©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 77-kDa Protein Associates with pp125 in Mast Cells and Becomes Tyrosine-phosphorylated by High Affinity IgE Receptor Aggregation (*)

Majed M. Hamawy (§) , Kenji Minoguchi , William D. Swaim , Stephan E. Mergenhagen , Reuben P. Siraganian

From the (1) Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-1188

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The focal adhesion kinase, pp125, is a novel non-receptor protein tyrosine kinase expressed in different cells including mast cells. Here we report that a 77-kDa protein associates with pp125 in the mast cell analog, rat basophilic leukemia (RBL-2H3) cells. When pp125 immunoprecipitates were subjected to an in vitro kinase assay, there was prominent phosphorylation on tyrosine of pp125 and of a 77-kDa protein. By V8 protease digestion mapping and by immunoblotting with two different anti-pp125 antibodies, the 77-kDa protein was distinct from pp125. This Fak Associated Protein or FAP was detected in RBL-2H3 cells but not in fibroblasts. The aggregation of the high affinity IgE receptor, FcRI, induced the in vivo tyrosine phosphorylation of FAP. However, there was a marked decrease in the in vitro phosphorylation of FAP in the immunoprecipitates from FcRI aggregated cells. Both of these FcRI-mediated effects were enhanced by cell adhesion. There was strong association of FAP with non-tyrosine-phosphorylated pp125. Thus this interaction does not appear to be mediated by the Src homology 2 domain. Together the data indicate that FAP associates with pp125 and suggest that FAP may play a role in FcRI signaling.


INTRODUCTION

pp125 is a non-receptor protein tyrosine kinase that accumulates at focal adhesion sites, although it has no Src homology (SH)() 2 and 3 domains (1, 2) . This kinase has a central catalytic domain and an amino- and a carboxyl-terminal non-catalytic regions. It is a cytosolic protein that lacks membrane localization signals. pp125 is widely expressed in different cell lines and tissues and appears to be involved in signal transduction from cell surface receptors for adhesion (integrins), immunoglobulins, neuropeptides, and growth factors (3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . Thus, pp125 may be a point of convergence for signals transduced from different receptors. In Src-transformed chicken embryo cells, pp125 is complexed with the activated pp60, whereas in normal chicken embryo cells it associates with pp59(13, 14) . pp125 is a substrate for activated pp60(5) . In vitro, pp125 phosphorylates paxillin, a focal adhesion protein (15).

Mast cells and basophils are secretory cells that play an important role in inflammation (16) . Both cells can be activated for secretion by several stimuli including the aggregation of the high affinity IgE receptor (16, 17) . Although FcRI lacks intrinsic kinase activity, its aggregation initiates intracellular biochemical reactions including the tyrosine phosphorylation of several proteins (3, 4, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . Recently, we reported that the aggregation of FcRI induces tyrosine phosphorylation of pp125 in adherent rat basophilic leukemia (RBL-2H3) cells, a mast cell line (3) . In this report we show that a 77-kDa protein associates with pp125. We also demonstrate the effect of cell activation on the in vivo and on the in vitro tyrosine phosphorylation of the 77-kDa protein.


EXPERIMENTAL PROCEDURES

Materials

All reagents unless otherwise specified were obtained as described previously (3, 4, 29) .

Immunoglobulin

Mouse monoclonal anti-trinitrophenyl IgE (TNP-142) has been described previously (30) . Several different anti-pp125 antibodies were used for these studies. The monoclonal antibody (mAb) 2A7 and the rabbit polyclonal antibodies raised against the COOH-terminal non-catalytic domain (651-1028 amino acids) of chicken pp125 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse mAb raised against a fragment consisting of amino acids 354-533 and anti-paxillin mAb were from Transduction Laboratories (Lexington, KY). The rabbit polyclonal antibody raised against the COOH-terminal 150 amino acid residues of mouse pp125 was a generous gift of Dr. Steven K. Hanks (Vanderbilt University, Nashville, TN). Affinity-purified rabbit anti-phosphotyrosine antibodies were prepared as described previously (31) and coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech Inc.). The anti-p72 rabbit antibody has been described previously (23) . Normal mouse IgG was from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY-20 was from ICN Immunobiologicals (Lisle, IL).

Cell Culture and Activation

The RBL-2H3 cells and the Swiss mouse 3T3 fibroblasts were maintained as described previously (32, 33) . IgE-mediated stimulation of RBL-2H3 cells was performed as described previously (3, 4, 29) . The role of cell adhesion was examined as described previously with some modification (3, 4, 29) . Briefly, RBL-2H3 cells grown as monolayers were detached by trypsinization, washed, and sensitized with anti-TNP IgE in suspension (10 cells/ml) for 90 min at room temperature. The cells were then washed with EMEM containing 0.1% bovine serum albumin, suspended in the same medium (10 cell/ml), and divided into 4 aliquots. Two aliquots were added to two tissue culture dishes that were coated with 15% fetal calf serum (adherent cells), whereas the other aliquots remained in tubes (non-adherent cells). Following a 30-min incubation at 37 °C, antigen or medium alone was added to the adherent and non-adherent cells. After an additional 30 min at 37 °C, the supernatants from the dishes were aspirated and the cells were lysed, while the cells in tubes were centrifuged and then lysed. Lysates were cleared by centrifugation and used for immunoprecipitation as described below.

Immunoprecipitation

Lysates of 10 cells were mixed with 5 µg of anti-pp125 antibody, and the mixture was then added to 10 µg of rabbit anti-mouse immunoglobulin that had been preincubated for 2 h at 4 °C with 25 µl of protein A coupled to agarose beads. After 2 h at 4 °C, the beads were washed five times with lysis buffer, then resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiled for 5 min.

Affinity purification of tyrosine-phosphorylated proteins was as described previously (3) . Briefly, lysates from 2 10 cells were incubated for 2 h at 4 °C with 2 mg of polyclonal rabbit anti-phosphotyrosine antibody coupled to Sepharose 4B beads. The supernatants (unbound proteins) were collected, and the beads were then transferred to a column and washed with 200 column volumes of lysis buffer. The tyrosine-phosphorylated proteins were eluted with 40 mM phenyl phosphate. The unbound proteins (supernatant) or the tyrosine-phosphorylated proteins eluted with phenyl phosphate were then reprecipitated with anti-pp125 mAb 2A7 as above.

Immunoblotting

Aliquots from total cell lysates or immunoprecipitates were separated by SDS-PAGE (10%) and electrotransferred onto nitrocellulose membranes. Immunoblotting with the horseradish peroxidase-conjugated anti-phosphotyrosine antibody PY-20 was as described previously (3) . Membranes were also probed with 1 µg/ml anti-pp125 using either the mAb from Transduction Laboratories or the rabbit polyclonal antibody from Upstate Biotechnology followed by horseradish peroxidase-coupled goat anti-mouse or anti-rabbit immunoglobulin (1:25,000 dilution). The signals were visualized using an enhanced chemiluminescence kit (ECL, Amersham Corp.) as described previously (3) .

In Vitro Kinase Assay

This was performed as described previously (34) . The immunoprecipitation with anti-pp125 was as described in the previous sections. The beads were then washed five times with lysis buffer, once with 50 mM Tris-HCl buffer containing 100 mM NaCl, pH 7.2, and once with kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl, and 2 mM MnCl). The immunoprecipitates were resuspended in 50 µl of kinase buffer, and the reaction was initiated by the addition of 5 µCi of [P]ATP (3000 Ci/mmol, DuPont NEN). After 10 min at room temperature, the beads were washed twice with ice-cold lysis buffer. Bound proteins were eluted by boiling in sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by autoradiography. Phosphoamino acid analysis was performed as described previously (23) .

Two-dimensional Electrophoresis

pp125 precipitates were subjected to two-dimensional gel electrophoresis as described previously (31) .

V8 Protease Digestion

The digestion was carried out as described previously with some modifications (35) . Briefly, pp125 precipitates after in vitro kinase assay were fractionated in a first-dimension SDS-PAGE (10% gels). Each lane was cut out, equilibrated in protease buffer (125 mM Tris-HCl, pH 6.8, 0.1% SDS) for 30 min, placed on the top of a second-dimension SDS-PAGE (4-20% linear gradient), and sealed in 1% agarose. The top of the gel was then overlayered with 0-6 µg of Staphylococcus aureus strain V8 protease (Sigma) in SDS-PAGE sample buffer. The proteins were allowed to enter slowly into the stacking gel, and the current was turned off for overnight digestion by the protease. The migration was then resumed until the dye front reached the bottom of the gel. Intact and digested proteins were detected as described above.

RESULTS

Association of a 77-kDa Protein with pp125

As we have reported previously (3) , the anti-pp125 mAb 2A7 precipitated a 115-kDa tyrosine-phosphorylated protein from RBL-2H3 cell lysates (Fig. 1A). When these pp125 immunoprecipitates were subjected to in vitro kinase assay, there were prominent phosphorylated 115-kDa and 77-kDa proteins (Fig. 1B). The extent of the in vitro phosphorylation of pp125 was variable in different experiments; however, the phosphorylation of the 77-kDa protein was always prominent in these immune kinase assays. Under these same conditions, neither protein was seen in precipitates with normal mouse IgG (Fig. 1B). By phosphoamino acid analysis, the 77-kDa protein was phosphorylated only on tyrosine in vitro (data not shown).


Figure 1: Co-immunoprecipitation of a 77-kDa protein with pp125. Proteins were immunoprecipitated from RBL-2H3 cell lysates with either normal mouse IgG (lane1) or with anti-pp125 mAb 2A7 (lane2). The immunoprecipitates either were directly boiled in SDS-PAGE sample buffer (A) or were subjected to in vitro kinase assay (B-D) before boiling. Eluted proteins were fractionated by 10% SDS-PAGE and then transferred to nitrocellulose membranes. A, the precipitates were immunoblotted with anti-phosphotyrosine mAb. B, the precipitates were subjected to an in vitro kinase assay. C, immunoblotting of the proteins in B with mAb raised to NH-terminal amino acids 354-533 of pp125. D, identical membrane to the one shown in B was immunoblotted with polyclonal antibodies raised to the COOH-terminal amino acids 651-1028 of pp125.



The experimental evidence strongly suggested that the 77-kDa protein was not a fragment of pp125. The immunoprecipitates were blotted with two different anti-pp125 antibodies that react with either the NH-terminal or COOH-terminal half of the molecules (Fig. 1, C and D). The rabbit polyclonal antibody (from Upstate Biotechnology) is against amino acids 651-1028 (which are in the COOH-terminal half of the molecule), whereas the mouse mAb (from Transduction Laboratories) reacts with amino acids 354-533 (in the NH-terminal half of the molecule). Although both antibodies strongly recognized pp125, neither antibody bound to the 77-kDa protein (Fig. 1, C and D). To further rule out that the 77-kDa protein is a fragment of pp125, we utilized a proteolytic digestion approach. Due to the very small amounts of proteins precipitated by pp125, the proteins could not be metabolically labeled. Therefore, pp125 immunoprecipitates after in vitro kinase assay were used for proteolytic digestion (Fig. 2). The 77-kDa protein was readily digested into at least three fragments by V8 protease. However, under the same conditions, pp125 was digested only to high molecular weight fragments. Similar results were obtained by varying the concentrations of the V8 protease or by using different digestion conditions. Furthermore, similar data were obtained when pp125 immunoprecipitated from mouse fibroblasts was digested with V8 protease. Together, these data indicate that the 77-kDa protein is not a fragment of pp125.


Figure 2: V8 protease digestion of the in vitro phosphorylated pp125 and the 77-kDa protein. pp125 precipitates from RBL-2H3 cell lysates were subjected to in vitro kinase assay, and the proteins were then separated by SDS-PAGE (10% gels). Each lane was cut out, equilibrated in protease buffer, placed on the top of a second-dimension SDS-PAGE (4-20% linear gradient), and sealed in 1% agarose. The top of the gel was then overlayered with 200 µl of sample buffer without (A) or with (B) 4 µg of S. aureus strain V8 protease. The proteins were allowed to slowly enter the stacking gel, and the current was turned off for an overnight digestion by the protease. The migration was then resumed until the dye front reached the bottom of the gel. Arrow indicates pp125; solidarrowhead is the 77-kDa protein.



There was specific association of this 77-kDa protein with pp125. In one set of experiments, the amount of the anti-pp125 mAb 2A7 used for immunoprecipitation was varied from 0 to 5 µg while keeping constant the secondary antibody and the protein A-coupled beads. After in vitro kinase assay, both the 77-kDa protein and pp125 were detected with 1 µg of mAb 2A7 and maximal amounts of both proteins were observed with 3 µg of mAb 2A7 (data not shown). When the quantity of the anti-pp125 mAb was kept constant but the amount of beads was doubled, there was no change in the in vitro phosphorylation of the 77-kDa and 115-kDa proteins. Thus, the 77-kDa protein was not precipitated nonspecifically by these experimental conditions.

Different anti-pp125 antibodies were used for immunoprecipitation followed by in vitro kinase assay to further confirm the specific association of this 77-kDa protein with pp125. The monoclonal antibody 2A7 and two polyclonal rabbit antibodies bind to the COOH-terminal non-catalytic domain of the molecule, whereas another monoclonal antibody binds to the NH-terminal half of the molecule (amino acids 354-533). The 77-kDa protein was detected by in vitro kinase assays in the precipitates of the antibodies that bind to the COOH-terminal but not to the NH-terminal half of the pp125 (data not shown).

Together, the data indicate that the 77-kDa protein is not a fragment of pp125 but associates specifically in vivo with pp125. We have called this 77-kDa protein Fak Associated Protein or FAP.

FAP Was Not Detected in pp125Precipitates from Fibroblasts

The adhesion of fibroblasts to fibronectin through integrins induces tyrosine phosphorylation of pp125 and increases its in vitro kinase activity (1, 2) . However, in none of these studies with fibroblasts was there a 77-kDa protein coprecipitated with pp125. Therefore, we examined whether the discrepancy between the present findings and the results with fibroblasts was due to the use of different methods. Although FAP was present in pp125 immunoprecipitates from RBL-2H3 cells, it was not detected in the precipitates from mouse 3T3 fibroblasts (Fig. 3). There was also approximately 10-fold less pp125 in RBL-2H3 cells than in the fibroblasts. Thus, either FAP may not be present in fibroblasts or it may not associate with pp125 in fibroblasts.


Figure 3: The pp125-associated 77-kDa protein is not detected in mouse 3T3 fibroblasts. Lysates from 10 fibroblasts (lane1) or from 10 RBL-2H3 cells (lane2) were immunoprecipitated with the anti-pp125 mAb 2A7 and then subjected to an in vitro kinase assay (upperpanel). The same proteins were also blotted with anti-pp125 (lowerpanel).



FcRI Aggregation Induces in Vivo Tyrosine Phosphorylation of FAP

The aggregation of FcRI on adherent RBL-2H3 cells increases tyrosine phosphorylation of pp125(3) . Therefore, we investigated if FcRI aggregation would also induce tyrosine phosphorylation of FAP. In pp125 immunoprecipitates from lysates of activated RBL-2H3 cells, there was a 77-kDa protein that was tyrosine-phosphorylated in vivo (Fig. 4A). Although in some experiments other tyrosine-phosphorylated proteins were coprecipitated with pp125 from lysates of activated cells (Fig. 5), only the 77-kDa protein was consistently detected. By two-dimensional electrophoresis, this in vivo tyrosine-phosphorylated protein migrated with the same characteristics as the in vitro phosphorylated FAP detected in kinase assays (data not shown). Therefore, FAP was also tyrosine-phosphorylated in vivo following FcRI aggregation.


Figure 4: FcRI aggregation induces tyrosine phosphorylation of pp125-associated 77-kDa protein. RBL-2H3 cells sensitized with antigen-specific IgE were incubated for 30 min at 37 °C with either antigen (Ag+) or buffer (Ag-). The cell lysates were then used for immunoprecipitation with anti-pp125 mAb 2A7. Precipitated proteins were either directly boiled in SDS-PAGE sample buffer (A) or were subjected to an in vitro kinase assay (B) before boiling. Eluted proteins were fractionated in SDS-PAGE (10% gels) and then transferred to a nitrocellulose membrane. A, anti-phosphotyrosine blotting of pp125 precipitates. B, in vitro phosphorylation of pp125 precipitates. C, immunoblotting of the proteins in B with anti-pp125 mAb. Arrow indicates pp125; solidarrowhead is the 77-kDa protein.




Figure 5: Time course of FcRI-induced tyrosine phosphorylation of the pp125-associated 77-kDa protein. IgE-sensitized cells were stimulated for the indicated times at 37 °C with antigen. At each time point the lysates were immunoprecipitated with anti-pp125 mAb 2A7 and either immunoblotted with anti-phosphotyrosine antibodies (A) or subjected to in vitro kinase assay (B). C, immunoblotting of the proteins in B with anti-pp125 mAb. Arrow indicates pp125; solidarrowhead is the 77-kDa protein.



Although the in vivo tyrosine-phosphorylated FAP was detectable only in stimulated cells, by in vitro kinase assay FAP was present in the pp125 immunoprecipitates from the lysates of both unstimulated and stimulated RBL-2H3 cells (Fig. 4B). However, after FcRI aggregation there was markedly less FAP detectable by in vitro kinase assay (Fig. 4B). As we could not detect these proteins in the pp125 immunoprecipitates by either metabolic labeling or by in vitro biotinylation, it was impossible to determine whether there was an actual decrease in the amounts of FAP or only changes in its in vitro phosphorylation. However, pp125 was equally precipitated from activated and from non-activated cells (Fig. 4C). In time-course studies there was a correlation between the increased in vivo tyrosine phosphorylation of FAP and the decrease in its in vitro phosphorylation (Fig. 5). These changes in the phosphorylation of FAP were observed within 1-3 min of FcRI aggregation. Thus, the extent of the decrease in the in vitro tyrosine phosphorylation of FAP correlated with the degree of cell activation. Therefore, FcRI aggregation induced the in vivo tyrosine phosphorylation of FAP and decreased the amount of FAP detected by in vitro kinase assay.

The aggregation of FcRI induces tyrosine phosphorylation of several proteins in the 70-80-kDa range such as paxillin, Btk, and p72. However, by immunoblotting, none of these proteins were detected in anti-FAK precipitates (data not shown). Thus, FAP is distinct from paxillin, Btk, and p72.

Adhesion of RBL-2H3 Cells Enhances FcRI-induced Tyrosine Phosphorylation of FAP

Adhesion of cells to the extracellular matrix is important for cellular development and function (36, 37, 38) . The adhesion of RBL-2H3 cells to surfaces coated with either fibronectin or fetal calf serum markedly enhances FcRI-mediated secretion (29) . This adhesion also enhances FcRI-induced tyrosine phosphorylation of several proteins including that of pp125(3, 4) . Therefore, we examined the effect of cell adhesion on FcRI-induced tyrosine phosphorylation of FAP. FcRI aggregation induced the in vivo tyrosine phosphorylation of FAP in adherent cells (Fig. 6A). By in vitro kinase assay, there was equal phosphorylation of FAP in the immunoprecipitates from lysates of unstimulated adherent and non-adherent cells (Fig. 6B). However, there was a greater decrease in the in vitro phosphorylation of FAP when the lysates were from FcRI-activated adherent compared to non-adherent cells (Fig. 6B). Together, the data demonstrate that cell adhesion enhances both FcRI-induced tyrosine phosphorylation of FAP and FcRI-induced decrease in the in vitro phosphorylation of FAP. Furthermore, the data confirm the observation (Fig. 5) that the decrease in the in vitro phosphorylation of FAP, which was detected by in vitro kinase assays, correlates with the extent of cell activation.


Figure 6: Adhesion enhances FcRI-induced in vivo tyrosine phosphorylation of the pp125-associated 77-kDa protein. RBL-2H3 cells grown as monolayers were detached by trypsinization and sensitized with IgE in suspension (10 cells/ml) for 90 min at room temperature. The cells were then washed with EMEM containing 0.1% bovine serum albumin, suspended in the same medium, and divided into 4 aliquots. Two aliquots were added to two tissue culture plates that were coated with 15% fetal calf serum (Ad+, adherent cells), whereas the other aliquots remained in tubes (Ad-, non-adherent cells). Following a 30-min incubation at 37 °C, antigen (Ag+) or medium (Ag-) was added to the adherent and to the non-adherent cells. After 30 min at 37 °C, the cells were lysed and the proteins precipitated with anti-pp125 mAb 2A7 were either boiled in sample buffer (A) or subjected to an in vitro kinase assay (B) before boiling. The proteins were analyzed by immunoblotting with anti-phosphotyrosine antibodies (A) or by in vitro kinase assay (B). The precipitated proteins were also immunoblotted with anti-pp125 mAb (C). Arrow indicates pp125; solidarrowhead is the 77-kDa protein.



FAP Associates with Non-tyrosine-phosphorylated pp125in Vivo

The association of FAP with pp125 in lysates from non-adherent RBL-2H3 cells (Fig. 6, lane 3) suggested that this interaction did not require the tyrosine phosphorylation of pp125. To further examine this question, proteins were precipitated with anti-phosphotyrosine antibodies to obtain tyrosine-phosphorylated and non-phosphorylated pp125. Each fraction was then reprecipitated with anti-pp125. Anti-phosphotyrosine antibodies precipitated at least 50% of tyrosine-phosphorylated pp125 (Fig. 7A). However, this tyrosine-phosphorylated pp125 represented only a minor fraction of the total cellular pp125 (Fig. 7C). Although similar amounts of tyrosine-phosphorylated pp125 were precipitated from both fractions, FAP was detected only in the immunoprecipitates that contained the non-tyrosine-phosphorylated pp125 (Fig. 7B). These results indicate that FAP associates with non-tyrosine-phosphorylated pp125 and suggest that tyrosine phosphorylation of pp125 may not play a role in its association with FAP.


Figure 7: The 77-kDa protein associates with non-tyrosine-phosphorylated pp125. Lysates from 2 10 cells were incubated for 2 h at 4 °C with 2 mg of polyclonal rabbit anti-phosphotyrosine antibody coupled to Sepharose 4B beads. The unbound proteins were collected, and the beads were washed extensively. The bound tyrosine-phosphorylated proteins were eluted with 40 mM phenyl phosphate. The unbound proteins (supernatants) or the tyrosine-phosphorylated proteins eluted with phenyl phosphate were then reprecipitated with anti-pp125 mAb 2A7. The precipitates were either analyzed by immunoblotting with anti-phosphotyrosine antibodies (A) or by in vitro kinase assay (B). The precipitated proteins were also immunoblotted with anti-pp125 mAb (C). Lane1, precipitation with anti-pp125 from cell lysate supernatants that did not bind to anti-phosphotyrosine affinity beads; lane2, precipitation with anti-pp125 from the proteins eluted from the anti-phosphotyrosine affinity beads. Arrow indicates pp125; solidarrowhead is the 77-kDa protein.



DISCUSSION

The following experimental evidence indicates a specific association of the 77-kDa protein, FAP, with pp125. First, there was association of FAP with pp125 when different antibodies to pp125 were used for immunoprecipitation. Second, varying the amount of the antibody used for immunoprecipitation or the amount of protein A beads did not effect the extent of the association. Third, FAP was not detected in the precipitates of normal mouse IgG or of several other protein tyrosine kinases including Lyn and Syk (data not shown). Fourth, FAP co-immunoprecipitated with pp125 from RBL-2H3 cells but not from fibroblasts. The results also indicate that FAP is a protein distinct from pp125. Thus, antibodies to the NH-terminal and to the COOH-terminal non-catalytic domains of pp125 did not bind to FAP and proteolytic digestion confirmed that FAP and pp125 were different proteins. Although only pp125 and FAP were detected in the anti-FAK immunoprecipitates, it is still possible that the association of these two molecules is mediated by other non-tyrosine-phosphorylated protein(s).

There was strong association of FAP with non-tyrosine-phosphorylated pp125. Therefore, it seems unlikely that this association is mediated by an SH2 domain on the FAP molecule. In contrast, Src family kinases bind through their SH2 domains to tyrosine-phosphorylated pp125(13, 14) . The pp125 molecule lacks both SH2 and SH3 domains that are commonly used by tyrosine kinases to bind their substrates. It also lacks proline-rich regions that interact with the SH3 domains of other proteins. Therefore, the interaction between FAP and pp125 is not due to SH2 or SH3 domains. Previous studies suggested that pp125 may interact with other proteins by mechanisms independent of SH2 or SH3 domains (39, 40, 41) . For example, an alternatively spliced form of pp125 that includes the carboxyl-terminal non-catalytic domain localizes at focal adhesion sites. Mutated forms of pp125 lacking a part of this COOH-terminal non-catalytic domain fail to localize to focal adhesion sites. Thus, the COOH-terminal non-catalytic domain of pp125 contains sufficient information to direct pp125 to focal adhesion sites. Since the COOH-terminal non-catalytic domain of pp125 lacks tyrosine residues that are phosphorylated in vivo, pp125 must be localizing to focal adhesion sites by interacting with other proteins in an SH2/SH3 independent manner. The association of FAP with pp125 may depend on similar interactions.

Aggregation of FcRI results in tyrosine phosphorylation of several proteins including pp125(3, 4, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . Here we observed that aggregation of FcRI induced the tyrosine phosphorylation of FAP. The time course of the tyrosine phosphorylation of both FAP and pp125 was similar. The in vivo tyrosine phosphorylation of pp125 by FcRI aggregation did not result in increased in vitro autophosphorylating activity. Previous studies suggested that the activity of pp125 is not modulated by tyrosine phosphorylation (42, 43) . The increased in vivo tyrosine phosphorylation of FAP correlated with a decrease in its in vitro phosphorylation. The mechanism by which FcRI cross-linking is modulating the in vitro phosphorylation of FAP is unclear because we could not determine the amount of FAP in the immunoprecipitates. Thus, FcRI aggregation may lead to the dissociation of FAP from pp125 and/or to an actual decrease in the in vitro phosphorylation of FAP.

Phosphorylation of proteins on tyrosine residues is a mechanism by which receptors transduce and propagate their signals. Once proteins are tyrosine-phosphorylated, they can interact with other proteins that have SH2 domains (44, 45) . Several SH2-containing proteins are involved in FcRI signaling (18, 21, 22, 23, 25, 46, 47) . The aggregation of FcRI results in tyrosine phosphorylation of pp125 and FAP, which could in turn cause interactions of these molecules with SH2-containing proteins. Thus, both pp125 and FAP can play important roles in propagating FcRI signaling. FAP was consistently observed in pp125 precipitates from RBL-2H3 cells but not from fibroblasts. Hence, FAP may be a mast cell protein that plays a role in FcRI signaling. However, the precise function of FAP is currently under investigation.


FOOTNOTES

*
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: Laboratory of Immunology, Bldg. 10, Rm. 1N106, NIDR, NIH, Bethesda, MD 20892. Tel.: 301-496-5105; Fax: 301-480-8328.

The abbreviations used are: SH, Src homology; mAb, monoclonal antibody; EMEM, Eagle's minimal essential medium with Earle's salts; FcRI, the high affinity receptor for IgE; RBL-2H3 cells, rat basophilic leukemia cells; PAGE, polyacrylamide gel electrophoresis; FAP, Fak Associated Protein.


ACKNOWLEDGEMENTS

We thank Drs. William Hook and Mark Swieter for helpful discussion and criticism of the manuscript. We thank Dr. Volker Stephan for the preparation and purification of the polyclonal rabbit anti-phosphotyrosine antibodies. We also thank Adizat A. Eko for technical assistance.


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