphorylated proteins in GST-CDw150ct-PY
immunoprecipitate were the same size as the proteins coprecipitated
with native CDw150 (Fig. 1A, lane 2). These
proteins were 145 kDa and a triplet of 53/56/59 kDa. In vitro kinase
assays followed by phosphoamino acid analysis showed that all of these
proteins and the fusion protein itself were phosphorylated in vitro on
both tyrosine and serine (Fig. 1B). Anti-phosphotyrosine
Western blotting of precipitates also revealed these
tyrosine-phosphorylated proteins coprecipitated with GST-CDw150ct-PY
but not with control fusion proteins (Fig. 2A). A 145-kDa protein was the
main tyrosine-phosphorylated protein that specifically bound to
GST-CDw150ct-PY (Fig. 2A).
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C). Anti-Blk
and anti-Shc rabbit sera also did not reprecipitate this band (data
not shown). At the same time, Western blotting of immunoprecipitates of
native CDw150 revealed only Fgr associated with CDw150 in vivo
(Fig. 1D), but not Lyn, Fyn, Lck, or Shc (data not shown).
To identify the 145-kDa band coprecipitated with CDw150 and
GST-CDw150ct-PY, we performed sequential immunoprecipitation of the
145-kDa phosphoprotein labeled by in vitro kinase assays using Abs to
the following molecules about 120–150 kDa: PLC1, PLC2, Jak1,
Jak2, Jak3, FAK, CD22, CD21, PKCµ, 120-kDa substrate for Src,
p120cbl, and SHIP. Only Abs to SHIP were able to
reprecipitate this band (data not shown). To confirm this result, we
used Western blotting with anti-SHIP sera. SHIP was detected in
both GST-CDw150ct-PY (Fig. 2B) and in actual CDw150 in vivo
immunoprecipitates (Fig. 2C). Thus, in B cells CDw150 is
associated with Fgr and SHIP and potentially may also bind Lyn.
Ligation of CDw150 induces tyrosine dephosphorylation of SHIP
Ligation of the BCR results in rapid activation of protein
tyrosine kinases (PTK) including Src-family kinases and rapid
phosphorylation of Ig-
/Ig-ß and receptors such as CD22 and CD19
(2, 29, 30). Because the Src-family kinases Fgr and Lyn associate with
CDw150, we decided to check whether CDw150 can be
tyrosine-phosphorylated after BCR cross-linking. Anti-phosphotyrosine
blots of CDw150 immunoprecipitates revealed that BCR cross-linking
rapidly induces tyrosine phosphorylation of CDw150 within 20 s,
while ligating CDw150 itself has the opposite effect (Fig. 3A). Furthermore, ligation of
CDw150 with either whole mAb or F(ab')2 of IPO-3 followed
by F(ab')2 of goat anti-mouse Ig sera, unlike BCR
ligation, did not lead to new protein tyrosine phosphorylation; rather
it led to the dephosphorylation of proteins about 145 and 56–60 kDa in
size (Fig. 3B). One possibility was that ligation of CDw150
led not to its dephosphorylation (see Fig. 3A), but to its
internalization or shedding; however, this appeared not to be the case
because the level of CDw150 surface expression remained constant as
measured by flow cytometry (data not shown).
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B), we tested whether CDw150
cross-linking affects tyrosine phosphorylation of PTKs and SHIP.
Ligation of CDw150 did not induce any changes in the level of tyrosine
phosphorylation of either Fgr, Lyn, Fyn, Lck, Csk, Btk, or Syk. Also,
when we used enolase and myelin basic protein as substrates, we
detected no changes in the activities of these PTKs after CDw150
ligation (data not shown). On the other hand, ligation of CDw150
induced tyrosine dephosphorylation of SHIP, whereas BCR cross-linking
resulted in tyrosine phosphorylation of SHIP (Fig. 3C). This
decreased level of tyrosine phosphorylation of SHIP was detected as
early as 1 min after CDw150 ligation and was not restored to initial
levels until 1 h later (Fig. 4).
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). Fgr also associated with SHIP, but only 30–60
min after CDw150 ligation (Fig. 4). In summary, ligation of CDw150
induces 1) the dephosphorylation of both SHIP and CDw150 and 2) the
association of Lyn with SHIP. Coincident with the recovery of SHIP
tyrosine phosphorylation after CDw150 ligation, Fgr is found associated
with SHIP. CDw150 associates with PTPase CD45
Because CDw150 can induce tyrosine dephosphorylation of itself and
intracellular proteins, we next tested whether CDw150 has a PTPase
associated with it. In a preliminary study using assays with three
different substrates for evaluating PTPase activity, we found that in B
cell lines CDw150 does have PTPase activity associated with it (38).
Surface biotinylation of the B cell lines CESS and MP-1 revealed a
200-kDa band coprecipitated with CDw150 in Nonidet P-40 lysates (Fig. 5A). This protein was not
detectable when immunoprecipitates were washed with high-salt buffers.
A broad 60- to 95-kDa band is actually CDw150, which after EndoF
treatment had size of 41 kDa (Fig. 5A). Using Western
blotting with anti-CD45 mAb we found that a 200-kDa surface
molecule, coprecipitated with CDw150, was the PTPase CD45 (Fig. 5B). Control immunoprecipitates of a surface molecule,
Bgp95, expressed at similar levels as CDw150 on these cell lines, did
not contain CD45 (Fig. 5B).
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A and 2A). Because the SH2-containing
PTPase SHP-1 and SHP-2 have been reported to associate with SHIP (33, 37), and SHP-2 may associate with CDw150 transfected in COS cells (16),
we tested whether the 65- to 68-kDa band was one of these phosphatases.
Indeed, sequential reprecipitation showed that this protein bound by
CDw150ct-PY could be specifically reprecipitated with SHP-1 mAb (data
not shown); however, we were unable to detect SHP-1 and SHP-2
specifically associated with native CDw150 precipitated from MP-1 cells
(data not shown). Kinetics experiments showed that up to 60 min after
CDw150 ligation, the level of tyrosine phosphorylation of either SHP-1
or SHP-2, which is an indicator of PTPase activity (39), did not change
(data not shown). Thus, the PTPase activity constitutively associated
with CDw150 in B cells is most likely to be mediated by CD45. CDw150 regulates CD95-mediated apoptosis
Many of the molecules associated with CDw150 are involved in the regulation of apoptosis (16, 19, 34, 40, 41), and a defect in one of these, DSHP/SAP, leads to B cell lymphoproliferative disease, XLP (16, 17). Moreover, three-color staining of tonsillar B cells and analysis of the expression of CDw150 on B cell lines revealed that CDw150 invariably is coexpressed on cells together with CD95/Fas (data not shown). Therefore, we decided to test whether CDw150 is involved in the regulation of CD95-mediated apoptosis.
A number of cell lines express both CDw150 and CD95, including the B
cell lines Raji, Namalwa, CESS, RPMI-1788, and MP-1 and the T cell line
HPB-ALL. Each of these lines was incubated with CDw150 mAb, CD95 mAb,
or a combination of the two mAb. The MOPC 21 myeloma protein (IgG1)
served as a negative control. The level of apoptotic cells was
evaluated using annexin V, as illustrated with the MP-1 line in Fig. 6. These results were confirmed using
fluorescent dye Hoechst 33342 (data not shown). Anti-CDw150 alone
(1–20 µg/ml) did not induce apoptosis (spontaneous apoptosis =
2–6%). However, anti-CD95 (0.1–5.0 µg/ml, optimal dose 1
µg/ml) induced apoptotic death within 4 h that peaked by 24
h (Fig. 6). Preincubation of cells with anti-CDw150 for at least 30
min before cross-linking CD95 resulted in a synergistic effect both in
the number of apoptotic cells and the rate of apoptosis (Fig. 6). These
effects were detected in the B cell lines Raji, RPMI-1788, and MP-1,
and also in the immature T cell line HPB-ALL.
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), ligation of CD40 for
30 min before CD95 stimulation rescued cells from CD95-mediated
apoptosis. At the same time co-cross-linking of both CDw150 and CD40
before ligation of CD95 restored the level of apoptotic cells. Kinetics
experiments revealed that the rescue effect of CD40 cross-linking was
evident as early as 2 h before CD95 ligation (Fig. 6B),
while coligation of CDw150 completely eliminated this protective effect
of CD40. Thus, in the human B cell lines MP-1, RPMI-1788, and Raji,
signaling via CD40 rescues cells from CD95-mediated apoptosis, but
ligation of CDw150 can block CD40-mediated protection. Conversely, CD40
ligation cancels out the synergistic effect of CD95 and CDw150.
Because CDw150 associates with SHIP and induces its tyrosine
dephosphorylation, and SHIP has been implicated in the regulation of
Akt/PKB (43, 44), we tested whether activation of Akt and/or tyrosine
phosphorylation of SHIP correlate with CDw150-mediated regulation of
CD95-induced death. The phosphorylation of Ser473 is one indicator of
Akt activation (45), so we evaluated the level of Akt activation by
Western blotting with phospho-Akt antisera. Stimulation of MP-1 cells
via either the BCR, CDw150, CD40, or CD40 plus CDw150 led to
phosphorylation of Akt (Fig. 7A). The highest level of Akt
phosphorylation was detected after BCR or CDw150 cross-linking.
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and 4). CD40 cross-linking
did not induce significant changes in tyrosine phosphorylation of SHIP,
but ligation of CDw150 alone or in combination with CD40 resulted in
dephosphorylation of SHIP (Fig. 7A). Ligation of CD95 did
not affect either Akt phosphorylation (data not shown) or SHIP tyrosine
phosphorylation (Fig. 7B). Dephosphorylation of SHIP was a
consistent feature specific for CDw150 cross-linking alone and was
evident when CDw150 was ligated in combinations with either CD40 or
CD95 (Fig. 7B). Thus serine phosphorylation of Akt did not
correlate with an anti-apoptotic signal via CD40 or a pro-apoptotic
signal via CDw150. But dephosphorylation of SHIP did correlate with the
pro-apoptotic effect induced via CDw150. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2), and the XLP gene
product, DSHP/SAP (16, 17). The cytoplasmic tail of CDw150 has two
pYxxV motifs. Phosphopeptides of this motif could bind SH2 domains of
both Fgr and SHIP (34, 46, 47). Interestingly, phosphopeptides with
this motif block the association of SHIP and Shc (34). Moreover, a
similar motif in the HS1 protein, after tyrosine phosphorylation, can
activate Fgr via binding to its SH2 domain (47). We are currently
testing the hypothesis that the pYxxV motifs in the cytoplasmic tail of
CDw150 can be bound by the SH2 domains of Fgr and SHIP and that these
interactions may be modulated by DSHP/SAP. If so, CDw150 could function
to regulate Fgr as well as to compete with Shc for binding to SHIP. To
clarify how defective DSHP/SAP leads to B cell proliferation (16, 17),
it will be important to determine whether and when DSHP/SAP interacts
with CDw150 and/or affects SHIP or Fgr activities. The fact that both
DSHP/SAP (17) and CDw150 (11, 12) are expressed in germinal center B
cells and B lymphomas suggests that a CDw150-DSHP/SAP pathway may
normally regulate B cell growth.
CDw150 is associated with both protein tyrosine and serine/threonine
kinases (8), which can phosphorylate it both in vitro (Fig. 1) and in
vivo, especially after BCR cross-linking (Fig. 3A). These
results suggest that CDw150 may regulate BCR signaling pathways as do
other coreceptors such as CD22 and CD19, which like CDw150 are
phosphorylated after BCR ligation (2, 30). We do not yet know which
kinases are responsible for CDw150 phosphorylation. In vitro kinase
assays using the nonphosphorylated tail of CDw150 (GST-CDw150ct) as a
substrate did not provide an answer because all the PTKs tested,
including the Src-family kinases Lyn, Fyn, Fgr, and Lck, as well as the
Syk PTK, phosphorylated the cytoplasmic tail of CDw150 in vitro (data
not shown). The phosphorylated tail of CDw150 is able to bind the
Src-family kinase Lyn (Fig. 1). However, this interaction may occur
indirectly via SHIP because 1) Lyn was not found to be associated with
native CDw150 in vivo and 2) Lyn always coprecipitated with SHIP (Fig. 4).
SHIP also binds to the tyrosine-phosphorylated tail of CDw150 (Figs. 1, 2). Unlike most 5'-phosphatases, this enzyme selectively hydrolyzes the
5'-phosphate from inositol-1,3,4,5-tetraphosphate (IP4) and
phosphatidylinositol-3,4,5-trisphosphate (PIP3), two inositol
phosphates implicated in signal transduction (48, 49). SHIP possesses
an SH2 domain critical for its tyrosine phosphorylation and association
with Shc (34). Not only does CDw150 bind SHIP, ligation of CDw150
induces tyrosine dephosphorylation of SHIP. This is the first evidence
of receptor-induced tyrosine dephosphorylation of SHIP. SHIP becomes
tyrosine-phosphorylated in response to stimulation via, e.g., a number
of cytokines, high-affinity IgE receptors, or coligation of BCRs with
FcRIIB (31, 46, 48, 50, 51, 52, 53, 54). Tyrosine phosphorylation of SHIP on Tyr
917 and Tyr 1020 is required for SHIP to be bound by Shc through its
PTB domain (32, 34, 55). Furthermore, the SH2-domain of SHIP is
required in order for SHIP to be translocated to membranes or to be
tyrosine-phosphorylated and in order for SHIP to affect cell fate (34, 36, 51, 56). Together these results have led to a model suggesting that
the tyrosine phosphorylation of SHIP is essential for its functions
(50). According to this model, CDw150-induced dephosphorylation of SHIP
would reduce its activity. CDw150-triggered dephosphorylation of SHIP
occurs even in the presence of signals via CD40 and/or CD95. The fact
that SHIP-/- cells, like CDw150-stimulated cells, are
more susceptible to activation-induced cell death (41) is consistent
with this possibility.
Ligating CDw150 promoted CD95/Fas-mediated cell death, and overall
increased cell death correlated with SHIP dephosphorylation, implying
that SHIP may play a role in CDw150-regulated cell death. SHIP could
regulate apoptosis via a number of possible mechanisms. First, through
hydrolysis of PIP3 to phosphatidylinositol-3,4-bisphosphate (PI3,
4-P2), SHIP might promote the activation of Akt (43, 44), which may
prevent apoptosis (57), e.g., by serine phosphorylation of BAD, a
pro-apoptotic member of the Bcl-2 family (58, 59). However, in this
study, the phosphorylation of Akt on Ser473 did not correlate with
either promotion or protection from cell death (Fig. 7A).
A second possibility is that SHIP may affect B cell fate through competition with Grb2 to bind to tyrosine-phosphorylated Shc so that activation of the Ras pathway is prevented (32, 34, 35). We have not detected Shc associated with CDw150 or SHIP before or after CDw150 ligation (data not shown). Shc is not required for BCR-induced activation of the mitogen-activated protein kinase family kinase, Erk2, in some B cells (60); thus, it is unclear to what extent Shc affects B cell fate. Finally, reduction of SHIP activity in B cells through CDw150 may increase [Ca2+]i and thereby promote cell death. SHIP-/- B cells display enhanced mobilization of [Ca2+]i after BCR ligation, suggesting that SHIP normally functions in B cells to inhibit [Ca2+]i (41, 61). Scharenberg et al. (62) recently reported that SHIP can inhibit a PIP3/Btk-dependent calcium signaling pathway. Ligation of CDw150 in B cells both increases [Ca2+]i (8) and reduces SHIP tyrosine phosphorylation; thus, it is possible that CDw150 ligation increases levels of PIP3 and Btk activity. Btk has been shown to promote sustained increases in [Ca2+]i (63) and to be essential for BCR-induced cell death (64). Increasing [Ca2+]i in B cells can promote cell death (65, 66), and blocking [Ca2+]i with bis(2-aminophenoxy)ethane-N,N,N[prime,N'-tetraacetate (BAPTA-AM) can prevent BCR-induced death (67). Furthermore, CD95/Fas-induced B cell death also requires a change in [Ca2+]i (68).
Taken together, just as the BCR, CD40, and CD95/Fas can induce positive or negative signals to B cells depending on the context (4, 42), it is likely that CDw150 has a dual function as well. Ligation of CDw150 stimulates T cell proliferation (9) and also in B cells has a costimulatory effect with CD40 and IL-4 (8). However, signaling via CDw150 also modulates CD95-mediated apoptosis, and the lack of a key CDw150-binding protein, DSHP/SAP, leads to susceptibility to uncontrolled B cell growth (16, 17). Further insights into how CDw150 regulates T and B cell fate will come as we learn more about how the various signaling components interacting with CDw150 affect each other.
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Acknowledgments |
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Footnotes |
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2 S.V.M. and L.M.S. contributed equally to this work.
3 Current address: Xcyte Therapies, 2203 Airport Way South, Suite 300, Seattle, WA 98134.
4 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195. E-mail address:
5 Abbreviations used in this paper: BCR, B cell antigen receptor; [Ca2+]i, intracellular free calcium; CDw150ct, cytoplasmic tail of CDw150; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; PY, phosphotyrosine; SAP, SLAM-associated protein; SH2, Src-homology 2; SHIP, SH2-containing inositol phosphatase; SLAM, signaling lymphocytic activation molecule; XLP, X-linked lymphoproliferative disease.
Received for publication December 10, 1998. Accepted for publication February 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk. Mol. Cell. Biol. 16:1305.[Abstract]
1 upon B cell activation. J. Exp. Med. 183:547. immunoreceptor tyrosine-based inhibition motif peptide motif. Mol. Cell. Biol. 17:4305.[Abstract]
RIIb. Curr. Opin. Immunol. 10:306.[Medline]
RIIB1 and is tyrosine phosphorylated during ’negative’ signaling. Immunol. Lett. 54:77.[Medline]
RIIB. Nature 383:263.[Medline]
receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell. Immunol. Lett. 57:159.[Medline]
2 in ERK, JNK and p38 MAP kinase activation by the B cell antigen receptor. J. Exp. Med. 188:1287.2. J. Exp. Med. 184:31.This article has been cited by other articles:
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 |
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  Q. Chen, J. L. Cannons, J. C. Paton, H. Akiba, P. L. Schwartzberg, and C. M. Snapper A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in Response to Intact Streptococcus pneumoniae versus Pneumococcal Conjugate Vaccine J. Immunol., December 15, 2008; 181(12): 8258 - 8266. [Abstract] [Full Text] [PDF]
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 G. Roncador, J.-F. G. Verdes-Montenegro, S. Tedoldi, J. C. Paterson, W. Klapper, E. Ballabio, L. Maestre, S. Pileri, M.-L. Hansmann, M. A. Piris, et al. Expression of two markers of germinal center T cells (SAP and PD-1) in angioimmunoblastic T-cell lymphoma Haematologica, August 1, 2007; 92(8): 1059 - 1066. [Abstract] [Full Text] [PDF]
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 J. L. Cannons, L. J. Yu, D. Jankovic, S. Crotty, R. Horai, M. Kirby, S. Anderson, A. W. Cheever, A. Sher, and P. L. Schwartzberg SAP regulates T cell-mediated help for humoral immunity by a mechanism distinct from cytokine regulation J. Exp. Med., June 12, 2006; 203(6): 1551 - 1565. [Abstract] [Full Text] [PDF]
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 M. Morra, R. A. Barrington, A. C. Abadia-Molina, S. Okamoto, A. Julien, C. Gullo, A. Kalsy, M. J. Edwards, G. Chen, R. Spolski, et al. Defective B cell responses in the absence of SH2D1A PNAS, March 29, 2005; 102(13): 4819 - 4823. [Abstract] [Full Text] [PDF]
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 S. V. Mikhalap, L. M. Shlapatska, O. V. Yurchenko, M. Y. Yurchenko, G. G. Berdova, K. E. Nichols, E. A. Clark, and S. P. Sidorenko The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells Blood, December 15, 2004; 104(13): 4063 - 4070. [Abstract] [Full Text] [PDF]
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 G. G. Welstead, E. C. Hsu, C. Iorio, S. Bolotin, and C. D. Richardson Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein J. Virol., September 15, 2004; 78(18): 9666 - 9674. [Abstract] [Full Text] [PDF]
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S. G. Tangye, K. E. Nichols, N. J. Hare, and B. C. M. van de Weerdt Functional Requirements for Interactions Between CD84 and Src Homology 2 Domain-Containing Proteins and Their Contribution to Human T Cell Activation J. Immunol., September 1, 2003; 171(5): 2485 - 2495. [Abstract] [Full Text] [PDF]
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D. Howie, S. Okamoto, S. Rietdijk, K. Clarke, N. Wang, C. Gullo, J. P. Bruggeman, S. Manning, A. J. Coyle, E. Greenfield, et al. The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production Blood, September 26, 2002; 100(8): 2899 - 2907. [Abstract] [Full Text] [PDF]
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 S. Ek, C.-M. Hogerkorp, M. Dictor, M. Ehinger, and C. A. K. Borrebaeck Mantle Cell Lymphomas Express a Distinct Genetic Signature Affecting Lymphocyte Trafficking and Growth Regulation as Compared with Subpopulations of Normal Human B Cells Cancer Res., August 1, 2002; 62(15): 4398 - 4405. [Abstract] [Full Text] [PDF]
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 M. Leitges, K. Gimborn, W. Elis, J. Kalesnikoff, M. R. Hughes, G. Krystal, and M. Huber Protein Kinase C-{delta} Is a Negative Regulator of Antigen-Induced Mast Cell Degranulation Mol. Cell. Biol., June 15, 2002; 22(12): 3970 - 3980. [Abstract] [Full Text] [PDF]
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P. Chu, D. Deforce, I. M. Pedersen, Y. Kim, S. Kitada, J. C. Reed, and T. J. Kipps Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia PNAS, March 19, 2002; 99(6): 3854 - 3859. [Abstract] [Full Text] [PDF]
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D. Howie, M. Simarro, J. Sayos, M. Guirado, J. Sancho, and C. Terhorst Molecular dissection of the signaling and costimulatory functions of CD150 (SLAM): CD150/SAP binding and CD150-mediated costimulation Blood, February 1, 2002; 99(3): 957 - 965. [Abstract] [Full Text] [PDF]
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M. Kruse, E. Meinl, G. Henning, C. Kuhnt, S. Berchtold, T. Berger, G. Schuler, and A. Steinkasserer Signaling Lymphocytic Activation Molecule Is Expressed on Mature CD83+ Dendritic Cells and Is Up-Regulated by IL-1{beta} J. Immunol., August 15, 2001; 167(4): 1989 - 1995. [Abstract] [Full Text] [PDF]
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M. J. Czar, E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, A. Sher, C. S. Duckett, R. Ahmed, et al. Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP PNAS, June 7, 2001; (2001) 131193098. [Abstract] [Full Text] [PDF]
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C. Erlenhoefer, W. J. Wurzer, S. Löffler, S. Schneider-Schaulies, V. ter Meulen, and J. Schneider-Schaulies CD150 (SLAM) Is a Receptor for Measles Virus but Is Not Involved in Viral Contact-Mediated Proliferation Inhibition J. Virol., May 15, 2001; 75(10): 4499 - 4505. [Abstract] [Full Text]
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L. M. Shlapatska, S. V. Mikhalap, A. G. Berdova, O. M. Zelensky, T. J. Yun, K. E. Nichols, E. A. Clark, and S. P. Sidorenko CD150 Association with Either the SH2-Containing Inositol Phosphatase or the SH2-Containing Protein Tyrosine Phosphatase Is Regulated by the Adaptor Protein SH2D1A J. Immunol., May 1, 2001; 166(9): 5480 - 5487. [Abstract] [Full Text] [PDF]
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J. E. Damen, M. D. Ware, J. Kalesnikoff, M. R. Hughes, and G. Krystal SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation Blood, March 1, 2001; 97(5): 1343 - 1351. [Abstract] [Full Text] [PDF]
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N. Ono, H. Tatsuo, K. Tanaka, H. Minagawa, and Y. Yanagi V Domain of Human SLAM (CDw150) Is Essential for Its Function as a Measles Virus Receptor J. Virol., February 15, 2001; 75(4): 1594 - 1600. [Abstract] [Full Text]
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 Z. Korade-Mirnics and S. J. Corey Src kinase-mediated signaling in leukocytes J. Leukoc. Biol., November 1, 2000; 68(5): 603 - 613. [Abstract] [Full Text]
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A. G. Castro, T. M. Hauser, B. G. Cocks, J. Abrams, S. Zurawski, T. Churakova, F. Zonin, D. Robinson, S. G. Tangye, G. Aversa, et al. Molecular and Functional Characterization of Mouse Signaling Lymphocytic Activation Molecule (SLAM): Differential Expression and Responsiveness in Th1 and Th2 Cells J. Immunol., December 1, 1999; 163(11): 5860 - 5870. [Abstract] [Full Text] [PDF]
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 M. E. March, D. M. Lucas, M. J. Aman, and K. S. Ravichandran p135 Src Homology 2 Domain-containing Inositol 5'-Phosphatase (SHIPbeta ) Isoform Can Substitute for p145 SHIP in Fcgamma RIIB1-mediated Inhibitory Signaling in B Cells J. Biol. Chem., September 22, 2000; 275(39): 29960 - 29967. [Abstract] [Full Text] [PDF]
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N. Mavaddat, D. W. Mason, P. D. Atkinson, E. J. Evans, R. J. C. Gilbert, D. I. Stuart, J. A. Fennelly, A. N. Barclay, S. J. Davis, and M. H. Brown Signaling Lymphocytic Activation Molecule (CDw150) Is Homophilic but Self-associates with Very Low Affinity J. Biol. Chem., September 1, 2000; 275(36): 28100 - 28109. [Abstract] [Full Text] [PDF]
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M. J. Czar, E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, A. Sher, C. S. Duckett, R. Ahmed, et al. Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP PNAS, June 19, 2001; 98(13): 7449 - 7454. [Abstract] [Full Text] [PDF]
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