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(Received for publication, December 26, 1995) From the
This study shows that aggregation of U937 cell high affinity IgG
Fc receptor (Fc
Aggregation controls signaling through Fc receptors
(FcR)( For Fc Evidence presented from an earlier report (20) and the
present report is consistent with the second model. We used an assay
system that allowed us to examine the effect on one FcR type of
cross-linking another FcR type on the same cell. Western blots of
precipitated FcR showed that normally there is no detectable kinase
activity for nonaggregated FcR. However, in the presence of a
phosphatase inhibitor, aggregation of one FcR type induced rapid and
extensive phosphorylation of tyrosine motifs on noncross-linked FcR.
The data in this report implicate phosphatases as necessary to prevent
global FcR involvement and suggest that normal intracluster restriction
of
Figure 1:
Signaling by Fc
Triggering under the same conditions resulted in a transient burst
of O
Figure 2:
Absence of phosphorylation of
nonaggregated FcR
Figure 3:
Tyrosine phosphorylation of nonaggregated
Cross-linking of Fc
Figure 4:
Ligand-blocked nonaggregated Fc
In the same
experiment, Fc To
examine phosphorylation kinetics, we measured the onset and maximal
phosphorylation times for
Figure 5:
Time courses of phosphorylation of
aggregated and nonaggregated FcR. Cells were incubated with
HB63(-) or 197 (+) at 37 °C for the indicated times and
then solubilized with cholate lysis buffer. Paired aliquots were
precipitated through anti-murine antibody (left panel) or A77 (right panel), and the nonreduced precipitates were analyzed
by sequential anti-phosphotyrosine (upper panels) and
anti-
Figure 6:
Evidence against
To determine whether
phospho-
Figure 7:
Phosphorylation involving Fc
Figure 8:
Lack
of vanadate-induced co-aggregation of Fc
Other indirect evidence suggests a lack of
co-aggregation. As shown in Fig. 3, Fig. 4, and Fig. 5, nonaggregated FcR were deficient in tyrosine
phosphoproteins that co-precipitate with aggregated FcR. In several
experiments, the co-precipitating panel consisted of 32-, 52-66-,
and 72-kDa (Syk kinase) (
Figure 9:
Effect of pervanadate on
In
the presence of vanadate, there was an inhibition of protein tyrosine
phosphatase activity, and also an increased or sustained tyrosine
kinase activity. This was demonstrated in the observation that FcR
aggregation-dependent increase in cellular phosphotyrosine occurred
over a longer time than the normal peak activity would have predicted.
Though normally transient FcR-triggered tyrosine phosphorylations would
have declined after the first 3-5 min of stimulation,
phosphorylation in the presence of vanadate was greater during this
initial period and it continued to accumulate for 12-18 min.
Thus, vanadate prevented dephosphorylation of To further support this conclusion, evidence is
presented that argues against alternative explanations. We show that
This conclusion is
further supported by the observation that pervanadate treatment of
cells resulted in the phosphorylation of Thus, it is
clear from results in this report that kinase activity for Our central
conclusion is that there is a vanadate-sensitive mechanism that
prevents kinase activation and the tyrosine phosphorylation of the
nonaggregated FcR component chains. Normal intracluster restriction of
Vanadate subverted the normal cellular mechanism, but it is not
clear how that occurred. One difficulty in interpreting molecular
events is that we do not know in sufficient detail how kinases that
phosphorylate FcR tyrosine motifs become activated, with what
proportion of receptors they are preassociated, and what other
regulatory molecules are present. One interpretation of our data is
that vanadate blocked the deactivation (dephosphorylation) of kinases
in aggregates and caused their release. However, in view of the effect
of pervanadate, it is more likely that kinases are sufficiently
preassociated with, or recruited to, nonaggregated FcR and
phosphorylate receptors once activated. Yamashita et al.(14) estimate that
Volume 271,
Number 19,
Issue of May 10, 1996 pp. 11099-11105
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Chain Tyrosine Phosphorylation
Subverted by a Protein-tyrosine Phosphatase Inhibitor (*)
RI) results in the transient tyrosine
phosphorylation of FcRI -chain but not the phosphorylation of
-chains associated with nonaggregated IgA Fc receptors (Fc
R)
on the same cells. Thus, normally, tyrosine phosphorylation of
-chains is limited to FcR in aggregates. In contrast, aggregation
of FcRI in the presence of vanadate induced the sustained tyrosine
phosphorylation of FcRI -chains and the rapid and extensive
phosphorylation of nonaggregated FcR -chains and low affinity
IgG Fc receptors (FcRII). This global phosphorylation of motifs on
nonaggregated FcR was also detected upon aggregation of FcR or
FcRII, which induced the phosphorylation of nonaggregated
FcRI -chains. Vanadate prevented dephosphorylation of
proteins and increased kinase activity in stimulated cells. Evidence
failed to support alternative explanations such as acquisition of
phospho- through subunit exchange or a coalescence of
nonaggregated with aggregated FcR. It is likely, therefore, that
activated kinases interacted with nonaggregated FcR in stimulated
cells. Pervanadate induced the tyrosine phosphorylation of -chains
in the absence of FcR cross-linking, indicating that the kinases could
be activated by phosphatase inhibition and could react with
nonaggregated substrates. We conclude that under normal conditions
there is a vanadate-sensitive mechanism that prevents tyrosine
phosphorylation of nonaggregated FcR -chain motifs in activated
cells, restricting their phosphorylation to aggregates.
)(1, 2) . Cross-linking of high
affinity IgG Fc receptors (FcRI) or IgA Fc receptors (FcR) on
monocytic U937 cells results in the rapid generation of oxygen radicals (3, 4, 5, 6) and tyrosine
phosphorylation of their respective
-chains(6, 7) . Cross-linking of the low affinity
Fc receptor for IgG (FcRII), which lacks -chains (8, 9) results in the phosphorylation of tyrosine
motifs in the cytoplasmic domain of the receptor(10) . The
-chain immunoreceptor tyrosine activation motifs containing
YXXL sequences (11) are substrates for Src family
kinases (12) that are capable of reacting with the
nonphosphorylated motif and of binding through SH2 domains to the
tyrosine-phosphorylated product Y*XXL. Activity of Src family
kinases Hck and Lyn associated with FcRI are increased upon
FcRI cross-linking(13) . Binding of Lyn to high affinity
IgE Fc receptor (Fc
RI) -chains is increased by FcRI
aggregation(14) .RI, phosphorylation of tyrosine
motifs is restricted to aggregates, with little involvement of
``bystander'' nonaggregated
receptors(15, 16, 17) . Limitation to
intracluster units is also evidenced by sustained binding of Lyn to
FcRI in isolated aggregates and phosphorylation of aggregate
subunits in preference to an exogenously supplied
substrate(14, 18) . Both kinds of evidence suggest a
spatial restriction of kinase activity to aggregates. It has been
suggested that restriction is due to a requirement for aggregated
receptors as sites for kinase activation (14, 19) and
to a requirement that substrate be in the aggregated
state(18) . Another possibility is that kinase activity is
under a positive control preventing activity in nonaggregated
receptors. If this is the case, inhibition of the control should allow
tyrosine phosphorylation of nonaggregated as well as aggregated FcR. -chain phosphorylation may be due to this vanadate-sensitive
mechanism.
Cells, Antibodies, and Precipitants
U937 cells,
subclone 10.6 (also called A12.13), were cultured in RPMI containing
10% fetal bovine serum and interferon-, as described
previously(21) , to increase expression of
FcRI(22) , FcR(6) , and cell
functions(6, 21) . Antibodies and FcR ligands used for
experiments included anti-FcRI mAb 197 (Medarex, Annendale, NJ),
mAb HB63 (mIgG2a isotype control and high affinity FcRI ligand),
anti-FcRI mAb 32.2 (Medarex), anti-FcR mAbs A62 and A77
(gifts from H. Kubagawa, University of Alabama at Birmingham), mAb P3
(mIgG1 isotype control), anti-FcRII mAb and Fab IV.3 (Medarex),
human IgG1 (high affinity FcRI ligand), and the following
Sepharose-conjugated antibodies: 32.2, human IgG (Sigma), A77, and
IV.3. Anti-murine antibodies included F(ab)`
sheep
anti-mouse IgG F(ab)` preadsorbed against human IgG
(Organon Technika, Durham, NC), goat anti-mouse 
-chain antibody
(Pierce), FITC-conjugated F(ab)` goat anti-mouse IgG
(Caltag, San Francisco, CA), and Sepharose-conjugated goat anti-mouse
IgG (Organon Technika). Other precipitants included PY20-agarose
(Transduction Labs, Lexington, KY), protein A-Sepharose (Sigma), and
protein G-Sepharose (Genzyme, Boston, MA). Primary immunoblot
antibodies included rabbit anti-phosphotyrosine antibody (a gift from
G. Lienhard, Dartmouth Medical School, Hanover, NH), horseradish
peroxidase-conjugated PY20 (Transduction), rabbit anti- (a gift
from J.-P. Kinet, NIAID, National Institutes of Health), and rabbit
anti-Syk (Upstate Biotechnologies, Inc., Lake Placid, NY). Secondary
immunoblot antibodies included horseradish peroxidase-conjugated
anti-rabbit IgG and anti-murine IgG (Bio-Rad).FcR Activation and the Respiratory Burst
U937 10.6
cells in superoxide (O
) assay medium
were added to an equal volume of a second medium containing 10
µg/ml control antibodies or mAb 197, which cross-links FcRI
through Fc and Fab trivalent binding(23) . Alternatively, cells
were reacted with 5 µg/ml control or anti-FcR antibody (24) for 20 min at 22 °C, centrifuged, resuspended in
O assay medium, and added to an equal
volume of a second medium containing 40 µg/ml anti-murine antibody.
FcR aggregations were done at 37 °C. For assaying tyrosine
phosphorylations or O, the second
medium was 10
M luminol in
phosphate-buffered saline(24) . Orthovanadate
(Na
VO
), buffered and at a concentration of 200
µM, was present during FcR aggregation except where
indicated. To measure respiratory bursts, luminol-mediated
chemiluminescence was monitored on a Pharmacia 1250 luminometer and is
expressed in mV, as described previously(24) .Cellular Tyrosine Phosphoproteins
Cells reacted
with antibodies were rapidly chilled, washed twice with cold
phosphate-buffered saline, and boiled for 20 min in nonreducing SDS
sample buffer. For reduction, boiling was continued for 3 min following
the addition of 4% 2-mercaptoethanol. Proteins were separated by
SDS-PAGE and analyzed by Western blot. phospho- in cellular
proteins was distinguished from nonphosphorylated -chains through
the migration pattern on nonreducing gels. Unreduced phosphorylated
-chains migrate to a broad 
28-kDa position compared with
unphosphorylated bands at 22 kDa(6) .Immunoprecipitations and Western Blotting
Cells
reacted and washed as above were solubilized at a concentration of
10
/ml in cholate lysis buffer (15 mM sodium
cholate, 0.1% Nonidet P-40, 130 mM KCl, 200 µM CaCl, 200 µM MgCl, 10 mM NaF, 500 µM NaVO (pH 7.6), 5
mM sodium pyrophosphate, 5 mM NaHPO, 0.23 units/ml aprotinin, and 200
mM phenylmethylsulfonyl fluoride, pH 7.7). Lysates were
centrifuged for 10 min at 16,000 
g, and the
postnuclear supernatants were aliquoted for separate precipitations.
Precipitations for 60 min were performed with the following bead
conjugates: nonaggregated FcR on A77-conjugated beads; aggregated
FcR on anti-murine antibody-conjugated beads or protein
G-Sepharose, as indicated; nonaggregated FcRI on 32.2- or
hIgG-conjugated beads, as indicated; aggregated FcRI on
anti-murine antibody-conjugated beads; nonaggregated FcRII on
IV.3-conjugated beads. Precipitates were washed three times with lysis
buffer and boiled in nonreducing Laemmli sample buffer. For reduction,
bead supernatants were boiled with 4% 2-mercaptoethanol. Proteins were
separated on 12 or 16% SDS-polyacrylamide gels and transferred to
polyester-supported nitrocellulose membranes (Schleicher & Schuell,
Keene, NH). The membranes were blocked and immunoblotted as described
previously(6) . Bands were visualized by the ECL procedure.
Similar results were obtained using 0.5% Nonidet P-40, 0.5% digitonin
as lysis buffer(6) . For reblotting, membranes were stripped
twice at 50 °C for 30 min each with 2% SDS and 5%
-mercaptoethanol in 62 mM Tris, pH 6.8.Assay for in Vitro Subunit Exchange
Cells were
incubated with 197 or HB63 in activation medium, and solubilized at a
concentration of 5 10
/ml in 0.5% Nonidet P-40, 0.5%
digitonin lysis buffer(6) . Duplicate FcR precipitations
were conducted on lysates from 1.5 10 cells on
A77-conjugated beads. Also prepared were FcRI- and
FcR-depleted lysates from 2 10 cells. FcR were
depleted through successive adsorptions by bead-conjugated antibodies:
goat anti-murine for 3 h, goat anti-murine for 1 h, A77 for 16 h, goat
anti-murine for 1 h, hIgG for 1 h, and A77 for 1 h. Preadsorption was
verified through anti-phosphotyrosine immunoblot of adsorbed proteins
to detect FcR-associated phospho-. Duplicate FcR precipitates
were either kept on ice as controls or rotated with preadsorbed lysates
from 4 10 cells for 1 h. Control precipitations
were executed on lysate aliquots representing 2 10 cells to assess the presence of free phospho- following FcR
depletion. The control precipitations were performed on bead-conjugated
PY20 or on anti- antibody-coated protein A-Sepharose for 1 h. All
precipitates were washed three times with lysis buffer, and boiled in
SDS-sample buffer. Precipitates were separated by SDS-PAGE and analyzed
by Western blot.Cyofluorographic Assay for Measuring FcR
Aggregation/Internalization
Cells were incubated at 37 °C
for 18 min with RPMI alone or containing 197 or HB63 (5 µg/ml).
These cells were centrifuged, washed briefly, and incubated an
additional 10 min with 5 µg/ml A77 or P3. Vanadate (200
µM) was present or absent throughout. All samples were
washed three times with 0.1% bovine serum albumin in phosphate-buffered
saline, stained with FITC-conjugated F(ab)` goat anti-mouse
IgG at 4 °C, and analyzed by cytofluorography as described
previously(23, 25) . Results are expressed as
FITC-antibody binding sites/cell.
Transient Tyrosine Phosphorylations and the Effect of
Vanadate
An early response by monocytic cells triggered through
FcRI is the tyrosine phosphorylation of several proteins including
FcRI -chains(6, 7) . Triggering also induces
a transient respiratory burst that is tightly coupled to de novo receptor cross-linking(23) . To determine whether induced
tyrosine phosphorylations were also transient, we reacted U937 cells
with anti-FcRI mAb 197, which effectively cross-links because of
trivalent binding. The time course of induced tyrosine phosphorylations
was measured by Western blot. Under normal conditions (Fig. 1, right panel), tyrosine phosphorylations of pp72 (Fig. 1A), and -chains (Fig. 1B)
were transient, peaking by 3-5 min. Additional transiently
phosphorylated proteins were detected with longer exposures. The lack
of a sustained phosphorylation suggests that phosphatase activity is
present in aggregated receptors. In the presence of vanadate, however,
phosphorylations of 72-kDa proteins (Fig. 1A),
-chains (Fig. 1B), and other proteins (Fig. 1A) accumulated, reaching a maximum by 18 min.
This indicates that vanadate blocked normal dephosphorylation.
Incubation with vanadate alone did not increase tyrosine
phosphorylations (lane(-) 24`). The continued
accumulation of phosphoproteins (Fig. 1A) suggests
sustained activation of kinases that phosphorylate FcR tyrosines. A
shorter exposure of the left panel revealed a decrease in
total cellular unphosphorylated concomitant with an increase in
phosphorylated -chains. This clearly demonstrated a shift to the
phosphorylated form of a significant portion of total cellular .
RI is normally
transient. A and B, effect of vanadate on
FcRI-induced tyrosine phosphorylations of cellular proteins and
-chains. Cells were incubated with 197 (+) or medium(-)
in the presence or absence of vanadate (VO4). SDS extracts
containing total cellular proteins were separated on 12% reducing (A) or on 16% nonreducing (B) SDS-PAGE. Reduced
proteins were analyzed by anti-phosphotyrosine (anti-PY) and
nonreduced proteins by anti- (anti-) Western blot. Brackets denote positions of tyrosine-phosphorylated (P-) and unphosphorylated -chain bands. Exposure
times for panels were identical. A shorter exposure of the samples
stimulated 18 min with vanadate revealed that band intensities for
phosphorylated -chains increased and nonphosphorylated
-chains decreased relative to zero time. C, the transient
respiratory burst. Cells were incubated with 197 in the absence of
vanadate as above, and the production of O was monitored by chemiluminescence
(mV/s).
production (Fig. 1C). Respiratory burst kinetics were similar to
the tyrosine phosphorylation response in the absence of vanadate.Absence of Bystander Involvement during FcR
Cross-linking
According to reports, FcRI aggregation does
not result in the phosphorylation of nonaggregated (bystander)
FcRI- associated
-chains(15, 16, 17) . To determine
whether nonaggregated FcR in monocytic cells become phosphorylated, we
used an assay system in which FcR of one class were aggregated and
nonaggregated FcR of another class were examined for phosphorylation of
their associated -chains. We aggregated FcRI in the absence
of vanadate for an optimal time (5 min) (Fig. 1) and examined
aggregated FcRI and nonaggregated FcR by
immunoprecipitating the receptors from lysates of the cells. As shown
in Fig. 2, nonaggregated FcR contained only a trace of
phosphotyrosine compared with aggregated FcRI. In the converse
experiment, FcR were aggregated with little effect on
FcRI (Fig. 2). Longer incubations did not increase
phosphorylation of nonaggregated receptors (not shown). These results
indicate that nonaggregated bystanders were not significantly targeted
by aggregation-activated kinases.
-chains in activated cells. Some cells were
reacted with 197 (+) (lanes 1 and 3) or HB
63(-) (lanes 2 and 4) for 5 min in the absence
of vanadate. Other cells were reacted with A77 (lane 5) or P3 (lane 6) followed by sheep anti-murine antibody (lanes 5 and 6) to cross-link (+) or not(-) FcR.
Washed cells were lysed with cholate buffer, and postnuclear
supernatants were subjected to immunoprecipitation procedures.
Nonaggregated FcR (lanes 1 and 2), aggregated
FcRI (lanes 3 and 4), and nonaggregated
FcRI (on 32.2-conjugated beads, lanes 5 and 6)
were precipitated, and the nonreduced precipitates were separated by
SDS-PAGE on 16% gels and analyzed by anti-phosphotyrosine Western blot.
The bracket denotes nonphosphorylated , and bars denote phosphorylated .
Tyrosine Phosphorylation of Bystander FcR in the Presence
of Vanadate
Because FcRI triggering in the presence of
vanadate resulted in extensive phosphorylation of cellular -chains (Fig. 1), we examined the possibility that this may have
included the phosphorylation of nonaggregated FcR. We cross-linked
FcRI and examined FcRg in receptor immunoprecipitates. As
shown by Western blot (Fig. 3A), -chains
co-precipitating with nonaggregated FcR were extensively
phosphorylated. Blotting with anti- antibodies confirmed this and
demonstrated similar intensities of phospho- bands in
nonaggregated FcR and aggregated FcRI (Fig. 3A). In the same experiment, we cross-linked
FcR and examined nonaggregated FcRI in receptor
immunoprecipitates (Fig. 3B). As shown (Fig. 3B), nonaggregated FcRI was
phosphorylated in anti-FcR-activated but not in nonactivated
cells. Recoveries of receptors in precipitates in all cases were
assessed by anti--chain blots.
-chains in the presence of vanadate. A, cells were
incubated for 15 min with 197 (+) or HB 63(-) and with 200
µM vanadate present. FcR and FcRI were
precipitated from the lysates through A77 (lanes 1 and 2) and anti-murine antibody (lanes 3 and 4),
respectively. B, cells were preincubated with A59 (lane
5) or P3 (lane 6) for 20 min, washed, and incubated with
anti-murine -chain antibody in the presence of vanadate for 15
min. FcR (lane 5) and nonspecific proteins (NS, lane
6) were precipitated via protein G-Sepharose. FcRI was
precipitated via hIgG-conjugated beads (lanes 7 and 8). Nonreduced precipitates were electrophoresed and analyzed
by sequential anti-phosphotyrosine and anti- Western blot.
Anti- blots of aggregated FcR show that this precipitate was
inefficiently recovered (lane 5). Brackets denote
phospho-.
R was
also executed in the presence of hIgG1 to block a potential Fc
interaction of anti-FcR with FcRI. The results show that
-chains in the hIgG1-FcRI complexes had become phosphorylated (Fig. 4, A and B, lane 1).
Furthermore, the possibility of anti-murine antibody co-cross-linking
and stimulating via bound hIgG1 was also eliminated by an oxidase assay
in which cells preincubated with hIgG1 or not and incubated with the
same set of antibodies were found to be activated only through IgA
receptors. Values from the oxidase assay (in mV) were 4451 ± 228
for A77-hIgG1-coated cells, 4414 ± 752 for A77-coated cells, and
19 ± 9 and 9 ± 2 for P3-hIgG1- and P3-reacted cells,
respectively. All received second antibody. These results eliminated Fc
bridging as the source of nonaggregate involvement.
RI are
efficiently phosphorylated. A, immunoprecipitation of
FcRI after FcR cross-linking in the presence of hIgG1. Cells
were preincubated for 20 min with 10 µg/ml hIgG1 and A77 (lanes
1 and 2) and then incubated for 18 min with sheep
anti-murine antibody (lanes 1 and 2) in the presence
of 200 µM vanadate. FcRI and FcR were
precipitated from lysates via 32.2 (lane 1) and anti-murine
antibody (lane 2), respectively. B, efficient
transphosphorylation of FcRI. Cells were incubated as above with
197 (+) or HB63(-). FcRI were precipitated via
anti-murine antibody (lanes 5 and 6). Nonreduced
precipitates were analyzed by sequential anti-phosphotyrosine and
anti- Western blot. Band intensities for phospho- (brackets)
in lanes 1 and 5 indicate similar efficiencies of
phosphorylation. FcR precipitates (lanes 3 and 4) are shown for comparison.
RI were cross-linked (Fig. 4, A and B, lane 5), causing the extensive phosphorylation of
FcR. Anti- immunoblots of aggregated (lane 6)
compared with nonaggregated FcRI (lane 1) show similar
intensities of phospho- bands, suggesting that similar numbers of
chains in nonaggregated ligand-occupied FcRI were phosphorylated.
Noticeable decreases in unphosphorylated and increases for
phospho- within individual samples (lanes 1 and 6) imply an efficient shift in state. Similar mobility
patterns for phospho- in each case are consistent with equivalent
site modifications by kinases. These results demonstrate the efficient
phosphorylation of tyrosines on nonaggregated FcR -chains.-chains of aggregated FcRI and
nonaggregated FcR. As shown by anti-phosphotyrosine and anti-
Western blot (Fig. 5), phosphorylation of FcR was
detectable by 6 min and plateaued by 18 min (Fig. 5).
FcRI phosphorylation was detectable by 3 min and plateaued
between 12 and 18 min. Similar maximal intensities were observed, and
anti- blots show similar amounts of FcR in precipitates. These
results show the rapid and prolonged phosphorylation of aggregated and
nonaggregated FcR -chains.
(lower panels) Western blot. Brackets denote the position of phospho-.
Phospho-
To determine whether nonaggregate phosphorylation could
be an artifact of immunoprecipitation in which phospho- Is Not Acquired through Subunit
Exchange exchanged
for unphosphorylated , or vice versa, immunoprecipitates
of nonaggregated FcR (containing phospho--chains) were
incubated for the usual time with an unstimulated lysate precleared of
endogenous FcR -chains. Exchange was judged by comparing the
original with lysate-incubated precipitates. As shown in Fig. 6A, these two were identical, indicating that
FcR did not exchange its associated phospho- during
immunoprecipitation. In the converse experiment (Fig. 6B), unphosphorylated FcR in precipitates
were incubated in lysates of FcRI-stimulated cells. The lysates
had been precleared of FcRI and FcR (Fig. 6B, lower panel) but contained free phospho- chains (lane
7). Exchange was again judged by comparing original with
lysate-incubated precipitates. The results show that unphosphorylated
in FcR precipitates was not exchanged for phospho-.
Collectively, the results show that phospho- was not acquired
through subunit exchange in vitro.
-subunit exchange
during immunoprecipitation. Cells were incubated with 197 or HB63, and
FcR precipitates and lysates cleared of FcRI and FcR
were prepared as described under ``Materials and Methods.'' A, lack of FcR phospho- exchange with
nonphosphorylated in the lysate. FcR precipitates from
197-stimulated (S/FcR; lanes 2 and 4)
or HB63-reacted nonstimulated (NS/FcR; lanes 1 and 3) cells were rotated with a lysate from
nonstimulated cells (NS/lysate) (lanes 3 and 4)) or kept on ice (lanes 1 and 2).
Precipitates were washed and separated by nonreducing SDS-PAGE.
phospho- retained by FcR precipitates was assessed by
anti-phosphotyrosine and anti- (not shown) Western blot. B, lack of FcR exchange with phospho- in the
lysate. 197-stimulated and HB63-reacted nonstimulated cell precipitates
were exposed to FcR-cleared lysates from stimulated cells (S/lysate) (lanes 1-4) or kept on ice (lanes 5 and 6). Precipitates were washed and
separated by reducing SDS-PAGE. Analysis for exchange of FcR
-chains for phospho- in the lysate was by
anti-phosphotyrosine Western blot. A control precipitate to assess free
phospho- remaining in the FcR-depleted lysate is shown in lane
7. Depletion from the stimulated lysate (S-lysate) was
verified by anti-phosphotyrosine Western blot for phospho- in
preadsorbed proteins (lower
panel).
exchange in vivo explains the appearance of
phospho- in nonaggregated FcR, we triggered FcRI and
precipitated from the cell lysate nonaggregated FcRII. FcRII
lack -chains but are phosphorylated in cytoplasmic domain motifs
upon cross-linking(10) . Fig. 7A shows that
nonaggregated FcRII in FcRI-activated but not in nonactivated
cells were phosphorylated on tyrosines. Similarly, upon triggering
through FcRII, -chains for FcRI and FcR became
phosphorylated (Fig. 7B). These data indicate that FcR
lacking exchangeable -chains are phosphorylatable bystanders and,
with cross-linking, are able to induce -chain phosphorylation.
This suggests that direct kinase activity rather than subunit exchange in vivo explains bystander -chain phosphorylation.
RII. A, FcRI aggregation induces phosphorylation of
FcRII. Cells were incubated for 15 min with 197 (lanes 3 and 4) or HB63 (lanes 1 and 2) with
vanadate present. FcRI and FcRII were precipitated and
electrophoresed under reducing conditions. B, FcRII
aggregation induces phosphorylation of -chains associated with
FcRI and FcR. Cells were preincubated with Fab IV.3 (lanes 5-8) or medium (lanes 1-4) and
incubated for 18 min with sheep anti-mouse (lanes 1-8)
in the presence of vanadate. Cholate lysis buffer extracts were
prepared and FcRI were precipitated on hIgG (lanes 2 and 6) or 32.2 beads (lanes 4 and 8), or
FcR were precipitated (lanes 3 and 7) from 2
10 cell equivalents. SDS extracts representing
10
cells were also separated for total cellular proteins (lanes 1 and 5). Proteins were electrophoresed under
nonreducing conditions and analyzed by sequential anti-phosphotyrosine (upper panels) and anti- (lower panels) Western
blot.
Bystander Phosphorylation Is Not Due to FcR
Co-aggregation
We investigated the possibility that vanadate may
have induced co-aggregation of nonaggregated with aggregated FcR,
making nontargeted FcR available to aggregate-docked kinases.
Aggregation was assessed by measuring internalization of receptors.
Following aggregation and a predetermined interval for internalization
of FcRI, the cells were fluorescently labeled to quantitate
FcRI and FcR remaining on the surface. The results (Fig. 8) show that 197-FcRI aggregates were effectively
internalized (>60%) without a concomitant reduction in surface
FcR. As similar results were obtained in the presence and absence
of vanadate (Fig. 8), the data do not support a vanadate-induced
co-aggregation.
R. Aggregation was
assessed by measuring induced internalization, and all incubations were
conducted in the presence or absence of 200 uM vanadate. Cells
were incubated for 18 min with HB63 or 197 to occupy(-) or
cross-link (+) FcRI, and then for an additional 10 min with
P3 or A77. After washing, cells were stained with FITC-second antibody
and analyzed by cytofluorography. Cells reacted with HB63, then P3 or
with 197, then P3 were stained for surface FcRI sites. Surface
FcR sites were obtained by subtracting data from 197 or HB63, then
P3 reacted cells from that of 197 or HB63, then A77, reacted cells.
Data represent the mean of FITC-second antibody binding sites/cell
± the standard deviation.
)bands. Discrete co-precipitations
are consistent with a lack of co-aggregation of FcR types.Tyrosine Phosphorylation of Nonaggregated
Treatment of nonactivated
cells with vanadate prereacted with H by
Treatment of Cells with PervanadateO in order
to produce pervanadate induced the tyrosine phosphorylation of .
This did not occur upon treatment with vanadate or HO alone. These data (Fig. 9) show that -chains can
become phosphorylated in the absence of any FcR cross-linking. Based on
this, it appears that the kinases that interacted with nonaggregated
FcR were negatively regulated by tyrosine phosphatases.
-chains in
nonactivated cells. Cells were incubated at 37 °C with
O medium containing 250 µM vanadate premixed with 750 µM HO (lane 1), 250 µM vanadate (lane
2), 750 µM HO (lane
3), or phosphate-buffered saline (lane 4). After 5 min of
incubation, cells were harvested, and total cell protein samples were
separated on a nonreducing gel. Results were obtained by
anti-phosphotyrosine and anti- Western blot. Results obtained
after 15 min of incubation (not shown) agreed with data shown. Brackets
show the position of phospho-.
Transient and Cluster-restricted Tyrosine
Phosphorylation of Aggregated FcR
Evidence is presented showing
that under normal conditions FcRI-induced tyrosine
phosphorylations are transient. Similar observations have been reported
by Duchemin et al.(7) and by Swieter et al.(26) for aggregated FcRI in rat basophilic leukemia
cells. In our experiments, peaks of phosphorylation occurred by
3-5 min. Importantly, even at the peak of this activity,
phosphorylation of -chains triggered by FcRI aggregation
occurred on subunits of the aggregated receptors but was absent from
noncross-linked FcR on the same cells. Similarly, cross-linking of
FcR did not cause phosphorylation of -chains on FcRI.
This absence of nonaggregated FcR involvement indicates that tyrosine
phosphorylation in monocytes is normally restricted to FcR in
aggregates or clusters. As previously mentioned, this lack of bystander
involvement is normal for nonaggregated FcRI -chains in
suboptimally FcRI-triggered
basophils(15, 16, 17) .Restriction of Kinase Activity to Clusters Subverted by
Vanadate
Interestingly, we found that aggregation of FcRI
in the presence of vanadate resulted in tyrosine phosphorylations not
only of -chains associated with aggregated FcRI but also of
-chains associated with nonaggregated FcR and of the
cytoplasmic domain of nonaggregated FcRII. Phosphorylation of
nonaggregated FcR began shortly after the onset of phosphorylation
of tyrosines in aggregated FcRI, suggesting the rapid
activation or association of activated kinases with nonaggregated FcR.
Phosphorylation of nonaggregated FcR was efficient, comparing well with
phosphorylated -chains in overtly aggregated FcR. It was
extensive, as demonstrated by dramatic shifts of total cellular and
FcR-associated -chains from the nonphosphorylated to the
phosphorylated state. Collectively, the results show an
activation-dependent phosphorylation of nonaggregated FcR -chains
and motifs under conditions that inhibit phosphatase activity.-chains, and it
appears to have either prevented deactivation (and promoted release) of
kinases in aggregates or activated kinases preassociated with
nonaggregated FcR.-chain phosphorylation did not occur as a result of (i) anti-FcR
bridging of nontargeted FcR or (ii) vanadate-induced co-aggregation of
nonaggregated with aggregated FcR. We also demonstrated that (iii)
phospho- on nonaggregated receptors was not acquired by subunit
exchange in vitro. As for in vivo, (iv) -chain
exchange between aggregated and nonaggregated FcR would not account for
the ability of bystander FcRII to be phosphorylated or to induce
the phosphorylation of FcRI and FcR -chains when
cross-linked. Collectively, the evidence is consistent with direct
kinase activity on bystander component chains.-chains without any FcR
cross-linking. Pervanadate is a potent inhibitor of phosphotyrosine
dephosphorylation(27, 28) . It increases tyrosine
phosphorylations in a number of cell
types(28, 29, 30) , including T
lymphocytes(31, 32) . The effect of pervanadate in
this study suggests that relevant kinase activation can occur
independently of aggregation and, conversely, that -chains need
not be aggregated to be substrates. Since -chains do not require
the aggregated state for phosphorylation, this state is unlikely to
dictate restriction of kinase activity to clusters.-chains
is not obligatorily limited to clusters of FcR, although clustering is
the normal mechanism for kinase activation. It is also clear that
aggregation of -chains is not a physical requirement for kinase
interaction with their tyrosines. Pribluda et al.(18) have described clustered FcRI motifs as the
normal and necessary state of substrate for activated kinases to
phosphorylate in trans their nearest neighbor FcRI -chains.
The same group has also shown FcRI dimers are a sufficient size to
satisfy the requirements for trans-phosphorylation(33) .
Therefore, our observations are not inconsistent with the model of
Pribluda et al., since nonaggregated FcRI and FcR
may exist as dimers ()prepared to trans-phosphorylate paired
chains but needing something more for kinase activation.-chain phosphorylation may be due to this mechanism, and
phosphatases as regulatory molecules are implicated in the process. 25% of resting FcRI are
associated with Lyn kinase in rat basophilic leukemia cells. Wang et al.(13) identified Lyn and Hck associated with
resting FcRI. Kent et al.(34) found phosphatase
activity in FcRI aggregates and Swieter et al.(26) found it in monomers. In our experiments,
FcR-mediated oxygen radical production may have converted some vanadate
to pervanadate or, alternatively, stimulation may have caused
channeling of orthovandate intracellularly. Either way, it would appear
that phosphatases were inhibited that were functionally associated with
kinases in nonaggregated FcR. Thus, a reasonable hypothesis is that
nonaggregated FcR phosphorylation is normally negatively regulated by
phosphotyrosine phosphatases and that aggregation induces FcR
phosphorylation by transiently inactivating the phosphatases.
)RI, high affinity
IgG Fc receptor(s); FcRI, high affinity IgE Fc receptor(s);
FcR, IgA Fc receptor(s); FcRII, low affinity IgG Fc
receptor(s) on U937 cells; hIgG1, human IgG1; mAb, monoclonal antibody;
FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel
electrophoresis.))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
