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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-04-1058.
IMMUNOBIOLOGY
From the Department of Experimental Medicine and
Pathology, Istituto Pasteur-Fondazione Cenci Bolognetti, University
"La Sapienza," and Department of Clinical Biochemistry, Istituto
Superiore di Sanita', Rome, Italy; and Istituto Mediterraneo di
Neuroscienze "Neuromed," Pozzilli, Italy.
Membrane recruitment of the SH2containing 5' inositol
phosphatase 1 (SHIP-1) is responsible for the inhibitory signals that modulate phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. Here we have investigated the molecular mechanisms underlying SHIP-1 activation and its role in CD16-mediated cytotoxicity. We
initially demonstrated that a substantial fraction of SHIP-1-mediated 5' inositol phosphatase activity associates with CD16 CD16, the low-affinity receptor for the Fc fragment
of IgG (Fc CD16-mediated recognition of antibody-coated target cells triggers NK
cytotoxic response, which requires congregation of signaling molecules
into the supramolecular activation cluster that leads to the
polarization of lytic granules and raft microdomains into the area of
NK-target cell contact.3-6
Lipid rafts are specialized plasma membrane microdomains where
signaling complexes are nucleated after receptor engagement. Recent
studies have demonstrated that MIRR engagement results in receptor
enrichment in the raft compartment along with key signaling molecules,
such as protein tyrosine kinases, lipid kinases, adaptor proteins, and
phosphoinositides.2,7
Among the signaling pathways responsible for NK-cell cytotoxicity,
granule polarization, and secretion, a fundamental role for
phosphatidylinositol 3-kinase (PI3K) has been recently
reported.8,9 By mediating membrane recruitment of
pleckstrin homology (PH) domain-containing signaling proteins,
phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3) represents a critical upstream component of
major signaling pathways, and its synthesis and degradation require a
tight control.10 SH2-containing 5' inositol phosphatase
1 (SHIP-1) represents a major route for degradation of
PI3,4,5P3 through its conversion into
PI3,4P2.11,12 SHIP-1 activation has been
implicated mostly in the negative signaling mediated by the inhibitory
receptor Fc Reagents
Preparation of human NK cells
SHIP-1 5'-phosphatase assay To produce the substrate and standards for SHIP 5'-phosphatase assay, we prepared 32P-PI3,4,5P3 and 32P-PI3,4P2 using commercial sources of PI4,5P2 and PI4P, 32P -adenosine
triphosphate (ATP), and the recombinant constitutively active form of
PI3K (p110*) as described elsewhere.21 Briefly, 32P-P3,4,5P3 in chloroform/methanol (1:1,
vol/vol) was dessiccated and resuspended by sonication in SHIP
assay buffer (50 mM Tris [tris(hydroxymethyl)aminomethane],
pH 7.5, 0.125% NP40). SHIP-1,  chain, or shc was immunoprecipitated
from unstimulated or CD16-stimulated NK cells. One hundred microliters
of substrate in SHIP assay buffer was mixed with 50 µL 50 mM Tris, pH
7.5, and 30 mM MgCl2 containing the specific
immunoprecipitates, for 60 minutes at 37°C in the presence of a
mixture of protease inhibitors. After extraction of phospholipids with
a chloroform/methanol mixture (methanol/1 M HCl/chloroform, 10:7:20),
the organic phase containing SHIP substrate was dried, resuspended in
30 µL chloroform/methanol (1:1), and separated by thin-layer
chromatography (TLC) using aluminium-backed Silica gel 60 plates (Merck, Darmstadt, Germany) saturated with 1% potassium oxalate
in 50% methanol, as previously described.22 The plates
were developed in chloroform/acetone/methanol/acetic acid/water
(40:15:13:12:8) and the radioactive lipids visualized by
autoradiography. The identity of PI3,4,5P3 and
PI3,4P2 was confirmed by comparison with
32P-PI3,4,5P3 and
32P-PI3,4P2, prepared separately, and run on
the same TLC plate. PI3,4,5P3 and PI3,4P2 were
quantified by densitometric analysis. Ten million cell equivalents of
the immunoprecipitated samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by
anti-SHIP, anti-, or anti-shc immunoblot.
Raft isolation and immunoprecipitation Lipid rafts were isolated as reported23 with slight modifications. Briefly, NK cells stimulated with control mAb-, anti-CD16-, or human IgG-coated beads (3 × 108) were gently sonicated in 1 mL ice-cold TNE buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA [ethylenediaminetetraacetic acid]) supplemented with a mixture of protease and phosphatase inhibitors. After centrifugation at 800g for 10 minutes at 4°C, the postnuclear supernatants were incubated with Triton X-100 at a final concentration of 0.05% for 1 hour at 4°C. The lysate was then mixed with an equal volume (1 mL) of 80% sucrose prepared in TNE buffer, placed in an ultracentrifuge tube, and carefully overlaid with 2 mL 30% sucrose and 1 mL 5% sucrose. Tubes were centrifuged at 48 000 rpm for 17 hours at 4°C. Then, 400-µL fractions were harvested from the top. Protein content determination in the isolated fractions was performed by colorimetric assay. SDS-PAGE was loaded with equal amounts of proteins recovered from each fraction. Ponceau S red staining of the nitrocellulose membrane confirmed an equivalent protein content between the lanes. Rafts were recovered mainly from the low-density fractions 3, 4, 5, and 6.For immunoprecipitation studies, equivalent amounts of proteins of raft
(pooled 3, 4, 5, 6 fractions) and soluble (pooled 10, 11, 12 fractions)
samples were treated with 60 mM N-octylglucoside for 1 hour at 4°C.
SHIP-1 and Confocal microscopy To analyze SHIP-1 localization in raft domains, NK cells were stained with Alexa Fluor 594-conjugated CTB (40 µg/mL) at 4°C for 40 minutes. In the last 20 minutes, anti-CD16 (B73.1) or anti-MHC class I control mAb was added. FcR+ P815 target cells were pretreated with Alexa Fluor 594-conjugated CTB as above. After washing, effector (E) and target (T) cells were resuspended in warm RPMI 1640 medium, mixed together (E/T ratio, 2:1), briefly pelleted, and incubated for 3 minutes at 37°C. The pellet was gently resuspended and spun onto ice-cold poly-L-lysine-coated glass slides. Cells were then fixed in 3.7% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in phosphate-buffered saline (PBS) bovine serum albumin (BSA) 1%. Permeabilized cells were stained using 1:100 dilution of anti-SHIP mAb for 60 minutes at room temperature, washed, and then incubated with 1:100 dilution of FITC-labeled GAM. To analyze SHIP and CD16 distribution, NK cells were treated with FITC-conjugated anti-CD16 or anti-CD56 mAb. NK cells were then left alone or allowed to form conjugates with P815 target cells, spun onto ice-cold poly-L-lysine-coated slides, fixed, and permeabilized, as above. Cells were then stained with 1:200 dilution of anti-SHIP polyclonal antibody for 60 minutes at room temperature, washed, and then incubated with 1:200 dilution of Texas red-labeled goat antirabbit IgG.To analyze CD16 distribution in the rafts, NK cells were treated with Alexa Fluor 594-conjugated CTB as above. In the last 20 minutes, FITC-conjugated anti-CD16 or anti-TfR mAb was added. NK cells were then left alone or allowed to form conjugates with CTB-labeled P815 target cells as above, spun onto ice-cold poly-L-lysine-coated slides, and fixed. The slides were then mounted in antifade reagent containing glycerol buffer and analyzed for rafts, SHIP-1, or CD16 cellular distribution using a confocal fluorescence microscope (Leika TCSD4, Norkfork, Germany). Fifty to 100 conjugates were evaluated per slide. Vaccinia virus infection Wild-type and recombinant vaccinia viruses encoding Flag-tagged wild-type (SHIP-WT) or a catalytic domain-deleted SHIP (SHIP- CAT) were kindly provided by Dr Andrew M. Scharenberg (University of Washington, Seattle).24,25 Viruses were amplified,
semipurified, and titrated using standard
techniques.26 Semipurified vaccinia viruses were used to
infect human NK cells for 1 hour in serum-free medium at a multiplicity
of infection of 20:1. Cells were then incubated for an additional 4 hours in RPMI containing 0.1% BSA and 25 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). After infection, dead cells were removed by
Ficoll-Hypaque density gradient centrifugation for 30 minutes at
1600 rpm.
Cytotoxicity assay The 51Cr release reverse antibody-dependent cellular cytotoxicity (ADCC) assay was performed as previously described.19 The murine FcR+ P815 mastocytoma cell line was used for reverse ADCC. Maximal and spontaneous releases were determined by incubating 51Cr-labeled target cells with 1 M HCl or medium alone, respectively.
SHIP-1-mediated inositol 5'-phosphatase activity associates with
CD16 chain through
the adaptor protein shc.18 We extended this initial observation and explored whether SHIP-1-mediated 5' phosphatase activity would actually associate with CD16 receptor complex. SHIP-1
selectively hydrolyzes the 5'-phosphate of PI3,4,5P3
leading to its conversion to PI3,4P2.11,12
Lysates from CD16-stimulated human NK cells were immunoprecipitated
with anti-shc, anti-, or control mAb and tested for the inositol
5'-phosphatase activity using radiolabeled PI3,4,5P3 as
substrate. As shown in the autoradiogram in Figure
1A, 5'-phosphatase activity leading to
almost complete dephosphorylation of PI3,4,5P3 to
PI3,4P2, is present in SHIP-1 immunoprecipitates, but not
in control mAb-precipitated samples. The catalytic activity detectable
in anti-SHIP immunoprecipitates from unstimulated cells was not
modulated following mAb-mediated CD16 stimulation (not shown). On the
other hand, CD16 ligation induced the appearance of 5'-phosphatase
activity in immunoprecipitates. Kinetic analysis of
receptor-associated catalytic activity shows a peak of substrate
conversion at 2 minutes, which declines almost completely at 10 minutes. CD16 cross-linking also results in increased 5'-phosphatase
activity in shc immunoprecipitates. In some donors we found low amounts
of basal substrate conversion that coprecipitates with shc, likely
attributable to basal levels of shc/SHIP-1 complexes detectable in
unstimulated NK cells (not shown). Anti-MHC class I mAb-treated NK
cells used as negative control showed the same activity observed in
unstimulated samples (not shown). As evaluated by blotting with
anti-shc or anti- mAb, equal amounts of protein were loaded in each
lane (Figure 1B).
The percentage of conversion of PI3,4,5P3 to
PI3,4P2 in the different samples was then calculated on the
basis of equivalent cell number, arbitrarily assuming as 100% the
catalytic activity in the SHIP-1 immunoprecipitates (Figure 1C). On
receptor triggering, almost 30% of total 5'-phosphatase activity was
found associated with CD16 These data indicate that CD16 ligation on NK cells induces the association of SHIP-1-mediated 5'-inositol phosphatase activity with the CD16 receptor complex. CD16 cross-linking induces the transient translocation of SHIP-1 to the raft fraction Increasing evidence has highlighted the critical role of recruitment and assembly of signaling complexes in the detergent-insoluble glycosphingolipid-enriched membrane microdomains, termed rafts.2,7Because cellular phosphoinositides, including the SHIP-1 natural
substrate PI3,4,5P3, are highly enriched in the raft
microdomains,26,27 we analyzed whether CD16 triggering
could induce SHIP-1 redistribution into this cellular compartment. To
this purpose, we fractionated NK-cell lysates on sucrose gradient
centrifugation into 12 fractions. The position of lipid raft-containing
fractions in the sucrose gradient was determined by Western blot
analysis of the presence of GM1 ganglioside using the GM1-specific
ligand CTB. As shown in Figure 2D, GM1 is
particularly enriched in fractions 3 to 6. In contrast, tubulin is
completely excluded from the lipid rafts (Figure 2B), as
described.22 Fractions 8 to 12 derived from the Triton
X-100-soluble compartment represent cytosolic and nonraft membrane
fractions. We examined the distribution of SHIP-1 before and after CD16
stimulation that was obtained either by specific anti-CD16 mAb (Figure
2A, right) or by human IgG-coupled polystyrene beads (Figure 2E,
right) to mimic CD16 natural ligand. We found very low basal levels of
SHIP-1 in the rafts of unstimulated or IgG F(ab')2-coupled
bead-stimulated NK cells (Figure 2A,E, left), but the phosphatase
accumulates in this compartment on CD16 stimulation (Figure 2A,E,
right). The amount of SHIP-1 detected in the rafts on receptor
ligation was a substantial portion of the protein detected in the
soluble fraction of unstimulated cells. As expected, the adaptor
protein LAT was also enriched in the raft fractions although its
content was not increased on CD16 stimulation (Figure 2C).
We then analyzed the kinetics of SHIP-1 translocation to the raft
domains on CD16 receptor triggering. Immunoprecipitation and Western
blot analysis of pooled sucrose-gradient fractions reveal an early and
transient translocation of SHIP-1 to the rafts that is detectable
already at 30 seconds, is maximal at 2 minutes, and completely recedes
at 10 minutes after stimulation (Figure 3).
To determine the cellular distribution of SHIP-1 and raft domains in NK
cells engaged by reverse ADCC, we performed confocal microscopy
analysis. FcR+ P815 target cells and anti-CD16 or control
mAb-treated human NK cells were stained with Alexa-Fluor
594-conjugated CTB, and coincubated for 3 minutes at 37°C. After
fixing and permeabilization, cells were stained with anti-SHIP mAb
followed by FITC-conjugated secondary mAb. The percentage of E/T
conjugates was close to 35% in anti-CD16-stimulated samples, whereas
it reached only 7% in control mAb-treated samples (not shown). Figure
4 shows representative photograms of NK
cells alone or conjugated with antibody-coated target cells. Like T
cells,28 NK cells alone as well as control mAb-treated NK
cells contacting P815 targets show a mostly cytoplasmic distribution of
SHIP (Figure 4A,D). In contrast, after CD16 cross-linking by means of
reverse ADCC, SHIP-1 undergoes marked redistribution toward the
NK/target cell contact area (Figure 4G). In such conditions, as
previously described,4 lipid rafts cluster and polarize in
"macroraft" structures (Figure 4H). Superimposition of the red and
green fluorophores gave a yellow area at the NK/target interface,
clearly demonstrating the colocalization of polarized SHIP-1 and
clustered rafts (Figure 4I). Polarization is observed in about 70% of
anti-CD16-treated NK/target cell conjugates (not shown) but not in NK
cells alone or in control mAb-treated NK cells contacting P815 targets
(Figure 4C,F), where no macroraft formation occurred (Figure 4B,E).
These results further support the evidence that the inhibitory signaling molecule SHIP-1 translocates to raft microdomains on CD16 engagement on NK cells. CD16 receptor complex mediates the recruitment of SHIP-1 to
the raft compartment via receptor We initially analyzed by confocal microscopy the relative distribution
of CD16 receptor and SHIP-1. To this purpose, NK cells were stained at
4°C with FITC-conjugated anti-CD16 or anti-CD56 mAb. Cells were
either directly fixed or allowed to bind P815 target cells for 3 minutes at 37°C and then fixed. After fixing and permeabilization,
cells were stained with anti-SHIP polyclonal antibody followed by Texas
red-conjugated secondary antibody. Representative images in Figure
5 show that both CD16 and CD56 are
distributed throughout the plasma membrane (Figure 5B,H). On CD16
stimulation by means of reverse ADCC, both SHIP-1 and CD16 undergo
marked redistribution toward the NK/target cell contact area (Figure
5J-K) where large receptor clusters are formed. Superimposition of the
red and green fluorophores gave a yellow area at the NK/target interface, demonstrating the colocalization of polarized SHIP-1 and
CD16 receptor (Figure 5L). Polarization is not observed in NK cells
alone or in control mAb-treated NK cells contacting P815 targets
(Figure 5A-I).
To better understand the mechanisms involved in SHIP-1 interaction with
CD16 receptor and whether such a complex is formed in the raft domains,
we analyzed the redistribution of Unphosphorylated
To analyze whether phospho- We also found that the relative amounts of SHIP-1, shc, and phospho- Time-course analysis of the formation of the SHIP-1/shc/ CD16 receptor accumulates in the lipid rafts after its engagement In human NK cells, chain has been recently shown to be part of
activating receptors other than CD16.30 We therefore
analyzed by confocal microscopy whether the CD16 receptor
ligand-binding subunit colocalizes with rafts also.
To this purpose, NK cells were stained at 4°C with FITC-conjugated
anti-CD16 or anti-TfR mAb and Alexa Fluor 594-conjugated CTB. Cells
were either directly fixed or allowed to bind CTB-labeled P815 target
cells for 3 minutes at 37°C and then fixed. Representative images in
Figure 7 show that both CD16 and rafts
are distributed throughout the plasma membrane and partially colocalize
(yellow points) in the absence of receptor cross-linking (Figure 7A-C). When anti-CD16-treated NK cells are engaged by P815 target cells, large receptor clusters are formed, mostly polarized toward the area of
target cell contact (Figure 7D), that colocalize with polarized
macrorafts (Figure 7E-F). As expected, in anti-TfR-treated control
NK cells either alone or conjugated with P815 cells, no TfR/GM1
colocalization was observed (Figure 7G-L).
Collectively, our results demonstrate the ligand-dependent selective accumulation of CD16 receptor complexes within the rafts. SHIP-1 catalytic activity is involved in the regulation of CD16-dependent NK cell-mediated cytotoxicity To directly define a functional role for SHIP-1 in CD16-mediated cytotoxicity, we used the vaccinia virus expression system to overexpress SHIP-WT or the SHIP-CAT mutant. Human NK cells were
infected with the recombinant vaccinia viruses and SHIP-1 expression
was assessed by Western blot analysis using anti-Flag mAb (Figure
8). Uninfected and SHIP-WT-,
SHIP-CAT-, or control vector-infected NK cells were tested in a
reverse ADCC assay using FcR-bearing P815 target cells in the presence
of anti-CD16 mAb. SHIP-WT-infected NK cells exhibited a significant
down-regulation of CD16-mediated killing when compared with the
cytotoxic activity of NK cells infected with the empty virus (Figure
8). The overexpression of the catalytic domain-deleted mutant SHIP
showed the same cytotoxicity as the control virus-infected cells, in
accordance with previous evidence showing that SHIP-CAT does not
behave as a dominant-negative construct.24 The reliability
of vaccinia virus expression system was indicated by the modest
decrease of specific lysis shown by control virus-infected NK cells
compared with the uninfected ones. We performed trypan blue exclusion
analysis to rule out the possibility that SHIP overexpression could
affect cell viability. No major differences in the number of viable
cells were detected in SHIP-WT-infected cells with respect to empty
virus-infected cells (not shown).
No detectable cytotoxicity against P815 targets was observed in the absence of anti-CD16 mAb at the indicated E/T ratios (not shown). Taken together, these results suggest that SHIP-1 is involved in the down-regulation of CD16-mediated NK cytotoxicity and that SHIP-1 inhibitory function depends on its enzymatic activity.
In the past few years since its original identification, SHIP-1 phosphatase has been shown to play a key role as a negative signaling molecule by its ability to reduce the levels of PI3,4,5P3, thus removing a membrane-targeting signal for PH domain-containing effector molecules.11,12 Although SHIP-1 has been demonstrated mainly to be responsible for the
inhibitory activity of Fc Here we provide novel insights on the mechanisms of SHIP-1 compartmentalization and activation induced by CD16 stimulation on NK cells as well as on its functional role in the modulation of ADCC function. Our data first demonstrate that CD16 receptor complex associates with SHIP-1-mediated phosphatase activity following receptor ligation, in that a substantial fraction (near to 30%) of SHIP-1 activity coprecipitates with the engaged receptor. Among the mechanisms required for SHIP-1 activation, plasma membrane localization of the enzyme directly contributes to lipid phosphatase-mediated substrate hydrolysis, whereas its tyrosine phosphorylation does not affect the enzymatic activity.22 Lipid rafts are specialized regions of the plasma membrane that provide an important scaffold for the assembly of functional signaling complexes.2,7 Raft clustering and polarization at the NK/target cell contact area allow the concentration and exclusion of specific membrane proteins and permit the enrichment of downstream mediators, thus orchestrating positive and negative signals crucial for the development of cytotoxicity.4-6 We show here that after CD16 engagement, SHIP-1 rapidly and transiently translocates to the raft fraction where most phosphoinositides, including PI3,4,5P3, are highly enriched.26,27 Accordingly, SHIP-1 translocation to lipid rafts has been described in response to B-cell receptor (BCR) stimulation.32 We have previously demonstrated that after reverse ADCC, a
SHIP-1/shc/ To explore the possibility that CD16 receptor complex could mediate
SHIP-1 recruitment to raft domains, we initially analyzed CD16
distribution in these domains, because no data are presently available.
We report here the presence of After its cross-linking, CD16 is clustered and colocalizes with lipid
macrorafts, and hyperphosphorylated p21-23 The kinetics of SHIP-1 recruitment to rafts is quite rapid and strictly
overlapping that of CD16-mediated PI3K activation.36 At
variance with our data, a recent report describing SHIP-1 translocation to rafts on BCR/Fc The activation of the cytolytic machinery is a tightly regulated process. Recent reports have highlighted a pivotal role of PI3K in the regulation of NK cytotoxicity.8,9 In this context, PI3K has been found to control the activation of the vav/Rac1 pathway and the downstream PAK and ERK1/2.9 Moreover, PI3,4,5P3 has been identified as a component of the signaling pathways coupling tyrosine kinases to Ca++ mobilization.38 Notably, rac1, ERK1/2 effectors, and Ca++ elevation critically control cytolytic granule polarization and exocytosis.9,19,39-43 Our results demonstrate that SHIP-1 could limit activation signals necessary for CD16-dependent cytotoxic function. Overexpression of SHIP-WT reduced CD16-mediated cytotoxicity and the functional catalytic domain of SHIP-1 is required, in that overexpression of the phosphatase inactive mutant did not affect the cytotoxic function. Collectively, our findings promote the view that recruitment of SHIP-1 to rafts is an important step that couples CD16 receptor complex to phosphoinositide turnover. By mediating SHIP-1 recruitment/activation, CD16 may quench the magnitude of PI3K-initiated signals, thereby restricting the duration or intensity (or both) of cytotoxic function. Among the negative signals controlling NK cytotoxicity, much work has been focused on the role of the tyrosine phosphatase Src homology 2 domain containing tyrosine phosphotase (SHP-1) coupled to a number of inhibitory NK receptors3; recently, however, a role of SHIP has emerged in regulating the NK repertoire and allogeneic bone marrow transplantation.44 Here we highlighted a functional role of SHIP-1 inositol phosphatase in the modulation of the molecular events regulating lymphocyte-mediated cytotoxicity. It is possible that the pharmacologic control of SHIP may allow for immunomodulation of ADCC function for therapeutic gain.
We thank D. Milana, A. M. Bressan, P. Birarelli, A. Procaccini, and A. Sabatucci for expert technical assistance.
Submitted April 8, 2002; accepted July 23, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-04-1058.
Supported in part by grants from Associazione Italiana per la Ricerca sul Cancro, Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica 40% and 60%, Ministero della Sanità, and Consiglio Nazionale delle Ricerche special project on Biotechnologies, Center for Excellence in Molecular Biology and Medicine.
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: Ricciarda Galandrini, Department of Experimental Medicine and Pathology, University "La Sapienza," Viale Regina Elena, 324, 00161 Rome, Italy.
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  S. V. Kondadasula, J. M. Roda, R. Parihar, J. Yu, A. Lehman, M. A. Caligiuri, S. Tridandapani, R. W. Burry, and W. E. Carson III Colocalization of the IL-12 receptor and Fc{gamma}RIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-{gamma} Blood, April 15, 2008; 111(8): 4173 - 4183. [Abstract] [Full Text] [PDF]
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 T. Tarasenko, H. K. Kole, A. W. Chi, M. M. Mentink-Kane, T. A. Wynn, and S. Bolland T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses PNAS, July 3, 2007; 104(27): 11382 - 11387. [Abstract] [Full Text] [PDF]
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 R. Parihar, R. Trotta, J. M. Roda, A. K. Ferketich, S. Tridandapani, M. A. Caligiuri, and W. E. Carson III Src Homology 2-Containing Inositol 5'-Phosphatase 1 Negatively Regulates IFN-{gamma} Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12 Cancer Res., October 1, 2005; 65(19): 9099 - 9107. [Abstract] [Full Text] [PDF]
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R. Galandrini, F. Micucci, I. Tassi, M. G. Cifone, B. Cinque, M. Piccoli, L. Frati, and A. Santoni Arf6: a new player in Fc{gamma}RIIIA lymphocyte-mediated cytotoxicity Blood, July 15, 2005; 106(2): 577 - 583. [Abstract] [Full Text] [PDF]
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R. Trotta, R. Parihar, J. Yu, B. Becknell, J. Allard II, J. Wen, W. Ding, H. Mao, S. Tridandapani, W. E. Carson, et al. Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation Blood, April 15, 2005; 105(8): 3011 - 3018. [Abstract] [Full Text] [PDF]
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M. I. Bonaparte and E. Barker Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules Blood, October 1, 2004; 104(7): 2087 - 2094. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
chain associated with homodimers or heterodimers of the
T-cell receptor 
RI).
tubulin (Tub 2.1) mAb were from Sigma-Aldrich (Milan, Italy).
Alexa Fluor 594-conjugated CTB subunit was from Molecular Probes
(Leiden, The Netherlands). Human recombinant PI3K p110 was from Alexis
(Nottingham, United Kingdom).
,
as assessed by immunofluorescence and cytofluorometric analysis. The
experiments were performed on NK-cell populations that were more than
90% pure. Antibody-mediated CD16 stimulation was performed as
described.
) or CD16-stimulated cultured NK cells
(3 × 108/sample) were fractionated by sucrose gradient
centrifugation as described in "Materials and methods." SHIP-1
immunoprecipitates were obtained from equal amounts of proteins from
solubilized raft and detergent-soluble fractions, and analyzed on 8%
SDS-PAGE by anti-SHIP-1 immunoblot (A). An aliquot of the pooled raft
or soluble fractions (as above) was loaded on 15% SDS-PAGE and
analyzed by Western blotting with antitubulin (B), anti-LAT (C), or
peroxidase-conjugated CTB (GM1) (D). An experiment
representative of 3 performed is shown.
) or with recombinant vaccinia virus encoding
Flag-tagged SHIP-WT (
) and SHIP-
) and were assayed in a
4-hour 51Cr release assay against P815 targets in the
presence of 5 µg/mL anti-CD16 mAb. Data are expressed as percent
specific cytotoxicity ± SD obtained from 3 independent
experiments. Overexpressed SHIP constructs in one representative
experiment are shown (inset).