One of the major signaling pathways by which
extracellular signals induce cell proliferation and differentiation
involves the activation of extracellular signal-regulated kinases
(ERKs). Because calmodulin is essential for quiescent cells to enter
cell cycle, the role of calmodulin on ERK2 activation was studied in cultured fibroblasts. Serum, phorbol esters, or active Ras induced ERK2
activation in NIH 3T3 fibroblasts. This activation was not inhibited by
calmodulin blockade. Surprisingly, inhibition of calmodulin prior to
fetal bovine serum addition prolonged activation of ERK2. Furthermore,
inactivation of calmodulin in serum-starved cells induced ERK2
phosphorylation that was dependent on MAP kinase kinase (MEK).
Inactivation of calmodulin in serum-starved cells also induced
activation of Ras, Raf, and MEK. On the contrary, tyrosine
phosphorylation of tyrosine kinase receptors was not observed. These
results indicate that calmodulin inhibits ERK2 activation pathway at
the level of Ras. Calmodulin inhibition induced overexpression of
p21cip1 which was dependent on MEK activity. We
propose that inhibition of Ras by calmodulin prevents the activation of
ERK2 at low serum concentration. Thus, entering into the cell cycle
after serum addition would imply the overcoming of the inhibitory
effect of calmodulin and consequently ERK2 activation. Furthermore,
down-regulation of Ras by calmodulin may be also important to determine
the duration of ERK2 activation and to prevent a high
p21cip1 expression that would lead to an
inhibition of cell proliferation.
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INTRODUCTION |
Cells have evolved signal transduction pathways that allow them to
respond to extracellular signals. Those signaling pathways will lead to
the regulation of effector proteins that will finally cause cell
proliferation or differentiation.
One of the major signal transduction pathways results in the activation
of a class of intracellular protein serine/threonine kinases termed
extracellular signal-regulated kinases
(ERKs),1 also known as
mitogen-activated protein kinases (MAPKs) (1). Two highly related
mammalian ERKs, ERK1 (p44MAPK) and ERK2
(p42MAPK) are activated in response to growth factors and
hormones (2, 3). Once activated, those kinases are able to
phosphorylate and activate numerous cytoplasmic proteins including
p90rsk (S6 ribosomal protein kinase) (4), phospholipase
A2 (5), and EGF receptor (6). After stimulation, ERKs
translocate into the nucleus (7) where they activate a number of
transcription factors such as Elk1 (8, 9), c-Ets1, and c-Ets2 (10), thus altering the pattern of gene expression (11).
ERKs are activated by a dual phosphorylation on threonine and tyrosine
residues. Their specific activators, called MAP kinase kinases (MAPKK)
or MEK, constitute a new family of dual-specific threonine/tyrosine
kinases (12). MEK1 and MEK2 are the kinases known to activate ERK1 and
ERK2. MEK1 and MEK2 are in turn activated by an upstream MAP kinase
kinase kinase, which has been identified as the product of the
proto-oncogene raf-1 (13). Raf-1 seems to integrate
different signals that will lead to ERK activation. Its activation is
complex and not completely well understood, with Ras activation being
an essential element in the pathway. Active Ras targets Raf-1 to the
cell membrane and becomes attached to it by an unknown mechanism (14).
However, recruitment of Raf to the membrane by binding to Ras cannot
account for full activation of Raf (15). Ras GTPases exist in two
conformations, an inactive GDP-bound form and an active GTP-bound
state. The cyclic interconversion of Ras is regulated by the activity
of the guanidine exchange factors (GEFs), which allow the replacement of Ras-bound GDP by GTP, and the Ras-GTPase activating proteins (GAPs),
which activate the intrinsic GTPase activity of Ras that converts GTP
back to GDP (16). Activation of Ras by tyrosine kinase receptors
involves recruitment of SOS, a guanidine exchange factor to the
membrane (2). Other GEFs non-activated by tyrosine kinase receptors
that activate Ras have been identified in mammalian cells such as
Ras-GRF/CDC25Mm (17).
Activation of the ERK pathway is transient, and it has been argued that
it is the duration of ERKs activation that determines whether a
stimulus elicits proliferation or differentiation (18, 19, 20). In
cells activated to proliferate, Raf is associated to the membrane only
for 5 min, and its activity decreases to basal levels 15 min after
growth factor addition in cultured fibroblasts (21). ERKs activity
after mitogenic stimuli shows a initial peak at 5-10 min followed by a
sustained phase of lower activity of 4 h (22). However, a more
prolonged or a high intensity activation of the MAPK pathway induces
growth arrest of the cells mediated by the cell cycle inhibitor
p21cip1 (20, 23, 24). Whereas much attention has
focused on the mechanisms leading to ERK phosphorylation by growth
factors and oncoproteins, few studies have turned to the question of
how the pathway is down-regulated. Several constitutive and inducible ERK phosphatases responsible for its down-regulation have been described (25), including Pyst1 (26), MKP1, 2 (27, 28), and PAC1 (29).
Expression of MKP1 has been shown to be ERK- and
Ca2+-dependent (30). Less is known about the
down-regulation of the pathway upstream of ERK. In some cells, ERK
mediates the phosphorylation of SOS to terminate
Ras-dependent activation of ERK (31).
Ca2+ and calmodulin (CaM) are known to act as second
messengers in signal transduction pathways and to regulate cell
proliferation (32-35). Through the action of CaM-binding-proteins like
CaM-dependent kinases II and IV, calcineurin, hnRNP A2,
hnRNP C, and others, they regulate a great variety of cellular
processes, such as gene expression, protein translation, and protein
phosphorylation (36). By using expression vectors capable of inducibly
synthesizing CaM sense or antisense mRNAs, it has been shown that
progression through G1 and mitosis exit is sensitive to
changes in the intracellular concentration of CaM (37). Furthermore,
the addition of specific anti-CaM drugs to cell cultures inhibits
reentry of growth-arrested cells into the cell cycle
(G0/G1 transition), the progression into and
through the S phase and the entry and exit from mitosis (35, 38-44).
During G1, CaM is essential to activate cdk4 and phosphorylate pRb (44, 45). Moreover CaM participates in the activation
of cdc2 during mitosis entry (33) and in its inactivation at the
metaphase/anaphase transition (46). Despite the evidence indicating
that CaM plays a role in cell cycle entry from quiescence (G0/G1 transition), not much is known about the
CaM-dependent steps essential for this transition. CaM has
been shown to play a role in the activation of the MAPK signaling
pathway (47-51). For example, the Ras-GRF exchange factor of cortical
neurons is a CaM-binding-proteins, and a Ca2+ influx in
these cells is able to activate Ras and ERKs (47). In other cellular
types, CaM-dependent kinases have been involved in the MAPK
activation pathway (50-51).
We have analyzed here the involvement of CaM in the ERK signaling
pathway in cell-cultured fibroblast. Surprisingly, results show that
CaM is not essential for the activation of the ERK pathway but for its
inactivation. This down-regulation of the pathway is due at least in
part to an inhibitory effect of CaM on Ras activation. Furthermore,
activation of ERK2 by CaM inhibition induces an increase in
p21cip1 expression together with a cell cycle
arrest.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
NIH 3T3 cells (ATCC) or NIH 3T3 cells
constitutively expressing active mutant N-Ras (lys61) under a CMV
promoter (NIH 3T3RasLys61) (gift of Dr. T. Thompson, Barcelona) or
normal rat kidney cells (NRK) were made quiescent by culturing them in
Dulbecco's minimum essential medium with 0.5% fetal bovine serum
(FBS) during 2 days. 10% FBS, 100 µM TPA, 25 ng/ml EGF,
15 µg/ml W13, 15 µg/ml W12, 5 µM KN93, or 2 µg/ml
cyclosporin A were added directly to the media, and for the time
indicated in the results.
Gel Electrophoresis and Immunoblotting--
Cells were lysed in
a buffer containing 2% SDS, and 67 mM Tris-HCl, pH 6.8. The same amount of protein of each extract was electrophoresed in
SDS-10% PAGE gels essentially as described by Laemmli (52). After
electrophoresis, the proteins were transferred to Immobilon-P strips
for 2 h at 60 V. The sheets were preincubated in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), 0.05%
Tween-20, and 5% BSA for 1 h at room temperature and then
incubated for 1 h at room temperature in TBS, 0.05% Tween 20, 1%
BSA, and 0.5% defatted milk powder containing antibodies against: ERK1
and ERK2 (03-6600 Zymed Laboratories Inc., 1:500
dilution), phospho-ERK1 and ERK2 (No. 9101S, New England Biolabs, 1:500
dilution), phospho-MEK (No. 9121S, New England Biolabs, 1:500
dilution), c-Raf-1 (R19120 Transduction Lab., 1:500 dilution),
phospho-Tyr (PY20, Transduction Lab., 1:750 dilution). After washing in
TBS, 0.05% Tween-20 (three times, 10 min each), the sheets were
incubated with either a peroxidase-coupled secondary antibody (1:1000
dilution) (Bio-Rad) or an alkaline phosphatase-coupled secondary
antibody (1:10000) (Promega) for 1 h at room temperature. After
incubation, the sheets were washed twice in TBS, 0.05% Tween 20, and
once in TBS. The reaction was visualized by ECL (Amersham Pharmacia
Biotech) or with BCIP/NBT (Promega). Control of protein loading and
transfer was done by stripping the gels and re-blotting them with
anti-ERK1 and -ERK2 antibodies.
Raf Immunoprecipitation and Kinase
Assay--
Immunoprecipitations were performed as described by
Morrison (53). Cells (5-10 × 107) were lysed in 1 ml
of radioimmune precipitation buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS,
0.5% sodium deoxycholate, 2 mM EDTA) containing 1 mM PMSF, 1 mM aprotinin, 20 µM
leupeptin, and 5 mM sodium vanadate. To immunoprecipitate
Raf-1 from cells lysates, 2.5 µg of anti-Raf-1 (R19120, Transduction
Lab) or 2.5 µg of a nonrelated monoclonal antibody were first
prebound to 20 µl of protein G-Sepharose beads (Sigma) in 1 ml of
radioimmune precipitation buffer for 1 h at room temperature. The
anti-Raf-coated beads were washed twice with radioimmune precipitation
buffer. Then, 500 µg of protein from the lysates were added and
incubated for 2 h at 4 °C. The immunoprecipitated complexes
were washed three times with 1 ml of cold Nonidet P-40 lysis buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 2 mM EDTA) containing 1 mM PMSF, 1 mM aprotinin, 20 µM leupeptin, and 5 mM sodium vanadate, resuspended, and incubated for 20 min
at 25 °C in 40 µl of kinase buffer (30 mM HEPES-Na, pH
7.4, 7 mM MnCl2, 5 mM
MgCl2, 100 mM NaCl, 1 mM
dithiothreitol, 15 µM ATP) plus 20 µCi of
[
-32P]ATP(3000 Ci/mmol; Amersham Pharmacia Biotech)
and 40 mg/ml pGST-MEK inactive fusion protein. Then, the samples were
electrophoresed on SDS-polyacrylamide gels, and the gels were stained
with Coomassie Blue, dried, and exposed to x-ray films at
80 °C.
Immunocytochemistry--
Quiescent cells were grown on glass
coverslips. To detect ERK1 and ERK2 cells were fixed in 4%
paraformaldehyde/phosphate-buffered saline (PBS) for 10 min at room
temperature and permeabilized at
20 °C for 10 min with methanol.
After washing three times with PBS, the nonspecific sites were
subsequently blocked with sheep serum in PBS (1:5) for 30 min at room
temperature. Cells were then incubated 1 h at 37 °C, with the
specific polyclonal antibodies anti-ERK1 and ERK2 (No. 06-182; Upstate
Biotechnology) in 1% BSA/PBS (1:200). Coverslips were then washed
three times (5 min each) in PBS and incubated for 45 min at 37 °C
with fluorescein-conjugated anti-rabbit antibody (dilution 1:50,
Boehringer) in 1% BSA/PBS. After two washes in PBS, coverslips
were mounted on glass slides with Mowiol (Calbiochem).
Measurement of Ras Activation--
The capacity of Ras-GTP to
bind to RBD (Ras-binding domain of Raf-1) was used to analyze the
amount of active Ras (54, 55). Cells were lysed in the culture dish
with 25 mM Tris-HCl, pH 7.5, 5 mM EGTA, 150 mM NaCl, 5 mM MgCl2, 1% Triton
X-100, 1% N-octyl glucoside, 1 mM PMSF, 1 mM aprotinin, and 20 µM Leupeptin. Cleared (10,000 × g) lysate (1 mg) was incubated with 30 µg
of GST-RBD bound to glutathione-Sepharose beads for 2 h at
4 °C. Beads were washed four times with the lysis buffer. Bound
proteins were solubilized by the addition of 30 µl of Laemmli loading
buffer and run on 12.5% SDS-PAGE gels. Proteins were then transferred
and immunoblotted as described above using pan-Ras monoclonal antibody
(Oncogene Sciences OP40, 1:100 dilution).
 |
RESULTS |
Effect of CaM Inhibition on ERK2 Activation--
Stimulation of
serum-starved NIH 3T3 fibroblast by 10% FBS, or 100 µM
TPA for 10 min, resulted in an activation of ERK2, as demonstrated by
the increase in ERK2 tyrosine phosphorylation analyzed by Western
blotting using antiphosphotyrosine antibodies (Fig.
1A) or phospho-specific
anti-ERK1 and -ERK2 antibodies (data not shown). To analyze if CaM was
essential for the signaling pathways leading to ERK2 phosphorylation,
quiescent cells were pretreated with the anti-CaM drug W13 (15 µg/ml)
for 20 min prior to stimulation with FBS or TPA. W13 has been
extensively used to inhibit CaM in cell cultures, and it is known to be
highly specific at the doses used in this work (40, 42, 56, 57). W12
was used as a control because it is a compound chemically similar to
W13 but with a much lower affinity for CaM (40). As shown in Fig.
1A, W13-pretreatment did not have any effect on the level of
phosphorylated ERK2, determined by Western blotting using
antiphosphotyrosine (PY20) antibody (Fig. 1A). The same results were obtained using the antiphospho-ERK2 specific antibodies (data not shown).

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Fig. 1.
Effect of W13 addition on ERK2
phosphorylation induced by FBS, TPA, or Ras. A, ERK2
phosphorylation in serum- and TPA-stimulated NIH 3T3 fibroblasts after
pretreatment with the anti-CaM drug W13. Quiescent NIH 3T3 cells
(Q) were stimulated for 10 min with 10% FBS or 100 µM TPA. In the indicated lanes, W12
or W13 (15 µg/ml) was added to the cultures 20 min prior to FBS or
TPA addition. B, quiescent NIH 3T3Nras(lys61) (Q)
cells, expressing the constitutively active N-ras (lys61) mutant, were
treated for 30 min with W12 or W13 (15 µg/ml). In panels A
and B, cells were lysed and ERK2 phosphorylation analyzed by
Western blotting using the antiphosphotyrosine (PY20) antibody as
indicated under "Experimental Procedures." P-ERK2 corresponds to a
band of 42 kDa that comigrates with ERK2 using anti-ERK1 and ERK2
antibodies.
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ERK2 phosphorylation was also induced at 0.5% FBS by expression of
activated N-Ras. Inhibition of CaM in NIH 3T3NRas(Lys61)-transformed cells by W13 treatment for 30 min did not have any effect on
Ras-induced ERK2 phosphorylation (Fig. 1B). Thus, CaM is not
essential for any of the studied signaling pathways leading to ERK2
activation in NIH 3T3.
Effect of CaM Inhibition on ERK2 Down-regulation--
Because the
duration of ERKs activation is also important for cell response, the
effect of CaM inhibition on the timing of ERKs phosphorylation was
analyzed. In nontreated cells (data not shown) or in cells preincubated
with W12 (for 20 min prior to 10% FBS addition) (Fig.
2), ERK2 phosphorylation was high at 10 min, started to decrease at 30 min, and was slightly higher than in
unstimulated cells by 2 h after FBS addition. Surprisingly, when
cells where preincubated with W13, ERK2 phosphorylation still remained
high 2 h after the stimulation (Fig. 2). Inhibition of CaM later
in G1 (between 4 and 10 h after serum addition), when ERK2 phosphorylation was already decreased, did not lead to a second
activation of ERK2 (data not shown). Thus, inhibition of CaM
prolonged the phosphorylation of ERK2, suggesting that CaM is involved
in its down-regulation.

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Fig. 2.
Effect of W13 addition on the duration of
ERK2 phosphorylation. Quiescent NIH 3T3 cells were preincubated
with either W12 or W13 (15 µg/ml) or without any drug ( ) and 20 min
later were stimulated with 10% FBS and harvested at the indicated
times (t). Cells were lysed and ERK2 phosphorylation was
analyzed by Western blot using the antiphospho-ERK1 and ERK2 antibody
as indicated under "Experimental Procedures." A representative
experiment of three different experiments is shown in the figure.
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The effect of W13 on ERK2 phosphorylation in serum-starved cells was
also analyzed. When serum-starved cells (0.5% FBS) were incubated with
W13 for 30 min, an increase in ERK2 phosphorylation was observed. This
increase was similar to that produced by addition of 10% FBS and was
not observed with W12 (Fig.
3A). This effect was not
mediated by CaM-dependent kinase II or calcineurin because treatment of serum-starved cells with KN93, an inhibitor of
CaM-dependent-kinase II, or cyclosporin A, an inhibitor of
calcineurin, did not induce ERK2 phosphorylation (Fig.
3A).

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Fig. 3.
W13 addition induces ERK2 phosphorylation at
low FBS concentration. A, quiescent (Q) NIH
3T3 cells (0.5% FBS) were treated for 10 min with 10% FBS or for 30 min with W12 (15 µg/ml), W13 (15 µg/ml), cyclosporin A (2 µg/ml)
(C.A.), or KN93 (5 µM). Then, cells were lysed
and ERK2 phosphorylation was analyzed by Western blotting using the
antiphosphotyrosine (PY20) antibody as indicated under "Experimental
Procedures." P-ERK2 corresponds to a band of 42 kDa that comigrates
with ERK2 using anti-ERK1 and ERK2 antibodies. A representative
experiment of three different experiments is shown in the figure.
B, quiescent NIH 3T3 cells were cultured for 10 h in a
media without FBS. Then, W12 ( ) or W13 ( ) (15 µg/ml) were added
to the media, and 20 min later, cells were treated for 10 min with
increasing concentrations of FBS. Cells were lysed and ERK2
phosphorylation was analyzed by Western blotting using the
antiphospho-ERK1 and -ERK2 antibodies as indicated under
"Experimental Procedures." Bands were quantified by an image
analysis system (Bio-Image, Millipore). Values in the graph are the
mean of three different experiments; standard deviations are lower than
10% of the mean.
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Cooperation between FBS addition and CaM inhibition to induce ERK2
phosphorylation was analyzed in quiescent cells that had been
maintained for the last 10 h in the complete absence of FBS. Cells
were pre-treated with W12 or W13 for 20 min, and ERK2 phosphorylation was studied 10 min after addition of increasing amounts of FBS. As
shown in Fig. 3B, W13 addition synergized with low
concentrations of FBS to induce ERK2 phosphorylation. Maximal increase
in ERK2 phosphorylation induced by CaM inhibition was observed at 0.5% FBS. In agreement with the results in Fig. 1A, when the
amount of FBS added to the media was higher than 2%, no additional
increase in ERK2 phosphorylation was induced by CaM inhibition (Fig.
3B). Similar results were obtained using NRK cells (data not
shown). These results suggest that CaM inhibits any of the signaling
pathways by which FBS activates ERK2.
We also analyzed whether ERK2 phosphorylation induced by CaM inhibition
in serum-starved cells correlated with a nuclear accumulation of ERKs.
As shown in Fig. 4, after 2 days of serum
starvation, ERK2 was localized in the cytoplasm. Treatment with W13 for
30 min induced a translocation of ERK2 into the nucleus similar to what
occurred with 10% FBS incubation. W13 treatment of serum-starved NIH
3T3 cells also induced an increase in fos mRNA levels
analyzed by Northern blotting (data not shown).

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Fig. 4.
Immunolocalization of ERK2 after W13
addition. Quiescent (Q) NIH 3T3 cells were treated with
10% FBS, W12 (15 µg/ml) or W13 (15 µg/ml) for 30 min. Then cells
were fixed with 4% paraformaldehyde, and subcellular localization of
ERK2 was analyzed by immunocytochemistry using anti-ERK1 and ERK2
antibodies as indicated under "Experimental Procedures." FBS and
W13 but not W12 induced the translocation of ERK2 from the cytoplasm
into the nucleus.
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Analysis of the Pathway Leading to ERK2 Activation by CaM
Inhibition--
The only kinases known to phosphorylate and activate
ERK2 are MEK1 and MEK2. Thus, we analyzed whether these kinases were involved in ERK phosphorylation induced by CaM inhibition. As shown in
Fig. 5A, ERK2 phosphorylation
induced by W13 addition to serum-starved cells was not observed when
cells were previously treated with the specific inhibitor of MEK,
PD98059 (Calbiochem). Furthermore, W13 addition to serum-starved cells
induced an increase in MEK phosphorylation as determined by Western
blotting using phospho-MEK-specific antibodies (Fig.
5B). These results indicate that W13 induces ERK2
phosphorylation by MEK and that CaM inhibits the activation of MEK at
low serum concentration.

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Fig. 5.
MEK is involved in the phosphorylation of ERK
induced by W13 addition in serum-starved cells. A,
quiescent NIH 3T3 cells (Q) were treated for 10 min with
10% FBS or for 30 min with W12 or W13 (15 µg/ml). In the indicated
lane (W13/PD), cells were incubated with the MEK
inhibitor PD98059 (100 µM) for 30 min prior to the
addition of W13. ERK1 and ERK2 phosphorylation was determined by
Western blotting using antiphospho-ERK1 and -ERK2 antibodies as
indicated under "Experimental Procedures." B, quiescent
NIH 3T3 cells were activated with 10% FBS for 10 min or treated with
W12 or W13 (15 µg/ml) for 30 min. MEK1 and MEK2 phosphorylation was
analyzed by Western blotting using antiphospho-MEK1 and MEK2 specific
antibodies as indicated under "Experimental Procedures." The same
blot was subsequently incubated with antiphospho-ERK1 and ERK2 specific
antibody.
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Next, the effect of CaM inhibition on Raf-1, the main kinase involved
in MEK1 and MEK2 activation, was analyzed. W13 addition to
serum-starved cells induced an increase in Raf-1 activity as determined
by immunoprecipitation followed by kinase assay using inactive MEK-GST
as substrate, compared with W12-treated cells and quiescent cells (Fig.
6A). CaM inhibition induced
also a gel-mobility shift of Raf-1, similar to that induced by FBS
(Fig. 6B) that has been related with its phosphorylation and
activation. Although Raf activation is not very well understood, the
increase in Ras-GTP seems to be an essential event for Raf activation.
Thus, the levels of Ras-GTP upon W13 treatment of serum-starved cells
were analyzed. As shown in Fig.
7A, CaM inhibition induced an
increase in the levels of Ras-GTP that was not observed in W12-treated
cells. The levels of Ras-GTP after W13 treatment were as high as those reached upon 10% FBS addition. A mechanism for Ras activation is
activation of tyrosine kinase receptors involving autophosphorylation of these receptors and the recruitment of SOS to the plasma membrane through the interaction with the adapter protein GRB2. After 10 min of
10% FBS addition to serum-starved cells, tyrosine phosphorylation was
increased in the area of the gel where the EGF receptors and PDGF
receptors move, 170-190 kDa (Fig. 7B). On the contrary, no increase in tyrosine phosphorylation in the same area of the gel was
observed after the addition of W13 or W12 to serum-starved cells (Fig.
7B). Thus, CaM inhibition induced activation of
Ras/Raf/MEK/ERK without any detectable activation of tyrosine kinase
receptors.

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Fig. 6.
Effect of W13 addition on Raf
activation. A, quiescent NIH 3T3 cells (Q)
were treated for 10 min with 10% FBS or for 30 min with W12 or W13 (15 µg/ml). Cells were lysed and immunoprecipitated with anti-Raf
antibody (anti-Raf-1) or with a control nonrelated
monoclonal antibody (mAb) and assayed for kinase activity
using MEK-GST as substrate as indicated under "Experimental
Procedures." A representative experiment of a total of three is shown
in the figure. B, Raf-1 phosphorylation after CaM inhibition
in serum-starved NIH 3T3 cells. Quiescent NIH 3T3 cells (Q)
were treated for 10 min with 10% FBS or for 30 min with W12 or W13 (15 µg/ml). Cells lysates and Western blot analysis using anti-raf-1
antibody were done as indicated under "Experimental Procedures."
The decrease in the electrophoretical mobility of Raf-1 corresponds to
its phosphorylation. The change in electrophoretical mobility in
FBS-stimulated and W13-treated cells was reproducible in a total of
three experiments.
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Fig. 7.
Effect of W13 addition on Ras activation and
growth factor receptor tyrosine phosphorylation. A,
quiescent NIH 3T3 cells (Q) were treated for 10 min with
10% FBS or for 30 min with W12 or W13 (15 µg/ml). Cells were lysated
and Ras-GTP was determined by precipitating with RBD-Sepharose followed
by Western blotting with anti-Ras antibody as indicated under
"Experimental Procedures." B, quiescent NIH 3T3 cells
(Q) were treated for 10 min with 10% FCS (FCS), 25 ng/ml
EGF, or for 30 min with W12 or W13 (15 µg/ml). Cells were lysated and
Western blots were performed as indicated under "Experimental
Procedures" using antiphosphotyrosine antibodies (PY20).
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CaM Inhibition Increased the Levels of the Cell Cycle Inhibitor
p21cip1 and Inhibited Cell Proliferation--
In agreement
with previous reports (38) when W13 was added to quiescent
serum-starved NIH 3T3 cells 20 min prior to 10% FBS addition, DNA
synthesis at 20 h was inhibited by 80%. As inhibition of CaM
prevents entry in S phase and prolongs ERK activation, we looked for a
relationship between these two effects. The expression of
p21cip1 has been shown to be dependent on ERK1
and ERK2 activity (20). Thus, the levels of this cell cycle inhibitor
were analyzed upon CaM inhibition. In W13-pretreated cells, a prolonged
expression of p21cip1 was observed upon FBS
addition (Fig. 8A), being the
p21cip1 protein still present 9 h after
serum addition. On the contrary, in control cells (W12-pretreated
cells), 10% FBS addition induced a transient increase in
p21cip1 that showed a maximum at 2 h (Fig.
8A). Thus, inhibition of CaM induced sustained ERK2
activation and increased p21 expression, in parallel with an inhibition
of cell cycle progression.

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Fig. 8.
Effect of W13 addition on
p21cip1 expression. A,
quiescent NIH 3T3 cells (Q) were treated with either W12 or
W13 (15 µg/ml) and 20 min later were stimulated with 10% FBS. Cells
were harvested at the indicated times after FBS addition. B,
quiescent NIH 3T3 cells (Q) were treated with 10% FBS, W12,
or W13 (15 µg/ml) for 2 h. In the indicated lane
(W13/PD), cells were incubated with the MEK inhibitor
PD98059 (100 µM) for 30 min prior to the addition of W13.
In both panels A and B, cells were lysed and
p21cip1 levels were analyzed by Western blotting
as indicated under "Experimental Procedures." Both panels
A and B are representative results of three different
experiments.
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CaM inhibition in serum-starved cells (0.5% FBS) also induced the
expression of p21cip1 (Fig. 8B). This
p21cip1 expression is MEK-dependent
because it was not observed when the cells were preincubated with the
MEK inhibitor PD98059 (100 µM) prior to W13 addition
(Fig. 8B).
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DISCUSSION |
ERK signaling pathway is very important for the cellular response
to extracellular signals. Signaling through this pathway has been shown
to mediate differentiation, proliferation, or oncogenic transformation
depending on the cellular context and the duration of the activation
(19, 58) Thus, in addition to activation of ERK, its down-regulation is
also decisive for cell response. Inactivation of the pathway is
important to prevent ERK phosphorylation at low serum concentration and
to prevent an excessively prolonged peak of ERK activity when cells are
stimulated by growth factors. In this sense, constitutive and
serum-inducible ERK phosphatases have been described, but inactivation
of the pathway upstream of ERK is not well understood. We have analyzed
the involvement of CaM in ERK activation and inactivation pathways in
cell-cultured fibroblasts.
Ca2+ and CaM are essential for the activation of ERK in
response to various stimuli in cortical neurons and lymphocytes;
however, we have shown here that CaM is not essential to activate ERK2 by serum, TPA or constitutive activation of Ras in cultured fibroblasts (NIH 3T3 or NRK cells).These results agree with the fact that in Rat-1
cells, buffering of Ca2+ does not prevent ERK activation
induced by LPA or EGF (31). Thus, although CaM is necessary for NIH 3T3
fibroblast to reenter cell cycle from quiescence, it is not essential
for ERK2 activation.
We have shown that CaM is involved in the down-regulation of the ERK2
activation pathway. CaM inhibition increases the duration of ERK2
phosphorylation when cells are stimulated with 10% FBS. ERK2
activation has a dual effect on cell proliferation. On one hand it
induces the expression of cyclin D1 which is essential for
G1 progression (59, 60), and on the other hand it induces p21cip1 expression (20).
p21cip1 seems to be necessary at low levels for
cdk4/cyclin D1 activation, but at high levels acts as an inhibitor of
cdk4/cyclin D1 and cdk2/cyclin E (61, 62). Thus, an intense or highly
sustained activation of ERK2 could induce an excessive increase of
p21cip1 expression and, as a consequence, cell
cycle inhibition. In fact, a high intensity Raf signal has recently
been shown to cause a cell cycle arrest mediated by
p21cip1 (23, 24). In agreement with that, the
sustained activation of ERK2 induced by CaM inhibition in
serum-stimulated cells correlates with a lengthened expression of
p21cip1 and an inhibition of cell proliferation.
At least two reports indicate an inhibitory role of Ca2+ on
ERK activity. First, expression of the ERK phosphatase, MKP1, is
Ca2+-dependent in Rat-1 cells; consequently, a
depletion of the intracellular Ca2+ in these cells induces
a more sustained increase in ERK1 activity (31). Second,
Ca2+ addition to cultures inhibits EGF-induced stimulation
of ERK2 activity in human primary keratinocytes (63). We have also
shown that CaM inhibition not only leads to a prolonged activation of ERK2 upon FBS addition but also to an activation of ERK2 at low serum
concentration. The fact that, in total absence of FBS, CaM inhibition
has almost no effect on ERK phosphorylation, but that a cooperation
exists between CaM inhibition and FBS addition for the activation of
ERK2, suggests that CaM inhibits any of the signal transduction
pathways leading to ERK2 phosphorylation that are activated by FBS. We
have analyzed the activation of different elements of the ERK signaling
pathway and shown that CaM inhibition leads also to Ras, Raf, and MEK
activation. Thus, we conclude that CaM is down-regulating the Ras
activation pathway, although a multiple effect of CaM at several levels
in the ERK signaling pathway cannot be excluded. We have also proved
that CaM-dependent kinase II or the
CaM-dependent phosphatase calcineurin are not involved in
the inhibitory effect of CaM on the ERK activation pathway.
The rate-limiting step in Ras activation is the exchange of bound GDP
for GTP, which is catalyzed by GEFs (16). Several guanidine exchange
factors are involved in Ras activation in response to different
stimuli. In response to tyrosine kinase receptors activation, Ras is
activated by the SOS nucleotide-exchange factor. Access of SOS to the
membrane where Ras is located is because of the binding of SOS to the
receptor through SH2 domain- and SH3 domain-mediated interactions
involving the adapter proteins GRB2 and SHC. Although it has been shown
that some tyrosine kinase receptors such as EGF receptor are able to
bind CaM (64, 65) we have not observed any increase in tyrosine
phosphorylation levels of the EGF receptor or any other protein of
170-190 kDa after CaM inhibition. Other Ras guanidine exchange factor
have been identified as Ras-GRF/Cdc25Mm (66) and Ras-GRF2 (67). Both
factors are most abundant in brain but are also expressed in other
tissues and a variety of cell lines (66, 68). Activation of these
factors is not very well understood, and they have been suggested to
respond to G protein-coupled receptors (69). Both factors contain IQ
motifs that allow their binding to CaM, and in both neurons and
epithelial cells, its activity seems to be stimulated by
Ca2+ (47, 67). Those results are controversial because
in vitro studies show that full-length Ras-GRF activity is
inhibited by CaM (70). Our results agree with the presence of Ras-GRF
or a homologous protein in NIH 3T3 cells whose activity is inhibited by
CaM. Studies to determine this are underway in our laboratory. Alternatively CaM could activate a GAP protein acting on Ras. In this
sense, it has been shown that IQGAP1 binds CaM and that its interaction
with cdc42 is inhibited by CaM (71).
We propose two possible physiological roles, which are not exclusive,
for the negative regulation of the ERK signaling pathway by CaM. First,
CaM is defining a threshold in the Ras/Raf/MEK/ERK signaling pathway to
prevent activation of the pathway at low serum concentration because of
the basal activity of some components of the pathway upstream of Ras.
When growth factor receptors are activated by serum addition, this
threshold is overpassed and ERK is activated even in the presence of
Ca2+ and CaM. Second, CaM down-regulation of Ras is
essential to regulate the duration and the intensity of ERK activation.
In consequence, CaM inhibition prolongs Ras/Raf/MEK/ERK activation and
p21cip1 expression, and thus induces inhibition
of cell cycle progression.
We thank Dr. F. R. McKenzie (Nice,
France), for the gift of GST-RBD plasmid and the advice in the Ras
activity analysis, and Dr. Timothy Thompson (Barcelona, Spain), for the
gift of the NIH 3T3RasLys61 cell line. We also thank Esther
Castaño for the help in the analysis of c-fos expression and Anna
Bosch for the technical assistance in confocal microscopy. We are also
grateful to Dr. N. Rocamora and Dr. J. Comella for fruitful
discussions.