(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) ()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 Fc
RIIB1 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.
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 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.
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 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.
Figure 1:
Association of PTP1C with Vav in
hemopoietic cells. A, cell lysates were prepared from 10 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.
The data shown in Fig. 1A also reveal isolated murine splenocytes to express two PTP1C
species (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.
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 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.
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 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.
To extend these data, Vav immunoprecipitates from
ConA-treated normal me and me 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.
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.