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

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: [email protected]
Abbreviations: ATK, arachidonyl trifluoromethylketone; cPLA2, cel-

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

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

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

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-

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

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

: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]

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

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

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.

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,

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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