From the Section on Chemical Immunology, Arthritis and Rheumatism Branch, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-1820
Received for publication, April 18, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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Chinese hamster ovary fibroblasts previously
transfected with the high affinity receptor for IgE (Fc Cellular responses mediated by the high affinity receptor for IgE
(Fc Two molecular models have been proposed to explain the role of
aggregation in stimulating the phosphorylation of the receptors. The
transphosphorylation model we have used to study this system is based
on the following experimental findings (4, 5): (a) Lyn
kinase is constitutively and weakly associated with a small fraction of
the receptors; b) in vivo and in vitro, the constitutively associated kinase is able to phosphorylate the receptors
but only when they are aggregated; and (c) the initial phosphorylation of the receptors, but not their aggregation per se, promotes their association with additional, more firmly bound, Lyn kinase. These findings suggest that aggregation stimulates the
phosphorylation of the receptors by stabilizing the juxtaposition of
the kinase with its substrate, thereby shifting the balance between
phosphorylation and dephosphorylation (6).
A second translocation model is based on the following observations:
(a) aggregates of receptors, particularly larger aggregates, become localized, at least transiently, in discrete detergent-resistant membranes (DRM)1 (7-9);
(b) such microdomains occupy only a small fraction of the
total plasma membrane but contain a substantial fraction of the total
Lyn kinase (7); (c) upon aggregation, those receptors that
become phosphorylated are largely found in the DRM; and (d) at least in some experiments (see "Discussion"),
cholesterol-depleting agents, which interfere with the formation of
such microdomains, inhibit the phosphorylation of the receptors (10).
It has been proposed that these findings indicate that the primary
effect of aggregation is to localize the receptors to regions of the membrane enriched in Lyn kinase (11). Neither of the two models predicates any aggregation-induced increase in the specific activity of
the kinase per se, consistent with the limited experimental data on this question (5).
We undertook the present studies for two reasons. First, to clarify
whether the translocation of the aggregated receptors to DRM was a
necessary and sufficient or simply an accompanying aspect of the
interaction between Lyn and the receptor. Second, to establish a system
with which we could analyze in as physiological a milieu as possible
the effect of genetically introduced changes in the structure of the
receptor or Lyn on their interaction.
We used genetic constructs of portions of the receptor similar to those
that had already been applied to this system by others (12-16).
Specifically, we used cDNA constructs in which the ecto- and
transmembrane domains of a subunit of interleukin-2 receptors (CD25 We performed complementary experiments by constructing chimeric
constructs consisting of the ecto- and transmembrane domains of Tac to
a construct of Lyn that had been mutated to remove the sites of
myristoylation and palmitoylation. We then examined the ability of such
constructs to influence the phosphorylation of the aggregated
receptors, much as we had previously studied normally anchored Lyn (17,
18).
Materials--
Avidin and polyclonal anti-Tac coupled to biotin
were purchased from R & D Systems (Minneapolis, MN). Extravidin
coupled to horseradish peroxidase was obtained from Sigma. Monoclonal
anti-phosphotyrosine antibody (anti-PY) coupled to biotin (4G10),
rabbit polyclonal anti-Lyn kinase, and Src family kinase substrate
peptide (19) were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Dr. T. Waldmann (NCI, National Institutes of Health)
provided a hybridoma producing monoclonal anti-Tac (clone HD245). The
hybridoma cells were gradually weaned off serum in successive tissue
cultures. The antibody was purified from the serum-free supernatants
using a kit containing immobilized protein A (Pierce) and labeled with carrier-free [125I]iodine using Iodogen tubes also from
Pierce. (Iodination of the anti-Tac with chloramine T resulted in the
complete loss of its bindability to Tac-expressing cells.) Monoclonal
anti-Tac coupled to biotin (clone B1.49.9) was from Cappel (Durham,
NC). Goat anti-mouse IgE, mouse monoclonal anti-DNP IgE (iodinated as
appropriate), and covalently cross-linked oligomers of IgE were
prepared as described previously (17). Polyclonal anti-caveolin was
from Santa Cruz (Santa Cruz, CA). SpinZyme® phosphocellulose filters
were from Pierce.
Preparation of Chimeric Constructs--
Table
I shows selected sequences of the
constructs we used for our studies. Dr. J. Oliver (University of New
Mexico) generously provided the cDNAs for constructs consisting of
the ecto- and membrane spanning domains of Tac fused to the C-terminal
42 residues of the
For the Lyn-based chimeric constructs, we generated fusion proteins by
polymerase chain reaction. cDNA coding for Lyn kinase mutated at
its N terminus was prepared as follows: sense and antisense primers for
the desired mutations of the intact wild-type short form of Lyn
(5'-CTCGAGATTAAATCAAAAAGGAAAGACAATC-3' and 5'-CTCGAGCCACTATGGCTGCTGCTGATACTGCCCTTCCGTGGCACTG-3') were synthesized and used in polymerase chain reaction with the
wild-type Lyn as template. The sense primer encodes a XhoI
site at its N terminus that excludes the N-terminal methionine and
glycine. The primer also substitutes the codon for cysteine with one
for glutamic acid. A construct of Tac coding for the ecto- and
transmembrane domain was generated by polymerase chain reaction with an
XhoI site at its C terminus. The two cDNAs were digested
with XhoI and then ligated to generate the fusion chimera
(TT-Lyn) and subsequently subcloned into the expression vector pZeo.
TT-LynKR was generated similarly using the Lyn cDNA
that encodes the catalytically inactive Lyn generated previously (17).
Analysis of each construct was determined using the Big Dye® kit
obtained from Applied Biosystems (Foster City, CA) and shown to be as planned.
Stable CHO Transfectants--
Plasmid expression constructs (10 µg) were electroporated (0.4 cm gap, 200 volts, 500 millifarad) into 1 × 107 CHO-B12 cells, a cell
line stably expressing 170,000 Fc Quantitation of Fc Stimulation of Cells--
Stimulation of CHO transfectants with
IgE plus antigen or with preformed covalently cross-linked oligomers
IgE was conducted as described previously (17). Where aggregation of
Tac was employed, the cells were incubated first with 2 µg/ml
monoclonal anti-Tac-biotin (clone B1.49.9) at room temperature and then
with 25 µg/ml avidin, for 10 min at 37 °C. Fc Immunoprecipitation and Western Blotting--
Fc Isolation of Plasma Membranes--
For monitoring of Fc Isolation of DRM--
The method we used is similar to that
described by Rodgers and Rose (24) and by Field et al. (8).
Briefly, cells were detached with 3 mM EDTA in PBS and
resuspended in growth medium at 5 × 106/ml. After the
desired treatment, 107 cells in 0.5 ml were diluted 2-fold
with 0.5 ml of 0.1% Triton X-100. The lysate was kept at 4 °C with
gentle agitation for 30 min. It was then mixed with an equal volume of
85% sucrose, transferred to a Beckman 344060 clear centrifuge tube and
overlaid with 6 ml of 30% sucrose prepared in a pH 7.5 buffer
containing 0.05% Triton X-100, 10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4, and 2 mM sodium iodoacetate
supplemented with protease inhibitors leupeptin, pepstatin, aprotinin,
each at 1 µg/ml, and 5 mM 4-(2-aminoethyl)-benzensulfonyl fluoride or phenylmethylsulfonyl fluoride (buffer A). The 30% layer
was overlaid with 3.5 ml of 5% sucrose, again prepared with buffer A. Tubes were centrifuged in a swinging bucket rotor at 38,000 rpm
for12-14 h. Successive 1-ml fractions were removed from the top of the
gradient. All fractions were counted and then diluted with Triton X-100
to a final concentration of 0.5% prior to immunoprecipitations of
Fc In Vitro Assays for Associated Kinase--
Triplicate
immunoprecipitates with anti-Lyn, anti-Tac, or control antibodies were
incubated in 25 µl of kinase assay buffer (25 mM Pipes,
150 mM NaCl, 5 mM KCl, pH 7.2, 5 mM
MnCl2, 2 mM CHAPS, 0.5 mM
Na3VO4) containing 2 mM substrate
peptide. Reactions were initiated by addition of ATP (100 µM ATP, 10 µCi of [ Transfection with Tac- Fc
Although the peptide molecular mass of the constructs is only
TT-
Additional studies examined the relationship between the ratio of
TT-
Clones containing the TT- TT- Inhibitory Constructs Are Not Phosphorylated--
In experiments
similar to those reported previously (17), we tested whether the
inhibitory constructs themselves became phosphorylated. Cells
transfected with TT- Mechanism of Inhibition--
Because a fundamental aim of this
study was to investigate further the role of membrane microdomains in
promoting the interaction of Lyn kinase with Fc
Wilson et al. (16) had previously shown that TT-
The results are described in Fig. 6
(top panel). In the unstimulated cells (open
symbols) both 100% of the recovered TT-
Two similar analyses of a TT-
In separate experiments we exposed cells to either
125I-labeled IgE or anti-Tac and then fractionated Dounce
homogenates of the TT- Interaction of Kinase with Constructs--
The simplest
explanation for the results described so far is that the chimeric
constructs competed with the Fc
We applied the same procedure to assay the endogenous Lyn in
immunoprecipitates from the TT- Transfection with Tac-Lyn Constructs--
Wild-type rat Lyn has
the N-terminal sequence MGCIKSK ... (17, 26). During its
biosynthesis the methionine is removed, the glycine is myristoylated,
and the cysteine is palmitoylated (27). The Tac-Lyn chimeric construct
we prepared (TT-Lyn) (Table I) truncates the Tac protein after its SGL
sequence, substitutes a glutamic acid residue for the N-terminal
glycine-cysteine sequence of the mature Lyn, and then continues with
the remaining 489 residues of the short form of the rat enzyme (Lyn B
(17, 28)). The fourth construct (TT-LynKR), is exactly like
Tac-Lyn except that the lysine in the catalytic site (residue 279 in
wild-type Lyn) was replaced by arginine, rendering the kinase inactive.
Table II lists the clones examined most intensively. As can be seen,
the apparent expression of either Fc Enhanced Response in Transfectant with Chimeric Construct of Lyn
(TT-Lyn)--
We previously reported that Fc
Clone C4L was assessed multiple times (Fig. 8, left middle
panel). To compare the experiments, the amount of
anti-phosphotyrosine bound to the Inhibitory Activity of Inactive Construct of Lyn
(TT-LynKR)--
In our previous studies, we observed an
inhibition of aggregation-induced phosphorylation of Fc Topological Distribution of Tac-Lyn Constructs and Effect of
Aggregation--
Several of these transfectants were also examined
with respect to the distribution of both the Tac constructs and the
Fc
Aggregation of either the enhancing TT-Lyn or inhibitory
TT-LynKR constructs failed to affect phosphorylation of
Fc Previous Studies--
Several studies have utilized chimeric
constructs in which the cytoplasmic domain of either the
We were particularly interested in the observations of Wilson et
al. (16), which suggested an experimental way of distinguishing between the two models referred to in the Introduction. Wilson et
al. noted that in transfected RBL-2H3 cells, the TT-
The system we used differs only somewhat, but we think significantly
from that used by Wilson et al. (16). First, rather than RBL
cells, we used CHO cells transfected with Fc TT-
TT- Mechanism of the Inhibition--
We found direct support for the
hypothesis that TT-
What was unexpected was that the ability of the constructs
to inhibit the response did not require and was not enhanced by the
secondary aggregation of the construct with biotinylated anti-Tac and
avidin as had been found by Wilson et al. (16). Again, we believe this difference results from the sensitivity of the system we
employed and the more limited aggregation of Fc
Our studies with the chimeric constructs of Lyn yielded complementary
results. In previous studies on CHO-B12 cells, we observed enhanced
aggregation-induced phosphorylation of tyrosines on the
The experiments with the Lyn constructs were in part aimed to overcome
some of the difficulties in interpreting prior experiments in which the
interaction of a Src family kinase with a multi-subunit immune response
receptor was probed. In a prior study in which we utilized both the
yeast two-hybrid methodology and CHO cell transfectants, we obtained
evidence that the constitutive interaction occurs between the membrane
proximal, unique domain of Lyn and principally the C-terminal
cytoplasmic domain of the receptor
A related study employed a chimeric construct of CD8 fused to the
cytoplasmic domain of the T cell receptor
We also wanted to test whether the interaction of the kinase with the
receptor could be examined independently of their co-localization in
specialized microdomains or "rafts." All our results suggest that
they can be. By employing biochemical fractionation we clearly demonstrated that under the conditions in which TT-Lyn or
TT-LynKR amplify or inhibit the phosphorylation of Fc
It could be argued that in view of the small amount of endogenous Lyn
in the CHO cells, the relevant fraction responsible for the
phosphorylation of Fc
In the experiments of Sheets et al. (10) in which they
reported a sharp decline in the initial phosphorylation of FcRI) were
further transfected with the
subunit of the receptor for
interleukin 2 (Tac) or with chimeric constructs in which the
cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic
domain of either the
subunit or the
subunit of Fc
RI. Whereas
native Tac failed to affect the aggregation-induced phosphorylation of
Fc
RI, both chimeric constructs substantially inhibited this
reaction. Alternatively, the Fc
RI-bearing fibroblasts were
transfected with two chimeric constructs in which the cytoplasmic
domain of Tac was replaced with a modified short form of Lyn kinase.
The Lyn in both of the chimeric constructs had been mutated to remove
the sites that are normally myristoylated and palmitoylated,
respectively; one of the constructs had in addition been altered to be
catalytically inactive. The catalytically active construct enhanced,
and the inactive construct inhibited, aggregation-induced
phosphorylation of the receptors. All of the chimeric constructs were
largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their
aggregation was neither necessary nor enhanced their effects. These
results and others indicate that the receptor and Lyn interact through
protein-protein interactions that neither are dependent upon either the
post-translational modification of the kinase with lipid moieties nor
result exclusively from their co-localization in specialized membrane domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI) begin with the phosphorylation of the tyrosines in
each of the three immunoreceptor tyrosine activation motifs of the
receptor (1). This phosphorylation is stimulated by aggregation of the
receptors and in the cells studied in greatest detail, the RBL-2H3 line
(2), is effected by the Src family kinase Lyn (3).
,
Tac) were fused to the cytoplasmic domains of the
or the
chains of Fc
RI, alternatively. The constructs were stably transfected into Chinese hamster ovary (CHO) fibroblasts that we had
previously transfected with Fc
RI (17). The latter transfectants contain minimal amounts of endogenous Lyn kinase and, therefore, are
particularly sensitive to manipulations that either enhance or inhibit
the interaction of active kinase with the receptor (17, 18).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or of the
subunit of Fc
RI (TT-
or
TT-
) in pcDL-SRa296 (16). They were digested with BamHI
and EcoRI and subcloned into pZeo-2SV (minus orientation of
the multiple cloning site), which had been digested with the same
enzymes. The cDNA for wild-type Tac (TT-T), which contains the Tac
cytoplasmic domain of 13 residues (20) in pGM, was obtained from W. Leonard (NHLBI, National Institutes of Health) and similarly subloned
into pZeo-2SV. We also prepared a construct in which a stop codon was
inserted after that coding for leucine residue 238 so that no
cytoplasmic domain would be expressed (TT-0).
Characteristics of Tac and chimeric constructs
).
Columns 4 and 5 show the number for the 5' base in the 5' codon of the
open reading frame and for the corresponding N-terminal amino acid
residue from the cytoplasmic domains of the Tac protein, the
subunit (C-terminal), the
subunit, and the Lyn protein,
respectively. The junctional sequence is given in the last column with
the joined residues shown in bold type. In the construct
Tac-LynKR, arginine substitutes for lysine at residue 279.
RI under G418 selection (Ref. 17
and Table II). After 72 h, the
transfected cells were placed under selection with 250 µg/ml zeocin.
Clones were picked after 5-7 days, expanded, and screened for
expression of Fc
RI and the Tac construct. A second set of
transfectants for TT-
and all of the TT-Lyn transfectants were
prepared using LipofectAMINE (Life Technologies, Inc.). In a 6-well
plate, 3 × 105 CHO-B12 cells were plated and grown
overnight to 60-70% confluence. 1 µg of plasmid DNA was diluted
into 100 µl of Opti-MEM serum-free medium (Life Technologies, Inc.)
and mixed with a second solution containing 6 µl of LipofectAMINE in
100 µl of Opti-MEM serum-free medium. The mixture was incubated at
room temperature for 30 min., diluted with 0.8 ml of Opti-MEM, and
overlaid onto the cells. After 5 h at 37 °C, 1 ml of CHO-B12
growth medium containing 20% (2×) fetal bovine serum was added to the
cells. The transfectants were then selected with zeocin, cloned and
expanded as for transfectants generated by electroporation. Duplicate
wells of each clone to be tested were grown to confluence in 24-well
plates (
2.5 × 105 cell/well). The cells in one set
of wells were incubated with 1 µg/ml of [125I]IgE in
0.1 ml for 1 h at 37 °C, washed five times with
phosphate-buffered saline (PBS), and then solubilized with boiling hot
1% SDS, in a 62.5 mM Tris, 0.5× PBS buffer. The lysates
were then
-counted. For assessing expression of the Tac constructs,
cells were incubated with 0.5 µg/ml of [125I]anti-Tac
in 0.5 ml, at 4 °C for 30 min, washed four times with ice-cold
Iscove's medium, and then solubilized and counted as for the cells
labeled with IgE. To maintain the integrity of cell surface Tac, cells
were harvested using 3 mM EDTA in PBS rather than trypsin
for passaging and stimulation. In addition, all CHO transfectants were
cryopreserved in growth media supplemented with 5%
(CH3)2SO, and a new vial of cells was routinely
thawed every 2 months to assure a consistent ratio of Tac to
Fc
RI.
Expression of Tac and FcRI on transfected CHO-B12 cells
RI by
electroporation and selected with G418 (CHO-B12 (last row)), was
further transfected by electroporation or LipofectAMINE either with
vector containing the zeocin antibiotic resistance marker only (pZeo)
or in addition with Tac chimeras (TT-
, TT-
, TT-Lyn, or
TT-LynKR) or the wild-type or truncated Tac (TT-T and TT-0),
and selected with zeocin. Fc
RI were quantitated using
[125I]IgE in duplicate incubations two to seven times for
each transfectant except those transfected with TT-0. Tac was
quantitated using [125I]anti-Tac from clone HD245 in
duplicate incubations two to seven times for each transfectant except
those transfected with TT-0. We assumed each molecule of anti-Tac bound
two molecules of Tac (22). The values shown are the means ± S.E.
RI and Tac Constructs--
CHO
transfectants were harvested and incubated at 5 × 106
cells/ml with 5 µg/ml [125I]IgE for 1 h at
37 °C. Nonspecific binding was assessed by preincubating the cells
with 50 µg/ml unlabeled IgE for 30 min. Cells were then centrifuged
through a mixture of phthalate oils (21), and the radioactivity in the
pellets was counted. Alternatively, CHO transfectants were incubated at
5 × 106 cells/ml with 0.5 µg/ml
[125I]anti-Tac for 30 min at 4 °C. Nonspecific binding
was measured in duplicate incubations with 5 µg/ml unlabeled
anti-Tac. Cells were then isolated as above, and the pellets were
counted. The number of molecules of Tac was calculated from the
recovered radioactive counts and the specific activity of the labeled
anti-Tac, assuming one molecule of anti-Tac binds two molecules of Tac
protein (22).
RI were aggregated
with oligomers of IgE for 30 min at 37 °C when appropriate.
Alternatively, Tac constructs were aggregated using a monoclonal
anti-Tac followed by a polyclonal anti-mouse IgG.
RI were
solubilized and immunoprecipitated with anti-IgE, and phosphorylation
of tyrosines on their
, and the
subunits was determined as
described previously (18). Tac constructs were extracted with 1%
Nonidet P-40 and immunoprecipitated with monoclonal anti-Tac.
Immunoprecipitates were analyzed on 10% Tricine gels followed by
immunoblotting first with biotinylated polyclonal anti-Tac or 4G10 and
then with horseradish peroxidase-coupled avidin.
RI,
CHO transfectants were sensitized for 1 h at room temperature with
5 µg/ml IgE, 10% of which had been labeled with 125I.
For monitoring of Tac constructs, the CHO cells were labeled with
[125I]anti-Tac for 30 min, at 4 °C. After washing the
cells three times with buffer A (4), plasma membranes were isolated by Dounce homogenization and sedimented in 30% isotonic Percoll (23). Successive 1-ml fractions were removed from the top of the gradient, the location of the visible band was noted, and the radioactive counts
in each fraction were determined in a
-counter.
RI or 1% Triton X-100 for immunoprecipitation with anti-Tac.
-32P]ATP
(PerkinElmer Life Sciences)) at 25 °C and vortexed every 10-15 min.
The reactions were quenched by the addition of 10 µl of 50% (v/v)
acetic acid. The peptide was then isolated using phosphocellulose
filters. The filters were washed twice with 0.5 ml of 0.075 M phosphoric acid and then counted in a scintillation counter in Filtron-X mixture.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI Subunit Constructs--
CHO cells
previously transfected with Fc
RI (17) were further transfected by
electroporation with Tac (TT-T) or a chimeric construct containing the
extracellular and transmembrane domains of Tac and the C-terminal
cytoplasmic domain of either the
chain (TT-
) or the
chain of
Fc
RI (TT-
). Stable clones that had been selected with zeocin and
characterized for their expression of Fc
RI and the Tac constructs
are listed in Table II. The ratio of TT-
to Fc
RI ranged from 0.3 to 2.2, and the that of TT-
to Fc
RI ranged from 0.2 to 2.5. When
analyzed by Western blotting, anti-Tac immunoprecipitates of the
detergent extracts of such transfectants revealed a diffuse band of 55 kDa (Fig. 1, lanes 1,
4, and 6).
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Fig. 1.
Expression of Tac and
Tac-Fc RI subunit constructs in CHO-B12
cells. CHO cells (4 × 106 cell equivalents)
previously transfected with Fc
RI (CHO-B12) were further transfected
either with Tac (clone 21A4E), pZeo vector (4E), TT-
(2A3E), or
TT-
(A3E). Tac constructs were solubilized in 1% Triton X-100,
immunoprecipitated (IP) with monoclonal anti-Tac
(T) or an irrelevant control, anti-hemagglutinin
(C) antibody and Western blotted with polyclonal anti-Tac.
The apparent molecular mass (in kDa) of the upper band is shown on the
left.
30 kDa, glycosylation of the ectodomain of Tac is known to retard
its mobility. The bands with apparently lower molecular mass (38, 40 kDa) likely represent non- or hypo-glycosylated constructs not
expressed on the cell surface. Such species were previously described
for chimeric constructs of Tac fused to the cytoplasmic portion of the
T cell receptor
chain or the
chain of Fc
RI (13).
Inhibits the Phosphorylation of Fc
RI--
Fig.
2 shows the results of an experiment in
which cells from clone 2A3E expressing on their surface approximately
0.9 TT-
per Fc
RI (Table II) were first reacted with anti-Tac, or
not, to aggregate the Tac constructs. They were then reacted with 500 ng/ml of either monomeric IgE or a mixture of trimeric and tetrameric IgE for 30 min. Cells transfected with receptors only (CHO-B12) were
treated similarly. The receptors were immunoprecipitated and Western
blotted with anti-phosphotyrosine. The odd numbered lanes show that
there is only minimal phosphorylation of the receptors on cells reacted
with the monomeric IgE, whereas substantial phosphorylation of the
and
subunits is apparent in cells reacted with the oligomers.
Similar results were obtained in the cells transfected with TT-
, but
in this and repeated similar experiments analyzed quantitatively, the
phosphorylation was substantially less (
70% in the experiment
illustrated). Prior aggregation of the Tac-
constructs neither
enhanced nor diminished the inhibition (lane 6 versus lane
8).
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Fig. 2.
Effect of aggregation on the inhibitory
action of TT- . CHO cells transfected with
Fc
RI alone (CHO-B12) or co-transfected with TT-
(clone 2A3E) were
treated with 2 µg/ml anti-Tac-biotin in growth media (+) or media
alone (
) for 20 min at 25 °C. Cells were washed and stimulated
with 25 µg/ml avidin or buffer for 10 min at 37 °C and then
stimulated with 0.5 µg/ml monomeric (M) or oligomeric
(O) IgE for 30 min at 37 °C. Fc
RI were solubilized,
immunoprecipitated (IP) with anti-IgE, resolved by PAGE, and
Western blotted with anti-PY. The results are representative of three
experiments conducted, one with TT-
clone 2A3E and two with clone
2C9E.
to Fc
RI and the inhibition of phosphorylation. The IgE
receptors on cells with TT-
to Fc
RI ratios of either 0.3 or 0.9 were aggregated with increasing concentrations of antigen, and the
phosphotyrosine on the receptor was compared with identically stimulated control cells (cells transfected solely with the zeocin marker-containing plasmid). A clone expressing a TT-
to Fc
RI ratio of 0.3 (clone 2C9E) and stimulated with 80, 150, or 300 ng
antigen/ml showed 40, 60, and 60% less phosphotyrosine in their
chains and 60, 40, and 30% less phosphotyrosine in their dimer of
chains compared with the control cells (
versus
, Fig. 3). Somewhat greater expression of the
construct (clone 2A3E, TT-
to Fc
RI ratio of 0.9) showed further
inhibition (Fig. 3,
), the corresponding reductions being 50, 70, and 70% for
and 60, 70, and 50% for the dimer of
chains.
However, the clones expressing a TT-
to Fc
RI ratio of
~2.0 showed no substantially greater inhibition (results not
shown).
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Fig. 3.
Effect of expression of
TT- on phosphorylation of
Fc
RI. CHO-B12 cells transfected with
vector containing only the pZeo antibiotic resistance marker (clones 4E
and 5E (
)) or with TT-
(clones 2A3E (
) and 2C9E (
)) were
sensitized with anti-DNP-specific IgE (trace-labeled with
[125I]IgE), washed, and stimulated with
DNP6-BSA for 2 min. Fc
RI were solubilized,
immunoprecipitated, resolved by PAGE, and Western blotted with anti-PY.
The blots were stripped, reprobed with antibody to the Fc
RI
chain, and scanned to quantitate the PY per receptor
(upper
panel) and
chain dimer (lower panel). A fixed
amount of phosphorylated human Lyn was loaded onto each gel and the
densitometric values for PY normalized to each other. The results shown
are the means ± S.E. from five experiments conducted with clones
2A3E and 2C9E.
construct were also compared with clones
transfected with unmodified Tac (TT-T). Compared with clone 21A4E,
clone 2A3E, which showed comparable expression of the Tac epitope
(Table II), showed only 1/2 to 1/4 as much
phosphorylation in response to a paucivalent antigen (Fig.
4). Cells transfected with the construct
coding for a truncated Tac (TT-0) did show repeated although quite
variable decreases in phosphorylation of Fc
RI, but notably the cells
grew quite poorly compared with all the other transfectants.
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Fig. 4.
Specificity of inhibitory action of
TT- . Clone 2A3E containing the TT-
construct (
,
) was compared with clone 21A4E expressing a
comparable amount of transfected unmodified CD25
(TT-T) (
,
).
The open symbols represent the data for the dimer of
subunits; the filled symbols represent the data for the
subunit. The results shown are the averages of three determinations
from two separate experiments, i.e. single samples in one
experiment and duplicate samples in the second experiment. All the data
have been normalized relative to the PY on the
subunit of the
Fc
RI in the TT-T transfectants stimulated with 75 ng/ml
antigen.
Inhibits the Phosphorylation of Fc
RI--
Analogous
studies were performed on cells transfected with the corresponding
chimeric constructs containing the cytoplasmic extension of the Fc
RI
subunit (Table II). When stimulated with 75, 150, 300, or 600 ng/ml
antigen, clones expressing a TT-
to Fc
RI ratio of 0.2-0.7 (A3E
and B1E) revealed 3,
2, 25, and 22% inhibition of
antigen-induced phosphorylation on the
chain, and 8, 5,
14, and 51% inhibition on the dimer of
chains relative to
clones transfected with the plasmid containing solely the zeocin marker
(Fig. 5,
versus
). The
corresponding inhibitions observed on clones expressing equal numbers
of TT-
and Fc
RI (clones A1L, C1L, and D6E; Table II) were 30, 70, 60, and 80% on the
subunit and 30, 70, 40, and 70% on the dimer
of
subunits (Fig. 5,
versus
). The time
dependence of inhibition was unremarkable. Compared with cells
transfected with TT-T (clone 21A4E), the TT-
transfectant C1L showed
40-50% less phosphorylation over the 4-min time period studied (data
not shown).
View larger version (16K):
[in a new window]
Fig. 5.
Effect of expression of
TT- on phosphorylation of Fc
RI.
Fc
RI from pZeo transfectants (clones 4E and 5E (
)) and TT-
Fc
RI transfectants (clones A1L, C1L, D6E, B1E and A3E (
,
))
were aggregated with DNP6-BSA for 2 min. The relative
amounts of phosphotyrosine on the
(top panel) and
chains (bottom panel) of Fc
RI are depicted (means ± S.E.) for the five experiments conducted. The data for clones A1L, C1L,
and D6E were averaged (
), as were the data for clones A3E and B1E
(
).
were sensitized with IgE, and aliquots were
stimulated with 0, 50, or 150 ng/ml DNP-BSA for 2 min. Duplicate
samples were either solubilized with 1% Triton X-100 and
immunoprecipitated with anti-TAC or solubilized with 0.5% Triton X-100
and immunoprecipitated with anti-IgE. Both sets of immunoprecipitates
were then analyzed by Western blotting with anti-phosphotyrosine. In
the cells stimulated with antigen, tyrosines on the subunits of the
receptor became phosphorylated, but no phosphorylation of the chimeric
construct was observed (data not shown). Stripping the blots and
reprobing them with anti-Fc
RI
or anti-TAC antibodies confirmed
the equivalent immunoprecipitation in the samples. Western blots of
immunoprecipitated TT-
from either stimulated or unstimulated cells
likewise failed to show any phosphotyrosine on the chimeric constructs.
The experiments also showed that there was no co-precipitation of the
Tac constructs with the transfected Fc
RI.
RI, we analyzed the
distribution of the inhibitory chimeric constructs. TT-
transfectants were sensitized with [125I]IgE and then
stimulated or not with antigen.
and
TT-
chimeric constructs are expressed independently on the surface of RBL transfectants by failing to observe co-localization when one or the other fluorescently labeled species was aggregated. We
observed a similar independence on the CHO cell transfectants. In one
type of experiment we tested whether the constructs
co-immunoprecipitated with the Fc
RI and failed to observe any
co-immunoprecipitation. We also looked for co-distribution on sucrose
gradients in which the detergent-insoluble plasma membrane domains were
separated from the remainder of the cellular components after treating
the cells with 0.05% Triton X-100 (8). The gradient fractions were
-counted to quantitate the IgE receptors and then adjusted to 1%
Triton X-100 and divided equally. One aliquot was precipitated with
anti-Tac and Western blotted with anti-TAC to localize the TT-
.
Samples from the second set were directly Western blotted for both Lyn
and caveolin.
and 98% of the
recovered Fc
RI were found in the detergent-sensitive plasma membrane
fractions (9-12). Similarly, 100% of the endogenous Lyn kinase was
recovered in these fractions (not shown). After stimulation with
antigen (filled symbols), the percentage of Fc
RI in
fractions 3-5 containing the DRM increased from 2 to 17% without a
corresponding movement of TT-
or Lyn kinase.
View larger version (16K):
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Fig. 6.
Distribution in membranes of
Tac- and and TT-
constructs and Fc
RI. CHO-B12 cells
transfected with TT-
(clone 2A3E) and TT-
(clone C1L) were
sensitized with [125I]IgE (
,
, and stimulated or
not with 100 ng/ml DNP6-BSA for 2 min. The low speed
supernatant from cells lysed with 0.05% Triton X-100 was fractionated
on sucrose gradients. Successive 1-ml fractions were removed from the
top of the gradient, the location of the visible band was noted, and
each fraction was measured in a
-counter. Aliquots of each fraction
were then further solubilized in 1% Triton X-100, immunoprecipitated
with anti-Tac, and analyzed by Western blotting with either anti-Tac
(
,
), anti-Lyn (not shown), or anti-caveolin (not shown).
Top panel, TT-
unstimulated with antigen (open
symbols) or stimulated (filled symbols). Lower
panel, duplicate experiments with TT-
.
transfectant (clone C1L) gave similar
results (Fig. 6, lower panel). In the unstimulated cells, 100% of TT-
and 96% Fc
RI were confined to fractions 9-12; the corresponding values after stimulation were unchanged for both TT-
and Fc
RI. For purposes of clarity the distribution of the caveolin
is not shown in the figure, but approximately 60% of the caveolin was
localized in fractions 3-5 and 30% in fractions 9-12 in each of the
gradients analyzed.
transfectants or CHO-B12 cells on Percoll
gradients. The visible band observed after centrifugation of the
Percoll gradients contained >90% of TT-
and Fc
RI, verifying
that the corresponding proteins were largely in the plasma membrane.
RI for the limited amount of
endogenous Lyn kinase in the CHO cells. To investigate further the
ability of Lyn kinase to interact with TT-
, we modified a protocol
we previously used for assaying co-immunoprecipitated Lyn and Fc
RI
(4). To test several variables we first assayed the kinase
immunoprecipitated from CHO cells expressing transfected rat Lyn kinase
(clone A11 (17)) using rabbit polyclonal anti-Lyn serum or serum from
unimmunized rabbits as a control. The immunoprecipitates were washed as
described under "Experimental Procedures" and then subjected to an
in vitro kinase assay in the presence of
[
-32P]ATP and a peptide that is a relatively specific
substrate for Src family kinases (19). The incubation mixtures were
applied to phosphocellulose spin filters, which were washed and counted in a
counter. The activity associated with the anti-Lyn
immunoprecipitates led to a linear increase in phosphorylation of the
substrate peptide between 0.5 and 2 h, whereas control
immunoprecipitates induced only negligible modification (data not
shown). More importantly, the amount of phosphorylated peptide
recovered was linearly proportional to the amount of cell extract
utilized, whereas there was no significant increase in the amount of
activity detected in the control preparations. Notably also, in the
absence of peptide, the recovery of counts was only equal to that found
in the assay blanks containing no lysate. This shows that the
radioactivity we monitored was due to phosphorylation of the peptide
and not due to autophosphorylation of the kinase.
or TT-T transfectants solubilized under conditions that gave a ratio of micellar detergent to lipid (
)
(25) of 3. These conditions were previously shown to stabilize the
association of the kinase with Fc
RI in RBL cells (4). To minimize
nonspecific precipitation of kinase with the protein A-Sepharose beads
used for the isolation, the immunoprecipitating or control antibody was
prebound to the beads, and the lysates were extensively precleared
prior to the specific immunoprecipitation. Fig.
7 (upper panel) shows
the averaged results of two in vitro kinase
assays of anti-Tac immunoprecipitates from TT-
and TT-T transfectants, respectively. Substantially more kinase activity co-precipitated with the TT-
than with the TT-T. Control
immunoprecipitates using an irrelevant antibody showed only a small
difference between the two lysates (Fig. 7, lower
panel).
View larger version (14K):
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Fig. 7.
Kinase activity associated with
TT- . Lysates from 3 × 106 CHO-B12
cells transfected with Tac (TT-T, clone 21A4E (
), or TT-
, clone
2A3E (
)) were reacted with anti-Tac or an isotype-matched control
antibody, and the immunoprecipitates were assayed for co-precipitating
endogenous kinase using a Src family kinase substrate. The average of
two such experiments with each time point of the assay measured in
triplicate is shown. Top panel, anti-Tac immunoprecipitates.
Lower panel, control (anti-hemagglutinin)
immunoprecipitates. Two of the six samples assayed after 4 h gave
aberrantly high values, and these data were not included in the
averages. Error bars show ranges of values.
RI, Tac, or the chimeric Tac was
variable even within a clone (relatively high standard errors), but
there was no marked tendency for the expression of Fc
RI to be
affected differentially by any of the constructs. As noted in Table
II, we assumed that one molecule of anti-Tac bound two molecules
of the surface expressed Tac construct (22).
RI on CHO cells
transfected with receptor alone (CHO-B12) show virtually no
phosphorylation of tyrosines when exposed to dimerized IgE and only a
modest response to small doses of a paucivalent dinitrophenylated
antigen (DNP6-BSA) after sensitization with anti-DNP IgE.
Both the responses to the dimers of IgE and the responses to antigen
were progressively enhanced in the cells express increasing amounts of
transfected wild-type Lyn kinase (17, 18). The top panels in
Fig. 8 shows a similar comparison between
CHO-B12 cells and the two clones of cells expressing the highest amount
of the transfected catalytically active or inactive Tac-Lyn constructs
(clones C4L, 34A3E; Table II). In these panels, the extent of
phosphorylation of the
and
subunits of the receptors 3 min
after the addition of a variable amount of DNP6-BSA are
depicted separately. All the data were normalized to the amount of
anti-phosphotyrosine antibody bound to the
subunit on receptors
from the cells reacted with 200 ng/ml of antigen. It is clear that at
this dose the extent of phosphorylation of the
to
2
ratio is
1:2, as expected from prior studies (29). Within
experimental error, this was equally true at other doses and with the
alternative clones depicted in the figure. It can be seen that at all
doses of antigen, there is a roughly 2-fold enhancement in the amount
of phosphorylation of both the
and
subunits of the receptors in
clone C4L compared with the cells not transfected with the Tac-Lyn
construct.
View larger version (20K):
[in a new window]
Fig. 8.
Aggregation-induced phosphorylation of
tyrosines on Fc RI in CHO-B12 cells and
transfectants. Top panels, phosphorylation of the dimer
of
subunits and of the
subunit of Fc
RI, 3 min after addition
of several different doses of DNP-BSA to clone C4L (
), 34A3E (
),
and CHO-B12 (
) cells. All of the data have been
normalized to the amount of phosphotyrosine determined for the
subunit on receptors of the CHO-B12 cells exposed to 200 ng/ml antigen.
Each data point shows the mean of duplicate assays. Left middle
panel, phosphorylation of the combined
and
subunits of
Fc
RI, 3 min after addition of several different doses of DNP-BSA in
five separate experiments on clones C4L(
), 34A3E (
), and CHO-B12
(
) cells. The data have been normalized to the amount of
phosphotyrosine determined for the receptors on the CHO-B12 cells
exposed to 200 ng/ml antigen. The error bars show the
positive standard error of the mean; where error bars are
not shown they were smaller than the symbol. Inset, summary
of five experiments in which clone 21A4 (TT-T) (
) and CHO-B12 (
)
were compared. Right middle panel, comparison of multiple
cloned transfectants. Phosphorylation of the combined
and
subunits of Fc
RI, 3 min after addition of several different doses of
DNP-BSA to 10 different clones (Table II) transfected either with
TT-Lyn (C4L,
; A5L,
; C6L,
; A6L, B1L, and D6L,
) or with
TT-LynKR (34A3E,
; 31A4E,
; 33A4E,
); CHO-B12,
) cells. The data have been normalized to the amount phosphotyrosine
on the Fc
RI of CHO-B12 cells exposed to 200 ng/ml antigen. The
error bars show the positive standard error of the mean;
where error bars are not shown they were smaller than the
symbol. Bottom panel, time course of phosphorylation of the
combined
and
subunits of Fc
RI. Results are from 5-12
separate experiments on clones C4L (
), 34A3E (
), and CHO-B12
(
) cells 1.5, 3, and 6 min after addition of 200 ng/ml of DNP-BSA.
The data have been normalized to the amount phosphotyrosine on the
Fc
RI of CHO-B12 cells exposed to antigen for 3 min.
and
subunits combined from
the cells untransfected with the chimeric construct and stimulated with
200 ng/ml antigen was set at 1, and the amount of phosphorylation
determined on all the other samples was normalized to it. Although
there was a moderate variability, it is apparent that clone C4L
reproducibly shows an enhanced phosphorylation of the receptors
compared with the clone untransfected with the Lyn construct. Five
additional clones of cells transfected with the same construct (Table
II) were examined. The two clones expressing the next highest amounts of the construct (A5L and C6L) showed enhancements closely similar to
those observed with clone C4L (Fig. 8, right middle panel). However, three clones expressing still lesser amounts (clones B1L, A6L,
and D6L) showed only irregular increases in phosphorylation of their
receptors at any dose (Fig. 8) or time (not shown). The enhancement was
not limited to a particular point in time. As seen in the bottom
panel of Fig. 8, stimulation with 200 ng/ml of the antigen led to
a progressively enhanced phosphorylation of the receptor over the time
period examined time.
RI in cells
that had been transfected with a construct of Lyn kinase in which the
lysine in the catalytic site had been mutated to arginine. Analogous transfections using Tac-LynKR were assessed in the present
study. The data shown by the filled circles and dashed
lines in the top panels of Fig. 8 are for clone 34A3E,
which expressed the highest amount of the construct and somewhat more
than the clone expressing the catalytically competent TT-Lyn. It is
apparent that in clone 34A3E the receptors were only one-third or less
phosphorylated than in the cells transfected with the catalytically
active TT-Lyn construct. In six further experiments this clone gave
similar results (Fig. 8, left middle panel). The time course
of phosphorylation of the receptors in this clone is shown in the
bottom panel of Fig. 8. Finally, the right middle
panel of Fig. 8 shows our findings with the three clones
transfected with Tac-LynKR studied in greatest detail as a
function of dose of antigen.
RI, by density gradient centrifugation in sucrose of Triton X-100 extracts of the cells. To some cell samples 0.2 µg/ml of
125I-labeled murine anti-Tac was added, and the cells were
incubated for 20 min on ice and then washed in buffer A. They were
resuspended at 1 × 107 cells/ml and incubated further
with or without 25 µg/ml of anti-mouse IgG Fab fragment for 10 min at
37 °C. Similarly, cells were examined with or without having their
receptor-bound IgE aggregated by addition of 200 ng/ml DNP-antigen for
2 min at 37 °C. In the absence of anti-Tac, the TT-Lyn and
TT-LynKR constructs, like those containing cytoplasmic
domains of the receptor, were distributed virtually exclusively in the
fractions containing both soluble and membrane proteins outside the DRM (Fig. 9,
). For those four clones,
83% of the Tac was within fractions 9-12 and <5% in fractions
3-5. The distribution was very similar in the cells stimulated with
antigen (96 and 1% in the same fractions). As expected the Tac
constructs partially translocated to the DRM when they themselves were
aggregated either with only the monoclonal anti-Tac (Fig. 9,
) and
even more so when reacted in addition with a second anti-antibody (Fig.
9,
).
View larger version (13K):
[in a new window]
Fig. 9.
Distribution on membranes of Tac-Lyn
constructs. Transfected cells were lysed in 0.05% Triton X-100,
and the extracts were separated on sucrose gradients. Individual
fractions were either counted for radioactivity or analyzed by Western
blotting. , composite results from four separate experiments on
cells transfected either with TT-Lyn or with TT-LynKR. The
cells were otherwise not treated. The points show the average of the
densitometric readings of Western blots, and the error bars
show the standard deviations.
, distribution of counts from extract
of TT-T transfectant reacted with radioiodinated monoclonal anti-Tac.
, distribution of counts from extract of TT-T transfectant reacted
with radioiodinated anti-Tac and then a polyclonal anti-mouse
Fab.
RI. Table III shows a detailed
analysis of one such experiment in which two clones transfected with
LynKR constructs were compared with clone 42C2E. Although
transfected with Lyn, the latter clone expressed little of it (Table
II) and showed no enhanced phosphorylation of Fc
RI compared with
those transfected with TT-T or vector alone (data not shown). If, in the penultimate column of Table III, one first compares the relative amounts of phosphorylation of the receptors in the cells in which only
the Fc
RI were aggregated, it is apparent that the inhibition observed in clones 31AE (sample 2B) and 34AE (sample 2C) was about 35 and 55%, respectively. The ratio of the relative phosphorylation of
the receptors in the respective samples 3 versus 2 (column 9) is in each case not appreciably different than 1, indicating that
aggregation of the chimeric constructs did not appreciably enhance
their effect.
Effect of aggregation on the inhibitory action of construct
TT-LynKR
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or
subunits of Fc
RI were fused to the ecto- and transmembrane domain of
an irrelevant protein, such as CD4 (12) or CD25
(13-16). The
chimeric constructs generally have been transfected into the rat
mucosal type mast cell line RBL-2H3 and yielded the following
observations. Aggregation of the
-containing construct stimulates a
variety of cellular responses that resemble an anemic version of the
responses induced by aggregation of Fc
RI. Tyrosines on downstream
proteins, but neither the construct nor the unaggregated Fc
RI are
phosphorylated following aggregation of the construct (15, 16).
Although there is reason to think that the action of the
constructs
is mediated via Lyn, under the conditions used, Lyn is not detectable
in immunoprecipitates of that construct (15). Experiments with the
construct containing the C-terminal cytoplasmic domain of the
subunit gave virtually the opposite results; that is, although
aggregation of the
construct failed to trigger phosphorylation of
it or of other cellular components, immunoprecipitates of it revealed
associated Lyn. The amount of Lyn recovered appeared to be independent
of whether the
-construct had been aggregated or not. These results
were consistent with earlier observations that the C-terminal domain of
both the
and
subunits are required for full activation but that
-less receptors can nevertheless stimulate responses (30, 31). Yeast two-hybrid studies also showed a weak interaction between Lyn kinase
and the C terminus of the
chain; contrariwise, reaction of the
cytoplasmic domain with Lyn, if any, was too feeble to be observed by
this method (17).
construct could inhibit responses stimulated by aggregation of Fc
RI. They hypothesized that although it could not independently activate downstream events, the
-construct sequestered a critical component required by Fc
RI, likely Lyn. Their suggestion anticipated our subsequent findings that the Lyn available to the Fc
RI in RBL cells
is in short supply and shuttles between different Fc
RI (32). We were
particularly intrigued that Wilson et al. (16) only observed
inhibition when the TT-
construct was aggregated. We hypothesized
that this might be explained using the model proposed by Sheets and
co-workers (11) as follows: The monomeric TT-
construct would be
distributed outside the specialized microdomains but like wild-type
CD25
would translocate to the microdomains upon aggregation (33).
Then if the interaction between aggregated Fc
RI and Lyn
preferentially occurred in these domains, that could explain why the
aggregated but not the monomeric TT-
inhibited the responses
triggered by Fc
RI. The experiments described in this paper were
designed to test this explanation.
RI. The only response to
aggregation of the receptors exhibited by these transfectants is
phosphorylation of the receptors themselves. Likely this is because the
cells contain no Syk kinase, an essential component for all downstream
responses (34). The CHO cells contain only a small amount of Lyn
kinase, and therefore their response to aggregation of Fc
RI is
particularly sensitive to manipulations that affect the availability of
active Lyn to the receptors (17, 18). Furthermore, by using either
small oligomers or relatively low doses of antigens modified only
lightly with haptenic groups, we stimulated the cells less vigorously
than is generally done. Thus, the aggregates of receptor that form are
likely to be smaller than with more vigorous stimuli.
and TT-
Inhibit the Phosphorylation of Fc
RI--
Like Wilson et al. (16), we observed inhibition of
the Fc
RI-induced response in those cells transfected with TT-
.
The inhibition was specific for the
portion of the construct
because the responses of cells transfected with plasmids containing
only the antibiotic marker were indistinguishable from those
transfected with CD 25
, i.e. TT-T.
also inhibited the phosphorylation of Fc
RI, but three times
more TT-
than TT-
was required to see more or less equivalent inhibition. As already noted, prior studies appeared to yield contradictory results with respect to the capacity of the
chains to
interact with Lyn. That Fc receptors lacking
as well as chimeric constructs of
can stimulate responses mediated by Lyn strongly favors such coupling, but evidence for a direct interaction could not
be obtained from either in vitro kinase assays (15) or the yeast two-hybrid complementation studies (17). Likewise, with TT-
,
Wilson et al. (16) failed to see the same inhibitory effect they observed with TT-
. We believe that all of the results are consistent with this interaction being relatively weak. One likely reason we were able to observe such an interaction in our current studies is that our in vivo assay preserves the normal high
mutual concentration and co-localization of Lyn and the cytoplasmic
domain of
at the cytoplasmic face of the plasma membrane. A likely second reason is that as already noted, various features of our protocol make the system particularly sensitive to perturbations of the
available Lyn kinase.
inhibits the action of Fc
RI because it
associates with Lyn by comparing the kinase activity in
immunoprecipitates of chimeric and control constructs. Utilizing a Src
family kinase-specific substrate, the results strongly support such a
specific interaction and, being consistent with related studies by
others (15, 16), were not surprising.
RI we used to
stimulate the cells.
and
subunits of the receptors in cells that had been transfected with
catalytically active Lyn kinase (17, 18). On the other hand, cells
transfected with the catalytically inactive constructs of the kinase
showed diminished aggregation-induced phosphorylation of Fc
RI. We
interpreted those results in the context of a model in which the
initial aggregation-dependent phosphorylation of Fc
RI
results from a transphosphorylation by Lyn kinase constitutively associated with a small percentage of receptors. The transfection with
wild-type Lyn was thought to have enhanced the probability that any
given aggregate included at least one molecule of active kinase,
whereas transfection with incompetent Lyn would decrease that probability.
chain (17). Those results were
consistent with analogous observations by others (16, 31). In a related
study in which the association of p59Fyn with T-cell receptors was
explored, it was concluded that the N-terminal 10 amino acid residues
in the unique domain of the kinase were critical for the coupling.
However, the role of certain of these residues in the protein-protein
interaction could not be distinguished from those residues involved in
localizing the enzyme correctly (35). Thus, the myristoylated glycine
anchors the enzyme to the inner leaflet of the plasma membrane, and the palmitoylated cysteine appears to localize it preferentially within microdomains (7).
chain and examined its
interaction with p59Fyn (36). However, that work examined the
interaction between the kinase and phosphorylated
immunoreceptor tyrosine activation motifs, whereas what we wish to
define is an earlier interaction involving the constitutive association between a Src family kinase and the unphosphorylated receptor. Varying
the sequence of the Tac-Lyn chimeras used here should now allow us to
examine the role of the sequence of the unique domain of the kinase
without concern about the extent of localization on the plasma
membrane, which can confound the interpretation of the results.
RI,
the critical components are largely localized outside the DRM (Fig. 9
and Table III). Wilson et al. (16) did not examine the
phosphorylation of Fc
RI per se, and we cannot rule out
that the later events that they monitored involved receptors localized
to the DRM. Possibly, they were able to inhibit these distal events
only by driving the chimeric constructs into the DRM through vigorous
aggregation. Alternatively, we think it more likely that the more
extensive aggregation of Fc
RI they employed, coupled with the
greater supply of Lyn available to the receptors in the RBL cells they
transfected, made it more difficult to observe the inhibition by the
unaggregated constructs.
RI we monitored was in the DRM but went
undetected. Likewise, that it was the tiny amounts of the transfected
constructs in the DRM that were solely responsible for the effects we
observed. In principle, such a thesis is virtually impossible to
reject. Nevertheless, our experimental findings cannot be explained
simply by the co-localization per se and are only explicable
on the basis of protein-protein interactions.
RI following disruption of the microdomains, large correction factors were
required to compensate for major depletions of the receptors that
accompanied the experimental procedures. Our observations are more
consistent with the recent observations by Yamashita et
al.2 They found that
later events, but not the initial phosphorylation of the
receptor tyrosines, were inhibited when DRM were disrupted by depleting
the cells of cholesterol. Likewise, using different approaches to
prevent localization of Lyn kinase to DRM, Kovarova et
al.3 observed no
reduction in the initial phosphorylation of the receptor or in Syk
kinase. Taken together, the data suggest that the constitutive association between Lyn and Fc
RI can occur outside discrete membrane microdomains and that it is mediated principally by the protein-protein interactions rather than principally by induced co-localization based
on surrounding or covalently attached lipids. On the other hand, it
seems likely that under normal conditions these critical interactions
can, and perhaps more often do, occur in the specialized membrane domains.
![]() |
FOOTNOTES |
---|
* 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.
These authors contributed equally to this work.
§ Present address: Allergy and Clinical Immunology Division, John Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6108.
¶ Present address: 16 Franklin Woods Dr., Somers, CT 06071.
To whom correspondence should be addressed: Bldg. 10, Rm.
9N-228, 10 Center Dr., MSC-1820, NIAMS, NIH, Bethesda, MD 20892-1820. Tel.: 301-435-6126; Fax: 301-402-0012; E-mail:
metzgerh@exchange.nih.gov.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M003397200
2 T. Yamashita, T. Yamaguchi, K. Murakami, and S. Nagasawa, submitted for publication.
3 M. Kovarova, L. Draberova, J. Rivera, and P. Draber, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DRM, detergent-resistant membrane; DNP, 2,4-dinitrophenyl; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Pipes, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin.
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REFERENCES |
---|
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