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 of extracellular signal-regu
Trang 1Synergistic activation of signalling to extracellular signal-regulated
kinases 1 and 2 by epidermal growth factor and 4b-phorbol
12-myristate 13-acetate
Jorrit J Hornberg1, Marloes R Tijssen1and Jan Lankelma1,2
1
Department of Molecular Cell Physiology, Institute of Molecular Cell Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, the Netherlands;2Department 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 and quantitative data on 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 of extracellular signal-regulated kinase (ERK)
phos-phorylation upon epidermal growth factor (EGF)
stimula-tion in normal rat kidney fibroblasts This profile is a major
determinant for the cellular response that is evoked We
found that EGF stimulation leads to a biphasic ERK-PP
pattern, consisting of an initial peak and a relaxation 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 pattern, but the ERK-PP concentration
in the quasi-steady state-phase was synergistically higher
than after stimulation with either EGF or 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 to ERK, without increasing its sensitivity to low EGF concentrations Furthermore, PKC inhibition did not affect the ERK-PP time profile upon EGF stimulation and a cellular phospholipase A2 (cPLA2) inhibitor did not decrease the synergistic effect of EGF and PMA This indicates that the positive feedback loop from ERK to Raf via cPLA2and PKC does not contribute sig-nificantly to signalling from EGF to ERK in normal rat kidney cells Taken together, we provide a quantitative description of which reported interactions in this network affect the time profile of ERK phosphorylation
Keywords: EGF; MAPK; PMA; signaling network; syner-gism
The increase in knowledge of the building blocks of living
cells (genes, proteins) will stimulate the development of
integrative biology [1,2] Cellular signalling provides an
interesting platform for this integrative or systems
approach Many signalling proteins have been identified
and how they communicate with each other through signal
transduction pathways has been extensively researched These pathways can interact at many levels (e.g by direct interaction of the molecules or by regulation of gene transcription), which gives rise to large signalling networks
In order to fully understand how such networks operate, it is necessary to integrate experimental data and to understand how (qualitatively) and to what extent (quantitatively) interactions in the network take place By using biomathe-matical models, predictions can be made about the beha-viour of signalling 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), can be activated by various extracellular stimuli, e.g epidermal growth factor (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 Grb2 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: j.lankelma@vumc.nl
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 http://www.bio.vu.nl/vakgroepen/mcp/
(Received 22 June 2004, accepted 6 August 2004)
Trang 2phosphorylation and activation Although Raf1 can be
phosphorylated by many kinases, the exact mechanism by
which it is activated after EGF stimulation is not entirely
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 activated by ERK-PP that induce 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 pathway, 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 transduction pathways
[10], which may lead to emergent 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
A2 (cPLA2) [27], causing the release of arachidonic acid,
which, together with calcium and diacylglycerol (DAG),
activates PKC (reviewed in [28]) Furthermore, PKC can
phosphorylate EGFR [29] This 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 (IP3) and DAG IP3 brings about
calcium release, which together with DAG activates PKC
(reviewed in [28]) Taken together, all these interactions
constitute a very complex signalling network (Fig 1) We
hypothesized that this network is capable of
quasi-intelligent behaviour, for instance by making the output
(ERK phosphorylation) dependent on the integration of
two signal inputs Therefore, we measured signalling to
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 quasi-steady state-concentrations of ERK-PP at
different EGF concentrations and found 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 kidney (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)1streptomycin in a humidified 5% (v/v) CO2 incubator at 37C For serum-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), 100 lgÆmL)1 penicillin and 100 lgÆmL)1 streptomycin
Stimulation experiments Cells grown in culture dishes (for 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 G0-phase of the cell cycle Cells were stimulated with various concentrations of EGF (Becton Dickinson) and/or PMA (Calbiochem) for different periods
of time as indicated PKC was inhibited by preincubation with 5 lM bisindolylmaleimide I (also referred to as GF109203X; Calbiochem) for 1 h [34] and cPLA2 was inhibited by preincubation for 1 h with 10 l arachidonyl
Fig 1 The complex structure of the signalling network to ERK Depicted are the MAPK and PKC signalling modules (blue and yellow boxes, respectively) Activated EGFR signals via Ras through the MAPK cascade to ERK, which leads to the activation of various transcription factors (TFs) EGFR can also activate PKC through PLCc These two signalling modules communicate with each other via several mechanisms: (a) ERK activates cPLA 2 , which releases arachi-donic acid (AA) that, together with calcium, can activate PKC; (b) PKC directly phosphorylates Raf; (c) PKC also phosphorylates EGFR Internalized EGFR (iEGFR), although still capable of sig-nalling to Ras, is normally degraded over time [57] PKC-mediated phosphorylation of iEGFR causes it to recycle back to the cell surface.
In addition it causes a decreased EGF binding affinity and tyrosine kinase activity of the receptor Also shown are the stimulators and inhibitors (depicted in red) used in this study EGF was used to activate the EGFR, PMA to activate PKC Bisindolylmaleimide was used to block PKC activity and ATK to block cPLA 2 activity.
Trang 3trifluoromethylketone (ATK) [35] or 4-bromophenacyl
bromide [36]
Western blot analysis
After stimulation, cells were washed twice with ice-cold
phosphate-buffered saline (NaCl/Pi; 17 mM NaH2PO4,
38.5 mM Na2HPO4, 68 mM NaCl, pH 7.4) and incubated
on ice with lysis buffer [10 mMTris/HCl, pH 7.5, 150 mM
NaCl, 0.1% (v/v) SDS, 0.1% (v/v) octylphenolpoly(ethylene
glycolether) (Nonidet P40), 0.1% (w/v) sodium
deoxycho-late, 50 mM NaF, 1 mM Na3VO4, 1· Complete protease
inhibitor mix (Roche)] for 20 min Cell lysates were scraped
in lysis buffer using a cell scraper (25 cm/1.8 cm, Costar),
collected, vortexed for 10 s, frozen in liquid nitrogen and
stored at)80 C Protein contents in the cell lysates were
determined with the bicinchoninic acid assay (Pierce)
Proteins were separated by SDS/PAGE For each sample,
exactly 10 lg of total protein was loaded on the gel in
loading buffer (250 mMTris/HCl, pH 7.6, 8% (w/v) SDS,
40% (v/v) glycerol, 0.05% (w/v) bromophenol blue, 20 mM
dithiothreitol) Proteins were electrotransferred to
Immuno-BlotTM poly(vinylidene difluoride) membranes (Bio-Rad)
using 400 mA overnight at 4C Membranes were washed
in Tris-buffered saline (TBS: 20 mM Tris/HCl, pH 7.6,
150 mM NaCl) supplemented with 0.05% (v/v) Tween-80
(TBS-T), preincubated for 1 h at room temperature with
blocking buffer [5% (w/v) skimmed milk powder (Oxoid) in
TBS-T], supplemented with 0.5 mMNa3VO4, and incubated
overnight at 4C with monoclonal mouse
anti-(phospho-p42/44 MAP kinase) Ig (Cell Signalling) in blocking buffer
(1 : 2000), supplemented with 0.5 mM Na3VO4 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 were washed again and then incubated for
5 min with Lumi-LightPLUS Western Blotting Substrate (Roche) Signals were detected with a FluorSTM MultiI-mager (Bio-Rad) and quantified using theMULTI-ANALIST software (Bio-Rad) All measurements were performed in the linear detection range of this method
All time curves for Fig 2 were measured in independ-ent experimindepend-ents, and as we wanted to compare them to each other and calculate the standard errors for each time point, the individual time curves had to be scaled to each other Scaling the curves with respect to the maximal 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 real 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) representative curve Therefore, we applied a multivariate least squares approximation [37,38] of the type Y¼ X b + e, in which
Y is the representative curve, X is the matrix of all other curves (which were treated similarly), b represents the regression coefficients (one for each curve X) and e is the error vector (zero mean, common variance [37,38]) For the three different conditions [stimulated with (a) EGF (b) PMA or (c) EGF + (PMA)], the scaled time curves were drawn (i.e Xb) in Fig 2 The standard errors were calculated as described previously [39]
Immunocytochemistry After stimulation, cells were washed twice with ice-cold NaCl/Pi, fixed by incubation for 30 min at 4C with ice-cold 4% (v/v) paraformaldehyde in NaCl/Piand 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 times (x-axis) with 10 ngÆmL)1EGF (n), 100 n M PMA (h) or both EGF and PMA (s) Cells were harvested and ERK-PP was measured 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 independent experiments, that were scaled to each other using a multivariate least squares approximation (see Experimental procedures) Error bars represent the standard error of the mean.
Trang 4with TBS-Triton (TBS, supplemented with 0.1% (v/v)
Triton X-100) Cells were then incubated with 100% (v/v)
methanol for 10 min at )20 C in order to permeabilize
cellular membranes and washed Cells were then incubated
for 1 h at room temperature with 5% FBS in TBS-Triton
and subsequently incubated overnight at 4C with
mono-clonal mouse anti-(phospho-p42/44 MAP kinase) Ig (Cell
Signalling) in 5% (w/v) BSA in TBS-Triton (1 : 400) Cells
were washed for 15 min with TBS-Triton, for 15 min with
0.1% (w/v) BSA in TBS-Triton and incubated for 2 h at
room temperature with Cy5TM-labeled goat 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 placed
in Vectrashield for fluorescence (Vector) on a microscope
slide and stored in the dark at 4C Fluorescence was
detected using a confocal scanning laser microscope (Leica
TCS 4D) A krypton-argon laser line (647 nm) was used for
excitation of the Cy5TM-label, and a long pass filter (665 nm)
was used for detection of the emitted light (with beam
splitter at 660 nm) Obtained images were quantified using
theSCION IMAGEsoftware (Scion Corporation)
Results
Biphasic time profile of ERK-PP by EGF or PMA
stimulation
We first determined the dynamic profile of phosphorylated
ERK after stimulation with EGF in NRK fibroblasts by
quantitative Western blotting Cells were serum-starved and
subsequently stimulated with 10 ngÆmL)1EGF for various
periods of time We observed a biphasic ERK-PP profile
(Fig 2) Upon EGF stimulation, the ERK-PP
concentra-tion rose from a background level to 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 increase was followed by a
decrease to a relatively low level, after about 1 h of EGF
stimulation This ERK-PP profile suggests the possibility of
damped oscillatory behaviour, consistent with complex
behaviour of the complex circuitry regulating ERK-PP
We also determined the ERK phosphorylation dynamics
induced by PKC activation by addition of 100 nMPMA to
serum-starved NRK cells The profile resembled that
induced by EGF stimulation (Fig 2) After about 4 min,
a peak concentration was reached, followed by a rapid
decline to a very low concentration that sustained for several
hours We refer to this as a quasi steady-state, as 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) The first peak
concentration induced by PMA was always lower than that
induced by EGF
EGF and PMA activate signalling to ERK synergistically
In order to determine whether the different signal inputs to
ERK (via EGFR and via PKC) affect each other, we
stimulated serum-starved NRK cells with both 10 ngÆmL)1
EGF and 100 nM PMA and again determined the time
profile of the ERK-PP concentration (Fig 2) We 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 and PMA act synergistically on the quasi-steady state-phase of the profile, but not on the initial peak
As the individual peak shapes may have been lost during averaging of the curves, we titrated EGF at 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 in the second phase
of the time profile further, we measured the 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 nMPMA The results (Fig 3) show that the (quasi-steady state) ERK-PP concentration depends on the EGF concentration used and 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) or presence (m) of PMA The 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 obtained using a Michaelis–Menten type equation (Eqn 1) The fitting parameters are shown in the table inset (their standard deviations are indicated between brackets) [ERK-PP] basal : the
ERK-PP concentration without EGF present; [ERK-ERK-PP] max : the maximum steady state ERK-PP concentration that can be induced by EGF; K, EGF concentration needed to obtain the half-maximum ERK-PP concentration In addition, a representative image of the immunoblots
is depicted.
Trang 5ERK-PP concentration was similar to that obtained with
high EGF concentrations, but when PMA and EGF were
added simultaneously, it reached a much 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-PPsteady state
¼ ½ERK-PPbasalþ½ERK-PPmax ½EGF
Kþ ½EGF ðEqn 1Þ [ERK-PP]basal is the ERK-PP concentration in
serum-starved cells before 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 resulted in a two- to threefold increase of
the additive [ERK-PP]max, reflecting the synergistic
activa-tion Interestingly, K with respect to EGF was 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 EGF
concentra-tions above 1 ngÆmL)1 The synergistic effect is only found in
the concentration range 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 and stimulated for 1 h with 100 ngÆmL)1 EGF, 100 nM PMA or both, after which ERK-PP was stained with a fluorescent label and visualized using laser scanning microscopy (Fig 4A), as described in Experimen-tal procedures The fluorescent signal per image was, after subtracting the background, divided by the number of cells
in the picture The average 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 4B), which is consistent with the results discussed in the previous section
Positive feedback circuit via cPLA2and PKC is not involved in EGF-mediated ERK phosphorylation
in NRK cells nor in the synergistic activation According to schemes available in the literature, after EGF stimulation, PKC may be activated via PLC-c and, via the positive feedback loop, by cPLA2(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 stimulated for 1 h with 100 ngÆmL)1EGF, 100 n M PMA or both and detected using a scanning laser microscope Representative images of four independent experiments are shown (B) Quantification of the immunostaining The average fluorescent signal per cell in four independent experiments is depicted; the error bars represent the standard error of the mean Please note that the method applied produces a relatively high background, which hampers the quantification The EGF and PMA costimulation produces a signal that significantly emerges from this background, whereas stimulation with either EGF or PMA only does not.
Trang 6NRK 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 ERK-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 stimulation,
indi-cating that PMA 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 inhibition in the early peak and was reduced in the
quasi-steady state-phase to a level comparable to that after
stimulation with EGF only These results show that in our
system, PKC had no significant effect on ERK
phosphory-lation after EGF stimuphosphory-lation This implies that the positive
feedback loop via cPLA2, which caused sustained ERK
phosphorylation upon stimulation with platelet-derived
growth factor (PDGF) [8], was not activated by EGF
stimulation in these cells To investigate this further, we
blocked cPLA2activity by the inhibitor ATK The ERK-PP
concentration again did not decline, even if the stimulation
was carried out with EGF and PMA together (Fig 5)
Similar results were obtained with 4-bromophenacyl
bro-mide, which is a different, general PLA2inhibitor (results
not shown) This shows that the positively regulating circuit
is not involved in the synergistic ERK phosphorylation
caused by EGF and PMA
Discussion
The architecture of signal transduction networks is often
highly complex, due to the large number of participating
protein complexes, cross interactions between pathways and
the functioning of regulatory circuits It is this complexity
that makes the understanding of cellular signalling a
difficult task For example, the features of a whole network
cannot be understood simply as the sum of features of its parts; the network as such may give rise to system or
emergent properties [4,40] To obtain reliable computer models that can calculate the outcome of signalling events, the interactions between signalling modules need 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 measured the dynamic time profile of ERK phosphorylation after stimulation by EGF, and observed a biphasic pattern consisting of a first rapid peak and relatively low and a broad second peak developing into what we refer to as a quasi-steady state The first peak has been described by others in many 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 causes Sos to dissociate from the growth factor receptor complex Thus,
as the local Sos concentration at the inner surface of the membrane decreases, Ras activation is impaired as well as subsequent activation of downstream signalling molecules 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 and behave as an on/off-switch [42] The biphasic pattern we observe here may reflect sustained oscillations that are initially synchronized in all cells in the culture but that become desynchronized over time Alternatively, damping may be caused by MAPK phosphatases (MKPs) that are up-regulated within
30 min after initial MAPK signalling [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 days and then 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 ATK (10 l M ; 1 h preincu-bation) ERK-PP was measured in the cell lysates by quantitative Western blotting The PKC inhibitor abolished PMA-induced ERK phosphorylation, whereas EGF-induced ERK phosphorylation was unaffected The
ERK-PP concentration induced by EGF and PMA costimulation declined only in the quasi-steady state-phase Inhibition of cPLA 2 did not affect the peak or the quasi-steady state-phase Shown are the mean results of three independent experiments, the error bars rep-resent the standard error of the mean.
Trang 7of 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, but 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 bisindolylmaleimide I
Simultaneous stimulation with PMA and EGF yielded a
remarkably synergistic effect in the quasi-steady state phase
of the time profile Interestingly, in the presence of PMA,
the ERK-PP quasi-steady state-concentration could still be
elevated further by EGF concentrations above 1 ngÆmL)1,
whereas this was not the case in the absence of PMA
(Fig 3) Furthermore, the ratio between 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 signalling [43,45], indicating that the response is
probably cell type-dependent It has been reported
previ-ously that ERK1 was synergistically activated in hamster
fibroblasts after stimulation with serotonin and basic
fibroblast growth factor for 120 min [46] In platelets,
pretreatment with thrombopoietin and stimulation with
a-thrombin led to a 30% phosphorylation of ERK2 in
the early phase (1–5 min), whereas thrombopoietin or
a-thrombin alone activated ERK2, either, not at all or to a
very low degree, respectively [47] In another study,
simultaneous stimulation of human embryonic kidney cells
with carbachol, which activates PKC via a G-protein
coupled receptor, and EGF, did not exert additive effects on
ERK activity after 10 min [48], which supports our finding
that synergism is not present in the initial peak (Fig 2) We
believe the latter is caused by saturation of the
phosphory-lation of all cellular ERK Indeed, it has been shown
previously that in NRK cells, 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 cPLA2and PKC In fact, cPLA2
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, resulting 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 suggested to cause Ser259
autophos-phorylation and activation [24] Ser259 was also identified as
a major Raf phosphorylation site upon growth factor
stimulation [50] The synergism might also originate
up-stream of Sos, as ERK activation by PKC has been shown to
depend both on Sos51 and on Ras-GTP-Raf complexes [52]
A different explanation could be that PKC has an
inhibitory effect on one of the down-regulating mechanisms
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 surface, instead of degradation [33], and EGF might then cause 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 same 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 EGF signal to ERK, also increases the EGF concentration that is needed for half-maximum ERK phosphorylation (Fig 3) Clearly, control of PKC-mediated EGFR phosphorylation on signalling to ERK is distributed over these processes Another, more speculative, candidate target could be an 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 EGF, this may lead to sustained ERK phosphorylation
In conclusion, we have investigated the interaction between the MAPK and PKC signalling modules in a quantitative manner and found that they affect each other in multiple ways EGF stimulation and PKC activation caused
a synergistic activation of ERK in the quasi-steady state-phase PKC here acts as a signal amplifier for growth factor signalling We found no evidence for the functioning of a positive feedback mechanism via cPLA2 The interactions between MAPK and PKC apparently serve to facilitate quasi-intelligent signal integration, which may 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 hypothesize that the synergistic ERK phosphorylation, which results in pro-longed signalling, has implications for the outcome of signalling This may be of importance in the constitutive ERK activation often found in human 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 manuscript We also thank K Krab and
H Dekker for excellent technical advice on curve fitting and immunofluorescence microscopy, respectively.
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