From the Department of Pharmacology, Brain Research
Institute, and Brain Korea 21 Projects for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea and the
§ Department of Anatomy and Neurobiology, University of
Maryland School of Medicine, Baltimore, Maryland 21201
Received for publication, November 26, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Growth factors bind to their specific receptors
on the responsive cell surface and thereby initiate dramatic changes in
the proliferation, differentiation, and survival of their target cells. In the present study we have examined the mechanism by which growth factor-induced signals are propagated to the nucleus, leading to the
activation of transcription factor, cis-acting cAMP
response element (CRE)-binding protein (CREB), in immortalized
hippocampal progenitor cells (H19-7). During the differentiation of
H19-7 cells by basic fibroblast growth factor (bFGF) a critical
regulatory Ser133 residue of CREB was phosphorylated
followed by an increase of CRE-mediated gene transcription. Expression
of S133A CREB mutants blocked the differentiation of H19-7 cells by
bFGF. Although the kinetics of CREB phosphorylation by EGF was
transient, bFGF induced a prolonged pattern of CREB phosphorylation.
Interestingly, bFGF-induced CREB phosphorylation and subsequent
CRE-mediated gene transcription is not likely to be mediated by any of
previously known signaling pathways that lead to phosphorylation of
CREB, such as mitogen-activated protein kinases, protein kinase A,
protein kinase C, phosphatidylinositol 3-kinase-p70S6K,
calcium/calmodulindependent protein kinase, and casein kinase 2. By using in vitro in gel kinase assay the
presence of a novel 120-kDa bFGF-inducible CREB kinase was identified.
These findings identify a new growth factor-activated signaling pathway
that regulates gene expression at the CRE.
Growth factors act by binding to cell surface receptors to elicit
the regulation of cell growth and differentiation. This, in turn,
triggers a variety of intracellular signaling pathways that ultimately
control cell physiology. Activation of signaling cascades results in
the pattern of gene expression through the functional modulation of
various transcription factors.
Basic fibroblast growth factor
(bFGF)1 is a potent mitogenic
factor that is also known to initiate changes important for neural differentiation, survival, and plasticity (1). Mechanisms underlying these diverse actions of bFGF are not well understood but may result,
at least in part, from distinct signaling pathways controlling gene
expression. The bFGF receptor belongs to the tyrosine kinase class of
membrane receptors. The binding of bFGF to its receptor results in the
activation of Ras-dependent mitogen-activated protein kinase (MAPK) cascade (2). Coupled phosphorylation events induce the
sequential activation of Raf-1 kinase, MAPK kinase (MEK), and MAPK (or
extracellular signal-regulated protein kinase (ERK)) (3).
Multiple signaling pathways converge at the level of the cyclic AMP
response element (CRE)-binding protein (CREB), a transcriptional factor
that regulates expression of CRE-containing genes (4). CREB mediates
cellular responses to a variety of physiological signals, including
neurotransmitters, depolarization, synaptic activity, mitogenic and
differentiating factors, and stressors (5-9). Upon phosphorylation at
Ser133, CREB can facilitate transcriptional activation of
the genes containing the CRE motif (10). The activity of CREB is
regulated after various kinds of stimulation by multiple kinases,
including protein kinase A (PKA), protein kinase C (PKC), isozymes of
calcium/calmodulin-dependent kinase (CaM kinase) (11-13),
p90RSK (14), p70S6K (15), and MAPK-activated
protein (MAPKAP) kinase-2 (7). Moreover, phosphorylation of CREB at
Ser133 regulates expression of the c-fos,
somatostatin, and tyrosine hydroxylase genes in PC12 cells (10,
16).
To identify signaling pathways transmitting extracellular FGF signal to
nucleus, the induction mechanism of immediate early gene
pip92 by bFGF was previously studied in rat hippocampal
progenitor H19-7 cells. Although signaling through Raf-1 occurs
exclusively through the SRE, bFGF can also activate a region of the
pip92 promoter that contains a CREB binding site near the
site of transcription initiation (17). Given the role for CREB in
regulating genes that mediate a multitude of cellular responses and to
investigate the signaling mechanisms by which bFGF regulates gene
expression, we examined the effect of bFGF-induced neuronal
differentiation on the activation of CREB and subsequent CRE-mediated
gene transcription in neuronal H19-7 cells. The present study suggests
that CRE-mediated gene transcription appears to be important during the
differentiation of neuronal H19-7 cells induced by bFGF and the
activation of novel protein kinase signaling pathway is required for
the bFGF responsiveness.
Materials--
Fetal bovine serum, Dulbecco's modified Eagle's
medium, hygromycin, and geneticin were purchased from Life
Technologies, Inc. PD98059, SB203580, Ro-31-8220, LY294002, rapamycin,
KT5720, and KN-62 were purchased from Calbiochem (La Jolla, CA).
Rp-cAMPS, 2-hydroxy-5-(2,5-dihydroxybenzylamino)benzoic acid,
herbimycin A, and tyrphostin 47 were purchased from Biomol (Plymouth
Meeting, PA). EGF and bFGF were purchased from Bachem (Bubendorf,
Switzerland). The assay kits of PKC and CaM kinase 2 were purchased
from Upstate Biotechnology, and a colorimetric PKA assay kit (Spinzyme
format) was purchased from Pierce. Anti-phosphoCREB and CREB antibodies were purchased from Upstate Biotechnology (Lake Placid, NY), and anti-phospho-p70S6K was from New England Biolabs (Beverly,
MA). All other chemicals were purchased from Sigma. Plasmids encoding
wild type and mutant GST-CREB were provided by M. Comb. Mammalian wild
type and mutant CREB expression plasmids (pCG-CREB, PCG-L, and
pCG- Cell Culture--
The rat neuronal hippocampal progenitor cell
line (H19-7) was generated by transduction with the retroviral vectors
containing temperature-sensitive simian virus 40 large T antigen that
is functionally active at 33 °C and inactive at 39 °C as
described elsewhere (18). Transient Transfection and Luciferase Assay--
pCRE-TK-Luc, A
reporter plasmid was transiently transfected by using a LipofectAMINE
reagent (Life Technologies, Inc.) either alone or with kinase-inactive
SEK or MEKK mutant plasmid, as indicated. Plasmid pCMV-EGFP
(CLONTECH), which contains jellyfish green
fluorescent protein gene driven by the cytomegalovirus promoter, was
used as an internal control to determine transfection efficiency. In every transfection experiment, the CRE-lacking thymidine kinase (TK)
promoter construct (pTK-Luc) was used as a negative control. Luciferase
activity was measured by using a luciferase assay kit (Promega) and
luminometer (EG & G Berhold, Germany). When specified, the cells were
stimulated with 10 µM forskolin as a positive control for
CRE-mediated gene transcription.
Western Blot Analysis--
Cells were solubilized with lysis
buffer A containing 20 mM Tris, pH 7.9, 137 mM
NaCl, 5 mM Na2EDTA, 10% glycerol, 1.0% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, 20 µM leupeptin, 1 mM
Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM tetrasodium pyrophosphate, and 1 mM Measurement of PKC, PKA, and CaM Kinase Activity--
PKA
activity was measured using the colorimetric PKA assay kit. The treated
cells were washed with ice-cold phosphate-buffered saline twice,
resuspended in lysis buffer (50 mM Tris-HCl, 2.5 mM EDTA, 1 mM MgCl2, 10 mM NaF, 10% glycerol, pH 7.2), sonicated, and centrifuged.
Supernatants were subsequently taken as lysates. 30 µg of cell lysate
was used to measure PKA activity. The activities of PKC and CaM kinase
2 were measured by using PKC and CaM kinase 2 assay kit (Upstate
Biotechnology) as described in the manufacturer's protocol. The final
volume of each incubation sample was 60 µl, and reaction was
terminated by transfer of 25 µl onto phosphocellulose filter.
The radioactive phosphopeptide bound to the filter was quantitated
after 1% phosphoric acid wash.
CK2 Assay--
The assay for phosphotransferase activity of CK2
was conducted in a reaction mixture containing 20 mM
Tris-HCl, pH 7.5, 120 mM KCl, 10 mM
MgCl2, and 100 µM [ In Vitro in Gel Kinase Assay--
A 12.5% gel for SDS-PAGE was
prepared by using 50 µg of bacterially expressed wild type or mutant
GST-CREB/ml as the substrate for phosphorylation. The cell extracts
stimulated with bFGF in the absence or presence of various protein
kinase inhibitors were applied to the gel. All gel renaturation and
phosphorylation protocols were performed as described elsewhere (20).
CREB Activates CRE-mediated Gene Transcription in Response to bFGF
in H19-7 Cells--
To assess whether bFGF exerts its stimulatory
effect on the activation of transcription factor CREB and subsequent
CRE-mediated gene transcription, we assayed the gene expression of
CRE-containing TK promoter-reporter construct. Treatment of H19-7 cells
with bFGF resulted in the increase of CRE-mediated gene transcription in a time-dependent manner, and it reached a plateau after
2 h (Fig. 1). To test the role of
CREB phosphorylation on CRE-mediated gene transcription, cells were
transfected transiently with pCRE-TK-Luc reporter plasmid plus mutant
CREB expression vector, such as pCG-L, in which RRPSY amino acids
130-134 of CREB are replaced by RRSLY, or pCG- Expression of S133A CREB Mutant Blocks bFGF-induced Neurite
Outgrowth in H19-7 Cells--
The functional role of CREB activation
during FGF-induced differentiation of H19-7 cells was further examined.
Treatment of H19-7 cells with bFGF induces differentiation at 39 °C
at which large T antigen is inactivated (21). Differentiated cells are resistant to mitogenic stimulation by serum and express neuronal markers, such as neurofilament and brain type II sodium channel. A
dominant-negative construct encoding CREB protein with
Ser133 phosphorylation site mutated to Ala133
(pRSV-CREB S133A) was used to block the activation of CREB, and a
jellyfish green fluorescent protein gene (GFP) (pCMV-EGFP) was used as
a marker for the transfected cells, respectively. Either empty or
mutant CREB expression vector (pRSV-CREB S133A) was cotransfected along
with pCMV-EGFP, and subsequently the formation of neurite outgrowth in
GFP-positive cells was analyzed in H19-7 cells. Previously, it was
determined that the efficacy for expressing cotransfected plasmids in
the same cell is about 80% in H19-7 cells (22). As shown in Fig.
2, untransfected control cells not
expressing GFP had a similar percentage of differentiated cells
(~63%) in two separate transfection experiments. However, cells that
express GFP in the mutant CREB-transfected population had only 27%
differentiated cells, in contrast to 58% differentiated cells in the
pCMV-transfected population. Taken together, these results suggest that
a relatively stable CREB phosphorylation by bFGF is likely to play a
role in the differentiation of neuronal H19-7 cells.
EGF and bFGF Stimulates CREB Phosphorylation at Ser133
Residue in a Distinct Kinetic Pattern in H19-7 Cells--
To confirm
the previous finding that bFGF exerts its stimulatory effect on the
activation of CREB during CRE-mediated gene transcription, Western blot
analysis was performed by using an antibody specific for the
Ser133-phosphorylated form of CREB. During the
differentiation of H19-7 cells by bFGF, Ser133 residue in
the CREB protein was phosphorylated rapidly and sustained for 1-2 h
after growth factor stimulation (Fig. 3).
Differences in CREB phosphorylation are known to be critical in the
determination and regulation of EGF-mediated proliferation and
NGF-induced differentiation of neuronal PC12 cells (5). Like PC12,
H19-7 cells respond differentially to EGF and bFGF. Although EGF
treatment induces a proliferation at the permissive temperature
(33 °C), the addition of bFGF, but not EGF, induces differentiation
at the nonpermissive temperature (39 °C) (21). Based on this
finding, it was examined whether CREB phosphorylation occurred
differentially by EGF and bFGF in H19-7 cells. In contrast to prolonged
CREB phosphorylation by bFGF, EGF treatment, leading to proliferation
but not differentiation, induced transient CREB phosphorylation, and it
was declined to basal levels within 30 min (Fig. 3). Taken together,
these results suggest that stable CREB activation by bFGF is important
to make a decision on the fate of hippocampal progenitor cell to
terminally differentiate to neuronal cells.
FGF-dependent CREB Phosphorylation Does Not Require the
Activation of Extracellular Signal-regulated Kinases in H19-7
Cells--
Upon binding to bFGF, its receptor dimerizes and activates
an intrinsic tyrosine kinase activity leading to the increase in intracellular calcium, phosphoinositide turnover, phosphorylation of
intracellular proteins, and activation of immediate early genes, such
as c-fos and myc (23). To clarify the downstream
signaling pathways of bFGF, the effect of receptor tyrosine kinase
inhibitors on bFGF-induced CREB phosphorylation was examined. As shown
in Fig. 4A, herbimycin A, a
benzoquinoid ansamycin antibiotic, which irreversibly and
selectively inhibits receptor tyrosine kinases by reacting with thiol
groups, completely blocked the CREB phosphorylation by bFGF in H19-7
cells. Pretreatment of the cells with tyrphostin 47, another selective
inhibitor of receptor tyrosine kinases, also decreased CREB
phosphorylation to the basal levels, compared with that of bFGF alone
(Fig. 4A). Stimulation of bFGF receptors is known to
activate Ras and a subsequent kinase cascade culminating in the
activation of p42 and p44 ERKs (2). To identify early events in the
signaling pathway leading to CREB phosphorylation, the role of Ras
activation was examined. Transient transfection of dominant-negative
Ras mutant (pMT3RasN17) significantly inhibited the activation of ERK,
but not CREB phosphorylation by bFGF (Fig. 4B), suggesting
that CREB is phosphorylated via Ras-independent signaling pathway
during the differentiation of H19-7 cells by bFGF.
In neuronal PC12 cells, neurogenic NGF activates the ERKs, which in
turn activate pp90 ribosomal S6 kinase family of Ser/Thr kinases
(p90RSK), all three members of which were found to catalyze
CREB Ser133 phosphorylation (14). A major pathway by which
p90RSK is activated by growth factor receptors involves
sequential activation of Raf, MEK, and ERK. It was previously shown
that the stimulation of H19-7 cells with bFGF induced the activation of
the Raf-MEK-ERK pathway, resulting in the differentiation (22). To
analyze the initial signals generated by Raf-1, H19-7 cells were
transfected with a vector expressing Activation of Stress-activated Protein Kinases, such as JNK, and
p38 Kinase Is Not Involved in the bFGF-induced CREB Phosphorylation and
the Differentiation of H19-7 Cells--
In addition, MAPK-activated
protein kinase-2 (MAPKAP kinase-2), an enzyme that lies immediately
downstream of p38 kinase, was recently shown to mediate CREB
Ser133 phosphorylation in neuroblastoma SK-N-BE cells
exposed to bFGF (7). To investigate the effect of stress-activated
protein kinases, such as JNK and p38 kinase, on bFGF-induced CREB
phosphorylation, H19-7 cells were pretreated with chemical p38 kinase
inhibitor, SB203580 or transiently transfected with kinase-deficient
SEK mutant cDNA, and subsequently bFGF-dependent CREB
phosphorylation and subsequent induction of luciferase activity by
CRE-thymidine kinase promoter were measured. As shown in Fig.
6, 30 µM SB203580 or
kinase-deficient SEK mutant had no effect on the ability of bFGF to
induce CREB phosphorylation (Fig. 6A) and to stimulate CRE-mediated luciferase activity (Fig. 6B), suggesting that
CREB phosphorylation is not mediated by the activation of
stress-activated JNK or p38-MAPKAP kinase-2 pathway. As a control, we
previously observed that pretreatment of SB203580 or transient
transfection of kinase-inactive SEK results in a significant inhibition
of p38 induced by anisomycin (25) or JNK activity by NMDA in
H19-7 cells (26). In addition, neither JNK nor p38 was shown to be significantly activated by activated Raf-1 (17) or
bFGF2 within the first few
hours of stimulation in
Consistent with the result in Fig. 5, pretreatment of the cells with 30 µM MEK inhibitor PD98059 for 30 min did not inhibit the
activation of reporter luciferase by bFGF (Fig. 6). As a positive control, the addition of adenylate cyclase activator, 10 µM forskolin, increased the luciferase activity ~5-fold
greater than that of FGF. Taken together, these results indicated that
bFGF-induced CREB phosphorylation is not mediated via the activation of
stress-activated JNK or p38 signaling pathways.
bFGF-dependent CREB Phosphorylation Is Not Likely to Be
Mediated by PKA, PKC, CaM Kinase, PI-3K/p70S6K, and CK 2 in
H19-7 Cells--
The binding of bFGF to its receptor is known to
induce receptor dimerization, autophosphorylation at
Tyr766, and activation of phospholipase C, which in turn
activates PKC. Because Ser133 residue of CREB is contained
within a consensus sequence of PKC phosphorylation and is
phosphorylated by PKC (27), we examined whether TPA-sensitive isoforms
of PKC affect the levels of CREB phosphorylation during the FGF-induced
differentiation of H19-7 cells. Pretreatment of the cells with
competitive PKC inhibitor, Ro-31-8220, or chelerythrine chloride failed
to inhibit bFGF-induced phosphorylation of CREB (Fig.
7A), suggesting that bFGF
stimulates CREB phosphorylation via a pathway distinct from that
activated by PKC. In addition, bFGF-induced CREB phosphorylation was
not changed significantly with other PKC inhibitors, such as calphostin C and hypericine (data not shown). As a control, the PKC activation by
phorbol 12-myristate 13-acetate was inhibited to the basal level by
Ro-31-8220 and chelerythrine at a similar concentration (Fig.
7B).
Many growth factors activate p70S6K, a protein kinase that
is activated by a Ras-independent pathway (28) and that appears to be
triggered by the activation of PI-3K (29, 30). Both CREB and CRE
modulator, another member of the CREB family, were efficiently
phosphorylated in vitro by p70S6K (15). The
macrolide rapamycin is an efficient and specific inhibitor of the
mitogen-induced activation of p70S6K (31). LY294002, a
selective PI-3K inhibitor, inhibits mitogenesis, glucose transport, and
activation of p70S6K (30, 32). When the H19-7 cells were
pretreated with 50 ng/ml rapamycin or 10 µM LY294002, the
levels of CREB phosphorylation induced by bFGF were not changed
remarkably (Fig. 7C). As a control, addition of LY294002 or
rapamycin at the same concentration blocked the activation of
p70S6K by serum in H19-7 cells (Fig. 7D).
Ser133 residue of CREB is phosphorylated in response to an
increase in intracellular cAMP and Ca2+ concentration by
PKA (10, 11) and/or by CaM kinase (12, 13, 33). When H19-7 cells were
pretreated with 0.5 µM KT5720, specific PKA inhibitor, or
0.1 µM KN-62, CaM kinase antagonist (34, 35),
serum-induced PKA activation (Fig.
8B.), and the activation of
CaM kinase 2 by ionomycin (Fig. 8C) were remarkably blocked,
respectively. However, pretreatment of the cells with KT5720 or KN62
did not change the levels of FGF-induced CREB phosphorylation significantly, compared with that of bFGF alone (Fig. 8A)
In addition, pretreatment of the cells with other specific inhibitors
for PKA and CaM kinase 2, such as K-252a and
2-hydroxy-5-(2,5-dihydroxybenzylamino)benzoic acid, did not cause a
remarkable decrease of CREB phosphorylation by bFGF (data not
shown).
CREB is known to be a substrate of CK2 (36). Recently, CREB was
reported to be phosphorylated in a cell cycle-dependent manner, and the pDE-1 domain (Ala106-Gln122) of
CREB is phosphorylated by CK2 (37). Although the addition of specific
CK2 inhibitors,
5,6-dichloro-1-
Furthermore, when the cells were pretreated with rapamycin, KT5720,
DRB, and/or KN-62 together for 30 min prior to bFGF stimulation, the
levels of CREB phosphorylation were not changed noticeably (data not
shown). Overall, these results suggested that
bFGF-dependent CREB phosphorylation does not require the
activation of previously known CREB kinase pathways, such as PKA,
PKC, PI-3K/p70S6K, CaM kinase, or CK2.
Identification of Novel CREB Kinase(s) Activated by bFGF during the
Differentiation of H19-7 Cells--
To identify bFGF-inducible CREB
kinase(s), in vitro in gel kinase assays were performed by
using either wild type or mutant GST-CREB proteins as the substrates.
Extracts containing equal protein from H19-7 cells that had been
stimulated with 10 ng/ml bFGF in the absence or presence of combined
various protein kinase inhibitors were resolved by SDS-PAGE, renatured,
and assayed for CREB phosphorylation in the gel. As shown in Fig.
9, a slight and constitutively active
kinase of ~36-kDa was observed. In addition, the results showed two
previously unreported CREB kinases of ~76 and 120 kDa are activated
markedly by bFGF (Fig. 9A). Although pretreatment of the
cells with the inhibitors of PI-3K, PKC, PKA, and CaM kinase does not
affect the activity of 76-kDa kinase, it has a high basal activity
without bFGF. In contrast, the 120-kDa kinase is greatly inducible by
bFGF treatment. When the protein kinase inhibitors for PKA and PKC were
added during the kinase assay as well as the treatment of H19-7 cells,
the activation pattern of two novel kinases was not affected (data not
shown), implying that the novel CREB kinases are not regulated by
protein dissociation. Furthermore, no significant kinase activity was detected when the mutant GST-CREB S133A, in which Ser133
residue of CREB had been mutated to Ala133 (Fig.
9B), or GST protein (data not shown) was used as a
substrate. These results suggested that a novel bFGF-inducible CREB
kinase of 120 kDa phosphorylates the Ser133 residue and is
likely to play an important role in the differentiation of immortalized
hippocampal neuronal cells.
The present study demonstrated that bFGF stimulates the
phosphorylation of CREB at Ser133, which plays an important
role during the differentiation of neuronal H19-7 cells. This
post-translational modification leads to an increase in the
transcriptional activity of CREB, as shown by using a CRE-TK-Luc
reporter system. CREB was shown to play an important role in neuronal
differentiation. NGF induces CREB phosphorylation (38), and a
dominant-negative ATF1 blocks neurite outgrowth in a subcell line of
PC12 (39). In F11 neuroblastoma cells, cAMP induced neurite outgrowth
and activated CREB (40).
A number of different kinases may be capable of mediating CREB
phosphorylation under different circumstances, although the relative
contributions of particular kinases in cells have not been clearly
determined. PKA, PKC, CaM kinase, CK2, MAPKAP kinase-2, p60S6K, and p90RSK have been reported to
phosphorylate CREB. By using specific inhibitors or kinase-deficient
mutant cDNA, we demonstrated that bFGF-mediated CREB activation
appears not to require any of the signaling pathways leading to the
phosphorylation of CREB in H19-7 cells. We do not rule out the
possibility that a redundant combination of two known pathways is
responsible for CREB phosphorylation, and the use of inhibitors are not
sufficiently exhaustive and detailed to allow definite conclusion
regarding the identity of the isoforms involved. For example, a
particular p38 isoform, such as p38 In addition, cAMP activated the transcription factor Elk-1 and induced
neuronal differentiation of PC12 cells via its activation of the MAPK
cascade (41). These cell type-specific actions of cAMP require the
expression of the serine/threonine kinase, B-Raf, and activation of the
small G protein, Rap1. Rap1, activated by mutation or by PKA, is a
selective activator of B-Raf and an inhibitor of Raf-1. Thus, it is
possible that the differentiating signal of bFGF is transmitted through
the activation of B-Raf, but not by Raf-1, which will also be important
for the differentiation of H19-7 cells. However, based on the finding
that combined treatment of the cells with various protein kinase
inhibitors altogether did not attenuated the levels of phosphorylated
CREB by FGF (Figs. 8 and 9), and the size of novel CREB kinase obtained
from an in gel kinase assay does not correspond to the reported sizes
of those protein kinases, our current study shows the presence of a new
growth factor-activated signaling pathway that regulates gene
expression at the CRE.
The 15-min delay of initial CREB phosphorylation peak in the presence
of PD980509 initially made us think that bFGF signals are transmitted
through two pathways, which was implicated in FGF-induced induction of
pip92: a transient one for Raf-1-MEK-ERK that parallels the
time-dependent curve of EGF and the other one for novel
kinase (17). A loss in the signal during initial time points when ERK
is suppressed could be explained with this interpretation. However, the
results in Figs. 5 and 6 and from the previous pip92 deletion analysis (17) indicated that the activation of Raf-1-MEK-ERK pathway is not required for the CREB phosphorylation by bFGF. Inconsistent with these findings, transient CREB phosphorylation by EGF
is not suppressed considerably in the presence of
PD98059.2
Recently, Akt was shown to promote phosphorylation of CREB and activate
cellular gene expression via a CRE-dependent mechanism (42). Akt is rapidly and specifically activated by diverse ligands, such as platelet-derived growth factor, EGF, and FGF, and promotes cell
survival (43). Several lines of evidence support the possibilities that
ligand-induced activation of Akt is mediated through PI-3K signaling
and that Akt may represent novel PI-3K targets. Based on the findings,
it was tested whether the activation of Akt is involved in the
activation of CREB by bFGF in H19-7 cells. The finding that PI-3K
inhibitor, LY294002, was not blocking bFGF-induced CREB phosphorylation
by bFGF, and the molecular mass of novel CREB kinase did not
correspond to that of Akt (60 kDa) suggests that the activation of Akt
is not necessary for CRE-mediated gene induction by bFGF. Similar to
our findings, exposure of PC12 cells to physiological levels of hypoxia
rapidly induced a phosphorylation of CREB, and this effect was not
mediated by any of the previously known CREB activation pathways
(44).
Although EGF and bFGF initially induce very similar intracellular
signaling pathways, these two growth factors ultimately elicit very
different cellular responses in H19-7 cells. Likewise, PC12 cells, when
exposed to NGF, traverse the cell cycle several times and then
differentiate into postmiotic cells that in many ways resemble
sympathetic neurons (45). In contrast to NGF, EGF is a mitogen for PC12 cells.
By controlling the phosphorylation of transcription factors such as
Elk-1 and CREB and thereby regulating the expression of immediate early
gene and possibly delayed response gene, MAPK pathway was suggested to
transmit the signals of divergent cell fates, including proliferation
and differentiation. FGF, NGF, or EGF induces the MAPK pathway with
different time courses, and the difference in kinetics accounts for the
differential response of PC12 and H19-7 cells to these two agents (17,
22, 46). In both cells exposed to NGF (PC12) or bFGF (H19-7), sustained activation of the MAPK lasts for several hours. In contrast, treatment with the mitogenic EGF leads to transient activation of the MAPK signaling pathways, lasting only minutes after the initial EGF stimulus
(47, 48). In support of this finding, when EGF receptors are
overexpressed in PC12 cells, the time course of EGF-induced Ras-MAPK
activation is prolonged, and the overexpressing PC12 cells
differentiate along a neuronal pathway in response to EGF (49).
Segal and Greenberg (50) proposed that sustained activation of the Ras
signaling pathway in NGF-treated PC12 cells can result in the sustained
phosphorylation of transcription factors such as CREB. This may allow
CREB to selectively activate delayed response genes that have
CREB-binding sites within their regulatory regions. Such delayed
response genes would be activated in response to NGF, but not EGF, and
might encode proteins that contribute to the acquisition of a neuronal
phenotype (5). Consistent with this possibility, CREB-binding sites
have been found within the promoters of genes that respond to NGF with
delayed kinetics, such as tyrosine hydroxylase (51), transin (52), and
neuronal secretary protein, VGF (53).
The current model for neuronal differentiation based on PC12 cells is
that prolonged activation of ERK is both necessary and sufficient for
differentiation. However, recent reports have shown that this model is
not generally applicable in other neuronal cells. For example,
prolonged MAPK is not sufficient for the differentiation of
H19-7 cells, even though prolonged activation of Raf-1 is sufficient (22), indicating that under physiological conditions MAPK is neither
necessary nor sufficient. Furthermore, our current finding that
Raf-MAPK pathways are not required for CREB phosphorylation by bFGF in
H19-7 cells suggests that mitogenic and differentiating signals
transmit through distinct pathways within PC12 and H19-7 cells, respectively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CREB S119A), pCRE-TK-Luc, and parental pTK-Luc vectors were
kindly provided by K. Saeki. A plasmid encoding mutant CREB with
Ser133 phosphorylation site mutated to Ala133
(pRSV-CREB S133A) was provided by J. Eberwine. Dominant-negative SEK
mutant, pSEK1(AL)/EE-CMV, and pEE-CMV DNAs were kindly provided by D. Templeton, and dominant inhibitory Ras mutant was provided by C. Marshall.
Raf-1:ER cells were made by stable
transfection of H19-7 cells with estradiol-regulated Raf-1 generated by
fusing a constitutively active, oncogenic portion of human Raf-1 to the hormone-binding domain of human estrogen receptor as described elsewhere (19). The proliferating cells were cultured at 33 °C in
medium containing 10% fetal bovine serum and 200 µg/ml of geneticin
to maintain selection on the transduced immortalization vector.
Raf-1:ER cells were also grown in hygromycin. Prior to differentiation, cells were shifted to 39 °C in N2 medium for 2 days
(H19-7 cells) or 1 day (
Raf-1:ER cells). The H19-7 cells were
differentiated with 10 ng/ml bFGF.
Raf-1:ER cells were
differentiated with 1 µM estradiol. As a measure of
neuronal differentiation, morphological changes were quantitated by
measuring the length of processes in differentiated cells, and the
expression of neuronal markers were measured by immnuocytochmistry and
immunoblotting. Differentiated cells are defined as those cells
containing at least one neurite longer than the diameter of cell body.
When specified, cells were pretreated with 30 µM of p38
kinase inhibitor, SB203580, or MEK inhibitor, PD98059, 30 min prior to
bFGF stimulation to block the activation of p38 or MEK. When indicated,
the cells were pretreated with LY294002, rapamycin, KT5720, or KN62 for 30 min to block the activation of PI-3K, pp70S6K, PKA, or
CaM kinase 2, respectively.
-glycerophosphate. Then cell extracts were resolved
by SDS-PAGE and transferred to a nitrocellulose membrane. After
blocking, the membranes were incubated with a suitable antibody,
according to the manufacturer's protocol. The membrane was then
incubated with a peroxidase-conjugated secondary antibody, and the
bands were visualized by ECL (Amersham Pharmacia Biotech).
-32P]ATP
in the presence of 5 mg/ml
-casein in a total volume of 30 µl. The
reaction was started by the addition of cell lysate including enzyme
and incubated at 37 °C for 1 h. The reaction was terminated by
spotting 10 µl of the reaction mixture on to P-81 phosphocellulose
paper. The paper was washed in 100 mM phosphoric acid, and
the radioactivity was measured by scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CREB S119A, in which
amino acids 88-101 of CREB are spliced out and Ser119, a
target of protein kinase A that corresponds to Ser133 of
CREB is replaced to Ala119. Expression of CREB proteins
with the mutation of critically regulatory Ser133 residue
significantly inhibited the activation of luciferase activity by bFGF
(Fig. 1). These results imply that the stimulation of H19-7 cells with
bFGF caused CREB activation, possibly through the phosphorylation of
its Ser133 residue followed by the activation of
CRE-mediated gene transcription.
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Fig. 1.
bFGF-induced activation of CRE-mediated gene
transcription via cis-regulatory CRE motif. 1 µg of DNA of pCRE-TK-luciferase reporter plasmid was transiently
transfected into immortalized hippocampal H19-7 cells with 5 µg of
either parental vector, pCG or mutant CREB expression vector pCG-L, or
pCG- CREB S119A. Then the cells were stimulated with 10 ng/ml bFGF
for the indicated time, and the luciferase activity of reporter plasmid
was measured. Data are plotted as the percentages of maximum luciferase
activity and represent the means plus the ranges of the samples from
three independent experiments in triplicate.
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Fig. 2.
Effect of dominant-negative CREB on
bFGF-induced neurite outgrowth in H19-7 cells. A, the
cells were either untreated (Control) or cotransfected with
empty parental control vector (P) or construct encoding
dominant-negative CREB (mCREB), plus pCMV-EGFP vectors
(GFP). The cells were subsequently stimulated with bFGF
under differentiation condition, and the GFP-expressing cells were
scored for differentiation as judged by morphological changes. The
differentiation percentage of transfected cells was obtained by
dividing the number of differentiated and GFP-expressing cells by total
number of GFP expressing cells. The total numbers of GFP-expressing
cells counted were 200-250 for empty and mutant CREB vectors,
respectively, and the numbers of untransfected cells counted were
~200. B, micrographs of the H19-7 cells from A
that were cotransfected with S133A mutant CREB and pCMV-EGFP vectors
and subsequently treated with bFGF. The GFP-positive cells were
observed by fluorescence microscopy (GFP), and the same
field of the cells was visualized by phase-contrast microscopy
(Morphology). The GFP-positive cells are indicated by
arrows.
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Fig. 3.
Differential kinetic pattern of CREB
phosphorylation by either bFGF or EGF and its functional role during
the differentiation of H19-7 cells. The cells were stimulated with
10 ng/ml bFGF or 10 nM EGF for the indicated time. The
phosphorylated and endogenous CREB proteins were identified by Western
blot analysis (A). These results are representative of two
independent experiments. In B, phosphorylated CREB bands
were quantified with PhosphorImager analyses.
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Fig. 4.
Inhibition of bFGF-inducible CREB
phosphorylation by the receptor tyrosine kinase inhibitors but not by
dominant-negative Ras in H19-7 cells. H19-7 cells were stimulated
with 10 ng/ml bFGF for 30 min in the absence or presence of 1 µmol/ml
of herbimycin A (Her), or 10 µmol/ml of tyrphostin 47 (Tyr), respectively (A). When specified, cells
were transiently transfected with 5 µg of dominant-negative Ras
expression plasmid, pMT3RasN17 (Ras N17) (B). Phosphorylated
ERK and CREB proteins were identified by Western analysis. These
results are representative of two independent experiments. As a control
for equal protein loading, the amounts of nonactivated CREB were
measured.
Raf-1:ER (24). Upon exposure to
estradiol (E2),
Raf-1:ER is activated within minutes, enabling one
to monitor downstream signaling events after Raf-1 activation. As shown
in Fig. 5A, stimulation of
Raf-1:ER cells with bFGF caused a rapid and prolonged CREB
phosphorylation, which was similar to that in H19-7 cells. To test
whether CREB was phosphorylated by the Raf-1-MEK-ERK-p90RSK
signaling pathway,
Raf-1:ER cells were pretreated with 30 µM MEK inhibitor PD98059 for 30 min before bFGF
stimulation. Although PD98059 completely blocked the activation of ERK
by bFGF in
Raf-1:ER cell (19), there was no significant inhibition
of CREB phosphorylation (Fig. 5B), interestingly with a
delay of initial CREB phosphorylation by ~15 min. When
Raf-1:ER
cells were stimulated with 1 µM estradiol leading to the
activation of Raf-1 kinase followed by the sequential activation of ERK
pathway and the differentiation of the cells (19), CREB was not
phosphorylated at all (Fig. 4). The ERK induction by estradiol is due
to selective Raf-1 activation, because no induction of ERK by estradiol
was observed in the parent H19-7 cells lacking the
Raf-1:ER fusion
protein (17). Taken together, these data indicated that bFGF-induced
signals leading to the CREB phosphorylation are not transmitted through
Raf-MEK-ERK-p90RSK pathway in H19-7 cells.
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Fig. 5.
bFGF-inducible CREB phosphorylation is
independent of the activation of Raf-1 kinase in neuronal H19-7
cells. Raf-1:ER cells were untreated (NoT) or
stimulated with (A and B) 10 ng/ml bFGF
(FGF) or (C and D) 1 µM
estradiol (E2) for the indicated time, respectively. When
specified, cells were pretreated with 30 µM MEK
inhibitor, PD98059 (MI) for 30 min before bFGF or E2
stimulation (B). Phosphorylated CREB and ERK proteins were
measured by using Western analysis. These results are representative of
three independent experiments. Cont, control.
Raf-1:ER and H19-7 cells.
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Fig. 6.
Effect of JNK and p38 kinase activity on
bFGF-induced CREB phosphorylation and subsequent CRE-mediated gene
transcription in H19-7 cells. A, where indicated, 5 µg of kinase-inactive SEK mutant DNA was transiently transfected into
the cells. The cells were stimulated with bFGF for 1 h in the
absence or presence of 30 µM SB 203580, and subsequently
CREB phosphorylation was measured. B, 1 µg of DNA of
pCRE-TK-luciferase reporter plasmid was transiently transfected into
H19-7 cells either alone or with 5 µg of a kinase-inactive mutant SEK
(mSEK*). Where indicated, cells were pretreated with 30 µM PD98059 or 50 µM SB203580 for 30 min.
Then the cells were stimulated with 10 ng/ml bFGF for 1 h, and the
luciferase activity of reporter plasmid was measured. Data are plotted
as the percentages of maximum luciferase activity and represent the
means plus the ranges of the samples from two independent experiments
in triplicate.
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Fig. 7.
Effect of specific protein kinase inhibitors,
such as PKC, PI-3K, or S6 kinase on bFGF-induced CREB phosphorylation
in H19-7 cells. In A and C, H19-7 cells were
either untreated (NT) or pretreated with 20 µM
chelerythrine chloride (CC) or 100 nM Ro-31-8220
(Ro), 50 ng/ml rapamycin (R), or 10 µM LY294002 (LY), for 30 min to block the
activation of PKC or PI-3K-p70S6K, respectively. The cells
were either untreated (C) or stimulated with 10 ng/ml bFGF
(FGF), and Western blot analysis was performed to identify
phosphorylated CREB bands. In B, after addition of 10 µM of phorbol 12-myristate 13-acetate (PMA) in
the absence or presence of 20 µM chelerythrine chloride
(C) or 100 nM Ro-31-8220 (Ro) for 30 min, the PKC activity was measured. In D, the activation of
p70S6K by 10% serum in the absence or presence of 50 ng/ml
rapamycin (R) or 10 µM LY294002
(LY) was measured. These results are representative of two
or three independent experiments.
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Fig. 8.
Effect of the specific inhibitors of PKA, CaM
kinase, or CK2 on bFGF-induced CREB phosphorylation. In
A and D, H19-7 cells were untreated
(NT or C) or pretreated with 0.5 µM
KT5720 (KT), 0.1 µM KN-62 (KN), 100 µM DRB or 300 µM heparin (Hep)
for 30 min to block the activation of PKA, CaM kinase 2, or CK2,
respectively. The cells were then stimulated with 10 ng/ml FGF
(F), and Western blot analysis was performed to identify the
phosphorylated CREB bands. These results are representative of two or
three independent experiments. In B, C, and
E, the cells were grown on DMEM containing 1% fetal bovine
serum at 33 °C for 24 h. Where indicated, the cells were
pretreated with KT5720 (B), KN62 (C), DRB, or
heparine (Hep) for 30 min, and 10% serum (PKA and CK2) or
500 nM ionomycin (CaM kinase 2 and Iono) was added directly
to the culture medium. The treated cells were harvested, and then PKA,
CaM kinase 2, or CK2 activity was measured. Values are the means ± S.E. of three independent experiments.
-D-ribofuranosylbenzimidazole (DRB) or
heparin (average molecular weight, 3000) completely blocked the
induction of CK2 activity by serum (Fig. 8E), it did not
attenuate bFGF-induced CREB phosphorylation, respectively (Fig.
8D).
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Fig. 9.
Identification of novel bFGF-inducible CREB
kinases in H19-7 cells. H19-7 cells were untreated (NT)
or pretreated with various protein kinase inhibitors (PKIs),
including LY294002, KT5720, KN-62, and Ro-31-8220 for 30 min, and then
stimulated with 10 ng/ml bFGF for 30 min. Cell extracts containing
40-50 µg of proteins were resolved by SDS-PAGE gel containing 50 µg/ml of bacterially expressed wild type GST-CREB (A) or
mutant GST-CREB S133A (B) as a substrate. The in gel
renaturation assay was performed, and the positions of protein
molecular mass markers are shown on the left-hand
side of figure. Two novel CREB kinases that are activated by bFGF
and are distinct from other already known CREB kinases are indicated by
filled arrows, and a constitutive and slight active CREB
kinase is depicted by an open arrow on the left-hand
side. These results are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, is not sensitive to SB203580 inhibition.
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ACKNOWLEDGEMENTS |
---|
We are deeply grateful to Marsha R. Rosner for support and for critically reading the manuscript. We thank J. Kim, J. Chung, M. G. Lee, and D. G. Kim for helpful discussions, B. Ross for reading the manuscript, and H. S. Kang, J. H. Kim, and Y. Bae for excellent technical assistance. We also thank M. Comb for providing the cDNA construct encoding GST-CREB, K. Saeki and J. Eberwine for wild type and various mutant CREB expression vectors, D. Templeton for dominant-negative SEK mutant, pSEK1(AL)/EE-CMV, and pEE-CMV, and C. Marshall for pMT3RasN17 plasmid.
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FOOTNOTES |
---|
* This work was supported by Brain Science Research Grant 98-J04-02-01-A-08 from Korea Institute for Science and Technology Evaluation and Planning (KISTEP) (to K. C. C.), Brain Korea 21 Projects for Medical Science in Yousei University (to J. Y. S.), and in part by National Institutes of Health Grant NS33858 (to Marsha R. Rosner at the University of Chicago where this work originated).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Present address: Dept. of Pharmacology, Yonsei University College of Medicine, Shinchon-Dong 134, Seodaemun-Gu, Seoul 120-752, Korea. Tel.: 82-2-361-5229; Fax: 82-2-313-1894; E-mail: kchung@yumc.yonsei.ac.kr.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010610200
2 K. C. Chung, et al., unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast
growth factor;
bFGF, basic FGF;
CaM kinase, calcium/calmodulin-dependent kinase;
CK2, casein kinase 2;
CRE, cAMP response element;
CREB, CRE binding protein;
DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole;
EGF, epidermal growth factor;
ERK, extracellular signal-regulated protein
kinase;
GFP, green fluorescent protein;
JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein kinase;
MAPKAP kinase 2, MAPK activated
protein kinase 2;
MEK, MAPK kinase;
NGF, nerve growth factor;
PI-3K, phosphatidylinositol 3'-kinase;
SEK, stress-activated protein kinase
kinase;
TK, thymidine kinase;
PKA, protein kinase A;
PKC, protein
kinase C;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione
S-transferase.
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