From the Division of Hematology, University of Washington School of Medicine, Seattle, Washington 98195
Received for publication, March 28, 2000, and in revised form, September 8, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thrombopoietin (TPO) is a recently
characterized member of the hematopoietic growth factor family that
serves as the primary regulator of megakaryocyte (MK) and platelet
production. The hormone acts by binding to the Mpl receptor, the
product of the cellular proto-oncogene c-mpl. Although many
downstream signaling targets of TPO have been identified in cell lines,
primary MKs, and platelets, the molecular mechanism(s) by which many of
these molecules are activated remains uncertain. In this report we
demonstrate that the TPO-induced activation of phosphoinositol 3-kinase
(PI3K), a signaling intermediate vital for cellular survival and
proliferation, occurs through its association with inducible signaling
complexes in both BaF3 cells engineered to express Mpl (BaF3/Mpl) and
in primary murine MKs. Although a direct association between PI3K and
Mpl could not be demonstrated, we found that several proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and association in BaF3/Mpl cells in response to TPO stimulation, complexes that recruit and enhance the enzymatic activity of PI3K. To verify the
physiological relevance of the complex, SHP2-Gab2 association was
disrupted by overexpressing a dominant negative SHP2 construct. TPO-induced Akt phosphorylation was significantly decreased in transfected cells suggesting an important role of SHP2 in the complex
to enhance PI3K activity. In primary murine MKs, TPO also induced
phosphorylation of SHP2, its association with p85 and enhanced PI3K
activity, but in contrast to the results in cell lines, neither Gab2
nor IRS2 are phosphorylated in MKs. Instead, a 100-kDa
tyrosine-phosphorylated protein (pp100) co-immunoprecipitated with the
regulatory subunit of PI3K. These findings support a model where PI3K
activity is dependent on its recruitment into TPO-induced
multiphosphoprotein complexes, implicate the existence of a scaffolding
protein in primary MKs distinct from the known Gab and IRS proteins,
and suggest that, in contrast to erythroid progenitor cells that employ
Gab1 in PI3K signaling complexes, utilization of an alternate member of
the Gab/IRS family could be responsible for specificity in TPO signaling.
The regulation of platelet production is a complex process.
Maintained within relatively normal limits, both reduced and excessive platelet production leads to pathologic bleeding or thrombosis. Much is
known of the cell biology of this process. Bone marrow megakaryocytes
(MKs)1 are the immediate
cellular precursors of blood platelets, and a hierarchy of stem cells,
multipotent progenitors, and progenitors committed to the MK lineage
are responsible for continuously renewing the marrow pool of MKs (1).
Recently, the primary regulator of this developmental pathway,
thrombopoietin (TPO), and its cellular receptor, the product of the
proto-oncogene c-mpl, were molecularly cloned and
characterized (2). In a number of cell culture systems TPO has been
shown to affect all aspects of MK development, supporting the survival,
proliferation, and differentiation of cells from the hematopoietic stem
cell to the mature MK (3-7). In keeping with these in vitro
findings, the genetic elimination of tpo or c-mpl
leads to greatly reduced levels of hematopoietic stem cells, multipotent and committed progenitors, mature MKs, and blood platelets (8-10), indicating that TPO is a general regulator of hematopoiesis and the major humoral factor controlling MK and platelet production in vivo.
The intracellular signaling pathways utilized by c-Mpl have been
extensively studied by many groups of investigators. After stimulation
with TPO the Mpl receptor is believed to change conformation resulting
in a homodimeric receptor complex capable of supporting the
transphosphorylation and activation of two tethered Janus kinases, JAK2
and TYK2 (11-14). However, only JAK2 appears to be essential for
subsequent signaling events (15). In contrast to these reports, one
series of experiments has called the importance of JAK2 activation into
question (16). Nevertheless, once activated, Janus kinases
phosphorylate a number of substrates, including the Mpl receptor itself
(providing a docking site for SH2-containing proteins), the latent
transcription factors signal transducers and activators of
transcription (STAT) 3 and STAT5, and a number of adapter proteins
including Shc and SHP2. Other signaling intermediates have also been
found to be activated by TPO in various cell lines including
mitogen-activated protein kinase (MAPK; see Refs. 17 and 18), protein
kinase C (19, 20), and PI3K (16, 21, 22), molecules that play vital
roles in MK development. For example, in previous studies we have shown
that MAPK is activated in primary MK and is essential for nuclear
endomitosis (18), a characteristic feature of MK development, and that
the protein kinase C Much recent work has focused on the role of PI3K in supporting cell
survival. One of the best studied mechanisms by which PI3K prevents
programmed cell death is mediated by its activation of Akt, leading to
phosphorylation and sequestration of the apoptosis-promoting Bcl
family member Bad (26). Many investigators have reported that several
hematopoietic cytokines, including interleukin (IL)-3 (26, 27),
erythropoietin (EPO; see Refs. 28 and 29), and IL-6 (30) can activate
PI3K and Akt. However, PI3K has also been shown to mediate several
other cellular events, including proliferation (31, 32). In addition to
its effects on Akt, PI3K can affect other signaling events, including
the Ras/Raf/MEK/MAPK and GSK-3 The mechanism of PI3K activation has been extensively studied in
several cytokine-responsive cell lines. For some receptors that induce
PI3K activation, such as the EPO, platelet-derived growth factor, or
Flt3 receptors, a 4-residue site of the form pYXXM or pYVAC
(where pY is phosphotyrosine) has been described to bind directly the
regulatory subunit of PI3K (33-35). Neither of these sites exists
within the cytoplasmic domain of the Mpl receptor. However, in other
receptors, complexes of p85 with scaffolding/adapter proteins have been
identified to induce PI3K activation by conformational changes in p85
and by bringing the catalytic subunit of PI3K, p110, to the cell
membrane in close proximity to its phospholipid substrates (36). This
adapter mechanism has also been studied in normal erythroid progenitor
cells, in which Gab1 has been identified to play a scaffolding role in
PI3K activation (37). However, although PI3K has been shown to be
activated by TPO in several cell lines (21, 36, 38), its activation has
never been reported in primary MKs, and the TPO-induced signaling
pathways leading to SHP2 and PI3K are still to be determined in cells
of this lineage. Therefore, in the present study the role of
scaffolding/adapter molecules in the TPO-induced activation of PI3K has
been explored using both BaF3/Mpl cells and primary murine MKs.
Insulin receptor substrates (IRS) and their related proteins,
Grb2-associated binders (Gabs), are
involved in many cytokine-signaling events (37, 39). Gab/IRS proteins
have a pleckstrin homology domain responsible for recruitment to the
plasma membrane and several protein-protein docking motifs that when
phosphorylated allow the assembly of multiprotein signaling complexes.
At least five IRS and three Gab adapters have been reported, but
additional members are likely to be identified. Included among the
signaling intermediates that associate with Gab/IRS proteins are the
p85 subunits of PI3K, SHP2, Grb-2, and Crk. However, the role(s) of Gab/IRS proteins in megakaryopoiesis have not yet been described. As
engagement of TPO by its receptor has been shown to activate a wide
variety of signaling pathways, we tested whether one or more of these
proteins is involved in TPO signaling in MKs. We found that several
proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and
association in BaF3/Mpl cells following exposure to TPO, complexes that
recruit and enhance the enzymatic activity of PI3K. Furthermore, SHP2
was found to be important for full PI3K activation suggesting the role
of Gab/IRS proteins in recruiting SHP2 and PI3K into the same complex.
In primary murine MKs, TPO also induced phosphorylation of SHP2, its
association with p85, and enhanced associated PI3K activity, but in
contrast to the results in cell lines or in erythroid progenitor cells,
none of the known Gab or IRS proteins were phosphorylated in MKs.
Instead, a 100-kDa tyrosine-phosphorylated protein (pp100)
co-immunoprecipitated with the regulatory subunit of PI3K. These data
provide further insights into the molecular mechanisms of
megakaryopoiesis by identifying several molecules upstream of PI3K
employed during TPO signaling.
Reagents--
Purified recombinant murine TPO was a generous
gift of Dr. Akihiro Shimosaka (Kirin Pharmaceuticals, Tokyo, Japan),
and human TPO was kindly provided by Dr. Donald Foster (ZymoGenetics,
Inc., Seattle, WA). Anti-IRS1 and anti-Grb2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IRS2, anti-SHIP, anti-PI3K (p85 subunit), and anti-phosphotyrosine (4G10) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). A rabbit
polyclonal anti-c-Mpl antiserum was the generous gift of Dr. Donald
Foster (ZymoGenetics), and the rat AMM2 monoclonal antibody against
murine c-Mpl was the kind gift of Dr. Takashi Kato (Kirin). Dr. Toshio
Hirano (Osaka, Japan) generously provided rabbit anti-Gab1 and
anti-Gab2 antibodies, and Dr. Larry Rohrschneider kindly provided the
anti-Gab3 antibody. Anti-phospho-Akt (Ser473)
antibody was obtained from New England Biolabs (Beverly, MA). Western
blot chemiluminescence reagents were purchased from PerkinElmer Life
Sciences, and all other reagents were purchased from Sigma.
Cell Lines and Cell Culture Conditions--
The murine
IL-3-dependent cell line BaF3 was engineered to express the
murine Mpl receptor (BaF3/Mpl; see Ref. 40) and was maintained in RPMI
1640 medium (BioWhittaker, Walkersville, MD) with 10% heat-inactivated
fetal calf serum (HyClone, Logan, UT) and murine IL-3 (0.2% (v/v)
conditioned medium from baby hamster kidney cells engineered to secrete
mIL-3). The human leukemic cell line UT-7/TPO (kindly provided by Dr.
Norio Komatsu) was maintained in Iscove's modified Dulbecco's medium
(IMDM, Sigma) with 10% fetal calf serum and 5 ng/ml human TPO. These
cell lines were serum- and growth factor-deprived by incubation
overnight in culture medium supplemented only with 0.5% bovine serum
albumin (BSA). To obtain primary murine MKs, BDF-1 mice (Jackson
Laboratories, Bar Harbor, ME) were subcutaneously injected with 2 µg
of human TPO daily for 5 days, and bone marrow cells were obtained by
flushing and were cultured for 3 days in IMDM supplemented with 1%
Nutridoma SP (Roche Molecular Biochemicals) and 250 ng/ml human TPO.
The cultured mature MKs were purified by unit gravity sedimentation on
a discontinuous BSA density gradient as described previously (41). The
purity of the collected cells was greater than 90% MKs, which were
identified by acetylcholinesterase staining. MKs were starved in IMDM
with 1% Nutridoma without cytokines for 7 h prior to signaling studies.
Immunoprecipitation and Western Blot Analysis--
Serum- and
cytokine-starved BaF3/Mpl cells and MKs were stimulated with 25 ng/ml
murine TPO for 10 min, washed once with ice-cold phosphate-buffered
saline (PBS), and lysed in a buffer composed of 20 mM
Tris-HCl, pH 8.0, 137 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, 10% glycerol, 100 mM NaF, and 0.5% Nonidet P-40. The protein concentration of lysates was measured by Protein/DC assay (Bio-Rad). Specific proteins were immunoprecipitated from cell lysates by overnight incubation at 4 °C with the indicated antibodies. Protein
A/G-conjugated agarose beads (Santa Cruz Biotechnology) were then added
and incubated for 2 additional hours at 4 °C. The pelleted beads
were then washed 3 times with lysis buffer, resuspended in gel
electrophoresis loading buffer, and heated to 90 °C for 5 min. The
immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis and Western blot analysis, as described previously
(11).
Phosphoinositol 3-Kinase Assay--
BaF3/Mpl cells were
incubated overnight in RPMI 1640 medium with 0.5% BSA and purified MKs
in 1% Nutridoma in IMDM for 7 h. The starved cells were
stimulated with 25 ng/ml murine TPO for 10 min, lysed, and
immunoprecipitated as detailed above. Two hours after adding protein A
or protein A/G-agarose beads, the immune complexes were washed three
times with the lysis buffer, three times with 0.1 M
Tris-HCl, pH 7.4, 5 mM LiCl, and 0.1 mM sodium orthovanadate, and twice with TNE buffer (10 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate). After the final wash, 50 µl of
TNE, 10 µl of 2 mg/ml sonicated phosphatidylinositol in 10 mM Tris-HCl with 1 mM EGTA, 10 µl of 0.1 M MgCl2, and 5 µl of ATP mix (0.88 mM ATP containing 30 µCi of [32P]ATP and 20 mM MgCl2) were added sequentially. The kinase
reaction mix was incubated at 37 °C for 10 min before stopping the
reaction by adding 20 µl of 6 N HCl. The radiolabeled
lipid was then extracted with 160 µl of 1:1
CHCl3:methanol and centrifuged for 10 min. Fifty µl of
the lower organic phase was spotted on a silicon TLC plate (EM Science,
Gibbstown, NJ) pretreated with 1% potassium oxalate and separated by
chromatography in
CHCl3:methanol:H2O:NH4OH (120:94:22.6:4) for 3 h. The plates were dried and visualized by
autoradiography and quantified by PhosphorImager (Molecular Dynamics,
Sunnydale, CA).
Dominant Negative SHP2 Experiments--
We obtained a dominant
negative SHP2 (DN SHP2) construct consisting of the pair of SH2 domains
of SHP2 (residues 1-216) in a eukaryotic expression vector, pCAGGS (a
generous gift from Dr. Hiroshi Maegawa, Shiga, Japan). Ten µg of DN
SHP2 plasmid was co-transfected with 1 µg of the pMX-puro plasmid (a
gift from Dr. Toshio Kitamura) into BaF3/Mpl cells by electroporation.
A control culture was transfected with pMX-puro plasmid alone.
Puromycin at a final concentration of 1 µg/ml was added 24 h
after transfection. Expression of the dominant negative protein was
determined by Western blot analysis of whole cell lysates probed with a
monoclonal antibody to the N terminus of SHP2 (Transduction
Laboratories, San Diego, CA). The experiments were performed in 2 pools
of cells from two separate transfections. Cells from the first
co-transfection were also plated at limiting dilution, and clones that
do and do not express DN SHP2 by Western blot analysis were obtained to
assess the effects of DN SHP2 on PI3K activation and complex formation.
Thrombopoietin Activates PI3K in BaF3/Mpl Cells and Murine
MKs--
Previous studies (18) in our laboratory revealed that TPO is
a potent stimulus of MAPK activation. As part of that work we found
that the PI3K inhibitors wortmannin and Ly294002 decreased MAPK
activation significantly, suggesting that PI3K was also activated in
cells exposed to TPO and that one of the downstream targets of PI3K was
MAPK. To explore more formally the activation of PI3K in response to
TPO, we conducted Western blot analyses to detect phosphorylation of
Akt, a known downstream target of PI3K activation, and we performed
direct PI3K functional assays on BaF3/Mpl cells and purified murine
MKs. We found that TPO induced the activation of Akt in both cell types
(Fig. 1) but not in the presence of the
PI3K inhibitor Ly294002, suggesting that PI3K is activated and acts on
PDK2, one of the immediate activating kinases for Akt. We also found
that total cellular PI3K activity was higher in TPO-stimulated BaF3/Mpl
cells than in control cells. The average PI3K activity of
TPO-stimulated cells was modestly increased, 120 ± 5.0% that of
unstimulated cells, as determined by PhosphorImager analysis of four
separate experiments and was higher than unstimulated cells in each
experiment performed. However, the amount of PI3K activity present in
membrane-localized signaling complexes was markedly increased by TPO
(see below).
Thrombopoietin Induces the Association of PI3K with Several
Phosphotyrosine-containing Proteins in Mpl-bearing Cell Lines--
To
begin to identify the molecular mechanism(s) of TPO-induced PI3K
activation, BaF3/Mpl cells were starved overnight and stimulated with
TPO for 10 min, and p85 PI3K immunoprecipitates were prepared and
size-fractionated, blotted, and probed with an anti-phosphotyrosine
(4G10) antibody. Prior to stimulation with TPO, two
phosphotyrosine-containing proteins of ~100 kDa were detected (Fig.
2). In the presence of TPO
tyrosine-phosphorylated proteins of ~170, 145, 116, 110, 100, and 70 kDa were detected in BaF3/Mpl cells, although the ~100-kDa protein
was dominant.
Since the molecular mass of members of the Gab family of
adapters is ~95-110 kDa, proteins known to associate with PI3K
following stimulation of the T cell and B cell receptors, IL-6 and EPO, we studied the expression of Gab proteins in BaF3/Mpl cells and primary
murine MKs. We found that Gab1, Gab2, and Gab3 were all expressed in
BaF3/Mpl cells, but only Gab1 and Gab3 were present in MKs (Fig.
3, A and C). In
addition to Gab proteins, members of the IRS family also play a
scaffolding role in many cell types. For example, IRS2 has been shown
to modulate PI3K activation following stimulation of erythroid cells
infected with Friend spleen focus-forming virus, which engages
the EPO receptor (42), and in a hematopoietic cell line expressing the
EPO receptor (43). Moreover, the molecular weight of the largest
phosphotyrosine-containing proteins that co-immunoprecipitated with p85
PI3K (~170 kDa) matches that reported for IRS1 and IRS2. Thus,
although IRS proteins have not been described as mediating TPO
signaling, we also investigated the expression of IRS1 and IRS2 in
BaF3/Mpl cells and MKs. BaF3/Mpl cells were found to express IRS2 but
not IRS1, and MKs displayed neither IRS1 nor IRS2 (Fig. 3, D
and E).
The formation of multiprotein signaling complexes in response to
hematopoietic cytokine receptor stimulation is dependent on the
phosphorylation of many of the constituent proteins. Thus, we next
assessed whether TPO induced tyrosine phosphorylation of several
adapters and signaling mediators and whether they associated with PI3K.
As shown in Fig. 4A, Gab2 is
prominently tyrosine-phosphorylated in BaF3/Mpl cells stimulated with
TPO. Although BaF3/Mpl cells express small amounts of Gab1 and Gab3,
they were not tyrosine-phosphorylated following exposure to TPO (data
not shown). In addition, a 70-kDa phosphoprotein co-immunoprecipitated
with Gab2 in these cells when stimulated with TPO; reprobing of the
same immunoblot revealed it to be SHP2 (Fig. 4B). The
reciprocal experiment, immunoprecipitation with anti-SHP2, and probing
for Gab2 confirmed the strong association of the two phosphoproteins
(Fig. 4, E and F). Gab2 was also found to be
associated with p85 (Fig. 4C). Although there are several reports describing the association of JAK2, Grb2, and SHIP with SHP2
and Gab proteins, none of these proteins were detected in our
immunoprecipitates of SHP2 and Gab2 (data not shown).
As noted above, we found that BaF3/Mpl cells express IRS-2 but not
IRS-1 (Fig. 3, D and E). Like Gab2,
phosphorylation of IRS2 was induced by TPO stimulation of BaF3/Mpl
cells (Fig. 5A). In addition,
IRS2 associated with p85 PI3K (Fig. 5B) upon exposure of
cells to TPO but not with SHP2 (data not shown). Moreover, IRS2 was
found to associate constitutively with the Mpl receptor (Fig.
5D). Similar results were found in the human megakaryocytic cell lines, UT-7/TPO, following stimulation with TPO (data not shown).
Thus, TPO induces the assembly of at least two multiphosphoprotein complexes in Mpl-bearing cell lines, one composed of Mpl, IRS2, and p85
PI3K and the other containing SHP2, Gab2, and p85 PI3K. We failed to
detect reproducibly an association of p85 PI3K and Mpl by
immunoprecipitation analysis.
PI3K Is Activated in TPO-induced Multiprotein
Complexes--
Previous work has shown that binding of the SH2 domain
of p85 PI3K with a phosphotyrosine-containing scaffolding protein can activate PI3K. Two non-mutually exclusive mechanisms for this event
have been proposed; one hypothesizes a Gab/IRS-induced conformational change in p85 allowing its activation of the p110 catalytic subunit, the other posits that the recruitment of PI3K to the membrane by the
pleckstrin homology domain containing scaffolding proteins brings it in
proximity of activated Ras, activating the p110 catalytic subunit, and
in contact with its lipid substrates. Consistent with the involvement
of this latter mechanism, TPO has been shown to activate Shc (11, 13),
an adapter protein linked to Ras activation, suggesting that the
multiphosphoprotein complex formed upon stimulation of BaF3/Mpl cells
with TPO is an important mechanism of PI3K activation. To test whether
the complexes that form in TPO -stimulated BaF3/Mpl cells were an
important mechanism of PI3K activation, the capacity to phosphorylate
phosphoinositol was measured in the multiprotein complexes formed in
TPO-treated BaF3/Mpl cells. BaF3/Mpl cells were stimulated with the
hormone for 10 min and lysed, and Gab2 or IRS2 immunoprecipitates were subjected to a direct functional kinase assay based on
[ SHP2 in the Complex Is Important for PI3K Activation--
To
investigate the physiological significance of SHP2-based complex
formation in BaF3 cells, a dominant negative SHP2 (DN SHP2) construct
was used to prevent SHP2-Gab2 association. The construct comprises the
N-terminal SH2 domains of SHP2 that theoretically can compete with the
endogenous wild type SHP2 to form the SHP2-Gab2-p85 complex.
This construct was shown to inhibit SHP2/IRS1 association in Rat 1 fibroblasts (44). BaF3/Mpl cells were co-transfected with DN SHP2 and
the pMX-puro plasmid as a selectable marker. Transfectants were then
selected to obtain stable cloned cell lines. Both nonexpressing (clone
1) and the highly expressing clones (clone 2) were chosen for
further studies (Fig. 7). TPO-induced Akt
phosphorylation was reduced in the DN SHP2 expressing BaF3/Mpl clone
(Fig. 7B) correlating with the markedly diminished
TPO-induced SHP2/Gab2 association. These data suggest that SHP2 in the
complex plays an important role in PI3K activation. This result is not caused by the variation between clones because similar results were
found in 2 pools of uncloned cells from two separate transfections. Compared with cells transfected with pMX-puro alone, BaF3/Mpl cell
pools expressing DN SHP2 consistently have decreased Akt phosphorylation after TPO stimulation (data not shown).
PI3K Is Recruited to a Multiphosphoprotein Complex and Is Activated
by TPO in MKs--
To determine the physiologic relevance of the
TPO-induced signaling complexes identified in BaF3/Mpl cells, we
extended our study to primary cells. Unstimulated purified mature
murine MKs were tested for the presence of Gab/IRS family members by
Western blotting; we found that mature murine MKs express Gab1 and Gab3 but not Gab2 (Fig. 3, A-C), although the latter was
detectable by reverse transcriptase-polymerase chain reaction (data not
shown). Neither IRS1 nor IRS2 was detected by Western blotting in MKs (Fig. 3, D and E). Next, freshly isolated
purified MKs were starved of serum and growth factors for 7 h,
stimulated with TPO for 10 min, and protein lysates prepared. As shown
in Fig. 8A, TPO induced tyrosine phosphorylation of SHP2 and its association with p85 PI3K in
MKs (Fig. 8C). To identify whether known Gab proteins were
involved in TPO signaling in MKs, the isoforms of Gab present in MKs
(Gab1 and Gab3) were immunoprecipitated from TPO-stimulated MK lysates
and probed for phosphotyrosine-containing proteins. We failed to find
P-Gab1 or P-Gab3 in MKs (Fig. 8, D and F),
despite probing lysates equivalent to all the cultured MKs from up to five mice for each experimental condition (Fig. 8) and finding clear
phosphorylation of Gab1 in M07e cells stimulated with stem cell
factor or granulocyte-macrophage colony-stimulating factor (data not
shown). However, when a p85 PI3K immunoprecipitate from TPO-stimulated
MKs was probed for phosphotyrosine-containing proteins, a 100-kDa
TPO-induced phosphoprotein (pp100) was detected (Fig. 8H).
It is unlikely that pp100 represents one of the known IRS proteins, as
the molecular mass of IRS-1, IRS-2, and IRS-4 are greater than 160 kDa
and that of IRS3 is 60 kDa. In contrast, its electrophoretic mobility
suggests that pp100 may be related to the Gab family of adapters.
Since PI3K was associated with SHP2 and pp100 after stimulation with
TPO in primary MKs, we studied whether the formation of this complex
enhances PI3K kinase activity. Since pp100 is an unknown protein, no
antibody is available for direct immunoprecipitation analysis. As pp100
is the main tyrosine-phosphorylated protein associated with p85 PI3K,
we immunoprecipitated total tyrosine-phosphorylated protein with the
4G10 antibody and assayed for PI3K activity with the
[ The new findings in this paper are that TPO binding to Mpl
triggers the following: 1) the association of p85 PI3K with two different adapter complexes in BaF3/Mpl cells, one composed of p85, the
SHP2 phosphatase, and Gab2, and the other composed of p85 and IRS2. 2)
SHP2 in the complex is important for PI3K activation. 3) A related
signaling complex forms in primary MKs, also involving SHP2 and p85,
but the adapter appears to be an ~100-kDa phosphoprotein distinct
from any of the known Gab or IRS proteins. 4) Each of these complexes
enhances the enzymatic activity of the associated PI3K lending
physiologic relevance to the findings. 5) This pattern of PI3K
activation is distinct from that seen in response to EPO, suggesting
that at least some of the signaling events downstream of EPO and TPO
may differ. We have also shown that Gab3 is present in both BaF3 cells
and MKs but is not involved in TPO signaling. Although previous work
has shown that TPO can activate PI3K in cell lines expressing the Mpl
receptor, the present results extend this finding to primary cells and
provide a molecular basis for the observation.
When engaged by ligand, growth factor receptors typically activate
numerous signaling pathways; the aggregate of signals generated from
activation of these pathways is critical for determining overall
cellular response. In previously reported work, we and others have
found that TPO stimulation of Mpl receptor bearing cell lines and
platelets leads to activation of PI3K. In the present work we extend
these findings to mature MKs. Several studies have also revealed a
similar activation of PI3K in erythroid cell lines and primary cells in
response to EPO, in which a direct association of the regulatory
subunit of the enzyme and the EPO receptor were readily demonstrated
(34). In contrast, we were unable to detect reproducibly a direct
association of Mpl and the p85 subunit of PI3K by Western blotting.
Although it is possible this negative result was due to interference of
the immunoprecipitating Mpl antibody with complex formation, or to the
transient nature of the association, the result also pushed us to seek
another mode of PI3K activation by Mpl.
By recruiting adapter proteins such as members of the Gab/IRS family to
the activated receptor complex, the biological effects of cytokine
receptor engagement can be amplified and diversified. For example,
SHP2, a phosphatase important for modulating growth factor signaling in
response to a variety of extracellular signals (45-46), possesses two
tandem repeats of SH2 domains at the N terminus. It has been proposed
that simultaneous binding of both SH2 domains is required for full
activation (47). Gab proteins contain two tyrosine residues with
putative SHP2-binding motifs near the C terminus of the molecule,
compared with a single site in activated Mpl. Thus, Gab recruitment can
serve as a more efficient means by which SHP2 can become activated
following stimulation of the Mpl receptor. In this study we have found
that TPO activates PI3K in BaF3/Mpl cells by generating a complex
composed of the p85 regulatory subunit of PI3K and phosphorylated SHP2
and Gab2 or of p85 and IRS2, presumably activating the kinase by
conformational changes and recruitment to the membrane allowing its
interaction with Ras. Although it appears that P-Gab2 is quantitatively
the more important scaffolding protein, as far more P-Gab2 associates with p85 PI3K than does P-IRS2, this conclusion requires that the
affinity of the 4G10 antibody for P-Gab2 and P-IRS2 is equivalent, and
both proteins contain equivalent numbers of sites of phosphorylation, and the antibody used to precipitate IRS2 does not interfere with its
association with p85. In fact, although not rigorously quantitative, our functional kinase analyses (Fig. 6) reveal nearly equivalent intensities of 32P incorporation into PI by Gab2 and IRS2
immunoprecipitates derived from identical numbers of cells, suggesting
both complexes are important for TPO-induced PI3K activation in these cells.
In contrast to our results in BaF3/Mpl cells, the composition of the
PI3K signaling complex responding to TPO in primary MKs differs
somewhat, apparently utilizing a distinct 100-kDa scaffolding protein
(Fig. 8H) along with SHP2. These results point out the importance of performing studies in primary cells, rather than relying
entirely on conclusions derived from transformed cell lines. The
relevance of the complex was tested in a functional PI3K assay; both
Tyr(P) and P-SHP2 immunoprecipitates derived from TPO-stimulated
MKs displayed enhanced activity in this assay (Fig. 9). Moreover, taken
together, our results raise the issue of what determines the
specificity of different hematopoietic cytokine receptors for the ever
increasing number of adapter proteins. For example, several cytokines
were previously shown to employ Gab1 to propagate cytokine signaling,
including IL-3, the T and B cell receptors, gp130-linked cytokines, and
epidermal growth factor (36, 48). However, with the cloning of Gab2,
many of these same cytokines were found to also utilize Gab2,
suggesting there was little specificity in adapter protein utilization.
In contrast, although insulin and insulin-like growth factors recruit both IRS1 and IRS2 into signaling complexes, leptin pretreatment of
cells enhances IRS1 association with PI3K but inhibits complex formation of IRS2 and PI3K (49), suggesting some specificity in adapter
utilization. Here, we provide further evidence that different cytokines
can employ different adapters. The EPO receptor utilizes Gab1 and Gab2
in the HCD57 cell line and Gab1 in primary erythroid progenitors (37).
We found that Gab2 and IRS2 are tyrosine- phosphorylated and associate
with p85 PI3K in response to TPO in BaF3/Mpl cells, but despite the
presence of Gab1 in MKs, an immunologically distinct 100-kDa protein is
phosphorylated and associates with p85 in these primary cells. Thus,
the EPO and TPO receptors appear to utilize distinct adapter proteins in their respective primary cells, despite the common use of JAK2 by
both receptors for signal initiation. These findings suggest that
another factor, in addition to JAK, is probably required for specific
patterns of Gab/IRS protein phosphorylation. Clearly, additional work
will be required to determine the nature of these modifying signals and
the identity of the ~100-kDa phosphoprotein that associates with SHP2
and p85 in primary MKs.
Although we have shown that the TPO and EPO receptors employ different
adapters in primary cells, the importance of the finding is dependent
on whether or not each Gab/IRS protein gives rise to a unique set of
signals. If so, then the differential use of adapters could provide a
mechanism for specificity in receptor signaling. IRS proteins contain a
phosphotyrosine binding domain and a larger number of putative
phosphotyrosine motifs, which are different from Gabs. The recruitment
of SHP2 stands as an excellent example of the potential for
differential signaling; the phosphatase has been shown to associate
with Gabs, a finding confirmed for the Mpl receptor in our study (Fig.
4, B and F), but does not interact with IRS-2 in
our studies or in those of others (43). In addition, a recent report
(50) demonstrated that IRS3 assumes a different subcellular
localization than IRS1 and IRS2, which could affect its function.
Furthermore, although these latter two scaffolding proteins are highly
homologous and share subcellular localization patterns, the
consequences of genetic elimination of IRS1 differ from that of IRS2
(51, 52). Thus, utilizing dissimilar subsets of Gab/IRS proteins and
the secondary mediators they recruit may provide a means to generate a
specific set of signals from seemingly related cytokine receptors. The reason why the cellular effects of EPO stimulation differ from that
induced by TPO in cells that bear receptors for both hormones (e.g. the combined erythroid-megakaryocytic colony-forming
cell) may relate to adapter protein signaling differences.
Another finding reported herein is that TPO induces the activation of
SHP2 in BaF3/Mpl cells, confirming the results of Saris and colleagues
(53) working in 32D cells and extending the observation to primary MKs.
SHP2 is a ubiquitous protein tyrosine phosphatase that is a positive
regulator of growth factor signaling (45). Furthermore, it has been
found to suppress the growth-inhibitory signals derived from activation
of the interferon receptor (54). In this study, we have found that SHP2
is phosphorylated in response to TPO and participates in a complex with
Gab/IRS-related proteins and PI3K. Since phosphorylated SHP2 can bind
to Grb2 (55), our demonstration of its TPO-induced recruitment and
phosphorylation could potentially couple the Mpl receptor to activation
of the SOS/Ras/Raf/MEK/MAPK pathway in an Shc-independent fashion,
helping to explain previous findings whereby truncated Mpl receptors
that fail to phosphorylate Shc nevertheless activated MAPK (24,
25).
To prove the physiologic relevance of the complex, a series of
experiments to disrupt complex formation was performed. By using a DN
SHP2 construct, we have shown that SHP2 is important for PI3K
activation as reflected by Akt phosphorylation. Previous studies of
insulin signaling have shown that this dominant negative form of SHP2
inhibits the capacity of insulin to activate PI3K in fibroblasts (44).
These data also suggest the physiologic role of the complex in bringing
SHP2 into close proximity with p85 to modulate the PI3K functions.
TPO-induced SHP2/p85 association was also found in primary MKs
suggesting that a similar mechanism may operate in primary cells. A
recent report revealed a novel tyrosine-phosphorylated protein, p90,
that may work downstream of SHP2 (56). However, we could not detect p90
in our complex. Therefore, the molecular mechanisms of SHP2 in
promoting growth factor signaling have to be clarified further.
Finally, our results also highlight the subtlety sometimes encountered
in studies of cytokine signaling. Class IA PI3Ks are heterodimeric
enzymes composed of regulatory and catalytic subunits that are
activated by the binding of numerous growth factor/cytokines to their
respective receptors. One of the downstream targets of PI3K is the Akt
(PKB) kinase, a protein that has been shown to play an important role
in cell survival by altering the metabolism of the pro-apoptotic
molecule Bad (26). More recently, activation of PI3K was also found to
be essential for IL-3 and TPO-induced cellular proliferation (31, 32).
However, we have found that the total PI3K activity in both BaF3/Mpl
cells and MKs is increased only very modestly by TPO stimulation.
Nevertheless, by testing the PI3K activity recruited into TPO-induced
signaling complexes, we found that the level of PI3K activation is
markedly enhanced. These results suggest that the main result of
TPO-induced PI3K activation is the translocation of the enzyme into the
signaling complexes assembled around the activated cytokine receptor.
By bringing together both enzyme and substrate, Mpl and other related cytokine receptors and their associated adapter proteins provide a
highly efficient mechanism for transmitting extracellular signals into
cellular responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform is essential for proplatelet formation
(23), the process by which platelets develop from MK cytoplasm.
Notably, TPO-induced MAPK activation has been shown to be both
Shc-dependent and Shc-independent in both cell lines and
platelets (24, 25). However, the Shc-independent pathway of MAPK
activation that follows TPO signaling remains undefined.
/
-catenin/LEF pathways, both of
which can play roles in cellular proliferation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
TPO induces serine phosphorylation of Akt in
BaF3/Mpl cells and primary MKs. BaF3/Mpl cells (A) or
purified murine MKs (B) were starved of serum and growth
factors, stimulated with a control buffer ( ) or one containing 25 ng/ml mTPO (+) for 10 min, lysed, and probed for the presence of
phospho-Akt with an antibody specific for phosphorylation at
Ser473. To demonstrate the role of PI3K in this
process, additional BaF3/Mpl cell preparations were pretreated with
either 16 µM Ly294002, a relatively specific PI3K
inhibitor, or an equal amount of Me2SO
(DMSO) diluent for 30 min before stimulation with TPO
(A). Following probing for phospho-Akt, the blots were
stripped and reprobed for total levels of Akt present in each lane.
Similar results were obtained in two additional experiments.
View larger version (22K):
[in a new window]
Fig. 2.
TPO induces the phosphorylation of several
p85 PI3K-associated proteins. BaF3/Mpl cells were starved and
stimulated with TPO (+) or control medium ( ) as described in the
legend to Fig. 1, lysed, and immunoprecipitated (IP) with an
antibody to p85 PI3K. The proteins were then size-fractionated by gel
electrophoresis, blotted, and probed for phosphotyrosine
(PY)-containing proteins. The relative mass in kDa is shown
to the left of the figure. Small arrows to the
right of the gel refer to phosphoproteins of ~170, 145, 116, 110, 100, and 70 kDa, and the large arrow indicates the
relative position of p85 when the gel was stripped and reprobed with a
p85-specific antibody, shown in the lower panel.
View larger version (18K):
[in a new window]
Fig. 3.
BaF3/Mpl cells and MKs display several Gab
and IRS-related proteins. Western blots were prepared from 100 µg of whole cell lysates of unstimulated BaF3/Mpl cells or purified
murine MKs and probed for Gab1 (A), Gab2 (B),
Gab3 (C), IRS1 (D), or IRS2 (E).
Lysates from NIH 3T3 cells served as a positive control in some of
these experiments (D and E). The molecular mass
of standard protein markers in kDa is shown to the left of
the blots. The weakly reactive band in anti-IRS1 antibody-probed
BaF3/Mpl cells (D) is not believed to be IRS1 due to its
slowed electrophoretic mobility but rather is thought to represent
cross-reactivity of the antibody with IRS2 (43).
View larger version (32K):
[in a new window]
Fig. 4.
TPO induces the formation of a
P-Gab2-P-SHP2-p85 complex in BaF3/Mpl cells. Cell lysates were
prepared from control ( ) and TPO-stimulated (+) BaF3/Mpl cells (as
described in Fig. 1) and immunoprecipitated with an antibody to Gab2
and sequentially probed for PY-containing proteins (A), SHP2
(B), p85 PI3K (C), or Gab2 (D). A
second Western blot was prepared with SHP2-immunoprecipitated
(IP) proteins and probed for PY (E), Gab2
(F), and SHP2 (G). Similar results were seen in
two additional experiments. The molecular mass of standard protein
markers in kDa is shown to the left of the blots.
View larger version (22K):
[in a new window]
Fig. 5.
TPO induces the formation of a P-IRS2-p85
complex in BaF3/Mpl cells. Cell lysates were prepared from
BaF3/Mpl cells as in Fig. 4, immunoprecipitated (IP) with an
anti-IRS2 antibody, and probed for PY (A), p85 PI3K
(B), and IRS2 (C). In a separate experiment, the
corresponding lysates were immunoprecipitated for murine c-Mpl and
probed for IRS2 (D) and Mpl (E). Similar results
were seen in three additional experiments.
-32P]ATP incorporation into phosphoinositol. In
contrast to the modest ~20% increase in total cellular PI3K activity
noted above, TPO greatly increased PI3K activity associated with both
Gab2 and IRS2 (Fig. 6). These results
indicate that both Gab2 and IRS2 can activate PI3K by recruiting PI3K
into their complexes in TPO-stimulated BaF3/Mpl cells.
View larger version (57K):
[in a new window]
Fig. 6.
TPO induces Gab2-SHP2-p85 and IRS2-p85
complexes to activate PI3K in BaF3/Mpl cells. Gab2 or IRS2
immunoprecipitates (IP) were prepared from BaF3/Mpl cells in
control ( ) or TPO medium (+) and then used in a
[
-32P]ATP phosphoinositol incorporation assay. An
autoradiogram of the thin layer chromatography plate is shown, with the
location of the origin and [32P]phosphatidylinositol
phosphate (PIP) indicated. This experiment has been
performed twice with similar results.
View larger version (29K):
[in a new window]
Fig. 7.
Dominant negative SHP2 inhibits PI3K
activation. BaF3/Mpl cells were co-transfected with DN SHP2 and a
plasmid containing a puromycin-selectable marker. Stable clones were
obtained and tested for expression by Western blot analysis
(A). Clone 1 expressed only wild type SHP2 (70 kDa,
WT) and clone 2 highly expressed the dominant negative form
(24 kDa, DN). Phospho-Akt (pAkt) was detected by
Western blot after 10 min of TPO stimulation (B). AKT
phosphorylation was markedly reduced in the DN SHP2-expressing clone
(2). The blot was then stripped and re-probed with anti-Akt
to verify equal amounts of protein. The lysates were also
immunoprecipitated (IP) with anti-Gab2 and probed for SHP2
to prove that the complex was disrupted. The blot was also stripped and
re-probed to verify the equal amounts of Gab2 per lane. These
experiments were performed at least twice. Similar results were found
in uncloned cells from two separate transfections (data not
shown).
View larger version (22K):
[in a new window]
Fig. 8.
TPO induces the formation of a
P-SHP2-pp100-p85 complex in MKs. Purified murine MKs were starved,
stimulated with TPO (+) or control medium ( ) for 10 min, lysed, and
immunoprecipitated (IP) with an anti-SHP2 antibody
(A-C) and probed sequentially for PY (A), SHP2
(B), and p85 PI3K (C), immunoprecipitated
(IP) with an anti-Gab1 antibody (D and
e), and probed for PY (D) and then Gab1
(E), immunoprecipitated with an anti-Gab3 antibody
(F and G), and probed for PY (F) and
then Gab3 (G), and immunoprecipitated with an anti-p85 PI3K
antibody (H and I) and probed for PY
(H) and then p85 PI3K (I). The molecular mass of
standard protein markers in kDa is shown on the left of the
blots. The experiment depicted in parts H and I
has been repeated five separate times, with the MKs derived from up to
10 mice in each experiment, with similar results.
-32P]ATP PI incorporation assay. After stimulation
with TPO for 10 min, PI3K activity was increased 2.1-fold in the
phosphotyrosine-containing protein immunoprecipitates following TPO
stimulation of MKs compared with unstimulated cell lysates (Fig.
9), suggesting that following TPO
stimulation PI3K was recruited to a pp100-containing complex leading to
activation of the kinase domain of the enzyme. In an independent
experiment, we also immunoprecipitated SHP2-containing complexes and
tested for PI3K activity. We found that compared with unstimulated MK
lysates, PI3K activity was increased 5.5-fold in SHP2
immunoprecipitates from TPO-stimulated MKs (Fig. 9).
View larger version (17K):
[in a new window]
Fig. 9.
TPO induces SHP2-pp100-p85 complexes to
activate PI3K in MKs. Phosphotyrosine-containing protein
(PY) or SHP2 immunoprecipitates were prepared from purified
murine MKs, incubated for 10 min in control ( ) or TPO medium (+), and
then used in a [
-32P]ATP PI incorporation assay. The
autoradiogram of the thin layer chromatography plate from one of two
experiments is shown, with the location of the origin and
[32P]phosphatidylinositol phosphate
(PIP). In two experiments, PI3K activity was increased a
mean of 2.1-fold in TPO-stimulated cells in phosphotyrosine-containing
proteins (left panel) and 5.5-fold in SHP2
immunoprecipitates (right panel) compared with one in
unstimulated MKs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Akihiro Shimosaka, Don Foster, Toshio Hirano, Takashi Kato, Toshio Hirano, Larry Rohrschneider, Hiroshi Maegawa, Norio Komatsu, and Toshio Kitamura for their gifts of reagents and Zenaida Sisk for help with the graphics.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Division of Hematology, Box 357710, University of Washington School of Medicine, 1959 N.E. Pacific St., Seattle, WA 98195. Tel.: 206-685-7868; Fax: 206-543-3560; E-mail: kkaushan@u.washington.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M002633200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MK, megakaryocyte; TPO, thrombopoietin; PI3K, phosphoinositol 3-kinase; STAT, signal transducers and activators of transcription; MAPK, mitogen-activated protein kinase; IL, interleukin; EPO, erythropoietin; IRS, insulin receptor substrates; Gab, Grb2-associated binder; BSA, bovine serum albumin; PI, phosphoinositol; DN SHP2, dominant negative SHP2; IMDM, Iscove's modified Dulbecco's medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Long, M. W., and Hoffman, R. (1995) in Hematology: Basic Principles and Practice (Hoffman, R. , Benz, E. J. , Shattil, S. J. , Furie, B. , Cohen, H. J. , and Silberstein, L. E., eds), 2nd Ed , pp. 274-286, Churchill Livingstone, New York |
2. |
Kaushansky, K.
(1995)
Blood
86,
419-431 |
3. |
Ku, H.,
Yonemura, Y.,
Kaushansky, K.,
and Ogawa, M.
(1996)
Blood
87,
4544-4551 |
4. |
Sitnicka, E.,
Lin, N.,
Priestley, G. V.,
Fox, N.,
Broudy, V. C.,
Wolf, N. S.,
and Kaushansky, K.
(1996)
Blood
87,
4998-5005 |
5. |
Debili, N.,
Wendling, F.,
and Katz, A.
(1995)
Blood
86,
2516-2525 |
6. | Kaushansky, K., Broudy, V. C., and Lin, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3234-3238[Abstract] |
7. |
Choi, E. S.,
Nichol, J. L.,
and Hokom, M. M.
(1995)
Blood
85,
402-413 |
8. | Gurney, A. L., Carver-Moore, K., de Sauvage, F. J., and Moore, M. W. (1994) Science 265, 1445-1447[Medline] [Order article via Infotrieve] |
9. |
Alexander, W. S.,
Roberts, A. W.,
Nicola, N. A.,
Li, R.,
and Metcalf, D.
(1996)
Blood
87,
2162-2170 |
10. |
Solar, G. P.,
Kerr, W. G.,
Zeigler, F. C.,
Hess, D.,
Donahue, C.,
de Sauvage, F. J.,
and Eaton, D. L.
(1998)
Blood
92,
4-10 |
11. |
Drachman, J.,
Griffin, J. D.,
and Kaushansky, K.
(1995)
J. Biol. Chem.
270,
4979-4982 |
12. | Sasaki, K., Odai, H., Hanazono, Y., Ueno, H., Ogawa, S., Langdon, W. Y., Tanaka, T., Miyagawa, K., Mitani, K., Yazaki, Y., and Hirai, H. (1995) Biochem. Biophys. Res. Commun. 216, 338-347[CrossRef][Medline] [Order article via Infotrieve] |
13. | Alexander, W. S., Maurer, A. B., Novak, U., and Harrison-Smith, M. (1996) EMBO J. 16, 6531-6540 |
14. |
Miyakawa, Y.,
Oda, A.,
Druker, B. J.,
Miyazaki, H.,
Handa, M.,
Ohashi, H.,
and Ikeda, Y.
(1996)
Blood
87,
439-446 |
15. |
Drachman, J. G.,
Millett, K. M.,
and Kaushansky, K.
(1999)
J. Biol. Chem.
274,
13480-13484 |
16. |
Dorsch, M.,
Fan, P. D.,
Danial, N. N.,
Rothman, P. B.,
and Goff, S. P.
(1998)
J. Exp. Med.
186,
1947-1955 |
17. | Rouyez, M. C., Boucheron, C., Gisselbrecht, S., Dusanter-Fourt, I., and Porteu, F. (1997) Mol. Cell. Biol. 17, 4991-5000[Abstract] |
18. |
Rojnuckarin, P.,
Drachman, J. G.,
and Kaushansky, K.
(1999)
Blood
94,
1273-1282 |
19. | Kunitama, M., Shimizu, R., Yamada, M., Kato, T., Miyazaki, H., Okada, K., Miura, Y., and Komatsu, N. (1997) Biochem. Biophys. Res. Commun. 231, 290-294[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Hong, Y.,
Dumenil, D.,
van der Loo, B.,
Goncalves, F.,
Vainchenker, W.,
and Erusalimsky, J. D.
(1998)
Blood
91,
813-822 |
21. | Sattler, M., Salgia, R., Durstin, M. A., Prasad, K. V., and Griffin, J. D. (1997) J. Cell. Physiol. 171, 28-33[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ritchie, A., Braun, S. E., He, J., and Broxmeyer, H. E. (1999) Oncogene 18, 1465-1477[CrossRef][Medline] [Order article via Infotrieve] |
23. | Rojnuckarin, P., and Kaushansky, K. (2001) Blood, in press |
24. |
Drachman, J. G.,
and Kaushansky, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2350-2355 |
25. |
Luoh, S. M.,
Stefanich, E.,
Solar, G.,
Steinmetz, H.,
Lipari, T.,
Pestina, T. I.,
Jackson, C. W.,
and de Sauvage, F. J.
(2000)
Mol. Cell. Biol.
20,
507-515 |
26. |
del Peso, L.,
González-Garcia, M.,
Page, C.,
Herrera, R.,
and Nuñez, G.
(1997)
Science
278,
687-689 |
27. |
Songyang, Z.,
Baltimore, D.,
Cantley, L. C.,
Kaplan, D. R.,
and Franke, T. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11345-11350 |
28. |
Bao, H.,
Jacobs-Helber, S. M.,
Lawson, A. E.,
Penta, K.,
Wickrema, A.,
and Sawyer, S. T.
(1999)
Blood
93,
3757-3773 |
29. |
Haseyama, Y.,
Sawada, K.,
Oda, A.,
Koizumi, K.,
Takano, H.,
Tarumi, T.,
Nishio, M.,
Handa, M.,
Ikeda, Y.,
and Koike, T.
(1999)
Blood
94,
1568-1577 |
30. |
Chen, R. H.,
Chang, M. C.,
Su, Y. H.,
Tsai, Y. T.,
and Kuo, M. L.
(1999)
J. Biol. Chem.
274,
23013-23019 |
31. |
Craddock, B. L.,
Orchiston, E. A.,
Hinton, H. J.,
and Welham, M. J.
(1999)
J. Biol. Chem.
274,
10633-10640 |
32. | Geddis, A. E., Miyakawa, Y., Rojnuckarin, P., Fox, N. E., and Kaushansky, K. (1999) Blood 94, 654 |
33. | Rottapel, R., Turck, C. W., Casteran, N., Liu, X., Birnbaum, D., Pawson, T., and Dubreuil, P. (1994) Oncogene 9, 1755-1765[Medline] [Order article via Infotrieve] |
34. |
Damen, J. E.,
Cutler, R. L.,
Jiao, H.,
Yi, T.,
and Krystal, G.
(1995)
J. Biol. Chem.
270,
23402-23408 |
35. | Klinghoffer, R. A., Duckworth, B., Valius, M., Cantley, L., and Kazlauskas, A. (1996) Mol. Cell. Biol. 16, 5905-5914[Abstract] |
36. |
Nishida, K.,
Yoshida, Y.,
Itoh, M.,
Fukada, T.,
Ohtani, T.,
Shirogane, T.,
Atsumi, T.,
Takahashi-Tezuka, M.,
Ishihara, K.,
Hibi, M.,
and Hirano, T.
(1999)
Blood
93,
1809-1816 |
37. |
Wickrema, A.,
Uddin, S.,
Sharma, A.,
Chen, F.,
Alsayed, Y.,
Ahmad, S.,
Sawyer, S. T.,
Krystal, G.,
Yi, T.,
Nishada, K.,
Hibi, M.,
Hirano, T.,
and Platanias, L. C.
(1999)
J. Biol. Chem.
274,
24469-24474 |
38. |
Dorsch, M.,
Danial, N. N.,
Rothman, P. B.,
and Goff, S. P.
(1999)
Blood
94,
2676-2685 |
39. |
Rother, K. I.,
Imai, Y.,
Caruso, M.,
Beguinot, F.,
Formisano, P.,
and Accili, D.
(1998)
J. Biol. Chem.
273,
17491-17497 |
40. | Kaushansky, K., Lok, S., Holly, R. D., Broudy, V. C., Lin, N., Bailey, M. C., Forstrom, J. W., Buddle, M., Oort, P. J., Hagen, F. S., Roth, G. J., Papayannopoulou, Th., and Foster, D. C. (1994) Nature 369, 568-571[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Drachman, J. D.,
Sabath, D. F.,
Fox, N. E.,
and Kaushansky, K.
(1997)
Blood
89,
483-492 |
42. |
Nishigaki, K.,
Hanson, C.,
Ohashi, T.,
Thompson, D.,
Muszynski, K.,
and Ruscetti, S.
(2000)
J. Virol.
74,
3037-3045 |
43. |
Verdier, F.,
Chretien, S.,
Billat, C.,
Gisselbrecht, S.,
Lacombe, C.,
and Mayeux, P.
(1997)
J. Biol. Chem.
272,
26173-26178 |
44. |
Ugi, S.,
Maegawa, H.,
Kashigawi, A.,
Adachi, A.,
Olefsky, J. M.,
and Kikkawa, R.
(1996)
J. Biol. Chem.
271,
12595-12602 |
45. |
Pazdrak, K.,
Adachi, T.,
and Alam, R.
(1997)
J. Exp. Med.
186,
561-568 |
46. |
Oh, E. S.,
Gu, H.,
Saxton, T. M.,
Timms, J. F.,
Hausdorff, S.,
Frevert, E. U.,
Kahn, B. B.,
Pawson, T.,
Neel, B. G.,
and Thomas, S. M.
(1999)
Mol. Cell. Biol.
19,
3205-3215 |
47. |
Pluskey, S.,
Wandless, T. J.,
Walsh, C. T.,
and Shoelson, S. E.
(1995)
J. Biol. Chem.
270,
2897-2900 |
48. |
Rodrigues, G. A.,
Falasca, M.,
Zhang, Z.,
Ong, S. H.,
and Schlessinger, J.
(2000)
Mol. Cell. Biol.
20,
1448-1459 |
49. |
Szanto, I.,
and Kahn, C. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2355-2360 |
50. |
Anai, M.,
Ono, H.,
Funaki, M.,
Fukushima, Y.,
Inukai, K.,
Ogihara, T.,
Sakoda, H.,
Onishi, Y.,
Yazaki, Y.,
Kikuchi, M.,
Oka, Y.,
and Asano, T.
(1998)
J. Biol. Chem.
273,
29686-29692 |
51. | Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J., Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G. I., Bonner-Weir, S., and White, M. F. (1998) Nature 391, 900-904[CrossRef][Medline] [Order article via Infotrieve] |
52. | Bruning, J. C., Winnay, J., Cheatham, B., and Kahn, C. R. (1997) Mol. Cell. Biol. 17, 1513-1521[Abstract] |
53. |
Mu, S. X.,
Xia, M.,
Elliot, G.,
Bogenberger, J.,
Swift, S.,
Bennett, L.,
Lappinga, D. L.,
Hecht, R.,
Lee, R.,
and Saris, C. J. M.
(1995)
Blood
86,
4532-4543 |
54. |
You, M., Yu, D. H.,
and Feng, G. S.
(1999)
Mol. Cell. Biol.
19,
2416-2424 |
55. | Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J., and Cooper, J. A. (1994) Mol. Cell. Biol. 14, 509-517[Abstract] |
56. |
Shi, Z. Q., Yu, D. H.,
Park, M.,
Marshall, M.,
and Feng, G. S.
(2000)
Mol. Cell. Biol.
20,
1526-1536 |