(Received for publication, October 23, 1996, and in revised form, March 11, 1997)
From the One of the earliest events after aggregation of
the high affinity receptor for IgE (Fc Mast cells and basophils accumulate at sites of inflammation and
play pivotal roles in the initiation of the allergic response. Aggregation of the high affinity receptor for IgE (Fc To understand the signaling cascade initiated by Fc PECAM-1 is a member of the Ig superfamily of cell adhesion molecules.
It is an integral membrane glycoprotein that is expressed on platelets,
endothelial cells, and cells of the myeloid lineage such as leukocytes
and basophils (reviewed in Refs. 10 and 11). PECAM-1 localizes to
intercellular junctions of endothelial cells or monolayer cultured
cells in which it is expressed. It functions in interendothelial cell
adhesion, leukocyte-endothelial interactions, transendothelial
migration, and angiogenesis (reviewed in Ref. 10). PECAM-1 can mediate
both homophilic (i.e. PECAM-1 on one cell interacting
with PECAM-1 on another cell) and
cation-dependent heterophilic binding. The heterophilic
ligands for PECAM-1 include cell surface glycosaminoglycans (12) and
PECAM-1 is a single chain molecule of 130 kDa containing six
extracellular Ig-like domains of the C2 subclass, one transmembrane region, and a cytoplasmic tail (14-18). The cytoplasmic domain of
PECAM-1 consists of 118 amino acids that include numerous serine, threonine, and tyrosine residues that could potentially become phosphorylated. In fact, the phosphorylation on serine residues of
PECAM-1 has been observed after activation of endothelial cells, platelets and T lymphocytes (19-21). This suggests that the
phosphorylation state of PECAM-1 could be important in regulating its
function. However, there have been no previous reports of
receptor-mediated tyrosine phosphorylation of PECAM-1.
Here we report that PECAM-1 is present on RBL-2H3 cells and becomes
tyrosine phosphorylated following Fc Pipes, Triton X-100, Nonidet P-40, protease
inhibitors, and protein A-agarose beads were from Sigma. CNBr-activated
Sepharose 4B beads were from Pharmacia Biotechn Inc. The materials for
electrophoresis were purchased from Novex (San Diego, CA),
polyvinylidene difluoride transfer membrane and Ultrafree PFL (low
binding cellulose) were from Millipore (Bedford, MA), and the sources
of other materials were as described previously (22).
The monoclonal antibody (mAb) R23 was generated
from the spleen of mice immunized with multiple injections of RBL-2H3
cells emulsified in adjuvant using methods described previously
(23-25). For these experiments mAb R23 was purified from ascites fluid by ammonium sulfate precipitation followed by ion exchange
chromatography on DE52. By immunodiffusion it was found to be of the
IgG1 isotype. For immunoprecipitation experiments it was coupled to
cyanogen bromide-activated Sepharose 4B. Anti-phosphotyrosine
monoclonal antibody PY-20 was from ICN Immunobiologicals (Lisle, IL).
Mouse monoclonal anti-Fc The RBL-2H3 cells
were maintained as monolayer cultures in Eagle's minimum essential
medium supplemented with 15% heat-inactivated fetal calf serum,
penicillin, streptomycin, and amphotericin (28). The Syk-deficient
TB1A2 variant cells and the 3A5 cells that have stably transfected Syk
have been described previously (29). Cells were activated with antigen
(dinitrophenol-coupled human serum albumin), anti-Fc For fluorescent microscopy, cells were cultured on coverslips and then
stimulated as described above. After rinsing with PBS, cells were fixed
with 2% paraformaldehyde (EM grade, Electron Microscopy Sciences) for
10 min. The coverslips were rinsed with PBS, incubated with 0.1 M glycine, and then permeabilized at After stimulation for the indicated
times, the monolayers were rinsed once with 12 ml of ice-cold PBS
containing 1 mM Na3VO4 and
solubilized in ice-cold lysis buffer (1% Triton X-100, 10 mM Tris, pH 7.4, 100 mM NaCl, 50 mM
NaF, 1 mM Na3VO4, 2 mM
phenylmethylsulfonyl fluoride, 21 µg/ml aprotinin). After incubating
on ice for 30 min, the cells were scraped off the plates, and the
lysates were centrifuged for 30 min at 16,000 × g at
4 °C. The post-nuclear supernatants were precleared by incubation
for 1 h at 4 °C with Sepharose 4B and then immunoprecipitated
with antibodies coupled to the same beads. After rotation at 4 °C
for 90 min, the beads were washed five times with ice-cold lysis
buffer, and the proteins were eluted by boiling for 5 min with sample
buffer as described previously (30).
Cell lysates and immunoprecipitated proteins
were separated by SDS-PAGE and electrotransferred to polyvinylidene
difluoride membranes (Immobilon P). The membranes were blocked for a
minimum of 4 h with 4% protease-free bovine serum albumin in
blotting buffer (10 mM Tris pH 7.4, 0.9% NaCl, 0.05%
Tween 20) and probed with 40 ng/ml anti-phosphotyrosine mAb PY-20
conjugated to horseradish peroxidase. For immunoblotting with mAb R23,
proteins were separated under nonreducing conditions, and the secondary
antibody was horseradish peroxidase-conjugated donkey anti-mouse IgG,
whereas for immunoblotting with the polyclonal anti-PECAM-1, proteins
were separated under reducing conditions, and the secondary antibody
was horseradish peroxidase-conjugated donkey anti-rabbit IgG. In some
experiments antibodies were stripped from the membranes, and the
membranes were reprobed with other antibodies as recommended by the
manufacturer. In all blots, proteins were visualized by enhanced
chemiluminescence (ECL Kit, Amersham Corp.) as described previously
(5).
Flat bottom Immunolon-2 assay wells
(Dynatech Laboratories, Inc., Chantilly, VA) were coated with 30 µg/ml of fibronectin or 30 µg/ml of mAb R23 by incubating at
37 °C overnight. Wells were washed and then blocked with PBS
containing 4% bovine serum albumin at 37 °C. After 3 h, the
wells were washed three times with Pipes buffer (25 mM
Pipes, 110 mM NaCl, 5 mM KCl, 5.6 mM glucose, 1 mM CaCl2, and 0.01%
bovine serum albumin, pH 7.4). RBL-2H3 cells grown overnight were
trypsinized from flasks, allowed to recover by incubation at 37 °C
in culture medium for 30 min. The cells were then washed three times
with the Pipes buffer and suspended in Pipes buffer at 2 × 106 cells/ml. 40 µl of this preparation was added to the
wells and incubated at 37 °C for 45 min. Then 20 µl of mAb BC4
solution (0.09 µg/ml) was added, and the cells were incubated at
37 °C for another 45 min. The cells were solubilized by adding 60 µl of 2 × lysis buffer.
The RBL-2H3 cells were grown as tumors in newborn rats
as described previously (28), and single cells were isolated (31). Lysates prepared from 20 × 109 cells were affinity
purified with 12 mg of mAb R23 coupled to beads and eluted with 0.5%
SDS, and the sample was concentrated to 150 µl. The purified proteins
were separated on 8% SDS-PAGE and electrophoretically transferred to
membranes. The major band stained with Ponceau S was excised and
subjected to N-terminal amino acid sequencing on a model 494 protein
sequencer (Applied Biosystems, Foster City, CA) employing the standard
blot protocol, which was optimized for this instrument. Amino acid
identification was obtained by manual observation with confirmation by
computer analysis (using the company's model 610A software) of the
chromatographic data.
To investigate the role of
tyrosine phosphorylated proteins in Fc
Although the relative migrations of the tyrosine phosphorylated protein
and the protein recognized by immunoblotting were similar, it was still
possible that these were two different proteins of similar size. To
directly demonstrate that the membrane protein was tyrosine
phosphorylated, proteins were immunoprecipitated from lysates of cells
that first had been stimulated by Fc Affinity purification was used to isolate enough of this
protein for amino acid sequence determination. Lysates of RBL-2H3 cells
were purified by affinity chromatography using immobilized mAb R23. By
Coomassie staining of an aliquot separated by SDS-PAGE, there was only
a broad 110-120-kDa band. Although this band migrated slightly faster
than that seen in the immunoprecipitates of cultured cells, it was
recognized by immunoblotting with mAb R23. The affinity purified
protein was resolved on 8% SDS-PAGE and electrophoretically transferred to membranes. This major band stained with
Ponceau S was excised and subjected to N-terminal amino acid
sequencing. The generated sequence of 10 amino acids was homologous to
that of both mouse and human PECAM-1 (Fig. 2). The
N-terminal sequence determined started at 40 amino acids after the
probable site of signal peptide cleavage, presumably as a result of
proteolysis during purification. These results strongly suggested that
the protein recognized by mAb R23 was PECAM-1.
Immunoprecipitation and immunoblotting studies were used to confirm
that the mAb R23 recognized PECAM-1. Three different polyclonal antibodies that react with human, rat, or mouse PECAM-1 all bound in
immunoblots to the 130-kDa protein immunoprecipitated by mAb R23 (Fig.
3A). These antibodies also recognized a
similar protein in cell lysates. In the reciprocal experiment, mAb R23
bound the same protein that was immunoprecipitated by the polyclonal
anti-rat PECAM-1 (Fig. 3B). In immunodepletion experiments,
the protein recognized by the polyclonal anti-rat PECAM-1 antibody was
dramatically decreased in lysates that were sequentially
immunoprecipitated with mAb R23 (Fig. 3C). These
results demonstrate that mAb R23 binds PECAM-1.
Protein tyrosine phosphorylation is an early event in
Fc
Some proteins are tyrosine phosphorylated early after Fc
The adherence of RBL-2H3 cells to extracellular matrix proteins,
mediated at least in part by integrins, regulates the Fc
Fc
Cells deficient in Syk protein tyrosine kinase were used to further
define at what point in the activation cascade the PECAM-1 is tyrosine
phosphorylated (Fig. 9). Fc
Using a monoclonal antibody to RBL-2H3 cells we identified the
adhesion molecule PECAM-1 as one of the substrates that is tyrosine
phosphorylated after Fc The present model for signaling by Fc The deduced amino acid sequences of the cytoplasmic domains of human,
murine, and bovine PECAM-1 are very similar and have four (human) or
five (bovine and mouse) tyrosine residues (16-18, 35). The murine
sequence 702YSEIR contains a potential tyrosine
phosphorylation site (18). This sequence is identical among the three
species except for a replacement of isoleucine with valine in the human
sequence. The cytoplasmic domain also has a number of serines that
could be phosphorylated. Stimulation of platelets and endothelial cells results in the phosphorylation of PECAM-1 on serine residues (19, 20).
In Jurkat T cells, activation with either phytohemagglutinin or phorbol
12-myristate 13-acetate induces serine phosphorylation and
down-regulation of the level of PECAM-1 on the cells but not in
tyrosine phosphorylation (19). The present results are the first report
that PECAM-1 is tyrosine phosphorylated in cells and that its
phosphorylation is regulated by cell surface receptors.
Basophils and mast cells have surface adhesion receptors that are
involved in the binding of these cells to other cells or to the
extracellular matrix (33). Binding results in aggregation of adhesion
receptors with the propagation of intracellular signals. RBL-2H3 cells
bind through integrin receptors to surfaces coated with fibronectin,
resulting in changes in the cytoskeleton, cell spreading, and a
redistribution of the granules to the periphery of the cells (36). At
sites of attachment to the extracellular matrix, the cytoplasmic
domains of the integrins form focal adhesion complexes that contain
proteins in noncovalent association. These include talin, vinculin,
The cytoplasmic domain controls the function of cell adhesion molecules
such as integrins, selectins, or members of the Ig superfamily. Thus
deletion of the cytoplasmic domain not only changes the capacity of
these receptors to interact with the cytoskeleton but also can result
in changes in ligand specificity (39-44). The activity of some
adhesion molecules is regulated by the state of phosphorylation of the
cytoplasmic domain. For example, the phosphorylation of LFA-1 by either
protein kinase C-dependent and/or -independent pathways
regulates the binding affinity of this molecule to intercellular
adhesion molecule 1 (45). Activation of endothelial cells, platelets,
or lymphocytes results in an increase in the phosphorylation of PECAM-1
on serine residues (19, 20) and association with the cytoskeleton.
Basophils and mast cells accumulate at sites of inflammation. For cells to leave the circulation and migrate into sites of inflammation they
require multiple adhesive interactions with the endothelium. Thus, cell
rolling is mediated by selectins, cell adhesion to the endothelium is a
function of integrins, and transmigration is due to PECAM-1.
PECAM-1 may exert both homophilic (PECAM-1 to PECAM-1) and
heterophilic (PECAM-1 to other molecules such as
glycosaminoglycan) interactions. These interactions may be important
for the transmigration of cells into tissues. Anti-PECAM-1 antibodies,
by binding to leukocytes or to endothelial cells, block the
transmigration of leukocytes without inhibiting the adherence of the
cells to the endothelium (46-49). Thus, phosphorylation of PECAM-1,
either by inducing conformational changes or by functioning as a
docking site, could modulate association of PECAM-1 with other
molecules, including the cytoskeleton, and regulate its function.
PECAM-1 may also be involved in regulating the function of other
cellular proteins. For example, aggregation of PECAM-1 regulates the
adhesive properties of In summary, we have found that PECAM-1 is present on RBL-2H3 cells and
is tyrosine phosphorylated after receptor aggregation. Such
modification of the molecule may be important for its physiological function. This is supported by evidence that the cytoplasmic domain of
PECAM-1 plays an important role. First, transfectants of PECAM-1 lacking the cytoplasmic domain are defective in aggregation (42). Second, alternatively spliced forms of PECAM-1 lacking the potential tyrosine phosphorylation site exhibit different aggregation properties (53). Third, phosphorylation of PECAM-1 on serines regulates its
down-regulation (19). Therefore, tyrosine phosphorylation of PECAM-1
may be crucial not only for the transmigration of basophils into
inflammatory sites but also for the regulation of degranulation.
We thank Drs. Majed Hamawy, Mark Swieter, and
Teruaki Kimura for helpful discussions and advice. We are grateful to
Greta Bader for technical assistance in histamine analysis.
Laboratory of Immunology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RI) on mast cells is the
activation of protein tyrosine kinases resulting in tyrosine
phosphorylation of numerous proteins. Using a monoclonal antibody
raised against the rat basophilic leukemia RBL-2H3 cells, we identified
that platelet/endothelial cell adhesion molecule 1 (PECAM-1 or CD31) was tyrosine phosphorylated in these cells. Aggregation of PECAM-1 did
not induce a detectable increase in its tyrosine phosphorylation, nor
did it result in degranulation. However, the minimal tyrosine phosphorylation of PECAM-1 in nonstimulated cells was dramatically increased after Fc
RI aggregation. This receptor-induced tyrosine phosphorylation of PECAM-1 was an early event, independent of Ca2+ influx or of the activation of protein kinase C
and of cell adhesion. PECAM-1 is an adhesion molecule that is required
for the transmigration of leukocytes across the endothelium into sites
of inflammation. Therefore tyrosine phosphorylation of PECAM-1 may
modulate its interaction with other molecules, thereby regulating the
migration of basophils into inflammatory sites.
RI) on these cells initiates a biochemical cascade that results in degranulation and
release of inflammatory mediators (1-3). The earliest event following
aggregation of Fc
RI is the phosphorylation of proteins on tyrosine,
an event that is critical for signal transduction in basophils or mast
cells (4-9).
RI aggregation,
we are attempting to identify molecules that become tyrosine phosphorylated after receptor activation. Rat basophilic leukemia RBL-2H3 cells provide a useful experimental model to study these signal
transduction pathways for degranulation in mast cells/basophils (2, 3).
Here we report that a monoclonal antibody raised to these cells
recognized a 130-kDa tyrosine phosphorylated protein. The
phosphorylation of this protein was dramatically increased after
Fc
RI aggregation. Amino acid sequence analysis of purified protein
suggested that this molecule was the rat homolog of the platelet
endothelial cell adhesion molecule-1
(PECAM-1),1 also called CD31. This was
confirmed by immunochemical studies.
v
3 integrin (13).
RI aggregation. Mast cells
and/or basophils accumulate and play a critical role at sites of
inflammation. This enhanced adhesion of activated cells to the
endothelium of capillaries and their migration into sites of
inflammation may be regulated by the tyrosine phosphorylation of the
PECAM-1 molecule.
Materials
RI
(mAb BC4) and
anti-trinitrophenol-specific IgE have been described previously (22,
26, 27). Polyclonal rabbit anti-human PECAM-1 and anti-rat PECAM-1 were
kindly provided by Dr. Kim Piotrowski (Blood Center of Southeastern
Wisconsin, Milwaukee, WI). Polyclonal rabbit anti-mouse PECAM-1 was
generously provided by Dr. Beat A. Imhof (Basel Institute for
Immunology, Basel, Switzerland). All other antibodies have been
described previously (24, 25).
RI
antibodies
(mAb BC4), calcium ionophore A23187 (0.5 µM), or phorbol
12-myristate 13-acetate (40 nM) essentially as described
previously (4). Briefly, 107 cells were seeded in Petri
plates (10-cm diameter), and after overnight culture, the cell
monolayers were washed once with 3 ml of Eagle's minimum essential
medium containing 0.1% bovine serum albumin and 10 mM
Tris, pH 7.5. The cells were then stimulated in the same medium. For
stimulation with antigen, the cells were cultured overnight with
antigen-specific IgE. After stimulation for the indicated times, the
medium was removed for histamine analysis. In experiments to deplete
extracellular Ca2+, the monolayers were washed with
calcium-free Eagle's minimal essential medium containing 10 µM EDTA and stimulated in this medium.
20 °C for 6 min
with cold methanol. After rinsing with PBS and blocking with 0.5%
bovine serum albumin/PBS, the cells were incubated with rabbit
anti-PECAM-1 (10 µg/ml) for 1 h at room temperature. The secondary antibody was fluorescein isothiocyanate-F(ab
)2
donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories). The
coverslips were mounted onto microscope slides and viewed under a Leica
4D TCS confocal microscope.
Characterization of mAb R23
RI-mediated signaling,
different monoclonal antibodies raised against rat basophilic leukemia
RBL-2H3 cells were tested to determine whether they immunoprecipitated
tyrosine phosphorylated proteins. One of these antibodies, mAb R23,
immunoprecipitated a 130-kDa protein that was weakly tyrosine
phosphorylated in nonstimulated cells but whose phosphorylation was
dramatically enhanced after Fc
RI aggregation (Fig. 1,
A and C). By immunoblotting, mAb R23 identified a
130-kDa protein in RBL-2H3 cells only under nonreducing conditions,
suggesting that binding of this antibody depends on the secondary
structure of the molecule (Fig. 1A). Immunofluorescence and
fluorescence-activated cell sorter analysis demonstrated binding of mAb
R23 to intact cells, suggesting that the antibody recognized the
extracellular domain of a transmembrane protein (Fig. 1B). These experiments suggested that this 130-kDa protein was tyrosine phosphorylated after Fc
RI aggregation.
Fig. 1.
The mAb R23 immunoprecipitates a 130-kDa
protein that is tyrosine phosphorylated after FcRI aggregation.
A, immunoblot analysis of the proteins recognized by R23.
Cell lysates were prepared from RBL-2H3 cells under nonreducing or
reducing conditions and analyzed by immunoblotting with mAb R23 (3 µg/ml). B, fluorescence-activated cell sorter analysis of
mAb R23 binding to RBL-2H3 cells. C, cells (5 × 106) were either nonstimulated (BC4
) or
stimulated for 30 min with 0.03 µg/ml of anti-Fc
RI
mAb BC4
(BC4 +). Proteins were immunoprecipitated with mAb R23
coupled to Sepharose 4B beads. The immunoprecipitates were analyzed by
immunoblotting with anti-phosphotyrosine antibodies and mAb R23. The
arrow indicates the protein detected with mAb R23. Molecular
mass markers (in kDa) represent migration of prelabeled standards.
[View Larger Version of this Image (17K GIF file)]
RI aggregation and then surface
labeled by biotinylation. By both one- and two-dimensional analysis the
tyrosine phosphorylated and the surface-labeled proteins were identical
(data not shown). Therefore, mAb R23 binds to the extracellular domain
of a 130-kDa membrane protein that is tyrosine phosphorylated after
receptor aggregation.
Fig. 2.
N-terminal amino acid sequence analysis of
the purified protein and comparison with the sequence of human and
mouse PECAM-1 (CD31).
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
The protein immunoprecipitated with mAb R23
is PECAM-1 (CD31). A, immunoprecipitates with mAb R23
(R23 IP) or total cell lysates (Lysate) were
separated by SDS-PAGE electrophoresis and then analyzed by
immunoblotting with mAb R23 (R23) or with polyclonal
antibodies that recognize human, rat, or mouse PECAM-1. B,
proteins from 5 × 106 cells were immunoprecipitated
with either mAb R23 coupled to Sepharose 4B or with a polyclonal rabbit
anti-rat PECAM-1 antibodies (SEW31). The immunoprecipitates were
analyzed by blotting with mAb R23 (R23) or with the
polyclonal anti-rat PECAM-1 (SEW31). C, lysates
from 107 cells were analyzed for PECAM-1 either as is
(R23 IP 0) or after removing PECAM-1 by a single (R23
IP 1) or double (R23 IP 2) immunoprecipitation with mAb
R23-coupled beads (50 µg of R23). The lysates were analyzed by
blotting with the polyclonal anti-rat PECAM-1 antibodies
(SEW31). For analysis by immunoblotting with mAb R23 the
precipitates were separated under nonreducing conditions, whereas for
analysis with polyclonal anti-PECAM the precipitates were separated
under reducing conditions. Note that in B there is binding
of the secondary antibody with nonreduced IgG (lane 1) and
with the heavy chain of reduced IgG (lane 4).
[View Larger Version of this Image (22K GIF file)]
RI-mediated signal transduction (7). Aggregation of Fc
RI
induced the time-dependent tyrosine phosphorylation of
PECAM-1 (Fig. 4). Tyrosine phosphorylation was
detectable at 1 min, peaked at 10 min, and was maintained at this level
for the duration of the experiment (30 min). In contrast, with antigen
stimulation it peaked at 10 min and was back to basal level by 30 min
(data not shown). Therefore, Fc
RI cross-linking induced tyrosine
phosphorylation of PECAM-1, the extent of which was influenced by the
aggregation signal. However, direct aggregation of PECAM-1 by the
addition of different concentrations of mAb R23 to adherent cells did
not induce degranulation and did not result in detectable changes in
total cellular tyrosine phosphorylation (data not shown). There was
also no change in the tyrosine phosphorylation of PECAM-1 itself.
Similarly, although RBL-2H3 cells attached to surfaces coated with mAb
R23, this did not induce tyrosine phosphorylation of PECAM-1.
Fig. 4.
Time course of FcRI-induced tyrosine
phosphorylation of PECAM-1 (CD31). Cells were either nonstimulated
(Blank) or stimulated for the indicated times; proteins were
immunoprecipitated with mAb R23 and analyzed by immunoblotting with
anti-phosphotyrosine antibodies followed by anti-PECAM-1
antibodies.
[View Larger Version of this Image (42K GIF file)]
RI
aggregation, whereas others are phosphorylated only after influx of
extracellular Ca2+ and/or activation of protein kinase C
(7, 9). Stimulation of cells to degranulate with either IgE and antigen
or the calcium ionophore A23187 resulted in a slight increase in
PECAM-1 tyrosine phosphorylation (Fig. 5). However,
there was no increase in PECAM-1 tyrosine phosphorylation after direct
activation of protein kinase C by the addition of phorbol 12-myristate
13-acetate. To further define the role of Ca2+ in
Fc
RI-mediated tyrosine phosphorylation of PECAM-1, cells were
stimulated in the presence or the absence of extracellular Ca2+ (Fig. 6). The absence of extracellular
Ca2+ did not affect Fc
RI-mediated tyrosine
phosphorylation of PECAM-1. Therefore, unlike other adhesion related
molecules such as pp125FAK, the tyrosine phosphorylation of
PECAM-1 is an early event that is upstream of the influx of calcium
and/or the activation of protein kinase C.
Fig. 5.
Tyrosine phosphorylation of PECAM-1 (CD31)
after RBL-2H3 stimulation. Cells (5 × 106) were
either nonstimulated (Blank) or stimulated for 10 min with the anti-FcRI
mAb BC4 (BC4), antigen (Ag),
calcium ionophore A23187 (Iono), or phorbol myristate
acetate (PMA). For the antigen-stimulated samples the cells
were cultured overnight with antigen-specific IgE as described under
"Experimental Procedures." Proteins from 5 × 106
cells were immunoprecipitated with mAb R23 antibodies coupled to
Sepharose 4B beads. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine and mAb R23 antibodies. The
arrow indicates the protein detected with mAb R23. Molecular mass markers (in kDa) represent migration of prelabeled
standards.
[View Larger Version of this Image (32K GIF file)]
Fig. 6.
Lack of an effect of Ca2+ in the
medium on FcRI-induced tyrosine phosphorylation of PECAM-1.
Cells grown as monolayers were either washed in regular
(Ca2+ +) or with medium lacking
Ca2+ (Ca2+
), and then some
were activated by anti-Fc
RI
mAb BC4 (BC4 +). Proteins
immunoprecipitated with mAb R23 were analyzed by immunoblotting with
anti-phosphotyrosine. Percent histamine release (%HR) is at
the bottom of each lane.
[View Larger Version of this Image (21K GIF file)]
RI-induced tyrosine phosphorylation of the focal adhesion kinase,
pp125FAK (32, 33). Thus, cell stimulation results in
minimal if any tyrosine phosphorylation of pp125FAK, unless
the RBL-2H3 are adherent (32). However, the Fc
RI-mediated tyrosine
phosphorylation of PECAM-1 was equally strong in nonadherent and
adherent cells (Fig. 7). Thus, unlike
pp125FAK, the Fc
RI-mediated tyrosine phosphorylation of
PECAM-1 is independent of cell adhesion. Activation of mast
cells also results in enhanced adherence (33). The increased tyrosine
phosphorylation of PECAM-1 in stimulated cells could therefore mediate
the enhanced adherence of activated mast cells. Both nonactivated and
activated RBL-2H3 cells adhered to surfaces coated with mAb R23 equally
well but did not adhere to purified recombinant PECAM-1 (data not
shown). Therefore, adherence as measured by these gross parameters of adherence through binding to PECAM-1 was not significantly modified after Fc
RI aggregation.
Fig. 7.
FcRI-induced tyrosine phosphorylation of
PECAM-1 in adherent and nonadherent cells. RBL-2H3 cells
nonadherent (FN
) or adherent to fibronectin (FN
+) were either nonstimulated or stimulated with anti-Fc
RI
mAb BC4 (BC4 +). Proteins immunoprecipitated with mAb R23
were analyzed by immunoblotting with anti-phosphotyrosine and
anti-PECAM-1 antibodies.
[View Larger Version of this Image (23K GIF file)]
RI aggregation induced a redistribution of PECAM-1 in cells (Fig.
8). As expected, in nonstimulated cells PECAM-1 was
membrane-associated. After 20 min of stimulation it had redistributed
to the ruffles near the apical surface of the cells. There was also an
increase in punctate staining just below the membrane at the apical
surface. Therefore, Fc
RI aggregation results in tyrosine
phosphorylation of PECAM-1 and in its redistribution on the cell
surface.
Fig. 8.
Localization of PECAM-1 in membrane ruffles
in stimulated cells by fluorescent confocal microscopy. A,
control nonstimulated cells. B, cells stimulated for 5 min
with anti-FcRI
mAb BC4. C and D, cells
stimulated for 20 min with anti-Fc
RI
mAb BC4. A,
B, and C, anti-PECAM-1 used as primary antibody.
D, normal rabbit IgG substituted for the primary antibody.
The images as optical sections were restacked as a single image for
output.
[View Larger Version of this Image (106K GIF file)]
RI aggregation with
anti-receptor antibodies induced minimal tyrosine phosphorylation of
PECAM-1 in Syk-deficient cells. This was increased in the cells that
had been reconstituted by the stable transfection of Syk. Therefore, although some tyrosine phosphorylation of PECAM-1 occurs upstream or
independent of Syk, it is predominantly dependent on the presence of
Syk in the cells. However, this does not necessarily mean that Syk
tyrosine phosphorylates PECAM-1.
Fig. 9.
FcRI-induced tyrosine phosphorylation of
PECAM-1 in Syk negative and Syk transfected cells. RBL-2H3
(Syk+, 2H3), TB1A2 (Syk
,
TB1A2), and Syk-transfected (3A5) cell lines were
either nonstimulated or stimulated with 0.3 µg/ml anti-Fc
RI
mAb
BC4 (BC4 +). Proteins immunoprecipitated with mAb R23 were
analyzed by immunoblotting with anti-phosphotyrosine and
anti-PECAM-1 antibodies.
[View Larger Version of this Image (36K GIF file)]
RI aggregation. The phosphorylation of
PECAM-1 was an early event after receptor activation and was not seen
when cells were stimulated with phorbol myristate acetate and did not
require cell adherence. The Fc
RI-induced tyrosine phosphorylation of
PECAM-1 was much stronger and persistent with anti-receptor antibodies
than with IgE antigen. However, there was still variation in the extent
of PECAM-1 tyrosine phosphorylation with different anti-Fc
RI
antibodies (data not shown). The varying efficiency of different
Fc
RI aggregation signals in inducing tyrosine phosphorylation of
PECAM-1 may be due to differences in the way these signals orient and
aggregate the receptor (34).
RI suggests the cooperation
between Lyn and Syk protein tyrosine kinases (29). Receptor aggregation
induces activation of a protein tyrosine kinase, probably Lyn, which
results in tyrosine phosphorylation of the receptor subunits. Syk is
then recruited by the tyrosine phosphorylated receptor subunits; its
activation then propagates downstream signals including the tyrosine
phosphorylation of phospholipase C-
and the rise in intracellular
calcium. Tyrosine phosphorylation of PECAM-1 in this cascade of events
was partly independent of Syk, although it was enhanced by the presence
of Syk. Tyrosine phosphorylation was also independent of the presence
of calcium in the medium. Although Fc
RI, Lyn, and Syk are all either
membrane proteins or associated with the membrane at different stages
of receptor activation, we could not detect any association by
immunoprecipitation of these molecules with PECAM-1, another membrane
protein (data not shown). Moreover, there was no kinase activity in the
PECAM-1 immunoprecipitates. Nevertheless, tyrosine phosphorylation of PECAM-1 may be due to Lyn, Syk, or other tyrosine kinases activated by
receptor aggregation.
-actinin, filamin, pp125FAK, and other phosphoproteins
(37). Formation of the complexes is accompanied by tyrosine
phosphorylation of proteins such as pp125FAK and of the
cytoskeletal protein paxillin (38). In contrast, aggregation of
PECAM-1, a member of the Ig superfamily of adhesion molecules, does not
induce the formation of such focal adhesion complexes nor does it
result in the tyrosine phosphorylation.
1 and
2 integrins on neutrophils, monocytes, and T cells (50-52). The interaction of endothelial PECAM-1
with leukocyte or basophil PECAM-1 could result in up-regulation of the
activity of integrins, which may provide the interaction necessary for
transmigration through the endothelial cell junctions. Similarly,
tyrosine phosphorylation of PECAM-1 could also influence its capacity
to regulate the adhesive activity of integrins.
*
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Immunology, Bldg. 10, Rm. 1N106, NIDR, NIH, Bethesda, MD 20892. Tel.:
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1
The abbreviations used are: PECAM-1,
platelet/endothelial cell adhesion molecule 1 (also called CD31); PAGE,
polyacrylamide gel electrophoresis; Pipes,
1,4-piperazinediethanesulfonic acid; mAb, monoclonal antibody; PBS,
phosphate-buffered saline.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.