From the
The focal adhesion kinase, pp125
pp125
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 Fc
Affinity purification of
tyrosine-phosphorylated proteins was as described
previously
(3) . Briefly, lysates from 2
Different anti-pp125
Together, the data
indicate that the 77-kDa protein is not a fragment of pp125
The aggregation of
Fc
There was strong association of FAP with
non-tyrosine-phosphorylated pp125
Aggregation
of Fc
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 Fc
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.
, 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, Fc
RI, 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 Fc
RI aggregated cells. Both of
these Fc
RI-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 Fc
RI signaling.
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).
RI 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 Fc
RI 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.
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.
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.
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).
but associates specifically in vivo with
pp125
. We have called this 77-kDa protein Fak Associated
Protein or FAP.
FAP Was Not Detected in pp125
The adhesion of
fibroblasts to fibronectin through integrins induces tyrosine
phosphorylation of pp125Precipitates from Fibroblasts
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).
Fc
The aggregation of FcRI Aggregation Induces in Vivo Tyrosine
Phosphorylation of FAP
RI on adherent
RBL-2H3 cells increases tyrosine phosphorylation of
pp125
(3) . Therefore, we investigated if Fc
RI
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 Fc
RI
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
Fc
RI 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 Fc
RI
aggregation. Thus, the extent of the decrease in the in vitro tyrosine phosphorylation of FAP correlated with the degree of cell
activation. Therefore, Fc
RI aggregation induced the in vivo tyrosine phosphorylation of FAP and decreased the amount of FAP
detected by in vitro kinase assay.
RI 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 Fc
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-induced
Tyrosine Phosphorylation of FAP
RI-mediated secretion
(29) . This
adhesion also enhances Fc
RI-induced tyrosine phosphorylation of
several proteins including that of
pp125
(3, 4) . Therefore, we examined the
effect of cell adhesion on Fc
RI-induced tyrosine phosphorylation
of FAP. Fc
RI 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 Fc
RI-activated adherent compared to
non-adherent cells (Fig. 6B). Together, the data
demonstrate that cell adhesion enhances both Fc
RI-induced tyrosine
phosphorylation of FAP and Fc
RI-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
pp125
The association of FAP with
pp125in Vivo
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).
. 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.
RI 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 Fc
RI 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 Fc
RI
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 Fc
RI
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, Fc
RI aggregation may lead to the
dissociation of FAP from pp125
and/or to an actual
decrease in the in vitro phosphorylation of FAP.
RI
signaling
(18, 21, 22, 23, 25, 46, 47) .
The aggregation of Fc
RI 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 Fc
RI
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 Fc
RI
signaling. However, the precise function of FAP is currently under
investigation.
RI, the high affinity receptor for IgE; RBL-2H3 cells, rat
basophilic leukemia cells; PAGE, polyacrylamide gel electrophoresis;
FAP, Fak Associated Protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.