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IMMUNOBIOLOGY
From the Terry Fox Laboratory, British Columbia Cancer
Agency, Vancouver, British Columbia, Canada.
The SH2-containing inositol-5'-phosphatase, SHIP, restrains bone
marrow-derived mast cell (BMMC) degranulation, at least in part, by
hydrolyzing phosphatidylinositol (PI)-3-kinase generated PI-3,4,5-P3 (PIP3) to PI-3,4-P2. To
determine which domains within SHIP influence its ability to hydrolyze
PIP3, bone marrow from SHIP Mast cells have a critical role in initiating acute
inflammatory responses against invading bacteria, helminthic parasites, and harmless allergens.1 On exposure to multivalent
antigens or allergens, these cells rapidly degranulate, releasing
preformed inflammatory mediators that act on the vasculature, smooth
muscles, connective tissue, mucous glands, and inflammatory cells to
cause immediate-type hypersensitivity reactions.2
Understanding how this degranulation process is regulated is key to our
being able to effectively treat inflammatory disorders such as
allergies, which affect as many as 20% of the Western population. We
recently demonstrated that the hemopoietic-specific Src-homology 2 containing inositol phosphatase, SHIP, is a key negative regulator or
"gatekeeper" of normal mast cell degranulation, setting the
threshold for and limiting IgE-induced degranulation3 as
well as preventing Steel factor (SF)-induced intracellular signaling
from progressing to degranulation.4 This protein was
originally identified as a 145-kd protein that became both tyrosine
phosphorylated and associated with the adaptor protein Shc in response
to multiple cytokines and to B- and T-cell antigen receptor engagement
in hemopoietic cells.5,6 Subsequent cloning revealed that
it possessed an amino terminal SH2 domain, a central inositol
polyphosphate 5'-phosphatase catalytic domain that could hydrolyze
phosphatidylinositol-3,4,5-trisphosphate (PIP3) in vivo, 2 NPXY sequences that, when phosphorylated, could bind
phosphotyrosine-binding (PTB) domains, and a proline-rich C-terminus
that was theoretically capable of binding to many SH3 domain-containing
proteins7 (Figure 1).
Initial studies showing that its enzymatic activity did not change
following cytokine stimulation,7 coupled with subsequent
studies indicating that SHIP exerted its effects in mast cells and B
cells by translocating to the plasma membrane,8 suggested
that it acts in large part, if not exclusively, by breaking down
antigen-, IgE-, or SF-induced PIP3 at the plasma membrane
to PI-3,4-P2. This reduces the ability of certain
pleckstrin homology (PH)-containing proteins (eg, protein kinase B
[PKB/Akt], phosphoinositide-dependent protein kinase 1 [PDK1],
Bruton's tyrosine kinase [Btk]) to target to the plasma membrane and
be activated.9-13 There is a good deal of support for this
mode of action. For example, it has been shown that SHIP breaks down
both cytokine- and antigen-induced PIP3 to
PI-3,4-P2 in mast cells and B cells4,10 and
this leads to a reduction in extracellular calcium entry4
and a decrease in the activation of the survival enhancing
serine/threonine kinase, PKB/akt.10-12 It has also been
shown in both mast cells and B cells that colocalization of the
inhibitory coreceptor, Fc
However, although there is much support for SHIP exerting its biologic
effects through its ability to hydrolyze PIP3, other data
show that SHIP acts in part by competing with Grb2 for Shc and thereby
reducing Ras activation.16,17 Because the Ras pathway has
been shown to be important for survival in interleukin
(IL)-3-stimulated cells,18 this competition may be
responsible, at least in part, for the reduced viability we observed in
SHIP overexpression studies in DA-ER cells.19 In fact,
strong evidence for this competition playing a role in
Fc Given SHIP's many protein-binding domains, it has the potential to
interact with many signal transduction intermediates and a number of
these have already been identified.5,6 For example, it has
been shown to bind to Shc via SHIP's SH2 and tyrosine phosphorylated NPXY motifs,19 SHP-2 via SHIP's SH2 domain20
and Grb2 in some cells (eg, B cells),21 but not in others
(eg myeloid cells)17 via SHIP's proline-rich regions.
SHIP also binds via its SH2 domain (which binds preferentially to
pY(Y/D)X(L/I/V))22 to the tyrosine phosphorylated ITIM of
certain inhibitory coreceptors (ie, the Fc To gain some insight into the role that SHIP's various domains play in
preventing SF-induced signaling from progressing to degranulation in
mast cells, we retrovirally infected bone marrow from
SHIP Construction of SHIP mutants
Mast cell isolation and FACS analysis
Northern Blot analysis The BMMCs of all SHIP-infected populations were washed once with phosphate-buffered saline (PBS) and then lysed at 1 × 107 cells/mL using TRIzol (Gibco BRL, Burlington, ON). Total RNA was isolated and separated on a 1% formaldehyde/agarose gel, transferred to a nylon membrane (Zeta-Probe, Bio-Rad, Mississauga, ON), prehybridized, hybridized, and washed as previously described.30 Probes used for hybridization were a 250-bp fragment of the most 5' part of the first exon of SHIP and the entire puromycin cDNA removed from the MSCVpac viral vector.Immunoprecipitation and immunoblotting Cells were starved overnight in IMDM, with 10% FCS and 150 µM MTG, washed, and stimulated at 37°C for various times with 400 ng/mL SF. The cells were then washed with ice-cold PBS and solubilized either by boiling 3 minutes with sodium dodecyl sulfate (SDS) sample buffer for total cell lysates or with 0.5% NP-40 in 4°C phosphorylation solubilization buffer17 at 4 × 107 cells/mL and subjected to immunoprecipitation as indicated. Western blotting was carried out as described previously.17 The anti-SHIP antibody (anti-N) was generated in rabbits using a glutathione S-transferase (GST) fusion protein containing the SH2 domain of murine SHIP (amino acids 7-133), the anti-GFP monoclonal antibody was from Clontech (catalogue no. 8362, Palo Alto, CA), the anti-Shc antibody was from Transduction Laboratories (catalogue no.14630, Lexington, KY), the anti-phosphoMAPK antibody was from New England BioLabs (Beverly, MA), the anti-MAPK antibody (Erk 1 CT) was a generous gift from Dr Steven Pelech and the antiphosphotyrosine antibody, 4G10, was purchased from Upstate Biotechnology Inc (Lake Placid, NY).PIP3 measurements Determination of PIP3 levels were performed essentially as described by Huber and coworkers.4Calcium measurements SHIP+/+ and SHIP / BMMCs, as well as
SHIP/ BMMCs expressing various SHIP constructs
(5 × 106 cells/mL), were incubated with 2 µM
fura-2/AM (Molecular Probes, Eugene, OR) in Tyrode
buffer31 at 23°C for 45 minutes. The cells were then
washed, resuspended in 1 mL of the same buffer at
5 × 105 cell/mL in a stirring cuvette. Following
stimulation by addition of 400 ng/mL SF, cytosolic calcium was measured
by monitoring fluorescence intensity at 510 nm, after excitation of the
sample with 2 different wavelengths (340 and 380 nm) using an MC200
monochromator from SLM AMINCO (Rochester, NY) with a 8100 V3.0 software program.
Degranulation assay Cells were washed with and resuspended in Tyrode buffer and then treated for 15 minutes with or without 400 ng/mL SF. The degree of degranulation was determined by measuring the release of -
hexosaminidase.32
Plasma membrane preparation The SHIP+/+ BMMCs, as well as SHIP/
BMMCs expressing WT and T1 SHIP constructs, were starved overnight in
IMDM with 1% FCS and 150 µM MTG, washed, and treated with and
without 200 ng/mL SF for 3 minutes at 37°C. Plasma membrane-enriched
membrane fractions were prepared as described by Huber and
colleagues.33 The final NP40 solubilized membrane
fraction, which was highly enriched for plasma membranes (as assessed
by biotinylating the cell surface of intact BMMCs (Hughes and Krystal,
manuscript in preparation), was then subjected either to Western blot
analysis with anti-SHIP or anti-HA antibodies and the blot reprobed
with anti-c-kit as a loading control.
Introduction of SHIP mutants into SHIP / BMMCs with SF or IgE results in substantially
more PIP3 and significantly less PI-3,4-P2 than
in SHIP+/+ BMMCs.5 To identify the domains
within SHIP that play a role in regulating both PIP3 levels
and degranulation, we initially constructed the N-terminal-HA and
C-terminal GFP-tagged SHIP constructs shown in Figure 1. Specifically,
in addition to the WT construct containing the full-length 1190 amino
acids of murine SHIP, we generated an R34G mutant, lacking a functional
SH2 domain, a D675G mutant in which a critical aspartic acid in its
catalytic domain was replaced with a glycine, rendering this enzyme
90% inactive (Ravichandran, personal communication, May 2000),
a 2NPXF mutant in which the tyrosines within the 2 PTB consensus
motifs, INPXY and ENPXY, were converted to phenylalanines, and a
C-terminally truncated form of SHIP containing 1 to 983 amino acids of
murine SHIP (T2).
In these initial studies, bone marrow from SHIP
Western blot analysis of SDS-lysed total cell lysates using anti-SHIP antibodies (Figure 2C) confirmed the GFP FACS results shown in Figure 2A, specifically, that the expression of all but the R34G construct was similar at the protein level, and also showed that their levels were about half that of the endogenous SHIP levels in SHIP+/+ BMMCs. Interestingly, in this regard, GFP FACS analysis of BMMC progenitors containing the R34G SHIP mutant revealed a low but detectable level of this protein at 3 weeks of suspension. However, it became totally undetectable by 6 weeks (data not shown). This is consistent with progenitors expressing high levels of this construct being selected against during BMMC maturation (as assessed by IgE R expression) and precluded our determining the role that SHIP's SH2 domain plays in regulating PIP3 levels and degranulation in this study. Expression of WT, but not D675G nor T2 SHIP, in
SHIP / BMMCs (data
not shown), we used this time point to determine if introducing WT-SHIP
into SHIP/ BMMCs reduced the SF-induced increase in
PIP3 levels to that observed in SHIP+/+ cells.
As can be seen in Figure 3A, the results
of several experiments revealed that although there was considerable
variation in the SF-induced PIP3 levels generated in
uninfected or vector control-infected SHIP/ BMMCs,
they were all significantly higher than those achieved in
SHIP+/+ BMMCs. Expressing the WT-SHIP construct in
SHIP/ BMMCs reduced the SF-induced PIP3
levels to very near the levels found in SHIP+/+ BMMCs. The
effect of the various SHIP mutants on SF-induced PIP3 levels was then determined and, as shown in Figure 3B, the D675G-SHIP was found incapable of reverting the PIP3 levels to those
seen in SHIP+/+ cells, as would be expected for a mutant in
which the enzymatic activity is severely compromised. The 2NPXF mutant
on the other hand was partially capable of reducing the
PIP3 levels to those observed in SHIP+/+ cells.
In fact, with longer time in suspension culture (approximately 5 months), cells expressing this mutant were capable of hydrolyzing PIP3 as well as WT-expressing cells (data not shown).
Intriguingly, the C-terminally truncated SHIP mutant, T2, was
completely unable to revert the PIP3 levels to
SHIP+/+ cell levels. This is especially interesting given
that Aman and coworkers very recently found that a C-terminally
truncated SHIP similar to T2 possessed full in vitro enzymatic
activity.34
T2-SHIP does not become tyrosine phosphorylated in response to SF To gain some insight, at the signaling level, into why the T2 SHIP mutant was incapable of hydrolyzing PIP3, we first compared the tyrosine phosphorylation patterns of the various SHIPs in response to SF. Specifically, SHIP/ BMMCs expressing the various
SHIPs were stimulated with SF for 0, 2, 5, and 10 minutes and then
subjected to anti-SHIP immunoprecipitation and Western blot analysis
with antiphosphotyrosine antibodies. As can be seen in Figure
4A, the tyrosine phosphorylation levels of WT, 2NPXF, and D675G SHIP were similar following SF stimulation. However, the T2 mutant was negligibly phosphorylated. Reprobing with an
anti-SHIP antibody demonstrated equal loading of the gel (Figure 4B).
These results suggest that SHIP's proline rich C-terminus is required
for its SF-induced tyrosine phosphorylation. Also, because the tyrosine
phosphorylation level of the 2NPXF mutant, following SF stimulation,
was close to that observed with the WT and D675G forms of SHIP, it
suggests that the tyrosines in the NPXYs are not the major
phosphorylation sites in SF-stimulated BMMCs.
In the present study we found that extraction of endogenous SHIP from
SHIP+/+ BMMCs or exogenously expressed full-length SHIPs
from SHIP To further elucidate the signaling capacity of T2 SHIP and the other
mutants we determined their capacity to associate, following SF
stimulation, with Shc. As a first step, we asked if Shc was tyrosine
phosphorylated in response to SF to the same degree in SHIP
Having established that Shc was tyrosine phosphorylated to the same
degree, following SF stimulation, in the various SHIP Because, on some occasions we observed a low but detectable level of T2
SHIP in our anti-Shc immunoprecipitates, we became concerned at this
point that the one remaining NPXY motif in our T2 mutant might confound
the interpretation of our results. We therefore constructed 2 more
truncated mutants, T1 (1-912 amino acids), which lacked both NPXY
motifs, and T3 (1-1027 amino acids), which contained both NPXY motifs
(Figure 6A) and generated a second set of
SHIP
C-terminally truncated SHIPs do not inhibit SF-induced calcium entry We had shown previously that SF stimulation of SHIP/ BMMCs leads to a far higher entry of
extracellular calcium than in SHIP+/+ BMMCs.4
We therefore compared the abilities of our various SHIP constructs to
revert the SF-induced intracellular calcium concentrations to that
observed in SHIP+/+ BMMCs. As shown in Figure
7, WT-SHIP effectively reduced the calcium levels to that observed in SHIP+/+ cells,
reflecting its effects on PIP3 levels (Figure 3A). The D675G, on the other hand, was totally incapable of lowering the SF-induced calcium levels, showing that the phosphatase activity of
SHIP is critical to this function in these cells (as was shown in
chicken DT40 B cells following BCR/Fc RIIB
stimulation8). Of interest, although the 2NPXF
SHIP mutant was also totally incapable of lowering calcium levels, it
did partially hydrolyze PIP3, suggesting that these 2 events may be uncoupled to some extent. Also of interest, the T1 and T3
truncated mutants were not only incapable of lowering the calcium
concentrations following SF stimulation to those in SHIP+/+
BMMCs, they consistently raised them above that seen in the
SHIP/ cells (Figure 7).
C-terminally truncated SHIPs are ineffective at reducing SF-induced degranulation SHIP prevents SF from inducing the degranulation of normal BMMCs.4 We therefore compared the abilities of our SHIP constructs to revert the SF-induced SHIP/ to a
SHIP+/+ BMMC degranulation phenotype. As can be seen in
Figure 8A, GFP-infected SHIP/ BMMCs degranulated to a far greater degree than
GFP-infected SHIP+/+ BMMCs, as expected (no differences
were observed between uninfected and empty (GFP) MSCVpac-infected
SHIP+/+ or SHIP/ BMMCs in terms of
PIP3, Ca++ or degranulation levels). As well,
SHIP/ BMMCs expressing WT-SHIP almost completely
reverted the degranulation phenotype to SHIP+/+ BMMCs,
whereas the D675G did not. The 2NPXY construct was partially capable of
reducing the SF-induced degranulation, but both truncated mutants were
totally ineffective. This degranulation pattern was directly
proportional to the PIP3 levels exhibited in these cells (Figures 3B and 6C).
T1-SHIP does not translocate to the plasma membrane on SF stimulation To gain some insight into why the C-terminally truncated SHIPs were ineffective at reducing SF-induced degranulation, we compared the ability of T1, WT, and endogenous SHIP to translocate, in response to SF stimulation, to the plasma membrane fraction of BMMCs. As can be seen in Figure 8B, WT-SHIP in SHIP/ BMMCs translocated
to the plasma membrane in a similar fashion to endogenous SHIP in
SHIP+/+ BMMCs. However, no increased levels of T1-SHIP were
apparent on SF-stimulation of SHIP/ BMMCs expressing
this construct. Reprobing with anti-c-kit established equal loading of
the stimulated and unstimulated lanes.
We show herein that introduction of WT-SHIP into
SHIP What is it that makes these last 163 amino acids of SHIP so crucial? It
is possible that one or more of the 3 resident PXXP sequences in this
region may participate in the recruitment of SHIP to a specific
location within the plasma membrane and/or submembranous cytoskeleton,
affecting both access to its substrate and its association with other
phosphotyrosine-binding proteins. The fact that we see the truncated
SHIP mutant T1 at the membrane, even though it does not translocate,
suggests that this mutant may not translocate to a specific subcellular
compartment where PI-3-kinase and the substrate PIP3 are
available. In support of this Petrie and coworkers38
have recently shown that SHIP and PI-3-kinase translocate into
a low-density insoluble fraction, lipid rafts, within 2 minutes
following BCR stimulation. It is possible therefore, that SHIP
recycling to lipid rafts may be compromised. Aman and
colleagues34 have also found incomplete restoration of
SHIP function in a SHIP deletion mutant (1-900 amino acids) fused to
the Fc Previous studies have suggested that SHIP's SH2 domain is integral to
the association of SHIP with the plasma membrane where it can hydrolyze
its membrane-associated substrate, PIP3. Our early
overexpression studies with WT and R34G SHIP demonstrated that SHIP's
SH2 domain was essential for SHIP's IL-3-induced tyrosine phosphorylation, its association with Shc, and its ability to increase
the apoptosis of confluent DA-ER cells.19 As well, SHIP's
SH2 domain has been shown to bind to several phosphorylated ITAM motifs
in vitro,22,27 the ITIM of Fc Our finding that the tyrosine phosphorylation levels of WT and 2NPXF
are similar after SF stimulation (Figure 4) was unexpected given that
the tyrosines within the 2 NPXY motifs have been reported to be the
major tyrosine phosphorylation sites in SHIP, at least in the
TCR-stimulated murine T-cell hybridoma cell line, BYDP.36 Interestingly, in COS cells this 2NPXF mutant was only slightly less
tyrosine phosphorylated than WT-SHIP when the Src kinase, Lck, was
coexpressed36 and our results may therefore reflect a
greater contribution of Lyn to SHIP's overall tyrosine phosphorylation in BMMCs than in BYDP cells. Furthermore, in keeping with the very
recent results in BCR/Fc Our finding that Shc is tyrosine phosphorylated in response to SF to
almost the same degree in SHIP The fact that SHIP's C-terminus is critical to its function has an impact on the roles of the C-terminally truncated forms of SHIP that have been detected.35,42 Mounting evidence suggests that SHIP may localize to different cellular compartments38 and this localization may be regulated by the C-terminal PXXP motifs. Typically, when we SDS-lyse normal BMMCs we obtain more full-length SHIP than when solubilizing at 4°C with NP40 or TX100 (which yields primarily the 135-kd SHIP species). This is consistent with there being a pool of SHIP that is not accessible using nonionic lysis procedures. If we solubilize the NP40-insoluble, cytoskeletal fraction from these BMMCs with SDS sample buffer we also obtain full-length SHIP. Additionally, it has been shown in platelets that thrombin stimulates the tyrosine phosphorylation and translocation of SHIP to the actin cytoskeleton43 and synaptojanin 2, another inositol polyphosphate 5-phosphatase containing a proline-rich C-terminus, is located predominantly within the particulate fraction.44 This latter observation is of particular interest because a second isoform, synaptojanin 1, differs markedly in its proline-rich tail and resides primarily in the cytosol.44 In summary, we show herein that SHIP's C-terminal 163 amino acids are essential for SHIP to reduce SF-induced PIP3, intracellular Ca++, and degranulation levels in BMMCs. Although much remains to be done to elucidate how it regulates these effects, it is worthy of note that SHIP's C-terminus is very different from that of SHIP244 and this may allow for differential regulation. Incidentally, the type I 5-phosphatases and the type II INPP5P, which do not contain proline-rich tails, have C-terminal prenylation sites that mediate their membrane association similar to those found for the Rho/Rac family of signaling intermediates.44 It is tempting to speculate that SHIP and SHIP2 may use their proline-rich tails in lieu of prenylation to assist in their recruitment to a specific location within the plasma membrane, in a cooperative effort with their SH2 domains.
We thank Christine Kelly for typing the manuscript and Vivian Lam for excellent technical support.
Submitted July 3, 2000; accepted October 16, 2000.
Supported by the NCI-C and the MRC-C with core support from the BC Cancer Foundation and the BC Cancer Agency. J.K. and M.R.H. hold NSERC studentships. G.K. is a Terry Fox Cancer Research Scientist of the NCI-C and supported by funds from the Canadian Cancer Society and the Terry Fox Run.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gerald Krystal, Terry Fox Laboratory, 601 West 10th Ave, Vancouver, British Columbia, Canada, V5Z 1L3; e-mail: gerryk{at}terryfox.ubc.ca.
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© 2001 by The American Society of Hematology.
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  H. S. Ramshaw, M. A. Guthridge, F. C. Stomski, E. F. Barry, L. Ooms, C. A. Mitchell, C. G. Begley, and A. F. Lopez The Shc-binding site of the {beta}c subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis Blood, November 15, 2007; 110(10): 3582 - 3590. [Abstract] [Full Text] [PDF]
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 W.-H. Leung and S. Bolland The Inositol 5'-Phosphatase SHIP-2 Negatively Regulates IgE-Induced Mast Cell Degranulation and Cytokine Production J. Immunol., July 1, 2007; 179(1): 95 - 102. [Abstract] [Full Text] [PDF]
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J. Ai, A. Maturu, W. Johnson, Y. Wang, C. B. Marsh, and S. Tridandapani The inositol phosphatase SHIP-2 down-regulates Fc{gamma}R-mediated phagocytosis in murine macrophages independently of SHIP-1 Blood, January 15, 2006; 107(2): 813 - 820. [Abstract] [Full Text] [PDF]
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K. Gimborn, E. Lessmann, S. Kuppig, G. Krystal, and M. Huber SHIP Down-Regulates Fc{epsilon}R1-Induced Degranulation at Supraoptimal IgE or Antigen Levels J. Immunol., January 1, 2005; 174(1): 507 - 516. [Abstract] [Full Text] [PDF]
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 M. G. Tomlinson, V. L. Heath, C. W. Turck, S. P. Watson, and A. Weiss SHIP Family Inositol Phosphatases Interact with and Negatively Regulate the Tec Tyrosine Kinase J. Biol. Chem., December 31, 2004; 279(53): 55089 - 55096. [Abstract] [Full Text] [PDF]
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Y. Wang, R. J. Keogh, M. G. Hunter, C. A. Mitchell, R. S. Frey, K. Javaid, A. B. Malik, S. Schurmans, S. Tridandapani, and C. B. Marsh SHIP2 Is Recruited to the Cell Membrane upon Macrophage Colony-Stimulating Factor (M-CSF) Stimulation and Regulates M-CSF-Induced Signaling J. Immunol., December 1, 2004; 173(11): 6820 - 6830. [Abstract] [Full Text] [PDF]
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V. Hernandez-Hansen, A. J. Smith, Z. Surviladze, A. Chigaev, T. Mazel, J. Kalesnikoff, C. A. Lowell, G. Krystal, L. A. Sklar, B. S. Wilson, et al. Dysregulated Fc{epsilon}RI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells J. Immunol., July 1, 2004; 173(1): 100 - 112. [Abstract] [Full Text] [PDF]
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H. Fang, R. A. Pengal, X. Cao, L. P. Ganesan, M. D. Wewers, C. B. Marsh, and S. Tridandapani Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP J. Immunol., July 1, 2004; 173(1): 360 - 366. [Abstract] [Full Text] [PDF]
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 K. Nakamura, T. Kouro, P. W. Kincade, A. Malykhin, K. Maeda, and K. M. Coggeshall Src Homology 2-containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow J. Exp. Med., January 20, 2004; 199(2): 243 - 254. [Abstract] [Full Text] [PDF]
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V. Lam, J. Kalesnikoff, C. W. K. Lee, V. Hernandez-Hansen, B. S. Wilson, J. M. Oliver, and G. Krystal IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor Blood, August 15, 2003; 102(4): 1405 - 1413. [Abstract] [Full Text] [PDF]
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 E. Marion, P. J. Kaisaki, V. Pouillon, C. Gueydan, J. C. Levy, A. Bodson, G. Krzentowski, J.-C. Daubresse, J. Mockel, J. Behrends, et al. The Gene INPPL1, Encoding the Lipid Phosphatase SHIP2, Is a Candidate for Type 2 Diabetes In Rat and Man Diabetes, July 1, 2002; 51(7): 2012 - 2017. [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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J. Kalesnikoff, N. Baur, M. Leitges, M. R. Hughes, J. E. Damen, M. Huber, and G. Krystal SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-{kappa}B Activity J. Immunol., May 1, 2002; 168(9): 4737 - 4746. [Abstract] [Full Text] [PDF]
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 J. M. Dyson, C. J. O'Malley, J. Becanovic, A. D. Munday, M. C. Berndt, I. D. Coghill, H. H. Nandurkar, L. M. Ooms, and C. A. Mitchell The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin J. Cell Biol., December 10, 2001; 155(6): 1065 - 1080. [Abstract] [Full Text] [PDF]
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R. Xu, J. Abramson, M. Fridkin, and I. Pecht SH2 Domain-Containing Inositol Polyphosphate 5'-Phosphatase Is the Main Mediator of the Inhibitory Action of the Mast Cell Function-Associated Antigen J. Immunol., December 1, 2001; 167(11): 6394 - 6402. [Abstract] [Full Text] [PDF]
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E. Marion, P. J. Kaisaki, V. Pouillon, C. Gueydan, J. C. Levy, A. Bodson, G. Krzentowski, J.-C. Daubresse, J. Mockel, J. Behrends, et al. The Gene INPPL1, Encoding the Lipid Phosphatase SHIP2, Is a Candidate for Type 2 Diabetes In Rat and Man Diabetes, July 1, 2002; 51(7): 2012 - 2017. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
R1- and BCR-induced PIP3
levels and this, in turn, inhibits calcium influx sufficiently to
curtail degranulation and proliferation,
respectively.
) were preloaded with 2 µM fura-2/AM and
then stimulated with 400 ng/mL SF. Cytosolic calcium was measured by
monitoring fluorescence intensity at 510 nm, after excitation of the
sample with 2 different wavelengths (340 and 380 nm).
