From the
The c-cbl protooncogene product (c-Cbl) is a 120-kDa
protein that has been shown to bind to the Src homology 3
domains of various proteins, suggesting its involvement in signal
transduction pathways. We identified one of the most prominent
tyrosine-phosphorylated proteins in Fc
The c-cbl protooncogene product (c-Cbl) was first
identified as the cellular homologue of the viral transforming protein
of the murine Cas NS-1 retrovirus(1, 2) . This
retrovirus induces pre-B cell lymphomas and myeloid leukemia in
infected newborn and adult mice and transforms different fibroblast
cell lines(1, 2) . The v-cbl oncogene product
(v-Cbl) represents a truncated form of its cellular homologue
containing only the N-terminal 355 amino acids of c-Cbl. Structurally
it possesses a potential nuclear localization signal, and in
subcellular fractionation analysis it has been reported to localize to
both nuclear and cytoplasmic fractions (3). The protooncogene product,
c-Cbl, is a 120-kDa protein that contains additional structural
features including a high proportion of basic amino acids, a putative
leucine zipper at the C terminus, and a zinc finger-like motif,
indicating that it may act as a transcription factor(2) .
However, unlike v-Cbl, c-Cbl has been exclusively localized to the
cytoplasm, and immunohistochemical studies suggest that it may be
associated with the cytoskeleton(3) . Both in vivo and in vitro evidence demonstrates that c-Cbl is able to bind to
the Src homology 3 (SH3)
We
report here that c-Cbl becomes tyrosine-phosphorylated in response to
the activation of different tyrosine kinase signaling pathways
including stimulation by antibody clustering of the Fc
In this report we identify a protein, c-Cbl, that becomes
tyrosine-phosphorylated in response to a variety of tyrosine kinase
signaling events, including activation of receptors that possess
intrinsic tyrosine kinase activity (EGFR and CSF-1R), activation of
receptors that recruit cytoplasmic tyrosine kinases (Fc
In macrophages, rapid tyrosine phosphorylation of c-Cbl was
observed in response to Fc
In contrast, when tyrosine
phosphorylation of c-Cbl was induced by EGF stimulation of fibroblasts
overexpressing EGFR and by CSF-1 stimulation of macrophages,
co-immunoprecipitation experiments revealed that c-Cbl was physically
associated with activated EGFR and CSF-1R. Since the EGFR and CSF-1R
possess intrinsic tyrosine kinase activity(12) , our results
indicate that tyrosine phosphorylation of c-Cbl is induced in the
signaling pathways utilized by receptor-type tyrosine kinases as well.
Because c-Cbl has no putative binding sites for activated tyrosine
kinase receptors such as SH2 domains(2, 13) ,
association of c-Cbl to the receptors may occur indirectly via adaptor
molecules such as Grb2, Nck, and c-Crk, which are known to bind to
activated EGFR (14-18), and, for Nck, to also bind to
c-Cbl(4) .
Fibroblasts transformed by the v-src oncogene also displayed enhanced tyrosine phosphorylation of
c-Cbl, and under certain lysis conditions, the v-Src and c-Cbl proteins
associated in vivo. On the other hand, c-Cbl was not markedly
tyrosine-phosphorylated in normal lung fibroblasts and in a 3T3 cell
line that was engineered to overexpress the c-Src protein. Enhanced
tyrosine phosphorylation of c-Cbl may reflect the fact that the Src
protein in v-src-transformed fibroblasts has a higher tyrosine
kinase activity than the Src protein in c-src-overexpressing
fibroblasts(19) . Therefore, tyrosine phosphorylation of c-Cbl
may depend on Src kinase activation.
Immunofluorescence analysis has
revealed that c-Cbl was localized in the cytoplasm and showed a diffuse
and/or reticular pattern in unstimulated macrophages or HER14 cells.
Fc
While this report was being prepared,
tyrosine phosphorylation of c-Cbl has also been reported in Jurkat
cells activated via the T cell receptor(20) . Similar to what we
show here for Src family members, these investigators also showed that
c-Cbl associates with the SH3 domains of Grb2, at least in
vitro. More recently, it was reported that the expression of
v-abl induced the tyrosine phosphorylation of c-Cbl and that
Abl and Cbl associated in vivo(21) . Hence, c-Cbl is
tyrosine-phosphorylated in response to Fc
We thank Dr. J. S. Brugge for providing the GST-SH3
fusion proteins and the SRD cell line. We also thank Dr. J.
Schlessinger for his generous gift of HER14 cells, Dr. D. Shalloway for
the c-src 3T3 cell line, and Dr. W. C. Horne for his careful
reading of the manuscript and helpful advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor
(Fc
R)-stimulated macrophages to be c-Cbl. Tyrosine phosphorylation
of c-Cbl occurred within 20 s after stimulation and reached maximum
levels within 3-5 min. c-Cbl was also tyrosine-phosphorylated in
epidermal growth factor (EGF) receptor-overexpressing cells upon EGF
stimulation, in macrophages in response to CSF-1 treatment, and in
v-src transformed cells. Furthermore, we found that c-Cbl
associated with these kinases in vivo. In vitro, c-Cbl bound
to the Src homology 3 domains of Src, Fyn, and Lyn in both
unstimulated and Fc
R-stimulated macrophages. Examination of cells
by immunofluorescence revealed that c-Cbl is diffusely distributed in
the cytoplasm in both unstimulated macrophages and EGF
receptor-overexpressing cells and translocated to a more specific
compartment of the cell, consistent with the trans-Golgi region,
following Fc
R clustering and EGF stimulation, respectively. These
results suggest that c-Cbl is involved in the signaling pathways
utilized by different types of tyrosine kinases.
(
)domain of
Nck, an adaptor protein that mediates specific protein-protein
interactions in tyrosine kinase signaling pathways(4) .
II/III
receptors in murine macrophages, mitogen stimulation of fibroblasts and
macrophages by the growth factors EGF and CSF-1, respectively, and
v-src transformation in fibroblasts. Immunofluorescence
analysis revealed that Fc
R clustering and EGF stimulation also
induce the translocation of c-Cbl from its diffuse and reticular
cytoplasmic localization in unstimulated cells to a more specific
region of the cell, consistent with the trans-Golgi region. Overall,
these results suggest the possibility that c-Cbl is recruited in the
signal transduction pathways utilized by a variety of tyrosine kinases.
Cells and Antibodies
To prepare peritoneal
macrophages, CD-1 mice received intraperitoneal injections of
thioglycolate broth as previously reported(5) . Three days
following these injections peritoneal exudate cells were isolated by
washing the peritoneal cavity with phosphate-buffered saline. More than
90% of the cells were macrophages(5) . The murine macrophage
cell line P388D1 (American Type Culture Collection, Bethesda, MD,
TIB63) was maintained in RPMI 1640 medium supplemented with 15% fetal
bovine serum (Sigma). HER14 cells, an NIH 3T3 stable cell line
overexpressing the human EGF receptor (EGFR), were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% heat-inactivated calf serum. NIH 3T3 cells overexpressing c-src (c-src 3T3 cells) and Balb/c3T3 cells transformed by
v-src (SRD cells) were cultured in DMEM supplemented with 5%
calf serum. Anti-phosphotyrosine antibody, clone 4G10, and anti-CSF-1
receptor (CSF-1R) antibody were obtained from UBI (Lake Placid, NY).
Anti-murine FcRII/III monoclonal antibody (2.4G2) was purchased
from PharMingen (San Diego, CA). F(ab`)
fragments of goat
anti-rat IgG were from Jackson ImmunoResearch Laboratories Inc. (West
Grove, PA). Rabbit polyclonal anti-c-Cbl antibody (C-15) and anti-EGFR
antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Stimulation and Lysis
FcR clustering in
peritoneal macrophages and the macrophage cell line P388D1 cells was
performed as previously reported(5) . The cells were pelleted at
14,000
g and lysed in ice-cold TNE lysis buffer (10
mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA,
1% Nonidet P-40, 2 mM Na
VO
, 10 mM NaF, and 20 µg/ml aprotinin). In some experiments, P388D1
cells were stimulated with CSF-1 (25 nM) for 30 s at room
temperature and lysed with TNE buffer. HER14 cells were stimulated with
EGF (275 ng/ml) for 5 min at 37 °C and lysed with HNTG buffer (30
mM HEPES, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1
mM EGTA, 1 mg/ml bovine serum albumin, 2 mM Na
VO
, 10 mM NaF, and 20 µg/ml
aprotinin) as previously reported(6) . SRD cells were lysed with
either TNE buffer or CHAPS buffer (50 mM Tris-HCl, pH 7.4, 150
mM NaCl, 8 mM CHAPS, 2 mM EDTA, 2
mM Na
VO
, 10 mM NaF, and 20
µg/ml aprotinin).
Western Blotting and Immunoprecipitation
Western
blotting was performed using ECL Western blotting detection reagents
(Amersham Corp.) according to the conditions recommended by the
supplier. For immunoprecipitation of c-Cbl, cell lysates were
precleared with protein A-Sepharose (Sigma) for 1 h and then incubated
with 1 µg of anti-c-Cbl antibody for 1 h. Immune complexes were
recovered with protein A-Sepharose and extensively washed 3 times with
TNE buffer. The protein was eluted by boiling in 1 sample
buffer (62.5 mM Tris-HCl, pH 6.9, 2 mM EDTA, 3% SDS,
3.75% glycerol, and 180 mM
-mercaptoethanol) for 2 min.
SH3-binding Proteins
Two hundred µg of the
cell lysate prepared in TNE buffer was precleared by incubating with
GST protein non-covalently bound to glutathione-Sepharose (Pharmacia
Biotech Inc.) for 2 h at 4 °C. After removing the GST-Sepharose
beads by centrifugation, the supernatants were incubated with 15 µg
of the GST-SH3 fusion proteins and 40 µl of a 1:1 slurry of
glutathione-Sepharose for 1 h at 4 °C. Cellular proteins bound to
the GST-SH3 fusion proteins were washed 3 times with lysis buffer and
eluted by boiling in 1 sample buffer for 2 min.
Confocal Microscopy
For immunofluorescence
analysis, cells were plated on glass coverslips and cultured overnight
at 37 °C. After the indicated treatments, cells were fixed in
phosphate-buffered saline containing 3.7% formaldehyde for 10 min at
room temperature and stained with anti-c-Cbl antibody following the
method previously reported(7) . Microscopy was performed using a
confocal microscope (model MRC 600, Bio-Rad) with krypton/argon lasers.
Fc
Fig. 1indicates that stimulation of the Fc Receptor-mediated Tyrosine Phosphorylation of
c-Cbl
R by
antibody clustering in peritoneal macrophages (A) and the
murine macrophage cell line P388D1, which is known to have Fc
R (B), induced tyrosine phosphorylation of several proteins,
which is consistent with previous reports(5) . Enhanced tyrosine
phosphorylation of the majority of these proteins was apparent within
20 s after the addition of the secondary antibody, reached a maximum
level within 3-5 min, and significantly diminished by 30 min (Fig. 1A). Due to the appearance of a 120-kDa protein
that was rapidly tyrosine-phosphorylated upon Fc
R stimulation, we
determined whether the c-Cbl protein became tyrosine-phosphorylated in
response to Fc
R stimulation of macrophages. c-Cbl was
immunoprecipitated from unstimulated and stimulated peritoneal
macrophages, and the immunoprecipitated material was analyzed by
anti-phosphotyrosine immunoblotting. As shown in Fig. 1C (Anti-PY blot), Fc
R clustering rapidly induced the
tyrosine phosphorylation of c-Cbl. This increase in tyrosine
phosphorylation was not due to changes in the level of the c-Cbl
protein upon Fc
R stimulation (Fig. 1C; Anti-Cbl
blot). Similar results were obtained in Fc
R-stimulated P388D1
cells (data not shown).
Figure 1:
FcR clustering induces
tyrosine phosphorylation of c-Cbl in macrophages. A and B, Fc
R clustering induces tyrosine phosphorylation in
macrophages. Peritoneal macrophages (A) and the murine
macrophage cell line P388D1 (B) were incubated with
anti-Fc
RII/III antibody for 30 min at 4 °C. Thereafter, the
cells were treated with goat F(ab`)
anti-rat IgG for the
indicated times at 37 °C. Cells were lysed and immunoblotted with
anti-phosphotyrosine antibody, clone 4G10. The mobility of the size
markers is indicated. C, Fc
R clustering induces tyrosine
phosphorylation of c-Cbl in macrophages. c-Cbl was immunoprecipitated
from cell lysates and immunoblotted with anti-phosphotyrosine antibody (Anti-PY). The anti-phosphotyrosine blot was stripped
of antibody and reprobed with anti-c-Cbl antibody (Anti-Cbl).
The position of c-Cbl is indicated by the arrow.
c-Cbl Binds to the SH3 Domain of Src Family Tyrosine
Kinases
Members of the Src family of tyrosine kinases
are activated upon signaling via Fc receptors (8, 9, 10) and possess SH3 domains that mediate
interactions with various cellular proteins(11) . Since c-Cbl
has been shown to bind to the SH3 domain of Nck(4) , we then
determined whether the c-Cbl protein was able to bind to the SH3 domain
of different Src family members. The SH3 domains of Src, Fyn,
and Lyn were expressed as GST fusion proteins and purified by
glutathione-Sepharose chromatography. Lysates prepared from
unstimulated P388D1 cells or FcR-stimulated cells were incubated
with glutathione-Sepharose beads containing either GST alone or the
GST-Src-SH3, GST-Fyn-SH3, or GST-Lyn-SH3 fusion proteins. Cellular
proteins bound to the different GST fusion proteins were fractionated
by 8% SDS-PAGE and immunoblotted with anti-c-Cbl antibody. Anti-c-Cbl
antibody reacted with a Src, Fyn, and Lyn SH3-binding protein in both
unstimulated and stimulated cells (Fig. 2B). In both
unstimulated and Fc
R-stimulated cells, the GST fusion proteins
containing the Src, Fyn, and Lyn SH3 domains bound to a 120-kDa protein
that reacted with anti-c-Cbl antibody on Western blots (Fig. 2B). Even though Fc
R stimulation led to
enhanced tyrosine phosphorylation of c-Cbl over the low, basal level of
phosphorylation observed in unstimulated cells (Fig. 2A), binding of c-Cbl to the SH3 domains of Src,
Fyn, and Lyn appeared to be similar in unstimulated or stimulated
cells. These results suggest that binding of c-Cbl to the SH3 domains
is not dependent on the tyrosine phosphorylation induced by Fc
R
stimulation.
Figure 2:
c-Cbl associates with the SH3 domains of
three different Src family members in vitro. A, TCL
and c-Cbl immunoprecipitates (c-Cbl IP) from unstimulated and
FcR-stimulated P388D1 cells (3 min of stimulation) were
immunoblotted with anti-phosphotyrosine antibody. B, cell
lysates from unstimulated and Fc
R-stimulated P388D1 cells were
incubated with glutathione-Sepharose beads containing GST, GST-Src SH3,
GST-Fyn SH3, and GST-Lyn SH3 fusion proteins. After extensively washing
the beads, the bound proteins were eluted in SDS-sample buffer,
separated on SDS-PAGE, and transferred to nitrocellulose. Filters were
immunoblotted with anti-c-Cbl antibody. c-Cbl is indicated by the arrow.
Activation of Other Types of Tyrosine Kinases Also
Induces Tyrosine Phosphorylation of c-Cbl
We then examined
whether tyrosine phosphorylation of c-Cbl was specific to
FcR-mediated signaling or whether it could also be induced in
other tyrosine kinase signaling pathways. We first tested the ability
of c-Cbl to become tyrosine-phosphorylated in response to stimulation
of growth factor receptors that possess intrinsic tyrosine kinase
activity. For this purpose, a cell line, HER14, that expresses high
levels of the EGFR was stimulated with EGF, and the macrophage cell
line, P388D1, was stimulated with CSF-1. In total cell lysates (TCL),
increased tyrosine phosphorylation of a protein with an electrophoretic
mobility similar to c-Cbl was observed in response to both EGF and
CSF-1 stimulation (Fig. 3, A and B). c-Cbl was
then immunoprecipitated from cell lysates of unstimulated and
stimulated cells and immunoblotted with anti-phosphotyrosine antibody
as before. As shown in Fig. 3, A and B, both
EGF and CSF-1 induced the tyrosine phosphorylation of
immunoprecipitated c-Cbl in HER14 and P388D1 cells, respectively (anti-PY blot). The increase in tyrosine phosphorylation of
c-Cbl in EGF and CSF-1-stimulated cells was not due to an increase in
the amount of protein in the stimulated cells (Fig. 3, A and B, anti-Cbl blot). Hence, tyrosine
phosphorylation of c-Cbl is induced not only after Fc
R clustering
but also in the signal transduction pathways of various receptor-type
tyrosine kinases.
Figure 3:
c-Cbl is tyrosine-phosphorylated in
response to mitogen stimulation and in fibroblasts transformed by
v-src. A and B, mitogen stimulation induces
tyrosine phosphorylation of c-Cbl. Total cell lysates prepared from
unstimulated NIH3T3 cells (A) overexpressing the human EGFR
(HER14) and HER14 cells stimulated with 275 ng/ml EGF for 5 min at 37
°C and murine macrophage cells (B) (P388D1) unstimulated
or treated with 25 nM human recombinant CSF-1 for 30 s at 37
°C were fractionated on 8% SDS-PAGE and blotted with
anti-phosphotyrosine antibodies (TCL anti-PY). Utilizing 300
µg of lysate prepared from unstimulated and stimulated cells in A and B, c-Cbl was immunoprecipitated and Western
blotted with anti-phosphotyrosine antibody (c-Cbl IP;
anti-PY). The c-Cbl IP blot was then stripped of antibody
and reprobed with anti-c-Cbl antibody (c-Cbl IP; anti-Cbl).
The migration of c-Cbl is indicated by the arrows, and the
170-kDa band that is tyrosine-phosphorylated in response to EGF and has
a similar mobility to the EGFR is indicated by an arrowhead. C, c-Cbl is tyrosine-phosphorylated in
v-src-transformed cells. c-Cbl was immunoprecipitated from
cell lysates of normal lung fibroblasts (LungFB),
NIH3T3 cells overexpressing c-src (c-src3T3), and
Balb/c3T3 cells transformed by v-src (SRD), and the
immunoprecipitated material was blotted with anti-phosphotyrosine
antibodies (c-Cbl IP; anti-PY). Antibody was removed from
these blots and reprobed with anti-c-Cbl antibody (c-Cbl IP;
anti-Cbl).
We next determined whether the non-receptor
tyrosine kinase, Src, induces tyrosine phosphorylation of c-Cbl. As
shown in Fig. 3C (anti-PY), c-Cbl does not
appear to be tyrosine-phosphorylated in lung fibroblasts that possess
endogenous c-Src or in NIH3T3 cells that overexpress the c-Src protein.
In contrast, c-Cbl is heavily tyrosine-phosphorylated in
v-src-transformed fibroblasts (SRD cells). The level of c-Cbl
does not, however, appear to differ in the three types of cultures
examined (Fig. 3C, anti-Cbl). Therefore, c-Cbl
appears to be tyrosine-phosphorylated by activated non-receptor
tyrosine kinases such as the viral Src protein.
c-Cbl Associates with Tyrosine Kinases in Vivo
We
then examined the physical association of c-Cbl with various tyrosine
kinases. EGFR was immunoprecipitated from HER14 lysates with or without
EGF stimulation and immunoblotted with anti-c-Cbl antibody. As shown in Fig. 4A, c-Cbl co-immunoprecipitated with EGFR in
stimulated HER14 cells but not in unstimulated cells (Fig. 4A, anti-Cbl). c-Cbl was also physically
associated with CSF-1R in P388D1 cells, and the association was
enhanced by stimulating the cells with CSF-1 (Fig. 4B, anti-c-Cbl). In both experiments, the amount of the
immunoprecipitated receptors did not change upon stimulation with the
appropriate ligand (Fig. 4, A and B, anti-EGFR and anti-CSF-1R, respectively). c-Cbl was
co-immunoprecipitated with v-Src in SRD cells when cells were lysed in
CHAPS buffer (Fig. 4C, C), but the
co-immunoprecipitation was not observed when cells were lysed in TNE
buffer (Fig. 4C, N). Co-immunoprecipitation of
c-Cbl with Src, Fyn, Lyn, or Syk was not observed in macrophages with
or without FcR stimulation (data not shown).
Figure 4:
c-Cbl associates with tyrosine kinases in vivo. A, EGFR was immunoprecipitated from HER14
cells untreated or treated with 275 ng/ml EGF for 5 min at 37 °C. B, CSF-1R was immunoprecipitated from P388D1 cells untreated
or treated with 25 nM CSF-1 for 30 s. C, v-Src was
immunoprecipitated from SRD cells lysed in either TNE buffer (N) or CHAP buffer (C). Immunoprecipitated material
was Western blotted with anti-c-Cbl antibody (anti-c-Cbl).
Antibody was removed from these blots and reprobed with anti-EGFR
antibody (A, anti-EGFR), anti-CSF-1R antibody (B,
anti-CSF-1R), and anti-Src antibody (C, anti-Src),
respectively. TCL were included from unstimulated cells, cells
stimulated with EGF (A) and CSF-1 (B), and SRD cells
prepared in TNE lysis buffer (C) and were included in
immunoblot analysis with anti-c-Cbl
antibody.
Stimulation Induces Translocation of
c-Cbl
Finally, we determined whether the subcellular
localization of c-Cbl was altered after stimulation of the different
receptors described in the above studies. Utilizing immunocytochemical
techniques with anti-c-Cbl antibody in conjunction with confocal
microscopy, diffuse staining in the cytoplasm was observed in
unstimulated macrophages and in HER14 cells (Fig. 5, A and C, respectively). Whether we stimulated macrophages
by antibody clustering of the FcR or HER14 cells by EGF treatment,
c-Cbl was found to translocate similarly upon stimulation and to
concentrate in a more specific region in the Golgi area of the cell (Fig. 5, B and D, arrows). In both
P388D1 cells and HER14 cells, the translocation began at 3 min after
stimulation (data not shown) and was complete after 30 min (Fig. 5, B and D). These results suggest that
tyrosine phosphorylation of c-Cbl resulting from various types of
stimulation similarly alters its subcellular localization.
Figure 5:
Stimulation induces a change in the
subcellular localization of c-Cbl. Cells were plated on coverslips and
cultured for overnight at 37 °C. A and B, P388D1
cells. C and D, HER14 cells. P388D1 cells were
unstimulated (A) or stimulated by FcR clustering for 30
min at 37 °C (B). HER14 cells were further cultured in
DMEM with 0.5% calf serum for 16 h and then unstimulated (C)
or stimulated with 275 ng/ml EGF for 30 min (D). Cells were
fixed with 3.7% formaldehyde for 10 min at room temperature and stained
with anti-c-Cbl antibody. Bars = 5
µm.
R), and
intracellular tyrosine kinases that are constitutively activated
(v-Src).
R stimulation. Though Fc receptors have
no intrinsic tyrosine kinase or tyrosine phosphatase activity, two
classes of cytoplasmic tyrosine kinases, the Src family and
the Syk/ZAP70 family, have been shown to be implicated in signaling via
Fc receptors (for review see Ref. 10). With respect to the Src
family, it has been reported that Fc receptor stimulation activates Src family tyrosine kinases in various cell
systems(8, 9, 10) . In this study, we report
that c-Cbl is able to bind to the SH3 domains of Src, Fyn, and Lyn at
least in vitro in both unstimulated and stimulated cells via
an association that may be related to the presence of a proline-rich
domain in the C terminus of c-Cbl(2) . In spite of the rapid
tyrosine phosphorylation of c-Cbl in Fc
R-stimulated macrophages
and the in vitro binding of c-Cbl to the SH3 domains of Src family tyrosine kinases, we failed to demonstrate the
physical association of c-Cbl with Src, Fyn, or Lyn in
Fc
R-stimulated macrophages. In addition we could not demonstrate
the physical association of c-Cbl with Syk. This may either be due to
an unstable or very transient association of the kinases tested with
c-Cbl in vivo or to the involvement of other kinases in the
tyrosine phosphorylation of c-Cbl.
R stimulation of macrophages and EGF stimulation of HER14 cells
both induced the translocation of c-Cbl to a somewhat more restricted
area of the cell, which, at least morphologically, is reminiscent of
the trans-Golgi region. These results suggest that tyrosine
phosphorylation of c-Cbl induced by various types of stimulation is
associated with a change in the subcellular localization of c-Cbl.
Although c-Cbl has putative motifs that have been identified in
transcription factors and v-Cbl has been reported to have nuclear as
well as cytoplasmic localization(2, 3) , we have not
been able to detect c-Cbl in the nucleus of cells before or after
stimulation with different agents. It is interesting to point out that
v-Cbl, which localizes in the nucleus, lacks the C-terminal region of
c-Cbl, which contains a proline-rich sequence that could be responsible
for binding to SH3-containing proteins such as Nck (2, 4) and, as shown
here, members of the Src family. It is therefore possible that the
proline-rich domain of c-Cbl associates with the SH3-containing
molecules such as Src family tyrosine kinases, adaptor molecules, or
cytoskeletal proteins such as
-spectrin, resulting in the
retention of the c-Cbl protein in the cytoplasm. Further studies will
be required to clarify the physiological role of the tyrosine
phosphorylation of c-Cbl in the subcellular localization of the protein
as well as its function.
R clustering and CSF-1
binding in macrophages, to EGF binding in HER14 cells, and to v-src and v-abl transformation, as well as in response to T
cell receptor activation. For Fc
R and EGFR stimulation, this is
associated with the translocation of c-Cbl to the central region of the
cell, in the trans-Golgi area. The fact that c-Cbl is
tyrosine-phosphorylated and translocated in response to different
agents may indicate that this protein is involved in a general cellular
phenomenon, which follows activation of signal transduction pathways by
various types of tyrosine kinases. Further study will be necessary to
identify the putative biological role of c-Cbl in tyrosine
kinase-mediated cell signaling.
R, Fc
receptor; DMEM,
Dulbecco's modified Eagle's medium; EGF, epidermal growth
factor; EGFR, EGF receptor; CSF-1, colony-stimulating factor-1; CSF-1R,
CSF-1 receptor; GST, glutathione S-transferase; PAGE,
polyacrylamide gel electrophoresis; TCL, total cell lysates; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.