From INSERM U485, Institut Pasteur, 25 rue du Dr.
Roux, 75015 Paris, France and § INSERM U25, Hôpital
Necker-Enfants Malades, 161 rue de Sèvres, 75743 Paris,
France
Received for publication, January 29, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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To identify new effectors of IgE receptor
(Fc Plasma membranes have an asymmetric lipid distribution. Whereas
both plasma membrane leaflets are composed mainly of choline-containing phospholipids (phosphatidylcholine and sphingomyelin), the inner leaflet is enriched relative to the outer leaflet in primary
amine-containing phospholipids (phosphatidylserine
(PS),1 and
phosphatidylethanolamine). This asymmetry is actively maintained by two
ATP-dependent enzymes: a phospholipid translocase that selectively transports aminophospholipids from the outer to the inner
leaflet; and a floppase that moves all phospholipids in the opposite
direction (1). The physiological importance of the maintenance of this
energy-consuming unbalance in membrane composition is unveiled when
this asymmetry is disrupted. When cells undergo apoptosis, translocase
and floppase become inactivated, and a phospholipid scramblase (PLSCR),
an enzyme that moves all phospholipids bi-directionally, becomes
activated (1). The consequence is a loss of phospholipid asymmetry with
a resulting increase in PS expression in the outer leaflet of the
plasma membrane. The apoptotic cell is then recognized and engulfed by
phagocytes expressing receptors for PS that confer both recognition and
phagocytic capabilities to these cells (2).
Yet, the role of phospholipid scrambling is not restricted to
apoptosis. In blood platelets and vascular endothelial cells, activation induces an increase in the surface expression of PS that in
turn activates coagulation enzymes (3). In at least one instance of
bleeding disorder known as Scott syndrome, a defect in
coagulation was related to a defect in scramblase activity in intact
cells (4, 5). Another role for phospholipid scrambling is suggested by
the increased surface expression of PS after mast cell activation (6,
7). These cells are involved in innate immunity; they are able to
phagocytose and kill pathogens, recruit leukocytes and lymphocytes
through cytokine production, and present antigen to immunocompetent
cells (8). These cells are also involved in allergic reactions; they
express receptors with high affinity for IgE (Fc So far, four PLSCR (numbered 1-4) have been cloned in humans and three
(numbered 1-3) in the mouse, demonstrating the existence of a family
of PLSCR (10). A comparison of the amino acid sequence of all the
members of the PLSCR family shows that they are type II membrane
proteins with strong homology for the transmembrane domain and for most
of the intracellular domain. This strongly suggests that these
domains represent the portion of the molecule that accounts for
scramblase activity. The extracellular domain is not conserved among
members and is extremely short or absent, suggesting it plays no, or
little, role in scrambling activity. Interestingly, the intracellular
amino terminus is extremely variable in length and composition among
members of the PLSCR family. It is thus possible that it serves
as a regulatory domain for the scrambling activity or that it serves
additional functions unrelated to scrambling. In this regard, it should
be noted that counterstructures for Src homology 3 (SH3) domains are
present in this region of the molecule for most members of the family.
Original studies focusing on the regulation of PLSCR activity
demonstrated that it is dependent on calcium (11). Yet most of these
studies were performed with purified PLSCR reconstituted into liposomes
(11-13) or into inside-out vesicles (13), i.e. away from
other potential regulatory elements. Today many data from the
literature point to other regulatory mechanisms of PLSCR activation. PS
exposure correlates with the level of PLSCR expressed in cells
stimulated with a calcium ionophore (14) but not when the cells are
stimulated by a more physiologic stimulus (15). The calcium
concentration required for the onset of scramblase activity is higher
when this enzyme is reconstituted into inside-out vesicles than into
liposomes (13) suggesting that elements associated with the plasma
membrane might negatively regulate the enzyme. In Scott syndrome,
although phospholipid scrambling is impaired, the level of PLSCR1 in
blood cells is normal, the nucleotide sequence of PLSCR1 shows no
mutation, and its activity after purification and reconstitution into
liposomes is normal (16-18). Additional data suggest that regulatory
elements rather than PLSCR itself are impaired in this syndrome (19,
20). In mast cells, externalization of PS induced by Fc Here, we have cloned a new member of the PLSCR family expressed in rat
mast cells using specific mouse monoclonal antibodies (mAbs). We
demonstrate that, upon aggregation of Fc Purification of Phosphoproteins for mAb Production--
Fifteen
150-cm2 culture flasks containing ~5 × 108 RBL-2H3 cells altogether were washed twice with HBT
(Hanks' buffer saline solution (Life Technologies, Inc.)
supplemented with 0.1% bovine serum albumin (BSA, Sigma-Aldrich) and
50 mM Tris, pH 7.2) and stimulated with 100 ng/ml anti-rat
Fc Hybridoma Production--
A BALB/c mouse was hyperimmunized with
the phosphoproteins by several injections of the eluates into the
footpads. A new batch of eluate was prepared immediately before each
injection. The first injection was in 200 µl of a 1:1 mixture with
complete Freund's adjuvant, the second was a week later in a 1:1
mixture with incomplete Freund's adjuvant, and the next four
injections were at 1-week intervals in PBS. One day after the last
injection the mouse was sacrificed, the inguinal and popliteal lymph
nodes were excised, the cells were fused with Ag8.653 myeloma cells,
and the resulting hybridomas were selected as described (24). After
selection, hybridomas were frozen and their culture supernatants
conserved at 4 °C in 0.1% NaN3 for screening.
Screening of the Hybridomas--
Screening was completed by an
immunoblotting approach. Purified phosphoproteins were resolved in
4-15% polyacrylamide two-dimensional minigels (Bio-Rad), and
transferred onto a polyvinylidene difluoride membrane (Millipore,
Bedford, MA). The membrane was incubated for 1 h in TTBS (10 mM Tris, 150 mM NaCl, 0.05% Tween 20)
containing 4% BSA and inserted in the mini-Protean® II
Multiscreen apparatus (Bio-Rad), and each channel was filled with 600 µl of a 1:30 dilution of hybridoma supernatant in TTBS containing
0.5% BSA (TTBS-BSA). Fifteen supernatants were screened per
membrane, and each supernatant was tested with phosphoproteins resolved
under reducing and nonreducing conditions. After a 1-h incubation, each
channel was washed twice with 10 ml TTBS. The membranes were then
incubated for 1 h with a 1:30,000 dilution of horseradish
peroxidase-conjugated goat anti-mouse antibody (Sigma-Aldrich) in
TTBS-BSA. After three washes in TTBS, proteins were visualized with the
SuperSignal chemiluminescence kit from Pierce, and the membranes were
exposed for autoradiography with X-OMAT films (Eastman Kodak Co.).
Hybridomas corresponding to selected positive supernatants were thawed
and subcloned, each set of subclones being screened likewise.
Immunoglobulin G in cell culture supernatants or in ascitis fluid
supernatants from selected positive subclones were purified on protein
G-coupled beads, and a fraction was coupled to CNBr-activated Sepharose
4B beads (both beads from Amersham Pharmacia Biotech) according to the
manufacturer's recommendations. The mAbs used in this study (129.2, 17.3, and 9.18) were all IgG1.
Construction and Screening of the Library--
Total RNA was
extracted from RBL-2H3 cells with TRIZOLTM (Life
Technologies, Inc.). mRNA was purified with the Messenger RNA Isolation kit from which 5 µg were used to construct a cDNA
library in
Screening with purified mAb 129.2 of 4.5 × 105
cDNA clones in
Positive clones were plugged out from the agar plates and sequentially
subjected to two additional rounds of screening to obtain complete
isolation of the clones. The cDNA were excised in pBlueScript from
the
Alignments were done with the Dialign 2.1 program (25) and edited with
Boxshade 3.33c.
RBL-2H3 Cell Culture and Stimulation and Peritoneal Mast Cell
Purification--
RBL-2H3 cells were maintained as published (26).
Routine stimulation was as follows. 1 × 106
cells/well were plated in 6-well plates overnight with or without a
1:1000 dilution of an ascitic fluid supernatant of
anti-2,4-dinitrophenol monoclonal IgE, DNP-48 (a kind gift of Dr. R. Siraganian, National Institutes of Health). After two washes in HBT,
the cells were stimulated with 1 µg/ml 2,4-dinitrophenol-human serum
albumin in HBT for 30 min at 37 °C.
Peritoneal mast cells were purified to 95% purity from 250-g male
Harlan Sprague-Dawley rats (Janvier, Le Genest St. Isle, France)
according to Ref. 27.
Immunoprecipitations and Immunoblot Analyses--
After
stimulation and two washes in ice-cold PBS, the cells were lysed on ice
in 200 µl of lysis buffer. After 10 min of incubation on ice, the
cell lysate was recovered by scraping, and the post-nuclear supernatant
was immunoprecipitated for 2 h at 4 °C with 10 µl of the
indicated anti-rat PLSCR mAb-coupled beads. After six washes of the
beads with 1 ml of ice-cold lysis buffer, the immunoprecipitated material was eluted by boiling in 50 µl of Laemmli loading buffer. Half the eluates were analyzed by immunoblotting following procedures already described (28) with mAb PY20 coupled to horseradish peroxidase
(dilution 1:2500; Transduction Laboratories) or with mAb 129.2, 17.3, or 9.18 at 1 µg/ml followed by goat anti-mouse Ig coupled to
horseradish peroxidase. In some cases the membranes were stripped by
three washes for 5 min in methanol before reprobing. Where indicated,
the samples were not transferred and the gel was subjected to silver staining.
For reprecipitation experiments, immunoprecipitated proteins were first
eluted by boiling for 5 min in 50 µl of 50 mM Tris, pH
7.2, containing 1% SDS. The eluate was quenched by the addition of 950 µl of lysis buffer and reprecipitated for 2 h with 20 µl of
mAb 129.2-coupled beads. After six washes with 1 ml of lysis buffer,
the final elution was in 50 µl of boiling Laemmli loading buffer.
Immunofluorescence Studies--
Anti-PLSCR mAb 129.2 binds the
intracellular portion of rat PLSCR. Therefore, cells had to be
permeabilized in immunofluorescence studies carried out with this mAb.
RBL-2H3 cells grown on slides were rinsed twice with ice-cold PBS and
fixed by a 20-min incubation in cold 3% paraformaldehyde. After 2 washes for 10 min each in PBS, the cells were incubated for 10 min in
0.1 M glycine in PBS. Cells were then permeabilized by a
30-min incubation in 0.05% saponin in PBS containing 0.2% BSA at room
temperature. Cells were labeled thereafter by the addition of 50 µg/ml anti-rat PLSCR mAb 129.2 or control IgG1. One hour
later, cells were washed three times in PBS/saponin/BSA before a 45-min
incubation with fluorescein isothiocyanate-labeled anti-mouse
IgG1 (Southern Biotechnology Associates, Birmingham, AL).
Cells were subsequently washed three times in PBS/BSA without saponin
and incubated for 5 min with 2 µg/ml Texas Red-labeled wheat germ
agglutinin (Molecular Probes, Eugene, OR). This latter incubation was
performed to visualize membrane labeling. After three final washes in
PBS/BSA, the slides were fixed and the cells were examined in a
confocal laser microscope (LSM 510 Carl Zeiss, Jena, Germany).
As a means of identifying new protein targets for tyrosine kinases
in FcRI) signaling, we purified proteins from Fc
RI-stimulated
RBL-2H3 rat mast cells on anti-phosphotyrosine beads and generated
mouse monoclonal antibodies (mAb) against these proteins. Two mAbs
bound to a protein that was identified as a new isoform of phospholipid
scramblase (PLSCR) after screening an RBL-2H3 cDNA expression
library. This isoform differed from PLSCR1 by the absence of an exon
3-encoded sequence and by an insert coding six QGPY(P/A)GP
repeats. The PLSCR family of proteins is responsible for a
redistribution of phospholipids across the plasma membrane. Although
rat PLSCR is a 37-kDa protein, anti-phosphotyrosine immunoblots
revealed the presence of 37-49 kDa phosphoproteins in the material
immunoprecipitated with either anti-PLSCR mAb but not with unrelated
monoclonal or polyclonal antibodies. Depletion of PLSCR resulted in the
absence of these phosphoproteins. Additional experiments led to the
identification of these phosphoproteins as phospho-PLSCR itself.
Stimulation of RBL-2H3 cells upon Fc
RI engagement resulted in a
dramatic increase in PLSCR tyrosine phosphorylation. A comparison of
the relative amounts of phospho-PLSCR and nonphosphorylated PLSCR demonstrated that only a tiny fraction was thus modified, indicating a
finely targeted involvement of PLSCR in Fc
RI signaling. Thus, this
study reports the cloning of a new isoform of PLSCR, as well as the
first observation that a member of the PLSCR family is a target for
tyrosine kinases and is involved in signaling by an immune receptor.
These findings open new perspectives on the role of phospholipid
scramblases and to the mechanisms involved in their regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RI) that, when
aggregated by specific IgE and allergen, trigger mast cell
degranulation with the release of vasoactive amines (such as histamine)
and proteases, and production of proinflammatory mediators (such as
leukotrienes and prostaglandins) and cytokines (9).
RI-mediated
cell activation is transient (7), revealing the action of
down-regulatory elements. Furthermore, in these cells, the increase in
free intracellular calcium after Fc
RI aggregation is at most 1 µM (21), far from the millimolar range required to
activate purified PLSCR in vitro (13). More direct evidences
for regulations that are not limited to calcium have been recently
reported. Thus, PLSCR is a target for protein kinase C (PKC)
; when
phosphorylated by the latter, the former has an increased activity
(22). In addition, it has been reported that palmitoylation of PLSCR
potentiates its activation by calcium (23).
RI, PLSCR shows dramatic
increase in tyrosine phosphorylation. Thus, PLSCR is a target for
tyrosine kinase activity and is involved in signaling by an immune
receptor. The significance of these observations is discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RI
mAb BC4 (a generous gift from Dr. R. Siraganian, National
Institutes of Health, Bethesda, MD) in HBT for 10 min. After two washes
in ice-cold phosphate-buffered saline solution (PBS), the cells were
lysed in 2 ml/flask of lysis buffer (50 mM Tris, pH 7.2, containing 1% Triton X-100, 0.1% SDS, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4,
250 kallikrein-inactivating unit/ml aprotinin and 50 µg/ml
leupeptin). After 10 min on ice, the cell lysate was recovered by
scraping, and the post-nuclear supernatant was immunoprecipitated with
3 ml of a mixture of anti-phosphotyrosine mAbs 4G10 coupled to agarose
beads (Upstate Biotechnology, Lake Placid, NY) and PY20 coupled to
agarose beads (Transduction Laboratories, Lexington, KY). After 4 h on a rotating wheel at 4 °C, the beads were washed six times at
4 °C with 10 ml of lysis buffer, followed by two washes with 10 ml
of PBS, and two washes with 10 ml of PBS containing 30 mM
octyl-glucopyranoside (PBS-O, Sigma-Aldrich). The beads were
then eluted twice by a 30-min incubation at 4 °C on a rotating wheel
in 3 ml of PBS-O containing 40 mM phenylphosphate. The
eluates were pooled and concentrated to 50-100 µl. After each purification, the quality of the material was controlled in
anti-phosphotyrosine immunoblots. The eluates were found to be specific
because, in contrast to phenylphosphate elution, no protein was eluted
by PBS-O alone as observed after silver staining. After elution, the
beads were regenerated by two 30-min elutions with 1 M NaCl and conserved at 4 °C in 0.2 M borate-buffered saline
solution, pH 8.
ZAP with the ZAP-cDNA®
Gigapack® III Gold Cloning kit, both kits from Stratagene
(La Jolla, CA). All procedures were done according to the corresponding
manufacturer's recommendations. Three million cDNA-independent
clones were obtained.
ZAP was performed after infection of XL1
Blue-MRF' bacteria, spreading onto agar plates, induction of protein
synthesis by isopropyl
-D-thiogalactopyranoside, and
protein transfer by adsorption onto cellulose membrane. After extensive
washings in TTBS containing 0.1% NaN3, membranes were
saturated by an overnight incubation in TTBS containing 4% BSA and
blotted for 1 h with 10 µg/ml mAb129.2 in TTBS-BSA. After
washings of the membranes, the latter were incubated for 1 h with
a 1:30,000 dilution of horseradish peroxidase-conjugated goat
anti-mouse IgG antibodies in TTBS-BSA. After several washes, the
positive clones were visualized with the SuperSignal kit from Pierce
and exposed for autoradiography.
ZAP genome according to the manufacturer's instructions.
Sequencing of the two strands of the cDNA was performed by
automated sequencing by Genome Express (Paris, France).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RI-stimulated mast cells, the bulk of tyrosine-phosphorylated proteins from Fc
RI-stimulated RBL-2H3 mast cells was purified with
anti-phosphotyrosine-coupled beads and specifically eluted with phenyl
phosphate. MAbs were raised against these proteins and, after screening
of 750 hybridoma clones, 10 groups totaling 105 positive clones were
identified, each group recognizing a different phosphoprotein of
defined Mr. mAbs directed against a protein that
migrated at ~40 kDa in 4-15% polyacrylamide gradient gels were
selected first. One of these, mAb 129.2, was used to screen an RBL-2H3
cDNA expression library constructed in
ZAP. Seven independent
positive cDNA clones were isolated from 4.5 × 105
clones. One of these positive clones, clone 312, was fully sequenced. It was 1.6 kilobase pairs in length and included the complete 3'-untranslated region, the complete coding sequence of a 335-amino acid protein, and part of the 5'-untranslated region (Fig.
1A). A rat EST sequence was
found in the GenBankTM nucleotide data bank (accession
number AA924029) that was identical to the stretch extending from
nucleotide 1056 to 1436 in clone 312 (data not shown). Matching the
translated sequence from clone 312 against the translated
GenBankTM nucleotide data bank also showed that it was most
homologous to human (68% identity; GenBankTM accession
numbers AF098642 and AB006746) and mouse (84% identity;
GenBankTM accession number AF159593) PLSCR1 (Fig.
1B). Yet, two major differences are apparent. The human gene
for PLSCR1 is composed of 9 exons, the third one coding for the
sequence extending from Asn-5 to Gln-31 (10). This sequence is
absent in rat PLSCR (Fig. 1B). Rat PLSCR also contains an
additional sequence made of six QGPY(P/A)GP repeats (Fig.
1B). Although this sequence is reminiscent of repeats
present in the sequence of collagens, its function remains so far
unknown. Thus, it appears that the rat PLSCR that we have cloned is
related to, but distinct from, PLSCR1. Analysis of the six remaining
positive clones by partial sequencing and/or restriction digest
indicated that all were also full-length cDNA clones of the same
gene (data not shown). The deduced amino acid sequence analyzed with
the TMpred program at the ISREC server was consistent with a type II
transmembrane protein with a short extracellular domain (Fig.
1B). It contains an intracellular calcium-binding domain
(29), multiple cysteines that are potential targets for palmitoylation (23), and three
P-(X)4-P-(X)2-P
domains that could associate with SH3 domains (Fig. 1B). A
PKC target consensus sequence (RXXTXR) is
conserved at Thr-178. These combined features are found in the sequence
of many members of the PLSCR family (10, 30).
View larger version (48K):
[in a new window]
Fig. 1.
Sequencing of rat PLSCR. A,
nucleotide and deduced amino acid sequences of clone 312. B,
comparison between rat PLSCR and human and mouse PLSCR1 amino acid
sequences. Identical residues are boxed in
black, conservative substitutions are boxed in
gray. The potential transmembrane domain identified with the
TMpred program at the ISREC server is indicated by a thick
underline. The calcium binding domain is indicated by a
dotted underline, the three proline-rich domains
(P-(X)4-P-(X)2-P)
with a thin underline, the three cysteine-rich regions with
a broken underline, and the threonine residue that is a
consensus target for PKC is shown with an arrowhead. The
exon 3-encoded region of human (and possibly mouse) PLSCR1 that is
missing in rat PLSCR is indicated by a double
underline.
Immunoprecipitation and immunoblotting of rat PLSCR showed a single
band of Mr 37,000 (Fig.
2A) corresponding to the size expected from the deduced amino acid sequence. In some instances, a
doublet was observed that could be the result of degradation by
proteases. Identical results were obtained from purified rat peritoneal
mast cells, further confirming that rat PLSCR indeed is expressed in
rat mast cells (Fig. 2A). Immunofluorescence analysis showed
that, in contrast to control IgG1, anti-rat PLSCR mAb 129.2 labeled RBL-2H3 cells with a pattern that was consistent with a
membrane location of the protein (Fig. 2B, panel
D). This was further confirmed by colocalization experiments
carried out with wheat germ agglutinin, a lectin that binds
extracellular domains of glycosylated membrane proteins (Fig.
2B, panels E and F).
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Since the anti-rat PLSCR antibodies had been generated by immunization
with proteins purified with anti-phosphotyrosine antibodies, we wished
to know whether this protein is phosphorylated in RBL-2H3 cells. To
that effect, immunoprecipitations were performed with mAb 129.2, and
the eluted phosphoproteins were detected by immunoblotting with
anti-phosphotyrosine mAb PY20. Heterogeneous phosphoproteins of 37-49
kDa were observed that did not comigrate with PLSCR as observed in
anti-rat PLSCR immunoblots of the precipitates (Fig. 3). These phosphoproteins were also
precipitated with another anti-rat PLSCR mAb from the same group, mAb
17.3 but not with mAb 9.18 that recognizes a 130-kDa phosphoprotein
(Fig. 3) and not with numerous other mAb or polyclonal mouse IgG of
irrelevant specificities (data not shown). In addition, when the cell
lysate was first depleted of its PLSCR content by immunoprecipitation with mAb 129.2 followed by a second immunoprecipitation procedure with
either mAb 17.3 (Fig. 3) or 129.2 (data not shown), the phosphoproteins were no longer detected. Thus, these proteins seemed to associate directly or indirectly with rat PLSCR.
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To determine whether FcRI-mediated cell activation affected the
phosphorylation of these phosphoproteins and of PLSCR, RBL-2H3 cells
were stimulated with IgE and antigen for 30 min. The material recovered
from anti-rat PLSCR immunoprecipitates was analyzed in
anti-phosphotyrosine immunoblots. A dramatic increase in the tyrosine
phosphorylation of the phosphoproteins was clearly observed (Fig.
4). This increase was observed in mAb
17.3 (Fig. 4) as well as in mAb 129.2 (data not shown)
immunoprecipitates. Yet, no clear-cut result was obtained regarding the
tyrosine phosphorylation of PLSCR because, here again, the
phosphoproteins did not comigrate with the latter.
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Thus, up to that point, we had not been able to determine whether rat
PLSCR was actually phosphorylated on tyrosine. To solve this question,
we first immunoprecipitated PLSCR with its supposedly associated
phosphoproteins, dissociated the complex by boiling for 5 min in 1%
SDS, and reprecipitated PLSCR from the eluted material. Analysis of the
tyrosine-phosphorylated proteins showed a pattern identical to that of
the phosphoproteins recovered without prior dissociation of the complex
(Fig. 5A). It became apparent therefore that the phosphoproteins were not precipitated through their
association with rat PLSCR but through direct binding of anti-rat PLSCR
mAb. Thus, the phosphoproteins could be PLSCR itself.
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To determine definitely whether the phosphoproteins were in fact phospho-PLSCR, tyrosine-phosphorylated proteins were purified from a large number of stimulated RBL-2H3 cells, concentrated and analyzed in anti-rat PLSCR immunoblots. An identical band of heterogeneous proteins of 37-49 kDa was observed in these samples and in anti-phosphotyrosine immunoblots of anti-rat PLSCR immunoprecipitates (Fig. 5B). As a control, mAb 9.18 immunoblots of anti-phosphotyrosine immunoprecipitates showed the expected 130-kDa phosphoprotein but not the 37-49 kDa proteins (Fig. 5B). Therefore, the phosphoproteins are in fact phospho-PLSCR.
Rat PLSCR was readily detected in silver staining of immunoprecipitates
from 2 × 106 cells, whereas phospho-PLSCR was not
(Fig. 5C), demonstrating that only a very small fraction of
this protein is phosphorylated on tyrosine. This explains the lack of
detection of phospho-PLSCR in mAb 129.2 (or 17.3) immunoblotting of
anti-rat PLSCR immunoprecipitates. This suggests that only finely
targeted PLSCR are involved in FcRI signaling. The difference
between the Mr of nonphosphorylated PLSCR and
the Mr of phospho-PLSCR could follow from
different causes. It is well known that phosphorylation of
proteins can result in retarded migration in gels. Thus, tyrosine
phosphorylation of rat PLSCR could result in a heterogeneous and
retarded apparent Mr, which could be observed
all the more if rat PLSCR is additionally phosphorylated on threonine
by protein kinase C
(22). Of interest, PKC
is involved in Fc
RI
signaling (31, 32). Another reason for the retarded
Mr observed could be the potential multiple
palmitoylations of rat PLSCR on the numerous clusters of cysteines that
are present in the molecule (Ref. 23 and Fig. 1B).
Additional studies will be necessary to determine the degree of
palmitoylation of PLSCR in rat mast cells. Thus, it is likely that
combined post-transcriptional modifications account for the large and
heterogeneous shift in the Mr of a fraction of
PLSCR after Fc
RI engagement.
The role of tyrosine phosphorylation of PLSCR could be related to the
known ability of this family of proteins to promote a redistribution of
phospholipids across the plasma membrane (11). Indeed, activation of
mast cells through FcRI leads to transient externalization of PS
that correlates with degranulation (6, 7). Although it is known that
externalization of PS is not dependent on tyrosine kinases in
erythrocytes stimulated by calcium ionophores (20), this dependence
could be true in mast cells stimulated through Fc
RI. Indeed, it is
also well known that tyrosine kinases are important for
IgE-dependent mast cell degranulation (33). Alternatively,
PLSCR tyrosine phosphorylation could be involved in the down-regulation
of Fc
RI-induced phospholipid scrambling, because the latter is
transient in mast cells (7). Another possible role for PLSCR
phosphorylation could be to make this protein serve as an adaptor for
other effector molecules. It contains three proline-rich domains that
could associate with SH3 domains on other proteins, and its location
could be used by the Fc
RI-generated signal to target SH2
domain-containing effector molecules at the plasma membrane. All of
these possibilities are under active investigation with the use of
mutant and chimeric proteins. In summary, this study reports the
cloning of a new member of the PLSCR family and the first observation
that a member of this family is a target for tyrosine kinases and is
involved in signaling by an immune receptor. This opens new
perspectives pertaining to the role of phospholipid scramblases and to
the mechanisms involved in their regulation.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Y. Goureau and B. Pasquier for expert assistance in confocal microscopy analyses. We are indebted to Dr. Reuben P. Siraganian (National Institutes of Health, Bethesda, MD) for the generous gift of reagents.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY024347 (rat PLSCR).
¶ To whom correspondence should be addressed. Tel.: 33.1.45.68.86.91; Fax: 33.1.45.68.87.03; E-mail: mben@pasteur.fr.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M100790200
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ABBREVIATIONS |
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The abbreviations used are:
PS, phosphatydylserine;
BSA, bovine serum albumin;
FcRI, IgE receptor
type I;
HBT, Hanks' buffer saline solution supplemented with BSA and
Tris;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline
solution;
PBS-O, PBS containing octyl glucopyranoside;
PLSCR, phospholipid scramblase;
SH, Src homology domain;
PKC, protein kinase
C;
TTBS, Tris-buffered saline solution containing Tween.
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REFERENCES |
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