(Received for publication, October 17, 1995)
From the
Although epidermal growth factor (EGF) activates
phosphoinositide (PI) 3-kinase activity in a number of types of cells
or cell lines, in most cases that we have investigated the p85
regulatory subunit of PI 3-kinase does not appear to bind directly to
the EGF receptor. Previously we demonstrated that EGF-dependent
activation of PI 3-kinase activity in A431 cells is accompanied by the
binding of p85 to ErbB3, an EGF receptor homologue. However, this
mechanism did not explain the large activation of PI 3-kinase activity
that was found in PC12 and A549 cells, which possess little or no
ErbB3. Here we provide evidence that the p120 proto-oncoprotein is an intracellular adapter protein that
associates with PI 3-kinase and thus is involved in the EGF-dependent
activation of this enzyme in these two cell lines. Using an
anti-p120
antibody, we immunoprecipitated the
EGF receptor from PC12 cells and PI 3-kinase activity from PC12 and
A549 cells in an EGF-dependent fashion. Treatment of PC12 cells with
nerve growth factor or insulin stimulated large increases in PI
3-kinase activity that was immunoprecipitated using anti-Tyr(P)
antibody but not using anti-p120
antibody. In
EGF-treated PC12 cells, the tyrosine phosphorylation of
p120
displayed similar kinetics to the
activation of PI 3-kinase as measured by both in vivo lipid
production and lipid kinase assays conducted using
anti-p120
and anti-Tyr(P) immunoprecipitates.
The use of glutathione S-transferase fusion proteins of
various domains of p85 demonstrated that p120
associated with both the SH2 and SH3 domains of p85.
p120
was also present in A431 cells and offers
an additional pathway by which EGF can activate PI 3-kinase in these
cells.
In a previous study we demonstrated that
EGF()-stimulated PI 3-kinase activity in A431, PC12, and
A549 cells was immunoprecipitated using an anti-Tyr(P) antibody
(Soltoff et al., 1994). In addition to the EGFR, A431 cells
express ErbB3, a
180-kDa protein that heterodimerizes with the
EGFR in response to EGF. We demonstrated that ErbB3 serves as an
adapter protein between the EGFR and PI 3-kinase in A431 cells, and
EGF-dependent PI 3-kinase activity was immunoprecipitated from these
cells using anti-ErbB3 antibodies. ErbB3 has multiple copies of the
consensus sequence for binding the SH2 domain of the p85 subunit of PI
3-kinase (Tyr-X-X-Met), and phosphorylation at these
sites presumably mediates the association. However, since PC12 and A549
cells have little or low levels of ErbB3, we hypothesized that another
protein must serve as an adapter between the EGFR and PI 3-kinase in
these cells.
We previously observed the EGF-dependent tyrosine
phosphorylation of a 120-130-kDa protein in PC12 and A549
cells (Soltoff et al., 1994). The cbl (c-cbl) proto-oncogene encodes a 120-kDa protein
(p120
) that is localized exclusively in the
cytoplasm. This protein was recently shown to be phosphorylated on
tyrosine in response to activation of the T cell antigen receptor
(Donovan et al., 1994). The overexpression of p120
does not lead to transformation. The protein encoded by a
retrovirus version of c-cbl (v-cbl) has 60% of the C
terminus deleted and is located both in the cytoplasm and in the
nucleus (Blake et al., 1991, 1993). The latter localization
was correlated with the transforming ability of cbl.
The
p120 protein has a Tyr-X-X-Met
site that could interact with the SH2 domains of p85 if phosphorylated
and a proline-rich sequence that could bind to the SH3 domain of p85.
Thus, we investigated the possibility that p120
binds PI 3-kinase in response to stimulation of PC12 or A549
cells with EGF.
Figure 1:
Time course of
EGF on PI 3-kinase activity in PC12 cells. Serum-starved cells were
left untreated (time 0) or were treated with EGF (100 ng/ml) for
various times up to 60 min. A, immunoprecipitation of PI
3-kinase activity from EGF-treated PC12 cells using
anti-p120 and anti-Tyr(P) (
P-Tyr)
antibody (Ab). Lysates were prepared in 1 ml of lysis buffer,
and PI 3-kinase activity was measured in immunoprecipitates (I.P.) of the cleared lysates. PI 3-kinase activity was
measured using exogenous PtdIns as a substrate. Proteins were
immunoprecipitated using anti-p120
(1 µg/ml)
or anti-Tyr(P) (6.6 µg/ml) antibodies. PtdIns-P (PIP), the
product of the lipid kinase assay, was separated using thin layer
chromatography. The radioactivity at the origin represents ATP or
related products. Note that the relative EGF-dependent increases were
similar in anti-p120
and anti-Tyr(P)
immunoprecipitations (see also Fig. 2). B, time course
of PI 3-kinase activity in EGF-treated PC12 cells immunoprecipitated
using anti-Tyr(P) antibody. PI 3-kinase was measured and quantified as
in A. n = 3 or 4 experiments. C, time
course of EGF on the levels of phosphoinositides of intact PC12 cells
labeled in vivo using
[
P]orthophosphate. EGF (100 ng/ml) was added at t = 0. The lipids were extracted and deacylated (see
``Materials and Methods'') and analyzed by HPLC. Levels are
presented in terms of the maximum levels. The averaged results from two
experiments are shown. The data were normalized to the total counts/min
of each compound at t = 0. The average maximum levels
of PI-3,4,5-P
, PI-3,4-P
, and PI-4,5-P
were 5327, 9706, and 154,672 dpm, respectively. For
PI-3,4,5-P
, the maximum value was reached at 1 min in one
experiment, and at 15 min in the other experiment. In terms of the
amounts of radioactivity incorporated into the different
polyphosphoinositides in unstimulated cells, PI-4,5-P
made
up the largest fraction.
Figure 2:
PI 3-kinase activity is immunoprecipitated
from lysates of EGF-treated but not insulin- or NGF-treated PC12 cells
using an anti-p120 antibody. Cells were treated
(or not) with EGF (100 ng/ml), insulin (100 µM), or NGF
(100 ng/ml) for 5 min. Lipid kinase assays were performed on
immunoprecipitated proteins obtained using anti-Tyr(P) antibody (6.6
µg/ml) or anti-p120
(1 µg/ml)
antibodies, and the production of PtdInsP in growth factor-treated
cells was normalized to that found in untreated cells. The number of
experiments is shown in parentheses at the bottom of
each bar. The absolute amount of growth factor-stimulated PI
3-kinase activity in anti-p120
immunoprecipitates relative to that found in anti-Tyr(P)
immunoprecipitates (IP) (in experiments in which both were
assayed simultaneously) was as follows for EGF-, insulin-, and
NGF-treated cells, respectively: 66.0 ± 10.0(11) , 3.2
± 1.7(4) , and 16.6 ±
2.9%(3) .
A more complete time course for PI 3-kinase activity in anti-Tyr(P) immunoprecipitates from EGF-treated PC12 cells demonstrates that elevated PI 3-kinase activity was maintained for at least 60 min after initial exposure to EGF (Fig. 1B).
To investigate the alterations in the
various phosphoinositide products in vivo, we measured the
levels of the phosphorylated polyphosphoinositides in
[P]orthophosphate-labeled cells. The lipids were
extracted and deacylated, and the glycerophospholipid polyphosphates
were identified and quantified by HPLC (Fig. 1C).
Within the first minute of exposure to EGF,
[
P]PtdIns-3,4-P
and
[
P]PtdIns-3,4,5-P
increased to
4-10 times the basal levels, and elevated levels of these lipids
were maintained for at least 30 min. After the first minute of EGF
exposure, the decrease in PI-3,4,5-P
levels and continued
increase in PI-3,4-P
levels are probably due to the
activity of a 5`-phosphatase that dephosphorylates PI-3,4,5-P
(Stephens et al., 1991; Carter et al., 1994;
Jackson et al., 1995). These results indicate that the time
course of the activation of PI 3-kinase in vivo is similar to
that observed in the in vitro kinase assay using the
anti-Tyr(P) and anti-p120
immunoprecipitates (Fig. 1, A and B).
A comparison of EGF-,
NGF-, and insulin-stimulated PI 3-kinase activities from PC12 cells
immunoprecipitated using anti-p120 and anti-Tyr(P)
antibodies is shown in Fig. 2. All three growth factors promoted
large increases in PI 3-kinase activities that were immunoprecipitable
using anti-Tyr(P) antibody. However, the relative increase in PI
3-kinase activity in the anti-p120
immunoprecipitates of
EGF-treated cells, unlike those of insulin- or NGF-treated cells, was
similar to the relative increase observed in anti-Tyr(P)
immunoprecipitates. In addition, only in EGF-stimulated cells was the
absolute amount of growth factor-stimulated PI 3-kinase activity in
anti-p120
immunoprecipitates similar to that which was
measured in anti-Tyr(P) immunoprecipitates. Studies by various
investigators have demonstrated that p85 associates with gp140
and insulin receptor substrate-1 (IRS-1) in response to NGF and
insulin, respectively (Soltoff et al., 1992; Sun et
al., 1993). In combination with previous studies, the results in Fig. 2indicate that each of the receptors for EGF, NGF, and
insulin stimulates PI 3-kinase by phosphorylating a different adapter
protein.
Since we had hypothesized that an adapter protein was also
responsible for the EGF-dependent PI 3-kinase activity found in A549
cells (Soltoff et al., 1994), we investigated the possibility
that PI 3-kinase associates with p120 in this cell line.
Similar to the results reported above with PC12 cells, treatment of
A549 cells with EGF (100 ng/ml for 1 min) resulted in a similar
increase of PI 3-kinase activity in anti-p120
and
anti-Tyr(P) immunoprecipitates (10.9 ± 4.3 (n =
3) and 7.5 ± 0.2 (n = 3) times basal activity in
anti-p120
and anti-Tyr(P) immunoprecipitates,
respectively). In these experiments the EGF-stimulated PI 3-kinase
activity in anti-p120
immunoprecipitates was 62.6
± 21.2% (n = 3) as large as that found in
anti-Tyr(P) immunoprecipitates, similar to that found in PC12 cells (Fig. 2, legend). These results are consistent with p120
serving as an adapter protein between the EGF receptor and PI
3-kinase in both PC12 and A549 cells.
EGF-dependent increases in PI
3-kinase activity were also found in anti-p120 immunoprecipitates from A431 cells. For cells treated with EGF
(100 ng/ml, 5 min), the activity in anti-p120
immunoprecipitates was 8.3 ± 3.2 (n = 5)
times that in unstimulated cells, and this value was 5.0 ± 1.8 (n = 5) in anti-Tyr(P) immunoprecipitates. These
results suggest that in addition to ErbB3, which is a transmembrane
receptor protein, the cytosolic protein p120
provides
another site for the recruitment of p85 in A431 cells.
Figure 3:
EGF stimulates the association of p85 with
p120 and the tyrosine phosphorylation of
p120
. PC12 cells were treated with EGF (100
ng/ml) for various times up to 15 min or were not treated (0), and
proteins were immunoprecipitated from cleared lysates using
anti-p120
antibody (Ab) (1 µg/ml)
or anti-Tyr(P) (
P-Tyr) (6.6 µg/ml) antibody. The
immunoprecipitated proteins were subjected to SDS-PAGE, transferred to
nitrocellulose filters, and probed overnight with various antibodies.
Filters were sequentially probed with: A, anti-p85 antibody
(1:8000 dilution); B, anti-p120
(0.2
µg/ml) antibody; and C, anti-Tyr(P) antibody (1.1
µg/ml). Molecular mass markers (in kilodaltons) are indicated on
the left. The arrows on the right indicate
p85 (A), p120
(B), the
locations of the EGF receptor (C, upper arrow) and
p120
(C, lower
arrow), and the EGF receptor (D). The time-dependent
variations in the levels of p85 in anti-p120
and
anti-Tyr(P) immunoprecipitates (I.P.) (A), and
p120
(C, lower arrow) in the
levels of p120
immunoprecipitated using
anti-Tyr(P) antibody (B), and in the tyrosine phosphorylation
of p120
in anti-p120
immunoprecipitates (C) are similar to the kinetics of
EGF-dependent activation of PI 3-kinase (Fig. 1).
When these
immunoprecipitates and accompanying lysates were reprobed for
p120, a
120-kDa protein was identified (Fig. 3B). In the anti-Tyr(P) immunoprecipitates, the
amount of p120
present peaked at 1 min after EGF
addition, in agreement with the peak in PI 3-kinase activity and p85 in
anti-Tyr(P) immunoprecipitates. When these blots were reprobed for
tyrosine-phosphorylated proteins, the time-dependent increases in
tyrosine phosphorylation of p120
in anti-p120
immunoprecipitates (Fig. 3C) showed similar
kinetics to increases in the amount of p120
protein in
anti-Tyr(P) immunoprecipitates (Fig. 3A), with the peak
amount of protein appearing after a 1-min exposure to EGF. In cell
lysates, proteins that co-localize with the EGF receptor and
p120
constitute the major proteins that are
tyrosine-phosphorylated in EGF-treated PC12 cells (Fig. 3C). The EGF receptor also was immunoprecipitated
from lysates of EGF-treated PC12 cells using anti-p120
antibody (Fig. 3D).
The concentration
dependence of EGF on PI 3-kinase activity immunoprecipitated using
anti-p120 antibody is shown in Fig. 4A.
The PI 3-kinase activity was increased in a concentration-dependent
manner between 1 and 100 ng/ml EGF (EC
10
nM). Consistent with these results, the association of PI
3-kinase (p85) with p120
displayed a similar
concentration dependence (Fig. 4B), suggesting that PI
3-kinase binds directly to p120
in response to EGF.
Figure 4:
Concentration dependence of EGF on PI
3-kinase activity and the association of PI 3-kinase with
p120. PC12 cells were treated with different
concentrations of EGF (0-100 ng/ml) for 1 min at 37 °C.
Proteins were immunoprecipitated from cleared lysates using
anti-p120
antibody (1 µg/ml) or anti-Tyr(P)
(
P-Tyr) (6.6 µg/ml) antibody (Ab). PI
3-kinase assays were performed on the immunoprecipitates (IP)
using exogenous PtdIns as a substrate. Subsequently, the
immunoprecipitated proteins were subjected to SDS-PAGE, transferred to
nitrocellulose filters, and probed overnight with anti-p85 antibody
(1:8000 dilution). A, PI 3-kinase activity in
anti-p120
immunoprecipitates, normalized to the
activity measured in response to 100 ng/ml EGF (n = 3).
In cells stimulated with 100 ng/ml EGF in these experiments, the PI
3-kinase activity in anti-p120
immunoprecipitates was 95.1 ± 13.1% (3) of that
found in anti-Tyr(P) immunoprecipitates. B, the
immunoprecipitation of p85 using anti-p120
and
anti-Tyr(P) immunoprecipitates. Duplicate samples from the same
experiment are shown. IB,
immunoblot.
Figure 5:
The association of p120 with GST fusion proteins of p85. A, lysates from
PC12 cells stimulated (+) or not(-) with EGF (100 ng/ml, 1
min) were incubated at 4 °C for 3 h with anti-Tyr(P) (
P-Tyr), anti-p120
antibody (Ab), or equimolar amounts of GST or various GST fusion
proteins of the p85 subunit of PI 3-kinase. These included the
C-terminal SH2 domain (GST-CSH2), the N-terminal SH2 domain (GST-NSH2), full-length p85 (GST-85), or the SH3
domain (GST-SH3). The two antibodies were cleared using
protein A-Sepharose beads, and the fusion proteins were cleared with
GSH-agarose beads. The samples were washed, boiled, subjected to
SDS-PAGE, and immunoblotted using anti-p120
or
anti-Tyr(P) antibodies. After longer ECL exposures of the
anti-p120
immunoblot (IB, not shown),
p120
was visualized in the GST-CSH2 precipitate
of EGF-treated cells at the position in which the
tyrosine-phosphorylated proteins co-localized (bottom). This
is demonstrated more clearly in a separate experiment shown in B. In A and B, the arrows designate
the location of
p120
.
The results presented here demonstrate that p120 becomes phosphorylated on tyrosine in response to EGF stimulation
of PC12 cells. In addition, p120
associates with PI
3-kinase in response to EGF stimulation. The tyrosine phosphorylation
of p120
(Fig. 3, B and C) and
the association of p85 with the p120
protein (Fig. 3A and Fig. 4B) are consistent
with the SH2 domain of p85 binding to tyrosine-phosphorylated
p120
(Fig. 5). The p120
protein has
two sites (Tyr
and Tyr
) with the
appropriate Tyr-X-X-Met motif that forms the
consensus binding site for p85 (Cantley et al., 1991); it was
identified as a likely binding protein for PI 3-kinase from a search of
the GenBank data base for proteins containing the PI 3-kinase SH2
binding motif (Songyang et al., 1993). p120
also
has proline-rich regions that could interact with the SH3 domain of
p85. The interaction of p85 with other proteins, including
pp60
, p56
, and p59
(Liu et al., 1993; Prasad et al., 1993; Kapeller et
al., 1994), through SH3-binding motifs has been observed, and SH3
domains of p120
were reported to bind to p47
(Rivero-Lezcano et al., 1994). When PC12 lysates were
probed with GST fusion proteins of p85, p120
bound to the
C-terminal SH2 domain in an EGF-dependent manner and constitutively
bound to the SH3 domain, suggesting that both domains contribute to the
association of p85 with p120
. The SH2 and SH3 domains of
p85 also bound to p120
in activated T cells (Fukazawa et al., 1995). The contrast between the ability of the SH3
domain of p85 to bind p120
from unstimulated cells and
the inability of full-length p85 to do this suggests that the SH3
domain may be sequestered in full-length p85. We previously suggested
that this occurs via binding to proline-rich sequences endogenous to
p85 (Kapeller et al., 1994). Thus, a model consistent with the
data is that engagement of the SH2 domains of p85 exposes the SH3
domain for further interaction with p120
to increase the
affinity. The specific mapping of the p85 binding site on p120
will require expression of cbl mutants lacking the
motifs predicted to bind these domains.
The interaction between
p120 and the EGF receptor appears similar to the insulin
receptor/IRS-1 interaction. IRS-1 is a cytoplasmic protein that has
multiple tyrosine residues that are phosphorylated by the insulin
receptor, and PI 3-kinase binds mainly to IRS-1 and not the insulin
receptor upon the addition of insulin to cells (reviewed in White and
Kahn(1994)). Our results suggest that p120
, a cytosolic
protein, binds PI 3-kinase but may not bind tightly to the EGFR.
p120
may be a substrate of the EGFR, since the sequence
around several tyrosine residues is in good agreement with the
predicted optimal motif for the EGFR (Songyang et al., 1995).
While this paper was in review, several other investigators reported
that p120
associated with the activated EGFR and became
tyrosine-phosphorylated in EGF-treated cells (Tanaka et al.,
1995; Galisteo et al., 1995), and it was suggested that
p120
binds both directly to the autophosphorylated
C-terminal tail of the EGFR as well as indirectly through an unknown
adaptor protein (Galisteo et al., 1995). p85 was also found to
associate with p120
in activated T cells (Meisner et
al., 1995; Hartley et al., 1995; Fukazawa et
al., 1995), and in vitro binding data indicated that this
association was mediated through both SH2 and SH3 domains of p85
(Hartley et al., 1995; Fukazawa et al., 1995).
As
a result of our previous study (Soltoff et al., 1994), we
concluded that there were at least two different adapter proteins
involved in the activation of PI 3-kinase by EGF (Soltoff et
al., 1994). We demonstrated that EGF stimulated the tyrosine
phosphorylation of ErbB3 in A431 cells and that PI 3-kinase was
recruited to the tyrosine-phosphorylated ErbB3 protein. Similar
findings were also reported by Kim et al.(1994). Since this
mechanism did not account for the activation of PI 3-kinase activity in
PC12 or A549 cells, we concluded that a second mechanism must occur in
these cells. The data presented in this paper indicate that
p120 serves as an adapter protein for the EGF-dependent
activation of PI 3-kinase in PC12 and A549 cells. The effects of EGF on
these cells were distinct from other growth factors (notably insulin)
that act via the stimulation of tyrosine kinases. In addition, the new
results obtained using A431 cells indicate that two complementary
systems (ErbB3 and p120
) involved in the EGF-dependent
activation of PI 3-kinase are present in the same cell. It remains to
be determined whether there are some cells that express ErbB3 and do
not express p120
.