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(Received for publication, May 3,
1995; and in revised form, November 3, 1995) From the
The association of the murine motheaten phenotype of severe
hemopoietic dysregulation with loss of PTP1C tyrosine phosphatase
activity indicates a critical role for this SH2 domain-containing
phosphotyrosine phosphatase in the regulation of hemopoietic cell
growth and differentiation. To explore the molecular basis for PTP1C
effects on hematopoiesis, we have investigated the possibility that
this enzyme interacts with the product of the Vav proto-oncogene, a
putative guanine nucleotide exchange factor expressed exclusively in
hemopoietic cells. Our data indicate that PTP1C physically associates
with Vav in murine spleen cells and in EL4 T lymphoma and P815
mastocytoma cells, and that this interaction is increased following
mitogenic stimulation and the induction of both PTP1C and Vav tyrosine
phosphorylation. The results also reveal tyrosine phosphatase activity
to be present in Vav immunoprecipitates from stimulated splenic and
P815 cells and suggest that a major portion of total cellular PTP1C
catalytic activity is associated with Vav. As Vav-associated tyrosine
phosphatase activity was not detected in PTP1C-deficient motheaten
splenic cells, it appears that PTP1C accounts for most, if not all,
Vav-coprecipitable tyrosine phosphatase activity in normal cells. The
data also demonstrate the capacity of the Vav SH2 domain alone to bind
to PTP1C in activated P815 cells, but suggest a role for the two Vav
SH3 domains in enhancing this interaction. In addition, the results
reveal PTP1C association with two other molecules implicated in Ras
activation, the Grb2 adaptor protein and mSos1, a GTP/GDP exchanger for
Ras. PTP1C therefore has the capacity to bind and potentially modulate
various signaling effectors involved in activation of Ras or
Ras-related proteins, and, accordingly, regulation of Ras activation
represents a possible mechanism whereby PTP1C influences hemopoietic
cellular responses. Among the phosphotyrosine phosphatases (PTP) ( In contrast to PTP1C association with specific cell
surface receptors, its interactions with downstream cytoplasmic
signaling effectors have not been defined. In this regard, one molecule
of potential interest is the 95-kDa product of the Vav proto-oncogene,
another SH2 domain-containing protein which, like PTP1C, has been
identified in all hemopoietic lineages and implicated by several lines
of evidence in the control of hemopoietic cell growth and
differentiation(18, 19, 20) . Inhibition of
Vav expression, for example, interferes with development of hemopoietic
cells from embryonic stem cells(21) , and, as is consistent
with its participation in a broad range of hemopoietic cell signaling
pathways, Vav has been shown to become tyrosine-phosphorylated
following cross-linking of antigen receptors on
lymphocytes(22, 23, 24) , Fc To
investigate this possibility, we evaluated the capacity of PTP1C to
interact with Vav in resting and activated mast cells and T
lymphocytes. As reported here, the results of this analysis reveal the
association of PTP1C protein and tyrosine phosphatase activity with Vav
and indicate that this interaction increases following mitogenic
stimulation of these cells and coincident with increases in tyrosine
phosphorylation of both Vav and PTP1C. The data also implicate both the
SH2 and SH3 domains of Vav in mediating the association of this protein
with PTP1C. Lastly, while a major portion of total intracellular PTP1C
activity appears to be contained in Vav-PTP1C complexes, the results of
this study demonstrate that PTP1C also binds to both Grb2 adaptor and
mSos1 GEF proteins. Together, these results suggest that PTP1C effects
on hemopoietic cell growth and development may be realized at least in
part through modulation of the signaling events linking receptor
stimulation to the activation of Ras or Ras-related proteins.
Figure 3:
Association of Vav SH2/SH3 domains with
activated PTP1C. A, schematic showing Vav sequences present in
the three GST-Vav fusion proteins used for in vitro binding
assays. Numbers below each construct refer to amino acid positions of
domain boundaries. B, cell lysates were prepared from 10
Figure 1:
Association of PTP1C with Vav in
hemopoietic cells. A, cell lysates were prepared from 10
The data shown in Fig. 1A also reveal isolated murine splenocytes to express two PTP1C
species (
Figure 2:
Increases in PTP1C-Vav association and
tyrosine phosphorylation following cell stimulation. A, cell
lysates were prepared from unstimulated(-) or ConA (20
µg/ml)-treated (+) EL4 cells and unstimulated(-) or
steel factor (100 ng/ml)-treated P815 cells, and 800 µg of lysate
proteins were immunoprecipitated with anti-Vav antibodies. Lysates were
also prepared from 10
In view of these findings, as well as previous data showing that Vav
association with another signaling effector in activated T cells, the
protein tyrosine kinase ZAP70, is mediated through binding of the Vav
SH2 domain to phosphotyrosine site(s) on ZAP70(40) , the
contribution of the Vav SH2 domain to Vav-PTP1C interaction was
investigated. To this end, GST fusion proteins containing the Vav SH2
domain alone and the Vav SH2 domain combined with the carboxyl-terminal
or both Vav SH3 domains (Fig. 3A) were coupled to
glutathione-Sepharose, incubated with steel factor-treated P815 cells,
and evaluated for PTP1C binding by immunoblotting with anti-PTP1C
antibody. As shown in Fig. 3B, the results of this in vitro analysis revealed PTP1C binding with the fusion
protein containing the Vav SH2 domain alone, but PTP1C binding was
observed considerably increased with the fusion proteins containing an
SH2 and SH3 domain and even more increased with the fusion protein
containing both Vav SH3 domains. These results indicate the capacity of
the Vav SH2 domain to interact with PTP1C in activated cells, and, as
has been demonstrated previously with respect to the SH2
domain-mediated intramolecular repression of Src activity(41) ,
the data also suggest that optimal binding of these molecules requires
the Vav SH3 domains as well. However, these results do not preclude the
possibility that the PTP1C SH2 domains and/or other sites within the
Vav protein contribute to the interaction of these proteins.
Figure 4:
Identification of tyrosine phosphatase
activity in Vav immunoprecipitates from stimulated splenic and P815
cells. A, cell lysates were prepared from steel factor-treated
P815 cells and from ConA-treated splenic cells obtained from me mice. Aliquots of 300, 600, and 900 µg of lysate proteins were
immunoprecipitated from P815 cells with anti-PTP1C antibody (IpPTP1C) and from both P815 and me splenic cells
with anti-Vav antibodies (IpVav and IpVav (me/me), respectively). The immunoprecipitates
were incubated with 2 mMp-nitrophenol phosphate at
37 °C for 4 h, and, after addition of NaOH, absorbance was measured
at 410 nm. The results shown are representative of three independent
experiments. B, cell lysates were prepared from 10
To extend these data, Vav immunoprecipitates from
ConA-treated normal me and me
Figure 5:
Association of PTP1C with Grb2 and mSos1
in P815 cells. A, cell lysates were prepared from unstimulated
P815 cells and 2000 µg of lysate protein immunoprecipitated with
anti-Grb2 (left panel) or anti-PTP1C (right panel)
antibodies. Duplicate samples of the precipitated proteins as well as
500 µg of lysate protein alone (L, left panel) or
1500 µg of lysate proteins immunoprecipitated with anti-Grb2
antibody (Ip:GRB2, right panel) were electrophoresed
through SDS-PAGE and immunoblotted with anti-PTP1C (left
panel) or anti-Grb2 (right panel) antibodies. B,
cell lysates were prepared from unstimulated P815 cells, and 2000
µg of lysate proteins were immunoprecipitated with either
anti-mSos1 or anti-PTP1C antibodies (as shown on top of each
panel), subjected to SDS-PAGE, and then immunoblotted with anti-PTP1C (left panel) or anti-mSos1 (right panel) antibodies. C, cell lysates were prepared from unstimulated(-) and
steel factor-treated (+) P815 cells, and 1000 µg of lysate
proteins were immunoprecipitated with anti-PTP1C, anti-Grb2, or
anti-mSos1 antibodies as indicated, electrophoresed through SDS-PAGE,
and immunoblotted with anti-PTP1C antibody. For all panels, the
positions of molecular mass markers are shown on the side, and arrows indicate the positions of PTP1C, mSos1, and
Grb2.
In
summary, we have shown that PTP1C associates with Vav, Grb2, and mSos1,
three cytosolic molecules expressed broadly among hemopoietic cells and
implicated in the activation of Ras or Ras-related signaling pathways.
The capacity of PTP1C to interact with and potentially modulate these
signaling proteins strongly suggests that PTP1C effects on hemopoietic
cell differentiation and growth are realized at least in part through
the regulation of Ras and/or Ras-related proteins. Similarly, the
association of Vav with PTP1C protein and phosphatase activity implies
a role for PTP1C in modulating Vav-induced transformation events. The
definition of the structural basis for and physiologic relevance of
PTP1C associations with these signaling effectors thus represents a
promising avenue toward elucidating the intracellular events regulating
downstream transmission of receptor-evoked activation signals in
hemopoietic cells.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3856-3862
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)identified to date, the cytosolic enzyme PTP1C is
distinguished by its predominant expression in hemopoietic cells and
the presence of two N-terminal located Src homology 2 (SH2) domains, a
motif found in only two other PTPs, Syp (PTP1D/SHPTP2) and the
Drosophila csw protein(1, 2, 3, 4, 5, 6) .
These properties, together with the recent data linking PTP1C gene
mutations to the profound hemopoietic dysregulation manifested by
motheaten (me) and viable motheaten (me
)
mice (7, 8, 9) , reveal a critical role for
PTP1C in modulating hemopoietic cell differentiation and growth. As
this PTP has been shown to associate with the activated c-kit,
erythropoietin, and IL-3 receptors(10, 11, 12) and, more recently, with the B cell antigen receptor
complex and the CD22 and FcRIIB1 receptors on
lymphocytes(13, 14, 15) , PTP1C appears to
subserve its regulatory role, at least in part, by modulating the
signaling capacities of membrane growth factor/antigen/cytokine
receptors. As is consistent with the marked overexpansion of multiple
hemopoietic cell types observed in PTP1C-deficient motheaten mice, the
data concerning PTP1C effects on the B cell antigen (13, 14, 15, 16) and IL-3 (11) receptors suggest that this phosphatase down-regulates
signaling cascades elicited by receptor engagement, presumably by
dephosphorylating and deactivating receptor components or
receptor-associated cytosolic protein tyrosine kinases. In conjunction
with the increased susceptibility of me
heterozygous mice to development of lymphoid malignancies (8, 17) and the implicit possibility that PTP1C has
tumor suppressor activity, these data suggest that the major influence
of PTP1C activity on hematopoiesis may be realized through the
suppression of signaling pathways that normally promote cell
activation. and
receptors on monocytes and mast cells,
respectively(24, 25) , and c-kit receptors on
multiple hemopoietic lineages(26, 27) . Vav contains a
number of structural motifs found in many signaling effectors,
including an SH2, a pleckstrin homology, and two SH3 domains as well as
a sequence motif (db1 homology domain) found in various proteins known
to function as guanine nucleotide exchange factors (GEF) for Ras and
Ras-related proteins(28, 29, 30) . On this
basis, it has been suggested that Vav represents a new class of
signaling substrates, the activation of which may provide a mechanism
for coupling cell surface receptors to Ras (22, 23, 24) . However, at present the
precise functions for Vav are unclear, as Vav has been shown to act as
a Ras GEF in T and B lymphocytes(31, 32) , but appears
to induce NIH3T3 transformation by mechanisms independent of Ras
activation(33, 34) . While the substrates for Vav GEF
activity remain to be defined, the cumulative data concerning Vav,
including its potential for oncogenic activation(35) , suggest
that the modulation of Vav signal transducing functions(s) represents
another possible mechanism whereby PTP1C might influence the
development and functions of multiple hemopoietic cell lineages.
Reagents
Polyclonal anti-PTP1C and anti-Syp
antibodies were generated in rabbits immunized with GST-PTP1C SH2
domain fusion proteins as described previously(5, 9) .
The monoclonal anti-phosphotyrosine 4G10 and anti-Vav antibodies as
well as rabbit polyclonal anti-murine son-of-sevenless 1 (mSos1)
antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid,
NY). Rabbit polyclonal antibody specific to Grb2 and a synthetic
peptide corresponding to residues 577-590 of the mouse Vav
protein were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA). Steel factor (SF) was obtained from Genzyme (Cambridge, MA), and
concanavalin A (Con A) as well as all chemicals for
immunoblotting/immunoprecipitation analyses were obtained from Sigma.Cells and Cell Lines
Fresh splenic cell
suspensions used in this work were prepared by standard procedures from
C57BL/6J-me/me and
+/+ and C3HeBFeJ-me/me and +/+
mice derived from breeding stock maintained at the Samuel Lunenfeld
Research Institute, Mount Sinai Hospital. The murine EL4 T lymphoma and
P815 mastocytoma lines were obtained from Dr. C. Paige and ATCC
(TIB64), respectively, and were maintained at 37 °C in Opti-MEM
(Life Technologies, Inc.) containing 100 µg/ml
penicillin/streptomycin and 10% fetal calf serum (Life Technologies,
Inc.). As controls for some experiments, we also used B16 melanoma
cells transfected with the vector pCMV4Neo or alternatively with a
construct containing the full-length PTP1C cDNA subcloned into the
pCMV4Neo vector (provided by Dr. B. Chan). These latter lines were
maintained under the same culture conditions as described above except
for the addition of 2 mg/ml Geneticin (Life Technologies, Inc.) to the
culture medium. For cell stimulations, cells were cultured in Opti-MEM
containing 0.5% fetal calf serum for 17 h, washed, and then cultured
for 10 min in the presence of 20 µg/ml ConA (EL4 and splenic cells)
or for 5 min in the presence of 100 ng/ml steel factor (P815 cells).Generation of GST-Vav Fusion Proteins
Glutathione S-transferase (GST)-Vav fusion proteins were generated by
subcloning polymerase chain reaction-amplified murine Vav sequences
into pGEX2T. The amplified fragments (illustrated in Fig. 3A) subcloned into this expression plasmid include
the Vav SH2 domain alone (amino acids 670-765), the Vav SH2 and
C-terminal SH3 domains (amino acids 670-942), and the Vav
N-terminal SH3, SH2, and C-terminal SH3 domains (amino acids
611-942). GST-Vav expression plasmids were transfected into Escherichia coli, and the fusion proteins were purified from
isopropyl-1-thio-
-D-galactopyranoside-induced bacteria
with glutathione-conjugated Sepharose beads (Pharmacia, Baie
d'Urf 130, Quebec).
steel factor-stimulated P815 cells and incubated for 2 h at 4
°C with 5 µg of purified GST-fusion protein immobilized on
glutathione-Sepharose beads. Complexes as well as lysate (L)
alone (i.e. no GST-fusion protein added) were electrophoresed
through SDS-PAGE and subjected to immunoblotting with anti-PTP1C
antibody. Numbers at the top represent the GST-Vav
expression protein used in duplicate samples. Molecular size markers
are indicated on the right, and the position of PTP1C is shown
on the left.
Immunoblotting Analysis
Protein lysates were
prepared by resuspending 10
-10 resting or
mitogen-treated splenic, EL4, and P815 cells in 1 ml of lysis buffer
(phosphate-buffered saline containing 1% Triton X-100, 1% Tween, 1
mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, and 0.001 mM dithiothreitol). 100 µg of cell
lysate protein was electrophoresed through 10% SDS-polyacrylamide gels
and electroblotted onto nitrocellulose, and the blots then were
incubated overnight at 4 °C in 10 mM Tris, pH 8.0, 150
mM NaCl, and 0.05% Tween 20 (TBST) containing 5% skim milk.
The proteins were then detected by incubating blots for 2 h at room
temperature with primary antibodies in TBST followed by I-protein A (Dupont, Canada). Blots were then washed with
TBST and exposed to Kodak XAR film at -70 °C. For
immunoprecipitations, cell lysates were prepared from 10
resting or mitogen-treated splenic, EL4, and P815 cells, and the
lysates were clarified by centrifugation for 10 min at 10,000 
g at 4 °C. 500-2000 µg of cell lysate protein was
incubated for 2 h at 4 °C with selected antibodies and then with
100 µl of protein A-Sepharose (Pharmacia) for 10 min at 4 °C.
The immune complexes were collected by centrifugation, washed three
times with lysis buffer, boiled for 5 min in SDS-sample buffer, and
then subjected to electrophoresis and immunoblotting as described
above.In Vitro Binding Assays
To evaluate PTP1C binding
to GST-Vav fusion proteins, protein lysates prepared from 10 steel factor-treated P815 cell lysates were incubated at 4 °C
for 2 h with 5 µg of fusion protein immobilized on
glutathione-Sepharose beads. After several washes in lysis buffer,
complexes were resuspended in sample buffer, boiled, and analyzed by
SDS-PAGE and immunoblotting with anti-PTP1C antibody and I-protein A.
Assays of Phosphatase Activity
To assay PTP1C and
Vav-associated phosphatase activities, PTP1C and Vav were
immunoprecipitated as described above from 300, 600, or 900 µg of
cell lysate proteins prepared from 10-10 P815 cells incubated for 5 min in medium containing 100 ng/ml
steel factor. Immunoprecipitates were washed three times in lysis
buffer and then incubated at 37 °C for 4 h in 200 µl of
phosphatase buffer (62 mM Hepes, pH 7.5, 6.25 mM EDTA, 12.5 mM dithiothreitol) containing 2 mMp-nitrophenyl phosphate (Sigma). Reactions were
terminated by addition of 1 ml of 0.2 M NaOH, and absorbance
was measured at 410 nm. Alternatively, tyrosine phosphatase activity
was measured in Vav and Syp immunoprecipitates prepared from cell
lysates of 10 motheaten or congenic wild-type ConA (20
ng/ml)-treated splenic cells. For this assay, the immunoprecipitated
proteins were incubated at 37 °C for 2 h in 25 µl of 10 mM Tris HCl, pH 7.4, containing 7.5 nM tyrosine
phosphopeptide (RRLIEDAEpYAARG), and the reaction was terminated by
addition of Malachite Green solution (UBI) as described previously (36) . Phosphate release was measured after 15 min by
evaluating absorbance at 605 nm.
PTP1C Interacts with Vav in Resting P815
Cells
To investigate the possible association of PTP1C with Vav
in unstimulated hemopoietic cells, Vav immunoprecipitates prepared from
P815 mastocytoma cell lines and from me, me, and wild-type control splenic cells were
examined by immunoblotting analysis for the presence of PTP1C. As shown
in Fig. 1, the 70-kDa PTP1C protein was coprecipitated with
Vav from both P815 cells (Fig. 1A) and resting normal
murine splenocytes (Fig. 1C). This association was
detected both by immunoblotting anti-Vav immunoprecipitates with
anti-PTP1C antibody and, conversely, by immunoblotting anti-PTP1C
immunoprecipitates with anti-Vav antibody (Fig. 1A and B). Moreover, the capacity of PTP1C to interact with Vav in
P815 cells was observed to be diminished markedly by preincubation of
the P815 lysates with a synthetic peptide representing a 13-amino-acid
segment of the Vav protein (Fig. 1A). As anticipated,
no PTP1C was detected in Vav immunoprecipitates from me splenic cells (Fig. 1C) which have been shown to
lack PTP1C protein(9) . By contrast, while the phosphatase
domain mutations found in me
PTP1C proteins
severely reduce this enzyme's catalytic activity, these mutant
proteins retain the capacity to interact with Vav (Fig. 1C).
unstimulated P815 cells and 1500 µg of lysate protein, then
immunoprecipitated with anti-Vav antibody plus Vav peptide (VAV
+ PEP), anti-Vav antibody alone, or anti-PTP1C antibody.
Lysates were also prepared from 10 C57B1 +/+
splenic cells (lane 1) and 10 EL4 lymphoma cells (lane 2), and the lysate proteins (1000 µg) were
resolved on SDS-PAGE and blotted with anti-PTP1C antibody and
I-protein A. B, cell lysates were prepared from
10
unstimulated P815 cells, and 800 µg (lane
1) or 1500 µg (lane 2) of lysate protein were
immunoprecipitated using anti-PTP1C antibody (left panel) or
anti-Vav (right panel) antibodies. Lysates (500 µg)
prepared from P815 cells (L) were also resolved on SDS-PAGE
and immunoblotted with anti-PTP1C antibody and I-protein
A. C, cell lysates were prepared from unseparated splenic
cells of C3HeBFeJ +/+ and me/me and
C57B1/6J +/+ and me
/me mice, and the lysate proteins (1500 µg) were
immunoprecipitated with anti-Vav antibody, resolved on SDS-PAGE, and
blotted with anti-PTP1C antibody. Aliquots (1000 µg) of lysate
alone (far left) and lysate plus Sepharose (beads)
were also blotted with anti-PTP1C antibody. In all three panels, the
positions of molecular mass markers are shown on the right;
positions of PTP1C and Vav are indicated by arrows. A
nonspecific band between 50 and 60 kDa is visible in some lanes and
represents Ig heavy chain variably retained on the
beads.
67 and 70 kDa, respectively), only one of which (the
latter species) is detectable in P815 cells. The existence of PTP1C
isoforms has been observed previously in other hemopoietic cell
populations (9) and, based on sequence analysis of PTP1C
transcript, ascribed to the alternative splicing of a 39-amino-acid
segment within the PTP1C C-terminal SH2 domain(7) . The
functional significance of PTP1C SH2 domain variants is not known, but
the expression of only one PTP1C species in P815 cells is consistent
with previously reported data indicating the two species to be
expressed variably in different hemopoietic and epithelial
lineages(9, 37) . Moreover, based on the exclusive
detection of the higher molecular weight PTP1C species in Vav
immunoprecipitates from murine splenic cells (Fig. 1C),
it appears that Vav may selectively interact with this single PTP1C
variant. While further studies are required to address this issue, the
data shown here reveal the capacity of PTP1C to associate with Vav in
both mast and unseparated splenic cells and suggest that the PTP1C
sequences which mediate Vav binding in resting cells map to regions
flanking the site of the me
phosphatase domain
mutation.PTP1C Binding to Vav Increases following Mitogenic
Stimulation and Appears to Be Mediated through the Vav SH2
Domain
It has been previously reported that PTP1C association
with both the c-kit and IL-3 receptors is markedly increased
following receptor engagement(10, 11) . To determine
whether PTP1C binding to Vav is also increased following cell
stimulation, Vav immunoprecipitates were prepared from resting,
ConA-treated EL4 and steel factor-treated P815 cells, and the
coprecipitation of PTP1C was assessed by immunoblotting analysis. As
shown in Fig. 2A, stimulation of both EL4 and P815
cells induced marked increases in the association of PTP1C with Vav.
This result cannot be ascribed to ConA/steel factor-driven increases in
expression of these proteins, as levels of PTP1C and Vav were not
appreciably different in resting versus stimulated cells (data
not shown). By contrast, steel factor and ConA treatment induced marked
increases in Vav tyrosine phosphorylation in P815 (Fig. 2C) and EL4 (data not shown) cells, respectively.
PTP1C tyrosine phosphorylation was also increased in association with
stimulation of these cells (Fig. 2, B and D).
These findings are consistent with previous data revealing the
induction of Vav(22, 23, 24, 25, 26, 27) and
PTP1C (13, 38, 39) tyrosine phosphorylation
following stimulation of a variety of cell surface receptors and
suggest that the enhanced association of PTP1C with Vav in activated
cells is mediated through an SH2 domain-phosphotyrosine interaction.
B16 melanoma cells stably transfected
with either pCMV4Neo vector alone (far left lane) or pCMV4Neo
ligated to the full-length PTP1C cDNA (second lane from the
left), and the lysates and immunoprecipitated proteins were
resolved by SDS-PAGE and immunoblotting with anti-PTP1C antibody. B, cell lysates were prepared from unstimulated(-) and
Con A-treated (+) EL4 cells and 800 µg of lysate proteins were
immunoprecipitated with anti-PTP1C antibody, resolved over SDS-PAGE,
and immunoblotted with the 4G10 anti-phosphotyrosine (pY) antibody. C, cell lysate proteins (500 µg) prepared from
unstimulated(-) and steel factor-treated (+) P815 cells were
immunoprecipitated with anti-Vav (left panel) or
anti-phosphotyrosine (right panel) antibodies, resolved over
SDS-PAGE, and immunoblotted with anti-phosphotyrosine (left
panel) or anti-Vav (right panel) antibodies. As a control (C), Vav immunoprecipitates prepared from P815 cell lysates
(800 µg) were immunoblotted with anti-Vav antibody. D,
cell lysates were prepared from unstimulated(-) and steel
factor-treated (+) P815 cells, and 800 µg of lysate proteins
were immunoprecipitated with anti-PTP1C (left panel) or
anti-phosphotyrosine (right panel) antibodies, resolved over
SDS-PAGE, and immunoblotted with anti-phosphotyrosine (left
panel) or anti-PTP1C (right panel) antibodies. The
positions of molecular mass standards are indicated in all four panels; arrows indicate the positions of PTP1C and
Vav.
Detection of Vav-associated Tyrosine Phosphatase
Activity
To evaluate the potential biologic relevance of PTP1C
association with Vav, Vav was immunoprecipitated from steel
factor-treated P815 cells and the immune complexes were assessed for
associated phosphatase activity. As evaluated by the dephosphorylation
of p-nitrophenol phosphate substrate, phosphatase activity was
clearly detected in these immunoprecipitates, the levels of activity
increasing linearly in proportion to the amount of cell lysate protein (Fig. 4A). To evaluate the extent to which PTP1C
contributes to Vav-associated phosphatase activity, this experiment was
repeated using ConA-treated splenic cells from me mice. As
shown in Fig. 4A, the level of Vav-coprecipitated
phosphatase activity in these PTP1C-deficient cells was negligible and
unaltered by the use of increasing amounts of cell lysate protein.
These findings suggest that PTP1C accounts for the majority of
Vav-associated phosphatase activity detected in steel factor-treated
P815 cells and thus imply that Vav or Vav-associated signaling
molecules may represent targets for PTP1C-induced tyrosine
dephosphorylation.
ConA-stimulated C3HeBFeJ wild-type (C3H) and me (me/me)
and C57BL/6J wild-type (C57B) and me (me/me) splenic cells, and the lysate
proteins (800 µg) were immunoprecipitated with anti-Vav antibody.
The immunoprecipitates were incubated at 37 °C for 2 h with a
synthetic tyrosine-phosphorylated peptide as described under
``Materials and Methods,'' and the reaction was terminated by
addition of Malachite Green. The amount of phosphate released was
determined spectrophotometrically by measuring absorbance at 605 nm.
The results shown are representative of two independent experiments,
and the bars indicate standard deviations for a single
experiment performed in duplicate.
splenic
cells were also assessed for their capacity to dephosphorylate a
tyrosine-phosphorylated synthetic peptide. As is consistent with the
contention that PTP1C accounts for the majority of Vav-associated
phosphatase activity, levels of tyrosine phosphatase activity detected
in Vav immunoprecipitates from me and me splenic cells were dramatically reduced relative to those
observed in splenic cell Vav immunoprecipitates from congenic wild-type
mice (Fig. 4B). By contrast, Syp-precipitable tyrosine
phosphatase activity was essentially the same in me and wild-type splenic cells (data not shown). Together, these
data indicate the association of Vav with tyrosine phosphatase activity
and strongly suggest that this activity is engendered by PTP1C. Based
on the relative levels of phosphatase activity contained in Vav versus PTP1C immunoprecipitates from stimulated P815 cells (Fig. 4A), it also appears that a considerable
proportion of cellular PTP1C activity is associated with Vav, an
observation which is consistent with previous data indicating that
PTP1C-ligand binding substantially enhances PTP1C catalytic
function(42) . By inference, these findings are highly
suggestive of a critical role for PTP1C in modulating the signal
transducing functions of Vav and/or Vav-associated proteins.Association of PTP1C with Grb2 and mSos1 in P815
Cells
While the precise relationship between Ras activation and
Vav-mediated transformation remains unclear, recent data suggest that
Ras proteins and Vav cooperate in a synergistic fashion to induce
cellular transformation(34) . The potential relevance of Vav to
Ras-related signaling events has also been suggested by the observed
capacity of Vav to associate physically with the Grb2
protein(43) , a ubiquitously expressed molecule known to
interact via its SH3 domains with proline-rich motifs in the Ras GEF
mSos1 and via its SH2 domain to phosphotyrosine residues in activated
growth factor receptors(44, 45, 46) . In view
of these findings and the current data revealing the association of
PTP1C with Vav, we next explored the possibility that PTP1C might also
interact with the Grb2-mSos1 complex in hemopoietic cells. To this end,
Grb2 and mSos1 or, alternatively, PTP1C immunoprecipitates were
prepared from resting and steel factor-treated P815 cells and subjected
to immunoblotting analysis with anti-PTP1C or anti-Grb2/mSos1
antibodies, respectively. As shown in Fig. 5, the results of
this analysis reveal the capacity of PTP1C to bind to both Grb2 (Fig. 5A) and mSos1 (Fig. 5B) in
unstimulated cells. As the interaction of Grb2 with Vav has also been
detected in unstimulated T cells(43) , it appears that PTP1C,
Vav, and the Grb2-mSos1 molecules may associate with one another as a
multimeric complex in resting hemopoietic cells. PTP1C interactions
with Grb2 and mSos1 were also examined in steel factor-treated cells,
and, as observed with respect to PTP1C binding to Vav, interactions
between this phosphatase and both Grb2 and mSos1 were increased,
following cell activation (Fig. 5C). These findings are
again consistent with the contention that PTP1C and Vav interactions
modulate receptor-evoked mitogenic signaling cascades and suggest that
this modulatory effect also involves the association of these proteins
with Grb2-mSos1. While the structural basis for the amalgamation of
these four signaling effectors requires further definition, recent data
revealing the capacity of the Grb2 carboxyl-terminal SH3 domain to
physically associate with the Vav amino-terminal SH3 domain (47) together with data implicating the Grb2 amino-terminal SH3
domain in mSos1 binding (45) provide some indication as to the
mode of Grb2-mSos1 association with PTP1C-Vav complexes. In addition,
the localization of a major site for PTP1C tyrosine phosphorylation
(Tyr-538) within a sequence (pYGNI) representing a consensus sequence
(pYXNX) for Grb2 SH2 domain binding (38, 39, 48) suggests a role for Grb2 SH2
domain interaction with PTP1C phosphotyrosine in the genesis of this
multimeric complex. In this context, PTP1C may act as an adaptor
linking activated receptors to Grb2-mSos1, a role already demonstrated
for the ubiquitously expressed Syp PTP in relation to the c-kit and platelet-derived growth factor
receptors(49, 50) . Whether or not PTP1C plays this
latter role in relation to Grb2-mSos1 and Vav, the interaction of this
phosphatase with these specific cytosolic signaling molecules suggests
a critical influence of this phosphatase on the signaling cascades
linking hemopoietic cell surface receptors to Ras activation.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
