From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and
erythropoietin (Epo) are hematopoietic growth factors that regulate
proliferation and differentiation of hematopoietic cells. They elicit
and control a cascade of biochemical events, the earliest of which is
tyrosine phosphorylation of several cellular proteins. Grb2/Ash is
composed of SH2 and SH3 domains. The SH2 domain binds to
tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins
containing proline-rich regions. It is considered that Grb2/Ash
functions as an adapter protein linking tyrosine kinases and Ras in
downstream of receptors for growth factors in fibroblasts. However, the
mechanisms of signal transduction through Grb2/Ash and the roles of
proteins associated with Grb2/Ash remain to be determined in
hematopoietic cells. By means of the binding experiments using the
glutathione S-transferase fusion protein including the
full-length Grb2/Ash, we have found that Shc and unidentified 130- and
135-kDa proteins are associated with Grb2/Ash and that they are
tyrosine phosphorylated by treatment with GM-CSF or Epo in a human
leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130)
using the glutathione S-transferase-Grb2/Ash affinity column.
The amino acid sequence analysis of the three peptides derived from the
in situ protease digestion of the purified pp130 showed that
the pp130 was identical to the human c- cbl proto-oncogene
product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of
Grb2/Ash both in vitro and in vivo but not to the SH2
domain of Grb2/Ash, and the binding of Grb2/Ash to c-Cbl or Sos was not
altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine
phosphorylated rapidly and transiently depending on GM-CSF or Epo
stimulation. These findings strongly suggest that c-Cbl is implicated
in the signal transduction of GM-CSF or Epo in hematopoietic cells and
that c-Cbl is involved in another signaling pathway different from the
Ras signaling pathway.
Growth factors elicit and control a cascade of biochemical
events including activation of Ras, Raf-1, and mitogen-activated
protein kinase in a tyrosine kinase-dependent manner, and the earliest
of them is tyrosine phosphorylation of several cellular proteins
through the activation of receptor-type and nonreceptor-type tyrosine
kinases
(1, 2) . Hematopoietic growth factors such as
GM-CSF and Epo also induce tyrosine phosphorylation of some cellular
proteins including the common
It is considered that a primary role of such
tyrosine kinases is to generate tyrosine-phosphorylated recognition
motifs for binding to SH2 domains and to form signal transduction
complexes
(4, 5) . Several SH2-containing proteins have
also catalytic or functional motifs as phospholipase C
The Grb2/Ash is a
25-kDa protein composed of only SH2 and SH3 domains in the order of
SH3-SH2-SH3
(7, 8) . The SH2 domain of Grb2/Ash binds to
tyrosine-phosphorylated proteins such as epidermal growth factor
receptor, Shc, IRS-1, and Syp
(9, 10, 11, 12, 13, 14, 15) .
On the other hand, the SH3 domains of Grb2/Ash bind to the proline-rich
domain of Sos
(16, 17) , dynamin
(18, 19) , and C3G
(20) , which regulate Ras or
Ras-related proteins. As a result of these associations, the Grb2/Ash
seems to be involved in coupling tyrosine kinases to the Ras
regulators. Hematopoietic growth factors induce the tyrosine
phosphorylation of Shc and form the complex of Shc and Grb2/Ash, and
this complex has a key role in Ras activation that is critical for the
proliferation
(14, 21, 22, 23, 24) . These
lines of evidence motivated us to search for signaling molecules that
interact with Grb2/Ash and participate in the signal transduction of
hematopoietic growth factors.
In this work, we have demonstrated
from the in vitro experiments that Grb2/Ash binds to a number
of cytosolic proteins that are tyrosine phosphorylated in response to
the GM-CSF stimulation in a human leukemia cell line, UT-7. Among these
proteins, we have purified pp130 and identified it as the product of
the c- cbl proto-oncogene. c-Cbl has been shown to be a
signaling molecule that binds to Grb2/Ash adapter protein through the
SH3 domain and is tyrosine phosphorylated by GM-CSF or Epo stimulation.
Recently, it was reported that c-Cbl was implicated in T cell receptor
signaling system
(25) , and these findings suggest that c-Cbl is
implicated in the signaling pathways triggered by GM-CSF or Epo in
hematopoietic cells and by stimulation of T cell receptor in T cells.
The c- cbl gene was cloned as the cellular homolog of
the v- cbl oncogene, which is the transforming component of a
murine tumorigenic retrovirus, CAS NS-1
(31) . The v- cbl oncogene is transforming in early B-lineage, and myeloid cells and
Gag-v-Cbl transforming protein lacks the C-terminal 62% of c-Cbl
containing the proline-rich region and leucine zipper motif but still
possesses high proportion of basic amino acids
(28) . However,
the biological roles of c-Cbl remain to be elucidated. In this work, we
have found that pp130 is constitutively associated with Grb2/Ash and
becomes tyrosine phosphorylated depending on GM-CSF or Epo stimulation
of UT-7 cells. Therefore, we purified the pp130 and identified it as
the proto-oncogene product of c- cbl.
We have shown that the
c-Cbl (pp130) is constitutively associated with Grb2/Ash through the
SH3 domain of Grb2/Ash in vitro and in vivo. This
association is supported from the observation that c-Cbl has
proline-rich sequences resembling the proposed consensus sequence for
the Abl SH3-binding region and the Grb2/Ash binding sites on mouse Sos
1 (Fig. 2 C). Moreover, it has been recently reported
that Nck, an adapter protein that has three SH3 domains, binds to the
product of proto-oncogene c- cbl in HL-60 promyelocytic
leukemia cell
(32) and that c-Cbl binds to the GST-Grb2/Ash
fusion protein in Jurkat T cell
(25) . The SH3 domain of
Grb2/Ash binds to the proline-rich domains of dynamin
(18, 19) , C3G
(20) , and Sos
(17) , which
regulate the small G-proteins. By forming these complexes, Grb2/Ash
plays important roles in Ras activation elicited by growth factors.
However, we could not find the homologous regions with guanine
nucleotide exchange factors or GTPase-activating protein in c-Cbl, as
described by Blake
(28) , unlike C3G and Sos. Moreover, tyrosine
phosphorylation of dynamin, C3G, and Sos was not reported, but c-Cbl
was tyrosine phosphorylated by GM-CSF or Epo stimulation, and, as shown
in Fig. 3, the binding of Grb2/Ash to c-Cbl or Sos was not
altered by GM-CSF stimulation. Therefore, it is likely that by the
association with c-Cbl, Grb2/Ash might also link a different signaling
pathway from the pathway regulating G-proteins and that these two
different pathways are mutually independent. By the association of
Grb2/Ash with Sos, Sos was translocated to the plasma membrane, where
Ras was anchored, and worked as a guanine nucleotide exchange factor
(33) . It is also possible that the localization of c-Cbl is
defined by its binding to Grb2/Ash. The product of gag-v- cbl lacks the proline-rich region of c-Cbl
(28) ; therefore, it
cannot bind to Grb2/Ash, resulting in the improper localization of the
oncogene product. This may contribute to its tumorigenicity.
As
shown in Figs. 1 and 4, c-Cbl (pp130) was tyrosine phosphorylated
depending on GM-CSF or Epo stimulation. The comparison of the
tyrosine-phosphorylated level of c-Cbl in c-Cbl immunoprecipitates in
Fig. 4
with that in GST-Grb2/Ash precipitates in
Fig. 1
suggests that nearly all of the tyrosine-phosphorylated
c-Cbl seems to be complexed with Grb2/Ash. It is interesting what
protein-tyrosine kinase is responsible for the phosphorylation of
c-Cbl. One candidate is c-Fes because we reported that GM-CSF, IL-3, or
Epo activates c-Fes in TF-1 human leukemia cells
(34, 35) . Jak2, Lyn, and Tec were also candidates
because these tyrosine kinases were tyrosine phosphorylated and
activated by hematopoietic growth factors including Epo, GM-CSF, or
IL-3
(36, 37, 38, 39) . However, we did
not find the potential association of c-Cbl with these kinases. The
role of tyrosine phosphorylation of c-Cbl is not clear, but several
reports suggest the importance of tyrosine phosphorylation of c-Cbl on
its tumorigenesis. In the 70Z/3 pre-B lymphoma, a mutant form of c-Cbl,
which deleted 17 amino acids, became tyrosine phosphorylated and
activated to have its tumorigenic potential. On the other hand, c-Cbl
was tyrosine phosphorylated in tumor cells expressing the product of
v- abl or bcr-abl and co-immunoprecipitated with
anti-Abl
(40) . These observations indicate that tyrosine
phosphorylation of c-Cbl could activate the signaling pathway
downstream of c-Cbl.
It is unique that c-Cbl is tyrosine
phosphorylated by the stimulation with GM-CSF and Epo because other
SH3-binding proteins including Sos, C3G, and dynamin are not tyrosine
phosphorylated. Therefore, it is possible that c-Cbl binds to
SH2-containing proteins by GM-CSF or Epo stimulation. Although we have
not detected the association of c-Cbl with the SH2 domains of Grb2/Ash
or Shc, c-Cbl has specific peptide sequences resembling the proposed
sequences of binding sites for the SH2-containing proteins YEEN (aa
141-144) for the Src family SH2, YDEV (aa 274-277) for the
Nck SH2, and NAIY (aa 671-674) for the Shc SH2
(5, 41) . This possibility was also suggested by the
report that showed the product of the c- cbl proto-oncogene can
bind to the GST-SH2 domain of Fyn, Lck, Blk, GTPase-activating protein,
and phospholipase C
UT-7 was a kind gift from Dr. Norio Komatsu (Jichi
Medical School, Tochigi, Japan). Polyclonal anti-Grb2/Ash antibody and
4G10 were generously provided by Dr. Tadaomi Takenawa (University of
Tokyo) and by Dr. Deborah K Morrison (NCI, National Institutes of
Health), respectively.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
chain of GM-CSF/interleukin-3
(IL-3)
(
)
/IL-5 receptors, Shc, Vav, Raf, and
mitogen-activated protein kinase
(3) . Therefore, certain
nonreceptor-type tyrosine kinases have to be activated by GM-CSF and
Epo because the GM-CSF and Epo receptors has no intrinsic activity of
tyrosine kinase.
or
GTPase-activating protein for Ras. However, other SH2-containing
proteins may serve simply as adapter molecules to form a complex with
signaling molecules such as Grb2/Ash, which binds to Sos leading to the
Ras activation, and p85, which binds to the catalytic p110 subunit of
the phosphatidylinositol 3-kinase
(6) .
Cell Lines and Growth Factors
UT-7
cells were maintained in RPMI 1640 medium containing 8% bovine serum
and 10 ng/ml GM-CSF. Recombinant human GM-CSF was supplied by Kirin
Brewery Co. Ltd. (Tokyo), and recombinant human Epo was delivered by
Chugai Pharmacy Co. Ltd. (Tokyo).
Antibodies
Polyclonal rabbit anti-c-Cbl
antibody (c-15), purchased from Santa Cruz Biotechnology (Santa Cruz,
CA), was used for the immunoprecipitation and the immunoblotting of
c-Cbl. Polyclonal rabbit antibody to Grb2/Ash (c-23) purchased from
Santa Cruz Biotechnology and monoclonal antibody to Grb2/Ash purchased
from MBL (Nagoya, Japan) were used for the immunoprecipitation and the
immunoblotting of Grb2/Ash, respectively. Protein A-purified polyclonal
rabbit anti-Sos antibody was generously provided by Dr. Y. Kaburagi
(University of Tokyo) and used for the immunoprecipitation and the
immunoblotting of Sos. A mouse monoclonal anti-phosphotyrosine antibody
(anti-Ptyr) 4G10 was used for the immunoblotting of
phosphotyrosine-containing proteins.
GST Fusion Proteins
The bacterial
expression plasmids coding GST fusion proteins containing the
full-length and the N-terminal SH3 domain or the SH2 domain of Grb2/Ash
(19) were generously provided by Dr. T. Takenawa (University of
Tokyo). These plasmids were transformed into XL I-Blue strain of
Escherichia coli, and the resulting transformants were induced
with isopropyl-1-thio--D-galactopyranoside to produce GST
fusion proteins. The bacteria were collected by centrifugation and
resuspended in the E. coli lysis buffer containing 40
mM Tris/HCl (pH 7.5), 5 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, and 1% Triton X-100. Vigorous sonication
was performed before centrifugation at 25,000
g for 20
min. The resulting supernatants were saved as crude extracts containing
GST fusion proteins.
Preparation of Cell Lysates
UT-7 cells
were incubated in RPMI 1640 medium containing 0.1% bovine serum albumin
without serum or growth factors for 8-15 h prior to stimulation
with growth factors and then resuspended in RPMI 1640 medium containing
100 µM NaVO
. The cells were
treated with 10 ng/ml GM-CSF or 20 units/ml Epo for 5 min at 37 °C
and then lysed at 4 °C in the lysis buffer containing 20
mM Tris/HCl (pH 8.0), 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 500 units/ml aprotinin, 2 mM
EDTA, 50 mM NaF, and 1 mM
Na
VO
. Unsolubilized materials were removed by
centrifugation at 15,000
g at 4 °C for 10 min.
Binding of Cellular Proteins to GST Fusion
Proteins
Lysates from 1 10
cells were
mixed with 40 µg of the fusion protein noncovalently coupled to
glutathione-agarose beads (Sigma) for 3 h at 4 °C. Beads were
washed with the lysis buffer before resuspension in Laemmli's
sample buffer.
Immunoprecipitation
To immunoprecipitate
c-Cbl, lysates from 1 10
cells were mixed with
polyclonal anti-c-Cbl antibody for 3 h at 4 °C The
immunoprecipitates were collected with protein A-Sepharose (Sigma). All
the immunoprecipitates were intensively washed with the lysis buffer
before resuspension in Laemmli's sample buffer.
Immunoblotting
Samples were subjected to
SDS-PAGE and electrotransferred onto polyvinylidene difluoride filters
(Millipore). Filters were blocked with the buffer containing 10
mM Tris/HCl (pH 7.4), 150 mM NaCl, 5% bovine serum
albumin, and 0.05% Triton X-100. For the immunoblotting of
phosphotyrosine-containing proteins or Grb2/Ash, filters were incubated
with 4G10 or monoclonal anti-Grb2/Ash antibody, respectively, and then
with goat alkaline phosphatase-conjugated anti-mouse IgG (Fc) antibody
(Promega). For the immunoblotting of c-Cbl, filters were sequentially
incubated with the polyclonal anti-c-Cbl antibody and with goat
alkaline phosphatase-conjugated anti-rabbit IgG (Fc) antibody
(Promega). After each incubation, filters were washed three times in
the buffer containing 10 mM Tris/HCl (pH 7.4), 150 mM
NaCl, and 0.05% Triton X-100. Color reaction was performed using nitro
blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Promega).
Purification of pp130
pp130 was purified
from UT-7 cell lysates by affinity columns. After pretreatment of
lysates from 1 10
cells with GST protein
noncovalently coupled to glutathione-agarose beads, lysates were mixed
with GST-Grb2/Ash fusion protein noncovalently coupled to
glutathione-agarose beads. Beads were washed with the lysis buffer and
eluted with 50 mM Tris/HCl (pH 9.6) containing 20 mM
reduced glutathione. The fractions containing pp130 were subjected to
SDS-PAGE and blotted onto a ProBlott membrane (Applied Biosystems,
Foster City, CA). After being visualized by Ponceau S, an isolated
130-kDa band (
5 pmol) was cut out and in situ digested
with Achromobactor protease I (lysylendopeptidase; Wako,
Tokyo). Sequences of three digested fragments were determined by the
amino acid sequence analysis. Homology search was carried out using the
Entrez program of NCBI, which includes Genbank, EMBL, DDBJ, PIR, and
SWISS-PROT data bases.
pp130 Binds to Grb2/Ash and GM-CSF Induces Tyrosine
Phosphorylation of pp130
As shown in
Fig. 1A, several tyrosine-phosphorylated proteins from
UT-7 cell lysates bound to Grb2/Ash in vitro, and this
phosphorylation was depending upon GM-CSF stimulation. Using anti-Shc
polyclonal antibody, the 52-kDa (pp52) and 66-kDa proteins (pp66) were
identified as Shc, which is one of the well known molecules that bind
to Grb2/Ash and has an important role in the signal transduction of
epidermal growth factor, Epo, and IL-3
(14, 26) (data
not shown). However, it is shown that neither pp130 nor pp135 was Sos,
dynamin, or C3G, which is reported to bind to Grb2/Ash, from the
results of experiments using the specific antibodies to each protein
(data not shown). In addition, pp130 and pp135 was not the chain
of the GM-CSF receptor or phospholipase C
. We then have determined
which domain of Grb2/Ash bound to pp130 or pp135 using GST fusion
proteins with the SH2 or the N-terminal SH3 of Grb2/Ash. As shown in
Fig. 1B, both pp130 and pp135 were associated with the
N-terminal SH3 domain but not with the SH2 domain of Grb2/Ash.
Conversely, Shc (pp66 and pp52) was shown to bind to the SH2 domain of
Grb2/Ash as described before
(14, 26) . When pp130 and
pp135 bound not only to GST-full-length Grb2/Ash fusion protein but
also to the N-terminal SH3 domain of Grb2/Ash, tyrosine phosphorylation
of these proteins was dependent on the treatment with GM-CSF. In
another human leukemia cell line, F36E, which proliferates in an
Epo-dependent manner
(27) , Shc, pp130, and pp135 were also
associated with the Grb2/Ash and became tyrosine phosphorylated by the
treatment with Epo (data not shown).
Figure 1:
A, proteins
associated with Grb2/Ash in vitro and their tyrosine
phosphorylation. The lysates from UT-7 cells unstimulated ( lane2) or stimulated with GM-CSF ( lane1)
were mixed with the GST fusion protein including the full-length
Grb2/Ash noncovalently coupled to glutathione-agarose beads. Beads were
resuspended in Laemmli's sample buffer, subjected to SDS-PAGE,
and immunoblotted with anti-Ptyr (4G10). Molecular mass markers,
indicated at the left, are given in kDa. The arrows indicate the positions of pp52, pp66, pp130, and pp135.
B, in vitro association of pp130 with the SH3 domain
of Grb2/Ash and tyrosine phosphorylation of pp130. The lysates from
UT-7 cells unstimulated ( lanes1 and 3) or
stimulated with GM-CSF ( lanes2 and 4) were
mixed with GST fusion protein including the SH2 domain ( lanes1 and 2) or the N-terminal SH3 domain ( lanes3 and 4) of Grb2/Ash noncovalently coupled to
glutathione-agarose beads. The resulting precipitates were resuspended
in Laemmli's sample buffer, subjected to SDS-PAGE, and
immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated
at the left, are given in kDa. The arrows indicate
the positions of pp52, pp66, pp130, and
pp135.
Identification of pp130 as the c-cbl Proto-oncogene
Product
To identify the Grb2/Ash-binding protein, pp130, a
large scale affinity purification was carried out using GST-Grb2/Ash
fusion protein. After the lysates from 1 10
UT-7
cells were pretreated with GST protein to preclear proteins associated
with GST, they were mixed with the GST-Grb2/Ash fusion protein
noncovalently coupled to glutathione-agarose beads. Successively, the
beads were washed and eluted with the elution buffer containing reduced
glutathione. The fractions containing pp130 were subjected to SDS-PAGE
and blotted onto the polyvinylidene difluoride membrane. After being
visualized by Ponceau S, an isolated 130-kDa band (
5 pmol) was cut
out and in situ digested with lysylendopeptidase
Achromobactor protease I. The sequences of the three obtained
fragments determined by amino acid sequence analysis were (K)TIVPWK
(P1), (K)ALVIAQNNIEMA (P2), and (K)NILREFVS (P3). These sequences were
used to search for homology with other known proteins, and all three
peptides were shown to be identical to the equivalent sequence of the
c- cbl proto-oncogene product (P1 = aa 197-203, P2
= aa 876-888, and P3 = aa 889-897)
(28) . Therefore, we concluded that the Grb2/Ash-binding
protein, pp130, is the human c- cbl proto-oncogene product.
Constitutive Binding of the c-cbl Proto-oncogene
Product and Grb2/Ash through the SH3 Domain
We then tried
to confirm the association between the c- cbl proto-oncogene
product and Grb2/Ash. As shown in Fig. 2 A, Western
blotting analysis revealed that anti-c-Cbl antibody recognized a
130-kDa molecule both in the UT-7 cell lysates and in
immunoprecipitates with anti-c-Cbl antibody. The c-Cbl was detected
after incubating the lysates with GST-full-length Grb2/Ash fusion
protein but not detected when GST protein was used instead of
GST-Grb2/Ash. Moreover, as shown in Fig. 2 B, the GST
fusion protein including the N-terminal SH3 domain of Grb2/Ash also
bound to c-Cbl, but GST protein and the GST-SH2 domain of Grb2/Ash
fusion protein did not. The association of c-Cbl with the full-length
or the N-terminal SH3 domain of Grb2/Ash was independent on GM-CSF
stimulation. These results show that the association of the c- cbl proto-oncogene product and Grb2/Ash is constitutive through the
N-terminal SH3 domain of Grb2/Ash. The c-Cbl has a proline-rich domain
(28) , and we could find in this domain two sequences that
resemble the proposed consensus sequence for the Abl SH3-binding site
(29) and the Grb2/Ash-binding sites on mouse Sos1
(30) (Fig. 2 C). Therefore, it is considered that
c-Cbl binds to the SH3 domain of Grb2/Ash through these two
proline-rich sequences.
Figure 2:
A,
in vitro association of c-Cbl and Grb2/Ash. The lysates from
UT-7 cells unstimulated ( lanes1-4) or
stimulated with GM-CSF ( lane5) were prepared. After
the lysates were mixed with anti-c-Cbl ( lane2), GST
protein ( lane3), and GST fusion protein including
full-length Grb2/Ash ( lanes4 and 5), the
resulting precipitates and the total UT-7 cell lysate ( TCL,
lane1) were subjected to SDS-PAGE and immunoblotted
with anti-c-Cbl. Molecular mass markers, indicated at the
left, are given in kDa. The arrow indicates the
position of c-Cbl. B, in vitro association of c-Cbl
with the SH3 domain of Grb2/Ash. The lysates from UT-7 cells
unstimulated ( lanes3, 5, and 6) or
stimulated with GM-CSF ( lanes2 and 4) were
mixed with GST fusion protein including the N-terminal SH3 domain
( lanes2 and 3), the SH2 domain ( lanes4 and 5) of Grb2/Ash, and GST ( lane6). The resulting precipitates and the total UT-7 cell
lysate ( TCL, lane1) were subjected to
SDS-PAGE and immunoblotted with anti-c-Cbl. Molecular mass markers,
indicated at the left, are given in kDa. The arrow indicates the position of c-Cbl. C, alignment of the
proline-rich sequences of c-Cbl with the consensus sequence for Abl
SH3-binding region and four proline-rich GRB2/ASH binding sequences of
mouse ( m) Sos1. The amino acid sequences shown as Cbl 1 and
Cbl 2 are from aa 489-498 and aa 528-537, respectively.
and X in the consensus sequence represent hydrophobic
and nonconserved amino acid residues,
respectively.
In Vivo Association between the c-cbl Proto-oncogene
Product and Grb2/Ash
It is important to know whether c-Cbl
is associated with Grb2/Ash in vivo. Anti-c-Cbl
immunoprecipitates from lysates of the UT-7 cells unstimulated or
stimulated with GM-CSF were subjected to SDS-PAGE and immunoblotted
with anti-Grb2/Ash antibody (Fig. 3 A). Grb2/Ash was
shown to be co-immunoprecipitated with c-Cbl from both lysates of
GM-CSF-stimulated or unstimulated cells. Conversely, c-Cbl has not been
detected in immunoprecipitates with anti-Grb2/Ash antibody probably
because the antibody we used could immunoprecipitate Grb2/Ash and might
inhibit the association of Grb2/Ash and c-Cbl. Moreover, the amounts of
c-Cbl expression and Grb2/Ash bound to c-Cbl were not affected by
GM-CSF stimulation (Fig. 3 A). Sos is a well known
protein that binds to the SH3 domain of Grb2/Ash and regulates the
activation of Ras
(17) . Similarly to c-Cbl, Sos was
constitutively associated with Grb2/Ash, and the amounts of Sos
expression, Sos bound to Grb2/Ash, and Grb2/Ash bound to Sos were not
affected by GM-CSF stimulation (Fig. 3 B). These results
suggest that the binding of Grb2/Ash to c-Cbl or Sos was not altered by
GM-CSF stimulation.
Figure 3:
A, in vivo association of c-Cbl
with Grb2/Ash. The lysates from UT-7 cells stimulated with GM-CSF
( lanes3 and 6) or unstimulated ( lanes4, 5, and 7) were mixed with anti-c-Cbl
( lanes3, 4, 6, and 7) or
normal rabbit serum ( NRS, lane5). The
resulting precipitates were subjected to SDS-PAGE and immunoblotted
with anti-Grb2/Ash ( lanes3-5) or anti-Cbl
( lanes6 and 7). The total UT-7 cell lysate
( TCL, lane1) and the immunoprecipitates
with anti-Grb2/Ash ( lane2) from UT-7 cell lysates
were also applied for reference. Molecular mass markers, indicated at
the left, are given in kDa. The arrow indicates the
position of Grb2/Ash or c-Cbl. B, in vivo association
of Sos with Grb2/Ash. The lysates from UT-7 cells stimulated with
GM-CSF ( lanes1, 3, and 5) or
unstimulated ( lanes2, 4, and 6)
were mixed with anti-Grb2/Ash ( lanes1 and
2) or with anti-Sos ( lanes3-6). The
resulting precipitates were subjected to SDS-PAGE and immunoblotted
with anti-Sos ( lanes1-4) or anti-Grb2/Ash
( lanes5 and 6). The arrow indicates the position of Sos or
Grb2/Ash.
GM-CSF and Epo Stimulation Induce Tyrosine
Phosphorylation of c-Cbl
Next, we examined whether the
tyrosine residues of c-Cbl were phosphorylated and whether the
phosphorylation was dependent on the factor stimulation. Lysates from
UT-7 cells with or without the stimulation with GM-CSF or Epo were
immunoprecipitated with anti-c-Cbl antibody, and the immunoprecipitates
were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibody (4G10). As shown in Fig. 4 A, c-Cbl was tyrosine
phosphorylated, and this phosphorylation was induced by the stimulation
with GM-CSF or Epo. As shown in Fig. 4 B, c-Cbl was
tyrosine phosphorylated within 2 min after GM-CSF stimulation of the
cells at 37 °C, attained the maximum level of phosphorylation at 5
min, and was dephosphorylated within 30 min.
Figure 4:
A,
GM-CSF and Epo induce tyrosine phosphorylation of c-Cbl. The lysates
from UT-7 cells stimulated with GM-CSF ( lane3), Epo
( lane2), or unstimulated ( lane1)
were mixed with anti-c-Cbl, and immunoprecipitates were subjected to
SDS-PAGE and immunoblotted with anti-Ptyr (4G10). Molecular mass
markers, indicated at the left, are given in kDa. The
arrow indicates the position of c-Cbl. B, time course
of tyrosine phosphorylation of c-Cbl. UT-7 cells were stimulated with
GM-CSF for indicated times at 37 °C. The cell lysates were mixed
with anti-c-Cbl, and immunoprecipitates were subjected to SDS-PAGE and
immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated
at the left, are given in kDa. The arrow indicates
the position of c-Cbl.
fusion proteins after tyrosine phosphorylation
of c-Cbl in T cell receptor-mediated activation
(25) . In the
report, it was demonstrated that c-Cbl constitutively binds to Grb2/Ash
and becomes tyrosine phosphorylated by the T cell receptor-mediated
activation. It was also mentioned that phosphorylation of c-Cbl by the
activation might induce interaction with SH2-containing proteins and/or
affect associations mediated by SH3 domains as suggested by the
decrease in binding to the SH3 domain of Grb2/Ash
(25) . In our
studies, the stimulation with GM-CSF did not affect the association of
c-Cbl and Grb2/Ash in vitro and in vivo, and this may
be due to the difference in the associated molecules involved in the
signal transduction pathways between the T-cell receptor and the
GM-CSF/Epo receptor. These findings demonstrate that c-Cbl contributes
to the signal transduction triggered by hematopoietic growth factors
such as GM-CSF and Epo as well as T-cell receptor-mediated activation.
It is important to search for signaling molecules downstream of c-Cbl
and mechanisms of tumorigenesis by abnormalities of c-Cbl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.