©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracluster Restriction of Fc Receptor -Chain Tyrosine Phosphorylation Subverted by a Protein-tyrosine Phosphatase Inhibitor (*)

(Received for publication, December 26, 1995)

Lorraine C. Pfefferkorn (§) Sharon L. Swink

From the Department of Microbiology at Borwell, Dartmouth Medical School, Hanover, New Hampshire 03756

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (FcRI) results in the transient tyrosine phosphorylation of FcRI -chain but not the phosphorylation of -chains associated with nonaggregated IgA Fc receptors (FcalphaR) 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 FcalphaR -chains and low affinity IgG Fc receptors (FcRII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of FcalphaR 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.


INTRODUCTION

Aggregation controls signaling through Fc receptors (FcR)(^1)(1, 2) . Cross-linking of high affinity IgG Fc receptors (FcRI) or IgA Fc receptors (FcalphaR) 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 (FcRI) -chains is increased by FcRI 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 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.

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.


MATERIALS AND METHODS

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) , FcalphaR(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-FcalphaR 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)`(2) sheep anti-mouse IgG F(ab)`(2) preadsorbed against human IgG (Organon Technika, Durham, NC), goat anti-mouse kappa-chain antibody (Pierce), FITC-conjugated F(ab)`(2) 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(2)) 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(2) 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(2), the second medium was 10M luminol in phosphate-buffered saline(24) . Orthovanadate (Na(3)VO(4)), 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 10^7/ml in cholate lysis buffer (15 mM sodium cholate, 0.1% Nonidet P-40, 130 mM KCl, 200 µM CaCl(2), 200 µM MgCl(2), 10 mM NaF, 500 µM Na(3)VO(4) (pH 7.6), 5 mM sodium pyrophosphate, 5 mM NaH(2)PO(4), 0.23 units/ml aprotinin, and 200 mM phenylmethylsulfonyl fluoride, pH 7.7). Lysates were centrifuged for 10 min at 16,000 times g, and the postnuclear supernatants were aliquoted for separate precipitations. Precipitations for 60 min were performed with the following bead conjugates: nonaggregated FcalphaR on A77-conjugated beads; aggregated FcalphaR 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% beta-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 times 10^6/ml in 0.5% Nonidet P-40, 0.5% digitonin lysis buffer(6) . Duplicate FcalphaR precipitations were conducted on lysates from 1.5 times 10^6 cells on A77-conjugated beads. Also prepared were FcRI- and FcalphaR-depleted lysates from 2 times 10^7 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 FcalphaR precipitates were either kept on ice as controls or rotated with preadsorbed lysates from 4 times 10^6 cells for 1 h. Control precipitations were executed on lysate aliquots representing 2 times 10^6 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)`(2) 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.


RESULTS

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 .


Figure 1: Signaling by FcRI 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(2) was monitored by chemiluminescence (mV/s).



Triggering under the same conditions resulted in a transient burst of O(2) 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 FcalphaR by immunoprecipitating the receptors from lysates of the cells. As shown in Fig. 2, nonaggregated FcalphaR contained only a trace of phosphotyrosine compared with aggregated FcRI. In the converse experiment, FcalphaR 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.


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(-) FcalphaR. Washed cells were lysed with cholate buffer, and postnuclear supernatants were subjected to immunoprecipitation procedures. Nonaggregated FcalphaR (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 FcalphaRg in receptor immunoprecipitates. As shown by Western blot (Fig. 3A), -chains co-precipitating with nonaggregated FcalphaR were extensively phosphorylated. Blotting with anti- antibodies confirmed this and demonstrated similar intensities of phospho- bands in nonaggregated FcalphaR and aggregated FcRI (Fig. 3A). In the same experiment, we cross-linked FcalphaR and examined nonaggregated FcRI in receptor immunoprecipitates (Fig. 3B). As shown (Fig. 3B), nonaggregated FcRI was phosphorylated in anti-FcalphaR-activated but not in nonactivated cells. Recoveries of receptors in precipitates in all cases were assessed by anti--chain blots.


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. FcalphaR 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 kappa-chain antibody in the presence of vanadate for 15 min. FcalphaR (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 FcalphaR show that this precipitate was inefficiently recovered (lane 5). Brackets denote phospho-.



Cross-linking of FcalphaR was also executed in the presence of hIgG1 to block a potential Fc interaction of anti-FcalphaR 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.


Figure 4: Ligand-blocked nonaggregated FcRI are efficiently phosphorylated. A, immunoprecipitation of FcRI after FcalphaR 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 FcalphaR 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. FcalphaR 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 FcalphaR. 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.

To examine phosphorylation kinetics, we measured the onset and maximal phosphorylation times for -chains of aggregated FcRI and nonaggregated FcalphaR. As shown by anti-phosphotyrosine and anti- Western blot (Fig. 5), phosphorylation of FcalphaR 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.


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-.



Phospho- Is Not Acquired through Subunit Exchange

To determine whether nonaggregate phosphorylation could be an artifact of immunoprecipitation in which phospho- exchanged for unphosphorylated , or vice versa, immunoprecipitates of nonaggregated FcalphaR (containing phospho--chains) were incubated for the usual time with an unstimulated lysate precleared of endogenous FcR alpha-chains. Exchange was judged by comparing the original with lysate-incubated precipitates. As shown in Fig. 6A, these two were identical, indicating that FcalphaR did not exchange its associated phospho- during immunoprecipitation. In the converse experiment (Fig. 6B), unphosphorylated FcalphaR in precipitates were incubated in lysates of FcRI-stimulated cells. The lysates had been precleared of FcRI and FcalphaR (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 FcalphaR precipitates was not exchanged for phospho-. Collectively, the results show that phospho- was not acquired through subunit exchange in vitro.


Figure 6: Evidence against -subunit exchange during immunoprecipitation. Cells were incubated with 197 or HB63, and FcalphaR precipitates and lysates cleared of FcRI and FcalphaR were prepared as described under ``Materials and Methods.'' A, lack of FcalphaR phospho- exchange with nonphosphorylated in the lysate. FcalphaR precipitates from 197-stimulated (S/FcalphaR; lanes 2 and 4) or HB63-reacted nonstimulated (NS/FcalphaR; 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 FcalphaR precipitates was assessed by anti-phosphotyrosine and anti- (not shown) Western blot. B, lack of FcalphaR 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 FcalphaR -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 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 FcalphaR 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, 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 FcalphaR. 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 FcalphaR were precipitated (lanes 3 and 7) from 2 times 10^6 cell equivalents. SDS extracts representing 10^5 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 FcalphaR remaining on the surface. The results (Fig. 8) show that 197-FcRI aggregates were effectively internalized (>60%) without a concomitant reduction in surface FcalphaR. As similar results were obtained in the presence and absence of vanadate (Fig. 8), the data do not support a vanadate-induced co-aggregation.


Figure 8: Lack of vanadate-induced co-aggregation of FcalphaR. 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 FcalphaR 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) (^2)bands. Discrete co-precipitations are consistent with a lack of co-aggregation of FcR types.

Tyrosine Phosphorylation of Nonaggregated by Treatment of Cells with Pervanadate

Treatment of nonactivated cells with vanadate prereacted with H(2)O(2) in order to produce pervanadate induced the tyrosine phosphorylation of . This did not occur upon treatment with vanadate or H(2)O(2) 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.


Figure 9: Effect of pervanadate on -chains in nonactivated cells. Cells were incubated at 37 °C with O(2) medium containing 250 µM vanadate premixed with 750 µM H(2)O(2) (lane 1), 250 µM vanadate (lane 2), 750 µM H(2)O(2) (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-.




DISCUSSION

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 FcalphaR on the same cells. Similarly, cross-linking of FcalphaR 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 FcalphaR and of the cytoplasmic domain of nonaggregated FcRII. Phosphorylation of nonaggregated FcalphaR 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.

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 FcRII to be phosphorylated or to induce the phosphorylation of FcRI and FcalphaR -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 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 FcalphaR may exist as dimers (^3)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 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI29455. Cytofluorimetry was supported in part by Core Grant CA23108 of the Norris Cotton Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 603-650-4831; Fax: 603-650-6223.

(^1)
The abbreviations used are: FcR, Fc receptor(s); FcRI, high affinity IgG Fc receptor(s); FcRI, high affinity IgE Fc receptor(s); FcalphaR, 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.

(^2)
L. C. Pfefferkorn and S. L. Swink, manuscript in preparation.

(^3)
L. C. Pfefferkorn, unpublished data.


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