From the Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Cbl proto-oncogene product has emerged as a
novel negative regulator of receptor and non-receptor tyrosine kinases.
Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor- Cbl, the 120-kDa cytoplasmic polypeptide product of the
c-cbl proto-oncogene, is the cellular homologue of
v-cbl, a retroviral oncogene that induces pre-B lymphomas
and myeloid leukemias in mice (1). A number of recent studies have
established Cbl as a component of the signal transduction cascades
downstream of both the receptor protein tyrosine kinases
(PTKs)1 and surface receptors
noncovalently associated with PTKs (2-6). Cbl is rapidly and
prominently phosphorylated on tyrosine residues upon stimulation
through a number of receptors, resulting in the association of Cbl with
SH2 domain-containing proteins, such as the p85 subunit of PI 3-kinase,
the Crk adaptor protein family, and the Rac exchange factor VAV.
Recently, the phosphopeptide motifs that mediate these interactions
have been localized within the C-terminal third of Cbl (7-10).
Furthermore, a large proline-rich region (amino acids 481-690) in Cbl
mediates interactions with the SH3 domains of Src family PTKs and the
adaptor proteins Grb2 and Nck, thus promoting the formation of
signaling protein complexes that are present in cells prior to receptor
activation. Although these associations have promoted the notion that
Cbl functions as a complex adaptor protein to couple PTKs to downstream
signaling pathways, lack of evolutionary conservation of the C-terminal region suggests that the primary role of Cbl may be different. Indeed,
a number of recent biochemical and genetic studies have identified Cbl
as a novel negative regulator of receptor and non-receptor PTKs
(2-6).
The first evidence for the role of Cbl as a negative regulator of
tyrosine kinase signaling was provided by studies of vulval development
in Caenorhabditis elegans, a process that requires the
LET-23 receptor tyrosine kinase, a homologue of the mammalian epidermal
growth factor receptor (EGFR) (11). A negative regulator of signaling
downstream of the LET-23 receptor, the suppressor of lineage defect 1 (sli-1), was shown to encode a Cbl homologue (SLI-1).
Recently, a Drosophila Cbl homologue (D-Cbl) was
also identified and shown to function as a negative regulator of the Drosophila EGFR-mediated R7 photoreceptor development (12,
13). Notably, the loss of function mutations in SLI-1, including one missense point mutation (G315E), mapped to the evolutionarily conserved
N-terminal region (Cbl-N) which functions as a tyrosine kinase binding
(TKB)2 domain in Cbl (14).
The Cbl TKB domain was shown to bind to the negative regulatory
phosphorylation sites within the SH2 kinase linker of ZAP70 and Syk
PTKs (15-17). These binding activities were abrogated by a mutation
(G306E), equivalent to the loss of function mutation (G315E) in SLI-1.
Notably, Cbl overexpression in the rat basophilic leukemia cell line
RBL-2H3 led to a decrease in the autophosphorylation and kinase
activity of Syk and inhibition of degranulation in response to Fc A potential role for Cbl as a negative regulator of mammalian receptor
PTKs is suggested by a number of findings. Decreased autophosphorylation of the EGFR and lower JAK-STAT phosphorylation was
observed in NIH3T3 cells that overexpressed Cbl, and higher EGFR
autophosphorylation was seen in antisense Cbl-transfected cells (20).
Conversely, NIH3T3 cells expressing oncogenic forms of Cbl revealed a
hyperphosphorylation of PDGFR Although the above studies clearly implicated Cbl as a negative
regulator of PTKs, the biochemical and cell biological mechanisms mediating such an effect have been less clear. Recently, we showed that
Cbl overexpression in NIH3T3 cells led to the enhancement of the
PDGF-induced ubiquitination and degradation of the PDGFR Cell Lines and Culture--
Parental and transfected NIH3T3
cells were cultured in Antibodies--
The murine monoclonal antibodies used were as
follows: 4G10 (anti-phosphotyrosine, anti-Tyr(P)) (24) (gift of Dr.
Brian Druker, Oregon Health Sciences University) and 12CA5 (anti-HA epitope tag) (25). The polyclonal rabbit antibodies used were as
follows: anti-PDGFR PDGF Stimulation--
For PDGF stimulation, cells were first
cultured for 24 h in medium containing 0.5% FCS (serum
deprivation). Recombinant human PDGF-AA (for selective activation of
PDGFR Immunoprecipitation and Immunoblotting--
Optimal amounts of
antibodies (determined by preliminary titrations) were added to
aliquots of lysates equalized for protein content by the Bradford assay
(Bio-Rad; using the bovine serum albumin standard). After 1-2 h of
rocking at 4 °C, 20 µl of protein A-Sepharose 4B beads (Amersham
Pharmacia Biotech) were added, and incubation was continued for 45 min.
Beads were washed six times in lysis buffer, and bound proteins were
solubilized in Laemmli sample buffer with 2-mercaptoethanol and
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Resolved polypeptides were transferred to polyvinylidene
difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA) and
immunoblotted with the indicated antibodies. Horseradish
peroxidase (HRPO)-conjugated protein A (Cappel-Organon
Technika, Durham, NC) was used as a secondary reagent for blotting,
followed by enhanced chemiluminescence (ECL, NEN Life Science
Products). Membranes were stripped and reprobed, as described (21).
Graphics were generated by direct scanning of films using a
Hewlett-Packard ScanJet 4cTM scanner and Corel
DrawTM version 6 software.
In Vitro GST Fusion Protein Binding Experiments--
Six-mg
aliquots of cell lysates were incubated with glutathione-Sepharose
beads on which GST or GST fusion proteins of Cbl (Cbl-N, aa 1-357;
Cbl-N/G306E; or Cbl-C, aa 358-906) were noncovalently immobilized
(14). After 3 h incubation at 4 °C, the beads were washed six
times in cold wash buffer (0.1% Triton X-100, 50 mM Tris,
pH 8.0, 150 mM NaCl), and proteins were eluted by boiling in Laemmli sample buffer containing 2-mercaptoethanol. Samples were
resolved by SDS-PAGE and analyzed by Western blotting.
Biotin Labeling of Cell-surface PDGFR Assessment of PDGF-dependent Cell
Proliferation--
The cells were plated in triplicate at a density of
2 × 104 cells per 25-cm2 flask in TUNEL Assay for Assessment of Apoptosis--
The cells were
grown on coverslips in Efficient Tyrosine Phosphorylation of Cbl Requires an Intact TKB
Domain--
Given that the G306E mutation in the TKB domain of Cbl,
corresponding to a loss-of-function mutation in C. elegans
Cbl homologue SLI-1, abrogate the ability of an oncogenic Cbl mutant to
transform NIH3T3 cells and to induce hyperphosphorylation of the
PDGFR
In view of the role of the TKB domain in the physical association of
Cbl with other PTKs, such as ZAP70 and Syk (15, 16), we first compared
the association of wild type Cbl versus Cbl-G306E with
PDGFR
Interestingly, the Cbl-G306E mutant itself exhibited a reduced level of
PDGF-induced tyrosine phosphorylation compared with that of wild type
Cbl, even though the cell lines examined expressed similar levels of
the Cbl proteins (Fig. 2A, middle and bottom panel). The reduced phosphorylation of Cbl-G306E was not due to changes in the kinetics of phosphorylation, as revealed by a time course experiment (Fig. 2B). These results indicate that the
TKB domain is essential for efficient PDGF-induced tyrosine
phosphorylation of Cbl.
A Critical Role of Cbl TKB Domain in the Enhancement of
Ligand-induced Ubiquitination of PDGFR
As expected (23), the ligand-induced ubiquitination of the PDGFR Association of Cbl with PDGFR
Stimulation of Cbl-Ph cells with PDGF-BB was used to assess if
selective stimulation of the PDGFR
Stimulation of Ph-Cbl cells with PDGF-BB induced a prominent
time-dependent phosphorylation of Cbl, which peaked at 3 min (Fig. 4A). When anti-HA
and anti- PDGFR
Our previous studies showed that Cbl could physically interact with the
PDGFR
These analyses revealed that Cbl-N, but not its G306E mutant, could
bind to the PDGFR
To determine if Cbl overexpression affects ligand-induced
ubiquitination of the PDGFR Cbl Enhances the Ligand-dependent Degradation of
PDGFR
As shown in Fig. 6, a
time-dependent decrease in the intensity of the receptor
band was observed following ligand stimulation of all cell lines.
However, whereas a significant level of biotinylated PDGFR
The loss of cell-surface PDGFR Cbl Inhibits PDGF-dependent Cell Proliferation in a TKB
Domain-dependent Manner--
Given that Cbl overexpression
led to enhanced ubiquitination and degradation of both the
All cell lines showed relatively little proliferation when grown in low
serum medium (0.5% FCS) (Fig. 7A,
top left panel). To quantify PDGF-dependent
proliferation, cells were cultured in the low serum medium containing
20 ng/ml PDGF-AA and were counted every other day. The
Cbl-overexpressing clones showed a decrease in
PDGF-AA-dependent growth, with about half as many cells
harvested at each time point beyond day 3, when compared with the
vector-transfected NIH3T3 cells (Fig. 7A, top right panel).
The retarded growth was also observed in two additional
Cbl-overexpressing clones (data not shown). Importantly, the
Cbl-G306E-transfected cells showed little or no retardation of growth
in PDGF-AA (Fig. 7A, top right panel). Cbl-transfected
NIH3T3 cells also showed a reduction of proliferative response to
PDGF-BB, as compared with the vector-transfected cells; at early time
points (days 1-5), the rate of proliferation of Cbl-G306E-expressing
cells in response to PDGF-BB was similar to that of vector-transfected
cells, but a substantial reduction was noted at later time points (days
9-13) (Fig. 7A, bottom left panel).
The above results were contradictory to our earlier studies (21); we
did observe reduced proliferation in a Cbl-transfected clone of NIH3T3
cells (Cbl.8) compared with parental NIH3T3 cells. Since our earlier
studies had been carried out using a late passage of Cbl.8, we
reanalyzed an early passage of this clone that was comparable to other
clones analyzed above. Indeed, a reduction in PDGF-BB-induced
proliferation was also observed using this early passage of the Cbl.8
clone (Fig. 7A, bottom right panel). Furthermore, a late
passage of this clone, comparable to that used in our
earlier studies (21), did not show reduced proliferative response to
PDGF-AA (data not shown). Apparently, long term culture selects for
cells that have compensated for the Cbl-induced decrease in PDGF responsiveness.
Since NIH3T3 cells express both PDGFR
The viability of NIH3T3 transfectants cultured with PDGF, as measured
by trypan blue staining, was more than 95% until day 7 (Fig.
8, middle and bottom
panels, and data not shown), indicating that the effect of Cbl was
primarily mediated through reduced proliferation rather than increased
cell death. However, a higher proportion of Cbl-transfected NIH3T3
cells, as compared with parental or vector-transfected NIH3T3 cells,
underwent death at later time points. In the experiment shown in Fig.
7, the viability of Cbl transfectants decreased to about 50% by day 9, whereas the viability of control cells at the same time point was about
95% (Fig. 8). When NIH3T3 transfectants were cultured in low serum
(0.5% FCS) medium, the Cbl-transfected cells showed a higher degree of
cell death compared with control cells at day 9 (Fig., 8, top panel). All cell lines showed a decrease in
cell viability at later time points (day 11 and day 13). These results
suggest that, in addition to reduced proliferation, wild type Cbl
overexpression also enhances cell death in transfected cells at later
time points.
Morphological examination of Cbl overexpressing cells at day 9 (when
reduced viability was clearly observed) revealed a larger proportion of
cells with shrunken cytoplasm and condensed nuclei, whereas the vast
majority of vector-transfected cells grew as monolayers of flat
refractile cells (data not shown). These observations suggested that
the observed cell death may be due to apoptosis. A TUNEL assay was
performed to assess if this was the case.
Compared with relatively few TUNEL-positive cells in the cultures of
vector-transfected and Cbl-G306E-transfected cells (Fig. 9, A and E), a
larger proportion of TUNEL-positive cells was observed in cultures of
Cbl-transfected cells. The apoptotic cells are clearly visible with
their nuclear fragmentation (Fig. 9C, see arrows). In addition, several dead cells are visible as
condensed bright spots apparently reflecting late stage apoptosis (Fig. 9C). Propidium iodide staining demonstrated that lack of
TUNEL-positive cells in vector-transfected or Cbl-G306E-transfected
cells was not due to lower cell density on the coverslips (Fig. 9,
B, D, and F). Overall, these results indicate
that Cbl overexpression induces a TKB domain-dependent
reduction of PDGF-induced cellular proliferation and that
Cbl-overexpressing cells are sensitized to undergo apoptotic cell
death.
Recent studies have clearly identified the Cbl proto-oncoprotein
as a negative regulator of receptor and non-receptor tyrosine kinases.
However, the precise biological consequences of this negative
regulation, such as alterations in proliferation, differentiation, or
metabolic activity of a cell, have not been defined. Here, we provide
evidence that Cbl-dependent negative regulation of the PDGF
receptors provides a mechanism to down-regulate the cellular proliferation initiated by PDGF. We also demonstrate that Cbl physically interacts with and functionally regulates PDGFR A role for Cbl to regulate negatively the cellular proliferation in
response to growth factors has been implied previously but has not been
experimentally demonstrated. In fact, previous studies, including our
own analysis of one Cbl-transfected NIH3T3 clone (Cbl.8), failed to
detect a difference in the rate of proliferation between cells
overexpressing Cbl and the parental NIH3T3 cells (20, 21, 32). These
earlier findings had assessed both serum-dependent and
PDGF-induced proliferation. We confirmed the lack of an effect of Cbl
overexpression on the proliferation of the late-passage Cbl.8 clone
used in our earlier studies (data not shown). To clarify this
discrepancy, we derived an additional clone (Cbl.9) from the same bulk
transfectant that was used to derive Cbl.8, and in addition we derived
an independent Cbl-overexpressing NIH3T3 clone Cbl.2-8. These clones
were examined relatively early after cloning (typically 10-30
passages). These analyses revealed a substantial and reproducible
reduction in the PDGF-AA and PDGF-BB-induced proliferation of
Cbl-transfected NIH3T3 cells compared with those transfected with the
vector alone. A similar reduction in PDGF-induced proliferation was
also observed using an early passage of the Cbl.8 clone (Fig.
7A). Apparently, the growth disadvantage conferred by Cbl
overexpression is selected against, and continuous culture appears to
promote growth of cells that have compensated for the inhibitory effect
of Cbl on proliferation.
We have also observed a reduction in serum- and
EGF-dependent proliferation of the Cbl-overexpressing
NIH3T3 cells (data not shown), indicating that Cbl-mediated negative
regulation of PTKs indeed impinges on the proliferative outcome of
receptor activation. This conclusion is consistent with the increased
serum or PDGF-induced proliferation of NIH3T3 cells transformed with
oncogenic Cbl mutants (21). Our results support the likelihood that
increased hypercellularity in various organs such as thymus, spleen,
and mammary gland of Cbl Our earlier finding of a general up-regulation of the PDGFR The present study provides direct data to show that PDGFR Cbl overexpression also led to an enhancement of ligand-induced
ubiquitination and degradation of the PDGFR Recent studies have revealed the crucial importance of the N-terminal
tyrosine kinase-binding domain of Cbl for its negative regulatory
influence on PTKs. This domain is highly conserved during evolution (1,
11-13) and is the site of loss-of-function mutations in the C. elegans Cbl homologue SLI-1 (11). By itself, this region is
oncogenic and up-regulates PDGFR In conclusion, studies reported in this paper generalize the negative
regulatory role of Cbl for the PDGFR family and demonstrate that the
tyrosine kinase binding domain of Cbl is indispensable for the
functional effects of Cbl. Importantly, our studies demonstrate that
Cbl-mediated biochemical effects on the PDGFR lead to important biological consequences, as shown by a reduction in cellular
proliferation in responses to PDGFR ligands. Further mechanistic
studies of Cbl-mediated negative regulation of the PDGFR signaling
cascade are likely to elucidate a novel mode of biological control of cellular responses to extracellular stimuli.
(PDGFR
) and that the expression of
oncogenic Cbl mutants up-regulated the PDGFR
signaling machinery
strongly suggested that Cbl negatively regulates PDGFR
signaling.
Here, we show that, similar to PDGFR
, selective stimulation of
PDGFR
induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an
enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFR
, as observed with PDGFR
. We
show that Cbl-dependent negative regulation of PDGFR
and
results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More
importantly, the G306E mutation abrogated the ability of Cbl to enhance
the ligand-induced ubiquitination and degradation of the PDGFR and to
inhibit the PDGF-dependent cell proliferation and
protection from apoptosis. These results demonstrate that Cbl can
negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding
domain of Cbl.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R1
stimulation (18); the Cbl-induced reduction in Syk tyrosine kinase
activity requires an intact Cbl TKB domain (16). Overexpression of Cbl
in Jurkat T cells was shown to reduce Ras-dependent AP1
transcription factor activity, whereas overexpression of the oncogenic
70Z/3 Cbl mutant induced an enhancement of basal and Ca2+
ionophore-induced nuclear factor of activated T cell reporter activity
(9, 19). These studies have highlighted the role of Cbl as a negative
regulator of ZAP70/Syk PTKs.
and an up-regulation of signaling
downstream of this receptor (21). In addition, NIH3T3 cells expressing
the oncogenic forms of Cbl showed an increase in the
autophosphorylation and kinase activity of a transfected human EGFR,
both under serum-starved and EGF-stimulated conditions (22).
Apparently, the effect of the oncogenic mutants of Cbl on PDGFR
and
EGFR reflects a reversal of the negative regulatory role of endogenous
wild type Cbl.
(23). These
findings suggested that one mechanism of the negative regulatory
function of Cbl for receptor PTKs may be at the level of regulating the
ligand-induced turnover of PDGFR
. Here, we have extended these
initial observations along three major directions. First, we show that
Cbl regulates both the
and
PDGF receptors. Second, we show that
an intact TKB domain is required for Cbl-dependent enhancement of ubiquitination and degradation of the PDGFR. Finally, we
demonstrate that Cbl-mediated negative regulation of the PDGF receptors
is biologically relevant in that Cbl overexpression induces a TKB
domain-dependent reduction in PDGF-induced cell proliferation. These studies generalize the role of Cbl in regulating receptor PTK signaling and suggest a direct correlation between the
biological functions of Cbl and its ability to regulate ligand-induced receptor ubiquitination and degradation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium (
-MEM)
supplemented with 10% fetal calf serum (FCS, HyClone Laboratories,
Inc., Logan, UT). Transfectants of the PDGFR
-negative Patch mutant
mouse-derived 3T3 cell line (Ph) (28) were cultured in Dulbecco's
modified Eagle's medium containing 10% FCS. For all transfectants,
the medium was supplemented with 500 µg/ml G418 (Life Technologies,
Inc.). Transfectants were established by retroviral transfection using
pJZenNeo plasmid constructs, as described (21, 23). Clonal
transfectants of the NIH3T3 cells overexpressing the N-terminal
hemagglutinin (HA) epitope-tagged human Cbl (3T3-HA-Cbl.8 and
3T3-HA-Cbl.9, referred to here as Cbl.8 and Cbl.9, respectively) have
been described previously (21, 23). Additional NIH3T3 cell clones
expressing HA-Cbl (Cbl.2-8) or HA-Cbl-G306E (G306E.1-10 and
G306E.2-3) and the pJZenNeo vector-transfected clones (pJ.1 and pJ.8)
were derived using the same retroviral transfection approach. The
HA-Cbl expressing (Ph-Cbl.1-8, Ph-Cbl.2-8, and Ph-Cbl.2-10) or the
pJZenNeo vector-transfected (Ph-pJ.1 and Ph-pJ.2) Ph cell lines were
also established using retroviral transfection. The overexpression of
exogenous HA-Cbl in NIH3T3 (Fig. 1A) and Ph (Fig. 1C,
middle and bottom panels) transfectant clones was
demonstrated by anti-Cbl and anti-HA immunoblotting of whole cell
lysates. The expression of PDGF receptors in NIH3T3 transfectants was
quantified by anti-PDGFR
and anti-PDGFR
immunoblotting of serial
dilutions of whole cell lysates (Fig. 1B). The expression of
PDGFR
in Ph cell transfectants was assessed by anti-PDGFR
immunoblotting (Fig. 1C, top panel). Whereas different
clones varied in the level of transfected Cbl and PDGFR expression
under basal (unstimulated) conditions, no systematic correlation
between PDGFR levels and the nature of Cbl (wild type versus
G306E) or its level of expression was apparent.
(sc-431, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA); anti-PDGFR
(against the kinase insert; PharMingen, San
Diego, CA); anti-PDGFR
(Upstate Biotechnology, Lake Placid, NY, used
for Western blotting in Figs. 1, B and C, and
4C); and anti-ubiquitin (NCL-UBIQ, NovoCastra Laboratories,
Newcastle, UK; obtained from Vector Laboratories, Burlingame, CA).
Normal rabbit serum (negative control) was obtained from non-immunized rabbits.
) or PDGF-BB (both from Upstate Biotechnology Inc., Lake
Placid, NY) was then added at a final concentration of 20 ng/ml. At the
indicated time points, the medium was aspirated, and cell extracts were
prepared at 4 °C in a lysis buffer containing 0.5% Triton X-100
(Fluka, Buchs, Switzerland), 50 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl
fluoride, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 1 µg/ml each of leupeptin, pepstatin,
chymostatin, antipain, and aprotinin (Sigma).
and Assessment of Its
Ligand-induced Degradation--
Cell monolayers were washed with
ice-cold phosphate-buffered saline containing 20 mM HEPES
buffer solution, pH 7.35, and then incubated in the same buffer with
sulfo-N-hydroxysulfosuccinimide-biotin (Pierce) for 15 min
at 4 °C. After washing, the cells were incubated in
-MEM
containing 1 mg/ml bovine serum albumin (tissue culture grade; Sigma)
with PDGF-BB (7.5 ng/ml) at 37 °C for the indicated time points, and
lysates were prepared as described above. Anti-PDGFR
immunoprecipitates of these lysates were resolved by SDS-PAGE and
blotted with an avidin-HRPO conjugate (Vector Laboratories, Inc.,
Burlingame, CA) followed by enhanced chemiluminescence, as described
above. Densitometry was carried out using Scion Images for
WindowsTM software. Densitometric data are expressed in
arbitrary units.
-MEM
containing 0.5% FCS. After 18 h, the cells were switched to the
same medium supplemented with 20 ng/ml recombinant human PDGF-AA or
PDGF-BB, as appropriate. The culture medium, containing the indicated
PDGF isoforms, was changed on alternate days. The number of cells was
counted every other day from day 1 to day 13, or as indicated, using a hemacytometer.
-MEM containing 0.5% FCS and 20 ng/ml
PDGF-BB for 10 days, fixed in 4% paraformaldehyde in
phosphate-buffered saline, and permeabilized with 0.1% Triton X-100 in
0.1% sodium citrate. After a series of washes in phosphate-buffered saline, DNA fragmentation in apoptotic cells was determined by the
terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end
labeling (TUNEL) assay using the In Situ Cell Death
Detection Kit, POD (Roche Molecular Biochemicals), according to the
instructions of the manufacturer. Apoptotic cells were visualized and
photographed using a fluorescence microscope. Propidium iodide was used
to visualize the entire cell population on the coverslip.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(21), it appeared likely that a functional TKB domain would be
required for Cbl-mediated negative regulation of the PDGFR
. To
assess if this was the case, we established NIH3T3 clones expressing either wild type Cbl (Cbl.8, Cbl.9, and Cbl.2-8) or the Cbl-G306E mutant (G306E.1-10 and G306E.2-3) (Fig.
1).
View larger version (45K):
[in a new window]
Fig. 1.
Expression of HA-tagged Cbl proteins and PDGF
receptors in NIH3T3 and Ph cell transfectants. A, whole
cell lysates were prepared from NIH3T3 cells transfected with pJZenNeo
vector alone (pJ.1 and pJ.8, lanes 1 and 2), with
pJZenNeo constructs encoding hemagglutinin (HA) epitope-tagged wild
type Cbl (Cbl.8, Cbl.9, and Cbl.2-8, lanes 5-7), or with
Cbl-G306E (G306E.1-10 and G306E.2-3, lanes 3 and
4). 100 µg of each lysate was resolved by SDS-PAGE,
transferred to a PVDF membrane, and serially immunoblotted with
anti-Cbl (top panel) and anti-HA (bottom panel)
antibodies. B, 100, 50, or 25 µg of each cell lysate was
resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted
with anti-PDGFR (top panel) or anti-PDGFR
(bottom panel) antibodies. C, whole cell
lysates were prepared from Ph cells transfected with pJZenNeo vector
alone (Ph-pJ.1 and Ph-pJ.2) or with pJZenNeo constructs encoding HA
epitope-tagged wild type Cbl (Ph-Cbl.1-8, Ph-Cbl.2-8, and
Ph-Cbl.2-10). 100 µg of each lysate was resolved by SDS-PAGE,
transferred to a PVDF membrane, and serially immunoblotted with
anti-PDGFR
(top panel), anti-Cbl (middle
panel), and anti-HA (bottom panel) antibodies.
in PDGF-AA-stimulated cells. Following PDGF-AA stimulation, a
185-kDa phosphoprotein, corresponding to PDGFR
, was
co-immunoprecipitated both with Cbl and Cbl-G306E (Fig.
2A, top panel, lanes
2 and 4). Therefore, elimination of the TKB
domain function did not abolish the physical association between Cbl
and PDGFR
, similar to recent results with the EGFR (26). As expected
(23), the wild type Cbl-associated PDGFR band showed a substantial
proportion of the slower-migrating species, which represents the
ubiquitinated PDGFR
. In contrast, PDGFR
associated with the
Cbl-G306E mutant protein showed little of the slower-migrating species.
These results suggested that the TKB domain may be critical for
Cbl-dependent enhancement of the ubiquitination of PDGF
receptor, as fully described below. Because of the smearing of the
PDGFR as a result of enhanced ubiquitination, it was difficult to
quantify accurately the differences in the level of association between
PDGFR and wild type Cbl versus Cbl-G306E mutant. Therefore,
some loss of physical association as result of TKB domain inactivation
cannot be ruled out.
View larger version (52K):
[in a new window]
Fig. 2.
PDGF-AA-induced tyrosine phosphorylation of
wild type Cbl versus Cbl-G306E mutant and their
association with the PDGFR . A,
serum-deprived NIH3T3 transfectant lines Cbl.2-8 and G306E.1-10 were
either left unstimulated (
) or were stimulated with 20 ng/ml PDGF-AA
for 3 min (+), and lysates were prepared. 1-mg aliquots of each lysate
were subjected to immunoprecipitation (IP) with anti-HA
antibody 12CA5. Immunoprecipitated polypeptides were resolved by
SDS-PAGE, transferred to PVDF membrane, and subjected to anti-Tyr(P)
(4G10) immunoblotting (top and middle panels).
The membrane was stripped and reprobed with anti-HA antibody
(bottom panel). PDGFR
and Cbl are indicated.
B, wild type HA-Cbl (Cbl.8 and Cbl.2-8) and HA-Cbl-G306E
(G306E.1-10 and G306E.2-3) expressing NIH3T3 cells were either left
unstimulated (lanes 1, 5, 9, and 13) or were
stimulated with 20 ng/ml PDGF-AA for the indicated times (min), and
cell lysates were prepared. 1-mg aliquots of lysates were then
subjected to immunoprecipitation (IP) with anti-HA antibody
followed by immunoblotting with the same antibody (bottom
panel). The membrane was stripped and reprobed with anti-Tyr(P)
antibody 4G10 (top panel).
--
To assess directly the
role of the TKB domain in Cbl-dependent enhancement of the
PDGFR
ubiquitination, we compared NIH3T3 cells transfected with
vector alone (pJ.8) to cells expressing wild type Cbl (Cbl.2-8) or
Cbl-G306E (G306E.1-10 and G306E.2-3). Serum-deprived cells were
stimulated with PDGF-AA for various time points, and anti-PDGFR
immunoprecipitations were carried out on their lysates. These were
analyzed by anti-Tyr(P) and anti-ubiquitin immunoblotting (Fig.
3, A and B, top and
middle panels).
View larger version (50K):
[in a new window]
Fig. 3.
PDGF-AA-induced ubiquitination of the
PDGFR in wild type Cbl and Cbl-G306E mutant
overexpressing NIH3T3 cells. Vector-transfected NIH3T3 cells
(pJ.8) or cells overexpressing HA-Cbl (Cbl.2-8) (A) or
Cbl-G306E (G306E1-10, G306E.2-3) (B) were serum-deprived
and either left unstimulated (
) (lanes 1, 5, and
9) or were stimulated with 20 ng/ml PDGF-AA for the
indicated time points (min), and cell lysates were prepared. 1-mg
aliquots of each lysate were used for immunoprecipitation with
anti-PDGFR
antibody. The immunoprecipitated polypeptides were
resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by
anti-PDGFR
immunoblotting (bottom panels). The
membranes was stripped and serially reprobed with anti-ubiquitin
(middle panels) and anti-Tyr(P) antibodies (top
panels). A and B represent separate
experiments.
(seen as the upward-shifted smear in anti-Tyr(P) blot) in
Cbl-overexpressing NIH3T3 cells was substantially higher as compared
with that in the vector-transfected cells and was already maximal at 3 min of PDGF stimulation (Fig. 3A, lane 6) versus about 10 min in the control cells (Fig. 3A, lanes 2 and
3). Notably, the Cbl-dependent enhancement of
PDGFR
ubiquitination was essentially abrogated by the G306E mutation
in Cbl (Fig. 3B, compare lanes 2-4 to
lanes 6-8 and 10-12). These results reveal a
critical role for the TKB domain in Cbl-mediated facilitation of
ligand-induced PDGFR
ubiquitination.
and Enhancement of the
Ligand-induced Ubiquitination of PDGFR
by Cbl
Overexpression--
Elucidation of signaling pathways that are either
shared between
and
PDGF receptors, or are selectively recruited
to these receptors, is of significant biological interest (27). Our
previous studies of NIH3T3 cells expressing the wild type Cbl (23) or the oncogenic Cbl proteins (21) have established the PDGFR
as a
target of the negative regulatory effect of Cbl; this effect appears to
involve the Cbl-dependent enhancement of the ligand-induced ubiquitination and degradation of the PDGFR
. However, it has remained unclear whether PDGFR
is selectively regulated by Cbl or if
Cbl-dependent regulation may be a feature of both the
and
PDGF receptors. To address this question, we overexpressed HA-tagged wild type Cbl in PDGFR
-negative Patch 3T3 cells (Cbl-Ph) (28), using retroviral transfection (Fig. 1C). Similar to
parental Ph cells, the Cbl-Ph cells failed to respond to PDGF-AA by
tyrosine phosphorylation of cellular proteins (data not shown).
can induce tyrosine
phosphorylation of Cbl and its association with the activated receptor.
For this purpose, lysates prepared from unstimulated or
PDGF-BB-stimulated Cbl-Ph cells were subjected to anti-HA
immunoprecipitation and analyzed by anti-Tyr(P) immunoblotting.
immunoprecipitates were resolved side-by-side, the
185-kDa Cbl-associated band (Fig. 4B, lane 4) co-migrated
with PDGFR
. In addition, the 185-kDa band was immunodepleted with
anti-PDGFR
antibody (data not shown). These experiments indicated
that, similar to selective stimulation of the PDGFR
, selective
stimulation of the PDGFR
can also induce Cbl phosphorylation and its
association with the receptor.
View larger version (46K):
[in a new window]
Fig. 4.
Association of Cbl with
PDGFR and enhancement of ligand-induced
PDGFR
ubiquitination in Cbl-overexpressing
NIH3T3 cells. A and B,
PDGF-BB-dependent phosphorylation of Cbl and its
association with PDGFR
. HA-tagged Cbl-overexpressing Patch cells
were serum-deprived and either left unstimulated (
) or were
stimulated with 20 ng/ml PDGF-BB for 3 min (B) or the
indicated time points (min) (A), and cell lysates were
prepared. 1-mg aliquots of lysates were used for immunoprecipitation
with normal rabbit serum (NRS, control), anti-HA antibody,
or anti-PDGFR
antibody. Immunoprecipitated polypeptides were
resolved by SDS-PAGE, transferred to PVDF membrane, and subjected to
immunoblotting with anti-Tyr(P). A, the membrane was first
immunoblotted with anti-HA antibody (bottom panel).
C, binding of PDGFR
to GST fusion proteins of Cbl.
PDGFR
-negative Patch cells were either left unstimulated (
) or
were stimulated with 20 ng/ml recombinant human PDGF-BB for 10 min (+)
prior to lysis. Binding reactions were carried out by incubating
glutathione-Sepharose beads coated with 10 µg of GST, GST-Cbl-N (Cbl
aa 1-357), GST-Cbl-C (Cbl aa 358-906), or GST-Cbl-N-G306E with 6-mg
aliquots of cell lysates for 4 h. Bound polypeptides were resolved
by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with
anti-PDGFR
antibody (bottom panel). The membrane was
stripped and reprobed with anti-Tyr(P) antibody (top
panel).
through the Cbl TKB domain, which binds directly to
autophosphorylated PDGFR
(21). In addition, the C-terminal region of
Cbl can associate with the activated receptor, consistent with its
ability to bind the SH3 domains of Src family kinases and adaptor
proteins, such as Grb2, which interact with autophosphorylated PDGFR
(29-31). To assess if Cbl could bind to PDGFR
via similar mechanisms, in vitro binding assays were performed using GST
fusion proteins that incorporated the TKB domain (Cbl-N) or the
remaining C-terminal sequences of Cbl (Cbl-C). PDGFR
-negative
parental Patch cells were either left unstimulated or were stimulated
with PDGF-BB, and their lysates were incubated with GST fusion proteins followed by serial anti-PDGFR
and anti-Tyr(P) immunoblotting.
present in lysates of PDGF-stimulated cells (Fig.
4C, lanes 4 and 6); no binding was detected in
lysates of unstimulated cells, consistent with previous results with
the PDGFR
(21). Binding of PDGFR
to GST-Cbl-C was also observed (Fig. 4C, bottom panel, lane 8); no binding to
control GST was observed (Fig. 4C, lane 2). Together with
our earlier studies (21), these results indicate that similar
mechanisms mediate the association of Cbl with both
and
PDGF receptors.
, serum-deprived parental NIH3T3 cells or
wild type Cbl-transfected NIH3T3 cells (Cbl.8) were stimulated with
PDGF-BB for the indicated time points, and anti-PDGFR
immunoprecipitates were analyzed by anti-Tyr(P) and anti-ubiquitin
immunoblotting. This experiment revealed that the PDGFR
ubiquitination was substantially enhanced and peaked earlier (3 versus 10 min) in Cbl-overexpressing cells compared with
parental cells (Fig. 5A, top
and middle panels). Enhancement of the ligand-induced
ubiquitination of the PDGFR
was also observed using two additional
wild type Cbl-expressing clones (Cbl.9 and Cbl.2-8) but not in the
vector-transfected clone (pJ.8) of NIH3T3 cells (Fig. 5B, top
panel and middle panels, compare lanes 2, 6, and 10). Notably, similar to the ubiquitination of PDGFR
,
the G306E mutation abrogated the effect of Cbl on the ubiquitination of
the PDGFR
(Fig. 5B, top and middle panels, lanes 13-20). These results demonstrate that, similar to
PDGFR
, the PDGFR
is also a target of Cbl-dependent
enhancement of ligand-induced ubiquitination and that a functional Cbl
TKB domain is required for this effect.
View larger version (68K):
[in a new window]
Fig. 5.
Enhanced ligand-induced ubiquitination
of PDGFR in Cbl-overexpressing NIH3T3 cells
and the impact of G306E mutation on the effect of Cbl.
Serum-deprived parental NIH3T3 cells, vector-transfected NIH3T3 cells
(pJ.8), or NIH3T3 cells expressing wild type Cbl (Cbl. 8, Cbl.9,
Cbl.2-8) or Cbl-G306E mutant (G306E.1-10, G306E.2-3) were either
left unstimulated (
) or were stimulated with 20 ng/ml PDGF-BB for the
indicated times (min), and cell lysates were prepared. 1-mg aliquots of
each lysate were subjected to immunoprecipitation with anti-PDGFR
antibody, resolved by SDS-PAGE, transferred to PVDF membrane, and
subjected to anti-PDGFR
blotting (bottom panel). The
membranes were stripped and serially reprobed with anti-ubiquitin
(middle panel) and anti-Tyr(P) (top panel)
antibodies. A and B represent separate
experiments.
in a TKB Domain-dependent Manner--
Given our
previous findings that overexpression of Cbl facilitated
ligand-dependent degradation of the PDGFR
(23), we
tested the effect of Cbl overexpression on the turnover of the
PDGFR
. For this purpose, we quantified the PDGFR
levels in
vector-transfected NIH3T3 cells and cells overexpressing either wild
type Cbl or its TKB domain mutant (Cbl-G306E) at various times after
stimulation with PDGF-BB. To analyze selectively the ligand-responsive
cell surface-expressed PDGFR
, serum-starved cells were
surface-labeled with biotin for 15 min at 4 °C. After washing, the
cells were incubated with PDGF-BB at 37 °C for the indicated times.
Lysates were prepared at each time point, immunoprecipitated with an
anti-PDGFR
antibody, and blotted with avidin-HRPO to quantify the
level of biotin-labeled PDGFR
.
was still
detectable at 90 min after PDGF stimulation in vector-transfected
NIH3T3 cells and cells overexpressing the TKB domain mutant of Cbl
(Cbl-G306E) (Fig. 6A, lane 4), relatively little PDGFR
were detected in Cbl-transfected cells even at the 60-min time point
(Fig. 6A, lane 3).
View larger version (29K):
[in a new window]
Fig. 6.
Enhanced ligand-induced degradation of
PDGFR in Cbl-overexpressing NIH3T3 cells and
the impact of G306E mutation on the effect of Cbl. Serum-deprived
vector-transfected NIH3T3 cells (pJ.1) and NIH3T3 cells expressing wild
type Cbl (Cbl.9, Cbl.2-8) or Cbl-G306E mutant (G306E.1-10,
G306E.2-3; clone designations are indicated on left) were
surface-labeled with biotin. Labeled cells were incubated either
without (
) or with 7.5 ng/ml PDGF-BB at 37 °C for the indicated
times (min) and lysed. Anti-PDGFR
immunoprecipitates of 1-mg
aliquots of the lysates were subjected to blotting with avidin-HRPO
followed by ECL detection. A, representative blot is shown.
B, densitometry was performed, and the intensity of PDGFR
signal at various time points was expressed as a percentage of that in
unstimulated cells at zero time point.
in these cells was quantified by
densitometry of the streptavidin blot (Fig. 6B). This
analysis highlights a substantially earlier and more pronounced loss of surface-labeled PDGFR
upon PDGF stimulation of the
Cbl-overexpressing NIH3T3 cells as compared with that in the
vector-transfected NIH3T3 cells. The half-life of the surface-labeled
PDGFR
following PDGF-BB stimulation was 41-52% shorter in
Cbl-transfected NIH3T3 cells compared with that in parental cells
(Cbl.9, 21 min and Cbl.2-8, 26 min versus pJ.1, 44 min).
Notably, the G306E mutation abrogated the effect of Cbl on the
degradation of the PDGFR
; the half-life of the surface-labeled
PDGFR
of CblG306E transfectants was close to that detected in
vector-transfected NIH3T3 cells (G306E.1-10, 32 min; G306E.2-3, 39 min). Similar results were observed in two additional experiments (data
not shown). Together, these results show that the enhancement of
PDGFR
ubiquitination as a result of Cbl overexpression is associated
with a faster ligand-induced loss of the surface PDGFR
and that a
functional TKB domain is essential for this effect.
and
PDGF receptors, and the essential role of the evolutionarily conserved
TKB domain in this process, we wished to ascertain if the regulatory
influence of Cbl led to any biological consequences. For example, a
faster down-regulation of the activated PDGFRs could be expected to
decrease the mitogenic effects of PDGF. To test if this was indeed the
case, we compared the PDGF-induced proliferation of vector-transfected
NIH3T3 cells with cells overexpressing either the wild type Cbl or its
TKB domain mutant (Cbl-G306E).
View larger version (30K):
[in a new window]
Fig. 7.
Inhibition of PDGF-dependent cell
proliferation in Cbl-overexpressing NIH3T3 and Ph cell lines.
A, NIH3T3 cells transfected with the vector alone (pJ.1,
pJ.8), or cells overexpressing wild type Cbl (Cbl.9, Cbl.2-8 or Cbl.8)
or Cbl-G306E mutant (G306E.1-10, G306E.2-3) were plated at 2 × 104 cells per 25-cm2 flask in -MEM plus
0.5% FCS without or with 20 ng/ml PDGF-AA or PDGF-BB. Beginning on day
1, and every other day thereafter, the cells were released with
trypsin/EDTA treatment and were counted in a hemacytometer. The data
points represent the mean of triplicates; error bars
show ± 1 S.D. B, Ph cells transfected with the vector
alone (Ph-pJ.1, Ph-pJ.2) or cells overexpressing wild type Cbl
(Ph-Cbl.1-8, Ph-Cbl.2-8, Ph-Cbl.2-10) were plated at 2 × 104 cells per 25-cm2 flask in Dulbecco's
modified Eagle's medium plus 0.1% FCS without or with 20 ng/ml
PDGF-BB. The cells were counted as described in A above. The
data points represent the mean of triplicates; error bars
show ± 1 S.D.
and -
, and a
-selective
ligand does not exist, we could not selectively stimulate PDGFR
in
these cells. Therefore, to confirm further the effect of the
overexpression of Cbl on PDGFR
-dependent cell
proliferation, we compared the PDGF-induced proliferation of
vector-transfected Ph cells with that of Ph cells overexpressing the
wild type Cbl. All clonal cell lines expressed comparable levels of
PDGFR
(Fig. 1C, top panel). All of the cell lines showed
relatively little proliferation when grown in low serum medium (0.1%
FCS) (Fig. 7B, left panel). When cells were cultured in the
low serum medium containing 20 ng/ml PDGF-BB and counted every other
day, the Cbl-overexpressing Ph clones showed lower
PDGF-BB-dependent growth at each time point beyond day 3, when compared with the vector-transfected Ph clones (Fig. 7B,
right panel). These results clearly demonstrate that Cbl
overexpression results in a reduction of the PDGF-induced proliferative
response and that the TKB domain plays a critical role in the
regulation of PDGF-dependent cell proliferation by Cbl.
View larger version (32K):
[in a new window]
Fig. 8.
Loss of cell viability in Cbl-overexpressing
NIH3T3 cells. The viability of cells, cultured as described in
Fig. 6, was assessed by trypan blue staining at days 5 (solid
bars) and 9 (striped bars). The data points represent
the mean of triplicates; error bars show ± 1 S.D.
View larger version (128K):
[in a new window]
Fig. 9.
Apoptotic cell death in Cbl-overexpressing
NIH3T3 cells. The vector-transfected NIH3T3 cells (pJ.1;
A and B) or cells overexpressing wild type Cbl
(Cbl.9; C and D) or Cbl-G306E mutant
(G306E.1-10; E and F) were cultured with 20 ng/ml PDGF-BB for 10 days. Cells were washed, fixed in 4%
paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1%
Triton X-100 in 0.1% sodium citrate, and stained for apoptotic cells
using the TUNEL assay (A, C, and E). The
coverslips were stained with propidium iodide to visualize the entirety
of cells on the coverslips (B, D, and F). The
same fields are shown in left and right panels.
Arrows show the apoptotic cells with fragmented nuclear
material. The very bright spots observed in TUNEL assay are
an artifact due to staining of exposed nuclei of the dead cells. The
representative fields were photographed at a magnification of × 400.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, similar to our earlier observations with the PDGFR
, thus suggesting a general role for Cbl as a negative regulator of PDGF receptors. Finally, we demonstrate that the N-terminal TKB domain of Cbl, which is
highly conserved through evolution and is the site of loss-of-function
mutations in C. elegans Cbl homologue SLI-1, is essential
for a functional effect of Cbl on PDGF receptors.
/
mice (33, 34) may derive, at
least in part, from the loss of the negative regulatory effect of Cbl
on cellular proliferation triggered by extracellular growth factors.
signaling cascade in the oncogenic Cbl-transfected NIH3T3 cells (21)
suggested that the effect of Cbl on PDGF-induced cellular proliferation
is likely to involve a general reduction in the level of various
PDGFR-initiated signaling pathways. Extracellular growth factors,
including PDGF, are involved in the regulation of both mitogenesis and
cell survival (27). Thus, an overall reduction of signals emanating
from the PDGFR could coordinately reduce mitogenesis and cell survival
in Cbl-overexpressing cells. PDGF-dependent reduction in
proliferation in Cbl-overexpressing cells was detectable at relatively
early time points (e.g. 3-5 days) when no detectable cell
death was observed (Fig. 7A). However, clear evidence of
reduced cell survival was seen at later time points (after 7-9 days).
These data are consistent with an overall reduction of PDGFR-mediated
signals upon Cbl overexpression. However, it remains possible that Cbl
may selectively influence certain signaling pathways. In particular,
the tendency of Cbl-overexpressing cells to undergo cell death is
notable. Distinct signaling pathways have been implicated in the
regulation of cell survival and the induction of mitogenesis in
response to certain growth factors (35). Notably, the PI 3-kinase
pathway is thought to provide a key mechanism to promote cell survival
(36). Given the association of Cbl with the p85 subunit of PI 3-kinase,
via Cbl phosphotyrosine 731 (9, 10), it remains possible that Cbl may
sequester PI 3-kinase and reduce the efficiency of a signaling pathway
that ensures cell survival. However, more detailed mutational and
biochemical analyses will be required to precisely delineate the basis
of reduced proliferation and enhanced apoptosis observed upon Cbl overexpression.
is a
target of the negative regulatory function of Cbl. This result is
significant for a number of reasons. Our previous studies showed that
the expression of oncogenic Cbl proteins in NIH3T3 cells led to an
up-regulation of the PDGFR
signaling cascade, but limited analyses
of the PDGFR
revealed no obvious alterations in its signaling
cascade. These results raised the possibility that Cbl may
preferentially affect the PDGFR
signaling (21). An independent study
reported that PDGF-BB stimulation of NIH3T3 cells led to Cbl
phosphorylation, but Cbl-PDGFR
association was not detected (37).
PDGF-BB-induced phosphorylation of Cbl and its association with
PDGFR
in Patch cells (Fig. 4, A and B), which
lack the expression of PDGFR
, directly establishes that Cbl
participates in signaling via the PDGFR
. The reasons for the earlier
observation of apparently selective deregulation of PDGFR
signaling
in oncogenic Cbl-transfected NIH3T3 cells are not clear. It is possible
that the availability of PDGFR
ligand in the serum and/or the
relative levels of PDGFR
versus PDGFR
at the time of
the analysis were contributing factors. It is also likely that the
failure to detect the PDGFR
-Cbl association in a previous
investigation (37) reflects a combination of the relatively low
level of the endogenous Cbl in NIH3T3 cells and the
inefficiency of the available antibodies in immunoprecipitating PDGFR
(data not shown).
. Given the correlation of enhanced receptor ubiquitination with receptor degradation (38), Cbl
is therefore likely to provide a mechanism of negative regulation for
the PDGFR
similar to its role for the PDGFR
. Consistent with this
suggestion, mutations in the cytoplasmic tail of the PDGFR
that
reduced its ligand-induced ubiquitination also led to reduced
ligand-dependent degradation and a higher mitogenic
response to PDGF (39). Furthermore, a substantial reduction in
PDGF-BB-induced cell proliferation was observed in NIH3T3 cells
overexpressing Cbl, similar to their reduced response to PDGF-AA (Fig.
7A), and was also observed in PDGFR
-negative Ph cells
overexpressing Cbl. Overall, these results strongly suggest that Cbl
regulates both the PDGFR
and PDGFR
.
signaling, whereas a mutation
(G306E) corresponding to a loss-of-function mutation in SLI-1 abrogated
both activities. However, it was not clear if the TKB domain would be
required for the negative regulatory function of normal Cbl on PDGF
receptors. We demonstrate that the G306E mutation indeed abrogates the
effect of Cbl on PDGF-induced cell proliferation as well as on PDGFR
ubiquitination and degradation. In addition, Cbl phosphorylation was
dramatically reduced when the TKB domain was mutated. Importantly,
however, Cbl-G306E was still capable of physical association with the
PDGFR
, likely via Src family kinases, Grb2, or other unidentified
adaptor molecules. Thus, the binding of the TKB domain of Cbl to
activated receptor PTKs appears to be critical for a functional effect
rather than merely a mechanism for stable association with PDGF
receptors. Notably, a reduced EGF-induced phosphorylation of Cbl-G306E
with the retention of its physical association with the EGFR has also been observed (26). In contrast, we have observed that an intact TKB
domain is essential for the physical association of Cbl with the
cytoplasmic PTK Syk, as well as for the Cbl-dependent
negative regulation of Syk (16).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Daniel F. Bowen-Pope for providing the Patch cell line and for reading the manuscript. We thank members of the Band laboratory for helpful suggestions and Navin Rao for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA76118 and CA75075 and American Cancer Society Grant CIM-89513 (to H. B.).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.
Fellow of the Uehara Memorial Foundation, Japan.
§ Scholar of the Massachusetts Dept. of Public Health Breast Cancer Program.
¶ Recipient of the U. S. Dept. of Defense Breast Cancer Research Program Career Development Award DAMD 17-98-1-8038.
Fellow of Association pour la Recherche Contre le Cancer and
is a scholar of the Massachusetts Dept. of Public Health Breast Cancer Program.
** To whom correspondence should be addressed: Smith Bldg., Room 538C, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101, Fax: 617-525-1102 or 525-1010; E-mail: hband{at}rics.bwh.harvard.edu.
2 The region referred to as the TKB domain was previously referred to as a phosphotyrosine-binding (PTB) domain, but structural studies show that this region is an integrated phosphopeptide-binding platform composed of a four-helical domain, an EF hand, and an SH2 domain (40).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTK, protein
tyrosine kinase;
ECL, enhanced chemiluminescence;
EGFR, epidermal
growth factor receptor;
GST, glutathione S-transferase;
TKB, tyrosine kinase-binding;
HRPO, horseradish peroxidase;
PDGFR, platelet-derived growth factor receptor;
PVDF, polyvinylidene
difluoride;
PAGE, polyacrylamide gel electrophoresis;
Tyr(P), phosphotyrosine;
-MEM,
-minimal essential medium;
FCS, fetal calf
serum;
HA, hemagglutinin;
aa, amino acid(s);
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling;
PI
3-kinase, phosphatidylinositol 3-kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|