1 Department of Cell Biology, Nencki Institute of Experimental Biology, 3
Pasteur St., 02-093 Warsaw, Poland
2 Universität Bielefeld, Fakultät für Chemie, Biochemie II, 33615
Bielefeld, Germany
* Author for correspondence (e-mail: asobota{at}nencki.gov.pl)
Accepted 21 October 2002
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Summary |
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Key words: Fc receptor II, Membrane rafts, Lyn, Capping, Actin cytoskeleton
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Introduction |
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Tyrosine residues of the ITAM in FcRIIA are phosphorylated by
several protein tyrosine kinases (PTKs) of the Src family among which Lyn is
most likely to phosphorylate the clustered receptor in vivo
(Bewarder et al., 1996
;
Ibarrola et al., 1997
).
Several data demonstrate that Src family PTKs phosphorylate ITAMs, thus
triggering signaling pathways of other immunoreceptors, including T-and B-cell
antigen receptors (TCR and BCR, respectively), IgE receptor (Fc
RI) and
IgA receptor (Fc
R) (Jouvin et al.,
1994
; Saouaf et al.,
1994
; van Oers et al.,
1996
; Lang et al.,
1999
). Taking into account the importance of Src family PTKs for
immunoreceptor signaling it is of special interest that the kinases are
sequestrated in sphingolipid/cholesterol-rich domains of the plasma membrane
(rafts). Owing to the association of long, saturated fatty acyl chains of the
sphingolipids with intercalating cholesterol molecules, these lipid
microdomains acquire a liquid-ordered phase
(London and Brown, 2000
). Such
a lipid organization allows the separation of the sphingolipid/cholesterol
domains from the more liquid, glycerophospholipid-based environment of the
plasma membrane, thus rendering them insoluble in non-ionic detergents, a
property widely used for raft isolation (detergent-resistant domains, DRMs).
In the inner leaflet of DRMs, PTKs of the Src family are anchored through
double acylation of the N-terminal region with the saturated fatty acyl chains
of myristate and palmitate (Shenoy-Scaria
et al., 1993
; Rodgers et al.,
1994
; Kabouridis et al.,
1997
; van't Hof and Resh,
1997
). In the outer leaflet of the domains
glycosylphosphatidylinositol (GPI)-linked proteins are docked
(Friedrichson and Kurzchalia,
1998
). Immunoreceptors, including TCR, BCR, Fc
RI and
Fc
R, were shown to be recruited to rafts upon crosslinking and undergo
phosphorylation catalyzed by Src family PTKs residing in the rafts and/or
relocated to these sites beside the receptors
(Field et al., 1997
;
Montixi et al., 1998
;
Xavier et al., 1998
;
Lang et al., 1999
;
Cheng et al., 2001
). Recently,
merging of BCR- and Fc
RIIB1-bearing rafts was proposed to facilitate
negative regulation of BCR signaling by Fc
RIIB
(Aman et al., 2001
). It should
be noted, however, that there are data which do not support an involvement of
membrane rafts in tyrosine phosphorylation of Fc
RI but suggest a weak,
constitutive Fc
RI-Lyn interaction enabling the initial phosphorylation
of activated Fc
RI (Pribluda et al.,
1994
; Kovarova et al.,
2001
).
In previous work concerning FcRII of U937 monocytic cells, which
express Fc
RIIA/C isoforms, we established that upon crosslinking the
receptor associated with high molecular mass complexes containing Lyn kinase.
This association of Fc
RII with Lyn, as well as the accompanying
tyrosine phosphorylation of Fc
RII, was regulated by plasma membrane
cholesterol level (Kwiatkowska and Sobota,
2001
). These data suggested an involvement of membrane rafts and
Lyn in Fc
RII phosphorylation and were further underscored by studies of
Fc
RII in HL-60 and K562 cells
(Katsumata et al., 2001
). In
the present study we examined an engagement of membrane rafts in remodeling of
the actin cytoskeleton after activation of Fc
RIIA. It was found that
tyrosine phosphorylation of Fc
RIIA, catalyzed by raft-associated PTKs
controlled rearrangement of the actin cytoskeleton and assembly of receptor
caps. The C-terminal tyrosine residue in the ITAM of Fc
RIIA (Y298) was
critical for triggering of this signaling pathway.
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Materials and Methods |
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Capping of FcRII and cell spreading
U937 cells (1.2x106 cells/ml) were washed and maintained
in HEPES-buffered saline (HBS) containing 125 mM NaCl, 4 mM KCl, 10 mM
NaHCO3, 1 mM KH2PO4, 10 mM glucose, 0.2%
bovine serum albumin (BSA) and 20 mM Hepes, pH 7.4. BHK cells were plated with
a density 5x104/ml and 20 hours later were used for
experiments conducted in HBS buffer supplemented with 1 mM CaCl2
and 1 mM MgCl2. To induce crosslinking of FcRII (patching),
the cells were exposed for 30 minutes at 0°C to unlabeled or
biotin-conjugated anti-Fc
RII IgG clone IV.3 followed by unlabeled or
FITC-conjugated goat anti-mouse IgG (Calbiochem). The IV.3 antibody was
purified from hybridoma (ATCC) supernatants on a Protein A-agarose column
(Pierce). Subsequent warming of the cells for 10 minutes at 20°C induced
formation of Fc
RII caps. The cells were fixed with 3% formaldehyde in
PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl2, pH
6.9) for 10 minutes at 0°C and 20 minutes at 20°C and mounted in
mowiol containing 2.5% DABCO (Sigma). The capping efficiency was quantified in
100-150 cells per sample using a Nikon fluorescence microscope equipped with a
63x oil immersion objective. In U937 cells the cap was assumed to be
formed if the crosslinked Fc
RII was accumulated on less than half of
the cell surface (Kwiatkowska and Sobota,
1999b
; Kwiatkowska and Sobota,
1999c
). BHK cells with distinct, large conglomerates of
crosslinked Fc
RII formed at the cell margins were scored as `cap
positive'.
To induce BHK cell spreading, suspensions of 1x105 cells in 300 µl of DMEM/10% fetal bovine serum were plated onto precoated coverslips (18 mm in diameter) and incubated at 37°C in a 5% CO2 humidified atmosphere for up to 5 hours. The coverslips were precoated with either 20 µg/ml IV.3 antibody or with 20 µg/ml fibronectin (Sigma) for 25 minutes, washed in distilled water, air-dried and incubated with 30 mg/ml BSA for 15 minutes to block unspecific binding sites. Before use, the coverslips were washed and dried as above. In control samples, coverslips were exposed to 30 mg/ml BSA only. At various stages of spreading the cells were fixed with 3% formaldehyde, mounted in mowiol/DABCO and analyzed using a Nikon microscope and 20x objective. To quantify spreading efficiency, cells with irregular outlines and at least one prominent protrusion were scored per 100-150 cell population.
Drug treatment
Cells were pretreated for 30 minutes at 37°C in HBS medium with kinase
inhibitors: piceatannol (Alexis), wortmannin (Sigma) and for 10 minutes with
PP1; herbimycin A was applied overnight in serum-free growth medium
supplemented with 20 mM HEPES, pH 7.4. All drugs were also present during
FcRII crosslinking and capping or cell spreading. Control samples
contained up to 0.1% DMSO as the drug carrier. The content of unesterified
cholesterol in cells was measured by a fluorimetric method
(Drzewiecka et al., 1999
). In
U937 cells the level of plasma membrane cholesterol was depleted for 1 hour at
37°C using ß-cyclodextrin (CDX), and cholesterol was reincorporated
as described previously (Kwiatkowska and
Sobota, 2001
). Since cholesterol concentration in BHK
transfectants was nine times higher than in U937 cells, BHK cells were treated
for 1 hour at 37°C with methyl-ß-cyclodextrin (MCDX) owing to higher
efficiency of MCDX for cholesterol release
(Kilsdonk et al., 1995
). CDX
and MCDX were from Sigma. Cells were pretreated with cytochalasin B (Sigma)
for 45 minutes at 37°C in HBS medium. DL-
-hydroxymyristic acid
(2-hydroxytetradecanoic acid) (HMA) and 2-bromopalmitic acid
(2-bromohexadecanoic acid) (BPA), from Sigma and Aldrich, respectively, were
introduced to the cells as complexes with BSA
(Nadler et al., 1993
). To this
end, 1 mmol of HMA or BPA was mixed with 5 g of Celite (BDH) for 10 minutes.
After drying, Celite covered with the fatty acids was stirred with 60 mg/ml
delipidated BSA (Sigma) for 1 hour at room temperature. The Celite was removed
by centrifugation (2°C, 10 minutes, 15,000 g). In control
samples HMA and BPA were absent in the prepared mixture. The obtained
fatty-acid-BSA complexes and control solutions were diluted in growth medium
supplemented with 2% fetal bovine serum and 20 mM HEPES, pH 7.4. U937 cells
were suspended in the media at a concentration of 2x106/ml
and incubated overnight at 37°C prior to experiments. BHK cells
(1x105/ml) were plated and cultured for 12 hours before
exposure to the drugs. The viability of the drug-treated cells was over 90% as
estimated by Trypan Blue exclusion.
Immunofluorescence microscopy
To study the colocalization of FcRII with cell-surface proteins,
serum-starved U937 cells were incubated with IV.3 mouse anti-Fc
RII IgG
followed by goat anti-mouse IgG-lissamine rhodamine (Jackson ImmunoResearch)
supplemented with either mouse anti-CD55 IgM (clone MEM-118, provided by
Vaclav Horejsi) or human transferrin (10 µg/ml, Sigma) (30 minutes at
0°C of each incubation). At this stage (Fc
RII patching) the cells
were either fixed with 3% formaldehyde or warmed for 10 minutes at 20°C
(Fc
RII capping) and then fixed. Goat anti-mouse IgM-FITC (Sigma) or
rabbit anti-transferrin IgG (Boehringer) followed by goat anti-rabbit IgG-FITC
(Sigma) were applied after cell fixation to avoid patching of CD55 and
transferrin receptor (TfR) induced by the secondary antibodies. Samples were
mounted in mowiol/DABCO. Images were collected using an Olympus Fluoview
confocal laser microscope in the mode of sequential excitation of FITC and
rhodamine dyes to exclude crossover of their fluorescence. To quantify the
degree of protein colocalization, confocal images of cells were analyzed with
Quantity One software (Bio-Rad). In each cell, seven parallel lines of 1 µm
in width were drawn across a pair of the confocal cell sections generated for
crosslinked Fc
RII and CD55 or crosslinked Fc
RII and TfR. The
distance between the lines was calculated for each cell to divide the cell
diameter into constant sections. Two-colored fluorescence intensity profiles
obtained for a pair of lines were superimposed. Peaks of intensity of
Fc
RII labeling that corresponded to the receptor patches and overlapped
with peaks of fluorescence of CD55 or TfR were scored and expressed as a
percentage of total number of Fc
RII peaks found in the seven analyzed
profiles per cell. At least 10 cells were analyzed in each variant.
In BHK cells, studies of colocalization of FcRII and
tyrosine-phosphorylated proteins or actin filaments were performed after
labeling of the receptor with IV.3 mouse anti-Fc
RII IgG and donkey
anti-mouse IgG-Texas Red (Jackson ImmunoResearch) for 30 minutes at 0°C
each. The cells were fixed with 3% formaldehyde in PHEM buffer either after
binding of IV.3 anti-Fc
RII or after Fc
RII crosslinking or after
subsequent cell warming for 10 minutes at 20°C. The fixed cells were
permeabilized with 0.1% Triton X-100 in Tris-buffered saline (TBS) (5 minutes,
0°C), blocked in 3% BSA for 30 minutes at room temperature and exposed for
1 hour at room temperature either to rabbit anti-phosphotyrosine IgG
(Transduction Laboratories) followed by anti-rabbit IgG-FITC (Sigma) or
phalloidin-FITC (10 ng/ml, Sigma). In the case of labeling of
tyrosine-phosphorylated proteins, TBS was supplemented with a cocktail of
tyrosine phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF,
50 M µM phenylarsine oxide). To visualize actin filaments in spreading BHK
cells, the cells were fixed, permeabilized and incubated with phalloidin-TRITC
(2 ng/ml, Sigma) as described above. Samples mounted in mowiol/DABCO were
analyzed using a Nikon fluorescence microscope as described above.
Gradient ultracentrifugation
U937 cells, 1.2x107 per sample, were lysed for 30 minutes
at 0°C in 220 µl of Triton X-100 (TX-100) lysis buffer composed of 0.2%
TX-100, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 30 mM Hepes, pH 7.5, phosphatase
inhibitors: 1 mM Na3VO4, 50 µM phenylarsine oxide, 30
mM p-nitrophenylphosphate and a cocktail of protease inhibitors (Boehringer).
Cells were further sheared by passing them five times through a 25-G needle.
After clarification (1.5 minutes, 480 g, 4°C), 200 µl
of the cell lysate was adjusted to 40% OptiPrep (Sigma) and 10% sucrose. 600
µl of the mixture was transferred to RP55-S centrifuge tubes (Sorvall) and
overlaid with 400 µl of ice-cold solutions of 30%, 25%, 20% and 300 µl
of 0% OptiPrep in 0.2% TX-100 lysis buffer supplemented with 10% sucrose. In
experiments where phosphorylation of FcRII was analyzed,
4x107 cells were lysed in 600 µl of 0.2% TX-100 lysis
buffer containing 40% OptiPrep and 10% sucrose, clarified, placed on the
bottom of the ultracentrifuge tube and overlaid with an OptiPrep gradient as
above. Gradients were spun for 3 hours at 170,000 g, 4°C
(RCM 100 ultracentrifuge, Sorvall). Seven fractions of 300 µl were
collected from the top of the gradient.
Immunoprecipitation and in vitro kinase assay
FcRII was immunoprecipitated from whole U937 cell lysates and from
OptiPrep gradient fractions. For immunoprecipitation, cells were either
exposed only to IV.3 mouse anti-Fc
RII (30 minutes, 0°C) or
incubated with anti-Fc
RII followed by goat anti-mouse IgG (30 minutes,
0°C) or left untreated. Subsequently, the cells (1x107
per sample) were solubilized for 30 minutes at 0°C in 3 ml of
TX-100/Nonidet P-40 lysis buffer (1% TX-100, 0.5% Nonidet P-40, 100 mM NaCl, 2
mM EGTA, 2 mM EDTA, 30 mM Hepes, pH 7.4, protease and phosphatase inhibitors
as described above). The lysates were clarified by centrifugation (1.5
minutes, 480 g, 4°C) and supplemented with 50 µl of 10%
protein G-bearing Omnisorb (Calbiochem). When cells were treated only with
IV.3 antibody, Omnisorb preadsorbed with 1.5 µg of goat anti-mouse IgG was
added to the cell lysates to facilitate precipitation of non-crosslinked
Fc
RII. After incubation for 2.5 hours at 4°C and an additional 30
minutes at 20°C, the Omnisorb beads were collected by centrifugation and
washed five to seven times in lysis buffer containing 0.5% TX-100 and finally
in TBS. The precipitates were boiled in 30 µl of 2xSDS-sample buffer
and subjected to SDS-PAGE. To precipitate Fc
RII from OptiPrep gradient
fractions, 200 µl of each fraction were diluted twice with 0.2% TX-100
lysis buffer and supplemented with 30 µl of 10% Omnisorb.
Immmunoprecipitation was conducted overnight at 4°C. The precipitates were
washed seven times with 0.2% TX-100 lysis buffer and prepared for SDS-PAGE as
described above.
To estimate the activity of Lyn and Syk, the kinases were
immunoprecipitated from U937 cells (5x106 per sample)
pretreated with PP1 or piceatannol or DMSO and subsequently incubated in the
presence of the drugs for 30 minutes at 0°C with IV.3 mouse
anti-FcRII and goat F(ab)2 anti-mouse IgG and warmed for 30
seconds at 20°C. Cells were lysed in 750 µl of lysis buffer as
described for Fc
RII immunoprecipitation. However, Nonidet P-40 was
exchanged in the lysis buffer for 20 mM N-octylglucoside to release Lyn from
TX-100-insoluble membrane fragments. After clarification, lysates (4°C)
were supplemented with 3 µg of rabbit anti-Lyn IgG or rabbit anti-Syk IgG
(N-19 and C-20, 1.5 µg each) (Santa Cruz Biotechnology) and 3 hours later
50 µl of 10% protein A-bearing Pansorbin (Calbiochem) was added for a
additional 2 hours. The beads were washed four times in lysis buffer
containing 0.5% TX-100, once in TBS and once in kinase buffer (50 mM NaCl, 15
mM MnCl2, 2 mM DTT, 30 mM imidazole, pH 7.4) and suspended in 200
µl of the kinase buffer containing 2 mM ATP. The kinase assay was started
by transferring the samples to 37°C. After 30 minutes, the beads were
pelleted and boiled in 2xSDS-PAGE buffer.
Immunoblotting
Immunoprecipitates, OptiPrep gradient fractions of U937 cells and lysates
of BHK cells were separated by to 10% SDS-PAGE. For lysis, BHK cells
(3.4x106 per sample) were treated with 100 µl of the
TX-100/Nonidet P-40 lysis buffer containing the protease and phosphatase
inhibitors mentioned above (0°C), and 10 minutes later, 30 µl of
4xSDS-sample buffer were added. After electrophoresis, proteins were
transferred to nitrocellulose membranes
(Kwiatkowska and Sobota,
2001). The membranes were incubated with antibodies: mouse or
rabbit anti-Lyn IgG, rabbit anti-Syk IgG, rabbit anti-TfR IgG, mouse
anti-phosphotyrosine IgG, clone PY99 (all from Santa Cruz Biotechnology),
mouse anti-actin (Boehringer) followed by goat anti-mouse IgG or anti-rabbit
IgG labeled with peroxidase (Santa Cruz Biotechnology). Mouse
anti-phosphotyrosine, clone PY-20, conjugated with peroxidase (Transduction
Lab) was used to reveal autophosphorylation of immunoprecipitated Lyn and Syk.
Distribution of Fc
RII in the OptiPrep gradient fractions was analyzed
on immunoblots by detection of biotin-labeled IV.3 anti-Fc
RII IV.3
antibody with the use of goat anti-biotin peroxidase-conjugated IgG (Sigma).
As non-labeled IV.3 was applied to immunoprecipitate Fc
RII from whole
cell lysates, rabbit anti-Fc
RII serum, kindly provided by J.-L.
Teillaud, was applied to detect the receptor in a corresponding series of
blots. CD55 was detected in the OptiPrep gradient fractions on slot-blots with
the use of mouse anti-CD55 IgG (clone IA10, kindly provided by V. Horejsi).
These studies were performed on fractions obtained from cells untreated with
IV.3 mouse anti-Fc
RII to avoid crossreactivity of anti-mouse
IgG-peroxidase applied for detection of mouse anti-CD55 IgG. Immunoreactive
bands were visualized with SuperSignal West Pico Chemiluminescent substrate
(Pierce) in a Fluor-S MultiImager and quantified densitometrically using
Quantity One software (Bio-Rad). Data shown are the mean±s.e.m. from
n number of experiments. Prestained molecular mass standards were
from Bio-Rad.
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Results |
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To assess the role of DRMs in FcRII capping, the cells were exposed
to CDX. The drug removed cholesterol and reduced the formation of Fc
RII
caps in a dose-dependent manner, arresting the receptor in patches
(Fig. 1D,E,G). After
reincorporation of cholesterol, reconstitution of Fc
RII capping was
observed (Fig. 1F,G). The level
of Fc
RII capping in the cholesterol-reloaded cells reached, but did not
exceed, that in CDX-untreated cells, despite the cells acquiring twofold
higher amounts of cholesterol over their initial cholesterol content (4.2
µg/2x106 cells after 30 minutes of cholesterol
reincorporation versus 2.6 µg/2x106 control cells).
Incubation of the CDX-treated cells without cholesterol-delivering complexes
did not restore either Fc
RII cap assembly or cholesterol level
(Fig. 1G).
As DRMs are centers of anchorage for kinases of the Src family, we examined
an involvement of these kinases in the phosphorylation and capping of
FcRII. Fig. 1H shows
that crosslinking (patching) of Fc
RII induced an intensive
phosphorylation of the receptor while progressive dephosphorylation of
Fc
RII correlated with the formation of the receptor caps. Several
downstream proteins undergo a similar cycle of tyrosine
phosphorylation/dephosphorylation during patching and capping of Fc
RII,
as shown earlier (Kwiatkowska and Sobota,
1999c
). The phosphorylation of crosslinked Fc
RII was
completely blocked by depletion of the plasma cholesterol
(Fig. 1H, lane CDX). Further
studies indicated that activity of Src family kinases associated with DRMs was
required for both Fc
RII phosphorylation and receptor cap assembly. Two
Src-family-specific PTK inhibitors, PP1 at 15 µM and herbimycin A at 8
µM, were found to significantly reduce the assembly of Fc
RII caps
(Fig. 1I). Herbimycin A was
likely to exert its prominent inhibitory effect by degradation of the Src
family PTKs (June et al.,
1990
). An overnight treatment of cells with 8 µM of this drug
led to a 54.1±3.2% reduction in Lyn content, estimated in relation to
the actin level, in whole cell lysates by densitometric analysis of
immunoblots. Under these conditions, phosphorylation of crosslinked
Fc
RII was strongly attenuated (Fig.
1H, lane Herb) and assembly of Fc
RII caps was inhibited by
89.2±1.1% (Fig. 1I).
Since the Src family PTKs rely on dual acylation as a signal for DRM location
(Shenoy-Scaria et al., 1993
;
Kabouridis et al., 1997
;
van't Hof and Resh, 1997
), we
next altered the protein fatty acylation level in cells with the use of HMA
and BPA. HMA is a potent inhibitor of N-myristoyltransferase, blocking
co-translational attachment of myristate to proteins including Src family
kinases (Nadler et al., 1993
;
van't Hof and Resh, 1999
). BPA
preferentially disturbs protein palmitoylation although a negative influence
on Fyn myristoylation was also found (Webb
et al., 2000
). Both agents markedly suppressed the assembly of
Fc
RII caps; nearly 90% inhibition of capping by 1 mM HMA, accompanied
by abrogation of phosphorylation of crosslinked Fc
RII, was prominent
(Fig. 1H, lane HMA and 1I). The
overall data indicate a sensitivity of Fc
RII capping to DRM-targeting
agents and point to an involvement of Src family PTKs in this event, although
the effect exerted by HMA and BPA on capping owing to their influence on lipid
synthesis can not be excluded.
It is of interest that the negative influence of different concentrations
of piceatannol, a Syk kinase inhibitor, on FcRII capping was less
profound, reaching 48% of capping inhibition at 100-200 µM
(Fig. 1I); however, 200 µM
piceatannol affected cell viability. No reduction of Fc
RII cap assembly
occurred under the influence of 0.001-1 µM wortmannin, an inhibitor of
phosphatidylinositol 3-kinase (PI 3-kinase)
(Fig. 1I). Syk is proposed to
control actin rearrangement during phagocytosis mediated by Fc
Rs and
the moderate influence of piceatannol on actin-dependent capping of
Fc
RII was unexpected. Therefore, we examined Syk activity, reflected by
kinase autophosphorylation, in cells treated with the drug. In our hands, 25
µM piceatannol blocked Fc
RII-induced Syk activity by 67%, whereas at
100 µM the drug reduced the kinase activity by 83%
(Fig. 1J). The discrepancy
between the strong inhibition of Syk activity and weaker effect on
Fc
RII capping exerted by piceatannol indicated that Syk is not a key
kinase for cap assembly. In addition, piceatannol at concentrations above 25
µM may affect Fc
RII capping not only by Syk inhibition but also as
non-specific inhibitor of Src family PTKs
(Majeed et al., 2001
). In
fact, examination of Lyn activity revealed that 100 µM piceatannol
diminished autophosphorylation of this kinase by 61%, whereas PP1 at 15 µM
inhibited Lyn activity by 79% (Fig.
1J). This PP1 concentration also inhibited the enhancement of Syk
activity induced by Fc
RII crosslinking, which is in line with the
supposition that Src family PTKs act upstream of Syk in the signaling pathway
of Fc
RIIA (Cooney et al.,
2001
).
Phosphorylation of crosslinked FcRII within DRMs controls
assembly of Fc
RII caps
The majority of the total Lyn kinase in U937 cells, 78.2±3.9%
(n=3), was accumulated in DRMs as revealed by flotation of Lyn to
fractions 1-2 of an OptiPrep density gradient (buoyant density 1.08-1.13
g/ml). These fractions also contained 63.2±2.2% of the total CD55, a
GPI-anchored protein (Fig. 2A).
Depletion of cholesterol in cells with the use of 5 mM CDX evoked a
significant shift of Lyn and CD55 within the density gradient from DRMs to
high density fractions 6-7, in which 64.8±0.7% (n=3) of the
kinase content and total CD55 were found
(Fig. 2A). Fractions 6-7
(buoyant density 1.28-1.34 g/ml) contained detergent-soluble proteins,
including TfR. No traces of this receptor were found in DRM fractions 1-2
isolated either from control cells or cells cultured under inhibitory
conditions (Fig. 2A, bottom).
Pretreatment of cells with 1 mM HMA or 0.3 mM BPA diminished the amount of Lyn
associated with DRMs to 35.9±3.1% and 44.9±3.8% of the total
(n=4), respectively (Fig.
2A, left panel). The exposure of cells to BPA led to an enrichment
of Lyn in the intermediate fractions 4-5 of the gradient (buoyant density
1.21-1.24 g/ml). On the other hand, the HMA treatment caused a displacement of
Lyn toward high-density fractions 6-7. Moreover, under the influence of HMA,
the cellular level of Lyn estimated in relation to actin content was reduced
by 40.8±5.5% (n=5). As myristoylation of the Src family PTKs
is required for the palmitoylation to occur
(Shenoy-Scaria et al., 1994)
the non-acylated Lyn formed after HMA treatment is likely to undergo a rapid
degradation that leads to the detected reduction of the Lyn content in U937
cells. Such a phenomenon was described for Lck and Lyn kinases in HMA-treated
T and B cells (Nadler et al.,
1993
). In striking contrast to Lyn, an association of CD55 with
DRMs was not disrupted by preincubation of U937 cells with HMA and BPA
(Fig. 2A, right panel). These
data indicate that the impairment of protein fatty acylation by HMA and BPA
significantly affected PTKs of the Src family that are located in the inner
leaflet of DRMs without disturbing the organization of the outer leaflet of
the domains where CD55 is anchored. In addition, docking of CD55 in DRMs
relies on the GPI moiety, which is expected to be acylated with palmitate or
other long-chain fatty acids. As BPA did not diminish association of CD55 with
DRMs it seems that the drug affects mainly protein palmitoylation without
general reduction of the fatty acyl coenzyme A population in the cells.
|
FcRII in unstimulated cells, exposed only to biotin-labeled
anti-Fc
RII IgG, was fully released from the plasma membrane with 0.2%
TX-100 and was recovered in fractions 5-7 of the density gradient, as revealed
by the distribution of the anti-Fc
RII antibody
(Fig. 2B). However, upon
crosslinking (patching), 84.1±1.5% (n=3) of total Fc
RII
was redistributed to DRM fractions 1-2, whereas the rest of the receptor
population was found in fractions 3-4 of the gradient
(Fig. 2B). Depletion of plasma
membrane cholesterol with 5 mM CDX rendered the receptor susceptible to TX-100
solubilization again. This was observed as a clear shift in the crosslinked
Fc
RII toward fractions 4-7 of higher density in the gradient
(Fig. 2B). By contrast,
exposing the cells to 1 mM HMA or 0.3 mM BPA did not impair the appearance of
the crosslinked Fc
RII in the lightest region of the density gradient,
indicating that the fatty acylation of proteins, including that of Src family
PTKs, is not crucial for the association of crosslinked Fc
RII with DRMs
(Fig. 2B). Taken together with
the lack of influence of HMA and BPA on CD55 presence in DRMs, these data
suggest that the drugs had no non-specific effects on DRMs lipids and did not
impair association of the crosslinked Fc
RII with DRMs.
Immunofluorescence studies on FcRII and CD55 distribution on the
surface of U937 cells confirmed the interaction of crosslinked Fc
RII
with DRMs. Although in unstimulated cells a diffuse distribution of CD55 was
seen (data not shown), crosslinking of Fc
RII was found to evoke
concomitant clustering of CD55, and significant colocalization of both
proteins was detected (Fig.
3A-C). This pattern closely resembled copatching of Fc
RII
and Lyn kinase as described previously
(Kwiatkowska and Sobota,
2001
). Quantitative analysis of Fc
RII and CD55 distribution
revealed that 76.9±4.8% of the receptor patches colocalized with CD55
clusters (Fig. 4E). Apparent
colocalization of CD55 and Fc
RII was maintained during assembly of the
receptor caps, although a fraction of CD55 remained scattered outside the cap
region (Fig. 3D). Surprisingly,
during crosslinking of Fc
RII, aggregation of TfR also occurred;
however, the clusters of Fc
RII (red) and TfR (green) were clearly
segregated and only 15.7±3.2% of the clusters overlapped
(Fig. 3E-H). The clusters of
TfR are likely to reflect an accumulation of the receptor in coated pits.
|
|
A distinct subset of strongly tyrosine-phosphorylated proteins was found in
the buoyant gradient fractions 1-2 recovered from cells subjected to
FcRII crosslinking (Fig.
4A). These included proteins in the range of 53-75 kDa and
proteins of 40 kDa (Fig. 4A, arrowhead) and 20 kDa. The 40 kDa phosphoprotein, present in DRM fractions
1-2, was Fc
RII as demonstrated by its immunoprecipitation with the IV.3
anti-Fc
RII antibody (Fig.
4B, middle panel, arrowhead). Lyn kinase was found to
co-immunoprecipitate with the receptor from low density fractions 1-2
(Fig. 4B, lower panel), most
likely as a phosphorylated enzyme judging from the detection of the
corresponding 53/56 kDa doublet by antiphosphotyrosine antibody
(Fig. 4B, middle panel, small
arrows). No phosphorylation of Fc
RII was visible in the receptor
immunoprecipitates obtained from gradient fractions 3-7, and simultaneously
barely detectable amounts of Lyn were found in these complexes
(Fig. 4B).
An impairment of protein acylation by 1 mM HMA and an inhibition of the
activity of Src family PTKs with 15 µM PP1 resulted in an attenuation of
phosphorylation of the crosslinked FcRII and the accompanying proteins
(Fig. 4A). The amount of Lyn
kinase co-immunoprecipitated with Fc
RII from DRMs in HMA-treated cells
was substantially reduced (Fig.
4B). The residual phosphorylation of Fc
RII detected in
PP1-treated cells could either reflect a functional redundancy of multiple
endogenous Src family PTKs or could result from the activity of
PP1-insensitive Syk kinase. The latter possibility seems unlikely since
pretreatment of U937 cells with 100 µM piceatannol enhanced, rather than
diminished, the phosphorylation of the cross-linked Fc
RII and other
proteins located in DRMs (Fig.
4A). Accordingly, the co-immunoprecipitation of phosphorylated Lyn
kinase with the receptor from DRM fractions was also preserved under these
conditions (Fig. 4B). The
inhibition of Syk activity reduced the phosphorylation of some soluble
proteins in fractions 6-7 (Fig.
4A, dots), which could account for the moderate inhibition of
Fc
RII capping in the piceatannol-treated cells
(Fig. 1H). Tyrosine
phosphorylation of the crosslinked Fc
RII and other proteins residing in
DRMs, as well as soluble proteins of fractions 6-7, was abolished by depletion
of plasma membrane cholesterol with 5 mM CDX
(Fig. 4A). No phosphorylated
Fc
RII and no traces of Lyn kinase were seen in receptor
immunoprecipitates obtained under these conditions
(Fig. 4B).
Taken together, the results show that crosslinked FcRII is recruited
to DRMs where it coexists jointly with Lyn kinase and undergoes tyrosine
phosphorylation. These events are required for subsequent assembly of
Fc
RII caps.
Tyrosine residue 298 of ITAM in FcRIIA is required for the
receptor-triggered actin reorganization
Phosphorylation of crosslinked FcRII is likely to trigger signal
transduction cascades targeting the actin cytoskeleton which in turn controls
assembly of Fc
RII caps (Kwiatkowska
and Sobota, 1999b
). To further explore this pathway we examined
the actin rearrangement induced by wild-type Fc
RIIA and
Y298F-substituted Fc
RIIA expressed in BHK cells. By analogy to U937
cells, crosslinking of wild-type Fc
RIIA in BHK cells with IV.3 mouse
anti-Fc
RII IgG and anti-mouse IgG at 0°C induced an intense
phosphorylation of the receptor followed by its progressive dephosphorylation
after shifting of the cells to 20°C
(Fig. 5A). Furthermore, the
receptor phosphorylation was strongly inhibited by 10 µM PP1 and 1 mM HMA,
indicating an involvement of Src family tyrosine kinases in this process
(Fig. 5B). Substitution of
Y298, the C-terminal tyrosine residue of the ITAM of Fc
RIIA, by
phenylalanine completely abrogated phosphorylation of the receptor upon its
crosslinking (Fig. 5A).
Detection of receptor-bound IV.3-biotin IgG confirmed that cell clones
transfected with wild-type and Y298F-mutated Fc
RIIA expressed
comparable amounts of the receptors, estimated in relation to actin content in
the cells (Fig. 5A).
|
Immunofluorescence studies revealed that crosslinking of both wild-type and
Y298F-substituted FcRIIA was correlated with clear clustering of the
receptors (Fig. 5D,E). Before
the receptor clustering, minute amounts of tyrosine-phosphorylated proteins,
located mainly at focal contacts, were visible in both types of BHK
transfectants (Fig. 5D,E).
However, crosslinking of wild-type Fc
RIIA evoked strong tyrosine
phosphorylation of proteins, which colocalized with receptor patches
(Fig. 5D). It is of interest
that subsequent shifting of the cells from 0°C to 20°C for 10 minutes
led to accumulation of the crosslinked receptor as 1-4 large conglomerates
located at the edges of rounding cells. The conglomerates of Fc
RIIA
resembled the cap-like structures of crosslinked epidermal growth factor
receptor described previously in A431 cells
(Kwiatkowska et al., 1991
).
The cap-like structures of Fc
RIIA were formed in 64.8±2.4% of
BHK cells and were enriched in tyrosine-phosphorylated proteins as well as
actin filaments (Fig. 5C,D).
Disruption of microfilaments by pretreatment of cells with 10 µM
cytochalasin B inhibited translocation of the Fc
RIIA patches into the
cap-like aggregates (data not shown). In cells transfected with Y298F
Fc
RIIA, protein tyrosine phosphorylation remained at a low level upon
receptor crosslinking and some of the phosphotyrosine-bearing proteins were
concentrated at focal contacts (Fig.
5E). Formation of Y298F Fc
RIIA conglomerates was severely
impaired: 8.1±3.9% of the cell population formed small aggregates of
crosslinked receptor after 10 minutes at 20°C
(Fig. 5C).
Tyrosine-phosphorylated proteins remained concentrated in focal contacts, and
no accumulation of actin filaments was detected at the crosslinked Y298F
Fc
RIIA (Fig. 5E).
In another approach, we addressed the FcRIIA-actin cytoskeleton
relations by studying adhesion and spreading of BHK transfectants on
anti-Fc
RIIA IV.3-coated substratum. When seeded on such substratum,
wild-type Fc
RIIA-expressing cells adhered within 30 minutes and during
the next 1.5 hours 71.1±2.9% of the cells spread, forming many thin,
finger-like protrusions and flat lamellae
(Fig. 6A,B). The adhesion of
the cells depended on interaction between Fc
RIIA and anti-Fc
RIIA
since BSA-coated substratum did not promote the cell attachment
(Fig. 6A). Furthermore,
spreading of cells onto IV.3-coated substratum required participation of the
actin cytoskeleton because it was blocked by 10 µM cytochalasin B
(Fig. 6A). Immunofluorescence
studies of actin filament organization in spreading cells revealed prominent
actin ribs in the protrusions of the cells, abundant dotted staining of actin
dispersed through the cell and few, delicate stress-fibers
(Fig. 6A). For comparison,
cells spreading on fibronectin-coated substratum (81.3±3.4% of the cell
population) manifested a more elongated and polarized shape with stronger
stress-fibers (Fig. 6A).
|
To gain insight into signaling events governing the actin rearrangement
during FcRIIA-mediated cell spreading, Src tyrosine kinase-targeting
agents were applied. It was found that the spreading was strongly diminished
by 10 µM PP1 and 1 mM HMA and also affected by depletion of plasma membrane
cholesterol with 8 mM MCDX (Fig.
6A,B). Immunoblotting analysis demonstrated that spreading of
wild-type Fc
RIIA-expressing cells on IV.3-coated substratum was
correlated with strong tyrosine phosphorylation of the receptor, which
remained at the elevated level for at least 3 hours
(Fig. 6C). By contrast,
fibronectin-promoted spreading of these cells did not induce phosphorylation
of the wild-type Fc
RIIA confirming activation of different receptors
and signaling pathways by anti-Fc
RIIA and fibronectin. Finally, seeding
of BHK cells transfected with mutant Y298F Fc
RIIA on anti-Fc
RIIA
IV.3-coated substratum did not elicit any phosphorylation of the receptor
(Fig. 6C). Accordingly, there
was no spreading of the cells on the IV.3-coated substratum, although adhesion
of the cells was efficient (Fig.
6A,B). The Y298F Fc
RIIA-expressing cells remained round
without any visible major protrusions even 5 hours after plating
(Fig. 6A).
![]() |
Discussion |
---|
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---|
Recruitment of the crosslinked FcRII to DRMs was required, but not
sufficient, for receptor phosphorylation. The phosphorylation was diminished
by HMA, an agent that inhibits myristoylation of proteins, including Src
family PTKs (Nadler et al.,
1993
). Under these conditions the integrity of the rafts was not
disrupted as judged by CD55 location in low-density gradient fractions,
although Lyn kinase was displaced from DRMs and its coimmunoprecipitation with
crosslinked Fc
RII was impaired. At present, there is no direct evidence
that Lyn kinase phosphorylates Fc
RII in U937 cells, and participation
of other PTKs of the Src family in this process cannot be excluded. However,
Lyn exhibited co-distribution with crosslinked Fc
RII in intact U937
cells and in DRMs as revealed by immunofluorescence and electron microscopy
studies (Kwiatkowska and Sobota,
2001
). In U937 cells Lyn is a predominant kinase of the Src family
and, judging from immunoblotting analysis, we estimated that the level of its
expression in these cells exceeds that of Hck by about 50 times. [Hck is
another member of the Src family known to associate with Fc
RII in THP-1
cells (Ghazizadeh et al.,
1994
)]. Lyn was also found to associate with Fc
RII in
THP-1, HL-60 monocytic cells and transfected mouse B-cells
(Ghazizadeh et al., 1994
;
Katsumata et al., 2001
;
Bewarder et al., 1996
) as well
as in neutrophils (Ibarrola et al.,
1997
). In addition, in vitro studies indicated preferential
phosphorylation of Fc
RIIA by Lyn
(Bewarder et al., 1996
)
confirmed by potent Fc
RIIA phosphorylation in COS cells co-transfected
with Lyn and the receptor (Cooney et al.,
2001
). Fc
RIIA expressed in non-hematopoietic BHK-21 cells,
lacking Lyn, was likely to be phosphorylated by another member of the Src
family of PTKs as indicated by the sensitivity of the process to PP1 and
HMA.
Phosphorylation of FcRIIA was abrogated by substitution of a single
tyrosine residue 298 by phenylalanine, which is in agreement with previous
data on the crucial role of this tyrosine residue of the ITAM in receptor
signaling (Mitchell et al.,
1994
; Bewarder et al.,
1996
; Ibarrola et al.,
1997
). The Y298F-substituted Fc
RIIA, when expressed in BHK
cells, did not undergo translocation into cap-like structures after
crosslinking and failed to promote spreading of the cells on the
anti-Fc
RII-coated substratum. Similar inhibitory effects were achieved
both in wild-type Fc
RIIA-expressing BHK cells and in U937 cells when
activity of Src family PTKs was affected by PP1 and HMA or integrity of rafts
was disrupted by cholesterol depletion. Concomitantly, phosphorylation of the
crosslinked Fc
RII(A) in the cells was strongly impaired under the
influence of the DRM- and PTK-targeting agents. Taken together, these data
argue that phosphorylation of tyrosine residues of the ITAM in Fc
RIIA,
catalyzed by raft-anchored PTKs of the Src family, triggers signaling pathways
that target the actin cytoskeleton. It was previously demonstrated that actin
and spectrin actively participate in formation of Fc
RII caps
(Kwiatkowska and Sobota,
1999b
), and sensitivity of cell spreading to cytochalasin B
indicates that this process also relies on the participation of the
actin-based cytoskeleton. The assembly of caps in U937 cells and other
leukocytes is concomitant with their polarization as usually caps are formed
at the posterior uropod of the cells (de
Petris and Raff, 1972
) (see
Fig. 1). It is noteworthy that
DRMs were recently shown to also play a pivotal role in the acquisition of the
polarity needed for chemotaxis in adenocarcinoma cells and in T cells
(Manes et al., 1999
;
Gomez-Mouton et al., 2001
). By
analogy with the assembly of Fc
RII caps and Fc
RIIA-promoted
spreading of cells, polarization of T cells requires an involvement of the
actin cytoskeleton, pointing again to an interaction between DRMs and the
actin network (Gomez-Mouton et al.,
2001
). Despite extensive studies, the mechanism(s) of such
interactions remain elusive and are likely to be complex (Pierini and
Maxfiled, 2001). Recently, an involvement of CD44 in cytoskeleton
rearrangement and raft reorganization in T cells was reported
(Föger et al., 2001
). The
presence of actin at clustered Fc
RI was shown by immunoelectron
microscopy (Wilson et al.,
2000
). Corresponding to our results, Harder and Simons reported
that protein tyrosine phosphorylation by Src family PTKs, which accompanied
patching of DRM components, was a prerequisite for the accumulation of actin
filaments at the patches (Harder and
Simons, 1999
). On the basis of these data, the plasma membrane
domains are thought to serve as centers for activation of immunoreceptors
(Horejsi et al., 1999
;
Simons and Toomre, 2000
) and
for subsequent remodeling of the actin network, for example, by local
generation of phosphatidylinositol 4, 5-bisphosphate and/or activation of Rho
GTPase (Hirao et al., 1996
;
Pike and Miller, 1998
;
Rozelle et al., 2000
;
Caroni, 2001
). Later on, when
formation of Fc
RII caps proceeds, the actin cytoskeleton could be
responsible for separation of the receptor and Src family kinases
(Holowka et al., 2000
;
Wilson et al., 2000
),
facilitating the observed protein dephosphorylation. The persistent
phosphorylation of wild-type Fc
RIIA detected in spreading BHK
transfectants could result from progressive activation of subsequent receptors
interacting with the substratum coated by ani-Fc
RII antibody.
FcRIIA-mediated phagocytosis, cell spreading and receptor capping
are triggered by common events, such as clustering and phosphorylation of the
receptor. Despite this, it seems that various signaling pathways can lead from
activated Fc
RIIA to the actin cytoskeleton. During
Fc
RII-mediated phagocytosis, activation of Syk and PI 3-kinase takes
place (Cooney et al., 2001
).
Chimeric proteins composed of an extracellular domain of Fc
Rs and an
intracellular domain containing either Syk or the p85 subunit of PI3-kinase
were sufficient to mediate phagocytosis of IgG-coated particles
(Greenberg et al., 1996
;
Lowry et al., 1998
).
Fc
R-mediated phagocytosis was abrogated in Syk-deficient monocytes and
macrophages (Matsuda et al.,
1996
; Crowley et al.,
1997
; Kiefer et al.,
1998
). Similarly, a chimera of Fc
R-truncated p85, unable to
bind the p110 catalytic subunit of PI 3-kinase, did not trigger phagocytosis
(Lowry et al., 1998
). Other
reports showed that inhibition of Syk and PI 3-kinase activity by piceatannol
and wortmannin, respectively, markedly diminished phagocytosis mediated by
Fc
Rs, including Fc
RIIA
(Ninomiya et al., 1994
;
Araki et al., 1996
;
Cooney et al., 2001
),
confirming that both kinases are indispensable for the process. It was
considered that Syk controls actin assembly at ingested particles
(Greenberg, 1999
). However, in
Syk-deficient macrophages (also in wortmannin-treated cells), formation of
actin cups surrounding IgG-coated particles occurred
(Araki et al., 1996
;
Crowley et al., 1997
). By
contrast, in hck-/- fgr-/- lyn-/-
macrophages, formation of actin cups was delayed
(Fitzer-Attas et al., 2000
).
On the basis of the comparison of the phenotypes of hck-/-
fgr-/- lyn-/- and syk-/- cells it was
inferred that the Src family PTKs can govern actin polymerization in
phagocytic cups not only through Syk but also without Syk engagement
(Fitzer-Attas et al., 2000
).
Our data showing that the activity of Syk and PI 3-kinase is less important
for assembly of Fc
RII caps than activity of Src family PTKs are in line
with those of Fitzer-Attas et al.
(Fitzer-Attas et al., 2000
).
During phagocytosis the activity of PI 3-kinase is proposed to coordinate
insertion of intracellular vesicles, providing the membrane for pseudopod
extension (Greenberg, 1999
;
Lennartz, 1999
). Extensive
studies of the capping mechanism carried out between 1980 and 1990 indicated
that translocation of protein patches in the plane of the plasma membrane is
driven by the actin-based cytoskeleton and the process of vesicle flow does
not essentially contribute to cap assembly
(Heath and Holifield, 1991
).
This provides a clue as to why the activity of PI 3-kinase is not significant
for capping.
Studies of Karnovsky's group demonstrated that capping of surface
immunoglobulins on B cells was controlled by the cellular cholesterol level
(Hoover et al., 1983). This
capping was inhibited by removal of lymphocyte cholesterol with liposomes and
restored after cholesterol repletion. Following these changes in cholesterol
content, lipids of the plasma membrane were reversibly shifted from a rigid to
fluid-like state. The authors suggested that the `gel-like lipid domain' of
the plasma membrane was a place where `protein(s) involved in capping were
located'. Disordering this domain, evoked by diminishing the cholesterol
level, led to the attenuation of capping ability, whereas addition of
cholesterol led to restoration of the gel-like nature of the domain and
enabled capping to occur (Hoover et al.,
1983
). Our results indicate that the gel-like lipid domain
proposed by Karnovsky can correspond to DRMs, serving as sites of Lyn kinase
concentration, the activity of which is crucial for Fc
RII capping to
proceed.
![]() |
Acknowledgments |
---|
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