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into the Cell Membranes, and Their Association with Gß After Activation of NK Cells with Chemokines1
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Introduction
of inhibitory Abs to PI 3-K
but not to PI 3-K into streptolysin
O-permeabilized NK cells also inhibits chemokine-induced NK cell
chemotaxis. Biochemical analysis showed that within 2–3 min of
activating NK cells, pleckstrin is recruited into NK cell membranes,
whereas PI 3-K associates with these membranes 5 min after
stimulation with RANTES. Recruited PI 3-K generates
phosphatidylinositol 3,4,5 trisphosphate, an activity that is inhibited
upon pretreatment of NK cells with wortmannin. Further analysis showed
that a ternary complex containing the ß dimer of G protein,
pleckstrin, and PI 3-K is formed in NK cell membranes after
activation with RANTES. The recruitment of pleckstrin and PI 3-K
into NK cell membranes is only partially inhibited by pertussis toxin,
suggesting that the majority of these molecules form a complex with
pertussis toxin-insensitive G proteins. Our results may have
application for the migration of NK cells toward the sites of
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), CC (ß), C (), and
CX3C (
) chemokines. The importance of chemokines and NK
cells in the eradication of viral infection has been explored by
Salazar-Mather et al. 3 , who observed that
MIP-13 recruits NK cells
into the sites of CMV foci, resulting in reduced infection with this
virus. In addition, members of the CC chemokines induce the
polarization of NK cells, and facilitate NK/target cell conjugate
formation 4 . The intracellular signaling pathways induced by
chemokines in NK cells in order for these cells to polarize and
extravasate into various tissues are largely unknown.
Chemokine receptors are coupled to the heterotrimeric G proteins in
various cell types 5 . The heterotrimeric G proteins are composed of
three subunits, , ß, and . The chemotactic effect of chemokines
is mediated through pertussis toxin (PT) substrates 2, 5 . Similarly,
MIP-1-, MCP-1-, RANTES-, SDF-1-, IP-10-, and lymphotactin-induced
NK cell chemotaxis is mediated through the heterotrimeric G proteins in
NK cells (reviewed in 6 . We have suggested previously that the
ß subunit of G proteins is involved in IP-10-, or
lymphotactin-induced NK cell chemotaxis 7 . Recently, the involvement
of Gß subunits in the chemotaxis of transfected cell lines has
been reported 8, 9 . However, the downstream signaling molecules
important for this activity have not been examined.
In addition to G protein-coupled receptors (GPCR), receptor tyrosine
kinases transmit intracellular signals resulting in the activation of
various molecules, such as the phosphoinositide 3-kinase (PI 3-K),
among others. PI 3-K is present in three different forms, I, II, and
III. These enzymes phosphorylate the D3 hydroxy position of the
inositol ring of phosphatidylinositol 10 . IA is composed of a
catalytic subunit of p110 , p110 ß, or p110 . These catalytic
subunits are associated with a regulatory p85 subunit. The latter
contains SH2 domains, SH3 domain, and proline-rich segments, which
facilitate its interaction with multiple sites present in various
proteins. On the other hand, IB (p110 subunit) does not have a site
that binds the regulatory p85, but instead has a motif, presumed to be
pleckstrin homology (PH), that binds the ß subunit of G proteins
11 . PH domains are present in more than 100 proteins, including GTP
exchange factors such as SOS and ARNO; GTPase-activating proteins such
as Ras-GAP; and in proteins such as dynamin, spectrin, general
receptors for phosphoinositide, in kinases such as Bruton’s tyrosine
kinase, and in phospholipases such as PLCß, PLC, or PLC, among
other molecules 12 . The PH domains associate with phospholipids to
recruit the PH-containing proteins in the proximity of their substrates
present in the cell membranes. For example, PH domain of PLC
associates with phosphatidylinositol 3,4,5 trisphosphate
(PI3, 4, 5 P3) and is recruited into the membrane by this
phospholipid, where it hydrolyzes its substrate, the PIP2
13 . Also, the PH domain of SOS is recruited into the membranes, where
it activates Ras 14 . In addition to binding phospholipids, PH domains
are important for protein-protein interactions. For example, the PH
domains of the ß adrenergic receptor kinase (G protein-coupled
receptor kinase 2) bind the ß dimer of PT-sensitive G proteins
15 . Although Gß activates PI 3-K, resulting in the activation
of the mitogen-activated protein kinase pathway 16 , or Jun kinase
17 , the binding of PI 3-K-PH to Gß has been disputed 18 .
Pleckstrin, the major substrate for protein kinase C in platelets, is
composed of two PH domains (N-terminal and C-terminal) separated by 150
amino acids. Activation of platelets with thrombin results in the
phosphorylation of pleckstrin by protein kinase C. Phosphorylated
pleckstrin in turn inhibits PI 3-K activity as a result of
activating GPCR 19 . This inhibition was reversed upon the addition of
purified Gß, suggesting that phosphorylated pleckstrin may
interact with the ß dimer. However, a correlation of coupling
and/or function between Gß, pleckstrin, and PI 3-K has not been
reported. In this study, we explored the possibility that there is an
interaction among these components as a result of activating NK cells
with chemokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture medium consisted of RPMI 1640 supplemented with 10% human AB serum (Ulleval Hospital, Oslo, Norway), 10 U/ml penicillin, 100 µg/ml streptomycin, 1 mM L-glutamine, 1% nonessential amino acids (all from Life Technologies, Paisley, U.K.), and 5 x 10-5 M 2-ME (Sigma, St. Louis, MO). AIM-V medium was from Life Technologies.
Preparation of NK cell membranes
IL-2-activated NK cells were prepared by adherence to plastic flasks, as previously described 20, 21 . The majority of these cells (more than 85%) show the CD16+CD56+CD3- phenotype, as determined by flow-cytometric analysis. Before performing the biochemical assays described in this work, these cells were incubated overnight in a serum-free (AIM-V) medium. This was necessary since serum has been reported to induce the recruitment of PH-containing proteins into the plasma membranes of various cell types 14 . NK cell membranes were prepared by incubating the cells in a lysis buffer containing 10 mM HEPES, pH 7.5, 3 mM MgCl2, 40 µg/ml PMSF, 10 µg/ml leupeptin, 2 µg/ml pepstatin A, and 2 µg/ml aprotinin. After homogenization and sonication, the mixtures were centrifuged at 1000 x g for 10 min. The supernatants were transferred into Beckman tubes; ultracentrifuged in a buffer containing 10 mM HEPES, 3 mM MgCl2, and 2 mM EDTA; and snap frozen at -70°C.
Chemotaxis assay
Nucleopore blind well chemotaxis chambers with a lower well volume of 200 µl were used. A maximum volume of 200 µl medium containing RPMI plus 1% BSA was placed in the lower wells in the presence or absence of various agents. Cells (1 x 105) were placed in the upper compartments of Boyden chambers above the filters. The chambers were incubated for 2 h at 37°C in a 5% CO2 incubator. The filters were then removed, dehydrated, and stained with 15% Giemsa stain for 7 min and then mounted on glass slides using a drop of immersion oil between the filters and the slides. Cells in 10 high power fields from two filters were counted and averaged for each sample. Migration index was calculated as the number of cells migrating toward the concentration gradients of chemokines, divided by the number of cells migrating toward medium only.
Permeabilization with streptolysin O (SLO)
The methods of permeabilization and introduction of Abs have
been previously described 7, 21 . In brief, human NK cells (5 x
106/100 µl) were incubated with 200 ng/100 µl of
activated SLO at 4°C for 15 min in a buffer containing 150 mM
K+-glutamate, 5 mM nitrilotriacetic acid, 0.5 mM EGTA,
0.2% BSA, and 10 mM PIPES, pH 7.2. The cells were washed three times
in the cold, and then warmed to 37°C, incubated for an additional 15
min, and then washed with a buffer containing 150 mM KCl and 20 mM
PIPES, pH 7.2. Goat affinity-purified anti-PI 3-K, rabbit
affinity-purified anti-PI 3-K (Santa Cruz Laboratories, San
Diego, CA), or goat IgG were incubated with SLO-permeabilized NK cells
for 1 h at 4°C. The cells were extensively washed, and then
incubated in culture medium for 3 h at 37°C to allow the cells
to rest before examining their ability to migrate in the
microchemotaxis chambers.
Coimmunoprecipitation and immunoblotting
Coimmunoprecipitation assay was done as described 22 . Protein
samples were incubated with 1/100 dilution of the proper Ab (mouse
anti-pleckstrin-PH from Transduction Laboratories (Lexington, KY),
rabbit anti-Gß from NEN-DuPont (Boston, MA), and goat anti-PI
3-K from Santa Cruz) in the immunoprecipitation buffer (10 mM Tris,
pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2
mM PMSF, 1% Triton-X, and 0.5% Nonidet P-40) overnight at
4°C with gentle shaking. About 20 µl of protein A/G agarose (Santa
Cruz) was added to each sample and incubated for another 4 h,
centrifuged, and washed three times, and the pellets were suspended in
electrophoresis sample buffers before running on SDS-PAGE.
Immunoblotting was performed by running the samples on SDS-PAGE and
then electrotransferred into polyvinylidene difluoride (PVDF)
membranes, blocked with 5% skim milk in TBS buffer for 2 h,
washed, and incubated with 1/1000 of the primary Abs, and 1/500
dilution of the secondary Abs. Development was done by using either
horseradish peroxidase-color development reagents (Bio-Rad) or enhanced
chemoluminescence (ECL) reagents (Amersham, Arlington Heights, IL)
according to the manufacturer’s specifications.
Pretreatment with PT or wortmannin
Cells were incubated overnight in AIM-V medium. They were either left intact or were treated overnight with 100 ng/ml activated PT (Sigma). These cells were harvested and membranes were prepared as described above. Pretreatment with wortmannin (Sigma) was done by incubating NK cells (1 x 106) with various concentrations of this metabolite at 37°C for 30 min, or with the appropriate concentration of DMSO, washed, and examined as above.
Phosphatidylinositol kinase assay
NK cells were either left intact or pretreated with 100 nM of
wortmannin. These cells were incubated with 100 pg/ml RANTES for 5 min,
and membranes were prepared from these cells. The membranes were
immunoprecipitated with anti-PI 3-K overnight at 4°C in the
presence of protein A/G agarose. The immunocomplex was washed once with
ice-cold PBS and once with a buffer containing 0.5 M LiCl, 100 mM
Tris-HCl, pH 7.5, and 1 mM sodium orthovanadate. It was then washed
once with distilled water, and once with a washing buffer containing 20
mM Tris-HCl, pH 7.5, 0.5 mM EGTA and 100 mM NaCl. The immunocomplex was
suspended in 50 µl of washing buffer, and was added to 0.5 µl of
PI4, 5 P2 (20 mg/ml; Sigma). This mixture was incubated for
10 min at 25°C with 10 µCi/sample of [-32P]ATP
(Amersham). MgCl2 (20 mM) was then added to this mixture,
which was incubated for additional 15 min. The reaction was stopped by
the addition of 150 µl of chloroform/methanol/11.6 N HCl, with the
volume ratio of 100:200:2. The organic phase was separated by the
addition of 100 µl chloroform. The lipid in the organic phase was
separated on TLC aluminum silica gel 60 precoated sheets (Merck,
Darmstadt, Germany). TLC plates were developed and resolved in
chloroform/methanol/ammonium hydroxide/distilled water, with the volume
ratio of 124:114:15:21. Radioactive PIP products were visualized by
autoradiography.
Statistical analysis
Significant values were determined by using the two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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controls chemokine-induced NK cell chemotaxis
PI 3-K has been shown to play an important role in NK cell
activity upon perturbation of FcR 23, 24 . In addition, wortmannin, a
fungal inhibitor of PI 3-K activity, inhibits RANTES-induced T cell
chemotaxis 25 . To investigate the role of PI 3-K in chemokine-induced
NK cell chemotaxis, we pretreated NK cells with different
concentrations of wortmannin. Fig. 1
shows that the C chemokine lymphotactin (Ltn), the CC chemokines MCP-1
and RANTES, or the CXC chemokines IP-10 and SDF-1 induced the in
vitro chemotaxis of NK cells (p < 0.005, as
compared with cells migrating toward culture medium only). Low
concentration of wortmannin (10 pM) did not inhibit chemokine-induced
NK cell chemotaxis. However, 1 nM of wortmannin inhibited the
chemotactic activity induced by Ltn, MCP-1, or IP-10
(p < 0.02, as compared with the migration of
cells not treated with wortmannin), whereas higher concentrations (10,
100, and 1000 nM) inhibited RANTES and SDF-1
(p < 0.05), as well as Ltn-, MCP-1-, and
IP-10-induced NK cell chemotaxis. None of the concentrations of
wortmannin used affected the viability of NK cells, as determined by
trypan blue exclusion test (not shown). To establish which PI 3-K
isotype is involved in the chemokine activity, we introduced Abs to PI
3-K into SLO-permeabilized cells. Results in the upper panel
of Fig. 2 show that affinity-purified
goat anti-PI 3-K inhibited Ltn, MCP-1, RANTES, IP-10, or
SDF-1 induction of NK cell chemotaxis (p <
0.001, p < 0.003, p < 0.004,
p < 0.02, and p < 0.001,
respectively, as compared with the migration of cells treated with goat
IgG). Neither goat IgG, nor Ab to PI 3-K inhibited the chemotactic
activity of the five chemokines examined (Fig. 2, upper
panel). Because all five chemokines examined exerted similar
effects, we have utilized the CC chemokine RANTES for further studies.
Dose-response curve shows that 0.01 and 0.1 µg of anti-PI
3-K/1 x 106 permeabilized NK cells are not
inhibitory, whereas 1 and 12.5 µg doses are inhibitory
(p < 0.005) for RANTES-induced NK cell
chemotaxis. As a control, different concentrations of this Ab were
incubated with unpermeabilized cells. None of these concentrations
inhibited RANTES-induced intact NK cell chemotaxis, indicating that the
Ab must enter permeabilized cells before it exerts its inhibitory
effect.
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plays an important role in the
chemotaxis of NK cells. To biochemically address the nature of PI
3-K effect, we investigated its recruitment to the membranes after
stimulating NK cells with RANTES. Fig. 3A shows that PI 3-K is not
present in NK cell membranes. However, it is recruited into these
membranes upon stimulation of these cells with RANTES, as determined by
immunoblotting NK cell membranes with a specific Ab to the catalytic
p110 subunit (lane 2). No such recruitment was
observed in the absence of stimulation with RANTES (lane
1 in Fig. 3A). To examine the association of PI 3-K
with Gß, NK cells were activated with culture medium or with
RANTES for 5 min. Membranes were prepared from these cells,
immunoprecipitated overnight with anti-PI 3-K, and then
immunoblotted with Ab to the common ß subunit of G protein. The
results in lane 1 of Fig. 3B show that in the
absence of stimulation with RANTES, anti-Gß picked up a 37-kDa
band representing Gß in NK cell membranes. Importantly, an
association between PI 3-K and ß subunit of G proteins
occurred after stimulation with RANTES (lane 2 in
Fig. 3B). Taken together, the results clearly show that
chemokine activation of GPCR results in the recruitment of PI 3-K
into NK cell membranes, and its association with the Gß dimer.
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After incubation in serum-free medium for 18 h, NK cells were
activated for 5 min with culture medium, Ltn, MCP-1, RANTES, IP-10, or
SDF-1. Membranes were prepared from these cells, and were examined
for the presence of pleckstrin. Fig. 4A shows that all five
chemokines induced the recruitment of pleckstrin into NK cell membranes
(lanes 2–6). To investigate whether chemokines
recruit pleckstrin from the cytosol, NK cells were treated with either
culture medium or RANTES. The results show that pleckstrin is abundant
in NK cell lysates, but not in NK cell membranes (lane
1 in the left and right panels of Fig. 4B). However, it is recruited into the membranes after
stimulating the cells for 5 min with RANTES (lane 2
in the left and right panels of Fig. 4B), suggesting that stimulation with chemokines facilitates
the distribution of this protein from the cytosol into the membranes. A
physical association between pleckstrin and Gß dimer was then
examined. NK cells were stimulated with culture medium or with RANTES,
and membranes were prepared from these cells, immunoprecipitated with
anti-pleckstrin-PH, and then immunoblotted with anti-Gß Ab
(left panel in Fig. 4C). Reciprocally, the
membranes were immunoprecipitated with anti-Gß, and then
immunoblotted with anti-pleckstrin-PH (right panel in
Fig. 4C). Only after stimulation with RANTES
(lane 2 in both panels of Fig. 4C) and not
with culture medium (lane 1 in both panels), an
association occurred between pleckstrin and Gß.
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dimer of PT-resistant G proteins forms a complex with
pleckstrin and PI 3-K in NK cell membranes
To demonstrate the nature of G proteins involved in the
recruitment of pleckstrin and PI 3-K, NK cells were either left
untreated or pretreated with 100 ng/ml of PT. Results in Fig. 5, A and B, show
that RANTES induced the recruitment of pleckstrin and PI 3-K,
respectively, as compared with culture medium-treated cells
(lane 3 versus lane 1). However, there was
only a partial inhibition of the recruitment of pleckstrin
(lane 2 in Fig. 5A) or PI 3-K
(lane 2 in Fig. 5B) upon pretreatment of
NK cells with 100 ng/ml of PT. Higher concentrations of PT affected the
viability of the cells (data not shown).
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into NK cell membranes is
resistant to wortmannin treatment
To investigate whether PI 3-K plays a role in the recruitment
of pleckstrin into NK cell membranes, NK cells were pretreated with
culture medium or wortmannin, washed, and then stimulated with RANTES.
In lane 1 of Fig. 5
C, a faint band representing
pleckstrin was present in NK cell membranes in the absence of
stimulation. The existence of such a band was variable in cells
generated from different donors. More important, treatment with RANTES
resulted in the recruitment of pleckstrin into NK cell membrane
(lane 3 in Fig. 5C). Concentration of
wortmannin (100 nM), which inhibited RANTES-induced NK cell chemotaxis
(Fig. 1), did not inhibit the recruitment of pleckstrin into NK cell
membranes (lane 2 in Fig. 5C).
Similarly, pretreatment of NK cells with wortmannin failed to inhibit
the association of PI 3-K with NK cell membranes upon stimulation
with RANTES. Fig. 6A shows
that incubation of NK cells for 5 min with RANTES, and not with culture
medium resulted in the association of PI 3-K with NK cell membranes
(left versus right lanes; similar to the
results obtained in Fig. 2A). Pretreatment of NK cells with
100 nM wortmannin before incubation with RANTES did not inhibit the
association of PI 3-K with NK cell membranes (Fig. 6A,
middle versus right lane). To correlate the
inhibitory effect of wortmannin on chemokine-induced NK cell chemotaxis
with the recruitment of PI 3-K into NK cell membranes, we examined
the ability of membrane-associated PI 3-K to generate phospholipids.
After stimulation with RANTES, NK cell membranes were
immunoprecipitated with Ab to PI 3-K overnight. This immune complex
was mixed with PI4, 5 P2 in the presence of
[32P]ATP. PI 3-K activity and the generation of
PI3, 4, 5 P3 were only seen after stimulation with
RANTES and not with culture medium (Fig. 6B,
right and left lanes, respectively). The ability
of PI 3-K immunoprecipitated from NK cell membranes after
stimulation with RANTES to generate PI3, 4, 5 P3 was
inhibited upon prior pretreatment of NK cells with 100 nM of wortmannin
(Fig. 6B, middle versus right lane).
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To examine the association of these molecules in NK cell
membranes, NK cells were stimulated with RANTES for 1–20 min, and
membranes were prepared from these cells, immunoprecipitated with
anti-PI 3-K, and then immunoblotted with Abs to either PI 3K-
(Fig. 7A) or pleckstrin-PH
(Fig. 7B). An association between these two molecules
occurred after 3 min (B). The same association was observed
when NK cell membranes were immunoprecipitated with anti-pleckstrin
first, and then immunoblotted with anti-PI 3-K (C).
However, the majority of the band detected after 3 min may be due to
pleckstrin since there was only a low recruitment of PI 3-K at this
time (A). A strong association between pleckstrin and PI
3-K occurred 5 min after stimulation with RANTES (B).
This coincided with the robust recruitment of PI 3-K into NK cell
membranes at this time point (A). The association between
these two molecules was also apparent 10 min after stimulation. Both
pleckstrin and PI 3-K almost disappeared from this complex 20 min
after stimulation with RANTES (B and C),
indicating a transient recruitment of these molecules after stimulation
with RANTES. Time kinetic association between pleckstrin and Gß
was also examined. After RANTES stimulation of NK cells for various
times, membranes from these cells were immunoprecipitated with
anti-pleckstrin-PH and then immunoblotted with anti-Gß. Fig. 7D shows that an association between these molecules
occurred after 2 min and was strong after 3 and 5 min of stimulation.
However, after 10 min, most of the Gß dimer dissociated from
pleckstrin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), CC (ß), C (), and CX3C (). The CC chemokine
MIP-1, MCP-1, RANTES, macrophage-derived chemokine, MIP-3,
or MIP-3ß 20, 26, 27, 28, 29, 30 ; the C chemokine lymphotactin 7, 31, 32 ; the
CXC chemokine IP-10, or SDF-1 7, 27, 33 ; and the CX3C
chemokine fractalkine 30, 34 have been shown to induce the chemotaxis
of NK cells. Furthermore, MIP-1 recruits NK cells toward the livers
of CMV-infected mice, which resulted in increased inflammation and
decreased susceptibility to infection with this virus 3 .
Receptors for CC chemokines are coupled to the heterotrimeric G
proteins, which are composed of three subunits, , ß, and . The
presence and identity of the G proteins in human and rat NK cell
membranes have been investigated. It was reported that these membranes
expressed Gi, Go, Gs,
Gq, Gz, and G13. These G proteins
play vital roles as early transducers of various biological functions
in NK cells, such as chemotaxis (reviewed in 6 . However, the
downstream signaling molecules important for the chemotaxis of NK cells
are not known. It has been observed that wortmannin, a fungal inhibitor
of PI-3K, inhibits RANTES-induced T cell chemotaxis 25 . Because
wortmannin has effects other than inhibition of PI 3-K, it is not clear
whether this enzyme is involved in mediating chemokine-induced cellular
chemotaxis. In this study, we showed that wortmannin as well as
inhibitory Abs to PI 3-K but not PI 3-K inhibit C, CC, and CXC
chemokine-induced NK cell chemotaxis, suggesting that PI 3-K IB plays
an important role in chemokine activation of NK cells.
In addition, we showed that PI 3-K is recruited into NK cell
membranes within 5 min after stimulation of these cells with the CC
chemokine RANTES. At almost the same time, an association between PI
3-K and the ß dimer of G proteins occurred. In addition, we
observed that pleckstrin is recruited from the cytosol into NK cell
membranes within 2–3 min after stimulating NK cells with C, CC, or CXC
chemokines. The recruitment of pleckstrin was insensitive to wortmannin
pretreatment, suggesting that PI 3-K products such as
PI3, 4 P2 or PI3, 4, 5 P3 are not important for
the recruitment of pleckstrin into NK cell membranes shortly after
activating GPCR. These results contrast the recruitment of PLC-PH
into the cell membranes upon stimulation with growth factors, which
occurs as a result of binding the PLC-PH to phospholipids, which
facilitates its association with the cell membrane. This activity was
sensitive to wortmannin pretreatment 13 . Therefore, there may be
multiple mechanisms by which PH domain-containing proteins are
recruited into the membranes, depending on the pathway utilized by the
ligands.
In addition, we observed that wortmannin did not inhibit the
recruitment of PI 3-K into NK cell membranes. To correlate the
finding that wortmannin inhibits NK cell chemotaxis with the
recruitment of this kinase into the membranes of these cells, we
observed that wortmannin inhibited the activity of this kinase. Hence,
the generation of PI3, 4, 5 P3 (Fig. 6) or
PI3, 4 P2 (data not shown) was abrogated upon pretreatment
of NK cells with this metabolite. These results suggest that during
activation with RANTES, PI 3-K is recruited into NK cell
membranes, placing it in close proximity to its phospholipid
substrates, resulting in the phosphorylation of various
phosphatidylinositol lipids.
Interestingly, pretreatment of NK cells with PT only partially
inhibited the recruitment of either pleckstrin or PI 3-K into their
membranes, suggesting that the majority of pleckstrin or PI 3-K that
are recruited into NK cell membranes shortly after stimulation with
RANTES are associated with the ß subunit of PT-resistant G
proteins. This is not surprising, considering that receptors for RANTES
as well as other chemokines present on NK cells are coupled to the
PT-insensitive Gq and Gz and the PT-sensitive
Gi and Go 21 .
Taken together, it appears that shortly after activation of NK cells
with chemokines, the ß dimer of G proteins associates with
pleckstrin, most likely through the PH domain since the Ab we have
utilized is specific for PH domain of pleckstrin. PI 3-K is also
associated with the ß/pleckstrin-PH complex forming a ternary
complex in NK cell membranes as a result of activating NK cells with
the CC chemokine RANTES. It is possible that the Gß dimer may act
as a docking molecule for pleckstrin and PI 3-K. This is similar to
the function of Gß, which upon activation of the M2 and M3
muscarinic receptors, recruits G protein-coupled receptor kinase,
resulting in the phosphorylation and desensitization of these receptors
35 . However, recruitment of PI 3-kinase, as described in this study,
results in activation and chemotaxis. It appears that after forming
this ternary complex, the ß dimer dissociates, presumably
reassociating with the subunit in the membranes, whereas the PI
3-K mediates NK cell chemotaxis by phosphorylating phospholipids
such as phosphatidylinositol 4 phosphate (PI4 P) or
phosphatidylinositol 4,5 bisphosphate (PI4, 5 P2), which
are important for this process. These results may shed some light on
the biochemical events utilized by chemokines to induce the
polarization and the extravasation of NK cells into the sites of virus
infection, or tumor growth 3, 4 .
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. A. Maghazachi, Department of Anatomy, University of Oslo, P.O. Box 1105 Blindern, Oslo, Norway N-0317. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage-inflammatory protein-1; GPCR, G protein-coupled receptor(s); IP-10, IFN-inducible protein-10; Ltn, lymphotactin; MCP-1, monocyte-chemoattractant protein-1; PH, pleckstrin homology; PI 3-K, phosphoinositide 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4 bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5 trisphosphate; PI(4,5)P2, phosphatidylinositol 4,5 bisphosphate; PLC, phospholipase C; PT, pertussis toxin; SDF-1, stromal-derived factor-1; SLO, streptolysin O.
Received for publication August 20, 1998. Accepted for publication December 14, 1998.
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A. Aoukaty and R. Tan Association of the X-linked Lymphoproliferative Disease Gene Product SAP/SH2D1A with 2B4, a Natural Killer Cell-activating Molecule, Is Dependent on Phosphoinositide 3-Kinase J. Biol. Chem., April 5, 2002; 277(15): 13331 - 13337. [Abstract] [Full Text] [PDF]
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 B. Cambien, M. Pomeranz, H. Schmid-Antomarchi, M.-A. Millet, V. Breittmayer, B. Rossi, and A. Schmid-Alliana Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion Blood, April 1, 2001; 97(7): 2031 - 2037. [Abstract] [Full Text] [PDF]
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C. Cerdan, E. Serfling, and D. Olive The C-class chemokine, lymphotactin, impairs the induction of Th1-type lymphokines in human CD4+ T cells Blood, July 15, 2000; 96(2): 420 - 428. [Abstract] [Full Text] [PDF]
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M. Inngjerdingen, B. Damaj, and A. A. Maghazachi Human NK Cells Express CC Chemokine Receptors 4 and 8 and Respond to Thymus and Activation-Regulated Chemokine, Macrophage-Derived Chemokine, and I-309 J. Immunol., April 15, 2000; 164(8): 4048 - 4054. [Abstract] [Full Text] [PDF]
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P. H. Naccache, S. Levasseur, G. Lachance, S. Chakravarti, S. G. Bourgoin, and S. R. McColl Stimulation of Human Neutrophils by Chemotactic Factors Is Associated with the Activation of Phosphatidylinositol 3-Kinase gamma J. Biol. Chem., July 28, 2000; 275(31): 23636 - 23641. [Abstract] [Full Text] [PDF]
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