(Received for publication, December 26, 1995)
From the
This study shows that aggregation of U937 cell high affinity IgG
Fc receptor (FcRI) results in the transient tyrosine
phosphorylation of Fc
RI
-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 Fc
RI in the presence of vanadate induced the sustained tyrosine
phosphorylation of Fc
RI
-chains and the rapid and extensive
phosphorylation of nonaggregated Fc
R
-chains and low affinity
IgG Fc receptors (Fc
RII). This global phosphorylation of motifs on
nonaggregated FcR was also detected upon aggregation of Fc
R or
Fc
RII, which induced the phosphorylation of nonaggregated
Fc
RI
-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.
Aggregation controls signaling through Fc receptors
(FcR)()(1, 2) . Cross-linking of high
affinity IgG Fc receptors (Fc
RI) or IgA Fc receptors (Fc
R) 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 (Fc
RII), 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 Fc
RI are increased upon
Fc
RI cross-linking(13) . Binding of Lyn to high affinity
IgE Fc receptor (Fc
RI)
-chains is increased by Fc
RI
aggregation(14) .
For FcRI, 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
Fc
RI 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.
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 -chain phosphorylation may be due to this vanadate-sensitive
mechanism.
Figure 1:
Signaling by FcRI is normally
transient. A and B, effect of vanadate on
Fc
RI-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).
Triggering under the same conditions resulted in a transient burst
of O production (Fig. 1C). Respiratory burst kinetics were similar to
the tyrosine phosphorylation response in the absence of vanadate.
Figure 2:
Absence of phosphorylation of
nonaggregated FcR -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(-) Fc
R.
Washed cells were lysed with cholate buffer, and postnuclear
supernatants were subjected to immunoprecipitation procedures.
Nonaggregated Fc
R (lanes 1 and 2), aggregated
Fc
RI (lanes 3 and 4), and nonaggregated
Fc
RI (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
.
Figure 3:
Tyrosine phosphorylation of nonaggregated
-chains in the presence of vanadate. A, cells were
incubated for 15 min with 197 (+) or HB 63(-) and with 200
µM vanadate present. Fc
R and Fc
RI 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. Fc
R (lane 5) and nonspecific proteins (NS, lane
6) were precipitated via protein G-Sepharose. Fc
RI 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 Fc
R show that this precipitate was
inefficiently recovered (lane 5). Brackets denote
phospho-
.
Cross-linking of FcR was
also executed in the presence of hIgG1 to block a potential Fc
interaction of anti-Fc
R with Fc
RI. The results show that
-chains in the hIgG1-Fc
RI 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.
Figure 4:
Ligand-blocked nonaggregated FcRI are
efficiently phosphorylated. A, immunoprecipitation of
Fc
RI after Fc
R 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. Fc
RI and Fc
R were
precipitated from lysates via 32.2 (lane 1) and anti-murine
antibody (lane 2), respectively. B, efficient
transphosphorylation of Fc
RI. Cells were incubated as above with
197 (+) or HB63(-). Fc
RI 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. Fc
R precipitates (lanes 3 and 4) are shown for comparison.
In the same
experiment, FcRI were cross-linked (Fig. 4, A and B, lane 5), causing the extensive phosphorylation of
Fc
R. Anti-
immunoblots of aggregated (lane 6)
compared with nonaggregated Fc
RI (lane 1) show similar
intensities of phospho-
bands, suggesting that similar numbers of
chains in nonaggregated ligand-occupied Fc
RI 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.
To
examine phosphorylation kinetics, we measured the onset and maximal
phosphorylation times for -chains of aggregated Fc
RI and
nonaggregated Fc
R. As shown by anti-phosphotyrosine and anti-
Western blot (Fig. 5), phosphorylation of Fc
R
was
detectable by 6 min and plateaued by 18 min (Fig. 5).
Fc
RI
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.
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- (lower panels) Western blot. Brackets denote the position of phospho-
.
Figure 6:
Evidence against -subunit exchange
during immunoprecipitation. Cells were incubated with 197 or HB63, and
Fc
R precipitates and lysates cleared of Fc
RI and Fc
R
were prepared as described under ``Materials and Methods.'' A, lack of Fc
R phospho-
exchange with
nonphosphorylated
in the lysate. Fc
R precipitates from
197-stimulated (S/Fc
R; lanes 2 and 4)
or HB63-reacted nonstimulated (NS/Fc
R; 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 Fc
R precipitates was assessed by
anti-phosphotyrosine and anti-
(not shown) Western blot. B, lack of Fc
R
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 Fc
R
-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).
To determine whether
phospho- exchange in vivo explains the appearance of
phospho-
in nonaggregated FcR, we triggered Fc
RI and
precipitated from the cell lysate nonaggregated Fc
RII. Fc
RII
lack
-chains but are phosphorylated in cytoplasmic domain motifs
upon cross-linking(10) . Fig. 7A shows that
nonaggregated Fc
RII in Fc
RI-activated but not in nonactivated
cells were phosphorylated on tyrosines. Similarly, upon triggering
through Fc
RII,
-chains for Fc
RI and Fc
R 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.
Figure 7:
Phosphorylation involving FcRII. A, Fc
RI aggregation induces phosphorylation of
Fc
RII. Cells were incubated for 15 min with 197 (lanes 3 and 4) or HB63 (lanes 1 and 2) with
vanadate present. Fc
RI and Fc
RII were precipitated and
electrophoresed under reducing conditions. B, Fc
RII
aggregation induces phosphorylation of
-chains associated with
Fc
RI and Fc
R. 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 Fc
RI were precipitated on hIgG (lanes 2 and 6) or 32.2 beads (lanes 4 and 8), or
Fc
R 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.
Figure 8:
Lack
of vanadate-induced co-aggregation of FcR. 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 (+) Fc
RI, 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 Fc
RI sites. Surface
Fc
R 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.
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) ()bands. Discrete co-precipitations
are consistent with a lack of co-aggregation of FcR types.
Figure 9:
Effect of pervanadate on -chains in
nonactivated cells. Cells were incubated at 37 °C with
O
medium containing 250 µM vanadate premixed with 750 µM H
O
(lane 1), 250 µM vanadate (lane
2), 750 µM H
O
(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-
.
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 -chains, and it
appears to have either prevented deactivation (and promoted release) of
kinases in aggregates or activated kinases preassociated with
nonaggregated FcR.
To further support this conclusion, evidence is
presented that argues against alternative explanations. We show that
-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 Fc
RII to be phosphorylated or to induce
the phosphorylation of Fc
RI and Fc
R
-chains when
cross-linked. Collectively, the evidence is consistent with direct
kinase activity on bystander component chains.
This conclusion is
further supported by the observation that pervanadate treatment of
cells resulted in the phosphorylation of -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.
Thus, it is
clear from results in this report that kinase activity for -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 Fc
RI
motifs as the
normal and necessary state of substrate for activated kinases to
phosphorylate in trans their nearest neighbor Fc
RI
-chains.
The same group has also shown Fc
RI 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 Fc
RI and Fc
R
may exist as dimers (
)prepared to trans-phosphorylate paired
chains but needing something more for kinase activation.
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
-chain phosphorylation may be due to this mechanism, and
phosphatases as regulatory molecules are implicated in the process.
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 25% of resting Fc
RI are
associated with Lyn kinase in rat basophilic leukemia cells. Wang et al.(13) identified Lyn and Hck associated with
resting Fc
RI. Kent et al.(34) found phosphatase
activity in Fc
RI 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.