Tài liệu Báo cáo khoa học: Synergistic activation of signalling to extracellular signal-regulated kinases 1 and 2 by epidermal growth factor and 4b-phorbol 12-myristate 13-acetate pptx

Synergistic activation of signalling to extracellular signal-regulated
kinases 1 and 2 by epidermal growth factor and 4b-phorbol
12-myristate 13-acetate
Jorrit J. Hornberg
1
, Marloes R. Tijssen
1
and Jan Lankelma
1,2
1
Department of Molecular Cell Physiology, Institute of Molecular Cell Biology, Faculty of Earth and Life Sciences, Vrije Universiteit,
Amsterdam, the Netherlands;
2
Department of Medical Oncology, VU Medical Center, Amsterdam, the Netherlands
Signal transduction pathways are often embedded in com-
plex networks, which result from interactions between
pathways and feedback circuitry. In order to understand
such networks, qualitative information on which inter-
actions take place a nd quantitative data o n their strength
become essential. Here, we have investigated how the mul-
tiple interactions between the mitogen-activated protein
kinase cascade and protein kinase C (PKC) affect the time
profile o f extracellular signal-regulated kinase (ERK) phos-
phorylation upon epidermal growth f actor (EGF) stimula-
tion in normal rat kidney fibroblasts. This profile i s a major
determinant for the cellular response that is evoked. We
found that EGF s timulation leads to a biphasic ERK-PP
pattern, consisting of an initial peak and a r elaxation to a low
quasi-steady state-phase. Costimulation with the EGF and
PKC activator, 4b-phorbol 12-myristate 13-acetate (PMA)
resulted in a similar p attern, but the ERK-PP concentration

in the quasi-steady state-phase was synergistically higher
than afte r stimulation with either EGF o r PMA only . This
resulted in prolonged signalling to ERK. PMA increased the
EGF concentration sufficient to obtain half-maximum ERK
phosphorylation. These data suggest that PKC amplifies
EGF-induced signalling t o E RK, w ithout incre asing its
sensitivity to l ow EGF c oncentrations. Furthermore, P KC
inhibition did not affect the ERK-PP time profile upon EGF
stimulation and a cellular phospholipase A2 (cPLA
2
)
inhibitor d id not decrease the synergistic effect of EGF and
PMA. This indicates that the positive feedback loop from
ERK to Raf via cPLA
2
and PKC does not contribute sig-
nificantly to signalling from EGF to ERK i n normal rat
kidney cells. Taken together, we provide a quantitative
description of which reported interactions in this network
affect the time p rofile of ERK phosphorylation.
Keywords:EGF;MAPK;PMA;signalingnetwork;syner-
gism.
The increase in knowledge of the building blocks of living
cells (genes, proteins) will stimulate t he development of
integrative biology [1,2]. Cellular signalling provides an
interesting platform for this integrative or Ôsystems
approachÕ. Many signalling proteins have been identified
and how they ÔcommunicateÕ with each other through signal
transduction pathways has b een extensively researched.
These pathways can interact at many levels (e.g. by direct

interaction of the molecules or by regulation o f gene
transcription), w hich gives rise to large signalling networks.
In order to fully understand how such networks operate, it is
necessary to integrate experimental d ata a nd to understand
how (qualitatively) and to w hat extent (quantitatively)
interactions in the network take place. By using biomathe-
matical models, p redictions can be made about the beha-
viour of si gnalling networks or, ultimately, of whole cells or
organisms [1,3–8].
Among the most intensively studied signal transduction
pathways are the mitogen-activated protein kinase (MAPK)
cascades, which are involved in many cellular processes,
such as proliferation, differentiation and apoptosis [9,10].
The mitotic MAPK pathway, via extracellular signal-
regulated kinase (ERK), c an be act ivated by various
extracellular stimuli, e.g. epidermal growth f actor (EGF),
which bind to dedicated receptors. Upon EGF binding, its
receptor (EGFR) dimerizes, leading to autophosphoryla-
tion of tyrosine residues on the cytoplasmic domain of the
receptor, thereby creating docking sites for adaptor pro-
teins, such as Shc and G rb2. The latter protein recruits Sos
to the plasma membrane, which causes the activation of
Ras by exchanging GDP, bound to Ras, for GTP [11–13].
Ras-GTP can bind cytoplasmic Raf1 leading to its
Correspondence to J. Lankelma, Department of Molecular Cell
Physiology Faculty of Earth and Life Sciences Vrije Universiteit
Amsterdam, De Boelelaan 1085 1081 HV Amsterdam, the Nether-
lands. Fax: +31 20 4447229, Tel.: +31 20 4447248,
E-mail:
Abbreviations: ATK, arachidonyl trifluoromethylketone; cPLA2, cel-

lular phospholipase A2; DAG, diacylglycerol; DMEM, Dulbecco’s
modified Eagle’s medium; EGF, epidermal growth factor; EGFR,
EGF receptor; ERK, extracellular signal-regulated kinase; ERK-PP,
doubly phosphorylated ERK; Grb2, growth factor receptor binding
protein 2; IP3, inositol triphosphate; MAPK, mitogen-activated pro-
tein kinase; MEK, MAPK/ERK kinase; MKP, MAPK phosphatase;
NRK, normal rat kidney; PDGF, platelet-derived growth factor;
PKC, protein kinase C; PMA, 4b-phorbol 12-myristate 13-acetate;
PLC-c, phospholipase C-g; Shc, Src homology and collagen domain
protein; TBS, Tris-buffered saline.
Note: a website is available at h ttp://www.bio.vu.nl/vakgroepen/mcp/
(Received 2 2 June 2004, accepted 6 August 2004)
Eur. J. Biochem. 271, 3905–3913 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04327.x
phosphorylation and activation. Although R af1 c an be
phosphorylated by many kinases, the exact mechanism by
which it is activated after EGF stimulation is not entire ly
clear [14]. Subsequently, Raf1 phosphorylates MAPK/ERK
kinase (MEK) 1 and 2, which in turn phosphorylate ERK1
and ERK2. Phosphorylated ERK (ERK-PP) has several
different cytoplasmic and nuclear targets. Transcription
factors a ctivated by ERK-PP that indu ce expression of
genes involved in cell cycle progression include Elk1, c-fos,
c-Jun and c-myc [9,15]. The duration of ERK activation
(transient or sustained) determines the repertoire of target
genes expressed [16], and also affects the type of cellular
response that is evoked [17,18]. Activation of ERK is
required for proliferation of fibroblasts [19] and constitutive
ERK activation frequently occurs in human primary
tumours and tumour cell lines [20]. The latter is often
caused by mutations in the genes encoding the constituents

of the p athway, such as Ras21, rendering them over-
activated. Signalling pathways are generally not simple
linear chains, but have several feedback mechanisms and
cross-reactivity with other signal t ransduction pathways
[10], w hich may lead t o e mergent properties such as
sustained oscillations and bistability [4,8,22].
We have investigated the interaction between the ERK
cascade and protein kinase C (PKC). PKC is also
involved in processes like proliferation, differentiation and
cell death [23]. Several different interactions between these
signal transduction modules have been reported. PKC
can directly activate the MAPK pathway by phosphory-
lating Raf [24–26]. It has also been implicated in a
positive feedback loop of the MAPK pathway [4,8].
Therein, ERK-PP phosphorylates cytosolic phospholipase
A
2
(cPLA
2
) [27], causing the release of arachidonic acid,
which, together with calcium and diacylglycerol (DAG),
activates PKC (reviewed in [28]). Furthermore, PKC c an
phosphorylate E GFR [ 29]. T his inhibits tyrosine kinase
activity of the receptor and causes the decrease of EGF
binding affinity [30–32]. It also results in diversion of the
internalized EGFR from the regular degradative pathway
to the recycling endosome [33]. EGFR is also capable of
signalling to PKC, via phospholipase C -c (PLC -c)
phosphorylation. PLC- c catalyses the production of
inositol triphosphate (IP

3
) a nd DAG. IP
3
brings about
calcium release, which together with DAG activates PKC
(reviewed in [ 28]). Taken together, all t hese interactions
constitute a very complex signalling network (Fig. 1). We
hypothesized that this network is cap able of quasi-
intelligent behaviour, for instance by making the output
(ERK phosphorylation) dependent on the integration of
two signal inputs. Therefore, we measured signalling t o
ERK after stimulation with EGF, PKC activator,
4b-phorbol 12-myristate 13-acetate (PMA) and a combi-
nation of both. We show that, when both signal inputs
were given simultaneously, ERK phosphorylation was
synergistically activated, leading to a prolonged active
quasi-steady state. Furthermore, we determined the
relative qua si-steady state-concentrations of ERK-PP at
different EGF concentrations and f ound that PKC not
only affects the maximum level of the stimulus-response
curve, but surprisingly also causes an increase in the EGF
concentration sufficient for half-maximum ERK phos-
phorylation.
Experimental procedures
Cell culture
Normal rat k idney ( NRK) fibroblasts were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Biowhit-
taker Europe), supplemented with 10% (v/v) foetal
bovine serum (FBS, Gibco), 100 lgÆmL
)1

penicillin and
100 lgÆmL
)1
streptomycin in a humidified 5% (v/v) CO
2
incubator at 3 7 °C. For s erum-starvation, cells were
washed once with 1· Hank’s buffered salt solution
(Gibco) and cultured in DMEM, supplemented with
0.5% (w/v) BSA, (AppliChem), 1 00 lgÆmL
)1
penicillin
and 100 lgÆmL
)1
streptomycin.
Stimulation experiments
Cells grown i n culture dishes (fo r Western blot analysis) or
on glass cover slips (for immunocytochemistry) to subcon-
fluency were serum-starved for 3 days in order to be
arrested in the G
0
-phase of the cell cycle. Cells were
stimulated with various concentrations of EGF (Becton
Dickinson) and/or PMA (Calbiochem) f or different periods
of time as indicated. PKC was inhibited by preincubation
with 5 l
M
bisindolylmaleimide I (also referred to as
GF109203X; Calbiochem) for 1 h [ 34] and c PLA
2
was

inhibited by preincubation for 1 h with 10 l
M
arachidonyl
Fig. 1. The complex str ucture of the signalling network to ERK.
Depicted are t he MAPK and PKC sign alling modules (blue a nd yellow
boxes, r esp ective ly). A c tivated EGFR signals via Ras through the
MAPK cascade t o ERK, which leads to the activation of various
transcription factors (TFs). EGFR can a lso activate PKC through
PLCc. These two signalling modules communicate with each other via
several m echanisms: (a) E RK activates cPLA
2
, which releases arachi-
donic acid (AA) that, togethe r with calcium, can ac tivate PKC; (b)
PKC d irectly phosphorylates Raf; (c) PKC also p hospho rylates
EGFR. Internalized EGFR (iEGFR), although still capable of sig-
nalling t o Ras, is n ormally degraded over t ime [57]. PKC-mediated
phosphorylation of iEGFR causes it to recycle back to the cell su rface.
In addition it cau ses a decreased EGF bin ding affinity and tyrosine
kinase activity of the r eceptor. Also shown are the stimulators and
inhibitors(depictedinred)usedinthisstudy.EGFwasusedtoactivate
the EGFR, PMA to activate P KC. B isindolylmaleimide was used to
block P KC activity an d ATK t o block cPLA
2
activity.
3906 J. J. Hornberg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
trifluoromethylketone (ATK) [35] or 4-bromophenacyl
bromide [36].
Western blot analysis
After stimulation, cells were washed twice with i ce-cold
phosphate-buffered saline (NaCl/P

i
;17m
M
NaH
2
PO
4
,
38.5 m
M
Na
2
HPO
4
,68m
M
NaCl, pH 7.4) and incubated
on ic e with lysis buffer [10 m
M
Tris/HCl, pH 7.5, 150 m
M
NaCl, 0.1% (v/v) SDS, 0.1% (v/v) octylphenolpoly(ethylene
glycolether) (Nonidet P40), 0.1% (w/v) sodium deoxycho-
late, 50 m
M
NaF, 1 m
M
Na
3
VO

4
,1· Complete protease
inhibitor mix (Roche)] for 20 min. Cell l ysates were scraped
in lysis buffer using a cell scrape r (25 cm/1.8 cm, Costar),
collected, v ortexed for 10 s, frozen in liquid nitrogen and
stored at )80 °C. Protein contents in the cell lysates were
determined with the bicinchoninic a cid assay (Pierce).
Proteins were separated by SDS/PAGE. For each sample,
exactly 10 lg of total protein w as loaded on the gel in
loading buffer (250 m
M
Tris/HCl, pH 7.6, 8 % (w/v) SDS,
40% (v/v) glycerol, 0.05% (w/v) b romophenol blue, 2 0 m
M
dithiothreitol). Proteins were electrotransferred to Immuno-
Blot
TM
poly(vinylidene difluoride) membranes (Bio-Rad)
using 400 mA overnight at 4 °C. Membranes were w ashed
in Tris-buffered saline (TBS: 2 0 m
M
Tris/HCl, p H 7.6,
150 m
M
NaCl) s upplemented w ith 0 .05% (v/v) Tween-80
(TBS-T), preincubated for 1 h at r oom temperature w ith
blocking buffer [5% (w/v) skimmed milk powder (Oxoid) in
TBS-T], supplemented with 0.5 m
M
Na

3
VO
4
, and incu bated
overnight at 4 °C with mo noclonal mouse anti-(phospho-
p42/44 MAP kinase) Ig (Cell Signalling) in blocking buffer
(1 : 2000), supplemented with 0.5 m
M
Na
3
VO
4
.After
washing, membranes were incubated for 1 h at room
temperature with horseradish peroxidase-conjugated goat
anti-(mouse IgG) Ig (Bio-Rad) in blocking buffer (1 : 3000).
Membranes w ere washed a gain a nd then incubated for
5 m in with Lumi-Light
PLUS
Western Blotting Substrate
(Roche). Signals were detected with a FluorS
TM
MultiI-
mager (Bio-Rad) and quantified using the
MULTI
-
ANALIST
software (Bio-Rad). All measurements were performed in
the linear detection range of this method.
All time c urves for Fig. 2 were m easured in i ndepend-

ent experiments, and as we wanted to compare them to
each other and calculate the stan dard errors for each time
point, the individual time curves had to be scaled to each
other. Scaling the curves with re spect to the m aximal
ERK-PP concentration was not possible, as, due to the
relatively dynamic nature of the curve around this time
point, the maximal ERK-PP concentration measured for
a certain time curve could not be exactly equal to the ÔrealÕ
maximum that is reached in the cells. The maximal
ERK-PP concentration measured for independent curves
can therefore differ and scaling the whole curve to this
time point would introduce errors at other time points. To
avoid such problems, we chose to use all time points to
scale all curves to one (arbitrarily chosen) ÔrepresentativeÕ
curve. Therefore, we applied a multivariate least squares
approximation [37,38] of the type Y ¼ X b +e,inwhich
Y is the representative curve, X is the m atrix of all other
curves (which were treated similarly), b represents the
regression coefficients (one for each curve X)ande is the
error vector (zero mean, common variance [37,38]). For
the t hree different conditions [stimulated with (a) EG F
(b) PMA or (c) EGF + (PMA)], the scaled time curves
were drawn ( i.e. Xb) in Fig. 2 . The s tandard errors were
calculated as described previously [39].
Immunocytochemistry
After stimulation, cells were washed twice w ith ice-cold
NaCl/P
i
, fixed by i ncubation for 30 min at 4 °Cwithice-
cold 4% (v/v) paraformaldehyde in NaCl/P

i
and washed
Fig. 2. Biphasic ERK-PP time profile induced by EGF or PMA alone and synergistic ERK phosphorylation induced by EGF and PMA together. Cells
were serum-starved for three days and subsequently stimulated for the indicated t imes ( x-axis) with 10 ng ÆmL
)1
EGF (n), 100 n
M
PMA (h) or both
EGF and PMA (s). Cells were harvested a nd ERK-PP was m easured in the cell lysates by quantitative Western blotting. EGF or PMA stimulation
leads to a biphasic time profile, with a high peak that decreases to a low quasi-steady state-level. EGF and PMA costimulation leads to synergistic
ERK phosphorylation in this second phase. The curves shown are the result of five ind epende nt experiments, that were scaled to each other u sing a
multivariate l east squares approximation (see E xperimental procedures). E rror bars represent the standard error o f the me an.
Ó FEBS 2004 Synergistic ERK activation by EGF and PMA (Eur. J. Biochem. 271) 3907
with TBS-Triton (TBS, supplemented with 0.1% (v/v)
Triton X-100). Cells were then incubated with 100% (v/v)
methanol for 10 m in at )20 °C in order to permeabilize
cellular membranes and w ashed. C ells were then incubated
for 1 h a t room temperature with 5% FBS in TBS-Triton
and subsequently incub ated overnight at 4 °C with mono-
clonal mouse anti-(phospho-p42/44 MAP kinase) Ig (Cell
Signalling) in 5% (w/v) BSA in TBS-Triton (1 : 400). C ells
were washed for 15 min with TBS-Triton, for 15 min with
0.1% (w/v) BSA in TBS-Triton and incu bated for 2 h at
room temperature with Cy
5
TM
-labeled g oat anti-(mouse Ig)
(Amersham) in 3% (w/v) BSA in TBS/Triton (1 : 400).
Next, cells were washed with TBS/Triton and then with
demi-water. The glass slides were air-dried, inversely p laced

in Vectr ashield for fluorescence (Vector) on a microscope
slide and stored in the d ark at 4 °C. Fluorescence was
detected using a confocal scanning laser microscope (Leica
TCS 4D). A krypton-argon laser line (647 nm) w as used for
excitation of the Cy
5
TM
-label, and a long pass filter (665 nm)
was used f or detection o f the emitted light (with b eam
splitter at 660 nm). Obtained images were quantified using
the
SCION IMAGE
software (Scion Corporation).
Results
Biphasic time profile of ERK-PP by EGF or PMA
stimulation
We first determined the dynamic profile of phosphorylated
ERK a fter stimulation w ith E GF in NRK fibroblasts by
quantitative Western blotting. Cells were serum-starved and
subsequently stimulated with 10 ngÆmL
)1
EGF for various
periods of time. W e observed a biphasic ERK-PP p rofile
(Fig. 2 ). Upon EGF s timulation, the ERK-PP concentra-
tion rose from a b ackground level t o a high peak
concentration after about 4 min and then returned to the
prestimulation level (after about 12 min) before increasing
slightly again. This second in crease was f ollowed by a
decrease to a relatively low level, after about 1 h of EGF
stimulation. This ERK-PP profile suggests t he possibility of

damped oscillatory behaviour, c onsistent with co mplex
behaviour of the complex circuitry r egulating ERK-PP.
We also determ ined the E RK phosphorylation d ynamics
induced by PKC activation by addition of 100 n
M
PMA to
serum-starved NRK cells. The profile resembled that
induced by EGF s timulation (Fig. 2). After about 4 min,
a peak c oncentration was reached, followed b y a rapid
decline to a very low concentration that sustained for several
hours. We refer t o this as a quas i steady-state, a s the ERK-
PP concentration remains at approximately the same level
for a relatively long period of time (compared to the time
that was needed to attain this concentration). T he first peak
concentration induced by PMA was always lower than t hat
induced by EGF.
EGF and PMA activate signalling to ERK synergistically
In order to determine whether the different signal inputs t o
ERK (via E GFR a nd via PKC) affect each other, we
stimulated serum-starved NRK cells with both 10 ng ÆmL
)1
EGF and 100 n
M
PMA and again determined the time
profile of the ERK-PP concentration (Fig. 2). W e observed
a biphasic pattern, with the first high peak being identical
to that obtained during stimulation by EGF alone. After
this first peak, the ERK-PP concentration reaches a quasi-
steady state-concentration of 2–3· the sum of the concen-
tration obtained after 1 h of stimulation with only EGF

and the concentration obtained with PMA only. Appar-
ently, EGF a nd PMA act synergistically on the quasi-
steady state-phase of t he profile, but not on the initial peak.
As the individual p eak shapes may have been lost during
averaging of the c urves, we t itrated EGF a t a fixed time
point of 60 min in order to measure accurately the
synergistic effect.
EGF concentration-dependency of synergistic activation
To investigate the synergistic activation i n the second phase
of the time profile further, w e measured t he ERK-PP
concentration after 1 h stimulation with different EGF
concentrations (ranging from 0 to 100 ngÆmL
)1
), both in the
absence and presence of 100 n
M
PMA. The results (Fig. 3)
show that the (quasi-stead y state) E RK-PP concentration
depends on the EGF concentration used a nd reaches a
maximal level. After stimulation with PMA alone, the
Fig. 3. Stimulus–response curves of ERK-PP to EGF. ERK-PP was
measured by quantitative Western blotting in cell lysates that were
harvested after 1 h of EGF-stimulation with the indicated concentra-
tions, in the absence ( n)orpresence(m) of PMA. T he data are
averages of four independent experiments, the error bars represent the
standard error of the mean. The drawn lines represent the curve fits
that were ob tained using a Michaelis–Menten type equ ation (Eqn 1).
The fitting parameters are shown in the table inset (th eir standard
deviations are i ndicated betwe en brackets). [ERK-PP]
basal

:theERK-
PP concentration without EGF present; [ERK-PP]
max
: the maximu m
steady state ERK-PP concentration th at can be induced by EGF; K,
EGF concentration needed to ob tain the half-maxim um ERK-PP
concentration. In additio n, a representative image o f the immunob lots
is depicted.
3908 J. J. Hornberg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
ERK-PP concentr ation was similar to that obtained with
high EGF concentrations, but when PMA and EGF were
added simultaneously, i t reached a m uch higher level, again
in an EGF concentration-dependent manner (Fig. 3). To
draw a stimulus–response curve, we fitted these data points
to the following equation (cf. the Michaelis–Menten equa-
tion):
½ERK-PP
steady state
¼½ERK-PP
basal
þ
½ERK-PP
max
½EGF
K þ½EGF
ðEqn 1Þ
[ERK-PP]
basal
is the ERK-PP concentration in s erum-
starved cells befo re EGF addition , [ERK-PP]

max
is the
maximum concentration of ERK-PP in the quasi-steady
state-phase (after 1 h of stimulation) and K is the EGF
concentration at which ERK-PP is 50% of its maximal level.
Addition of PMA r esulted in a two- to threefold increase of
the a dditive [ERK-PP]
max
, reflecting the synergistic activa-
tion. Interestingly, K with respect to EGF w as also remark-
ably higher when PMA was present. This indicates that,
when PKC is activated, the signalling pathways to ERK still
respond to EGF at higher growth factor concentrations
while, without PMA, they are saturated at EG F concentra-
tions above 1 ngÆmL
)1
. The synergistic effect is only found in
the concentration r ange above 1 ngÆmL
)1
.
Qualitative visualization of ERK-PP in fixed cells
In addition to the quantitative measurement of ERK-PP by
Western blotting, we qualitatively visualized ERK-PP in
fixed NRK fibroblasts by immunocytochemistry. Cells were
serum-starved a nd stimulated for 1 h with 100 ngÆmL
)1
EGF, 100 n
M
PMA or both, after which ERK-PP was
stained w ith a fluorescent labe l and visualized using laser

scanning microscopy (Fig. 4A), as described in Experimen-
tal procedures. The fluorescent signal per image was, after
subtracting t he background, divided by the number of cells
in the picture. T he ave rage signal intensities of four images
showed that EGF or PMA stimulated cells were compar-
able to untreated cells, whereas cells treated with both
stimulators showed a considerably higher signal intensity
(Fig. 4 B), which is consistent wi th the results discussed i n
the previous section.
Positive feedback circuit via cPLA
2
and PKC is not
involved in EGF-mediated ERK phosphorylation
in NRK cells nor in the synergistic activation
According t o schemes available in t he literature, after E GF
stimulation, PKC may be activated via P LC-c and, via the
positive feedback loop, by cPLA
2
(Fig. 1). To monitor the
effect of PKC on ERK phosphorylation, we stimulated
Fig. 4. Qualitative visualization of synergistic ERK phosphorylation by EGF and PMA using immunofluorescence and quantification of the
immunostaining. (A) ERK-PP was detected with a fluorescent label in fixed cells (for details see Experimental procedures) that were unstimulated
(control) or s timulated f or 1 h with 100 ng ÆmL
)1
EGF, 100 n
M
PMA or both and detected using a scanning laser microscope. Representative
images of four independent experiments are shown. (B) Quantification o f the immunostainin g. The average fluorescent signal per cell in four
independent e xperiments is depicted; the error bars represent t he s tandard e rror o f the mean . P lease not e that t he met hod app lie d prod uces a
relatively high background, which hampers the quantific ation. The EGF and PMA costimulation produces a signal that sign ificantly emerges from

this background, whereas stimulation with e ither EGF or PMA only d oes not.
Ó FEBS 2004 Synergistic ERK activation by EGF and PMA (Eur. J. Biochem. 271) 3909
NRK cells with EGF, also in the presence of the PKC
inhibitor bisindolylmaleimide I, and determined the ERK-
PP concentration after 6 and after 60 min. In three
independent experiments, we could not find an inhibitor-
induced change in the E RK-PP concentration, neither in
terms of the initial peak nor with respect to the quasi-steady
state-phase (Fig. 5). Bisindolylmaleimide I completely abol-
ished ERK phosphorylation after PMA s timulation, indi-
cating that PM A does not have a PKC-independent effect
on ERK-PP. The ERK-PP concentration induced by
costimulation with both EGF and PMA was unaffected
by PKC i nhibition in the early peak and w as reduced in the
quasi-steady state-phase to a level comparab le to that after
stimulation with EGF only. These r esults show that in our
system, P KC had no s ignificant e ffect o n ERK phosphory-
lation after E GF stimulation. This implies that t he positive
feedback loop via cPLA
2
, which caused sustained ERK
phosphorylation upon stimulation with p latelet-derived
growth factor (PDGF) [8], was not activated by EGF
stimulation in these cells. To investigate this further, we
blocked cPLA
2
activity by the inhibitor ATK. The ERK-PP
concentration again did not decline, even if the stimulation
was carried out with EGF and PMA t ogether (Fig. 5).
Similar results were obtained with 4-b romophenacyl bro-

mide, which is a different, general PLA
2
inhibitor (results
not shown). This shows that the positively regulating circuit
is not involved in the s ynergistic ERK phosphorylation
caused by EGF and PMA.
Discussion
The architecture of signal transduction networks i s often
highly complex, due to the large number of participating
protein co mplexes, cross interactions between pathways and
the functioning of regulatory circuits. It is this complexity
that makes th e understanding of cellular signalling a
difficult task. For example, the features o f a whole network
cannot be understood simply as the sum of features of its
parts; the network as s uch may give rise t o s ystem o r
Ôemergen tÕ properties [4,40]. To obtain reliable computer
models that can calculate the outcome of signalling e vents,
the interactions between signalling m odules nee d to be
experimentally measured in a quantitative manner [3,7].
We have assessed the output of the EGF-activated
MAPK pathway (ERK1/2) and its cross-talk with protein
kinase C. We have measur ed the dynamic time profile of
ERK phosphorylation a fter stimulation by EGF, and
observed a biphasic pattern consisting of a first rapid peak
and relatively l ow and a broad s econd peak developing into
what we refer to as a quasi-steady state. The first peak has
been described by o thers in m any cell types, but a biphasic
pattern, that could be attributed to the existence of damped
oscillations, seems to have escaped experimental resolution
thus far. Sustained oscillations have been predicted in a

theoretical study, in which they were explained by a
combinatory effect of negative feedback and ultrasensitivity
[22]. Both of these features have been demonstrated
experimentally. Negative feedback is constituted by the
phosphorylation of Sos by ERK, which c auses Sos to
dissociate from the growth factor receptor complex. Thus,
as the local Sos concentration at the inner s urface of the
membrane decreases, Ras activation is impaired as well as
subsequent activation of downstream signalling m olecules
such as ERK [41]. Ultrasensitivity has been demonstrated in
oocyte extracts, showing a steep stimulus/response curve for
ERK-PP as a function of activated Raf (Hill coefficient:
4–5), which led to the suggestion that the pathway was
equipped to filter out noise a nd behave as an Ôon/off-switchÕ
[42]. The biphasic pattern we o bserve h ere may reflect
sustained oscillations that are i nitially synchronized in all
cells in the culture but that become desynchronized over
time. Alternatively, damping may be c aused by MAPK
phosphatases (MKPs) that are up-regulated within
% 30 min after initial MAPK s ignalling [43]. Independent
Fig. 5. The positive feedback loop via cPLA
2
and PKC plays no significant role in ERK
phosphorylation. Cells were serum-starved for
3 d ays and t hen stimulated for either 6 or
60 min with EGF or PMA in the absence or
presence of the PKC inhibitor Bisindolyl-
maleimide I (5 l
M
; 1 h preincubation) or the

cPLA
2
inhibitor A TK (10 l
M
;1hpreincu-
bation). ERK-PP was measured i n the ce ll
lysates by q uantitative Western blotting. The
PKC inhibitor abolished PMA-induced ERK
phosphorylation, whereas EGF-in duced ERK
phosphorylation was unaffected. The ERK-
PP concentration induced by EG F and PMA
costimulation declined only in the quasi-
steady st ate-phase. In hibition of cPLA
2
did
not affect the peak o r the quasi-steady state-
phase. S hown are the mean results o f three
independent experiments, the erro r bars r ep-
resent the s tandard error of the mean.
3910 J. J. Hornberg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
of its precise mechanism, the transient oscillation is one
aspect of complexity that is observed in the dynamics of this
signal transduction chain.
PKC was not found to be involved in the EGF-mediated
ERK phosphorylation, bu t activation of PKC by PMA did
result in a transient ERK-PP profile. Although phorbol
ester receptors other than PKC have been reported [42,44],
we have shown that these do not affect signalling to ERK in
NRK cells, as PMA did not induce ERK phosphorylation if
PKC had been inhibited by b isindolylmaleimide I.

Simultaneous stimulatio n w ith PMA and E GF yielded a
remarkably synergistic effect in the quasi-steady s tate phase
of the time profile. In terestingly, in the presence of P MA,
the ERK-PP quasi-steady state-concentration c ould still be
elevated further by EGF concentrations above 1 ngÆmL
)1
,
whereas t his w as not the case in the absence of P MA
(Fig. 3). Furthermore, the ratio betw een the ERK-PP
concentration after stimulation with EGF and PMA
together on the one hand, and the sum of the ERK-PP
concentrations after stimulation with EGF and PMA
separately on the other hand, was not constant, but
increased with the EGF concentration, which confirmed
synergistic activation. In osteoblastic cells, PMA did not
affect EGF s ignalling [43,45], indicating that the response i s
probably cell type-dependent. It h as been reported previ-
ously that ERK1 was synergistically activa ted i n h amster
fibroblasts after stimulation with serotonin and basic
fibroblast growth f actor f or 120 min [46]. In platelets,
pretreatment with thrombopoietin and stimulatio n with
a-thrombin led to a 30% phosphorylation of ERK2 in
the early phase (1–5 min), whereas thrombopoietin or
a-thrombin alone activated E RK2, either, not at all or t o a
very low degree, respectively [47]. In another study,
simultaneous stimulation o f human embryon ic k idney cells
with carbachol, which activates PKC via a G-protein
coupled receptor, and EGF, did not exert additive effects on
ERK activity a fter 10 min [48], which supports our finding
that synergism i s not present in the initial peak (Fig. 2). We

believe the latter is caused by s aturation of the phosphory-
lation of all cellular ERK. Indeed, it h as been shown
previously that in NRK c ells, this EGF concentration
causes virtually all ERK to become doubly phosphorylated
([49], and our unpublished observation].
As to what molecular interactions underlie the observed
synergism, we have obtained some indications. We have
excluded the possibility that the synergism arises from a
positive feedback loop via cPLA
2
andPKC.Infact,cPLA
2
inhibition did not alter the ERK-PP concentration upon
EGF and PMA costimulation. Recently, this loop was found
active after PDGF stimulation in NIH-3T3 cells, r esulting in
prolonged ERK phosphorylation [8]. The synergism we find
here might arise at a site where the two signal inputs
converge, for instance at Raf. PKC phosphorylates Raf at
Ser499, which was s uggested to cause Ser259 autophos-
phorylation and activation [24]. Ser259 was also identified as
a major Raf phosphorylation s ite upon growth factor
stimulation [50]. The synergism might also originate up-
stream of Sos, as ERK activation by P KC has been shown to
depend both on Sos51 and on Ras-GTP-Raf complexes [52].
A different explanation could be that P KC has a n
inhibitory effect on one of the down-regulating mechanism s
of the pathway from EGF to ERK. One possibility could be
that EGFR down-regulation is affected by PKC. EGFR
phosphorylation on Thr654 by PKC has been shown to
cause recycling of internalized EGFR to the cell s urface,

instead of degradation [33], a nd EGF might then caus e a
second, prolonged ERK phosphorylation phase. This could
also explain why, during EGF and PMA costimulation,
ERK-PP rises again after returning to a low level. On the
other hand, the s ame phosphorylation of EGFR by PKC is
known to cause decreased EGFR tyrosine kinase activity,
its binding affinity for EGF [30–32] and mitogenic signalling
[53]. In fact, we show in this study that activated PKC,
although amplifying the E GF signal to ERK, also incr eases
the EGF concentration that is needed for half-maximum
ERK phosphorylation (Fig. 3). Clearly, con trol of PKC-
mediated EGFR phosphorylation on signalling to ERK is
distributed over these processes. Another, more speculative,
candidate target could be a n MKP which is up-regulated
about 30 min after ERK activation [43]. The activity of
MKPs is known to be regulated by phosphorylation, both
positively and negatively [54,55]. If PKC can inactivate an
MKP that is up-regulated after stimulation with E GF, this
may lead to sustained ERK phosphorylation.
In conclusion, we have investigated the interaction
between the MAPK and PKC s ignalling m odule s in a
quantitative manner and found that they affect each other in
multiple ways. EGF stimulation a nd PKC activation caused
a synergistic a ctivation o f ERK in the quasi-steady state-
phase. PKC here acts as a signal a mplifier for growth factor
signalling. We found no evidence for the functioning of a
positive feedback mechanism via cPLA
2
. The interactions
between MAPK and PKC apparently serve to facilitate

quasi-intelligent signal i ntegration, w hich m ay be necessary
to assure that certain responses are induced only when more
than one criterion needs to be met. DNA synthesis was
shown previously to be synergistically activated by fibro-
nectin and insulin [56]. As the duration of ERK signalling
influences the repertoire of influenced target genes [ 16] and
the cellular response [17,18], we h ypothesize that the
synergistic ERK phosphorylation, which results in pro-
longed signalling, has i mplications for the outcome of
signalling. This may be of i mportance in the constitutive
ERK activation often found in hu man tumour cells.
Acknowledgements
We thank W.P.H. de Boer and J.A. Ferreira for statistical advice.
G.S.A.T. van Rossum is indebted for the kind gift of the cPLA
2
inhibitors ATK and 4-bromophenacyl bromide and advice on the
manuscript. We are thankful to F.J. Bruggeman for stimulating
discussions and advice on the manus cript. W e also thank K. Krab an d
H. Dekker for excellent technical advice on curve fitting and
immunofluorescence microscopy, r esp ectively.
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