From the Department of Pathology,
§ University of Colorado Cancer Center, University of
Colorado Health Sciences Center, Denver, Colorado 80262 and the
¶ Department of Pathology, University of Western Australia,
Nedlands, Australia
Received for publication, October 13, 2000
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ABSTRACT |
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The v-Cbl oncogene induces myeloid and B-cell
leukemia; however, the mechanism by which transformation occurs is not
understood. An oncogenic form of c-Cbl (Cbl- The c-Cbl proto-oncogene has attracted considerable attention in
recent years, since it becomes phosphorylated on tyrosine residues
following activation of a wide variety of cell surface receptors.
Receptors whose activation induces the phosphorylation of Cbl include
the T cell receptor (1-6), the B cell receptor (7, 8), the Fc receptor
(9), and the receptors for numerous growth factors including epidermal
growth factor (10-14), colony-stimulating factor-1 (15),
erythropoietin (16, 17), interleukin-3
(IL-3)1 (16, 18),
granulocyte-macrophage colony-stimulating factor (17), thrombopoietin
(19), and prolactin (20). Cbl is a 120-kDa protein with 22 tyrosine
residues, an amino-terminal region able to bind to phosphotyrosine (21,
22), and a proline-rich region with numerous Src homology 3 binding
sites. The large size of the protein suggests that it may function as a
large adapter or scaffolding molecule that could regulate the
activation of other downstream signaling molecules in a manner similar
to that of insulin-receptor substrate-1 (23). Other studies have
suggested that Cbl functions as a negative regulator of signaling,
perhaps by stimulating the down-regulation of growth factor receptors (24, 25). Several recent studies have suggested that down-regulation of
growth factor receptors requires the RING finger motif of Cbl functioning as an E2-dependent ubiquitin-protein ligase inducing the ubiquitination of growth factor receptors (24, 26-29). Src-like kinases (3, 7, 18, 30-32), Syk and/or ZAP-70 (4, 21, 33),
phosphatidylinositol 3-kinase (PI 3-kinase) (2, 3, 7, 18, 31,
34, 35), and several small adapter proteins including Crk, Shc, and
Grb2 (1, 3, 7, 8, 35-37) have been observed to be associated with Cbl
in either a constitutive or ligand-induced manner. It has been
suggested that either the Syk/ZAP-70 family of tyrosine
kinases (38) or Src-like kinases (38-41) might be responsible for the
phosphorylation of Cbl. We have suggested that the phosphorylation of
Cbl could regulate the activation of PI 3-kinases in response to
certain stimuli such as cytokines like IL-3 and prolactin (18, 20, 41). Since phosphatidylinositol 3-kinase is thought to regulate the antiapoptotic protein kinase Akt (42-48), Cbl might represent an integration point for proliferative and antiapoptotic signaling pathways. Evidence demonstrating that Cbl plays a critical role in
these functions is lacking, particularly since mice bearing homologous
loss of both alleles encoding c-Cbl have a relatively mild phenotype
(49).
Cbl was first discovered as the oncogene present in the Cas NS-1
retrovirus, which has been shown to induce B-cell lymphomas and myeloid
leukemia in mice (50). The v-cbl oncogene is also capable of
transforming NIH3T3 cells in vitro, and these transformed cells are tumorigenic when injected into nude mice (51). The v-Cbl
oncogene consists of the amino-terminal end of the gag gene fused to the amino-terminal end of c-Cbl encompassing the
amino-terminal phosphotyrosine binding region but lacking the ring
finger motif, the proline-rich region, and the C-terminal end, which
contains several major phosphorylation sites (50, 52). Other oncogenic forms of c-Cbl have been discovered (51). The murine pre-B cell line
70Z expresses an oncogenic form of Cbl in which 17 amino acids, from
366 to 382, have been deleted (51). Studies by Langdon and colleagues
have demonstrated that deletion of either of two single amino acids
within this 17-amino acid region results in the oncogenic activation of
c-Cbl (51). In this study, we have utilized one of these mutants,
Cbl The expression of oncogenes in hematopoietic cells results in numerous
changes, including induction of growth factor-independent proliferation
(53-56), inhibition of terminal differentiation (53, 57, 58), and
suppression of apoptosis (59, 60). Recent studies have demonstrated
that the expression of oncogenic forms of Cbl in fibroblasts results in
an increase in the number of tyrosine-phosphorylated proteins,
suggesting that one or more tyrosine kinases have been activated by
v-Cbl (61). We have previously studied the ability of two activated
tyrosine kinases, v-Src and BCR-ABL, to induce growth
factor-independent proliferation of growth factor-dependent
cell lines (53, 54). In both cases, we were able to provide evidence
that these oncogenes induced the autocrine production of growth factors
such as IL-3 or granulocyte-macrophage colony-stimulating factor. These
oncogenes are also able to block the ability of granulocyte
colony-stimulating factor to induce terminal differentiation of
hematopoietic progenitor cell lines such as 32Dcl3 (53, 57). Therefore,
we hypothesized that oncogenic forms of Cbl would induce growth
factor-independent proliferation of factor-dependent cells
in a manner like that observed with v-Src. Contrary to our expectation,
we observed instead that the Cbl Cells and Cell Culture--
The 32Dcl3 cell line was obtained
from Dr. Joel Greenberger (University of Pittsburgh), and their
cultivation has been described recently (18). Fetal calf serum was from
Summit Biotechnology (Fort Collins, CO). Recombinant murine IL-3
(mIL-3) was obtained from Becton-Dickinson/Collaborative Biotechnology,
Inc. (Franklin Lakes, NJ). All other media components were from Life
Technologies, Inc.
32Dcl3 cells expressing the various mutants of c-Cbl were generated by
introducing the plasmid DNAs of interest into 32Dcl3 cells with a
Cell-Porator (1000 V/cm at 800 microfarads) (Life Technologies).
Electroporation chambers used had a 0.4-cm gap between the electrodes,
and cells were resuspended in Dulbecco's modified Eagle's medium
without any additional supplements at room temperature. The Cbl
Cells were cultured for 16 h in media supplemented with 7.5%
fetal calf serum to reduce the basal level of tyrosine-phosphorylated proteins prior to stimulation with recombinant mIL-3 for the indicated periods of time. In one set of experiments, cells were removed from
IL-3 and starved for 0-8 h to determine the kinetics with which
phosphorylated proteins disappeared.
Immunoprecipitation and Immunoblotting--
Cells to be
immunoprecipitated were lysed in EB (50 mM NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA, 50 mM
NaF, 1% Triton X-100, 1 mM sodium orthovanadate with 100 units/ml Kallikrein inhibitor), and the lysates were clarified by
spinning at 13,000 rpm in a Savant RCF13K refrigerated microcentrifuge
for 30 min. A 1-µg amount of the indicated antibody was added to a
cell lysate made from 2 × 107 32Dcl3 cells in a final
volume of 1 ml and placed on a rocking platform for 1 h at
4 °C. The immune complexes were collected by adding 30 µl of
Pansorbin (Calbiochem) to each immunoprecipitate for 1 h.
The bound proteins were washed three times with lysis buffer, and the
immunoprecipitated proteins were resolved by SDS-polyacrylamide gel
electrophoresis. The resolved proteins were electrotransferred to
Immobilon membranes (Millipore Corp., Bedford, MA). Detection of
proteins by immunoblotting was conducted using the enhanced chemiluminescence lighting (ECL) system according to the
manufacturer's recommendations (Amersham Pharmacia Biotech).
Agarose-conjugated anti-phosphotyrosine monoclonal antibody 4G10,
rabbit anti-JAK2, and sheep anti-Akt were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-Akt was obtained
from New England Biolabs (Beverly, MA), and anti-phospho-ERK was
obtained from Promega Biotechnology (Madison, WI). Polyclonal
antibodies directed against Cbl, ERK1/2, Bcl-2, Bax,
Bcl-XL, Bak, and A1 were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibodies to Mcl1 and
Bad were obtained from Transduction Laboratories (Lexington, KY). A
monoclonal antibody directed against the influenza HA epitope tag
(clone 12CA5) was obtained from Roche Molecular Biochemicals. A
monoclonal antibody 4G10 directed against phosphotyrosine was kindly
provided by Dr. Brian Druker (University of Oregon Health Sciences
Center, Portland, OR). A rabbit polyclonal antibody directed against
STAT5 was provided by Andrew Larner (Cleveland Clinic, Cleveland, OH).
Nonimmune rabbit serum was obtained from our own nonimmunized animals.
Proliferation Assays--
Cultures were initiated at 2 × 105 cells/ml in medium that either contained or lacked 50 units/ml recombinant murine IL-3 (rmIL-3). The proliferation of 32Dcl3
cells was monitored by direct counting of viable cells, which excluded
trypan blue. Results were plotted as the number of viable cells
at each time point examined. In one study, 100 nM
wortmannin (Calbiochem) was added to each set of cells, and the
proliferation of cells was monitored as described. Controls for this
study included cultures with the same concentration of ethanol, the
solvent in which the wortmannin was dissolved, as that present in the
wortmannin-treated cultures.
Cell Cycle Analysis--
Cells were cultured in the presence or
absence of 50 units/ml rmIL-3 for varying periods of time. Samples of
cells were withdrawn at varying time points, and the cells were
pelleted by centrifugation at 1,500 rpm for 5 min in a refrigerated
centrifuge. The supernatant fluid was removed, and the pellet was
washed once with phosphate-buffered saline. The supernatant fluid was
removed, and the pellet was vortexed briefly. A 0.5-ml volume of
saponin/propridium iodide solution was added (PBS containing 0.3%
saponin, 25 µg/ml propidium iodide, 10 mM EDTA,
0.2 mg/ml RNase) and incubated for 10 min at room temperature.
Stained cells were stored for up to 24 h at 4 °C before
analysis. Ten thousand stained cells were analyzed with a Coulter XL
flow cytometer (Hialeah, FL). Cell cycle modeling was performed with
the ModFit software package (Verity House Software, Topsham, ME).
Assay for Caspase-3 Activity--
The Caspase-3 Cellular
Activity Assay Kit PLUS (Catalogue number AK-703) from BIOMOL (Plymouth
Meeting, PA) was used according to the manufacturer's suggestions. At
varying time points, a sample of cells used was removed and pelleted by
centrifugation. The cells were lysed in cell lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol). Following complete lysis of the cells, the lysate was clarified by
centrifugation at 10,000 × g for 10 min at 4 °C,
and the supernatant was frozen at To determine whether oncogenic forms of Cbl could induce 32Dcl3
cells to become growth factor-independent, these cells were transfected
with vectors encoding a panel of Cbl mutants, and single cell clones
were isolated following growth in soft agar. The oncogenic forms used
included v-Cbl, which is present in the Cas-NS-1 retrovirus (50); the
70Z form of c-Cbl, which lacks a 17-amino acid region at the N-terminal
of the ring finger motif (51); and two deletion mutants lacking either
tyrosine residue Tyr368 or Tyr371, which are
also oncogenic (51) (Fig. 1). In this
study, we focused upon the CblY371) was expressed in
the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to
determine whether it was able to induce growth factor-independent
proliferation. We were unable to isolate clones of transfected 32Dcl3
cells expressing Cbl-
Y371 that proliferated in the absence of IL-3.
In contrast, 32Dcl3/Cbl-
Y371 cells did not undergo apoptosis like
parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/Cbl
Y371 cells arrested in G1 when cultured
in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured
in the absence of IL-3 for 24 h were present in a
sub-G1 fraction, while only 4% of the 32D/Cbl-
Y371 and
2% of the 32D/Bcl-2 cells were found in a sub-G1 fraction.
There was no difference in the pattern of tyrosine-phosphorylated
proteins observed following stimulation of either cell type with IL-3.
The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical
in both cell types. No differences were detected in the activation of
Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-
Y371
cells compared with parental 32Dcl3 cells. Likewise, there was no
difference in the pattern of phosphorylation of JAK2, STAT5, ERK1,
ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from
medium containing IL-3. The protein levels of various Bcl-2
family members were examined in cells grown in the absence or presence
of IL-3. We observed a consistent increased amount of Bcl-2 protein in
five different clones of 32D/Cbl-
Y317 cells. These data
suggest that the Cbl-
Y371 mutant may suppress apoptosis by a
mechanism that involves the overexpression of Bcl-2. Consistent with
this result, activation of caspase-3 was suppressed in 32D/Cbl-
Y371
cells cultured in the absence of IL-3 compared with 32Dcl3 cells
cultured under the same conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Y371, in which Tyr371 has been deleted.
Y371 deletion mutant suppressed
apoptosis induced by growth factor withdrawal. The mechanism by which
apoptosis is suppressed appears to involve an increase in the level of
the antiapoptotic protein Bcl-2.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Y371
mutant was expressed in the pZEN-Neo vector (51), which contains the
neomycin resistance marker as a dominant selectable marker. A Bcl-2
expression vector was constructed in which the coding sequence of Bcl-2
was inserted into the pcDNA3 expression vector (InVitrogen,
Carlsbad, CA). Following electroporation, the cells were cultured in
the presence of IL-3 for 2-3 days. Transfected cells were then
selected by the addition of 1.0 mg/ml G418 (Life Technologies).
G418-resistant cells were allowed to expand in the presence of IL-3 for
3 days, after which the drug was removed. Conditions that allow the
isolation of growth factor-independent cells have been previously
described (53, 54). Single cell clones were then isolated following
growth of the cells at limiting dilution in semisolid media containing
0.6% SeaPlaque agarose (FMC Corp., Freeport, ME). Isolated colonies
were picked from soft agar, expanded in liquid culture, and used in the
described experiments. Multiple clones of 32D/Cbl
Y371cells were
obtained from two separate electroporations to eliminate the
possibility of examining "sister" clones. All clones used in these
studies were negative for mycoplasma; this is important, since a recent report indicates that mycoplasma can suppress apoptosis of 32Dcl3 and
induce growth factor-independent proliferation (62).
70 °C until all the samples were
collected. Caspase assays were performed as outlined by the
manufacturer, and the cleavage of the substrate,
N-acetyl-Asp-Glu-Val-Asp-p-nitroanaline), was
monitored at 1-min intervals on a microtiter plate reader at 405 nm.
Data were plotted as A405 versus time
for each sample. The initial time period over which change in OD
versus time was linear was used to calculate the specific
activity of the caspase present in each sample, in comparison with
standard samples of caspase-3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Y371 mutant, although it appears that
the other oncogenic mutants behave in a similar manner (data not
shown). Expression of the epitope-tagged Cbl
Y371 protein could be
observed by probing an immunoblot of whole cell lysates with an anti-HA monoclonal antibody (Fig. 2).
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Fig. 1.
Structure of Cbl. The structural domains
of c-Cbl are noted. From the N-terminal end these motifs include a
glycine rich-region, seven histidines, a putative nuclear localization
signal within a basically charged region, a ring finger motif, two
acidic domains that flank a proline-rich region, and a leucine zipper
motif near the C-terminal end of the protein. Amino acids 1-357 encode
a phosphotyrosine-binding domain with a cryptic Src homology 2 domain-like structure (80). The end of the v-Cbl oncogene is noted; the
amino-terminal region of Cbl usptream of this stop site is included in
v-Cbl. Also noted is the location of the 17 amino acids (aa)
that are deleted in the 70Z form of Cbl. The location of tyrosine 371, which is deleted in the oncogenic form of Cbl used in this study, is
indicated by the boldface Y at the
bottom. Plus signs, the basic region;
minus signs, the acidic region; PP,
the proline-rich region.
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Fig. 2.
Expression of the
Cbl Y371 mutant in 32Dcl3 cells. 32Dcl3
cells were transfected with the pZEN-neo vector encoding the Cbl
Y371
mutant, neomycin-resistant cells were selected by growth in
medium containing 1 mg/ml G418, and single cell-derived clones were
isolated by growth in soft agar. The Cbl
Y371 mutant contained an
epitope tag allowing for the detection of protein expression by
immunoblotting with an anti-HA monoclonal antibody. Cell lines are
indicated at the top, lane numbers at the bottom,
and the position of the Cbl
Y371 protein on the
right.
Our attempts to isolate cells that were able to proliferate in growth
factor-free media were completely unsuccessful; however, we did notice
that 32D/CblY371 cells did not die as rapidly as the untransfected
control 32Dcl3 cells. Normally, we cannot detect any viable 32Dcl3
cells after 3 days of culture in media lacking IL-3; however, viable
32D/Cbl
Y371 cells could be found after 2 weeks, when cultured in the
absence of IL-3. This observation suggested that we quantitate the
proliferation of these cells in media that either contained or lacked
exogenous IL-3. As shown in Fig. 3,
32Dcl3 cells proliferate when cultured in the presence of 50 units/ml
recombinant murine IL-3; however, there is a rapid decrease in the
number of viable cells when they are cultured in the absence of IL-3.
The 32D/Cbl
Y371 clone 9 cells cultured in the presence of IL-3
proliferated in a manner similar to that seen with 32Dcl3 cells;
however, when cultured in the absence of IL-3, the number of viable
32D/Cbl
Y371 clone 9 cells did not decrease as rapidly. As a control
for these studies, we compared the proliferation of the 32D/Cbl
Y371
cells to 32Dcl3 cells transfected with a Bcl-2 expression vector. Bcl-2
has been reported to suppress apoptosis induced by growth factor
withdrawal (63-65). The 32D/Bcl-2 clone 2 cells behaved in a manner
similar to that observed with the 32D/Cbl
Y371 clone 9 cells in that
they proliferated in the presence of IL-3, and the number of viable
cells did not rapidly decrease when these cells were cultured in the
absence of IL-3. This suggests that the Cbl
Y371 mutant is able to
suppress apoptosis induced by growth factor withdrawal in a manner
reminiscent of that observed in cells that overexpress Bcl-2; however,
there must be some difference in the effect of Bcl-2
versus Cbl
Y371, since there was a transient 2-fold
increase in the number of 32D/Bcl-2 cells when cultured in the absence
of IL-3 that was not observed in 32D/Cbl
Y371 cells. The data shown
in Fig. 3 are representative of three different studies, and although
only one clone of 32D/Cbl
Y371 cells is shown in Fig. 3, similar
results were obtained with four other independently derived clones
(data not shown). 32Dcl3 cells transfected with vector alone (pLJ,
pZEN-Neo, or pcDNA3) behaved in a manner identical to that observed
with parental 32Dcl3 cells; i.e. they underwent apoptosis
when cultured in the absence of IL-3 (53, 54, 66) (data not shown). A
recent study has suggested that mycoplasma can suppress apoptosis of
32Dcl3 cells induced by growth factor withdrawal and allows these cells
to grow in the absence of IL-3 (62). All of the different clones used
in this study have been demonstrated to be free of mycoplasma (data not
shown), and none of the 32D/Cbl
Y371 cells proliferate in the absence
of IL-3 (Fig. 3). Therefore, the results we have described cannot be
due to mycoplasma contamination.
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Similar studies were conducted with three other oncogenic forms of
c-Cbl: v-Cbl, the 70Z mutant of c-Cbl, and CblY368. All of these
oncogenic forms of c-Cbl were also able to suppress apoptosis as
described above (data not shown). Similar results have also been
obtained with v-Cbl in the laboratory of S. J. Corey.2 It is of interest to
note that the G306E mutant of v-cbl, which is not
able to transform NIH3T3 cells (61), was able to suppress apoptosis of
32Dcl3 cells following IL-3 withdrawal as effectively as
v-cbl (data not shown). This point mutation inactivates the phosphotyrosine-binding activity of v-Cbl, suggesting that binding to
phosphotyrosine-containing proteins is not required for suppression of apoptosis.
To determine the effect of IL-3 withdrawal upon the cell cycle status
of the cells examined in Fig. 3, 32Dcl3, 32D/CblY371 clone 9, and
32D/Bcl-2 clone 2 cells were cultured in the absence of IL-3 for 0-24
h. Cells were withdrawn at 8, 16, and 24 h, fixed, and stained with saponin/propridium
iodide for flow cytometric analysis. As shown in Fig. 4 and Table
I, the withdrawal of IL-3 caused
an increase in the number of cells in the G1/G0
phase of the cell cycle for all three cell lines. There was a very
dramatic increase in the number of 32Dcl3 cells present in the
sub-G1 fraction after 24 h, compared with the number
of 32D/Cbl
Y371 clone 9 and 32D/Bcl-2 clone 2 cells present in this
fraction at the same time. ModFit analysis indicates that 18% of the
32Dcl3 cells were apoptotic after 24 h in the absence of IL-3,
while only 4 and 2% of the 32D/Cbl
Y371 clone 9 and 32D/Bcl-2 clone
2 cells, respectively, were apoptotic at that same time. This provides
additional evidence that Cbl
Y371 functions to suppress apoptosis
induced by IL-3 withdrawal.
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Oncogenic Cbl Does Not Induce an Increase in
Phosphotyrosine-containing Proteins in 32Dcl3 Cells--
Bonita
et al. (61) have reported that the expression of oncogenic
forms of Cbl in NIH3T3 cells results in the dysregulation of cellular
tyrosine kinases and that one of the kinases that was activated by
oncogenic Cbl was the receptor for platelet-derived growth factor. They
suggested that there may be other tyrosine kinases that are activated
by oncogenic Cbl in these cells (61). Based upon these results, we were
interested in determining whether there was an increase in the number
of phosphotyrosine-containing proteins in 32Dcl3 cells expressing the
CblY371 mutant or an alteration in IL-3-induced phosphorylation of
cellular proteins. Both 32Dcl3 and 32D/Cbl
Y371 clone 9 cells were
cultured overnight in the absence of IL-3 and then stimulated with 100 units/ml recombinant murine IL-3 for 0-30 min. The cells were then
lysed, immunoprecipitated with anti-phosphotyrosine monoclonal
antibody, and immunoblotted with a monoclonal antibody to
phosphotyrosine (Fig. 5). There was
essentially no difference in the number of phosphotyrosine-containing proteins present in 32Dcl3 cells from the number in 32D/Cbl
Y371 cells, although there was a slight decrease in the phosphorylation of a
band with an approximate molecular weight of 120,000 in the latter
cells (Fig. 5). It does appear that phosphorylation of the major
proteins that appear following IL-3 stimulation may be slightly delayed
in the 32D/Cbl
Y371 cells compared with the 32Dcl3 cells (Fig. 5). It
is also clear that the expression of the Cbl
Y371 deletion mutant did
not result in an increase in the basal level of
phosphotyrosine-containing proteins present in unstimulated cells (Fig.
5, lane 1 versus lane
6). This suggests that in contrast to the results of Bonita
et al. (61), expression of Cbl
Y371 in 32Dcl3 cells may
not result in dysregulation of tyrosine kinases.
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Activation of Signaling Molecules Downstream of the IL-3 Receptor
Is Not Altered in Cells Expressing the CblY371 Mutant--
Previous
studies on oncogenic forms of Cbl have demonstrated that they are able
to enhance the kinase activity of the receptors for epidermal growth
factor (13) and platelet-derived growth factor (61). Activation of
cytokine receptors has been shown to activate the Janus kinase
(JAK)/signal transducer and activator of transcription (STAT) pathway
(67, 68). Therefore, it seems logical to determine whether the
Cbl
Y731 mutant would enhance activation of the JAK2/STAT signaling
pathway. The activation/phosphorylation of both JAK2 and STAT5 was
examined by anti-phosphotyrosine immunoblotting. Activation of JAK2
requires the phosphorylation of tyrosine residues in the activation
loop of JAK2 (69), and therefore the tyrosine phosphorylation of JAK2
can be directly correlated with catalytic activation of this kinase.
There was no difference in the amount of phosphorylated JAK2 or the
kinetics with which phosphorylated JAK2 appeared in 32D/Cbl
Y371 when
compared with 32Dcl3 cells (Fig.
6A). Two different clones of
32D/Cbl
Y371 cells are examined in Fig. 6A. Compared with
32Dcl3 cells, there was no difference in the amount of JAK2 protein as
shown by reprobing the immunoblot with anti-JAK2 antibody (data not
shown). Although not shown here, overexpression of Bcl-2 in 32Dcl3
cells did not alter IL-3-induced activation of JAK2 or the amount of
JAK2 protein in these cells (data not shown). Thus, the observed
resistance of 32D/Cbl
Y371 cells to apoptosis induced by growth
factor withdrawal cannot be explained by an increase in basal JAK2
kinase activity.
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In addition to JAK2, cytokines such as IL-3 also induce activation of
Src-related tyrosine kinases such as Fyn, Hck, and Lyn (70, 71).
Although we have not observed the activation of the Syk-related
tyrosine kinases by IL-3, other investigators have observed its
activation by cytokines such as granuclocyte colony-stimulating factor
(72). We have examined that activation of both Fyn and Syk in
IL-3-stimulated 32Dcl3 and 32D/CblY371 cells, and, consistent with
the results shown with JAK2 above, we have not detected any alteration
in the activation of either Fyn or Syk (data not shown). Although we
have not conducted an exhaustive examination of all different tyrosine
kinases present in 32Dcl3 cells, our data indicate that there is no
alteration in at least three different tyrosine kinases, which
represent three different classes of tyrosine kinases (JAK2, Fyn, and Syk).
Activation of JAK family tyrosine kinase is required for activation of
STAT molecules (67, 68). The activation of STAT5 was examined in three
different clones of cells expressing the CblY371 mutant and two
clones of cells overexpressing Bcl-2 (Fig. 6B). Consistent
with the results shown in Fig. 6A, there was very little
difference in the amount or the kinetics of STAT5 phosphorylation in
any of the different cells examined. Although it appears that there may
be significantly less phosphorylated STAT5 in 32D/Cbl
Y371 clone 15 cells at the 5-min time point, the anti-STAT5 immunoblot indicates that
there is less STAT5 protein in this sample (Fig. 6B,
bottom panel, lane 11).
These data suggest that the activation of both JAK2 and STAT5 is
unaltered in 32Dcl3 cells expressing the Cbl
Y371 mutant and
in cells overexpressing Bcl-2, compared with the parental 32Dcl3 cell
line. Thus, the diminished apoptosis observed in Figs. 3 and 4 above
does not result from an alteration in the JAK/STAT signaling pathway as
detected by these approaches.
Numerous studies have demonstrated that activation of extracellular
signal-regulated kinases (ERKs) is required for proliferation and
morphological transformation by activated oncogenes. We therefore wanted to determine whether expression of the CblY371 mutant altered
IL-3-induced activation of ERKs in 32Dcl3 cells. ERK activation was
examined by immunoblotting whole cell lysates of IL-3-stimulated cells
with an antibody that was specific for the activated forms of ERK1 and
ERK2. Stimulation of 32Dcl3 cells with IL-3 was observed to result in
the rapid activation of ERK1 and ERK2 as shown in lanes
1-3 of Fig. 6C. The activation of ERK in these
cells generally peaks between 5 and 15 min after stimulation and
declines after this time (data not shown). The IL-3-induced activation
of ERKs was examined in three different clones of 32D/Cbl
Y371 cells
(Fig. 6C, lanes 4-12). The activation
of ERK in clones 13 and 15 appeared to be identical, with a peak of
active ERK at 5 min and then a decline after that time. The amount of
activated ERK in both clone 13 and clone 15 cells appeared to be
equivalent to that present in 32Dcl3 cells (compare lanes
2, 8, and 11 in Fig. 6C).
In contrast, the activation of ERK in 32D/Cbl
Y371 clone 9 cells
appeared to be more transient, and the amount of activated ERK did not
appear to be as great as that detected in the other cell lines (Fig. 6C, lanes 4-6). IL-3-induced
activation of ERK was also examined in one clone of 32D/Bcl-2 cells
(Fig. 6C, lanes 13-15). ERK
activation in these cells did not appear to differ significantly from
that detected in the 32D/Cbl
Y371 clone 13 and 15 cells. These
results suggest that the expression of the Cbl
Y371 protein in 32Dcl3 did not induce growth factor-independent activation of ERK and did not
block IL-3-induced ERK activation, although the activation of ERK might
be somewhat more transient in some clones of Cbl
Y371 cells.
Apoptosis is a genetically regulated cell death process that involves a
series of biochemical changes to a cell (73-75). Several molecules
have been identified that are able to regulate this process by
suppressing cellular death; these include the antiapoptotic members of
the Bcl-2 family (60), and the serine/threonine kinase Akt (also known
as protein kinase B, or PKB) (43, 46-48, 76). Recent studies have
indicated that cytokines such as IL-3 activate Akt and that activation
of Akt is critical for suppression of apoptosis induced by growth
factor withdrawal (43, 46-48, 76). Thus, an increase in the amount of
activated Akt could explain the decreased apoptosis observed with
32D/CblY371 cells. Activated Akt can be quantitated using an
antibody that recognizes the phosphorylated activated form of Akt.
The amount of activated Akt in unstimulated and IL-3-stimulated 32Dcl3
and 32D/CblY371 clone 9 cells was examined by immunoblotting whole
cell lysates with an anti-phospho-Akt antibody. As shown in Fig.
6D, activation of Akt was maximal 5 min after stimulation with IL-3 in both the 32Dcl3 and 32D/Cbl
Y371 clone 9 cells and declined after that time. The decline in phosphorylated Akt appears to
be slightly faster in the 32D/Cbl
Y371 clone 9 cells compared with
the 32Dcl3 cells, although this was not consistently observed in all
studies with these cells. There was no difference in the amount of
activated Akt present in either cell line over the time course examined
in this study or in the total amount of Akt (Fig. 6D,
bottom panel). It is clear from this study that
there is not a pool of activated Akt present in unstimulated
32D/Cbl
Y371 clone 9 cells that could account for the lower amount of
apoptosis observed in these cells following their cultivation in media
lacking IL-3. This suggests that some molecule other than Akt is responsible.
Activation of Akt is dependent upon activation of PI 3-kinase and can
be inhibited by treatment of cells with wortmannin (42, 43, 45, 46,
48). The ability of wortmannin to inhibit PI 3-kinase may explain in
part why wortmannin treatment can induce apoptosis of several cell
types. As a second means to determine whether Akt activation
contributed to the resistance of the 32D/CblY371 cells to apoptosis
induced by growth factor withdrawal, these cells were treated with 100 nM wortmannin upon removal from IL-3-containing media. When
the parental 32Dcl3 cells were cultured in the absence of IL-3, they
underwent apoptosis regardless of whether wortmannin was added to the
media (Fig. 7). Consistent with the
results shown in Fig. 3, 32D/Cbl
Y371 clone 9 cells remained viable
when cultured in the absence of IL-3; however, the addition of 100 nM wortmannin to the media resulted in the 2-3-fold
decrease in the number of viable cells as measured by trypan blue
exclusion (Fig. 7). This suggests that there may be a PI
3-kinase-dependent pathway that contributes to the
suppression of apoptosis in these cells. Therefore, it is possible that
Akt could contribute to this process; however, the results of the
wortmannin study clearly indicate that there must also be an
Akt-independent component to this process. The fact that there is a
wortmannin-sensitive component suggests that either there is a small
pool of activated Akt in 32D/Cbl
Y371 cells that cannot be detected
by immunoblotting with the anti-phospho-Akt antibody or that PI
3-kinase can activate antiapoptotic molecules other than Akt.
|
Expression of CblY371 Does Not Alter the Phosphorylation of
Signaling Molecules following IL-3 Withdrawal--
The data presented
in Figs. 5 and 6 indicate that Cbl
Y371 does not increase the
phosphorylation/activation of signaling molecules following IL-3
stimulation. It is possible, however, that Cbl
Y371 could increase or
prolong the phosphorylation of these signaling molecules following IL-3
withdrawal. To examine this point, 32Dcl3 and 32D/Cbl
Y371 clone 9 cells that had been cultured in IL-3 were removed from IL-3-containing
media, and cell lysates were prepared at times varying from 0 to 8 h. The phosphorylation of JAK2, STAT5, ERK1, ERK2, and Akt was examined
at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h
following IL-3 withdrawal. To demonstrate that these proteins could
still be phosphorylated/activated after an 8-h starvation, a sample of
both cells was stimulated with 100 units/ml IL-3 and also analyzed.
There was no difference in the time at which phosphorylated JAK2,
STAT5, ERK1, ERK2, and Akt disappeared from 32Dcl3 cells versus 32D/CblY371 clone 9 cells (Fig.
8, A, B,
C, and D, top panels). Withdrawal from
IL-3-containing media had no effect upon the amounts of these proteins
over the time period examined (Fig. 8, A, B,
C, and D, bottom panels). Furthermore,
all of these signaling molecules could be phosphorylated after 8 h
of withdrawal from IL-3, by stimulation of these cells with IL-3 for 15 min (Fig. 8, A, B, C, and
D, lanes 6 and 12). These data
indicate that the ability of Cbl
Y371 to suppress apoptosis induced
by growth factor withdrawal cannot be explained by the prolonged
phosphorylation/activation of these signaling molecules.
|
Elevated Levels of Bcl-2 Are Present in 32D/CblY371 Clone 9 Cells--
As noted above, some members of the Bcl-2 family of
proteins are able to suppress apoptosis (Bcl-2, Bcl-xL,
Mcl1, A1, and Bak), while others are able to induce apoptosis (Bad,
Bcl-xS, and Bax) (60). Immunoblot analysis was used to
determine whether the presence of the Cbl
Y7371 protein altered the
level of different Bcl-2 family members. For each cell line to be
analyzed, one set of cells was cultured in the presence of IL-3 for
16 h, while the second group of cells was cultured in the absence
of IL-3 for the same period of time. Whole cell lysates were prepared and subjected to immunoblot analysis. There was a dramatic increase in
the amount of Bcl-2 in 32D/Cbl
Y371 clone 9 cells compared with that
present in 32Dcl3 cells (Fig. 9,
lanes 3 and 4 versus lanes 1 and 2). Under the conditions
used in this study and the time exposure used, Bcl-2 was barely
detectable in 32Dcl3 cells grown in the presence or absence of IL-3
(Fig. 9A, lanes 1 and 2).
In contrast, the amount of Bcl-2 present in 32D/Cbl
Y371 clone 9 cells was almost the same as that detected in 32D/Bcl-2 clone 2 cells
(Fig. 9A, lanes 3 and 4 versus lanes 5 and 6). In
other studies, we have observed that the Bcl-2 protein can only be
detected when 32Dcl3 cells are grown in the presence of IL-3 and that
IL-3 stimulation of 32Dcl3 cells that had been cultured in the absence of IL-3 resulted in the appearance of Bcl-2 protein (data not shown).
This was not observed in Fig. 9A, perhaps because the time
the film was exposed was too short to detect the presence of Bcl-2 in
32Dcl3 cells grown in the presence of IL-3. In this context, it is
interesting to note that Bcl-2 was detected in both the 32D/Cbl
Y371
clone 9 and 32D/Bcl-2 clone 2 cells cultured in the absence of IL-3.
The presence of Bcl-2 in the latter cell line is not surprising, since
the Bcl-2 cDNA is overexpressed through the use the cytomegalovirus
promoter present in the pcDNA3 expression vector. The high level of
the Bcl-2 protein in the 32D/Cbl
Y371 clone 9 cells was not expected,
however, and could potentially represent a gene whose expression is
induced by the Cbl
Y371 mutant protein.
|
There was no Mcl1 protein detected in any of the three cell lines grown
in the absence of IL-3; however, the protein could be detected in all
three cell lines cultured in the presence of IL-3. Slightly more Mcl1
protein was detected in both the 32D/CblY371 clone 9 and 32D/Bcl-2
clone 2 cells compared with that detected in 32Dcl3 cells (Fig.
9C). The significance of this difference is not clear at
this time. There did not appear to be any significant difference in the
amount of either Bcl-xL or Bax between the three cell lines
(Fig. 9, B and D). The levels of these latter two
proteins were not altered when any of the three cell lines were
cultured in the absence of IL-3. These data suggest that the apparent
resistance of the 32D/Cbl
Y371 cell lines to apoptosis induced by
growth factor withdrawal could be explained by altered levels of the antiapoptotic protein Bcl-2. To demonstrate that the increased level of
Bcl-2 protein was not unique to just the 32D/Cbl
Y371 clone 9 cells,
we also examined the level of Bcl-2 protein in three other independent
clones of 32D/Cbl
Y371 cells (Fig. 9E). An elevated level
of Bcl-2 protein was observed in 32D/Cbl
Y371 clones 3, 4, and 5 when
they were compared with the parental 32Dcl3 cells (Fig. 9E).
Thus, this observation is not unique to a single clone of cells.
Transfection of 32Dcl3 and 32D/CblY371 with a
bcl-2 promoter reporter vector did not reveal any
significant difference in the expression of the bcl-2
promoter between these two cell lines (data not shown). Although it is
possible that the promoter did not include the required responsive
region of the bcl-2 promoter, we believe that this suggests
that transcriptional induction of the bcl-2 gene does not
explain the data presented in Fig. 9. Other possibilities include that
there is an increase in the half-life of the bcl-2 mRNA
or the half-life of the Bcl-2 protein.
Caspase Activation Is Suppressed in 32D/CblY371 Clone 9 Cells
Cultured in the Absence of IL-3--
As noted above, apoptosis is a
genetically controlled process that involves many different molecules.
A central event in the apoptotic process is the activation of a
specific set of proteases referred to as caspases (77). At least nine
different caspases are currently known. Some caspase family members are
activated very early during apoptosis, while others are activated late
in the death process. We have examined the activation of caspase 3 as a
function of the time in which the different cells were cultured in the
absence of IL-3. The 32Dcl3, 32D/Cbl
Y371 clone 9, and 32D/Bcl-2
clone 2 cells were cultured in the presence or absence of 50 units/ml
rmIL-3 for up to 48 h. Four hours after placing all three cell
types in growth factor-free media, the levels of caspase 3 was
essentially the same in all cultures. After this time point, however,
there was a dramatic increase in the amount of caspase activity in the
32Dcl3 cells but not in 32D/Cbl
Y371 clone 9 or 32D/Bcl-2 clone 2 cells (Fig. 10). Although there was a
slow but detectable increase in the amount of caspase detected by this
assay in 32D/Cbl
Y371 clone 9 and 32D/Bcl-2 clone 2 cells cultured in
the absence of IL-3 for 48 h, there was still at least 4-fold more
caspase activity in the 32Dcl3 cells cultured in the absence of IL-3
for 48 h (Fig. 10). This suggests that there is a dramatic
difference in the response of these different cell types to growth
factor withdrawal and that the responses of 32D/Cbl
Y371 clone 9 and
32D/Bcl-2 clone 2 cells are quite similar.
|
![]() |
DISCUSSION |
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---|
In this study, we have examined the effects of an oncogenic form of the c-Cbl proto-oncogene upon the proliferation of the growth factor-dependent murine myeloid cell line, 32Dcl3. The oncogenic form of Cbl we have used was constructed by Andoniou et al. (51) and contains a deletion of a single tyrosine residue, Tyr371. This activated form of Cbl has been previously reported to induce the transformation of NIH3T3 cells, and these transformed cells were tumorigenic when injected into nude mice (51). The deletion of this single tyrosine residue must result in a dramatic change in the conformation of this protein such that it becomes oncogenic. Mutation of the tyrosine to a phenylalanine residue, which cannot be phosphorylated, did not result in the oncogenic activation of this protein. The structure of a c-Cbl-UbcH7 complex has recently been published and indicates that both Tyr368 and Tyr371 lie in a linker region between the phosphotyrosine-binding domain and the RING finger motif and that these residues are in a buried environment and make multiple van der Waals contacts with hydrophobic residues in the phosphotyrosine-binding domain (78). Therefore, it is likely that deletion of either Tyr368 or Tyr371 would result in a conformational change.
Prior to beginning these studies, it had been demonstrated that
expression of an oncogenic form of Cbl in NIH3T3 cells resulted in the
disregulation of one or more tyrosine kinases present in those cells,
and one of the deregulated kinases appeared to be the platelet-derived
growth factor receptor (61). We therefore expected that expression of
the CblY371 protein in 32Dcl3 cells would result in the activation
of one or more tyrosine kinases in these cells. Based upon our studies
that indicated that two different activated tyrosine kinases, v-Src and
BCR-ABL (53), could induce growth factor-independent proliferation of
factor-dependent cells, we predicted that the Cbl
Y371
mutant would also induce growth factor-independent proliferation.
Contrary to our predictions, we did not observe an increase in the
basal level of phosphotyrosine-containing proteins in
32D/Cbl
Y371 cells; nor was the phos- phorylation/activation of
signaling molecules potentiated in 32D/Cbl
Y371 cells following withdrawal from IL-3-containing media. We also did not identify any
constitutively activated tyrosine kinases, and the cells remained growth factor-dependent for cellular proliferation.
Although we have not examined all tyrosine kinases present in 32Dcl3
cells, we have examined three different kinases that represent three different classes of tyrosine kinases: JAK2, Fyn, and Syk.
To our surprise, we observed that expression of the CblY371 protein
in 32Dcl3 cells resulted in a suppression of apoptosis. Suppression of
apoptosis was characterized by the continued viability of transfected
cells when cultured in the absence of IL-3, arrest of the cells in the
G1 phase of the cell cycle when cultured in the absence of
IL-3, and decreased caspase activation when the cells were cultured in
the absence of IL-3. The mechanism by which apoptosis was suppressed
appears to result from an increase in the level of the
antiapoptotic Bcl-2 protein. We have not detected an increase in
the level of any other antiapoptotic Bcl-2 family member; nor have we
detected a decrease in the amount of a proapoptotic Bcl-2 family
member. However, we have not exhaustively examined all of the members
of this family of proteins, since we have not been able to obtain good
antibodies to each of the different Bcl-2 family members. We have also
not detected the prolonged phosphorylation/activation of signaling
molecules in 32D/Cbl
Y371 following withdrawal from IL-3-containing media.
The bcl-2 oncogene was discovered because it mapped to a chromosomal breakpoint present in B-cell leukemia (79). In this case, the translocation of a promoter for the IgG upstream of the coding region of the bcl-2 gene resulted in the disregulated expression of the bcl-2 gene. The juxtaposition of the IgG promoter upstream of the bcl-2 gene explains why there is a specific increase in the amount of Bcl-2 in B-lymphocytes. The tissue specificity of this promoter also explains why neoplasia is not observed in other tissues. Based upon the observation that disregulated expression of bcl-2 results in the B-cell leukemia described above, it would be expected that the ability of oncogenic forms of Cbl to increase Bcl-2 protein levels could partially explain the leukemogenic potential of oncogenic forms of c-Cbl.
At this time, we do not understand the basis for the increased level of
Bcl-2 protein in 32Dcl3 cells expressing the CblY371 mutant protein.
Transfection of a bcl-2 promoter reporter vector into
32D/Cbl
Y371cells did not reveal an increase in promoter expression
in these cells compared with normal 32Dcl3 cells. This suggests that
other mechanisms may explain the increased level of Bcl-2 protein.
Possibilities include that there is an increase in the half-life of the
Bcl-2 mRNA, or an increase in the half-life of the Bcl-2 protein.
To our knowledge, this is the first example of an oncogene inducing a
change in the level of an antiapoptotic Bcl-2 family member.
The results obtained in our study are very similar to those described
by Corey and colleagues.2 These investigators have
expressed an oncogenic truncation mutant of c-Cbl, which is similar to
v-Cbl, in 32Dcl3 cells, and they have determined that it can suppress
apoptosis in a manner similar to that described in this
paper.2 In addition, they have shown that their
oncogenic form of Cbl blocks the terminal different of 32Dcl3 cells
induced by granulocyte colony-stimulating factor.2 If it
can be demonstrated that oncogenic forms of Cbl are all able to
suppress apoptosis by modifying the levels of Bcl-2 family members,
then another mechanism by which oncogenes can transform cells will be
established. It will also be of importance to determine whether
mutation of c-Cbl plays a role in the genesis of human cancer or leukemia.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kathy Barzen for conducting the caspase 3 assay as well as Seija Hunter, Pamela Garl, and Celicia Lemons for contributions to this project. We also thank Dr. Mary E. Reyland and Kathryn L. Schwertfeger for comments on the manuscript and Dr. Seth Corey for communicating results from his laboratory prior to the submission of this paper.
![]() |
FOOTNOTES |
---|
* This study was sponsored by National Institutes of Health Grants CA45241 and GM55754 (to S. M. A.). The University of Colorado Cancer Center Flow Cytometry Facility is supported by NCI, National Institutes of Health, Grant CA46934.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.
To whom correspondence should be addressed: University of
Colorado Health Sciences Center, Department of Pathology, Box B216, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-4787; Fax: 303-315-6721; E-mail: steve.anderson@uchsc.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009386200
2 S. J. Corey, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL-3, interleukin-3; Bcl-2, B-cell lymphoma 2; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; PI 3-kinase, phosphatidylinositol 3-kinase; STAT, signal transducer and activator of transcription; mIL-3, murine IL-3; rmIL-3, recombinant murine IL-3; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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