Transformation potency of ErbB heterodimer signaling is determined by B-Raf kinase

Article (PDF Available)inOncogene 23(29):5023-31 · July 2004with15 Reads
DOI: 10.1038/sj.onc.1207664 · Source: PubMed
Abstract
Cellular transformation occurs only in cells that express both ErbB1 and ErbB4 receptors, but not in cells expressing only one or the other of these receptors. However, when both receptors are coexpressed and ligand-stimulated, they interact with virtually the same adaptor/effector proteins as when expressed singly. To reveal the underlying regulatory mechanism of the kinase/phosphatase network in ErbB homo- and heterodimer receptor signaling, extracellular signal-regulated kinase (ERK) and Akt activities were evaluated in the presence of several enzyme inhibitors in ligand-induced cells expressing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4) receptor. The PP2A inhibitor okadaic acid showed receptor-specific inhibitory profiles for ERK and Akt activities. Moreover, B-Raf isolated only from E1/4 cells could induce in vitro phosphorylation for MEK; this B-Raf kinase activity was abolished by pretreatment of the cells with okadaic acid. Our study further showed that the E1/4 cell-specific B-Raf activity was stimulated by PLC gamma and subsequent Rap1 activation. The present study suggests that B-Raf kinase, which was specifically activated in the cells coexpressing ErbB1 and ErbB4 receptors, elevates total ERK activity within the cell and, therefore, can induce cellular transformation.
Transformation potency of ErbB heterodimer signaling is determined by
B-Raf kinase
M Hatakeyama*
,1
, N Yumoto
1
,XYu
1
, M Shirouzu
2
, S Yokoyama
2,3,4
and A Konagaya
1
1
Bioinformatics Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan;
2
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045,
Japan;
3
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan;
4
Cellular Signaling Laboratory and Structurome Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kohto,
Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Cellular transformation occurs only in cells that express
both ErbB1 and ErbB4 receptors, but not in cells
expressing only one or the other of these receptors.
However, when both receptors are coexpressed and
ligand-stimulated, they interact with virtually the same
adaptor/effector proteins as when expressed singly. To
reveal the underlying regulatory mechanism of the kinase/
phosphatase network in ErbB homo- and heterodimer
receptor signaling, extracellular signal-regulated kinase
(ERK) and Akt activities were evaluated in the presence of
several enzyme inhibitors in ligand-induced cells expres-
sing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4)
receptor. The PP2A inhibitor okadaic acid showed
receptor-specific inhibitory profiles for ERK and Akt
activities. Moreover, B-Raf isolated only from E1/4 cells
could induce in vitro phosphorylation for MEK; this B-
Raf kinase activity was abolished by pretreatment of the
cells with okadaic acid. Our study further showed that the
E1/4 cell-specific B-Raf activity was stimulated by PLCc
and subsequent Rap1 activation. The present study
suggests that B-Raf kinase, which was specifically
activated in the cells coexpressing ErbB1 and ErbB4
receptors, elevates total ERK activity within the cell and,
therefore, can induce cellular transformation.
Oncogene (2004) 23, 5023–5031. doi:10.1038/sj.onc.1207664
Published online 5 April 2004
Keywords: ErbB; B-Raf; transformation; PP2A; PKA
Introduction
ErbB receptors are a family of membrane receptor
tyrosine kinases that activate and recruit intracellular
signaling pathways after the binding of growth factors.
These receptors play essential roles in cellular prolifera-
tion and differentiation, and their overexpression and
mutation are implicated in a variety of mammalian
cancers (Olayioye et al., 2000; Yarden and Sliwkowski,
2001). The ErbB receptor family comprises the ErbB1/
EGF receptor (EGFR), ErbB2, ErbB3, and ErbB4. The
ErbB proteins share conserved intrinsic tyrosine kinase
domains and extracellular ligand-binding domains
distinguish their binding properties and affinities with
several types of epidermal growth factor (EGF)-like
ligands (Riese et al., 1996; Tzahar et al., 1996; Jones
et al., 1999). These distinct binding properties result in
diverse biological outputs. Further, the binding affinity
of the ligand with an ErbB receptor and the cellular
transformation potency are also modulated by coex-
pression of another ErbB receptor. For example, the
binding of EGF with EGFR is enhanced by the
coexpression of ErbB2 or ErbB3, and the binding of
heregulin (HRG) with ErbB3 or ErbB4 is enhanced
by the coexpression of EGFR or ErbB2 (Riese et al.,
1995; Cohen et al., 1996; Zhang et al., 1996; Wang et al.,
1998).
The ErbB1 and ErbB4 receptors possess strong
affinities for EGF and HRG, respectively, and induce
the MAPK cascade (Raf-MEK-ERK pathway) and
PI3K-Akt/protein kinase B pathway (Yarden and
Sliwkowski, 2001) upon ligand binding. However
ligand-induced transformation of 3T3-7d cells takes
place only when both receptors are cotransfected in the
same cells (Cohen et al., 1996). The mechanism
responsible for this biological response induced by the
coexpression of different ErbB receptors remains
unclear. Even when ErbB1 and ErbB4 are coexpressed
and form a heterodimer upon ligand binding, the two
receptors interact with the same signaling molecules
with which each receptor interacts when expressed
singly. For example, the ErbB1–ErbB4 heterodimer
interacts with the Grb2, Shc, the p85 subunit of
phosphatidylinositol 3
0
-kinase (PI3K), Cbl, and PLCg
in response to EGF in the same way that the ErbB1
homodimer does. Similarly, the same heterodimer
behaves identically to the ErbB4 homodimer in binding
to Grb2, Shc, and p85 in response to HRG (Cohen et al.,
1996; Olayioye et al., 1998). Thus, the increase in
biological response elicited by the coexpression of ErbB
receptors cannot be simply explained by the common
signaling cassettes recruited to the same ligand–receptor
Received 20 August 2003; revised 22 December 2003; accepted 13
February 2004; published online 5 April 2004
*Correspondence: M Hatakeyama; E-mail: marikoh@gsc.riken.jp
Oncogene (2004) 23, 5023–5031
&
2004 Nature Publishing Group
All rights reserved 0950-9232/04 $30.00
www.nature.com/onc
complex. Rather, it seems that some quantitative
(strength or duration of the initial or intermediate
signals) or qualitative differences (e.g., different regula-
tion systems on the same signaling cascades are
activated in the cells) influence the signal amplitudes
(Simon, 2000; Heinrich et al., 2002) and thereby produce
dissimilar biological responses in vivo. To understand
the mechanism of cellular transformation in ErbB signal
transduction, it is required to identify the factors that
influence the cellular transformation potency produced
by coexpression of different ErbB receptors.
In a previous study, we found that HRG-activated
ErbB4 receptor induced ERK and Akt activation, and
that Akt negatively regulated Raf-1 and the subsequent
ERK activity in CHO cells expressing ErbB4 receptor
(Hatakeyama et al., 2003). Furthermore, a computer
simulation of the ErbB4 signal transduction suggested
that the dynamics of ERK and Akt activities were
modulated by protein phosphatase 2A (PP2A). The
major regulatory signaling cassettes in ligand-stimulated
ErbB4 receptor signaling are Shc-MAPK and PI3K-Akt
pathways, and PP2A targets the dephosphorylation of
MEK and Akt for signal attenuation within these
pathways. In contrast, PP2A is known to activate Raf-
1 in the MAPK pathway by dephosphorylating the
inhibitory phosphoserine residue of the molecule (Dhil-
lon et al., 2002). Thus, PP2A modulates the activation
and attenuation of kinase activities in ErbB4 receptor
signaling pathways.
In the present study, we observed distinctive regula-
tion of PP2A on ERK and Akt activities in ligand-
induced cell lines expressing ErbB1 (E1), ErbB4 (E4),
and ErbB1/ErbB4 (E1/4) receptors. As for sensitivity
toward a PP2A inhibitor okadaic acid, E4 and E1/4 cells
showed virtually the same response and resulted in the
inhibition of ERK activities. In addition, specific
activation of B-Raf kinase, where PP2A acts as a
positive regulator, was observed only in E1/4 cells and
not in E1 or E4 cells. Further study showed that this E1/
4 cell-specific B-Raf kinase activity was followed by
PLCg and Rap1 activation upon EGF stimulation. This
phenomenon was consistent with data showing that the
highest net ERK activity and cellular transformation
was observed in E1/4 cells, and that B-Raf kinase
seemed to contribute to the rise in overall ERK activity
within the cells. Our results suggest that a difference in
the distinct regulation of kinase/phosphatase in the
signaling pathways results in different amplitudes in the
conclusive kinase activities in ErbB1-, ErbB4 homo-,
and heterodimer signaling, and determines cellular
transformation potency.
Results
Only E1/4 cells induce cellular transformation
First, we compared the transformation abilities of CHO
cells that express ErbB1 (E1), ErbB4 (E4), and ErbB1-
ErbB4 (E1/4) in the presence of EGF or HRG. Foci
formation was observed in ligand-stimulated E1/4 cells,
but not in E1 or E4 cells (Figure 1). This result was
consistent with a former study using ErbB1 and ErbB4
expressing NIH3T3 cells (Cohen et al., 1996), and it
confirmed that cellular transformation occurs only when
different ErbB receptors are coexpressed.
Receptor phosphorylation is a specific event asso-
ciated with ligand-induced activation of ErbB protein
tyrosine kinases. In our study, significant tyrosine
phosphorylation was observed on the ErbB1 receptor
in EGF-stimulated E1 and E1/4 cells and the ErbB4
receptor in HRG-stimulated E4 and E1/4 cells, respec-
tively (Figure 2a). The data showed that the ligands
specifically induced tyrosine phosphorylation on their
high-affinity receptors.
To confirm the heterodimer formation in E1/4 cells,
we isolated ErbB1, ErbB4, and tyrosine-phosphorylated
proteins from ligand-stimulated E1/4 cells using the
corresponding specific antibodies followed by Western
blot analysis. Isolated proteins were detected with
ErbB1 and ErbB4 receptor antibodies. Stronger band
intensities of ErbB1 and ErbB4 proteins were observed
after ligand stimulation in the ErbB4 and ErbB1
immunoprecipitates, respectively (Figure 2b). EGF
treatment of E1/4 cells for 30 s enhanced ErbB1
phosphorylation by 1.6870.54-fold and ErbB4 phos-
phorylation by 1.6770.02-fold. Similarly, HRG treat-
ment enhanced ErbB1 phosphorylation by 1.6770.54-
fold and ErbB4 phosphorylation by 2.4370.37-fold
(based on two independent experiments). This result is
consistent with published data showing that coexpressed
receptors form heterodimers in response to ligand
binding (Tzahar et al., 1997; Wang et al., 1998). In this
test, the wild-type CHO cells did not show detectable
ErbB receptors (data not shown); accordingly, we
estimated that the endogenous ErbB receptor level in
the wild-type cells is extremely low.
Figure 1 Focus formation assay for wild-type CHO cells, E1, E4,
and E1/4 cells. Wild-type CHO cells (WT) and CHO cells
expressing ErbB1 (E1), ErbB4 (E4) and both ErbB1 and ErbB4
receptors (E1/4) were incubated in the presence or absence of 1 nM
EGF or 1 nM HRG in a serum-free medium. After incubation for
14 days, the cells were rinsed with PBS and fixed with 10 %
formalin and stained with Giemsa reagent. Cont, absence of ligand;
EGF, incubation with EGF; HRG, incubation with HRG
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
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Effect of wortmannin on ERK and Akt activities
Next, to observe the underlying regulatory mechanism
of kinases and phosphatases in ErbB signaling, we tested
the effect of several protein kinase and phosphatase
inhibitors on ERK and Akt activation in wild-type
CHO cells, E1, E4, and E1/4 cells. Activated ErbB1 and
ErbB4 receptors have been shown to stimulate the
MAPK cascade through the binding of Shc or Grb2
adaptor proteins. Subsequently, these receptors activate
the PI3K-Akt cascade through the binding of the p85
subunit of PI3K to the receptor directly (in the case of
ErbB4) (Cohen et al., 1996) or indirectly via the docking
protein Gab1 (for ErbB1) (Rodrigues et al., 2000). As
PI3K is an upstream regulator of Akt, the PI3K
inhibitor wortmannin was added to observe the effect
of PI3K on ERK and Akt activation in the cells. The
addition of wortmannin suppressed the EGF-induced
ERK activation in E1 cells. However, wortmannin
treatment did not induce a remarkable change in ERK
activation in the EGF-treated E4 and E1/4 cells and in
the HRG-treated cells (Figure 3). Under these condi-
tions, the Akt activity was suppressed in all of the cell
lines tested. The suppression of ERK by wortmannin in
EGF-induced E1 cells seemed to result from the PI3K-
dependent MAPK activation (Oehrl et al., 2003);
however, our data were insufficient to evaluate the
direct PI3K-Ras interaction in E1 cells. On the other
hand, our study showed Akt and ERK activation within
the E4 cells where ErbB4 receptor phosphorylation was
not detected by anti-phosphotyrosine antibody (PY20).
Similar EGF-induced ERK activation in the ErbB4
receptor-expressing cell line was also observed in an
earlier study (Shelly et al., 1998; Tzahar et al., 1998).
ERK and Akt were distinctly regulated by PP2A in
different sets of ErbB receptors
In all cell lines, the MEK inhibitor PD98059 suppressed
ERK activity but showed no effect on Akt activity
Figure 2 Ligand-induced receptor phosphorylation in E1, E4, and
E1/4 cells and formation of heterodimer in E1/4 cells. (a) E1, E4,
and E1/4 cells were treated with 10 n
M EGF or HRG for 1, 5, and
10 min. Cell lysates containing equal amounts of protein were
subjected to immunoprecipitation (IP) using anti-ErbB1 or ErbB4
receptor antibody. After SDS–PAGE and membrane transfer,
resolved protein bands were immunoblotted with anti-phosphotyr-
osine (PY20) antibody and later re-blotted with the ErbB1 or
ErbB4 receptor antibody. (b) E1/4 cells were incubated in the
presence or absence of 10 n
M EGF or HRG for 30 s. Cell lysates
containing equal amounts of protein were subjected to IP using
anti-ErbB1, ErbB4 or PY20 antibodies, respectively. After Western
blot analysis, resolved protein bands were immunoblotted with
anti-ErbB1 or anti-ErbB4 antibody. Data show the representative
figure of two independent experiments
Figure 3 Inhibitory profile of wild-type CHO cells, E1, E4, and
E1/4 cells by a PI3K inhibitor. Wild-type CHO cells, E1, E4, and
E1/4 cells were pretreated in the presence or absence of a PI3K
inhibitor wortmannin (Wort; 100 m
M) 10 min prior to the addition
of the growth hormone. After treatment with growth hormone
(10 n
M EGF or HRG) for 10 min, cell lysates were subjected to
Western blot analysis, and blotted with antibodies to detect
phospho-p44/42 ERK (pERK) or phospho-Ser473 Akt (pAkt),
and later re-blotted with anti-ERK (ERK) or anti-Akt (Akt)
antibody, respectively. Data show the representative figure of three
independent experiments
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
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(Figure 4), indicating that the MAPK pathway did not
activate the PI3K-Akt pathway. When the PP2A
inhibitor okadaic acid was tested, ERK and Akt
activation were upregulated in the wild-type and E1
cells irrespective of the type of ligands (Figure 4).
However, okadaic acid inhibited ERK activity in the E4
and E1/4 cells. As for Akt activity, EGF and HRG
treatment resulted in different responses in E4 and E1/4
cells; in both cell lines, okadaic acid promoted Akt
activity in response to EGF but showed no effect in
response to HRG. The PP2A regulation of ERK activity
in E4 and E1/4 cells was distinct from that in wild-type
and E1 cells; we therefore inferred that expression of the
ErbB4 receptor stimulated a specific pathway for ERK
activation. Furthermore, our data showed that treat-
ment with the PP2A inhibitor promoted Akt phosphor-
ylation in the absence of ligands in the all cell lines
tested. These results suggested that the PP2A action sites
in the regulation of Akt and ERK activation are distinct
from each other. We used two different ErbB-expressing
clones to test the reproducibility of sensitivities toward
these enzyme inhibitors. As these clones showed similar
inhibitory patterns, we concluded that the expression
level of the receptor in the cells did not alter the
inhibitory profiles. The Akt activity observed in Figure 3
was not observed in the EGF-stimulated E1 cells in
Figure 4, but this difference was due to the shorter
exposure time used in Figure 4.
In MAPK (Raf-MEK-ERK) and PI3K-Akt path-
ways, PP2A is known to attenuate the activities of MEK
(Keyse, 2000; Zhou et al., 2002) and Akt (Andjelkovic
et al., 1996; Ivaska et al., 2002). These previous findings
are consistent with our results showing PP2A inhibition
of ERK and Akt in wild-type and E1 cells. However,
this regulatory mechanism is unlikely to explain the
positive stimulatory effect of PP2A on ERK activity in
E4 and E1/4 cells. Accordingly, we speculated that the
Raf activation by PP2A (Dhillon et al., 2002; Kubicek
et al., 2002; Strack, 2002) might be responsible for the
positive regulatory effect of PP2A on ERK activity.
B-Raf kinase is specifically activated in E1/4 cells
Earlier studies showed that dephosphorylation of Raf-1
by PP2A enhances the membrane translocation of Raf-
1, which, in turn, facilitates Raf-1 kinase activation
(Dhillon et al ., 2002; Kubicek et al., 2002). Furthermore,
the PKA-catalysed phosphoserine/threonine residues on
Raf-1 were understood to be PP2A action targets
(Peraldi et al., 1995). The mechanism of action of
Figure 4 Inhibitory profile of wild-type CHO cells, E1, E4, and E1/4 cells by MEK inhibitor and PP2A inhibitor. Wild-type CHO
cells, E1, E4, and E1/4 cells were pretreated in the presence or absence of PD98059 (PD; 50 m
M) or okadaic acid (OA; 2 mM) 10 min
prior to the addition of the 10 n
M growth hormone (EGF or HRG). After treatment for 10 min, the cell lysates were subjected to
Western blot analysis, blotted with antibodies to detect phospho-p44/42 ERK (pERK) or phospho-Ser473 Akt (pAkt), and later re-
blotted with anti-ERK (ERK) or anti-Akt antibody (Akt), respectively. Data show the representative figure of three independent
experiments
ErbB heterodimer-specific B-Raf activation
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B-Raf is not as clear as that of Raf-1; however, it has
been shown in the earlier study that B-Raf is also
positively regulated by PP2A (Strack, 2002). Since Raf-1
is a common mediator of the MAPK cascade in ErbB
receptor signaling (Yarden and Sliwkowski, 2001), we
tested B-Raf activity. Since, B-Raf is also activated, it is
likely that okadaic acid causes the inhibition of ERK
in ErbB4-expressing cells as a result of Raf-1 and
B-Raf inhibition even though MEK is activated by the
same reagent.
Briefly, we tested EGF-induced B-Raf phosphoryla-
tion using a phospho-(Ser/Thr) PKA substrate motif
antibody capable of detecting RXXT/S (PKA recogni-
tion site) phosphorylation. Our results showed that
PKA phosphorylation in B-Raf was decreased after
EGF administration in E1/4 cells, but increased in E1
and E4 cells (Figure 5a). Pretreatment of the cells with
okadaic acid inhibited this de-phosphorylation (data not
shown). Furthermore, B-Raf immunoprecipitates iso-
lated from the EGF-treated E1/4 cells induced in vitro
phosphorylation of MEK, and this B-Raf activity was
inhibited by pretreatment with okadaic acid, whereas
those from the E1 or E4 cells induced no such effect
(Figure 5b). Thus, it was inferred that B-Raf kinase
activity in E1/4 cells is suppressed by PKA phosphor-
ylation at the basal state, and promoted by PP2A
dephosphorylation after ligand stimulation.
It has been reported that B-Raf is activated
through PLCg and subsequent small GTPase Rap1
activation (York et al., 1998; Zwartkruis et al., 1998).
Rap1-induced B-Raf activation is known to stimulate
sustained activation of the MAPK cascade (York et al.,
1998; Kao et al ., 2001). To elucidate the B-Raf
activation mechanism in E1/4 cells, we analysed in vitro
B-Raf kinase activity in the presence of the PLCg
inhibitor U73122. Figure 6a shows that the kinase
activity of B-Raf isolated from E1/4 cells was suppressed
by this inhibitor. Similarly, Rap1 activity was specifi-
cally elevated in E1/4 cells in response to EGF
administration and diminished by the same inhibitor
(Figure 6b). Under these conditions, EGF did not
elevate Rap1 activity in E4 cells. Basal Rap1 activity,
however, was inhibited by U73122. Overall, B-Raf
activation seemed to be a specific event for E1/4 cells
and to induce an additional route for MEK-ERK
activation (Guo et al., 2001). These findings suggest that
this E1/4 cell-specific B-Raf activation by PLCg as well
as PP2A contributes to the elevation of total ERK
activity in E1/4 cells.
To confirm this, we compared the levels of EGF-
induced ERK phosphorylation between E1 and E1/4
cells and between E4 and E1/4 cells. ERK and phospho-
ERK proteins were isolated from the cells by immuno-
precipitation after treating the cells with 10 n
M EGF for
1, 5, 10, and 30 min, and placed side by side on the SDS–
PAGE gel. After membrane transfer, both ERK and
phospho-ERK proteins were detected with anti-ERK
antibodies (Figure 7a). The signal intensity ratios
showing phospho-ERK versus ERK were compared
between the E1 and E1/4 cells (Figure 7b) and between
the E4 and E/4 cells (Figure 7c). The results showed that
ERK originating from the EGF-induced E1/4 cells was
much more highly phosphorylated than that from the
E1 and E4 cells (Figure 7). However, this tendency was
specific to EGF and could not be observed in associa-
tion with the HRG-induced event, which results in
identical levels of ERK activation in E4 and E1/4 cells
(data not shown).
Discussion
Cells that coexpress different ErbB receptors tend to
undergo cellular transformation more frequently than
cells that express a single receptor (Cohen et al., 1996;
Zhang et al., 1996). In this study, we compared cells
expressing ErbB1, ErbB4, and ErbB1–4 and found
that distinct PP2A and PLCg regulatory mechanisms
exist among these cell lines. We also discovered that
cells coexpressing both the ErbB1 and ErbB4 recep-
tors could activate B-Raf kinase and induce cellular
transformation.
Ligand-stimulated ErbB1 and ErbB4 receptors acti-
vate the MAPK cascade and PI3K-Akt pathway. Raf-1,
located at the first step in the MAPK cascade,
determines the amplitude of ERK activity. Three types
of Raf have been identified in mammals: A-Raf, B-Raf,
and Raf-1 ( ¼ C-Raf). All the three Raf isoforms share
MEK as a common downstream substrate (Kolch, 2000;
Chong et al., 2003), and PP2A has been shown to be a
Figure 5 E1/4 cell-specific B-Raf dephosphorylation and activa-
tion. (a) E1, E4, and E1/4 cells were treated in the presence or
absence of 10 n
M EGF for 3, 5, and 10 min. Cell lysate was
immunoprecipitated (IP) with anti-B-Raf antibody and blotted
with phospho-(Ser/Thr) PKA substrate motif antibody, and later
re-blotted with anti-B-Raf antibody. (b) E1, E4, and E1/4 cells were
incubated in the presence or absence of 10 n
M EGF for 10 min with
or without pre-incubation with okadaic acid (OA; 2 m
M). After the
EGF treatment, B-Raf was isolated from the cell lysates using B-
Raf-specific antibody with the Catch-and-Release immunoprecipi-
tation system. The obtained soluble B-Raf fraction was incubated
with recombinant MEK-1 and [g-
32
P] ATP in the kinase buffer for
30 min at room temperature. Finally,
32
P incorporation of the
MEK-1 substrate was detected by autoradiogram after SDS–
PAGE. Data show the representative figure of two independent
experiments
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
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common positive regulator for Raf-1 (Abraham et al.,
2000; Jaumot and Hancock, 2001) and B-Raf (Strack,
2002). The ubiquitous Raf-1 is the most studied Raf
isoform, but the role of B-Raf in ErbB signaling has not
been intensively studied, notwithstanding the impor-
tance of B-Raf in human cancer (Brose et al., 2002).
Since B-Raf is an upstream regulator of MEK (Busca
et al., 2000) and displays higher MEK kinase activity
than Raf-1 (Papin et al., 1998), B-Raf activation should
greatly facilitate final ERK activity.
In our study, cellular responses to several enzyme
inhibitors, including okadaic acid, were virtually the
same in both E4 and E1/4 cells; however, B-Raf kinase
activity was specifically elevated in E1/4 cells. B-Raf is
known to be activated (MacNicol and MacNicol, 1999)
or inhibited (Peraldi et al., 1995) by PKA in a cell-
specific manner. In our study, the basal inhibitory
phosphorylation of B-Raf by PKA and PP2A-induced
stimulatory dephosphorylation of B-Raf seemed to be
observed only in E1/4 cells. On the other hand, in E1
and E4 cells, EGF-stimulated PKA seemed to phos-
phorylate B-Raf, thereby inhibiting its kinase activity.
The difference in the roles of PKA and PP2A for B-Raf
activation in these cell lines is not clear. B-Raf activation
in E1/4 cells seems to be more relevant than Raf-1
activation (e.g. activation by small G-protein binding
and PP2A dephosphorylation), while the suppression
mechanism of B-Raf activity in E1 and E4 cells seems to
be more complex.
Phosphorylation and dephosphorylation regulate and
determine the function of the proteins in the signal
transduction cascade in response to extracellular stimuli.
If we focus on an MAPK (Raf-1-MEK-ERK) cascade,
there are two PP2A action sites. One is MEK, where
PP2A acts as a signal attenuator by dephosphorylating
the protein. The other is Raf-1; however, at this site,
PP2A acts as a positive regulator. Accordingly, whether
PP2A stimulates or inhibits the entire MAPK cascade
depends on the contribution made by the ratio of the
Raf and MEK kinase in the cascade. It is clear from
Figure 4 that PP2A acted as a negative regulator of the
MAPK cascade in wild-type and E1 cells, and therefore
it is inferred that MEK makes a major contribution to
activation of the cascade in these cell lines. With respect
Figure 6 Inhibition of B-Raf kinase activity by PLCg inhibitor and E1/4 cell-specific Rap1 activation. (a) E1/4 cells were incubated in
the presence or absence of EGF (10 n
M) and PLC ginhibitor U73122 (10 mM). B-Raf kinase activity was assayed as described in the
Materials and methods section. The band intensities were quantified using a densitometer. (b) E1, E4, and E1/4 cells were incubated in
the presence or absence of EGF and U73122. Cell lysates were incubated with GST-Ral GDS-RBD agarose to isolate Rap1. Agarose
beads were subjected to Western blot analysis, and protein bands were detected using anti-Rap1 antibody (upper panel). The band
intensities were quantified using a densitometer and are shown in arbitrary units (AU) (lower panel). Data show the representative
figure of two independent experiments
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
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to the cells expressing both ErbB1 and ErbB4, our data
showed that there were additional positive regulatory
sites for PP2A, B-Raf, in addition to Raf-1. Conse-
quently, it was inferred from Figure 4 that PP2A acts as
a positive regulator in the entire MAPK cascade in this
cell line beyond its inhibitory role on MEK. However,
since the ERK activity in E4 cells was inhibited by
the PP2A inhibitor but did not show B-Raf activity,
there remains a possibility that B-Raf is not a control
point for ERK activation in ErbB4-expressing cells.
More intensive study is needed to elucidate B-Raf acti-
vation mechanisms caused by different ErbB receptor-
expressing cells.
In earlier investigations of EGF-induced ErbB1
receptor signaling, PP2A was shown to suppress Akt
activity through direct dephosphorylation of the en-
zymes (Andjelkovic et al., 1996; Keyse et al. 2000;
Ivaska et al., 2002; Zhou et al., 2002). When we used
okadaic acid to suppress PP2A, Akt activation took
place in all cell lines tested in concert with a direct
phosphorylation signal after EGF stimulation when
detected by anti-PKA substrate motif (RXXT/S) anti-
body (data not shown). Therefore, we inferred that
constitutive PP2A suppressed this PKA-induced Akt
activity in the basal state. PKA substrate motifs are
located at Thr72, Ser124, and Ser246 in the Akt
molecule. A PKA recognition site, Arg69-X-X-Thr72,
was found to reside in the PH domain, a domain
essential for the interaction of Akt with lipid membrane
(Thomas et al., 2002). Therefore, we postulate that
Thr72 phosphorylation by PKA may facilitate the
localization of Akt, required for its activation (Aoki
et al., 1998; Sable et al., 1998).
There is also the unsolved question of why PLCg in
E1/4 cells can activate B-Raf through Rap1 even though
the ErbB1 receptor also induces PLCg activation in
response to EGF (Cohen et al., 1996; Olayioye et al.,
1998). One clear difference between E1, E4, and E1/4
cells is that E1/4 cells form receptor heterodimers, which
may facilitate the specific signaling that causes the PLCg
activation and PKA-PP2A regulation that is essential
for B-Raf activation. However, further study is needed
to find a direct link among this heterodimer formation,
specific PLCg activation, and PKA-PP2A regulation.
Overall, our results indicate that specific regulation of
the kinases and phosphatases promotes cellular trans-
formation of the cells that coexpress different ErbB
receptors. In CHO cells, coexpression of ErbB1 and
ErbB4 receptors specifically induced B-Raf activation
through PLCg, and this additional route for MAPK
cascade, in addition to the Raf-1 route, resulted in a rise
in total ERK activity. Even though the direct link
between heterodimer formation and PLCg activation
has yet to be discovered, we surmised that ErbB1/4
heterodimer formation triggers a specific signaling event
that activates PLCg. Furthermore, the present study
shows a specific example that the signaling amplitude of
conclusive kinase activities leading to cellular transfor-
mation may be determined by the underlying differential
regulatory mechanisms of PKA and PP2A on the
effector enzymes in cellular signal transduction.
Materials and methods
Materials
Recombinant human heregulin-b
176–246
(HRG) was purchased
from R&D Systems (Minneapolis, MN, USA). EGF was
purchased from PeproTech House (London, England). Anti-
bodies for detecting phospho-p44/42 ERK, phospho-Ser473
Akt, phospho-Ser/Thr PKA substrate motif, ERK, and Akt
were purchased from Cell Signaling Technology, Inc. (Beverly,
MA, USA). Anti-ErbB1 receptor, anti-ErbB4 receptor, anti-
phosphotyrosine (PY20), anti-Rap1 antibody, and anti-B-Raf
were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Human recombinant MEK1 was purchased from
Upstate Biotechnology (Lake Placid, NY, USA). Okadaic acid
(PP2A inhibitor), PD98059 (MEK inhibitor), U73122 (PLCg
inhibitor), and wortmannin (PI3K inhibitor) were obtained
from Calbiochem (San Diego, CA, USA). GST-Ral GDS-Rap
binding domain (RDB) agarose was purchased from Upstate
Biotechnology (Lake Placid, NY, USA).
A method for constructing Chinese hamster ovary (CHO)
cells expressing full-length human ErbB1 (EGFR) or ErbB4
receptor is described elsewhere (Kim et al., 2002). The cells
coexpressing ErbB1 and ErbB4 receptors were constructed as
follows: ErbB1/pcDNA3.1/Zeo was digested by NheI–XbaI,
and inserted into the NheI–XbaI site of the mammalian
expression plasmid pcDNA3.1/Zeo ( þ ) (Invitrogen Corp.,
Carlsbad, CA, USA). The plasmid was then transfected into
ErbB4/Zeo cells by FuGENE6 (Roche Diagnostics, Basel,
Switzerland), and stable transfectants were selected by G418
(neomycin).
Figure 7 Comparison of the amplitudes of EGF-induced ERK
activity in E1, E4, and E1/4 cells. E1, E4, and E1/4 cells were
treated in the presence or absence of 10 n
M EGF for 1, 5, 10, and
30 min. Immunoprecipitates against anti-ERK (IP: E) and anti-
phospho-ERK (IP: P) antibodies from E1 and E1/4 cells, or E4 and
E1/4 cells, at each time point, were placed side by side on the SDS–
PAGE gel. After Western blot analysis, the proteins were detected
using anti-ERK antibody. Finally, protein band intensities were
quantified using a densitometer. (a) Comparisons of phospho-ERK
levels in E1 and E1/4 cells (upper panel), and in E4 and E1/4 cells
(lower panel). (b) Graph representing the phospho-ERK/ERK
ratio in E1 (solid line) and E1/4 (dotted line) cells. (c) Graph
representing the phospho-ERK/ERK ratio in E4 (solid line) and
E1/4 (dotted line) cells
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
5029
Oncogene
Cell culture
CHO cells expressing ErbB1, ErbB4 or ErbB1–4 receptors (E1,
E4, and E1/4 cells, respectively) were routinely maintained in
DMEM/F12 (Gibco BRL, Githersburg, MD, USA) medium
supplemented with 10% bovine calf serum and antibiotics. For
detection of the effect of growth hormones, the cells were
starved in serum-free DMEM/F12 for 16–24 h prior to the
experiment. To test the effect of kinase and phosphatase
inhibitors, the cells were pretreated with the inhibitors 10 min
prior to the addition of the growth hormone.
Focus assay
E1, E4, and E1/4 cells were transferred to six-well dishes
and grown in phenol red-free DMEM supplemented with
5% calf serum that had been heat-inactivated and treated
with 0.25%. (w/v) dextran-coated charcoal (5% CDCS-
DMEM) to remove mitogenic compounds. When the cells
reached confluence, the medium was replaced with serum-free
DMEM and incubated for 14 days in the presence or absence
of 1 n
M EGF or 1 nM HRG. After the incubation, the cells
were fixed with 10% formalin and stained with Giemsa
reagent.
Western blot analysis
The growth hormone-stimulated cells were rinsed with ice-cold
PBS and lysed with cell lysis buffer (pH 7.4) containing 1%
Triton X-100, 0.5% deoxycholate, 0.1% SDS, PBS and
protease inhibitors. Cell lysate was cleared by centrifugation,
and the protein concentration of the supernatant was
determined using protein assay reagent (Bio-Rad laboratories,
Hercules, CA, USA). For the detection of phosphorylated-
ErbB receptors, cell lysate samples containing equal amounts
of protein were subjected to immunoprecipitation using each
anti-ErbB antibody and then analysed by Western blot
analysis. The resolved proteins were blotted with anti-
phosphotyrosine (PY20) antibody. Alternatively, the protein
bands were later re-blotted with the corresponding anti-ErbB
antibody. To test for receptor heterodimer formation, cell
lysates containing equal amounts of protein were immunopre-
cipitated with antibodies against ErbB1, ErbB4 receptor, or
phosphotyrosine (PY20) and detected using ErbB4 or ErbB1
receptor antibody, respectively.
We examined ERK phosphorylation as a downstream
marker of the MAPK cascade and Akt phosphorylation at
Ser473 as a downstream marker of the PI3K-Akt pathway
(Resjo
¨
et al., 2002). Since the doubly phosphorylated forms of
ERK (Payne et al., 1991) and Akt (Andjelkovic et al., 1999)
function as active enzymes, we used the phosphorylation of
these enzymes as a direct activation marker. To detect the
active form of ERK, protein was subjected to Western blot
analysis and detected using anti-phospho-p44/42 ERK anti-
body. To compare the level of ERK phosphorylation in EGF-
induced cells expressing ErbB1, ErbB4, and ErbB1–4, we
performed immunoprecipitation using anti-ERK and anti-
phospho-p44/42 antibodies for the same protein samples,
respectively, and placed the immunoprecipitates side by side
on the gel. After membrane transfer, both proteins were
detected using anti-ERK antibody and the band intensities
were quantified using a densitometer (Fuji Film Corp, Tokyo,
Japan). The ratio of phosphorylated-p44/42 ERK proteins to
total ERK proteins was calculated.
To detect the phospho-PKA substrate motif in B-Raf, B-Raf
was immunoprecipitated with the Catch-and-Release immu-
noprecipitation system (Upstate Biotechnology) according to
the manufacturer’s protocol and subjected to Western blot
analysis. Protein bands corresponding to B-Raf were detected
using phospho-PKA substrate motif antibody.
Rap1 pull-down assay
Rap1 activation assay was performed exactly as described
by Zwartkruis et al. (1998). Briefly, E1/4 cells were grown
to 70% confluency and serum-starved as described above.
Following EGF stimulation for 5 min, the cells were washed
twice with PBS and lysed in Ral-buffer (10% glycerol, 1%
Nonidet P-40, 50 m
M Tris, pH 7.4, 200 mM NaCl, 2.5 mM
MgCl
2
, protease inhibitors, 1 mM NaF, and 1 mM Na
3
VO
4
).
Lysates were clarified by centrifugation and the supernatants
were incubated with 15 mg GST-Ral GDS-RDB agarose
for 1 h to isolate Rap1. Agarose beads were washed three
times in Ral buffer and subjected to Western blot analysis.
Protein bands were detected using anti-Rap1 antibody. To
observe the effect of PLCg on Rap1 activation, 10 m
M U73122
was added to the incubation medium 10 min prior to the
addition of EGF. The band intensities were quantified by a
densitometer.
B-Raf immunoprecipitation and in vitro kinase activity assay
Cells were cultured in a serum-free medium and treated with
10 n
M EGF for 10 min with or without pre-incubation with the
enzyme inhibitor for 10 min, then lysed in a lysis buffer
containing 50 m
M HEPES (pH 7.5), 10 nM EDTA, 150 mM
NaCl, 10 mM sodium pyrophosphate, 2 mM sodium orthova-
nadate, 100 m
M NaF, 1% Triton X-100 (v/v), 100 U/ml
aprotinin, 20 m
M leupeptin, and 0.18 mg/ml PMSF. B-Raf
was immunoprecipitated using the B-Raf-specific antibody
with the Catch-and-Release immunoprecipitation system
according to the manufacturer’s protocol. The obtained
soluble B-Raf fraction was incubated with 1 mg of recombinant
MEK-1 and 160 m
M [g
32
-P] ATP (6000 Ci/mmol) in a kinase
buffer containing 20 m
M MOPS (pH 7.2), 500 mM ATP, 75 mM
MgCl
2
,25mM glycerophosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, and 1 mM dithiothreitol. After incubation of
the reaction mixture for 30 min at room temperature, the
B-Raf kinase reaction was stopped by the addition of
SDS–PAGE loading buffer and the proteins were subjected
to SDS–PAGE. The band corresponding to the
32
P-labeled
MEK protein was analysed using the BAS2000 system (Fuji
Film Corp., Tokyo, Japan).
Abbreviations
CHO, Chinese hamster ovary; EGFR, EGF receptor; EGF,
epidermal growth factor; HRG, heregulin; MAPK, mitogen-
activated protein kinase; MEK, extracellular signal-regulated
kinase kinase; ERK, extracellular signal-regulated kinase;
SH2, Src homology 2 domain; Grb2, growth factor receptor-
binding protein 2; Shc, Src homology and collagen domain
protein; PI3K, phosphatidylinositol 3
0
-kinase; PLCg, phos-
phoinositide-specific phospholipase C-g; PKA, protein kinase
A; PH domain, pleckstrin homology domain; PP2A, protein
phosphatase 2A.
Acknowledgements
We thank Ms Mihoro Saeki for constructing CHO cells
expressing ErbB1 and ErbB4 receptors. We also thank Dr
Takashi Naka for critically reading the manuscript and Mr
Akinobu Fukuzaki for preparing the manuscript.
ErbB heterodimer-specific B-Raf activation
M Hatakeyama et al
5030
Oncogene
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5031
Oncogene
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