(Received for publication, August 26, 1994; and in revised form, November 21, 1994)
From the
A mechanism by which voltage-sensitive Ca channel (VSCC) activation triggers c-fos transcription
has been characterized. Ca
influx through VSCCs
stimulates phosphorylation of the transcription factor cAMP response
element-binding protein (CREB) on serine 133 leading to an increase in
the formation of transcription complexes that can elongate through a
transcription pause site within the c-fos gene.
Ca
-stimulated CREB serine 133 phosphorylation is
mediated by a Ca
-activated kinase and is not
dependent on the cAMP-dependent protein kinase (PKA). While necessary
for c-fos transcriptional induction following VSCC opening,
CREB serine 133 phosphorylation is not sufficient for transcriptional
activation. A second, PKA-dependent event is required. Following
induction, c-fos transcription is rapidly down-regulated.
Dephosphorylation of CREB serine 133 parallels and likely mediates the
transcriptional shut-off event. These results suggest that the
phosphorylation and dephosphorylation of CREB controls its ability to
regulate transcription in membrane-depolarized cells and that multiple
pathways contribute to Ca
-activated gene expression.
An important mechanism by which neurotransmitters are believed
to trigger long term adaptive neuronal responses is by activating
specific programs of gene expression(1, 2) . Within
minutes of neurotransmitter release, the expression of a family of
genes termed immediate early genes (IEGs) ()is induced in
the post-synaptic neuron. Many IEGs encode transcription factors that
then induce subsequent waves of late response gene expression. Late
response genes encode proteins that are likely to be determinants of
neuronal plasticity. These proteins may include
neurotransmitter-synthesizing enzymes, neurotransmitter receptors,
neurotrophins, as well as structural components of the synapse.
One
critical issue that remains to be solved is how a signal that is
initiated by neurotransmitter release at the synapse is propagated to
the nucleus of a post-synaptic cell to activate IEG transcription.
Studies of one IEG, the c-fos proto-oncogene, have begun to
give insight into this process. A variety of neurotransmitters that
trigger Ca influx have been found to stimulate
c-fos transcription rapidly and transiently both in vitro in cell culture systems (3, 4, 5) and
also trans-synaptically in the intact nervous
system(6, 7, 8, 9) . The c-fos gene encodes a transcription factor, Fos, which forms a
heterodimer with members of the Jun family of transcription factors and
regulates the expression of late response genes that contain
Fos/Jun-binding sites (consensus 5`-TGAG/CTCA-3`) within their
promoters(10) .
The pheochromocytoma cell line PC12 has
proven to be a useful model for elucidating the mechanisms by which
neurotransmitters that trigger Ca influx activate
c-fos transcription. Exposure of PC12 cells to elevated levels
of extracellular KCl depolarizes the plasma membrane and stimulates the
opening of L-type voltage-sensitive Ca
channels
(VSCCs). The subsequent rise in the level of intracellular
Ca
induces c-fos transcription(4, 11) . A Ca
response element (CaRE), located 60 nucleotides 5` of the
initiation site for c-fos mRNA synthesis, plays an important
role in mediating the c-fos response to VSCC
activation(12) . The -60 CaRE (-TGACGTTT-) is similar in
sequence to a consensus cAMP response element (CRE) (-TGACGTCA-)
present within the regulatory regions of a variety of genes that become
activated when cells are exposed to agents such as forskolin that
activate adenylate cyclase and stimulate cAMP production(13) .
When the c-fos CaRE/CRE is inserted within the promoter of
reporter genes that are normally non-responsive to forskolin or VSCC
channel activators the c-fos CaRE/CRE confers responsiveness
to these agents. The -60 CaRE functions by interacting with the
cAMP response element-binding protein (CREB) which becomes newly
phosphorylated at a critical regulatory site, serine 133, within
minutes of exposure of PC12 cells to elevated levels of KCl or
forskolin(12) . Phosphorylation of serine 133 is critical for
CREB-mediated transcription inasmuch as a mutation that converts serine
133 to an alanine inhibits CREB's ability to activate
transcription in response to treatment with VSCC activators or
forskolin(14, 15, 16) .
How phosphorylation of CREB serine 133 stimulates the ability of this transcription factor to induce gene expression is not completely resolved. While phosphorylation of serine 133 may enhance the binding of CREB to CREs that might otherwise bind it with low affinity(17) , the major effect of serine 133 phosphorylation is to increase CREB's ability to activate transcription once bound to the CaRE/CRE(14) . Whether CREB phosphorylation regulates transcription by enhancing the rate of formation of new transcription complexes at the promoter (new initiation), or by affecting the efficiency with which transcription complexes transcribe the length of the c-fos gene (elongation) remains to be determined.
Within minutes of its activation by neurotransmitters,
expression of the c-fos gene is repressed(4) . The
rapid disappearance of the c-fos mRNA reflects the fact that
this mRNA has an extremely short half-life (18) but is also a
consequence of transcriptional repression. With respect to
Ca regulation of c-fos transcription, the
mechanism of transcriptional shut-off has not been characterized. One
possibility is that the -60 CaRE and its associated protein,
CREB, mediate both the transcriptional activation and the subsequent
shut-off event. Since the phosphorylation of CREB serine 133 triggers
c-fos activation, the dephosphorylation of phosphoserine 133
might lead to the shut-off of c-fos transcription. An
alternative possibility is that distinct promoter elements and
transcription factors control the initial c-fos activation and
subsequent shut-off events.
The kinase(s) that catalyzes the
phosphorylation event upon activation of the L-type VSCCs has been a
subject of controversy. It is well established that in
forskolin-treated cells elevated levels of cAMP lead to the activation
of the cAMPdependent protein kinase (PKA) which then translocates to
the nucleus where it almost certainly catalyzes the phosphorylation of
CREB serine 133(15, 19, 20) . However,
PKA's involvement in the Ca signaling pathway
remains unresolved. Several studies suggest that membrane
depolarization of PC12 cells does not effectively stimulate cAMP
accumulation, suggesting that PKA is not likely to be activated and is
probably not the enzyme that triggers CREB serine 133 phosphorylation
under these conditions (12, 21) . In this case, a
Ca
-activated enzyme, such as a
Ca
-calmodulin dependent (CaM) kinase, was
hypothesized to phosphorylate CREB in response to membrane
depolarization. However, in another study using PKA-deficient cell
lines, VSCC activation failed to effectively stimulate c-fos transcription, even though CaM kinases were
activated(22) . This finding suggested that PKA activity is
important for depolarization activation of c-fos transcription
and that it might mediate CREB serine 133 phosphorylation.
In this
report, using the c-fos CaRE/CRE as a model, we have
characterized further the signaling pathways by which VSCCs trigger
gene expression. We have investigated the mechanism of transcriptional
initiation and provide evidence suggesting that upon VSCC activation
CREB phosphorylation leads to an increase in the rate of formation of
new transcription complexes. In addition, these newly formed
transcription complexes appear to have an increased efficiency of
elongation through an intragenic transcription pause site. The
mechanism of transcriptional shut-off that occurs subsequent to VSCC
activation also has been characterized. Dephosphorylation of CREB was
found to play a role in the down-regulation event. Furthermore,
characterization of the signaling pathways by which VSCCs lead to CREB
phosphorylation demonstrated that CREB phosphorylation is not mediated
by PKA but is likely to be catalyzed by a
Ca-activated kinase. While CREB serine 133
phosphorylation is necessary for Ca
-induced
CaRE/CRE-dependent transcription, it is not sufficient. In addition to
CREB serine 133 phosphorylation, a second, PKA-dependent signaling
pathway is also required for CREB-mediated transcriptional activation
of c-fos.
1S, 1AS, 2S, and
2AS are sense (S) or antisense (AS) rat c-fos cDNA fragments
from the first (1S or 1AS) or second, third, and part of the fourth (2S
or 2AS) exons cloned into M13. To generate the 1S and 1AS constructs,
pSP65r-fos (gift from T. Curran, Nutley, NJ) was digested with BglII and EcoRI and the 290-base pair fragment was
then ligated into M13mp18 (for 1S) or M13mp19 (for 1AS) that had been
digested with BamHI and EcoRI. For the 2S and 2AS
constructs, pSP65r-fos was digested with BglI, blunted, and
then digested with BstYI. The 461-base pair fragment was then
ligated into either Mp13mp18 or Mp13mp19 digested with SmaI
and BamHI. M13MATA contains a 720-base pair fragment from the
mouse -tubulin inserted into M13(23) .
Figure 2:
A, transcription through c-fos exon 1 and exon 2 in membrane-depolarized PC12 cells. Nuclei were
isolated from PC12 cells that were untreated or treated for the
indicated time (minutes) with depolarization solution (60 mM KCl). Nascent RNA transcripts were labeled in an in vitro nuclear run-on transcription assay, and equal numbers of
incorporated counts/minute were hybridized to single strand probes. 1S, 1AS, 2S, and 2AS are the sense (S) or antisense (AS) strands of probes 1 and 2
cloned into M13. M13 is M13mp18. Tubulin is mouse
tubulin in M13. Tubulin serves as an internal control for the total
number of incorporated counts/minute added to each hybridization, as
the transcription rate of the tubulin gene does not change in membrane
depolarized cells(62) . Diagram of single-stranded probes to
rat c-fos exons 1 and 2. The number of nucleotides as well as
the number of radiolabeled uracil residues in each probe is indicated. Probe 1 contains the entire first c-fos exon (275
nucleotides (nt)) and 16 nucleotides of exon 2. The 16
nucleotides of exon 2 do not contribute significantly to the probe 1
signal in stimulated cells, since they are located at the end of the
first intron. The RNA synthesized during the nuclear run-on reaction
contains intron sequences which are not contained in the cDNA probe
sequences bound to the filter. Therefore, the 16 nucleotides in the
synthesized RNA follow a long stretch of RNA that cannot hybridize to
the filter. This short 16 nucleotide sequence is not expected to
hybridize to the filter well and is expected to be digested during the
subsequent RNase wash step. Nevertheless, in calculating the fold
induction of the probe 1 signal, 10% of the total probe 1 signal in
stimulated cells was subtracted to eliminate the maximum potential
contribution of the 16 nucleotides based on the number of uracil
residues. Probe 2 contains all exon 2 (236 nucleotides) and 3
(108 nucleotides) sequences and 117 nucleotides of exon 4. P indicates site of putative elongation pause sequences. B,
membrane depolarization activation of transcription through the
c-fos CaRE/CRE is not dependent on c-fos intragenic
sequences. PC12 cells were transfected with 71wtFosCAT, or 71
pm3FosCAT, and pSV-1 as an internal control for transfection
efficiency. Cells were stimulated for 30 min by membrane depolarization
with KCl. Cytoplasmic mRNA was isolated and assayed by RNase
protection. mRNA transcribed from the transfected FosCAT, and
-globin genes and the endogenous rat c-fos
gene are indicated with arrows. The FosCAT arrow indicates correctly initiated transcripts. The higher molecular
weight band represents incorrectly initiated transcripts from the
transfected FosCAT reporter. C, c-fos
intragenic sequences cannot mediate membrane depolarization
regulation of transcription initiated by GAL4VP16. PC12 cells were
transfected with the indicated GAL4 fusion and reporter constructs and
with pSV
-1. Cells were stimulated for the indicated times by
membrane depolarization, and mRNA was isolated and assayed by RNase
protection. Bands representing transcription of the transfected human
c-fos reporter (c-fos
),
-globin, and
the endogenous rat c-fos (c-fos
) are
indicated with arrows.
As shown in Fig. 2A,
when the run-on assay was carried out using nuclei from unstimulated
cells, the P-labeled transcripts hybridized to probe 1 (1AS), but not to probe 2 (2AS). This suggests that
in unstimulated PC12 cells a detectable level of c-fos transcription was initiated at the promoter and transcribed
through exon 1. However, no detectable transcription complexes
successfully transcribed through downstream exon sequences indicating a
pause site (block) in transcriptional elongation at a position 5` to
exon 2. Exposure of PC12 cells to elevated levels of KCl (final
concentration, 60 mM) for 15 min resulted in a 4.1-fold
increase in hybridization to probe 1 and in the appearance of
hybridization to probe 2. The increased hybridization was specific to
the antisense (AS) c-fos probes and occurred to only
a very minor extent with the c-fos sense (S) probes.
Hybridization to both the exon 1 and exon 2-4 probes decreased at
later time points (3 h) to the levels seen in unstimulated cells. Taken
together these results suggest that membrane depolarization leads to an
induction of c-fos transcription by increasing the rate of
initiation of complexes at the promoter (reflected by the increase in
P-labeled transcripts that hybridize to exon 1), and by
increasing the ability of transcription complexes to proceed through 3`
exons (reflected by an increase in KCl-treated cells in the level of
P-labeled transcripts that hybridize to exon 2). It seems
likely that the elongation block site is located within the first
c-fos intron since analogous elongation pause sites have been
identified in non-neuronal cells within the first introns of the murine
and human c-fos genes(36, 37, 38) .
We next asked if the -60 c-fos CaRE/CRE mediates transcription in KCl-treated cells by stimulating an increase in the initiation of transcription or by relieving the block to elongation. To test whether the CaRE/CRE might mediate the initiation of transcription, the depolarization induction of a heterologous gene construct which consists of the c-fos promoter fused to a reporter gene was assayed. This reporter gene consists of 71 nucleotides of the mouse c-fos promoter, including the CaRE/CRE, the TATA box, and the c-fos initiation site, fused to a CAT gene (71wtfosCAT) (25) (Fig. 1B). This construct lacks the previously characterized c-fos elongation pause site. PC12 cells were transfected with 71wtfosCAT and then depolarized by exposure to elevated levels of extracellular KCl. Cytoplasmic mRNA was analyzed by RNase protection. Fig. 2B shows that depolarization leads to an increase in correctly initiated fosCAT reporter transcripts within 30 min. When a similar construct (71pm3fosCAT) containing a mutation within the CaRE/CRE was tested, the level of fosCAT transcripts was barely detectable, indicating the necessity for an intact CaRE/CRE for membrane depolarization induction of reporter gene expression. Because the 71wtfosCAT construct contains the c-fos promoter and initiation site but not the c-fos elongation pause site, these results suggest that the presence of an intact CaRE/CRE stimulates an increase in the formation of initiation complexes in membrane depolarized cells and indicate that the c-fos elongation pause sequences are not required for transcriptional activation by membrane depolarization.
Figure 1:
A, structure of the GAL4 fusion
constructs. GAL4(1-147) is the DNA-binding domain of the
yeast GAL4 DNA-binding protein. GAL4CREB contains the complete
CREB protein (341 amino acids) fused to the COOH terminus of
GAL4(1-147)(40) . GAL4CREBLZ lacks the
COOH-terminal 29 amino acids of GAL4CREB, including the leucine repeat
dimerization motif (LZ). GAL4VP16 contains the activation
domain of the herpes simplex virus transcriptional activator VP16 fused
to the COOH terminus of GAL4(1-147). B, structure of
reporter genes. pAF4 is a human c-fos genomic clone
that contains 711 nucleotides 5` to the c-fos
mRNA
initiation site(26) . pAF42 (G
)
contains 42 nucleotides 5` to the c-fos
mRNA
initiation site. pAF42CRE contains the consensus CRE
(TGACGTCA) inserted at position -42 of pAF42, and pAF42G
contains nine GAL4-binding sites inserted
at position -42. The four c-fos exons are indicated by boxes. The mRNA initiation site and the putative elongation
pause site are indicated by the horizontal and vertical
arrows, respectively. pSP6c-fos represents the
c-fos
sequences in the RNA probe transcribed from
the pSP6c-fos plasmid. The thin line indicates probe sequences
that are not protected in the RNase protection assays, while the box indicates a 296 nucleotide sequence that is protected by
c-fos
mRNA. Endogenous rodent c-fos mRNA
has been reported to protect a 65 nucleotide sequence in this
probe(26) . 71wtFosCAT is a mouse c-fos CAT
fusion reporter that contains 71 nucleotides 5` to the
c-fos
initiation site and 109 nucleotides 3`. The
wild type mouse CaRE/CRE is indicated in capital letters. 71pm3FosCAT is identical to 71wtFosCAT except for the changes
in the CaRE/CRE that are indicated in lowercase letters. p149 represents the c-fos
and linker
sequences in the RNA probe transcribed from the p149 plasmid. The thin line indicates probe sequences that are unprotected. The box indicates a 128 nucleotide sequence protected by fosCAT
mRNA transcribed from the transfected fosCAT reporter plasmid and a 109
nucleotide sequence protected by the mRNA transcribed from the
endogenous rat c-fos
gene.
While VSCC
activation induces c-fos transcription by regulating the
CaRE-dependent initiation of transcription at the promoter,
depolarization-induced changes in transcription elongation also occur.
The control of elongation could be due to a second
Ca-mediated regulatory event that occurs at the
elongation pause site. Alternatively, the control of elongation may be
linked to events that occur at the promoter. To begin to distinguish
between these possibilities, we analyzed the effects of membrane
depolarization on the expression of a c-fos gene whose
transcriptional initiation was not controlled by a
Ca
-responsive element, but rather by the constitutive
viral transcriptional activator, herpes simplex virus
VP16(39) . Because VP16 is a constitutive activator,
depolarization would not be expected to affect transcriptional
initiation, and any changes in expression should be due soley to
effects on elongation through c-fos sequences. To place
c-fos expression under the control of VP16, the yeast GAL4
fusion and reporter system was employed(40) . The VP16
transcriptional activation domain was directed to the human c-fos reporter (GAL4-fos) promoter by fusing it to the DNA binding
portion of the yeast GAL4 protein. The GAL4 portion of this fusion
protein, GAL4VP16, mediates binding to GAL4-binding sites that were
inserted at position -42 in the human c-fos reporter
(GAL4-fos or G
, Fig. 1B).
Expression of the GAL4-fos reporter in the presence of the GAL4VP16
activator was compared to expression in the presence of GAL4CREB, which
is known to be regulated by Ca
. The c-fos elongation pause sequences that are present within the GAL4-fos
gene would be likely to function as a pause site when this gene is
transiently transfected into PC12 cells, since these elongation pause
sequences have been shown previously to function in an in vitro transcription system(41) , and since elongation pause
sites have been shown to function when genes are introduced into cells
by transient transfection(42) . As shown previously, when cells
were co-transfected with GAL4-fos and the GAL4CREB fusion construct,
and mRNA levels were monitored by RNase protection, human c-fos transcripts were detected in cells that had been exposed to
elevated levels of KCl, but were not detected in unstimulated cells (Fig. 2C, panel 2)(14) . In contrast
to GAL4CREB, when GAL4VP16 was expressed in PC12 cells, high levels of
reporter gene expression were detected both before and after membrane
depolarization (Fig. 2C, panel 1). Transcription was
dependent on the fused activators, as GAL4 alone (GAL4(1-147)) did not activate transcription (panel
2, lanes 3 and 4). The presence of a
GAL4-binding site was also required since no transcripts were
synthesized when the reporter gene pAF42 (also termed G
),
which lacks GAL4-binding sites, was cotransfected with GAL4VP16 (panel 3, lanes 2 and 3). If the c-fos transcriptional elongation block was effectively regulated by
Ca
by a mechanism that is completely independent of
transcriptional initiation, then no detectable cytoplasmic c-fos mRNA would be expected to accumulate in unstimulated cells as a
result of transcription from the GAL4-fos reporter in the presence of
GAL4VP16. The simplest explanation of these results is that for the
wild type c-fos gene the pause in the elongation of
transcription can be affected by the transcription complex that forms
at the promoter, and regulation at the elongation pause site may be
dependent on events occurring at the promoter. However, proof of this
hypothesis requires that it be demonstrated that the c-fos elongation pause sequences still function effectively in
transiently transfected genes.
Figure 3:
A and B, the c-fos CaRE/CRE can mediate transcriptional shut-off following membrane
depolarization induction. PC12 cells were co-transfected with pAF42CRE (A) or 71wtFosCAT (B) and pSV-1. Cells were
stimulated by membrane depolarization for the indicated times, and
cytoplasmic mRNA was assayed by RNase protection with either antisense
c-fos
or antisense FosCAT and antisense
-globin riboprobes. The correctly initiated
c-fos
, FosCAT,
-globin, and endogenous rat
c-fos
transcripts are indicated with arrows. C, CREB can mediate transcriptional shut-off
following membrane depolarization induction. PC12 cells were
co-transfected with GAL4CREB
LZ or GAL4(1-147) fusion
constructs, pAF42G
, and pSV
-1. Cells were stimulated
for the indicated times by membrane depolarization, and cytoplasmic
mRNA was assayed by RNase protection. The correctly initiated
c-fos
mRNA is indicated with an arrow.
The doublet of bands migrating above the correct band are transcripts
that are incorrectly initiated from the transfected reporter
gene.
We next investigated
the possibility that CREB mediates transcriptional shut-off as well as
activation. The expression of the GAL4-fos reporter in the presence of
a GAL4CREB fusion protein that has a deletion of the CREB leucine
zipper (GAL4CREBLZ, Fig. 1A) was examined
at various times before and after exposure of PC12 cells to elevated
levels of KCl. Deletion of the leucine zipper prevents potential
heterodimerization with endogneous CREB family members that might
affect the biological activity of the fusion protein(43) . Fig. 3C shows that as with pAF42CRE, in the presence of
GAL4CREB
LZ, the GAL4-fos reporter was transiently induced by
depolarization. This induction was dependent on the CREB sequences
fused to GAL4 as GAL4 alone (GAL4(1-147)) did not mediate the
induction event. These findings suggest that CREB bound near the
c-fos TATA box contributes to both transcriptional activation
and shut off in membrane-depolarized PC12 cells.
Figure 4:
A,
CREB serine 133 is transiently phosphorylated in membrane-depolarized
PC12 cells. Left panel, PC12 cells were labeled with
[P]orthophosphate and stimulated with KCl for
the indicated time. Lysates were collected in boiling SDS and
immunoprecipitated with anti-phosphoCREB. The arrow indicates
CREB. The lower molecular weight bands are believed to represent
CREB-related proteins that share amino acid homology with the peptide
used to generate anti-phosphoCREB. Right panels, PC12 cells
were depolarized for the indicated times and then collected in boiling
SDS lysis buffer. Samples were run on SDS-PAGE, transferred to
nitrocellulose, incubated with anti-phosphoCREB or anti-CREB, and
antibody-antigen complexes were detected with alkaline phosphatase as
described under ``Materials and Methods.'' The arrow indicates CREB. B, forskolin treatment of PC12 cells
leads to both prolonged CREB-dependent transcription and prolonged
phosphorylation of CREB serine 133. Top panel, PC12 cells were
transfected with GAL4CREB
LZ, pAF42G
, and pSV
-1.
Cells were treated for the indicated times with forskolin or TPA (T), and mRNA was assayed by RNase protection. The correct
bands representing transcription of the transfected and endogenous
genes are indicated with arrows. Bottom left panel,
PC12 cells were labeled with [
P]orthophosphate
and stimulated with forskolin for the indicated time. Lysates were
collected in boiling SDS and immunoprecipitated with anti-phosphoCREB. Bottom right panel, PC12 cells were treated with forskolin for
the indicated times and then collected in boiling SDS lysis buffer.
Samples were run on SDS-PAGE and transferred to nitrocellulose. Filters
were incubated with anti-phosphoCREB or anti-CREB antibodies. The arrow indicates CREB.
To determine whether the disappearance of phosphoCREB was due to either dephosphorylation or degradation of phosphoCREB, parallel immunoblot analyses were performed with anti-phosphoCREB antibodies and antibodies that recognize both unphosphorylated and phosphorylated forms of CREB (anti-CREB). Anti-phosphoCREB antibodies detected the 43 kDa phosphorylated CREB band in extracts of cells depolarized for 15 min (Fig. 4A, right panel) but not in extracts of cells depolarized for 4 h. When the same extracts were immunoblotted with anti-CREB antibodies, the 43 kDa CREB band was detectable at both time points (Fig. 4A, right panel). Taken together with the finding that the majority of CREB molecules within a cell becomes phosphorylated on serine 133 within minutes of membrane depolarization(35) , these observations suggest that the disappearance of serine 133-phosphorylated CREB in depolarized cells is not due to degradation, but rather due to dephosphorylation of serine 133.
To examine further the correlation between the level of
c-fos transcription and the extent of CREB serine 133
phosphorylation, the time courses of transcription and CREB
phosphorylation were analyzed in cells stimulated with forskolin to
elevate intracellular levels of cAMP. Fig. 4B shows
that in contrast to depolarization, stimulation with forskolin resulted
in an extended c-fos mRNA signal. c-fosH mRNA
expression was detected as late as 6 h after stimulation of cells
transfected with pAF42CRE or GAL4fos in the presence of GAL4CREBLZ (Fig. 4B, top panel, and data not shown).
Analysis of the phosphorylation state of CREB indicated that in
forskolin-treated cells, serine 133 remains phosphorylated for an
extended time, with phosphoCREB detectable as late as 6 h after
stimulation (Fig. 4B, bottom panels, and data
not shown). For both reporter gene transcription and the
phosphorylation of CREB, peak levels were seen at early times
(20-60 min) following forskolin addition with some decrease in
these levels detected at later times. The observation that CaRE driven
transcription and the phosphorylation state of CREB are correlated in
forskolin-treated cells further suggests that the transcriptional
shut-off event is linked to the dephosphorylation of CREB serine 133.
Figure 5: A, the activity of PKA is not required for CREB serine 133 phosphorylation in membrane-depolarized PC12 cells. The PC12 wild type (parental) and the PKA-deficient (PKA def.) 123.7 cell line were either untreated (O) or treated with forskolin (F) or with KCl (K) for 10 min. Cell lysates were collected in boiling SDS sample buffer, separated by SDS-PAGE, and filters were incubated with anti-phosphoCREB antibodies. The arrow indicates CREB. B, CREB serine 133 phosphorylation occurs within 30 s of membrane depolarization. TPA can also induce CREB serine 133 phosphorylation. Left panels, PC12 cells were stimulated with either KCl or forskolin for the indicated times. Cell lysates were collected in boiling SDS sample buffer, separated by SDS-PAGE, and filters were incubated with anti-phosphoCREB antibodies. The arrow indicates CREB. Right panels, PC12 cells were treated with either forskolin (F), KCl (K), or TPA (T) for 10 min. Lysates were analyzed as above. The arrow indicates CREB.
To
determine which of the Ca response elements within
the c-fos gene lose their ability to function in PKA-deficient
cells, PKA-deficient and wild type PC12 cells were transfected with
various human c-fos reporter constructs, and the levels of
human c-fos mRNA transcripts were determined after membrane
depolarization, forskolin treatment, or phorbol ester addition. Both
the endogenous rat c-fos gene and the human c-fos gene pAF4, which contains 711 base pairs of upstream regulatory
sequences including several Ca
response elements were
effectively induced upon membrane depolarization of wild type PC12
cells (Fig. 6A, top panel). However, these
genes were poorly induced by KCl treatment of PKA-deficient PC12 cells.
Likewise, pAF42CRE, which contains only a CREB-binding site upstream of
the TATA box, was activated to a much greater extent (42-fold) in wild
type PC12 cells than in the PKA-deficient cells (Fig. 6A, bottom panel). The loss of a
transcription response in the PKA-deficient cells was also seen when
these cells were exposed to forskolin to activate adenylate cyclase (Fig. 6A). However, another inducer, TPA, was able to
effectively induce expression of the pAF4 gene in the PKA-deficient
cells, indicating that the failure of the CaRE to respond to KCl and
forskolin was not a general phenomena (Fig. 6A). These
findings, taken together with the observation that CREB serine 133
becomes phosphorylated upon KCl treatment of these cells (Fig. 5A), demonstrate that although CREB
phosphorylation is critical, it is not sufficient for
depolarization-induced CaRE/CRE-dependent transcription. A second
PKA-dependent phosphorylation event is also required. This conclusion
is supported by the finding that in wild type or PKA-deficient PC12
cells TPA induced CREB serine 133 phosphorylation (Fig. 5B, right panel), but did not activate
GAL4CREB
LZ-mediated transcription or transcription of pAF42CRE (Fig. 4B, top panel, last lane; Fig. 6A, bottom panel, respectively).
Figure 6:
A, the
CRE cannot mediate a transcriptional response to membrane
depolarization in PKA-deficient PC12 cells. Wild type (parental) and PKA-deficient (PKA def.) PC12 cells
were transfected with either pAF4 (top panel) or pAF42CRE (bottom panel) and pSV-1. Cells were stimulated for 60
min with either forskolin (F) or KCl (K) or for 30
min with TPA (T). Cytoplasmic RNA was analyzed by RNase
protection. Arrows indicate protected transcripts from the
transfected c-fos
and
-globin genes and the
endogenous c-fos
gene. B, SRE-dependent
transcriptional induction by membrane depolarization is not compromised
in PKA-deficient cells. Wild type (parental) and PKA-deficient (PKA def.) PC12 cells were transfected with pAF42SRE and
pSV
-1. Cells were either unstimulated (O) or stimulated
with KCl (K) for 60 min. RNA was analyzed by RNase protection,
and arrows indicate c-fos
,
-globin,
and c-fos
protected
transcripts.
We
tested the ability of a second Ca response element,
the c-fos serum response element
(SRE)(3, 31) , to function in the PKA-deficient cells.
In contrast to the activation of pAF42CRE, depolarization resulted in
nearly equivalent activation of pAF42SRE in both wild type and
PKA-deficient PC12 cells (Fig. 6B). Membrane
depolarization resulted in a level of transcription in the wild type
cells that was only 4.5-fold greater than the level detected in the
mutant cells. This was a significantly reduced difference than the
42-fold difference detected for pAF42CRE (Fig. 6A).
Because the pAF42SRE construct contains the same minimal promoter
sequences and intragenic sequences as the pAF42CRE plasmid, these
findings suggest that the PKA-dependent phosphorylation event that is
necessary for depolarization-activation of pAF42CRE probably involves
the modification of factors that interact at the CRE rather than
components of the basic transcriptional machinery or factors that
regulate transcriptional elongation.
The regulation of gene expression in neurons is critical for
their long term responses to stimulation(1, 2) . A
major signaling mechanism in stimulated neurons involves the membrane
depolarization-activated opening of VSCCs and the resultant increase in
intracellular free Ca(44) . We have studied
the mechanism by which Ca
regulates c-fos expression in PC12 cells and have found that this regulation is
complex, involving multiple processes. In this study, we present
evidence suggesting that a rise in intracellular Ca
and the subsequent phosphorylation of CREB serine 133 result in a
transient increase in both the formation of new transcription complexes
at the promoter and the ability of transcription complexes to elongate
through 3` exons. We also present evidence that the dephosphorylation
of CREB serine 133 parallels and therefore may mediate the shut-off of
c-fos transcription that occurs following induction.
Furthermore, our findings support the idea that CREB is directly
phosphorylated by a Ca
-activated kinase in response
to VSCC opening. However, in addition to the
Ca
-dependent phosphorylation of CREB serine 133, a
second PKA-dependent function is necessary for CaRE/CRE-mediated
transcriptional activation. This additional event may be the
phosphorylation of a second site on CREB, the CREB-binding
protein(45) , or a distinct CaRE/CRE-binding protein. The
PKA-dependent function appears to be activated by Ca
,
but not by TPA, which nevertheless can induce CREB phosphorylation at
serine 133.
The transcriptional
pause site within the rat c-fos gene described here is
comparable to pause sites found in the first intron of the murine and
human c-fos genes(36, 37, 41) . The
observation that Ca influx through VSCCs induces the
formation of transcription complexes that can elongate through the
pause site in the rat gene is consistent with a previous report in
non-neuronal cells that Ca
is required for elongation
through the pause site in the murine and human c-fos genes (36, 37) .
The regulation of the initiation of
c-fos transcription most likely occurs independently of
regulation of elongation. This idea is supported by the observations
that membrane depolarization can induce transcription of the
CaRE-driven reporter genes, CAT (Fig. 2B) and
-globin(12) , both of which lack the c-fos pause
sequences (Fig. 2B). In contrast, the regulation of
c-fos transcriptional elongation appears to be affected by the
transcription factors that regulate c-fos transcriptional
initiation. GAL4VP16 was observed to stimulate transcription of the
GAL4-fos gene even in unstimulated PC12 cells, suggesting that the
function of the c-fos pause site can be bypassed by a strong
transcriptional activator. This interpretation is based on the
assumption that the c-fos pause sequences function in genes
transiently transfected into PC12 cells, and thus remains to be
demonstrated.
Several examples of transcription factor-dependent elongation have been described previously. For example in the c-myc gene, which contains two promoters, whether an elongation pause site is recognized by transcription complexes depends on which promoter is used to initiate transcription(46) . More recent analysis of the c-myc gene has revealed that the processivity of RNA polymerase II along this gene is increased when transcription complexes form at the promoter in the presence of a strong activator such as VP16 (47) . Taken together these findings are consistent with the possibility that transcription complexes that form in the presence of phosphorylated CREB (a strong activator) can elongate through the pause site, while those formed in the presence of unphosphorylated CREB (a weak activator) cannot.
Our results suggest a simple model that might explain
the transient nature of c-fos transcriptional induction that
occurs when VSCCs are activated. Following the phosphorylation of CREB
serine 133 and induction of transcription, the dephosphorylation of
CREB serine 133 may lead to a shut-off of transcription. A different
model has been proposed that is based on the observation that CREB is a
member of a large family of CRE-binding proteins, some of which are
repressors (49, 50, 51) . These repressors,
CREMs, or cAMP response element modulator proteins, can heterodimerize
with CREB and inhibit CRE-mediated, cAMP-induced transcription under
some circumstances(49, 52) . Thus, transcriptional
down-regulation has been proposed to involve the formation of a
CREB:CREM heterodimer or the replacement of a CREB:CREB homodimer at
the CRE with a CREM:CREM homodimer. However, our findings with
GAL4CREBLZ in PC12 cells suggest that while CREM function may
contribute to transcriptional down-regulation, it is not required.
GAL4CREB
LZ, which does not contain the CREB leucine zipper, and
therefore cannot heterodimerize with endogenous leucine
zipper-containing proteins, mediates both transcriptional activation
and shut-off of a GAL4-fos reporter (Fig. 3C). This
demonstrates that CREB-activated transcription can be shut-off without
a contribution by other family members, such as the CREM proteins. In
support of the idea that transcriptional shut-off is mediated at least
in part by dephosphorylation of CREB, there was a strict correlation
between the level of transcription of the CRE-fos reporter gene and the
extent of CREB serine 133 phosphorylation in both depolarized and
forskolin-treated cells. An alternative interpretation of these
findings is that shut-off of transcription is followed by CREB
dephosphorylation. However, in support of the idea that the
dephosphorylation of CREB serine 133 precedes and is critical for
transcriptional shut-off, we found that in KCl-treated cells the
phosphatase inhibitor, okadaic acid, blocks both the dephosphorylation
of CREB serine 133 and c-fos transcriptional shut-off.
Two protein phosphatases, PP1 and PP2A, have been shown to
dephosphorylate CREB serine 133 in
vitro(53, 54) . Both of these phosphatases have
been postulated to play a role in CREB serine 133 dephosphorylation
that occurs several hours following forskolin treatment and could also
play a role in CREB dephosphorylation that occurs at later time points
following membrane depolarization.
We conclude that CREB serine 133 phosphorylation is not sufficient for CRE-mediated transcription in KCl-treated cells, since this phosphorylation event occurs normally in PKAdeficient cells, but the CRE is incapable of conferring a depolarization response. Therefore, a second PKA-dependent phosphorylation event may exist that is critical for CaRE/CRE-dependent transcription. We propose that the PKA-dependent function acts on the CaRE/CRE, rather than on c-fos basal promoter elements or intragenic sequences since a different reporter (SRE-fos) that also contains the c-fos promoter and intragenic sequences is effectively activated in the PKA-deficient cells (Fig. 6B). The PKA-dependent event could involve phosphorylation of CREB on a site other than serine 133. Alternatively, it may reflect involvement of CRE-binding proteins in addition to CREB or the CREB-binding protein (CBP) which has been shown to mediate cAMP-dependent transcription in non-neuronal cells(45) .
We conclude that the precise control of c-fos expression is necessary for the conversion of extracellular
stimuli to long term functional changes in neurons. This study
illustrates the intricate nature of c-fos regulation by
membrane depolarization. Membrane depolarization leads to an increase
in the rate of formation of new initiation complexes, as well as an
increase in the passage of transcription complexes through an
elongation pause site. This Ca-dependent activation
of c-fos transcription not only requires CREB serine 133,
which is phosphorylated in response to depolarization, but also an
additional PKA-dependent function. Finally, c-fos transcription must shut-off. Dephosphorylation of CREB serine 133
is a likely mechanism by which this occurs.