(Received for publication, August 10, 1995; and in revised form, November 15, 1995)
From the
The ability of activating transcription factor-1 (ATF1) or the
cAMP response element-binding protein (CREB) to enhance transcription
can be stimulated by increases in intracellular Ca concentrations. To identify protein kinases which may mediate the
ability of Ca
to activate these transcription
factors, we compared the ability of constitutively active forms of
several Ca
/calmodulin-dependent protein kinases (CaM
kinases) to activate ATF1 or CREB. We find that constitutively active
CaM kinase I and IV can activate both ATF1 and CREB. In addition,
expression vectors for full-length CaM kinase I and IV were able to
augment the ability of Ca
influx to activate ATF1 or
CREB consistent with a role for these kinases in mediating
transcriptional responses to Ca
signaling. In
contrast, CaM kinase II was unable to activate either ATF1 or CREB.
These findings provide a potential mechanism that may permit variation
in the ability of ATF1 and CREB to respond to changes in intracellular
Ca
concentrations depending on differences in the
relative concentrations of specific CaM kinases.
The cAMP and Ca signaling pathways are used
widely to regulate the transcription of specific genes. In at least
some cases, cAMP and Ca
converge to regulate the
activity of a single transcription factor(1, 2) . A
key study which led to this view of convergent regulation by cAMP and
Ca
involved analysis of the DNA sequences required
for Ca
-regulated transcription of the c-fos proto-oncogene(1) . It was found that the regions of the fos gene that are required for responses to Ca
mapped to a previously identified cAMP response element. Other
studies provided evidence that cAMP response element-binding protein
(CREB) (
)can bind to the fos cAMP response element
and that elevations in intracellular Ca
result in
phosphorylation of CREB at sites similar to those that were
phosphorylated in response to elevated cAMP levels. The use of a
GAL4-CREB fusion provided strong evidence that the transcription
stimulating activity of CREB can be regulated by increases in
intracellular Ca
(2) . It seems likely that
convergent regulation of CREB activity by cAMP and Ca
permits integration of multiple extracellular signals in the
regulation of specific genes.
The ability of cAMP and Ca to regulate CREB activity involves phosphorylation of CREB by the
cAMP-dependent protein kinase (PKA) or
Ca
/calmodulin-dependent protein kinases (CaM
kinases). PKA phosphorylates CREB on a single major site at Ser
and phosphorylation of this residue is crucial for activation of
CREB by PKA(3) . CaM kinases I, II, and IV have been shown to
phosphorylate CREB in vitro, providing evidence that each of
these protein kinases is a candidate for mediating the effects of
Ca
on CREB
activity(2, 4, 5, 6) .
Interestingly, recent studies have demonstrated that specific CaM
kinases can have very different effects on the activation of
CREB(7, 8) . In particular, CaM kinase II can inhibit
activation of CREB while CaM kinase IV can activate CREB. The
inhibitory effects of CaM kinase II are due to phosphorylation of a
negative regulatory site located at Ser
of CREB. These
observations provide a mechanism that would permit the Ca
signaling pathway to be either antagonistic or additive with the
cAMP pathway for activation of CREB depending on the relative activity
of specific CaM kinases.
Although CaM kinase I has been shown to phosphorylate CREB in vitro, the ability of CaM kinase I to activate CREB has not been tested. CaM kinase I was originally identified in brain extracts as an activity that could phosphorylate a specific site of synapsin I, a protein associated with synaptic vesicles. The purified kinase was found to effectively phosphorylate only synapsin I and II(9) . This led to the view that CaM kinase I had a very restricted substrate specificity and was probably functionally restricted to the modulation of neurotransmitter release. However, CaM kinase I can be found in tissues other than the brain and it has been shown that CREB appears to be good substrate for CaM kinase I in vitro(2) .
In addition to CREB, at least one
other member of the CREB/ATF family has been shown to be responsive to
both the cAMP and Ca pathways. Transfection studies
have shown that ATF1 can be activated by increases in either cAMP or
Ca
(10, 11) . Although it is clear
that the activity of ATF1 can be regulated by Ca
influx, the role of specific CaM kinases in regulating ATF1
activity has not been explored. It is possible that specific CaM
kinases might have differential effects on the activity of CREB and
ATF1. For instance, although previous studies have shown that CaM
kinase II can block activation of CREB(7, 8) , it is
possible that ATF1 can be activated by CaM kinase II. If this were the
case, changes in the relative concentration of CREB and ATF1 would
permit changes in the interaction between the cAMP and Ca
pathways. Thus it is important to examine the ability of specific
CaM kinases to modulate ATF1 activity.
In the present study we have
compared the ability of CaM kinase I, II, and IV to alter the
transcription stimulating activity of ATF1 and CREB. The results
indicate that CaM kinase I and IV can activate ATF1 and CREB.
Phosphorylation of Ser of CREB or Ser
of
ATF1 was found to be essential for transcriptional activation by CaM
kinase I and IV. Thus, both CaM kinase I and IV may participate in
mediating Ca
-stimulated activation of ATF1 and CREB
in specific tissues. In contrast, we find that CaM kinase II cannot
activate either ATF1 or CREB. As observed previously with CREB, CaM
kinase II phosphorylates two sites in ATF1. Thus the negative
regulatory effects of CaM kinase II are observed for both ATF1 and
CREB.
Figure 1:
Regulation of ATF1 and CREB activity by
PKA, CaM kinase I, and CaM kinase IV. GH cells were
transfected with 2 µg of an expression vector for the DNA binding
domain of GAL4 (GAL4), GAL4-ATF1
b-zip (GAL4-ATF1), or GAL4-CREB
b-zip (GAL4-CREB) as
indicated. Cells also received 5 µg of an expression vector for
PKA, CaM kinase I
, or CaM kinase
IV
and 5 µg of the 5xGAL4-TATA-luciferase
reporter gene (A and B) or a reporter gene containing
the thymidine kinase promoter linked to luciferase (TK-lucif) (C). Luciferase activity was determined 24 h after
transfection. Values are means ± S.E. for three separate
transfections.
Previous studies have
shown that phosphorylation of Ser of CREB is crucial for
transcriptional activation by PKA and CaM kinase
IV(3, 8) . A large portion of the transcriptional
activation domain of ATF1 is similar to the transcriptional activation
domain of CREB, including a conserved PKA site in which Ser
of ATF1 corresponds to Ser
of CREB. Mutation of
Ser
of ATF1 to alanine (ATF1-S63A) essentially blocked the
ability of CaM kinase I and CaM kinase IV to activate GAL4-ATF1.
Similarly, mutation of Ser
of CREB to alanine
(CREB-S133A) blocked activation in response to PKA, CaM kinase I, and
CaM kinase IV. These findings demonstrate that CaM kinase I and CaM
kinase IV can enhance the ability of ATF1 and CREB to stimulate
transcription, likely involving a crucial phosphorylation event at
Ser
of ATF1 or Ser
of CREB.
As indicated
above, it was surprising that expression vectors for constitutively
active forms of CaM kinase I and CaM kinase IV were able to activate
GAL4-ATF1b-zip, but that a PKA expression vector was not able to
substantially increase the activity of this fusion protein. To examine
this issue further, we performed a titration experiment using
increasing concentrations of expression vectors for each of three
protein kinases (Fig. 2). We found that at all of the tested
concentrations of DNA, expression vectors for CaM kinase I and CaM
kinase IV enhanced the ability of GAL4-ATF1
b-zip to stimulate
reporter gene expression. At lower concentrations of the expression
vectors, CaM kinase I consistently resulted in greater activation of
ATF1 than CaM kinase IV. In contrast, PKA had little effect on
activation of GAL4-ATF1
b-zip at any of the tested concentrations
of expression vector. The failure of PKA to substantially activate
GAL4-ATF1 might be due to inhibitory effects of the kinase. To address
this issue, a PKA expression vector was co-transfected with a vector
for a constitutively active form of CaMKI (Fig. 3). As observed
above, the CaMKI expression vector enhanced the ability of
GAL4-ATF1
b-zip to stimulate reporter gene activity while the PKA
vector by itself had little effect. Co-transfection of the two kinase
expression vectors demonstrated that PKA does not block activation by
CaMKI. Overall these studies provide evidence that the transcription
stimulating activity of ATF1 appears to be responsive to CaM kinases I
and IV and relatively unresponsive to PKA.
Figure 2:
Dose-response analysis of the ability of
PKA, CaM kinase I, and CaM kinase
IV
to activate ATF1 in GH
cells.
GH
cells were transfected with 2 µg of an expression
vector for GAL4-ATF1
b-zip fusion protein, 5 µg of the
5xGAL4-TATA-luciferase reporter gene and increasing amounts (0, 3, 5,
10, or 20 µg) of the expression plasmid for PKA (circles),
CaM kinase I
(squares), or CaM kinase
IV
(triangles). The total amount of DNA
for each transfection was equalized using pBSSK(-). Luciferase
activity was determined 24 h after transfection. Values are the mean
for two separate transfections.
Figure 3:
Transfection of an expression vector for
PKA does not block the ability of CaM kinase I to activate ATF1.
GH cells were transfected with 2 µg of an expression
vector for GAL4-ATF1
b-zip (GAL4-ATF1) or
GAL4-CREB
b-zip (GAL4-CREB) and 5 µg of expression
vectors for PKA and CaM kinase I
as indicated, as
well as 5 µg of the 5xGAL4-TATA-luciferase reporter gene.
Luciferase activity was determined 24 h after transfection. Values are
means ± S.E. for three separate
transfections.
We also examined the
ability of endogenous kinases to activate GAL4-ATF1b-zip (Fig. 4). To stimulate endogenous CaM kinases, transfected
GH
cells were depolarized by KCl treatment, which results
in Ca
influx through voltage-dependent Ca
channels. We previously found that in GH
cells,
KCl-induced activation of GAL4-CREB is dependent on the presence of
extracellular Ca
(8) . KCl treatment enhanced
the ability of GAL4-ATF1 to stimulate reporter gene expression. These
findings provide evidence that endogenous
Ca
-responsive enzymes can activate GAL4-ATF1.
Treatment of transfected cells with chlorophenylthio-cAMP had a modest
effect on the ability of GAL4-ATF1
b-zip to stimulate reporter gene
expression.
Figure 4:
Activation of ATF1 by KCl-induced
Ca influx. GH
cells were transfected with
6 µg of an expression vector for GAL4-ATF1
b-zip as indicated
and 15 µg of the 5xGAL4-TATA-luciferase reporter gene. After
transfection the cells were divided into three dishes. Cells were
treated by the addition of 80 mM KCl or 0.5 mM chlorophenylthio-cAMP (cAMP) at 18 h after transfection.
The cells were collected and luciferase activity determined 6 h after
treatment. Values are means ± S.E. for three separate
transfections.
Figure 5:
Two-dimensional phosphopeptide mapping of
the phosphorylation of CREB and ATF1 by PKA and CaM kinase I.
Recombinant poly(His)-CREB or poly(His)-ATF1 were phosphorylated in
vitro by PKA or CaM kinase I in the presence of
[-
P]ATP as indicated. The phosphorylation
products were purified by denaturing polyacrylamide gel
electrophoresis, oxidized, and then digested with trypsin.
Phosphopeptides were separated by high voltage electrophoresis in the
first dimension and by thin layer chromatography in the second
dimension. The phosphopeptides were visualized by
autoradiography.
Figure 6:
Comparison of the amino acid sequence
surrounding phosphorylation sites in CREB and ATF1. The amino acid
sequence of residues 127-150 of CREB are aligned with the
sequence of residues 57-80 of ATF1. Positions where amino acids
are identical for CREB and ATF1 are indicated by a dot between
the two sequences. PKA and CaM kinase II phosphorylation sites at
Ser and Ser
of CREB (3, 8) are indicated with an asterisk, and
the predicted tryptic phosphopeptides are enclosed by boxes.
For ATF1, the PKA phosphorylation site at Ser
(11) is indicated as well as a putative CaM kinase II
site at Ser
and the predicted phosphopeptides are enclosed
by boxes.
Figure 7:
Expression vectors encoding full-length
CaM kinase I or CaM kinase IV augment activation of CREB in response to
depolarization-induced Ca influx. GH
cells were transfected with 2 µg of an expression vector for
either the DNA binding domain of GAL4 (GAL4
) or
the GAL4-CREB
b-zip fusion protein (GAL4-CREB) and 5 µg of the
5xGAL4-TATA-luciferase reporter gene. The cells also received
increasing amounts (0, 1, 5, or 10 µg) of an RSV expression vector
for full-length CaM kinase I or CaM kinase IV. The total amount of DNA
for each transfection was equalized using an RSV-globin expression
vector. After transfection the cells were divided into two 60-mm
dishes, one of which was treated by addition of 80 mM KCl to
the medium 18 h after transfection. Luciferase activity was determined
6 h after KCl treatment. Values are means ± S.E. for three
separate transfections. The numbers at the top of each
histogram indicate the -fold activation by KCl
treatment.
Figure 8:
An expression vector for a constitutively
active form of CaM kinase II does not activate ATF1 or CREB. GH cells were transfected with 2 µg of an expression vector for
GAL4-ATF1
b-zip (GAL4-ATF1) fusion protein (A) or
GAL4-CREB
b-zip (GAL4-CREB) fusion protein (B), 5
µg of the 5xGAL4-TATA-luciferase reporter gene, and either 5 µg
of pBSSK(-) as control (
) or 5 µg of an expression
vector for PKA (&cjs2112;), CaM kinase I
(&cjs2098;), CaM kinase II
(&cjs2110;), or
CaM kinase IV
(
). Luciferase activity was
determined 24 h after transfection. Values are means ± S.E. for
three separate transfections.
Figure 9:
Two-dimensional phosphopeptide mapping of
the phosphorylation of ATF1 by CaM kinase II. Recombinant
poly(His)-ATF1 (wild-type or mutants as indicated) were phosphorylated in vitro with CaM kinase II in the presence of
[-
P]ATP. The phosphorylation products were
purified by denaturing polyacrylamide gel electrophoresis, oxidized and
digested with trypsin. Peptides were separated by high voltage
electrophoresis in the first dimension and by thin layer chromatography
in the second dimension. The phosphopeptides were visualized by
autoradiography. The open arrows indicate the phosphopeptide
that appears to contains Ser
, and the closed arrows indicate the phosphopeptide that probably contains
Ser
.
Figure 10:
Mutation of Ser of ATF1
permits activation by CaM kinase II. GH
cells were
transfected with 2 µg of an expression vector for
GAL4-ATF1
b-zip (GAL4-ATF1) or a mutant in which the codon
for Ser
of ATF1 was replaced with a codon for alanine (GAL4-ATF1-S72A), 5 µg of a 5xGAL4-TATA-luciferase
reporter gene, and increasing concentrations of an expression vector
for a constitutively active form of CaM kinase II (CaMKII
). The total amount of
DNA for each transfection was equalized using pBSSK(-).
Luciferase activity was determined 24 h after transfection. Values are
means ± S.E. for three separate
transfections.
We have compared the ability of CaM kinases I, II, and IV to
alter the activity of ATF1 and CREB. We find that CaM kinase I or IV
can activate either ATF1 or CREB. In contrast, CaM kinase II cannot
activate either ATF1 or CREB. These findings provide further insight
into the signal transduction pathways that are important for mediating
the ability of Ca to activate CREB and ATF1. The
results provide strong evidence that CaM kinase I as well as CaM kinase
IV can activate CREB and ATF1. In titration experiments we found that
low concentrations of the CaM kinase I vector were considerably more
effective than the CaM kinase IV vector in activating CREB. While we
have not determined that equal concentrations of expression vector
produce equivalent amounts of CaM kinase I and IV, these studies at
least raise the possibility that CaM kinase I may be more effective
than CaM kinase IV in activating CREB. The finding that full-length CaM
kinase I or IV can augment the activation of ATF1 or CREB in response
to Ca
influx supports the view that both of these
enzymes are good candidates as physiological regulators of CREB and
ATF1 activity.
In contrast to previous studies of
ATF1(10, 11) , we found that PKA was unable to enhance
the ability of GAL4-ATF1b-zip to stimulate reporter gene activity.
The earlier studies utilized either full-length ATF1 or full-length
ATF1 fused to the DNA binding domain of GAL4. As ATF1 can form
heterodimers with CREB(11) , the activation observed with
full-length ATF1 may actually reflect phosphorylation and activation of
CREB as part of an ATF1
CREB heterodimer. Studies utilizing
leucine zipper variants of CREB which are engineered to form specific
heterodimers have shown that a hemiphosphorylated CREB dimer or a
CREB
CREM
heterodimer can mediate PKA-induced transcriptional
activation(27, 28) . Our use of a GAL4-ATF1
b-zip
construct would eliminate formation of CREB
ATF1 heterodimers, and
this might account for the failure to respond to PKA. The finding that
ATF1 has only a limited ability to mediate transcriptional responses to
PKA would be consistent with recent studies, which concluded that ATF1
is a specific antagonist of CREB-mediated transcriptional
activation(29) . On the other hand, we found that both CaM
kinase I and CaM kinase IV were able to activate GAL4-ATF1
b-zip.
This finding is quite surprising as phosphopeptide mapping experiments
demonstrated that PKA, CaM kinase I, and CaM kinase IV all appear to
phosphorylate ATF1 at the same site in vitro,
Ser
. How is it that all three kinases appear to
phosphorylate ATF1 on Ser
in vitro, yet only CaM
kinase I or IV can activate GAL4-ATF1
b-zip in a transfection
experiment? We have used co-transfection experiments to demonstrate
that PKA cannot block the ability of CaMKI to activate ATF1. Thus, it
seems unlikely that PKA phosphorylates a factor that inhibits responses
to ATF1. It may be that CaM kinase I and IV phosphorylate other
transcription factors or coactivators, which are required to mediate
responses to phospho-ATF1. Microinjection experiments have shown that
phosphorylation of CREB on Ser
is sufficient for
transcriptional activation and thus other PKA-dependent
phosphorylations are not required for a response to phospho-CREB.
Similar microinjection experiments might yield insight into the
differential activation of ATF1 by PKA and CaM kinase I or IV.
The
use of expression vectors for full-length CaM kinase I and IV
demonstrated that both of these kinases can participate in
Ca-regulated activation of CREB. Most of our studies
relied upon the use of truncated, constitutively active forms of these
enzymes. It is possible that the truncated forms of the kinase may have
enhanced access to the nucleus and therefore might alter nuclear events
which are not normally regulated by the holoenzyme. The finding that
full-length CaM kinase I and IV can activate CREB supports a role for
these enzymes in the physiological regulation of transcription. It is
interesting that transfection of an expression vector for full-length
CaM kinase I stimulated basal, CREB-mediated transcriptional activation
in the absence of depolarization-induced Ca
influx.
On the other hand, full-length CaM kinase IV appeared to be much more
Ca
-dependent, suggesting that there may be
differences in Ca
-mediated activation of the two
enzymes. There is evidence that activation of both CaM kinase I and IV
requires activator protein, which appears to be another protein
kinase(30, 31, 32, 33, 34) .
Our findings suggest that some step in the activation of CaM kinase I
may be more sensitive to Ca
than occurs for
activation of CaM kinase IV. It seems likely that this altered
Ca
sensitivity occurs at the level of the CaM kinase
activator kinase. Thus our findings suggest possible differences in the
enzymes which mediate activation of CaM kinase I and IV.
Our studies
have shown that CaM kinase II is unable to activate ATF1. This finding
extends our previous observation that CaM kinase II cannot activate
CREB (8) and demonstrates that the failure to respond to CaM
kinase is conserved for both proteins. Unlike CaM kinase I and IV,
which phosphorylate ATF1 or CREB at a single major site, CaM kinase II
phosphorylates both ATF1 and CREB at two sites. One of the sites,
Ser of CREB or Ser
of ATF1, is the same site
that is recognized by PKA, CaM kinase I, and CaM kinase IV.
Phosphorylation of Ser
of CREB or Ser
of
ATF1 is crucial for transcriptional activation. The second site in ATF1
that is phosphorylated by CaM kinase II appears to be Ser
,
which corresponds to Ser
, the inhibitory CaM kinase II
site in CREB. Neither Ser
of CREB nor Ser
of
ATF1 match a consensus CaM kinase II phosphorylation site, which is
Arg-X-X-Ser/Thr (35) . Phosphorylation at
non-consensus sites by CaM kinase II has been reported for several
proteins(36, 37) . It will likely require experiments
exploring the phosphorylation of an extensive set of peptides based on
these proteins to further define the alternative consensus sites
recognized by CaM kinase II. In any case, the present findings and our
previous studies (8, 17) suggest that the ability of
CaM kinase II to phosphorylate a second, non-consensus site in ATF1,
and CREB appears to inhibit activation of these transcription factors.
In conclusion, we have demonstrated differential activation of ATF1
and CREB by specific CaM kinases. Both CaM kinase I and IV can activate
ATF1 and CREB. CaM kinase II is unable to activate either transcription
factor. Based on these studies, it seems likely that tissue-specific or
developmental changes in the concentrations of individual CaM kinases
permits variations in the ability of the Ca signaling
pathway to regulate the activity of specific genes.