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INTRODUCTION |
Receptor tyrosine kinases
(RTKs)1 have been implicated
in numerous cellular processes such as cell fate specification and
differentiation (1), oncogenic transformation (2), and axonal guidance
(3). Tyrosine residues in the cytoplasmic domains, which become rapidly phosphorylated following ligand engagement, orchestrate the intrinsic properties of RTKs. The activated RTK (or in some cases, an associated phosphotyrosine-bearing docker protein) thus serves as a docking site
that attracts various signaling molecules to the membrane vicinity. The
assembly of such signaling complexes allows the RTKs to initiate the
transmission of signals from the membrane to the nucleus via the MAPK
cascade (4). This pathway uses a set of highly conserved signal
transduction molecules to link the activated receptors to the MAPK
cascade activator, the GTP-binding protein Ras (5). The increasing
number and complexity of proteins discovered to be involved in
modulating the MAPK cascade indicate that the transmission of signals
originating from the RTKs is under exquisite homeostatic control
(6).
In the last decade, a number of major RTK/MAPK regulators have been
isolated from genetic screens in developmental models, allowing for the
delineation of many key mammalian signaling pathways. Fibroblast growth
factor receptors and epidermal growth factor receptors (EGFRs)
are subsets of RTKs that are coupled to Ras via one or more adaptor
proteins that contain specific protein-protein interaction domains (7,
8). In the EGFR signaling system, one such protein that serves as
a direct link between the receptor and Ras is Grb2 (9), which binds
constitutively to and recruits the Ras-activating guanine nucleotide
exchange factor Sos (10). Ras is subsequently activated by Sos via
GDP/GTP exchange. Ras has an inherent, weak GTPase activity. Auxiliary
proteins with GTPase-activating properties interact with and enhance
the GTPase activity of Ras and hence are down-regulators of MAPK
signaling pathways (11). Fibroblast growth factor receptors activate
the MAPK cascade by essentially a similar mechanism as EGFR, except they make use of a surrogate receptor cytosolic domain in the form of
the constitutively associated FRS2 docker protein (12-14), where
tyrosine residues on FRS2 serve as substrates of fibroblast growth
factor receptor and attract the SH2 domains of Grb2 and Shp2.
A common component of many signaling modules is the multi-adaptor
protein Cbl, first identified as a retroviral transforming gene product
that induces pre-B cell lymphoma and myeloid leukemia (15). The product
of the mammalian c-cbl gene (p120Cbl) is a
widely expressed cytoplasmic protein with several distinctive domains,
including an SH2 domain, a Ring finger motif, and a large proline-rich
stretch at its C terminus (16). Genetic and biochemical studies have
implicated c-Cbl in the attenuation of RTK-mediated signaling cascades;
partial loss-of-function mutations of Caenorhabditis elegans
LET-23 (an EGFR equivalent) result in developmental defects that
are reversed by mutation of the c-Cbl ortholog SLI-1, which acts early
at the level of LET-23, and the Grb2 homolog Sem5 (15, 17). The
engagement of a variety of transmembrane receptors, including growth
factor, antigen, and integrin receptors, results in tyrosine
phosphorylation of c-Cbl and its association with numerous cytoplasmic
signaling proteins (18, 19). Recently, c-Cbl has been demonstrated to
elevate the rate of ligand-induced endocytosis (termed
"down-regulation") of EGFR by tagging the receptors with ubiquitin
and targeting them for destruction by the lysosomal/proteasomal system
(20).
In Drosophila, Sprouty (dSPRY) was first identified as a
down-regulator of the "Breathless" (the Drosophila
equivalent of fibroblast growth factor receptor) signaling cascade that
governs proper tracheal branching (21). The removal or loss-of-function mutations of dSPRY gave rise to a morphological "sprouting" effect. dSPRY was also isolated in a separate genetic screen for inhibiting DER
(the Drosophila equivalent of EGFR)-dependent
cell recruitment during eye development (22) and has been reported to
be expressed in the developing eye imaginal disc and other tissues
where EGFR signaling is known to exert its control (23). The C-terminal half of dSPRY was shown to localize the protein to the inner surface of
the plasma membrane of Drosophila S2 cells, whereas the
N-terminal portion interacted, at least in vitro, with
DRK (the Drosophila homolog of Grb2) and Gap1, a Ras
GTPase-activating protein (22). Sequestration of a docking protein like
DRK would constitute a plausible mechanism for inhibition of the early
phase of RTK signaling. Gap1 has been shown previously to be a negative
regulator of signaling for the Drosophila Sevenless RTK
(24). It is possible that dSPRY might interact with and augment the
GTPase activity of Gap1, thus acting as an attenuator of Ras activity.
More recent genetic evidence led to the postulation that dSPRY
intercepts the Ras/MAPK cascade downstream of Ras, at the level of Raf
or MEK (25). Composite studies thus suggest that dSPRY acts as a
general antagonist of the RTK signaling pathways (23, 25).
To date, four mammalian Sprouty homologs have been cloned, with no
known binding motifs or physiological functions (21, 26). The Sprouty
proteins are classified under the same gene family by virtue of their
characteristic cysteine-rich residues located in their carboxyl
termini. Parallel investigations on the role of murine SPRY2 (mSPRY2)
in the development of the embryonic mouse lung suggest a conservation
of function between dSPRY and mSPRY2 with respect to their negative
modulation of respiratory organogenesis (27). Furthermore,
overexpression of Sprouty constitutes a reduction in fibroblast growth
factor-induced limb bud outgrowth (28). We have also recently
identified a novel translocation domain that is responsible for the
general targeting of Sprouty proteins to membrane ruffles upon
fibroblast growth factor/EGF stimulation (29). We were interested in
further characterizing hSPRY2 and its possible involvement in RTK
signal down-regulation by identifying cellular proteins that interact
with hSPRY2. We demonstrate in this study that hSPRY2 and dSPRY
interact directly with c-Cbl and dCbl and that the hSPRY2-c-Cbl
association leads to an inhibition of the role of c-Cbl in enhancing
the rate of internalization and possibly the down-regulation of
EGFR.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
Monoclonal antibodies to
phosphotyrosine (PY20) and EGFR (E12020) and anti-GST polyclonal
antibody were purchased from Transduction Laboratories (Lexington, KY).
Rabbit anti-c-Cbl (C-15) and murine anti-Cbl-b (G-1) antibodies were
from Santa Cruz Biotechnology (Santa Cruz, CA). Protein G/protein
A-agarose was from Calbiochem. Secondary anti-mouse and anti-rabbit
antibodies conjugated to horseradish peroxidase were from Sigma.
Anti-Gal4 binding domain and anti-Gal4 activation domain antibodies
were from CLONTECH (Palo Alto, CA). Human
recombinant EGF was from Upstate Biotechnology, Inc. (Lake Placid, NY).
Radioisotopes were from Amersham Pharmacia Biotech (Buckinghamshire,
United Kingdom).
DNA Constructs--
hSPRY2 cDNA was amplified by polymerase
chain reaction (Expand Long Template polymerase chain reaction system,
Roche Molecular Biochemicals) from an adult human brain library
(CLONTECH) and subcloned into the pGEX4T1 vector
for bacterial expression or into the pXJ40HA and pXJ40FLAG mammalian
expression vectors (courtesy of Dr. E. Manser, Glaxo Laboratory,
Institute of Molecular and Cell Biology). The N-terminal (residues
1-177), C-terminal (residues 178-315), 30C (residues 30-315), and
53C (residues 53-315) DNA fragments of hSPRY2 (see Fig. 8B)
and deletion mutants
11-53,
36-53,
53-122,
123-177, and
178-194 of hSPRY2 (see Figs. 3A and 8B) were
generated using standard polymerase chain reaction and molecular
cloning methods. Drosophila Sprouty cDNA was a kind gift
of Dr. G. Martin (University of California). The full-length (residues
1-592), N210 (residues 1-210), 202C (residues 202-592), and
179-199 fragments of dSPRY (see Fig. 8D) were subcloned
into the pXJ40FLAG vector. Human c-Cbl cDNA was kindly provided by Dr. W. Langdon (University of Western Australia). The full-length c-Cbl
(residues 1-906), Cbl-NR (residues 1-436),
Cbl-NO (residues 1-379), Cbl-NS (residues
1-290), Cbl-NE (residues 1-252), Cbl-CO (residues 437-906), Cbl-CR (residues 380-906), and
Cbl-CZ (residues 362-906) fragments (see Fig.
5A) were subcloned into the pXJ40HA vector. The gene
sequences of full-length c-Cbl, Cbl-NR, and
Cbl-CO were subcloned into the pQE60 vector (courtesy of
Dr. B. L. Tang, Institute of Molecular and Cell Biology) for
bacterial production of histidine-tagged fusion proteins. The
Drosophila Cbl expression construct was obtained from Dr. H. Meisner (University of Massachusetts Medical Center), and the
full-length (residues 1-450) and
RF (residues 1-369) fragments of
dCbl (see Fig. 8D) were subcloned into the pXJ40HA vector.
The EGFR cDNA was a gift from Dr. A. Ullrich (Max-Plank-Institut
fur Biochemie).
Cell Culture--
293T human kidney epithelial and Chinese
hamster ovary cells were cultured in RPMI 1640 medium supplemented with
10% fetal bovine serum (Hyclone Laboratories), 2 mM
glutamine, 10 mM HEPES (pH 7.4), and 100 units/ml
penicillin/streptomycin. For growth factor stimulation, cells were
washed and maintained in serum-free medium for 24 h prior to
EGF treatment at 37 °C for 10 min. Quiescent or stimulated cells
were rinsed with phosphate-buffered saline (PBS) and lysed in
radioimmune precipitation assay buffer (50 mM Tris-HCl (pH
7.3), 150 mM sodium chloride, 0.25 mM EDTA (pH 8.0), 1% Triton X-100, 1% sodium deoxycholate, 0.2% sodium fluoride, 0.1% sodium orthovanadate, and protease inhibitors (Roche Molecular Biochemicals)).
Immunoprecipitation, Pull-down Assays, and Western
Blotting--
Protein concentrations of cell lysates were normalized
using a BCA protein assay kit (Pierce) before incubation with 2.5 µg of the appropriate antibody (for immunoprecipitation) or 10 µg of GST
fusion protein (for pull-down assays) overnight at 4 °C. Subsequently, 30 µl of protein G/protein A-agarose suspension beads
were added to capture the immunocomplex for 1 h. Eluted proteins
were resolved on SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were
blocked for 1 h in PBS containing 1% bovine serum albumin and
incubated for 1 h with 1 µg/ml primary antibody followed by 0.5 µg/ml secondary antibody linked to horseradish peroxidase. Immunoreactive protein bands were detected using the ECL
chemiluminescence reagent (Amersham Pharmacia Biotech).
Immunofluorescence Microscopy--
COS-1 monkey kidney cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and seeded in 6-well plates containing
sterilized glass coverslips. At 70% confluency, cells were transfected
with 1-2 µg of plasmid DNA using LipofectAMINETM 2000 reagent (Life Technologies, Inc.). At 4 h post-treatment, the
transfection medium was aspirated and replaced with complete medium
overnight, followed by incubation in serum-free medium for a further
16-24 h. Prior to fixation, quiescent cells were either left untreated
or were stimulated with 100 ng/ml EGF at 37 °C for 10 min. Cells
were subsequently rinsed with cold PBSCM (PBS containing 10 mM calcium chloride and 10 mM magnesium
chloride) and fixed with 3% paraformaldehyde in PBSCM at 4 °C for
30 min. Cells on coverslips were permeabilized with 0.1% saponin
(Sigma) in PBSCM for 15 min at room temperature. Anti-FLAG polyclonal antibody (OctA-Probe, Santa Cruz Biotechnology) was used at 1 µg/100
µl in fluorescence dilution buffer (7% fetal bovine serum and
2% bovine serum albumin in PBSCM) and incubated with the coverslip for
1 h at room temperature. For single and double stainings of FLAG-tagged hSPRY2, fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (Roche Molecular Biochemicals) was used. For double labeling, HA-tagged c-Cbl was detected using anti-HA monoclonal antibody (Roche Molecular Biochemicals) and Texas Red®
dye-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted in FDB. Coverslips were mounted in Crystal
Mount reagent (BiØmeda) and viewed by MRC-1024 laser scanning confocal microscopy (Bio-Rad). The microscopic images were processed with the aid of LaserSharp software (Bio-Rad) and Adobe Photoshop.
Mutagenesis--
Mutation of the first cysteine in the Ring
finger region of c-Cbl to alanine (C381A) was performed using the
QuikChange mutagenesis kit (Stratagene) and polymerase chain reaction
according to the manufacturer's instruction. The mutant product was
derived using wild-type c-Cbl in pXJ40HA as a template and has been
verified by sequencing.
EGFR Down-regulation Assay--
To quantify the rate of receptor
internalization, Chinese hamster ovary cells were seeded at 50%
density in 24-well plates. At 24 h after passage, cells were
transfected with a total 1.5 µg of various expression constructs
overnight before subsequent serum starvation. Cells were then incubated
in fresh serum-free medium containing 0.1% of the proteasome inhibitor
MG132 (N-benzyloxycarbonyl-Leu-Leu-Leu-aldehyde, prepared in dimethyl sulfoxide; Sigma) for 2 h. Cells were
subsequently stimulated with 100 ng/ml EGF in binding buffer (RPMI 1640 medium containing 0.5% bovine serum albumin) at 37 °C for various
time intervals in quadruplicates. At the end of the incubation, cells were rinsed with cold binding buffer, and unbound ligands were removed
by washing three times in ligand stripping buffer (150 mM
acetic acid and 150 mM NaCl (pH 2.7)). To determine the
relative number of EGFR molecules on the cell surface, duplicate
incubations with either 10 ng/ml 125I-EGF alone or
125I-EGF + 100-fold excess EGF (to account for nonspecific
binding) were set up and allowed to proceed for 2 h at 4 °C.
After treatment, cells were rinsed with binding buffer and solubilized
with 0.1 M NaOH and 0.1% SDS at 37 °C for 15 min prior
to counting on a
-counter.
In Vitro Translation and Pull-down--
The TNT® T7
Quick Coupled transcription/translation system (Promega) was used
according to the manufacturer's protocol. The translated
proteins were incubated with 10 µg of each GST fusion protein bound
to glutathione-Sepharose beads at 4 °C for 4 h. Beads were
collected and washed with cold radioimmune precipitation assay buffer,
and the eluted proteins samples were resolved by SDS-PAGE. The gel was
then dried and subjected to autoradiography.
Yeast Two-hybrid Interaction Assay--
The cDNAs encoding
full-length hSPRY2 (residues 1-315), hSPRY2-N (residues 1-177), and
hSPRY2-C (residues 178-315) were subcloned into the pAS vector for
yeast expression of Gal4 binding domain fusion proteins. Full-length
c-Cbl (residues 1-906), Cbl-NR (residues 1-436), and
Cbl-CO (residues 437-906) were subcloned into the pACT
vector for expression of Gal4 activation domain fusion proteins. The
binding domain constructs were individually transformed into Y190 yeast
host, and transformants were selected on Trp-free synthetic dextrose medium plates at 30 °C for 2 days. Following yeast protein extraction and SDS-PAGE, fusion protein-expressing clones were identified by immunoblot analysis. Positive single transformants then
underwent a successive round of transformation with activation domain
constructs, and double transformants were selected on
Trp/Leu-free synthetic dextrose medium plates. To detect protein
interactions,
-galactosidase assays (CLONTECH)
were performed on dual transformants according to the manufacturer's protocol.
In Vitro Protein Binding Assay--
GST-tagged hSPRY2 fusion
protein bound to glutathione-Sepharose 4B beads was eluted using 1 bed
volume of 10 mM reduced glutathione (Sigma) in 50 mM Tris-HCl (pH 8.0) at room temperature for 10 min. 5-µg
amounts of GST-hSPRY2 protein were incubated with 5 µg of each
His-tagged c-Cbl fusion protein bound to
Ni2+-nitrilotriacetic acid beads (QIAGEN GmbH) at 4 °C
overnight in 500 µl of radioimmune precipitation assay buffer. Beads
were collected and washed with cold radioimmune precipitation assay
buffer, and the eluted proteins samples were resolved by SDS-PAGE and
Western-blotted with anti-GST antibody.
The full-length sequences of human SPRY2
(GenBankTM/EBI accession number AF039843), mouse
SPRY2 (accession number NM011897), Drosophila SPRY
(accession number AACO4257), mouse SPRY1 (accession number AF176903.1),
mouse SPRY4 (accession number AF176906.1), human c-Cbl (accession
number X57110), Drosophila Cbl (accession number AJ223175),
and SLI-1 (C. elegans homolog of Cbl; accession number
X89223) were aligned using the ClustalW method under DNASTAR
application. Analogous Sprouty sequences spanning residues 36-53 of
hSPRY2 as well as the sequences encoding the Ring finger regions of Cbl
proteins were extracted as shown.
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RESULTS |
Human SPRY2 Associates Constitutively with Isoforms of Cbl--
To
identify binding partners of hSPRY2, we reasoned that potential binding
candidates could be substrates of RTKs. With this notion in mind, we
stimulated 293T cells with EGF and analyzed the tyrosine-phosphorylated
proteins that bound to GST-hSPRY2. As shown in Fig.
1A (upper panel),
there were three major protein groups of 190, 120, and 55 kDa in
EGF-stimulated cell lysates that bound to hSPRY2. There appeared to be
at least two bands at the 120-kDa region. These proteins were not
pulled-down by the negative control GST-BNIP-2 (30). The banding
pattern of tyrosine-phosphorylated proteins was reminiscent of that
reported when c-Cbl was immunoprecipitated from the lysates of
EGF-stimulated cells (31). Subsequent Western blot analysis revealed
that the 190-kDa band was most likely EGFR (data not shown), and the
double bands at 120 kDa correspond to c-Cbl and Cbl-b. The 55-kDa band is currently unidentified. We further observed that binding of the
Cbl proteins to hSPRY2 was independent of EGF stimulation and tyrosine phosphorylation; the 120-kDa bands were equivalent in
intensity in pull-down assays using both stimulated and nonstimulated cell lysates (Fig. 1A, middle and lower
panels).

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Fig. 1.
Human SPRY2 associates constitutively with
isoforms of Cbl. A, hSPRY2 binds two 120-kDa proteins
in a pull-down (PD) assay. 293T cells were serum-starved for
24 h and were either not stimulated (O) or stimulated
with 100 ng/ml EGF (E) at 37 °C for 10 min before lysis.
Lysates were incubated with 10 µg of the GST fusion protein form of
either full-length hSPRY2 (GST-hSPRY2) or BNIP-2 (GST-BNIP-2) bound to
glutathione-Sepharose beads. Bound proteins were separated by SDS-PAGE,
blotted onto membrane, and probed with anti-phosphotyrosine antibody
PY20 (upper panel). The presence of c-Cbl and Cbl-b isoforms
were determined by immunoblotting (IB) with anti-c-Cbl
(middle panel) and anti-Cbl-b (lower panel)
antibodies. B, hSPRY2 co-immunoprecipitates with c-Cbl in a
constitutive manner. 293T cells were transfected
(Tf) with the FLAG-hSPRY2 expression construct and
serum-starved overnight. Quiescent cells were either not stimulated or
stimulated with 100 ng/ml EGF for 10 min. Cell lysates were prepared
and analyzed by immunoblotting with anti-FLAG antibody (denoted as
whole cell lysates (WCL); middle panel); lysates
subjected to immunoprecipitation (IP) with anti-c-Cbl
antibody were probed with anti-FLAG antibody to show the presence of
FLAG-hSPRY2 in the complex. The lower panel shows the
amounts of endogenous c-Cbl (whole cell lysates) and immunoprecipitated
c-Cbl in nonstimulated and EGF-stimulated lanes.
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The results obtained indicate the existence of a complex consisting of
Cbl isoforms, EGFR, hSPRY2, and the 55-kDa unidentified protein. As
c-Cbl has been implicated as a down-regulator of RTK signaling and had
been previously shown to bind to EGFR, we wanted to characterize the
nature of the interaction of c-Cbl with hSPRY2 and to explore the
functional significance of their association. To confirm the
interaction between hSPRY2 and Cbl in vivo, FLAG-hSPRY2 was
expressed in 293T cells, and EGF-treated or untreated cell lysates were
immunoprecipitated using anti-c-Cbl antibodies. As shown in Fig.
1B (upper panel), the tyrosine-phosphorylated
190- and 120-kDa bands were present in both whole cell lysates and the
anti-c-Cbl immunoprecipitates as analyzed by Western blotting with
PY20. From the anti-FLAG immunoblot (Fig. 1B, middle
panel), it is apparent that equal amounts of hSPRY2 were present
in the c-Cbl immunoprecipitates from both nonstimulated and
EGF-stimulated cells; c-Cbl therefore interacts with hSPRY2 constitutively.
The N-terminal Half of hSPRY2 Interacts Directly with the c-Cbl
N-terminal Region--
To demonstrate possible direct binding between
hSPRY2 and c-Cbl, three different approaches were taken: binding of
in vitro translated proteins, yeast two-hybrid analysis, and
an in vitro protein binding study. In the first experiment,
hSPRY2 was [35S]methionine-labeled by in vitro
translation and assessed for its ability to bind to GST-c-Cbl, with
both GST alone and GST-BNIP-2 as negative controls. As shown in Fig.
2A (upper panel),
hSPRY2 bound to GST-c-Cbl, but not to GST alone or to GST-BNIP-2. In the reciprocal experiment, c-Cbl was in vitro translated and
incubated with GST-hSPRY2 and GST-Grb2 (used as a positive control, as
it has been previously shown to bind directly to c-Cbl (32)). Although in vitro translated c-Cbl bound to GST-hSPRY2 and GST-Grb2,
it did not bind to GST alone or to GST-BNIP-2 (Fig. 2A,
lower panel), further demonstrating specific and direct
binding between hSPRY2 and c-Cbl. In the second experiment to verify
direct binding, the yeast two-hybrid interaction assay was employed.
From Fig. 2B (table), it is apparent that c-Cbl
bound to full-length hSPRY2 and to the N-terminal half of hSPRY2, but
not to the C-terminal half (which is highly conserved among all Sprouty
proteins (21)). In the third experiment, eluted GST-tagged hSPRY2
protein was subjected to pull-down assays with His-tagged c-Cbl fusion
protein beads. Bound proteins were resolved by SDS-PAGE,
Western-blotted, and probed with anti-GST antibody to reveal the
presence of GST-tagged hSPRY2. As shown in Fig. 2C, both
His-tagged full-length c-Cbl and His-c-Cbl-NR fusion
proteins bound directly in vitro to hSPRY2, but not the
His-tagged vector alone or the His-c-Cbl-CO construct. We
conclude that hSPRY2 and c-Cbl bind directly to each other and that the
N-terminal half of hSPRY2 mediates its binding to the N-terminal region
of c-Cbl.

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Fig. 2.
N-terminal half of hSPRY2 interacts
directly with the c-Cbl N-terminal region. A, hSPRY2
interacts directly with c-Cbl. In vitro translated
(Ts) hSPRY2 proteins (upper panel) labeled with
[35S]methionine were incubated with GST, GST-BNIP-2, or
GST-c-Cbl bound to glutathione-Sepharose beads, whereas in
vitro translated c-Cbl proteins (lower panel) labeled
with [35S]methionine were incubated with GST, GST-BNIP-2,
GST-hSPRY2, or GST-Grb2 bound to glutathione-Sepharose beads in
pull-down (PD) assays. Bound proteins were eluted, separated
by SDS-PAGE, and visualized by autoradiography. 20% of total
[35S]Met-labeled hSPRY2 and c-Cbl are shown for visual
normalization (denoted product). The integrity of all GST
fusion proteins used in this study has been affirmed by checking their
migration profiles on Coomassie Blue-stained gels (data not shown).
B, N-terminal half of hSPRY2 interacts directly with the
c-Cbl N-terminal region. The yeast strain Y190 was successively
transformed with various combinations of binding domain and activation
domain fusion constructs as listed in the table. Dual
transformants were selected on Trp/Leu-free synthetic dextrose medium.
For binding domain constructs in the pAS vector, O
denotes vector alone, and hSpry2-FL, hSpry2-N,
and hSpry2-C represent the full-length, N-terminal (residues
1-177), and C-terminal (residues 178-315) fragments of hSPRY2,
respectively. For activation domain constructs in the pACT vector,
O denotes vector alone, and c-Cbl-FL,
c-Cbl-NR, c-Cbl-NO represent
full-length c-Cbl (residues 1-906), Cbl-NR (residues
1-436), and Cbl-CO (residues 437-906), respectively.
BNIP-2 denotes the pACT-BNIP-2 fusion plasmid. Colonies that
grew on double nutrient selection plates were subjected to a
qualitative -galactosidase activity assay. A positive interaction
(+) is scored by colonies that turned blue within 2 h; indicates a negative interaction. PRO, proline-rich domain;
LZ, leucine zipper motif. C, hSPRY2 binds
directly to the N-terminal half of c-Cbl. Equal amounts of eluted
GST-tagged hSPRY2 protein were incubated in pull-down assays with
His-tagged full-length c-Cbl (His-c-Cbl-FL; residues
1-906), His-tagged vector alone, His-c-Cbl-NR (residues
1-436), and His-c-Cbl-CO (residues 437-906) bound to
Ni2+-nitrilotriacetic acid beads. Bound proteins were
eluted, separated by SDS-PAGE, and immunoblotted (IB) with
anti-GST antibody to check for direct binding to GST-hSPRY2
protein.
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Amino Acids 11-53 in the N-terminal Region of hSPRY2 Bind to
c-Cbl--
To further define the region in the N-terminal half of
hSPRY2 that is involved in the interaction between hSPRY2 and c-Cbl, full-length FLAG-hSPRY2 and various N- and C-terminal deletion mutants
of hSPRY2 (Fig. 3A) were
expressed in 293T cells. Cell lysates were subjected to
immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates
were then analyzed for the presence of c-Cbl. As shown in Fig.
3B, all of the hSPRY2 deletion mutants bound c-Cbl, except
the
N11 mutant (lane 2), which lacks amino acids 11-53.
Therefore, the region of hSPRY2 spanning residues 11-53 is important
for its interaction with c-Cbl.

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Fig. 3.
Amino acids 11-53 in the N-terminal region
of hSPRY2 bind to c-Cbl. A, shown is a schematic
representation of FLAG-tagged full-length hSPRY2 (FL;
residues 1-315); truncation constructs hSPRY2-N (residues 1-177) and
hSPRY2-C (residues 178-315); and deletion mutants N11 (with a
deletion of amino acids 11-53), N53 (with a deletion of amino acids
53-122), N122 (with a deletion of amino acids 122-177), and
C178 (with a deletion of amino acids 178-194).
B, 293T cells were transfected (Tf) with
FLAG-tagged hSPRY2 constructs as represented in A. Whole
cell lysates (WCL) blotted for each transfection was probed
with anti-FLAG antibody to check the expression levels of the various
hSPRY2 proteins (upper panel). Equivalent amounts of cell
lysates were subjected to immunoprecipitation (IP) with
anti-FLAG antibody (lower panel shows amounts of
precipitated proteins) and immunoblotted (IB) with
anti-c-Cbl antibody to check for binding of the various forms of hSPRY2
to endogenous c-Cbl (middle panel). O, vector
alone.
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c-Cbl and hSPRY2, but Not hSPRY2
N11, Associate in
Vivo--
Immunofluorescence studies were performed to investigate the
cellular localizations of c-Cbl, hSPRY2, and the characterized non-binding mutant of hSPRY2 (hSPRY2
N11). COS-1 cells were employed for this study, as they have been previously characterized with respect
to hSPRY2 localization and translocation (29). Cells were singly
transfected with each construct or cotransfected with HA-c-Cbl and
either the FLAG-hSPRY2 or FLAG-hSPRY2
N11 mutant construct. As shown
in Fig. 4A, when expressed
alone, c-Cbl was mostly found diffused in the cytosol (33) in both
quiescent and EGF-stimulated cells (first column). As
previously reported (29), singly transfected hSPRY2 demonstrates a
mainly cytosolic disposition, where it aligns on microtubules in
nonstimulated cells and translocates to membrane ruffles upon EGF
treatment. When singly expressed in the quiescent state, hSPRY2
N11
(upper row, third cell) also showed a similar
disposition as wild-type hSPRY2 (upper row, second
cell), as expected since the microtubule-binding sequences of
hSPRY2 were not disrupted in the hSPRY2
N11 mutation. Additionally,
the hSPRY2
N11 mutant also translocated to membrane ruffles upon EGF
stimulation (lower row, third cell), as did
wild-type hSPRY2 (lower row, second cell).

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Fig. 4.
c-Cbl and hSPRY2, but not
hSPRY2 N11, associate in
vivo. COS-1 cells were singly transfected with
1 µg each of HA-c-Cbl, FLAG-hSPRY2, and FLAG-hSPRY2 N11
(A); with 1 µg each of HA-c-Cbl and FLAG-hSPRY2
(B); and with 1 µg each of HA-c-Cbl and FLAG-hSPRY2 N11
(C). O denotes cells in a quiescent state, and
E denotes EGF-stimulated cells. FLAG-tagged Sprouty
constructs were stained using an anti-FLAG polyclonal antibody and
fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG. HA-tagged
c-Cbl was detected with an anti-HA monoclonal antibody and Texas
Red® dye-conjugated AffiniPure goat anti-mouse IgG. In
c-Cbl- and hSPRY2-cotransfected cells, c-Cbl colocalized with hSPRY2 on
microtubules (broken arrows) in the cytosol in the quiescent
state (B, first and second cells);
c-Cbl was found to colocalize with hSPRY2 in membrane ruffles
(solid arrows) upon EGF stimulation (B,
third and fourth cells). In c-Cbl- and
hSPRY2 N11-cotransfected cells in the quiescent state, c-Cbl appeared
diffuse, but did not colocalize with hSPRY2 N11 on microtubules in
the cytosol (C, first and second
cells). With EGF stimulation, c-Cbl remained cytosolic and did not
appear with hSPRY2 N11 in the membrane ruffles (C,
third and fourth cells).
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Interestingly, we noted that with or without EGF stimulation, c-Cbl
appeared to colocalize with hSPRY2 in coexpressing cells (Fig.
4B). The pattern was visually different when c-Cbl and
hSPRY2
N11 were coexpressed in cells. In the quiescent state, the
mutant hSPRY2 protein retained the same microtubule location, whereas the staining pattern of c-Cbl appeared to be cytosolic and did not
coincide with that of hSPRY2
N11 (Fig. 4C,
first and second cells). In the EGF-activated
state, the hSPRY2
N11 protein translocated to the membrane
normally, whereas c-Cbl remained diffused in the cytosol (Fig.
4C, third and fourth cells). These
results provide further evidence that amino acids 11-53 of hSPRY2
interact with c-Cbl, and this interaction can influence the spatial
distribution of c-Cbl in the cell.
hSPRY2 Binds to the Ring Finger Domain of c-Cbl--
The previous
yeast two-hybrid analysis demonstrated that the N-terminal half of
c-Cbl was responsible for binding to hSPRY2 (Fig. 2B).
Experiments were then performed to further define the region of c-Cbl
that effects its binding to hSPRY2. Truncation constructs of c-Cbl,
depicted schematically in Fig.
5A, were subcloned into the
pXJ40HA vector and tested for their ability to bind to GST-hSPRY2. 293T
cells were transfected with full-length HA-c-Cbl and its truncation
constructs, and cell lysates were subjected to pull-down assays with
GST-hSPRY2. Bound proteins were separated by SDS-PAGE, Western-blotted,
and probed with anti-HA antibody to reveal the presence of c-Cbl. As
shown in Fig. 5B (lower panel), only full-length
c-Cbl (denoted F in lane 2) and those truncation mutants of c-Cbl that contain an intact Ring finger domain could bind
to hSPRY2 (namely Cbl-NR (lane 3),
Cbl-CZ (lane 8), and Cbl-CR (lane 9)). These results indicate that hSPRY2 interacts with
the Ring finger domain of c-Cbl.

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Fig. 5.
hSPRY2 binds to the Ring finger domain of
c-Cbl. A, shown is a schematic representation of
HA-tagged full-length c-Cbl (FL; residues 1-906) and
truncation constructs Cbl-NR (residues 1-436),
Cbl-NO (residues 1-379), Cbl-NS (residues
1-290) Cbl-NE (residues 1-252), Cbl-CO
(residues 437-906), Cbl-CR (residues 380-906), and
Cbl-CZ (residues 362-906). 4H, four-helix
bundle; EF, calcium-binding motif; PRO,
proline-rich domain; LZ, leucine zipper motif. B,
293T cells were transfected (Tf) with the HA-c-Cbl
constructs as represented in A (where F is
HA-tagged full-length c-Cbl), and lysates were subjected to pull-down
(PD) assays with GST-hSPRY2. The whole cell lysate
(WCL) blot was probed with anti-HA antibody to show
expression levels of the various proteins (upper panel).
Bound proteins were immunoblotted (IB) with anti-HA antibody
to check for binding of the various forms of c-Cbl to GST-hSPRY2
(lower panel). O, vector alone.
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hSPRY2, but Not hSPRY2
N11, Inhibits c-Cbl-mediated EGFR
Down-regulation--
Members of the SLI-1/Cbl and Sprouty families
have been demonstrated to be involved in down-regulation of Ras/MAPK
signaling by exerting their effects in close proximity to various RTKs
(17, 22). Following the initial characterization of the binding between hSPRY2 and c-Cbl and their apparent colocalization in nonstimulated and
RTK-stimulated cells, it was hypothesized that hSPRY2 might direct
cytosolic c-Cbl to a functional intracellular location. Recently, c-Cbl
has been reported to target various RTKs and non-RTKs for degradation
by catalyzing the polyubiquitination of such target proteins (20, 34);
more specifically, its Ring finger domain was shown to contain
ubiquitin ligase activity (35). It is therefore plausible that hSPRY2
functions also to regulate the ubiquitin ligase activity of c-Cbl via
its specific binding to the Ring finger domain of the latter.
An experiment was thus performed to investigate the effect of hSPRY2 on
c-Cbl-induced down-regulation of stimulated
EGFR. The kinetics of EGFR
turnover (a measure of the rate of receptor internalization) in Chinese
hamster ovary cells coexpressing EGFR with wild-type c-Cbl, c-Cbl-C381A
(a dominant-negative c-Cbl Ring finger mutant), c-Cbl + hSPRY2, or
c-Cbl + hSPRY2
N11 were quantitated according to the methodology of
Waterman et al. (36). Transfectants were stimulated with EGF
for various time points before pulse labeling with
125I-EGF. The amounts of 125I-EGF bound to
EGFRs remaining on the cell surface after treatment were then measured.
In line with the previous observation that the down-regulation of EGFR
is related to an increase in c-Cbl-catalyzed polyubiquitination and
subsequent destruction of the receptors (36), for cells coexpressing
c-Cbl, the EGFR population remaining on the cell surface was decreased
by almost 60% compared with the vector control after 30 min of EGF
stimulation (Fig. 6). Furthermore, consistent with the importance of
the Ring finger domain as previously observed (37), coexpression of the
catalytically inactive c-Cbl-C381A mutant showed no suppressive
activity. Coexpression of hSPRY2 alone also caused no significant
change in the rate of EGFR internalization. However, coexpression of
c-Cbl and hSPRY2 was shown to abrogate the down-regulatory effect
(i.e. an elevated rate of receptor internalization)
exhibited by c-Cbl alone on EGFR. Significantly, the non-binding
hSPRY2
11 mutant had no effect on c-Cbl-enhanced receptor
turnover. As postulated, the binding of hSPRY2 to the Ring finger
domain of c-Cbl inhibited the down-regulatory effect of c-Cbl on
EGFR.

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Fig. 6.
hSPRY2, but not
hSPRY2 N11, inhibits c-Cbl-mediated EGFR
down-regulation. Chinese hamster ovary cells were transfected with
1.5 µg of total EGFR expression construct together with plasmids
encoding the respective gene products (vector control ( ), c-Cbl
( ), Cbl-C381A ( ), hSPRY2 ( ), c-Cbl and hSPRY2 ( ), or c-Cbl
and hSPRY2 N11 ( )) in 24-well plates. At 48 h
post-transfection, duplicate wells were incubated with EGF (100 ng/ml)
at 37 °C for various time intervals as indicated. Unbound EGF was
then stripped off, and the levels of surface EGFR were determined by a
competitive binding assay with 125I-EGF (see
"Experimental Procedures"). There were an average of 70,000 cpm at
the 100% EGFR level.
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Drosophila Sprouty Associates with Drosophila Cbl--
Evidence
has been presented that mammalian SPRY2 may have a parallel function in
lung development compared with Drosophila Sprouty in
tracheal branching (21, 27). Therefore, it was of interest to
investigate if dSPRY could similarly bind to dCbl. Although the Ring
finger domain of Cbl from various species is highly conserved (Fig.
7A), the homology between the
N termini of Sprouty proteins is relatively low. To assess a possible
interaction between dCbl and dSPRY, HA-dCbl or HA-DRK (the
Drosophila Grb2 homolog) was ectopically expressed in 293T
cells, and lysates were subjected to pull-down assays with GST alone,
GST-dSPRY, or GST-dCbl. Bound proteins were probed with anti-HA
antibody. The data shown in Fig. 7B (upper panel)
indicate that HA-dCbl bound to GST-dSPRY. It has been previously shown
that DRK binds to dSPRY (21), but not to dCbl (38). Fig. 7B
(middle panel) illustrates the association of DRK and dSPRY,
but not of DRK and dCbl. The reciprocal binding experiment was
performed; and as shown (Fig. 7B, lower panel),
FLAG-dSPRY bound to GST-dCbl.

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Fig. 7.
Drosophila Sprouty associates with
Drosophila Cbl. A, shown is an amino
acid sequence alignment of the Ring finger regions of Cbl orthologs.
Identical residues are indicated in black. Dashes
indicate no corresponding amino acid at that position. B,
HA-dCbl and HA-DRK were expressed in 293T cells, and lysates were
subjected to pull-down (PD) assays with GST-dSPRY
(upper panel) and GST-dSPRY or GST-dCbl (middle
panel). FLAG-dSPRY was expressed in 293T cells, and lysates were
subjected to pull-down assay with GST-dCbl (lower panel).
Bound proteins were resolved by SDS-PAGE and immunoblotted
(IB) with anti-HA (upper and middle
panels) or anti-FLAG (lower panel) antibody to test for
reciprocal binding between dSPRY and dCbl. The whole cell lysate
(WCL) loadings for each transfection (Tf) are
shown in the left lane of each panel. C, shown is
the heterologous binding between Sprouty and Cbl proteins of mammalian
and Drosophila origins. HA-tagged dCbl, c-Cbl, and DRK were
transiently expressed in 293T cells. The upper panel shows
their protein expression levels. Cell lysates were subjected to
pull-down assays with GST-dSPRY and GST-hSPRY2 and immunoblotted with
anti-HA antibody to check for bound proteins (lower panel).
DRK binding to dSPRY was used as a positive control. D,
FLAG-tagged dSPRY and hSPRY2 were expressed in 293T cells, and cell
lysates were subjected to pull-down assays with GST-dCbl and GST-c-Cbl
fusion proteins. Immunoblots of the whole cell lysates (upper
panel shows the relative protein expression levels) and of the
bound proteins (lower panel) were probed with anti-FLAG
antibody. O, vector alone.
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Cross-species Interaction between Sprouty and Cbl--
We next
investigated whether Sprouty and Cbl proteins are capable of
heterologous cross-species interaction, i.e. if hSPRY2 could
bind to dCbl and dSPRY could bind to c-Cbl. 293T cells were transfected
with HA-dCbl, HA-c-Cbl, or HA-DRK. Lysates were subjected to pull-down
assays with GST alone, GST-dSPRY, or GST-hSPRY2. As shown in Fig.
7C (lower panel), dSPRY bound to both dCbl and c-Cbl, whereas hSPRY2 bound to c-Cbl as well as dCbl. A reciprocal binding experiment was performed in which 293T cells were transfected with FLAG-tagged dSPRY or hSPRY2 constructs, and cell lysates were
subjected to pull-down assays with GST alone, GST-dCbl, or GST-c-Cbl.
The results shown in Fig. 7D (lower panel) reinforce the
previous observation that heterologous binding occurs between human
c-Cbl and dSPRY and between hSPRY2 and dCbl. These results suggest
there may be a conserved domain in dSPRY that is homologous to the
c-Cbl-binding domain of hSPRY2.
N-terminal Amino Acids 36-53 of hSPRY2 Are Responsible for Binding
to c-Cbl--
In examining the sequence of dSPRY closely, we found a
region between amino acids 179 and 199 in dSPRY that shows a low
homology to residues 36-53 in hSPRY2 (Fig.
8A). Residues 36-53 lie
within region 11-53 (Fig. 8B), which we previously
determined to be important for c-Cbl binding (Fig. 3A). We
therefore investigated if we could refine the binding domain to a
smaller region. FLAG-tagged hSPRY2 truncation and deletion constructs
as depicted in Fig. 8B were expressed in 293T cells, and
cell lysates were immunoprecipitated with anti-FLAG antibody and probed
with anti-c-Cbl antibody. The data shown in Fig. 8C
(upper panel) indicate a lack of binding between c-Cbl and
the 53C (without residues 1-53; lane 3) and
N36 (lacking
amino acids 36-53; lane 4) mutants, whereas binding was
apparent between c-Cbl and the 30C (without residues 1-30; lane
2) and full-length (lane 5) constructs. A reciprocal
precipitation experiment was performed in which cell lysates from the
same transfections were subjected to immunoprecipitation with
anti-c-Cbl antibody and immunoblotted with anti-FLAG antibody (Fig.
8C, third panel). The result is in agreement with
the above data. Thus, we further delineated that the c-Cbl-binding
region of hSPRY2 is contained within sequence 36-53.

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Fig. 8.
Common SPRY Cbl-binding regions in mammalian
and Drosophila systems. A, shown is an
amino acid sequence alignment of the putative Cbl-binding regions of
various Sprouty orthologs. Identical residues are indicated in
black. Dashes indicate no corresponding amino
acid at that position. B, shown is a schematic
representation of FLAG-tagged hSPRY2 constructs to determine whether
amino acids 36-53 are responsible for binding to c-Cbl. Shown are the
full-length (FL; residues 1-315), 230C (residues 30-315),
53C (residues 53-315), and N36 (with a deletion of residues 36-53)
hSPRY2 fragments. C, 293T cells were transfected
(Tf) with the FLAG-tagged hSPRY2 constructs represented in
B. The upper panel shows the presence of
endogenous c-Cbl bound by immunoprecipitated (IP)
FLAG-hSPRY2 constructs. Precipitated amounts of FLAG-tagged proteins
are shown in the second panel. For reciprocal binding,
immunoprecipitated c-Cbl proteins (amounts shown in the lower
panel) were checked for their binding to various FLAG-hSPRY2
constructs by immunoblotting (IB) with anti-FLAG antibody
(third panel). O, vector alone.
D, shown is a schematic representation of full-length dCbl
(residues 1-592) and dCbl RF (residues 1-369) and various
FLAG-tagged dSPRY constructs to determine the region of binding between
dCbl and dSPRY. 4H, four-helix bundle; EF,
calcium-binding motif; PRO, proline-rich domain;
LZ, leucine zipper motif. Shown are the full-length
(SPRY-FL; residues 1-592), N210 (residues 1-210), 202C
(residues 202-592), and N179 (with a deletion of residues 179-199)
SPRY fragments. E, dSPRY binds to the Ring finger domain of
dCbl. HA-dCbl and HA-dCbl RF were transiently expressed in 293T
cells. A whole cell lysate (WCL) blot immunoblotted with
anti-HA antibody shows equal protein expression (upper
panel). Precipitated proteins from lysates subjected to pull-down
(PD) assays with GST-dSPRY and GST alone were probed with
anti-HA antibody (lower panel). F, the N-terminal
amino acids 179-199 of dSPRY are responsible for binding to dCbl.
FLAG-dSPRY constructs as represented in D were transiently
expressed in 293T cells, and lysates were subjected to pull-down assays
with GST-dCbl and GST alone. Immunoblots of whole cell lysates
(upper panel) and bound proteins (lower panel)
were immunoblotted with anti-FLAG antibody.
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N-terminal Amino Acids 179-199 of dSPRY Bind to the Ring Finger
Domain of dCbl--
To extend the binding investigation to dSPRY and
dCbl, various constructs as depicted in Fig. 8D were made.
First, to investigate whether the Ring finger domain of dCbl was
involved in binding to dSPRY, 293T cells were transiently transfected
with dCbl, dCbl
RF, or vector alone. Cell lysates were subjected to
pull-down assays with GST-dSPRY or GST alone. As shown in Fig.
8E (lower panel), whereas full-length dCbl bound
to dSPRY, dCbl
RF (lacking the Ring finger domain) did not. This
result is indicative of the Ring finger domain of dCbl being involved
in its binding to dSPRY. Second, binding domain analyses were performed
to ascertain the site in dSPRY that binds to the Ring finger domain of
dCbl. The possible involvement of residues 179-199 in dSPRY was
directly addressed. FLAG-tagged full-length dSPRY and the dSPRYN210
(amino acids 1-210), dSPRY202C (amino acids 202-592), and
dSPRY
N179 (mutant with a deletion of amino acids 179-199)
constructs were transiently expressed in 293T cells, and lysates were
incubated with either GST alone or GST-dCbl. The binding data shown in
Fig. 8F (lower panel) indicate that dSPRY-derived
proteins that contain residues 179-199 bound to dCbl, whereas those
that lack the sequence did not. The region comprising residues 179-199
of dSPRY is therefore responsible for its interaction with dCbl;
deletion of this region of dSPRY can similarly abolish its binding to
c-Cbl (data not shown).
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DISCUSSION |
Recently, the Cbl family proteins have attracted a considerable
amount of attention as down-regulators of both RTK and non-RTK signaling (34, 39). Genetic analysis implicated the orthologs of Cbl in
C. elegans and Drosophila in down-regulating the
EGFR growth-promoting function (17, 20, 38). Human c-Cbl has been
reported in a number of cell systems to be tyrosine-phosphorylated upon
receptor stimulation and was found to exert a negative regulatory role
in tyrosine kinase signaling, albeit by an as yet undefined mechanism
(31-33). The N-terminal fragment of c-Cbl essentially consists of two
functional domains: an unconventional SH2-like domain (which
incorporates the four-helix bundle and EF-hand) and a Ring
finger motif. Structure-based mutation studies in the four-helix
bundle, EF, and SH2 domains revealed that the three domains together
form an integrated phosphoprotein recognition module (16). Furthermore,
a critical role of the SH2 domain in c-Cbl function is demonstrated by
the localization of a loss-of-function mutation in SLI-1 (C. elegans homolog of Cbl) within a 17-amino acid deletion N-terminal
to the Ring finger (18); this structural alteration renders the 70Z-Cbl
mutant oncogenic and causes it to exhibit an enhanced level of
tyrosine phosphorylation as well as to abrogate the negative regulatory
function of wild-type c-Cbl. In our binding domain analysis, we showed
that neither disruption nor lack of the SH2 domain (Cbl-NS
and Cbl-NE, respectively) or deletion of the 70Z region in
the c-Cbl C terminus (Cbl-CR) was accountable for
any lack of association with hSPRY2. We have delineated the
hSPRY2-binding region specifically to the Ring finger domain of
c-Cbl.
Various groups have reported evidence that c-Cbl down-regulates growth
factor receptors by helping to ubiquitinate them, thereby marking them
for destruction by the lysosomal/proteasomal route (40-42). More
recently, the Ring finger domain of Cbl, an evolutionarily conserved
structure present in >200 proteins, was found to possess E3 ubiquitin
ligase activity (35, 43). Conjugation of ubiquitin proceeds via a
three-step mechanism (44). Initially, the ubiquitin-activating enzyme
(E1) activates the C-terminal glycine residue of ubiquitin to a high
energy thiol ester with an internal E1 cysteine residue. One of several
ubiquitin-conjugating enzymes (E2) transfers the activated ubiquitin to
the substrate that is specifically bound to a member of the E3
ubiquitin ligase family. E3 ligases catalyze the covalent attachment of
ubiquitin to the substrate. In one case, tyrosine residues on activated
EGFR become phosphorylated upon growth factor stimulation and bind to
the SH2 domain of c-Cbl; c-Cbl functions as an E3 to mediate endocytic
sorting of the target substrate by relaying activated ubiquitin
molecules to EGFR via its Ring finger. Although all three members of
the Cbl family (c-Cbl, Cbl-b, and Cbl-3) can enhance ubiquitination
(40), two oncogenic variants (70Z-Cbl and v-Cbl) whose Ring fingers are defective are unable to desensitize EGFR (18, 20). The oncogenic viral
counterpart (v-Cbl), which lacks a functional Ring finger, inhibits
down-regulation by shunting endocytosed receptors to the
recycling pathway. This has exciting implications because, in relation
to our findings, binding of hSPRY2 to the catalytic site of c-Cbl would
suggest an important modulatory role of hSPRY2 in the fate and
signaling potency of growth factor receptors. Additionally, we found
that hSPRY2 binding to the Ring finger domain of c-Cbl abrogates the
latter's ability to induce down-regulation of EGFR.
Our studies have demonstrated that hSPRY2 binds directly to the Ring
finger domain of c-Cbl (and possibly also Cbl-b) via a small N-terminal
region. A similar observation was made for dSPRY and dCbl, the binding
of which encompasses similar regions. This is indicative of a
conservation of the interaction domains throughout evolution. In this
respect, it is interesting to note that there is no Sprouty ortholog
found in C. elegans. Taken together, the constitutive nature
of the hSPRY2-c-Cbl association may highlight the requirement for some
extrinsic factors to displace hSPRY2 from the Ring finger domain of
c-Cbl and, by so doing, uplifts its suppressive effect on the latter.
A number of questions are posed by the data presented. First of all, do
the other mammalian Sprouty proteins also bind to the Ring finger of
c-Cbl? Preliminary analysis revealed that murine SPRY1 binds c-Cbl, but
not murine SPRY4 (data not shown). Hence, one might infer that mSPRY4
plays a different functional role from mSPRY1 and mSPRY2. We have
recently presented evidence that the conserved cysteine-rich C-terminal
domain in various Sprouty proteins is responsible for directing the
proteins to membrane ruffles when cells are stimulated with growth
factors (29). Given the high degree of conservation among the
C-terminal regions, this would suggest that Sprouty proteins have a
common target for membrane ruffle association. We further demonstrated
that c-Cbl colocalizes with hSPRY2 in coexpressing cells, but not with the non-binding hSPRY2
N11 mutant. It becomes interesting then to
question the roles of hSPRY2 and c-Cbl in each of the two localities. Does hSPRY2 translocate to ruffles to assume a different function by
virtue of different or additional binding partners, or does the
hSPRY2-mediated translocation serve to target c-Cbl in near proximity
to the membrane-anchored receptors? If the latter case is true, one
would expect hSPRY2 to synergistically promote the action of c-Cbl in
the ligand-induced down-regulation of EGFR, which differs from our
experimental observation. Although we do not know the proportion of
endogenous c-Cbl that is bound by hSPRY2 (and therefore
"nonfunctional" in terms of EGFR down-regulation), our results
suggest that the inhibition of EGFR internalization that we observed in
c-Cbl- and hSPRY2-coexpressing cells may be due to an exclusion of
prospective E2·ubiquitin complexes from the Ring finger domain of
c-Cbl, thereby preventing downstream ubiquitination events.
Alternatively, hSPRY2 may have a direct involvement in receptor endocytosis.
On the other hand, we cannot rule out the possibility that a fraction
of the c-Cbl population that hSPRY2 binds to is tyrosine-phosphorylated or not or that, upon growth factor stimulation, hSPRY2 initiates a
stronger preference for binding to other protein partners that might
mediate translocation of the hSPRY2·c-Cbl complex to ruffles. It is
interesting to note from the immunofluorescence studies on hSPRY2- and
c-Cbl-coexpressing cells that a significant pool of c-Cbl still retains
an association with microtubules in the cytosol, although a major
portion has been shown to colocalize with hSPRY2 at the membrane
ruffles in EGF-treated cells. Given the previously reported function of
dSPRY and mSPRY2 in the attenuation of RTK activation (21, 22, 27), it
is also possible that hSPRY2 associates with other signaling proteins
to down-regulate the MAPK cascade. It will be interesting to pursue any
substantial attenuation of MAPK signaling in hSPRY2- and
c-Cbl-coexpressing cells. It is noteworthy that other studies
done by overexpressing two forms of c-Cbl mutants (namely C381A and
70Z-Cbl) showed an inhibitory effect on EGFR down-regulation, but no
effect on ERK activity was detectable (18, 37).
Do the various Sprouty isoforms bind to the relatively well conserved
Ring finger domains of proteins other than c-Cbl? Despite the
increasing number of proteins known to be ubiquitinated, the identification of the corresponding ubiquitin ligases (E3) has been
lagging. E3 ligases for which the amino acid sequences are known
include the N-end rule E3 ligases of yeast and mammals (45); members of the HECT (homologous to E6-AP
C terminus) family (46); Mdm2 (47); APC, the
anaphase-promoting complex (48);
and other F-box- and cullin-containing complexes (49). In the last few years, the once discrete Ring finger motifs intrinsic to a large number
of proteins have surfaced as instrumental components that confer on E3
proteins a capacity for E2-dependent ubiquitination (50,
51). In view of this, the mode of action of Sprouty as a general RTK
inhibitor certainly encompasses several layers of complexity.
With the above possibilities and taking current evidence into account,
it is difficult to envisage a simplistic model to explain how the
interaction between Sprouty and Cbl manifests itself in the
down-regulation of RTK signaling. In addition to the observed binding
between hSPRY2 and Cbl-b, which has been shown to play other distinct
roles in EGF-mediated signaling (52), there are likely to be other
candidate proteins that interact with Sprouty and Cbl. We are screening
and characterizing those proteins that bind to the various Sprouty
family members in an effort to better understand their physiological function.