(Received for publication, May 25, 1995; and in revised form, July 7, 1995)
From the
The murine retroviral oncogene v-cbl induces pre-B cell
lymphomas and myelogenous leukemias. The protein product of the
mammalian c-cbl proto-oncogene is a widely expressed
cytoplasmic 120-kDa protein (p120) whose normal
cellular function has not been determined. Here we show that upon
stimulation of human epidermal growth factor (EGF) receptor,
p120
becomes strongly tyrosine-phosphorylated and
associates with activated EGF receptor in vivo. A GST fusion
protein containing amino acids 1-486 of
p120
, including a region highly conserved in
nematodes, binds directly to the autophosphorylated carboxyl-terminal
tail of the EGF receptor. Platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), or nerve growth factor (NGF)
stimulation also results in tyrosine phosphorylation of
p120
. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120
homologue, plays a negative regulatory role in control of
the Ras signaling pathway initiated by the C. elegans EGF
receptor homologue. Our results indicate that p120
is involved in an early step in the EGF signaling pathway
that is conserved from nematodes to mammals.
The cbl oncogene was identified as the transforming
gene of the Cas NS-1 murine leukemia virus(1) . The
transforming product of this virus is a Gag-v-Cbl fusion protein in
which 40 kDa are encoded by v-cbl. Cloning of the mouse and
human c-cbl proto-oncogenes (2) revealed that
v-cbl is a truncated form of c-cbl, encoding only 355
NH-terminal amino acids. The primary sequence of
p120
contains an NH
-terminal domain
with a putative nuclear localization signal, followed by a RING zinc
finger motif. The COOH-terminal half of the protein contains multiple
proline-rich stretches that may function as ligands for SH3 domains.
Both the RING zinc finger and the proline-rich domain are absent in
v-Cbl. Whereas v-Cbl could be found both in the cytoplasm and nucleus
of cells infected with the Cas NS-1 retrovirus, p120
is exclusively cytosolic(3) .
Recently,
p120 was found to be tyrosine-phosphorylated
upon activation of the T cell receptor, and an in vitro interaction with the adaptor protein Grb2, independent of T cell
receptor activation, has been reported(4) . In addition, an
interaction between the SH3 domains of the adaptor protein Nck and the
proline-rich domain of p120
has been
reported(5) . Detailed analysis of the primary structure of
p120
revealed the presence of the motif PPVPPR,
which is identical to the Grb2 binding site in the Ras guanine
nucleotide releasing factor ``Sos''(6) . This
observation prompted us to examine in detail a possible interaction
between p120
and Grb2 in the context of the Ras
signaling pathway initiated by the epidermal growth factor receptor
(EGFR). (
)In this report we demonstrate that
p120
becomes strongly tyrosine-phosphorylated
upon stimulation of EGFR, platelet-derived growth factor (PDGF), nerve
growth factor (NGF), and fibroblast growth factor (FGFR1) receptors. We
also demonstrate that p120
associates with
activated EGFR both in vivo and in vitro. These
results suggest that p120
plays a role in an
early step of the EGFR signaling pathway.
We examined the possibility that p120 was involved in the EGFR-mediated signaling pathway.
Untreated and EGF-stimulated HER14 cell lysates were immunoprecipitated
with anti-p120
antibodies, and
tyrosine-phosphorylated proteins were detected with antibodies against
phosphotyrosine (Fig. 1A). Two strongly
tyrosine-phosphorylated proteins with apparent molecular masses of
approximately 120 and 170 kDa, as well as a weak band of approximately
55 kDa, were detected in lysates from EGF-stimulated cells.
Immunoblotting with anti-p120
antibodies
revealed that the 120-kDa protein corresponds to p120
(Fig. 1B). Immunoblotting with anti-EGFR
antibodies or with anti-Shc antibodies demonstrated that the 170- and
55-kDa polypeptides represent the EGFR and Shc protein, respectively (Fig. 1B). Immunoblotting with anti-Grb2 antibodies
demonstrated that this protein was present in this complex (Fig. 1B). p120
could also be
detected in activated EGFR immunoprecipitates (Fig. 1C). These results show that p120
associates in vivo with activated EGFR.
Figure 1:
EGF-induced tyrosine phosphorylation
and association of p120 with activated EGFR. A, lysates from untreated and EGF-stimulated HER14 cells were
incubated with affinity-purified anti-p120
antibodies. Immunoprecipitated proteins were resolved by
SDS-PAGE on a 9% gel and analyzed by immunoblotting with anti-Tyr(P)
antibodies. B, the blot in A was stripped and
re-probed with anti-EGFR (
-C), anti-p120
,
anti-Shc, and anti-Grb2 antibodies. C, EGFR complexes from
untreated and EGF-stimulated HER14 cell lysates were immunoprecipitated
with anti-EGFR antibodies (RK2) and analyzed by immunoblotting with
anti-EGFR and anti-p120
antibodies. 70 µg of
whole cell lysates were also loaded in C.
We next
investigated whether p120 is phosphorylated upon
stimulation with other growth factors. Addition of NGF or aFGF to PC12
cells induced tyrosine phosphorylation of p120
(Fig. 2, A and B). However, we
could not detect association between p120
and
the corresponding activated receptors. Immunoblotting with Grb2 or Shc
antibodies failed to reveal the presence of these proteins in
p120
immunoprecipitates (not shown). PDGF also
induced tyrosine phosphorylation of p120
(Fig. 2C), whereas insulin stimulation of
CHO cells overexpressing the insulin receptor did not have any effect (Fig. 2D).
Figure 2:
Tyrosine-phosphorylation of
p120is induced upon stimulation with aFGF, NGF,
and PDGF. A, PC12 cells were left untreated or treated with
acidic FGF and heparin and lysates immunoprecipitated with
anti-p120
(C-15) antibodies or anti-FGFR1
antibodies. B, PC12 cells were left untreated or stimulated
with NGF and lysates immunoprecipitated with C-15 or anti-NGFR
antibodies. C, HER14 cells were left untreated or stimulated
with EGF or PDGF and lysates immunoprecipitated with C-15 antibodies. D, CHO-IR cells were left untreated or stimulated with insulin
and lysates immunoprecipitated with anti-p120
antibodies. All immunoprecipitates were analyzed by SDS-PAGE
and immunoblotting with R2 and anti-phosphotyrosine antibodies. PC12
NGFR1 and PC12 FGFR1 express approximately 2.5
10
NGF receptors (TrkA)/cell and 2
10
FGF
receptor/cell, respectively. CHO-IR cells express 1.2
10
insulin receptors/cell.
Several previously characterized EGFR
mutants were used to dissect the interaction between p120 and the EGFR. Tyrosine phosphorylation of p120
was examined in NIH 3T3 cells expressing an EGFR mutant
lacking the carboxyl-terminal tail (CD196) (7) , a
kinase-negative receptor mutant (K721A)(8) , or a receptor
lacking five tyrosine autophosphorylation sites (Y5F)(7) .
Background levels of tyrosine-phosphorylated p120
were detected with the EGF-stimulated kinase negative mutant (Fig. 3). This indicates that EGFR tyrosine kinase activity is
essential for p120
phosphorylation, either by
directly phosphorylating p120
or by activating other
protein tyrosine kinases. In the CD196 and Y5F mutants,
p120
was still phosphorylated upon growth factor
stimulation, although to a lesser extent as compared with wild-type
EGFR (Fig. 3). Thus, the tyrosine-phosphorylated EGFR tail
facilitates p120
phosphorylation, although
association with the EGFR tail is not essential for EGF-induced
tyrosine phosphorylation of p120
. Immunoblotting
with anti-Grb2 antibodies showed the absence of this protein in
p120
immunoprecipitates from any of the mutant
cell lines (not shown).
Figure 3:
Effect of EGFR mutants on tyrosine
phosphorylation of p120. Lysates derived from
the indicated cell lines, either unstimulated or EGF-stimulated, were
incubated with affinity-purified anti-p120
(C-15) antibodies. Immunocomplexes were analyzed by SDS-PAGE
and immunoblotted with anti-p120
(R2) or
anti-Tyr(P) antibodies.
Interaction between p120 and activated EGFR must occur either directly or through an
adaptor protein. We investigated the possibility of a direct
interaction between p120
and EGFR using filter
binding assays with GST-p120
fusion proteins.
-
P-Radiolabeled N-Cbl and C-Cbl, encompassing amino
acids 1-486 and 487-906 of human
p120
, respectively, were used to probe filters
containing immunoprecipitates of EGFR from untreated or EGF-stimulated
HER14, CD196, or Y5F cell lysates. In this assay, N-Cbl showed strong
binding to tyrosine-autophosphorylated wild-type EGFR (Fig. 4A). The probe also bound, although more weakly,
to activated Y5F mutant receptor, which becomes phosphorylated on
several new tyrosine residues upon activation (7) . No binding
to CD196 EGFR deletion mutant was detected. We also examined the
binding of N-Cbl to a purified cytoplasmic domain of the receptor
(EGFR-1C)(18) , which had been incubated with or without ATP,
rendering EGFR-1C heavily phosphorylated or unphosphorylated,
respectively. As expected, N-Cbl bound to the phosphorylated form of
EGFR-1C (Fig. 4A). These data show that
p120
is able to bind directly to activated EGFR in vitro. N-Cbl was not tyrosine-phosphorylated, thereby
demonstrating that tyrosine-phosphorylation of p120
is not essential for receptor binding. In contrast, a
-[
P]fusion protein of the same specific
activity containing amino acid residues 487-906 (C-Cbl) did not
bind to activated EGFR or any other proteins in the same assay (not
shown).
Figure 4:
A p120 deletion
mutant (N-Cbl) binds to tyrosine-autophosphorylated EGFR in
vitro. Untreated and EGF-stimulated cell lysates (HER14, CD196,
and Y5F) were incubated with monoclonal anti-EGFR antibodies. EGFR-1C
is a purified protein that contains the entire cytoplasmic domain of
EGFR. 1 µg of EGFR-1C was incubated with or without 1 mM ATP in 50 µl of Hepes, pH 7.5, for 10 min at room temperature,
and the autophosphorylation reaction was stopped by addition of 2
Laemmli buffer. 70 ng of each EGFR-1C sample, half of each
HER14 immunoprecipitate, half of each CD196 immunoprecipitate, and
one-fourth of each Y5F immunoprecipitate were electrophoresed through
two equivalent 9% acrylamide gels and transferred to nitrocellulose.
One filter was probed with
-[
P]N-Cbl (A), stripped, and re-probed with anti-EGFR (
-F)
antibodies (C). The second filter was immunoblotted with
anti-Tyr(P) antibodies (B).
In each of the EGFR mutants, the extent of binding of N-Cbl
to EGFR correlated with tyrosine autophosphorylation of the receptor (Fig. 4). This suggests that N-Cbl binds to the
phosphotyrosine-containing region of EGFR, similar to binding of SH2
domains(19) . However, p120 has no SH2
domains. It has been recently reported that a domain in the NH
terminus of Shc is able to bind directly to phosphotyrosine
containing sequences in EGFR (20, 21) and
NGFR(22) , but a similar sequence does not exist in
p120
. It is noteworthy that the amino terminus
half of p120
, which is the region responsible
for binding directly to activated EGFR, is highly conserved between
p120
and its Caenorhabditis elegans homologue, Sli-1(23) . Since most of this region is
present in the oncogenic v-Cbl protein, our findings raise the
possibility that v-Cbl might interact with EGFR in vivo. On
the other hand, the carboxyl terminus half of
p120
, which contains a number of proline-rich
sequences that may serve as SH3 binding sites(24) , is mostly
absent in Sli-1(23) .
Both the amino-terminal region
(1-486) and the proline-rich region (487-906) of
p120 were able to form a complex with activated
EGFR when incubated with EGF-stimulated HER14 lysates (not shown). This
raises the possibility that, in living cells, p120
can bind to activated EGFR directly and indirectly. We have
previously shown that Grb2 can bind both directly and indirectly (via
Shc) to the EGFR (12) and directly (25) and indirectly
(via PTP1D) to the PDGF receptor(26) . By analogy with other
cases, it is possible that complex formation between the proline-rich
region of p120
and activated EGFR is mediated by
a protein that contains both SH2 and SH3 domains. The SH2 domain could
mediate interaction with activated EGFR, and the SH3 domain may bind to
proline-rich sequences of p120
. It was recently
reported that p120
can interact with the adaptor
proteins Grb2 (4) and Nck(5) . We analyzed the sequence
of p120
for possible Grb2 SH2 binding sites, but
no sequences corresponding to the consensus for this binding were
found. Structural and functional studies on SH3 domains have allowed
the identification of proline-rich sequences as SH3 domain
ligands(24) . p120
contains a number of
proline-rich sequences that fit this consensus. One sequence in
particular, amino acids 494-499 (PPVPPR), is identical to a known
Grb2 binding site in the Sos protein(6) , although flanking
residues, which also play a role in binding to the SH3 domain, are
different. We have shown that Grb2 and Shc were present in
p120
immunoprecipitates from EGF-stimulated
HER14 cells. However, little or no Grb2 and Shc was present in
p120
immunoprecipitates of lysates from NIH 3T3
cells overexpressing CD196, Y5F, and K721A EGFR mutants or from PC12
cells overexpressing NGF or aFGF receptors (not shown). Morever,
co-immunoprecipitation of p120
with either Grb2
or Shc was detected only upon EGFR activation and autophosphorylation.
These data argue against a direct interaction in vivo between
p120
and Grb2 or Shc. Their presence in
p120
immunoprecipitates is probably due to
tertiary complex formation with activated EGFR molecules, rather than
with p120
. Attempts to identify a putative
adaptor protein which would interact with EGFR and p120
in HER14 cells, by means of co-immunoprecipitation experiments,
were unsuccesful for Nck, the p85 subunit of PI3-kinase, Crk,
p120
, and PLC
.
Recent genetic experiments (23) implicate the protein product of sli-1, a C.
elegans homologue of the mammalian p120, as
a negative regulator of the vulval induction pathway initiated by
Let-23, the C. elegans homologue of the mammalian
EGFR(27) . Genetic studies have shown that mutations in sli-1 can rescue weak loss-of-function alleles of let-23, sem-5, and, partially, let-60 and
suggest that Sli-1 may act as a negative regulator of the vulval
induction pathway at the Let-23/Sem-5 step (23) . The studies
presented here show that p120
is associated with
activated EGFR and becomes phosphorylated on tyrosine residues in
response to EGF, FGF, PDGF, and NGF stimulation. By analogy with Sli-1
modulating Let-23 function, p120
may negatively
regulate EGFR signaling, for example by directly blocking binding sites
of adaptor proteins in the receptor. Elucidation of the exact role of
p120
in signaling via receptor tyrosine kinases
will provide further information about the network of interactions that
control this important signal transduction pathway.