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A Tyrosine-Phosphorylated Protein of 140 kD Is Constitutively Associated
With the Phosphotyrosine Binding Domain of Shc and the SH3 Domains of
Grb2 in Acute Myeloid Leukemia Cells
By Manfred Jücker, Charles A. Schiffer, and Ricardo A. Feldman
The Shc gene encodes three proteins that have been implicated as mediators of signal transduction from growth factor
receptors and nonreceptor tyrosine kinases to Ras. Overexpression of Shc in established murine fibroblasts results in
oncogenic transformation, indicating that Shc has oncogenic potential. Shc proteins contain a carboxy terminal SH2
domain and a novel non-SH2 phosphotyrosine-binding (PTB)
domain that specifically recognizes a phosphorylated
NPXpY motif in target proteins such as the epidermal
growth factor receptor. We show here that Shc is constitutively tyrosine-phosphorylated in all primary acute myeloid
leukemias analyzed and that, in some of these leukemias,
Shc is associated through its PTB domain with a tyrosinephosphorylated protein of 140 kD (p140) in vivo. In factor-
dependent cells, this 140-kD protein can be tyrosine-phosphorylated in vitro in response to cytokines involved in
myeloid proliferation and differentiation, ie, granulocytemacrophage colony-stimulating factor and colony-stimulating factor-1. A similar or identical protein of 140 kD is constitutively bound to the C-terminal SH3 domain of Grb2 in the
same acute myeloid leukemias. In addition to p140, other
tyrosine-phosphorylated proteins of 61 and 200 kD are constitutively associated with Shc in some of the leukemias
analyzed. Our results implicate Shc, Grb2, p140, and additional tyrosine-phosphorylated proteins of 61 and 200 kD in
signalling of acute myeloid leukemia cells.
q 1997 by The American Society of Hematology.
A
ing factor (GM-CSF).14 An autocrine growth stimulation of
AML cells by GM-CSF has been shown in some cases of
AML.15-17 We have previously shown that coexpression of
the a and b subunits of the human GM-CSF receptor in
established murine fibroblasts can cause ligand-dependent
transformation.18 However, mice constitutively expressing
the GM-CSF or interleukin-3 (IL-3) gene develop myeloproliferative syndromes but not acute leukemia.19-21 These data
suggest that the deregulated stimulation of hematopoietic
cells with an HGF, eg, GM-CSF or IL-3, via autocrine or
paracrine mechanisms can contribute to the leukemic transformation but that their unregulated expression alone is not
sufficient to cause leukemia.
Mutated HGF receptors can have transforming activities
that contribute to the development of leukemia in mice, as
has been shown for the colony-stimulating factor-1 (CSF-1)
receptor22 and erythropoietin (EPO) receptor.23 Mutations in
the CSF-1 receptor gene have been described in about 16%
of primary AML24 and constitutive tyrosine phosphorylation
of c-Kit, which is the receptor for stem cell factor (SCF),
has been detected in 7 of 12 cases of AML.25 These data
suggest that constitutively activated HGF receptors may be
involved in the transformation of AML cells.
A deregulated growth stimulation of AML cells may also
result from the constitutive activation of signal transduction
pathways downstream of the HGF receptors. Mutations in
ras genes,26,27 most often in N-ras, have been detected in
about 25% of cases of AML.28-30 Ras is also activated in
hematopoietic cells transformed by the p210Bcr/Abl fusion protein that is found in all cases of chronic myeloid leukemia.31
The prevention of apoptosis in hematopoietic cells by cytokine receptor stimulation may be mediated by Ras-dependent
upregulation of bcl 2 gene expression.32,33 These and additional data implicate Ras proteins directly in leukemogenesis.34
Ras proteins become transiently activated after stimulation
of receptor tyrosine kinases, cytokine receptors without an
intrinsic tyrosine kinase domain, or by activated cytoplasmic
tyrosine kinases.35-37 An increase in the active GTP-bound
form of Ras can be induced either by activating the Ras
nucleotide exchange factor SOS or by inhibiting the Ras
CUTE MYELOID LEUKEMIA (AML) is characterized by the clonal expansion of hematopoietic progenitor cells that are blocked in terminal differentiation.1 The
full leukemic phenotype of AML appears to be the result of
several transforming events that cooperate in the process of
leukemogenesis.2-4 In the last few years, genes that may be
involved in the pathogenesis of AML have been identified by
different approaches. The molecular cloning of translocation
breakpoints associated with AML5,6 has identified the genes
involved in the breakpoint junctions as transcriptional regulatory proteins such as AML1, ETO, EVI1, PML, and
RARa.6-10 From these and additional data it has been concluded that transcription factors play an important role in
the development of acute leukemias including AML, probably by blocking normal hematopoietic cell differentiation.4,11
Another major step in identifying genes that may contribute to AML came from the extensive work with hematopoietic growth factors (HGFs) and HGF receptors, which regulate the complex network of hematopoiesis.12,13 Several
HGFs can induce or enhance the proliferation of AML cells
in vitro, including granulocyte-macrophage colony-stimulat-
From the Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD; the University
of Maryland Cancer Center, Baltimore, MD; the Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD.
Submitted June 17, 1996; accepted October 22, 1996.
Supported by National Cancer Institute Grant No. R29 CA 55293,
by the National Leukemia Association, and by UMAB DRIF award to
R.A.F. M.J. was a recipient of a Deutsche Forschungsgemeinschaft
fellowship from the Federal Republic of Germany.
Address reprint requests to Ricardo A. Feldman, PhD, University
of Maryland School of Medicine, Department of Microbiology and
Immunology, BRB 13-043, 655 W Baltimore St, Baltimore, MD
21201.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8906-0031$3.00/0
Blood, Vol 89, No 6 (March 15), 1997: pp 2024-2035
2024
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SHC/P140/GRB2 COMPLEXES ACTIVATED IN AML
2025
GTPase activating protein.38 Activation of Ras by SOS involves the relocalization of SOS from the cytoplasm to the
plasma membrane, which is mediated by the adaptor protein
Grb2.39-41 Grb2 can bind constitutively to SOS, but the association of Grb2 with SOS can also be regulated, as has been
shown after stimulation of the T-cell antigen receptor.42 Grb2
can bind directly to the activated epidermal growth factor
(EGF) receptor or bind indirectly via its association with
tyrosine-phosphorylated Shc.43,44 Tyrosine phosphorylation
of Shc proteins provides therefore an alternative mechanism
to couple activated receptors and cytoplasmic tyrosine kinases to the Ras signalling pathway.
Shc adapter molecules can bind to activated receptor tyrosine kinases by interaction of their SH2 domain with distinct
tyrosine-phosphorylated motifs in the receptor.44-46 Shc also
contains a novel non-SH2 phosphotyrosine binding domain
(PTB) located at its N terminus.47-50 Whereas binding of SH2
domains to specific motifs is controlled by residues carboxy
terminal to the phosphotyrosine,51 the PTB domain selects
its tyrosine-phosphorylated binding sites on the basis of residues amino terminal to the phosphotyrosine, ie, Asn-Pro-XTyr(P).46,50 The presence of a PTB domain in several otherwise unrelated regulatory proteins suggests a general role
for this domain in signal transduction.52
Overexpression of Shc proteins transforms immortalized
fibroblasts and increases the proliferative response of a myeloid leukemia cell line, indicating that Shc proteins have
mitogenic and transforming potential.44,53 Constitutive phosphorylation of Shc proteins has been found in cells transformed by oncogenic tyrosine kinases54 and in chronic myeloid leukemia cells expressing the p210Bcr/Abl oncoprotein.55
Stimulation of cells with IL-3, GM-CSF, CSF-1, EPO, and
SCF, which can sustain the proliferation of AML cells in
vitro, leads to a transient tyrosine phosphorylation of Shc
proteins.55-57 In this report, we present evidence that, in
freshly isolated AML cells, Shc proteins are constitutively
tyrosine-phosphorylated and associated with known and new
signalling proteins, including a recently described 140-kD
phosphorylated protein. We have further characterized the
interactions between Shc and the Shc-associated proteins
implicating the PTB domain in signalling of AML cells. Our
results suggest a mechanism whereby the Ras pathway may
be constitutively activated in AML.
MATERIALS AND METHODS
Cells. The GM-CSF–dependent human erythroleukemia cell
line TF-158 was obtained from A. Miyajima (DNAX Research Institute, Palo Alto, CA). These cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol)
fetal bovine serum and 3 ng/mL recombinant human GM-CSF
(hGM-CSF). Control and v-fes–transformed NIH 3T3 cells have
been described.59 BAC1.2F5 is a macrophage cell line that is dependent on CSF-1 for survival and proliferation.60 BAC1.2F5 cells were
maintained in a minimal essential medium (a-MEM) supplemented
with 10% (vol/vol) fetal bovine serum (GIBCO) and 36 ng/mL of
human recombinant CSF-1 (Chiron Corp, Emeryville, CA). Leukemia cells were obtained from the peripheral blood of patients with
AML and chronic lymphocytic leukemia. The diagnosis in each
case was made by morphology, cytochemistry, and surface antigen
analysis. Normal peripheral blood cells were obtained from a healthy
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donor. Mononuclear peripheral blood cells from leukemia patients
and from a healthy donor were fractionated by gradient sedimentation using Histopaque-1077 according to the manufacturer (Sigma,
St Louis, MO). After gradient sedimentation, the percentage of blasts
was more than 90% in each sample. Fractionated leukemia cells
were then lysed without further handling.
Antibodies. Rat monoclonal antibody (MoAb) CRS1 directed
against the b subunit of the hGM-CSF receptor has been previously
described61 and was obtained from A. Miyajima. An MoAb directed
against Grb2 was purchased from Transduction Laboratories (Lexington, KY). MoAb EL6 against Grb2 was obtained from E. Lowenstein and J. Schlessinger.62 Rabbit antisera directed against Shc
and Jak2 and MoAb directed against phosphotyrosine (4G10) and
phospholipase C-g (PLC-g) were obtained from UBI (Lake Placid,
NY).
Stimulation with hGM-CSF. TF-1 cells were starved for 18 hours
in RPMI 1640 medium containing 0.1% (wt/vol) bovine serum albumin (BSA; fraction V; Sigma) at a cell density of 5 1 105 cells/mL.
Cells were then resuspended in fresh medium containing 0.1% (wt/
vol) BSA at a cell density of 1 1 107 cells/mL and were then
incubated for 2 minutes at 377C in the absence or presence of 25
ng/mL hGM-CSF, unless otherwise indicated. After incubation, cells
were diluted into cold phosphate-buffered saline containing 1 mmol/
L sodium orthovanadate and cell lysates were prepared as described
below. The stimulation of BAC1.2F5 cells with 35 ng/mL CSF-1
was performed as previously described.63
Preparation of cell lysates and protein analysis. Untreated or
hGM-CSF–treated cells were lysed in NP-40 lysis buffer (50 mmol/
L HEPES, pH 7.4, 150 mmol/L NaCl, 1% [vol/vol] NP-40, 2 mmol/
L EDTA, 10% [vol/vol] glycerol, 50 mmol/L sodium fluoride, 10
mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate,
and 2% [vol/vol] Trasylol [FBA Pharmaceuticals, New York, NY]).
Insoluble material was removed by centrifugation at 47C for 10
minutes at 14,000g. Protein concentration was determined using
the BioRad protein assay according to the manufacturer (BioRad,
Melville, NY), and cell lysates were stored in liquid nitrogen until
used.
Cell lysates were immunoprecipitated with the indicated antibodies and the immunoprecipitates were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDSPAGE and Western blot analysis were performed as described previously.63 The concentration of the MoAbs or rabbit sera used for
Western blot analyses were 0.5 mg/mL of antiphosphotyrosine MoAb
4G10 and anti–PLC-g1 MoAb, 1 mg/mL of anti-Shc MoAb, and
1:500 dilution for anti-GRB2 MoAb. Filters were developed using
the enhanced chemiluminescence (ECL) system according to the
manufacturer (Amersham, Arlington Heights, IL). Reprobing of nitrocellulose membrane was performed as described previously.64
Purification of bacterial fusion proteins. The Shc-SH2 domain
was expressed as a fusion protein with glutathione S-transferase
(GST) after subcloning a DNA fragment corresponding to nucleotides 1214-1495 of the Shc cDNA into pGEX-2T. The N-terminal
domain of Shc (amino acids 1 to 273) containing the PTB domain
was expressed as a fusion protein with maltose-binding protein
(MBP). pGEX-3X control and pGEX-fusion proteins were induced
with 0.1 mmol/L Isopropyl-b-D-thiogalacto-pyranoside (IPTG), the
induced bacteria were lysed by sonication in a buffer containing 1%
(vol/vol) Triton X-100, 50 mmol/L HEPES (pH 7.4), and the induced
proteins were adsorbed with glutathione-agarose (Sigma), as described previously.63 Expression of MBP and MBP fusion proteins
was induced with 0.3 mmol/L IPTG in the presence of 0.2% (vol/
vol) glucose for 2 hours at 377C. Bacteria were lysed as described
above and the MBP fusion proteins were adsorbed with amylose
resin (New England Biolabs, Beverly, MA).
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2026
JÜCKER, SCHIFFER, AND FELDMAN
RESULTS
hGM-CSF induces tyrosine phosphorylation of Shc proteins and their association with a 140-kD phosphotyrosyl
protein. The Shc proteins are known to be transiently phosphorylated on tyrosine after ligand stimulation of cytokine
receptors present in myeloid cells. Therefore, we wanted to
compare the state of phosphorylation of Shc proteins in cells
in which its phosphorylation is ligand-dependent with that
in AML, in which autocrine or paracrine mechanisms may
result in deregulated phosphorylation of Shc. Our analysis
of Shc proteins in the GM-CSF–dependent human erythroleukemia cell line TF-1 showed that, after hGM-CSF treat-
ment, all three Shc proteins p46Shc, p52Shc, and p66Shc become
rapidly and transiently phosphorylated on tyrosine (Fig 1A).
The major tyrosine-phosphorylated Shc protein was p52Shc,
which was also the most abundant form in TF-1 cells (Fig
1B). Maximal tyrosine phosphorylation of all three Shc proteins occurred within 2 minutes, remained at the maximum
for up to 5 minutes, and declined to very low (p52Shc) or
undetectable (p46Shc and p66Shc) levels 2 hours after hGMCSF stimulation. Tyrosine phosphorylation of Shc proteins
was dependent on the concentration of hGM-CSF, with maximal stimulation at 5 ng/mL (Fig 1A). Anti-Shc immunoprecipitates from hGM-CSF–treated TF-1 cells contained a
140-kD phosphotyrosyl protein (p140; Fig 1A). p140 was
not directly recognized by anti-Shc antibody in immunoblots
(Fig 1B), indicating that p140 was an associated protein
different from Shc. The time courses of coprecipitation of
phosphotyrosyl p140 and tyrosine phosphorylation of Shc
proteins were identical, suggesting that the direct or indirect
association between Shc and p140 may involve phosphotyrosine-dependent interactions (Fig 1A).
We then determined whether tyrosine-phosphorylated
p140 could also be detected after stimulation of growth factor
receptors with intrinsic tyrosine kinase domains such as the
CSF receptor. CSF-1 treatment of BAC1.2F5 cells resulted
in tyrosine phosphorylation of a similar or identical 140-kD
protein and its association with tyrosine-phosphorylated Shc
(Fig 2, lanes 3 and 4).
To examine whether p140 was a known signalling protein,
we compared its electrophoretic mobility with that of other
signalling proteins in the same molecular weight range,
namely Jak2 and the b subunit of the hGM-CSF receptor
(hGMR), which also become tyrosine-phosphorylated in response to hGM-CSF treatment.65-67 In TF-1 cells, Jak2 was
tyrosine-phosphorylated after hGM-CSF stimulation (Fig
3A, lanes 1 and 2), but Jak2 migrated with an apparent
molecular weight of 120 kD, indicating that p140 was not
Jak2 (Fig 3A, lane 4). Stripping and reprobing of the filter
shown in Fig 3A with anti-Jak2 antibodies showed that similar amounts of Jak2 were precipitated from unstimulated
and hGM-CSF–stimulated cells (data not shown). Similarly,
tyrosine-phosphorylated b, which had an apparent molecular
R
Fig 1. Tyrosine phosphorylation of Shc proteins in hGM-CSF–
stimulated TF-1 cells and identification of an associated 140-kD phosphotyrosyl protein. (A) Starved TF-1 cells were stimulated for the
indicated times with 25 ng/mL hGM-CSF or with the indicated concentrations of hGM-CSF for 5 minutes at 377C. Cells were lysed and
aliquots containing 1 mg protein were immunoprecipitated with antiShc antibodies. Immunoprecipitates were subjected to Western blot
analysis with antiphosphotyrosine antibodies. The positions of the
Shc proteins (p46, p52, and p66) and p140 are indicated. The apparent
molecular weights of the Shc proteins p46Shc, p52Shc, and p66Shc in
our gel system were consistently 51, 56, and 68 kD. To avoid confusion, we will continue to name the Shc proteins as p46, p52, and p66
according to Pelicci et al.44 (B) Starved TF-1 cells were stimulated for
the indicated times with hGM-CSF. Cells were lysed and total cell
lysates containing 50 mg of protein were subjected to Western blot
analysis using anti-Shc antibodies. The positions of Shc proteins
(p46, p52, and p66) are indicated. The arrow above p66 indicates a
slower migrating form of p66 detected during hGM-CSF stimulation.
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SHC/P140/GRB2 COMPLEXES ACTIVATED IN AML
2027
Fig 2. Association of tyrosine-phosphorylated p140 with Shc in
response to different growth factors. Anti-Shc immunoprecipitates
(IP:Shc) of unstimulated (lane 1) or hGM-CSF–stimulated (lane 2) TF1 cells and from unstimulated (lane 3) or CSF-1–stimulated (lanes 4)
BAC1.2F5 cells were analyzed by antiphosphotyrosine immunoblotting (lanes 1 through 4). The positions of p140, p52Shc, and Ig heavy
chain (Ig) are indicated on the left.
weight of 130 kD, did not comigrate with p140 either (Fig
3B, lanes 2 and 4). Interestingly, a 130-kD protein that comigrated with the b subunit of hGMR was also detectable in
the anti-Shc immunoprecipitate (Fig 3B, lane 2), suggesting
that Shc might associate with the b subunit directly or indirectly.
We also determined that PLC-g, which has an apparent
molecular weight of 145 kD, was not tyrosine-phosphorylated in hGM-CSF–stimulated cells, did not associate with
Shc proteins after hGM-CSF stimulation, and did not comigrate with p140 (data not shown). These results exclude an
identity of PLC-g with p140 and suggest that PLC-g may
not be activated by hGM-CSF in these cells.
We conclude that p140 is a new phosphotyrosyl protein
involved in hGM-CSF and CSF-1 signalling. p140 may act
in concert with Shc and Grb2, as described below in more
detail.
Shc and p140 are constitutively tyrosine-phosporylated
in AML cells. The Shc proteins are constitutively tyrosine
phosphorylated in cells transformed by v-src and v-fps,54 and
their overexpression can cause transformation in NIH 3T3
cells,44 suggesting that Shc activation may be a step in neo-
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plastic transformation. Therefore, we wanted to examine the
state of phosphorylation of Shc proteins in myeloid leukemia
cells. Our analysis showed that p52Shc was constitutively
tyrosine phosphorylated at different levels in freshly isolated
peripheral blood cells from eight of eight AML patients but
not in peripheral blood cells from a chronic lymphocytic
leukemia (CLL) patient or in peripheral blood mononuclear
cells (PBMC) from a healthy donor (Fig 4A and B). Whether
p46Shc was also constitutively phosphorylated in the leukemia samples could not be determined because p46Shc comigrated with the Ig band. For AML-4 and AML-8, for which
bone marrow cells were available, constitutively tyrosinephosphorylated Shc proteins were detected in both the peripheral blood and the bone marrow cells (data not shown).
Reprobing of the stripped antiphosphotyrosine blots with
anti-Shc antibody showed that p46Shc and p52Shc were expressed in all samples analyzed, whereas the 66-kD isoform
of Shc was expressed in AML-3 only (data not shown).
In 4 of 8 AML samples (AML-1, AML-2, AML-3, and
AML-7), immunoprecipitation of Shc resulted in coprecipitation of a tyrosine-phosphorylated protein of 140 kD (Fig
4A and B). This 140-kD protein had the same electrophoretic
mobility as the 140-kD protein coprecipitating with Shc in
hGM-CSF–stimulated TF-1 cells (Fig 4A). Two additional
tyrosine-phosphorylated proteins of 61 kD and 200 kD coprecipitating with Shc in AML cell lysates were discovered
during this analysis (Fig 4B, lanes 2, 4, and 6). The tyrosinephosphorylated 61-kD protein (p61) was detected in one
leukemia sample (AML-3; Fig 4B, lane 2) and the 200kD protein (p200) was detected in two other AML samples
(AML-5 and AML-7; Fig 4B, lanes 4 and 6). In one of
these leukemias (AML-7), p200 and p140 were found to
coprecipitate with Shc (Fig 4B, lane 6). However, in AML5, p200 was the only coprecipitating tyrosine-phosphorylated
protein, suggesting that Shc and p200 can form a complex
without tyrosine-phosphorylated p140 (Fig 4B, lane 4).
These results indicate that Shc is constitutively tyrosinephosphorylated in all AML samples analyzed and that Shc is
constitutively associated with p140 and additional tyrosinephosphorylated proteins of 61 kD and 200 kD in some AML.
p140 is constitutively associated with the phosphotyrosine
binding domain of Shc in AML cells. To further analyze
the association between Shc and p140 in more detail, we
used bacterial fusion proteins. Two domains of Shc have
been shown to interact with phosphotyrosine, ie, the SH2
domain, and the N-terminus containing the PTB domain.
The SH2 domain was expressed as a GST fusion protein
and the PTB domain as a MBP fusion protein. First we used
TF-1 cells as a model system, in which hGM-CSF induced
association of Shc with p140 (Fig 1A). Cell lysates of unstimulated and hGM-CSF–stimulated TF-1 cells were incubated with the MBP fusion protein containing the PTB domain of Shc (MBP-N-Shc). Proteins bound to the fusion
protein were purified by affinity chromatography on amylose
resin and further analyzed by SDS-PAGE and antiphosphotyrosine immunoblotting. As shown in Fig 5A, a tyrosinephosphorylated protein of 140 kD was recognized by the
PTB domain of Shc in a hGM-CSF–dependent manner (Fig
5A, lanes 3 and 4), whereas MBP vector protein did not
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2028
JÜCKER, SCHIFFER, AND FELDMAN
Fig 3. p140 is a new phosphotyrosyl protein. (A)
Cell lysates from unstimulated (lanes 1 and 3) or
hGM-CSF–stimulated (lanes 2 and 4) TF-1 cells were
immunoprecipitated with anti-Jak2 (lane 1 and 2) or
anti-Shc (lanes 3 and 4) antibodies and analyzed by
antiphosphotyrosine immunoblotting (lanes 1
through 4). The positions of Jak2, Shc, p140, and Ig
heavy chain (Ig) are indicated. (B) Cell lysates from
unstimulated (lanes 1 and 3) or hGM-CSF–stimulated (lane 2 and 4) TF-1 cells were immunoprecipitated with antibodies directed against Shc (lanes 1
and 2) or the b subunit of the human GM-CSF receptor (b-GMR; lanes 3 and 4) and analyzed by antiphosphotyrosine immunoblotting (lanes 1 through 4).
bind any phosphotyrosyl proteins (Fig 5A, lanes 5 and 6).
This 140-kD protein comigrated with the 140-kD protein
detected in anti-Shc immunoprecipitates (Fig 5A, lane 2). A
similar or identical 140-kD protein was also found to bind
to the PTB domain of Shc in a macrophage cell line
(BAC1.2F5) after stimulation with CSF-1 (Fig 5A, lanes 7
and 8). When we performed similar experiments using a
GST fusion protein containing the SH2 domain of Shc, we
were not able to detect any binding of the Shc SH2 domain
to p140 (data not shown).
We then analyzed whether the constitutive association of
Shc to p140 in primary AML was mediated through the PTB
domain of Shc. After incubation of cell lysates from five
AML patients (AML-3 through AML-7) with the MBP-NShc fusion protein immobilized on amylose resin, a 140-kD
phosphotyrosine protein was detected in AML-7 (Fig 5B,
lane 6) and was weakly detected in AML-3 and AML-6 (Fig
5B, lanes 2 and 5, respectively). In the case of AML-6, we
were not able to detect p140 in anti-Shc immunoprecipitates
(Fig 4B, lane 5), suggesting that the amount of p140 that can
be purified with a bacterially expressed Shc fusion proteins is
higher than by coimmunoprecipitation with anti-Shc antibodies. These data indicate that in these three AML samples
Shc is constitutively associated with p140 and that this association is mediated through the PTB domain of Shc.
Tyrosine-phosphorylated Shc and p140 form a complex
with Grb2 after hGM-CSF stimulation. Grb2 is an SH2/
SH3 adapter protein that links receptor tyrosine kinases to
the Ras pathway.40 Tyrosine-phosphorylated Shc proteins
bind to the SH2 domain of Grb2 after stimulation of cells
with growth factors and in cells transformed by oncogenic
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tyrosine kinases.37,44 To determine if Shc also associates with
Grb2 after hGM-CSF stimulation, immunoprecipitates of
Shc were analyzed for the presence of Grb2 by immunoblot
analysis. Grb2 was present in anti-Shc immunoprecipitates
from hGM-CSF–stimulated but not from unstimulated TF1 cells (Fig 6, lanes 3 and 4). The amount of Grb2 present
in the Shc immunoprecipitates was a fraction of the total
Grb2 that was precipitated by Grb2 antibodies (Fig 6, lane
5 and 6), indicating that only part of Grb2 formed a complex
with Shc after hGM-CSF stimulation. Stripping of the filter
and reprobing with anti-Shc antiserum showed that equal
amounts of Shc were precipitated by the antibody (data not
shown). In v-fes–transformed NIH 3T3 cells, which were
used as a positive control, Shc was also associated with
Grb2, whereas no association was detected in control NIH
3T3 cells (Fig 6, lanes 7 through 10).
In EGF-stimulated cells, tyrosine-phosphorylated Shc
binds to Grb2 through the Grb2 SH2 domain.37 Using the
Grb2 SH2 domain expressed as a GST fusion protein, we
analyzed whether in hGM-CSF–stimulated TF-1 cells binding of Shc to Grb2 was also mediated through the SH2
domain of Grb2. The Grb2 SH2 domain recognized tyrosinephosphorylated proteins of 51, 56, and 68 kD (Fig 7A, lane
6), which had the same electrophoretic mobility as p46Shc,
p52Shc, and p66Shc (Fig 7A, lane 2). Their identity as the
three Shc proteins was confirmed by stripping and reprobing
of the same filter with anti-Shc antibodies (data not shown).
Two other unknown tyrosine-phosphorylated proteins of 80
and 120 kD were also found to bind to the SH2 domain of
Grb2 in hGM-CSF–stimulated cells (Fig 7A, lane 6).
During these experiments, we detected a tyrosine-phos-
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SHC/P140/GRB2 COMPLEXES ACTIVATED IN AML
2029
Fig 4. Shc and p140 are constitutively phosphorylated on tyrosine in AML cells. (A) Cell lysates from unstimulated (Ï) or hGM-CSF–
stimulated (") TF-1 cells and lysates of primary leukemia cells obtained from two patients with AML (AML-1 and AML-2), CLL, and a lysate
of PBMCs obtained from a healthy donor were immunoprecipitated with anti-Shc antibodies and subjected to antiphosphotyrosine immunoblotting (P.Tyr). (B) Anti-Shc immunoprecipitates of lysates from hGM-CSF–stimulated TF-1 cells (lane 1) and primary AML (AML-3 through AML8; lanes 2 through 7) were analyzed by antiphosphotyrosine immunoblotting. The positions of p52Shc, p66Shc, p61, p140, and p200 are indicated
on the left. Molecular weight marker bands are indicated on the right.
phorylated protein of 140 kD in anti-Grb2 immunoprecipitates from hGM-CSF–stimulated cells (Fig 7A, lane 4),
which comigrated with p140 detected in anti-Shc immunoprecipitates (Fig 7A, lane 2). As shown in lane 6 of Fig 7A,
tyrosine-phosphorylated p140 did not associate with the SH2
domain of Grb2 but associated with the C-terminal and more
weakly with the N-terminal SH3 domain of Grb2 (Fig 7B,
lanes 4 and 6, respectively). Therefore, in TF-1 cells, hGMCSF induced the association of phosphotyrosyl p140 with
Grb2, and this association was mediated by the Grb2 SH3
domains. Further analysis is required to determine if p140
binds simultaneously to Shc and Grb2 or whether separate
p140/Shc and p140/Grb2 complexes are formed in response
to hGM-CSF.
Tyrosine-phosphorylated p140 binds to the C-terminal
SH3 domain of Grb2 in some AML cells. In the previous
sections, we showed that tyrosine-phosphorylated p140 was
constitutively associated with Shc through the PTB domain
in three of five AML analyzed (Fig 5B) and that p140 associ-
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ated with Grb2 through its SH3 domains. We therefore analyzed whether in AML p140 was also constitutively associated with Grb2 and whether this association was mediated
through the SH3 domain of Grb2. Cell lysates from unstimulated and hGM-CSF–stimulated TF-1 cells and from six
AML (AML-3 through AML-8) were immunoprecipitated
with anti-Grb2 antibodies and analyzed by antiphosphotyrosine immunoblotting. As shown in Fig 8A, a tyrosine-phosphorylated protein of 140 kD coprecipitated with Grb2 in
hGM-CSF–stimulated TF-1 cells and in AML-7. These data
indicate that in AML-7, Grb2 and p140 were constitutively
associated in vivo.
We then incubated lysates from the same AML (AML-3
through AML-8) with the C-terminal SH3 domain of Grb2
expressed as a GST fusion protein. After adsorption on glutathione agarose and antiphosphotyrosine immunoblotting,
p140 was detected in two of the six AML analyzed, ie,
AML-3 and AML-7 (Fig 8B, lanes 2 and 6). In the case
of AML-3, we were not able to detect p140 in anti-Grb2
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JÜCKER, SCHIFFER, AND FELDMAN
Fig 5. The association between Shc and p140 is mediated through the PTB domain of Shc and this complex is constitutively activated in
some AML. (A) hGM-CSF and CSF-1 induce the association of tyrosine-phosphorylated p140 with the PTB domain of Shc. Cell lysates from
unstimulated (lanes 1, 3, and 5) or hGM-CSF–stimulated (lanes 2, 4, and 6) TF-1 cells or from unstimulated (lane 7) or CSF-1-stimulated (lane
8) BAC1.2F5 cells were immunoprecipitated with anti-Shc antibodies (lanes 1 and 2) or adsorbed with either MBP vector (lanes 5 and 6) or
MBP fusion proteins containing the N-terminal PTB domain of Shc (MBP-N-Shc; lanes 3, 4, 7, and 8). Associated tyrosine-phosphorylated
proteins were analyzed by antiphosphotyrosine immunoblotting. (B) p140 is constitutively associated with the PTB domain of Shc in AML.
Cell lysates of unstimulated primary AML were either incubated with MBP vector protein (AML-3; lane 1) or MBP-N-Shc fusion protein (AML
3 through AML-7; lanes 2 through 6), and the adsorbed proteins were analyzed by antiphosphotyrosine immunoblotting. The AML samples
analyzed in this experiment correspond to the AML-3 through AML-7 samples analyzed in Fig 4B, lanes 2 through 6.
immunoprecipitates (Fig 8A, lane 3), suggesting that the
amount of p140 that can be purified with a bacterially expressed Grb2 fusion protein is higher than by coimmunoprecipitation with anti-Grb2 antibodies. An additional tyrosine-phosphorylated protein of 61 kD was also recognized
by the SH3 domain of Grb2 in AML-3 cells (Fig 8B, lane
2), which may be the same 61-kD protein that was present
in anti-Shc immunoprecipitates from AML-3 (Fig 4B, lane
2).
DISCUSSION
The identification of constitutively activated signalling
pathways involved in transducing an oncogenic signal in
leukemia cells is likely to shed light on the mechanisms of
leukemogenesis and may show new targets for antileukemia
therapy. In this study, we have focused on the analysis of
two adaptor proteins, Shc and Grb2, that are both involved
in the transduction of signals from a variety of activated
receptors to downstream targets. The experiments reported
here show that (1) Shc is constitutively phosphorylated on
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tyrosine in all AML analyzed; (2) a 140-kD tyrosine-phosphorylated protein (p140) is constitutively associated with
Shc and Grb2 in some cases of AML in vivo; (3) the association of p140 with Shc and Grb2 is mediated through the
PTB domain of Shc and the SH3 domains of Grb2, respectively; and (4) additional tyrosine-phosphorylated proteins
of 61 kD and 200 kD become part of the Shc/Grb2 signalling
complex in some AML.
Shc proteins become rapidly tyrosine-phosphorylated
upon stimulation of receptor tyrosine kinases such as the
EGF receptor44 after stimulation of receptors without intrinsic tyrosine kinase activity, such as the EPO receptor, IL-3
receptor, and GM-CSF receptor,55,56,68 and by activated tyrosine kinases such as v-Src or p210Bcr/Abl.54,55 In normal unstimulated PBMCs, we did not detect any tyrosine phosphorylation of Shc, indicating that Shc is not activated in these
cells. The same was true for a factor-dependent erythroleukemia cell line (TF-1), in which no Shc phosphorylation was
detectable after starvation of the cells in factor- and serumdeprived medium. Stimulation of TF-1 cells with hGM-CSF
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SHC/P140/GRB2 COMPLEXES ACTIVATED IN AML
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Fig 6. hGM-CSF induces the association of tyrosine-phosphorylated Shc with Grb2. Cell lysates from unstimulated (lanes 1, 3, and
5) or hGM-CSF–stimulated TF-1 cells (lanes 2, 4, and 6) and from
control NIH 3T3 (lanes 7 and 9) or v-fes –transformed NIH 3T3 cells
(lanes 8 and 10) were analyzed either directly (lanes 1 and 2) or after
immunoprecipitation with anti-Shc (lanes 3, 4, 7, and 8) or anti-Grb2
(lanes 5, 6, 9, and 10) antibodies. Total cell lysates and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using antiGrb2 antibodies (lanes 1 through 10). The positions of Grb2 and the
Ig heavy chain (Ig) are indicated on the left. Molecular weight markers
are indicated on the right.
r
Fig 7. Grb2 associates through its SH2 domain with Shc and
through its SH3 domain with p140, and both interactions are hGMCSF–dependent. (A) Binding of Grb2 to tyrosine-phosphorylated Shc
is mediated through the SH2 domain of Grb2. Cell lysates from unstimulated (lanes 1, 3, and 5) or hGM-CSF–stimulated (lanes 2, 4,
and 6) TF-1 cells were immunoprecipitated (IP) with anti-Shc (lanes
1 and 2) or anti-Grb2 (lanes 3 and 4) antibodies or adsorbed with a
GST fusion protein containing the SH2 domain of Grb2 (Grb2 SH2;
lanes 5 and 6). The precipitates were analyzed by SDS-PAGE followed
by antiphosphotyrosine immunoblotting (lanes 1 through 6). Arrows
indicate the position of the detected phosphotyrosyl proteins and
the GST-Grb2-SH2 fusion protein. (B) Binding of Grb2 to tyrosinephosphorylated p140 is mediated through the SH3 domains of Grb2.
Cell lysates of unstimulated (lanes 1, 3, 5, and 7) or hGM-CSF–stimulated (lanes 2, 4, 6, and 8) TF-1 cells were immunoprecipitated with
anti-Shc antibodies (lanes 1 and 2) or adsorbed with either a GST
fusion protein containing the C-terminal SH3 domain of Grb2 (GST
Grb2 SH3-C; lanes 3 and 4), the N-terminal SH3 domain of Grb2 (GST
Grb2 SH3-N; lanes 5 and 6), or with GST vector protein (GST) (lanes
7 and 8). The precipitates were analyzed by antiphosphotyrosine immunoblotting (lanes 1 through 8).
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JÜCKER, SCHIFFER, AND FELDMAN
resulted in a transient phosphorylation of Shc that returned
to baseline levels after stimulation, indicating that, in this
factor-dependent leukemia cell line, the activation and inactivation of Shc can still be regulated. However, in freshly
isolated samples from AML, Shc proteins were found to be
constitutively phosphorylated on tyrosine in all cases analyzed. This is in agreement with a recent report that the
constitutive phosphorylation of Shc proteins occurs frequently in human tumors including acute leukemias.69 These
data indicate that Shc is activated in AML cells and may
be involved in the constitutive stimulation of a signalling
pathway. This signalling pathway may be the Ras pathway
as discussed below in more detail.
During this analysis we identified three different tyrosinephosphorylated proteins of 200, 140, and 61 kD that formed
a constitutive complex with Shc in AML cells. Constitutive
binding of the 140-kD protein (p140) to Shc was detected in
four of the eight AML analyzed. Comparison of the FrenchAmerican-British (FAB) class and karyotype of the four
AML showing constitutive association of Shc with p140 did
not show a correlation between the appearance of the Shc/
p140 complex and either the FAB class or the chromosomal
abnormalities observed (data not shown). A similar or identical 140-kD phosphoprotein was found to associate with Shc
transiently after stimulation of factor-dependent cell lines
with hGM-CSF or CSF-1, indicating that p140 is involved
in signalling by different types of receptors, ie, cytokine
receptors without intrinsic tyrosine kinase activity and receptor tyrosine kinases. Binding of Shc to a tyrosine-phosphorylated protein of 140-150 kD that might be identical with our
p140 protein has also been detected after stimulation of cells
with a variety of growth factors and cytokines, suggesting
that the association of p140 to Shc is a common event upon
receptor stimulation.53,55,57,70,71
p140 was not recognized by antibodies directed against
known signalling proteins in the same molecular weight
range, suggesting that p140 was a novel signalling protein.
To further characterize this protein, we analyzed the nature
of its interaction with Shc and with Grb2. Although the SH2
domain of Shc can bind to phosphotyrosine sites in activated
receptors and other proteins, the SH2 domain of Shc was
not involved in its association with phosphorylated p140
(data not shown). Using an MBP-N-Shc fusion protein, we
showed that the ligand-dependent binding of p140 to Shc in
factor-dependent cell lines and the constitutive binding of
p140 to Shc in AML cells was mediated instead by the PTB
domain of Shc. Because the PTB domain of Shc binds to an
NPXpY motif in several tyrosine phosphorylated proteins,
we expect that the p140 protein contains an NPXpY motif.
R
Fig 8. The C-terminal SH3 domain of Grb2 binds to tyrosine-phosphorylated p140 in AML. (A) p140 is constitutively associated with
Grb2 in AML cells. Cell lysates from unstimulated (lane 1) or hGMCSF–stimulated TF-1 cells (lane 2) and from unstimulated primary
leukemia cells obtained from five patients with AML (AML-3 through
AML-8; lanes 3 through 8) were immunoprecipitated with anti-Grb2
antibodies followed by antiphosphotyrosine immunoblotting (P.Tyr).
The position of p140 is indicated on the left. (B) The C-terminal SH3
domain of Grb2 binds to p140 in AML cells. Cell lysates from unstimulated primary AML cells were incubated with either GST vector protein (GST; AML-3; lane 1) or with GST fusion proteins containing the
C-terminal SH3 domain of Grb2 (GST-Grb2-SH3; AML-3 through AML8; lanes 2 through 7). Associated tyrosine-phosphorylated proteins
were analyzed by antiphosphotyrosine immunoblotting (lanes 1
through 7). The AML samples analyzed in this experiment correspond
to the AML-3 through AML-8 samples analyzed in (A), lanes 3 through
8, and in Fig 4B, lanes 2 through 7. The position of tyrosine-phosphorylated proteins is indicated on the left. Molecular weight markers are
indicated on the right.
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SHC/P140/GRB2 COMPLEXES ACTIVATED IN AML
2033
Recently, a protein of 145 kD that associates with the PTB
domain of Shc after stimulation with several cytokines has
been cloned and named SHIP for SH2 containing inositol
phosphatase.72,73 This protein contains two NPXpY motifs
and also binds to the C-terminal SH3 domain of Grb2.72
Whether SHIP is identical with our p140 protein has to be
shown in further experiments.
In hGM-CSF–stimulated TF-1 cells, tyrosine-phosphorylated Shc associated with the SH2 domain of Grb2, as has
been shown in other systems.37 During this analysis, we
detected a 140-kD tyrosine-phosphorylated protein in antiGrb2 immunoprecipitates that comigrated with the 140-kD
protein detected in anti-Shc immunoprecipitates, suggesting
that they are identical. Binding of the 140-kD protein to
Grb2 was mediated through both SH3 domains of Grb2. A
protein of 140 kD that coprecipitates with Shc and binds to
the SH3 domains of Grb2 before and after stimulation with
hGM-CSF has been described by Lanfrancone et al.53 However, in our experiments, tyrosine-phosphorylated p140 was
detectable in a complex with Grb2 after stimulation with
hGM-CSF, but not in unstimulated cells. Although the association of phosphorylated p140 with the SH3 domains of
Grb2 in TF-1 cells was dependent on hGM-CSF stimulation,
phosphorylated p140 was constitutively associated with the
SH3 domain of Grb2 in two of six AML analyzed. Interestingly, these were the same two AML in which p140 was
also constitutively bound to the PTB domain of Shc. These
data suggest that once p140 is phosphorylated, it becomes
associated with Shc and Grb2 and that these interactions
can occur either transiently in factor-dependent cell lines or
constitutively in AML cells.
Three major questions about the possible meaning of the
activated Shc complexes in AML cells arise from our results.
First, which are the receptors and/or cytoplasmic tyrosine
kinases that are involved in phosphorylating Shc? Secondly,
which signalling pathway(s) are triggered by the activated
Shc complex? Third, does the constitutively activated Shc
complex have a transforming capacity in myeloid cells that
may contribute to the pathophysiology of AML?
Regarding the identification of the tyrosine kinase that
phosphorylates Shc, it has been recently shown that Shc
proteins are common substrates of several constitutively activated tyrosine kinases in human tumors.69 Recently, we have
detected tyrosine-phosphorylated Shc and p140 in immunoprecipitates of Src family members, ie, Src/Yes and Lyn in
hGM-CSF–stimulated TF-1 cells.64,74 Thus, our data suggest
that members of the Src family of tyrosine kinases may be
involved in the phosphorylation of Shc and/or p140. The
identification of Shc as a Src-SH3 domain binding protein75
is consistent with the idea that Shc may be a direct substrate
of Src family member(s) in human AML.
An important question is which signalling pathway(s) are
triggered in AML by the activated Shc complexes. A large
body of evidence implicates Shc in activation of the Ras
signalling pathway, which plays a central role in relaying
downstream signals from activated receptors on the cell surface, or from activated tyrosine kinases to transcription factors in the nucleus.35,76,77 It has been shown that phosphorylated Shc proteins bind the Grb2-SOS complex that leads to
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activation of Ras37,41 and that the formation of Shc-Grb2
complexes is necessary to induce transformation of immortalized fibroblasts.78 In addition, overexpression of Shc proteins increased the ligand-dependent activation of the Ras
signalling pathway,53 and a dominant negative mutant of
Shc can inhibit the CD4/Lck-mediated activation of Ras.79
It seems therefore possible that the constitutively activated
Shc complexes in AML are involved in activation of the Ras
signalling pathway and that this mechanism of Ras activation
may contribute to the pathophysiology of AML. In preliminary experiments, we have detected activated Raf and MAP
kinases in one case of AML with a constitutive Shc/Grb2/
p140 complex and in another case with a constitutive Shc/
p200 complex, whereas Raf and MAP kinase activation was
not detected in four other cases with activated Shc proteins
(M.J. and R.A.F., unpublished results). These data suggest
that the activated Shc complexes may trigger the Ras signalling pathway in some cases of AML, whereas in other cases,
Shc may activate other signalling pathway(s) that have not
been identified so far. Whether the constitutively activated
Shc complexes have the capacity to transform myeloid cells
and play a role in the pathophysiology of AML will require
further analysis.
ACKNOWLEDGMENT
We thank J.C. Gutheil for generously providing some of the leukemia samples used in this study. We are grateful to M.A. Tate for
excellent technical assistance. We thank A. Miyajima for providing
TF-1 cells, MoAb directed against the b subunit of hGMR, and
recombinant human GM-CSF; J. Schlessinger, E. Lowenstein, and
A. Batzer for the anti-Grb2 MoAb EL6 and the Grb2 SH2 domain;
L. Lanfrancone and P.G. Pelicci for the SH2 domain of Shc; T.
Gustafson for the N-terminal domain of Shc; A.M. Pendergast for
the SH3 domains of Grb2; and E.R. Stanley for recombinant
CSF-1.
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blda
WBS: Blood
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1997 89: 2024-2035
A Tyrosine-Phosphorylated Protein of 140 kD Is Constitutively Associated
With the Phosphotyrosine Binding Domain of Shc and the SH3 Domains of
Grb2 in Acute Myeloid Leukemia Cells
Manfred Jücker, Charles A. Schiffer and Ricardo A. Feldman
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