From the
Gene transcription can be induced by cAMP and Ca
Much information has accumulated about the pathway by which cAMP
stimulates transcription of a number of genes that contain a
cAMP-responsive element (CRE)
Recently the induction
of gene transcription by membrane depolarization and Ca
It is unknown whether also cAMP
induction of transcription relies on calcineurin phosphatase activity.
The aim of the present study was to investigate in pancreatic islet
cells whether calcineurin is involved in cAMP induction of CRE-mediated
transcription. The results obtained suggest the existence of a
calcineurin-sensitive phosphorylation event that interferes with the
transcriptional competence of CREB phosphorylated on serine 119,
offering a new mechanism through which CREB/CRE-dependent transcription
could be regulated.
To test the
relationship between the inhibition of calcineurin activity and
CRE-directed transcription stimulated by cAMP, rapamycin and
calcineurin expression vectors were used. Rapamycin is an analogue of
FK506. Rapamycin binds to the cytosolic FK506 receptor, the
FK506-binding protein FKBP-12, but in contrast to the FK506/FKBP-12
complex, the rapamycin/FKBP-12 complex does not inhibit calcineurin,
resulting in a competitive antagonism by rapamycin of FK506 action with
respect to calcineurin inhibition
(33) . Rapamycin does not,
however, bind to cyclophilins, the intracellular receptors of
cyclosporin A
(33) . Rapamycin alone had no effect on
calcineurin activity in HIT cells (data not shown). Inhibition by
cyclosporin A (60 n
M) of calcineurin activity was not reduced
by rapamycin (1.1 µ
M); it was even somewhat enhanced (Fig.
6 A). In contrast, inhibition of calcineurin activity by FK506
(5 n
M) was completely reversed by rapamycin
(Fig. 6 A). This indicates that in HIT cells as well,
inhibition by FK506 of calcineurin depends on a FKBP that binds FK506
and rapamycin. Rapamycin had no effect on gene transcription stimulated
by cAMP through the CRE (data not shown) and did not prevent its
inhibition by cyclosporin A (Fig. 6 B); it was even
somewhat enhanced (Fig. 6 B). In contrast, the inhibition
by FK506 was disinhibited by rapamycin (Fig. 6 B). Thus,
disinhibition of calcineurin activity was accompanied by disinhibition
of CRE-mediated transcription.
This study shows that cyclosporin A and FK506 inhibit
cAMP-induced transcription mediated by the CRE in a pancreatic islet
cell line. At the same concentrations, these structurally unrelated
agents also inhibit calcineurin phosphatase activity. Furthermore, when
inhibition of calcineurin by FK506 or cyclosporin A was reduced by
rapamycin or overexpression of calcineurin, CRE-dependent transcription
was disinhibited. These data strongly suggest that cyclosporin A and
FK506 inhibit CRE-directed transcription after stimulation by cAMP
through inhibition of calcineurin phosphatase activity. This implies
that after stimulation by cAMP, CRE-directed transcription depends not
only on protein kinase A but also on calcineurin phosphatase activity.
Together with previous evidence that also after membrane depolarization
and Ca
The cAMP- and Ca
Calcineurin is a Ca
Cyclosporin A and FK506 are of
great clinical importance as powerful immunosuppressive drugs. Through
inhibition of calcineurin, these drugs interfere with the nuclear
translocation of a subunit of the T-cell-specific transcription factor
NF-AT, which enhances the transcription of genes essential for
T-cell-activation, such as the interleukin-2 gene
(25, 34) . This may explain part of cyclosporin
A/FK506-mediated immunosuppression
(33) . The functional
disruption of CREB/CRE-directed transcription at concentrations that
block T-cell activation represents a novel mechanism of cyclosporin
A/FK506 action, which may underlie some of their pharmacological
effects, both desired and undesired.
We appreciate the following generous gifts:
calcineurin expression vectors from Dr. Gerald R. Crabtree, Stanford;
expression vector for KCREB from Dr. Richard H. Goodman, Oregon;
anti-phosphoCREB antibody from Dr. Michael Greenberg, Boston; KN-62
from Dr. Hiroyoshi Hidaka, Nagoya. Insulin was measured by Dr. Arnold
Hasselblatt, Göttingen. Cyclosporin A was provided by Sandoz,
FK506 by Fujisawa, rapamycin by Wyeth-Ayerst, and verapamil by Knoll.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
through distinct protein kinases phosphorylating the
transcription factor CREB, which binds to cAMP response elements (CREs)
in various genes. Induction of gene transcription by Ca
has been shown recently to depend on the
Ca
/calmodulin-dependent protein phosphatase
calcineurin in pancreatic islet cells. This study investigates the role
of calcineurin in CRE-directed gene transcription after stimulation by
cAMP. Reporter fusion genes under the transcriptional control of CREs
were transiently transfected into the cell line HIT. Pharmacological
evidence suggests that cAMP stimulates CRE-mediated transcription
through a Ca
-dependent mechanism. The
immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated
transcription stimulated by cAMP. At the same concentrations they also
inhibited calcineurin phosphatase activity. Reversal of calcineurin
inhibition by rapamycin or overexpression of calcineurin led to
disinhibition of CRE-mediated gene transcription. Immunoblots with a
phosphoCREB-specific antibody showed that cyclosporin A and FK506 do
not interfere with CREB phosphorylation at serine 119 stimulated with
cAMP or membrane depolarization. These results indicate that in HIT
cells stimulation of CRE-mediated transcription depends not only on the
activity of protein kinases phosphorylating CREB but also on the
Ca
/calmodulin-dependent protein phosphatase
calcineurin that is necessary for the transcriptional competence of
phosphorylated CREB.
(
)
in their promoter
with the core octamer sequence TGACGTCA. cAMP binds to the regulatory
subunits of protein kinase A and thereby induces the dissociation of
two catalytic subunits that are translocated to the cell nucleus
(1, 2) . The catalytic subunit then phosphorylates the
CRE-binding transcription factor CREB
(3, 4) at serine
119 (in CREB-327, corresponding to serine 133 in CREB-341), which leads
to an increase in its transcriptional activity
(5) . The effect
of cAMP is limited as CREB is dephosphorylated at serine 119 by
phosphatase-1 or -2a
(6, 7) .
influx in neuronal PC12 pheochromocytoma and endocrine pancreatic
islet cell lines has also been mapped to the CRE of the c- fos (8, 9, 10) , tyrosine hydroxylase
(11) , and glucagon genes
(12) . Elevation of the
intracellular free Ca
concentration activates CREB
(10, 12) and leads to an increased phosphorylation of
CREB at serine 119, the same site that is also phosphorylated by
protein kinase A
(10) . A CaM kinase probably mediates the
effect of Ca
as CaM kinases are activated in the
course of membrane depolarization
(13) , CaM kinases I, II, and
IV can phosphorylate CREB in vitro (10, 14, 15) , and a pharmacological
blocker of CaM kinase activity decreases the effect of membrane
depolarization on gene transcription
(12, 16) . However,
a model according to which CaM kinase mediates the stimulatory effect
of membrane depolarization on CREB/CRE-directed transcription may be
incomplete. In pancreatic islet cells the immunosuppressive drugs
cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated
by membrane depolarization and Ca
influx
(17) . Evidence suggests that cyclosporin A and FK506 act
through inhibition of the Ca
/calmodulin-dependent
protein phosphatase calcineurin
(17) . Hence, in addition to CaM
kinase, the phosphatase calcineurin seems to be necessary for
stimulation of CRE-mediated transcription by membrane depolarization in
pancreatic islet cells
(17) .
Plasmid Construction
The plasmids
-350GluLuc, 5xGluCRET81Luc, 4xCGCRET81Luc, Gal4-Luc
(12, 17) , 4xSomCRET81Luc
(18) , and pT81
(19) have been described previously.
Cell Culture and Transfection
The pancreatic islet
cell line HIT-T15
(20) was grown and transiently transfected as
described
(12, 21, 22, 23) . GHcells were transfected in suspension by the DEAE-dextran method
(5 µg of indicator plasmid/6-cm dish). JEG-3 cells were transfected
by the calcium phosphate precipitation method (10 µg of indicator
plasmid/6-cm dish). Transfection efficiency was checked by
cotransfection of CMV-
Gal. When indicated, 2 µg of pBJ5-CNA
plus 2 µg of pBJ5-CNB
(24, 25) , 5 µg of
RSV-KCREB
(26) , or 5 µg of pZ1
(12) were
cotransfected as described
(12) . Cell extracts were prepared 48
h after transfection and 6 h after stimulation. Test drugs were added 1
h before stimulation. The luciferase assay was performed as described
previously
(12, 21) . The
-galactosidase activity
was measured using a commercial kit for chemoluminescent detection
(Serva, Germany).
Calcineurin Phosphatase Assay
The calcineurin
phosphatase activity in the cultured cells was measured as described
(27, 28) .
Immunoblots
HIT cell lysates (in 50 m
M Tris-HCl, pH 7.0, 1% SDS, 2% 2-mercaptoethanol) were resolved on a
10% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, rinsed in TBST (10 m
M Tris-HCl, pH 8.0, 150
m
M NaCl, 0.05% Tween 20), incubated in 10% nonfat dry milk
dissolved in TBST for 1 h, and then incubated with either anti-CREB
antiserum
(29) , diluted 1:50,000 in TBST, or with
anti-phosphoCREB antibodies (0.2 µg/ml in TBST)
(30) for 2
h. Antibody-antigen complexes were detected with ECL reagents (Amersham
Corp.).
Insulin Measurement
Insulin was measured by
radioimmunoassay
(31) .
Materials
FK506 and rapamycin were dissolved in
ethanol. KN-62 was dissolved in dimethyl sulfoxide. A stock solution of
cyclosporin A (10 mg/ml) was prepared in ethanol with 20% Tween 80 and
further diluted in medium. Controls received the solvent only.
Pharmacological Evidence That a Calcium-dependent
Enzyme Other than CaM Kinase Is Involved in cAMP-induced
Transcription
The CRE of the rat glucagon gene has the typical
consensus sequence TGACGTCA and confers responsiveness to cAMP
(21) and Cainflux following membrane
depolarization induced by high KCl concentration in the pancreatic
islet cell line HIT
(12) . A reporter gene under control of five
copies of the rat glucagon CRE in front of the truncated viral
thymidine kinase promoter (from -81 to +52) was transiently
transfected into HIT cells. The glucagon CRE did not increase basal
activity (data not shown). However, transcription was stimulated
7.3-fold by 1.5 m
M 8-Br-cAMP (Fig. 1). Lowering the
extracellular Ca
concentration by addition of EGTA
had no effect on basal gene transcription, but inhibited
8-Br-cAMP-induced transcription by about 80% and abolished the
stimulation by high KCl (Fig. 1). The effect of EGTA seems to be
specific since a dye exclusion test showed no change in cell viability
after EGTA treatment compared to controls. Furthermore, the effect of
EGTA was reversible, since EGTA treatment followed by washing of the
cells and re-addition of Ca
had no significant effect
on the subsequent stimulation by forskolin (10 µ
M) of
insulin secretion (120.5 ± 17.0 ng ml
h
versus 159.5 ± 26.0 ng
ml
h
in controls) or CRE-mediated
transcription (6.1 ± 0.7-fold versus 5.8 ±
0.6-fold in controls). Therefore, these data suggest that cAMP-induced
gene transcription depends on extracellular Ca
.
Verapamil (10 µ
M), a blocker of L-type voltage-dependent
Ca
channels, inhibited CRE-mediated transcription by
27% after stimulation by 8-Br-cAMP ( p < 0.05,
t test), whereas depolarization-induced transcription was
completely blocked (data not shown), indicating that the stimulation of
CRE-mediated transcription by cAMP depends in part on Ca
influx through voltage-dependent Ca
channels.
Figure 1:
cAMP stimulates
CRE-mediated transcription through a Ca-dependent
step. Figure shows effect of 1.5 m
M EGTA. 5xGluCRET81Luc was
transfected into HIT cells and transcription stimulated by either 1.5
m
M 8-Br-cAMP or 45 m
M KCl. Luciferase activity is
expressed as percentage of the mean value in each experiment of the
activity measured in the respective control (5 m
M KCl, no
drug). Values are mean ± S.E. of three experiments, each done in
duplicate.
, no EGTA;
, EGTA.
KN-62 is a selective inhibitor of CaM kinases that spares other
protein kinases, e.g. protein kinase A, and
calmodulin-dependent enzymes
(32) . KN-62 (1 µ
M)
had no effect on 8-Br-cAMP stimulation of CRE-mediated transcription,
whereas it completely abrogated the stimulatory effect of membrane
depolarization (not shown). Therefore, all these data suggest that a
Ca-dependent enzyme other than CaM kinase is involved
in cAMP-induced transcription through the CRE.
cAMP-induced Gene Transcription Is Inhibited by
Cyclosporin A and FK506
Cyclosporin A and FK506 are inhibitors
of the Ca/calmodulin-dependent protein phosphatase
calcineurin
(33) . Fig. 2 shows the effects of cyclosporin A and
FK506 on gene transcription stimulated by cAMP through the CRE. Both
drugs inhibited cAMP-induced transcription (Fig. 2); in parallel
experiments they also inhibited Ca
-induced
transcription (Fig. 2) as reported previously
(17) . The
effect was concentration-dependent. The IC
for cyclosporin
A was about 60 n
M or 15 n
M after 8-Br-cAMP or KCl
stimulation, respectively, for FK506 about 1 n
M after both
stimuli. The maximal inhibition by cyclosporin A or FK506 was about
60-70% after stimulation with 8-Br-cAMP and about 80% after
stimulation with KCl. The effective concentrations are consistent with
the reported affinities of both drugs to their immunophilin receptors
and are similar to those concentrations effective in T cells
(25, 34, 35) .
Figure 2:
Cyclosporin A and FK506 inhibit
CRE-mediated transcription after stimulation by cAMP as well as by
membrane depolarization. 5xGluCRET81Luc was transfected into HIT cells
and transcription stimulated by either 0.8 m
M 8-Br-cAMP
( left panel) or 45 m
M KCl ( right panel).
CsA, cyclosporin A. Luciferase activity is expressed as
percentage of the stimulation by KCl or 8-Br-cAMP in the absence of
cyclosporin A or FK506 in each experiment. There was no significant
effect of cyclosporin A or FK506 on basal luciferase expression. Values
are means ± S.E. of three independent experiments each done in
duplicate.
Cyclosporin A also reduced
transcription stimulated by forskolin through the well characterized
CREs of the rat somatostatin or the human choriogonadotropin
genes (Fig. 3 A). The somatostatin, choriogonadotropin
,
and glucagon gene CREs contain the CRE octamer consensus sequence but
differ in the bases flanking the CRE octamer. These data thus indicate
that susceptibility to cyclosporin A and FK506 is conferred by the CRE
octamer and does not depend on the sequence context. Although a number
of transcription factors can bind to the CRE consensus sequence, only
CREB and the highly homologous factors ATF-1 and CREM
are known to
mediate cAMP responsiveness
(5) . The notion that cyclosporin A
and FK506 interfere with the action of CREB or a homologous factor is
supported by the observation that overexpression of a dominant
repressor of CREB, KCREB
(26) , inhibited the cyclosporin
A-sensitive transcription stimulated by forskolin through the
somatostatin CRE (Fig. 3 B). The stimulation by cAMP of
transcription mediated by a GAL4-CREB fusion protein, which contains
the CREB transactivation domain
(12) , was inhibited by
cyclosporin A (Fig. 3 B), indicating that CREB is
responsive to cyclosporin A.
Figure 3:
Cyclosporin A ( CsA) inhibits the
stimulation by cAMP of transcription mediated by CREB and CREs
containing the CRE octamer motif in different sequence contexts.
A, HIT cells were transfected with constructs in which the CRE
of either the rat somatostatin gene (SMS-CRE, 4xSomCRET81Luc) or the
human choriogonadotropin gene (CG
-CRE, 4xCG
CRET81Luc)
direct luciferase transcription. Forskolin 10 µ
M.
Luciferase activity is shown relative to the mean activity in each
experiment measured without treatment. B, left panel, HIT cells were cotransfected with 4xSomCRET81Luc
and the expression vector RSV-KCREB (KCREB) as indicated.
Fors, forskolin (3 µ
M). Overexpression of KCREB
had no effect on basal transcription. Luciferase activity is shown
relative to the mean activity in each experiment measured without Fors
and KCREB. Right panel, a luciferase reporter gene under the
control of GAL4-binding sites (Gal4-Luc) was transfected into GH
cells together with an expression vector for a GAL4-CREB fusion
protein (pZ1). Fors, stimulation by forskolin (10
µ
M) and isobutylmethylxanthine (100 µ
M).
CsA, 5 µ
M cyclosporin A. Luciferase activity is
expressed as percentage of the mean value in each experiment of the
activity measured in the absence of forskolin and cyclosporin A. Values
are means ± S.E. of three independent experiments, each done in
duplicate.
Caand cAMP stimulate
CRE-mediated transcription synergistically
(12) . While
CRE-mediated transcription was stimulated by 8-Br-cAMP 8.3-fold and by
KCl 3.7-fold, the combination of both induced a 38.8-fold increase in
gene transcription (Fig. 4). This synergism was almost completely
inhibited by cyclosporin A (Fig. 4). Thus, the synergism, like
the effect of the individual stimuli, is sensitive to inhibition by
cyclosporin A.
Figure 4:
Cyclosporin A inhibits the synergistic
action of cAMP and membrane depolarization. 5xGluCRET81Luc was
transfected into HIT cells. Treatment with 0.8 m
M 8-Br-cAMP,
45 m
M KCl, and 3.8 µ
M cyclosporin A
( CsA) as indicated. Luciferase activity is shown relative to
the mean activity in each experiment measured without treatment. Values
are means ± S.E. of two independent experiments, each done in
duplicate.
Evidence for the Involvement of Calcineurin
To
test whether cyclosporin A and FK506 are effective inhibitors of
calcineurin phosphatase activity in HIT cells, calcineurin activity was
measured. Untreated cells had a calcineurin activity of 127.3 ±
8.4 pmol of phosphate released minmg
( n = 7). Cyclosporin A and FK506 inhibited
calcineurin phosphatase activity in HIT cells in a
concentration-dependent manner (Fig. 5). The IC
values for
calcineurin inhibition were about 3 and 40 n
M for FK506 and
cyclosporin A, respectively. Thus, at concentrations that inhibited
CRE-directed transcription, cyclosporin A and FK506 also inhibited
calcineurin phosphatase activity in HIT cells.
Figure 6:
Rapamycin antagonizes the inhibitory
effect of FK506, but not of cyclosporin A, on calcineurin phosphatase
activity and cAMP-stimulated transcription. A, effect of
cyclosporin A ( CsA, 60 n
M), FK506 (5 n
M),
and rapamycin (1.1 µ
M) on calcineurin activity in HIT
cells. Calcineurin activity is expressed as percentage of controls that
received the solvents only. B, 5xGluCRET81Luc was transfected
into HIT cells. Effect of cyclosporin A ( CsA, 150
n
M), FK506 (5 n
M), and rapamycin (1.1
µ
M) on the increase in reporter gene expression induced by
1 m
M 8-Br-cAMP. Luciferase activity is expressed as percentage
of the stimulation by 8-Br-cAMP in the absence of other drugs in each
experiment. Values are means ± S.E. of two to three
experiments.
If inhibition of calcineurin activity
is indeed the mechanism by which cyclosporin A and FK506 inhibit
CRE-directed transcription after stimulation by cAMP, then
overexpression of calcineurin should render HIT cells more resistant to
the action of these compounds. Fig. 7 shows the effect of
overexpression of the subunits A and B of murine calcineurin
(24, 25) on gene transcription stimulated by cAMP
through the CRE. Cotransfection of the expression vectors for
calcineurin had no effect on basal transcription (not shown) and
increased 8-Br-cAMP-induced gene transcription only slightly
(Fig. 7). However, after overexpression of calcineurin FK506 (1.6
n
M) or cyclosporin A (60 n
M) no longer inhibited the
8-Br-cAMP-induced increase in gene transcription (Fig. 7). These
results suggest that cyclosporin A and FK506 inhibit cAMP-induced
transcription through inhibition of calcineurin, implying that gene
transcription stimulated by cAMP through the CRE depends on calcineurin
phosphatase activity in HIT cells.
Figure 7:
Overexpression of calcineurin renders HIT
cells more resistant to cyclosporin A and FK506 after stimulation by
cAMP. The reporter plasmid 5xGluCRET81Luc was cotransfected together
with either the empty expression vector pBJ5 (-) or with pBJ5-CNA
plus pBJ5-CNB (calcineurin). Transcription was stimulated by 1 m
M 8-Br-cAMP. FK506 (1.6 n
M) or cyclosporin A ( CsA,
60 n
M) were added as indicated. Luciferase activity is
expressed as percentage of 8-Br-cAMP stimulation in the absence of
other drugs in each experiment. Values are means ± S.E. of three
independent experiments each done in
duplicate.
CREB Phosphorylation at Serine 119 Is Not Changed by
Cyclosporin A and FK506
The cAMP and Casignaling pathways converge on serine 119 of CREB-327
(corresponding to serine 133 in CREB-341), which is phosphorylated in
response to both signals initiating an increased transcriptional
activity. Since cyclosporin A and FK506 inhibit CRE-directed
transcription stimulated by Ca
(17) and cAMP
(this study), it was tested whether cyclosporin A and FK506 change the
phosphorylation of CREB at serine 119. The phosphorylation of CREB at
serine 119 was investigated by immunoblots of HIT cell lysates using an
antibody that recognizes CREB phosphorylated at serine 119 but fails to
recognize CREB that is not phosphorylated at serine 119
(30) .
After treatment of HIT cells with forskolin or KCl, a band of 43 kDa
comigrating with CREB as stained by another anti-CREB antiserum and a
band of 38 kDa were increased in intensity (Fig. 8). The 38-kDa protein
could correspond to ATF-1
(36) , which is highly homologous to
CREB and seems to cross-react with the anti-phosphoCREB antiserum
(30) . Pretreatment of HIT cells with cyclosporin A and FK506
did not change the intensities of the bands corresponding to
phosphoCREB or phospho-p38 after stimulation by cAMP as well as
Ca
(Fig. 8). Therefore, inhibition of
calcineurin by cyclosporin A and FK506 interferes with CRE-mediated
transcription without reducing cAMP- or Ca
-induced
CREB phosphorylation at serine 119.
Figure 8:
Cyclosporin A and FK506 do not interfere
with CREB phosphorylation at serine 119 induced by cAMP or membrane
depolarization and Ca influx. The phosphorylation of
CREB at serine 119 was investigated by immunoblots. HIT cells were
treated for 4 h with 167 n
M FK506 ( lanes 2,
5, and 8 in A) or with 3.8 µ
M cyclosporin A ( CsA, lanes 3,
6, and 9 in A). 3 h before lysis 45 m
M KCl ( lanes 4-6 in A) or 10 µ
M forskolin ( Fors, lanes 7-9 in A)
were added. HIT cell lysates were blotted and incubated with
anti-phosphoCREB antibody ( A, right panel, and
B). The same HIT cell lysates were also blotted and probed
with an anti-CREB antiserum that does not distinguish between
phosphorylated and nonphosphorylated CREB in order to control for CREB
loading ( A, left panel). A, immunoblots of a
typical experiment. B, optic density ( OD) of the
43-kDa band ( left panel) or the 38-kDa band ( right
panel) in the anti-phosphoCREB blot, expressed as percentage of
the intensity after forskolin stimulation in the absence of other
drugs. Values are means ± S.E. of three independent
experiments.
Requirement for Calcineurin of CRE-mediated Transcription
Stimulated by cAMP in Cell Types Other than HIT Cells
To
determine if the requirement for calcineurin occurs in cell types other
than HIT cells, JEG-3 and GHcells were tested; these have
been used for analysis of cAMP-dependent activation of CREB
(3, 5, 37) . In JEG-3 cells, FK506 severely
inhibited calcineurin phosphatase activity whereas CRE-directed
transcription induced by cAMP was not changed by FK506 (Fig. 9). EGTA
(3 m
M), added 1 h before cAMP stimulation, did also not change
cAMP-induced transcription (not shown). In GH
cells, FK506
inhibited calcineurin phosphatase activity and severely blunted
CRE-mediated transcription induced by cAMP (Fig. 9). EGTA
treatment (3 m
M) reduced transcription by about 80% (not
shown). Consistent with other experiments in which additional cell
lines were tested,
(
)
these data indicate that,
under the experimental conditions used, transcription stimulated by
cAMP through CREs depends on calcineurin phosphatase activity in some
but not all cell types.
Figure 9:
Effect of FK506 on calcineurin phosphatase
activity and CRE-directed transcription stimulated by cAMP in JEG and
GH cells. For transcription experiments, the cells were
transfected with the reporter plasmid 5xGluCRET81Luc and stimulated by
8-Br-cAMP (1 m
M, JEG cells) or forskolin (3 µ
M,
GH
cells). The concentration of FK506 was 1.35 µ
M (JEG cells) or 167 n
M (GH
cells). Luciferase
activity is expressed relative to the mean value measured in each
experiment in the untreated cells. Calcineurin activity is expressed as
percentage of activity in untreated cells. Values are means ±
S.E. of three independent experiments, each done in
duplicate.
influx transcription mediated through the CRE
depends on calcineurin
(17) , this suggests a central role for
calcineurin phosphatase activity in CRE-mediated transcription in
pancreatic islet cells.
-induced
signaling pathways converge on the transcription factor CREB,
phosphorylation of CREB at serine 119 by cAMP- or
Ca
/calmodulin-dependent kinases being the initial
event of CREB activation
(9, 10) . Activation of CREB is
thought to involve an increase in transcriptional activity
(5) and possibly DNA binding
(2, 38) . Although
the mechanism by which phosphorylation at serine 119 stimulates CREB
transcriptional activity remains elusive, this modification may
stimulate interaction with one or more of the general transcription
factors or, alternatively, allow recruitment of a co-activator
(39, 40, 41) . To date, it has not been possible
to determine whether the single phosphorylation of CREB at serine 119
is sufficient for CREB activation. Based on the finding that
recombinant CREB phosphorylated in vitro on serine 119 was
transcriptionally active upon its microinjection into fibroblast
nuclei, it was suggested that phosphorylation of CREB at serine 119 is
sufficient to stimulate CRE-dependent transcription in intact cells
(42) . In the present experiments, cAMP- and
Ca
-induced CREB phosphorylation at serine 119 was
unimpaired in the presence of cyclosporin A and FK506, while
CRE-dependent transcription was largely reduced. This provides direct
evidence for the notion that under these conditions the phosphorylation
of CREB at serine 119 is not sufficient for activity. Furthermore,
since cyclosporin A and FK506 seem to act through inhibition of a
phosphatase (calcineurin), these results suggest the existence of an
additional phosphorylation event that directly or indirectly interferes
with the transcriptional competence of serine 119-phosphorylated CREB;
this inhibitory phosphorylation can be removed by calcineurin
phosphatase activity. The nature of the kinase involved is unclear. Its
activity may be constitutive or regulated by stimuli including cAMP and
Ca
. Its substrate could be any protein that directly
or indirectly can inhibit transactivation by CREB phosphorylated on
serine 119. Serine 119 in CREB is flanked by multiple consensus sites
for potential phosphorylation by protein kinase C, casein kinase II,
and glycogen synthase kinase-3
(5) within a region of CREB that
is required for interaction with CBP
(39) , a potential CREB
co-activator that together with a dTAF
110-like protein
(40) may be required for the interaction of serine
119-phosphorylated CREB with the TFIID complex. Although the functional
significance of these phosphorylation sites has not yet been
demonstrated, mutation of serine 128 of CREB to alanine (CREB-327
corresponding to serine 142 in CREB-341) in overexpressed GAL4-CREB
fusion proteins enhanced transcriptional activation in response to CaM
kinase II
(43) , suggesting that
phosphorylation of CREB at serine 128 may inhibit its transcriptional
activity. Furthermore, activation by protein kinase C through phorbol
esters in hepatoma cells reduced the occupancy of the CRE in the
tyrosine aminotransferase gene in vivo (44) , and
overexpression of p34 kinase, which also can phosphorylate CREB in
vitro, repressed transactivation by CREB in JEG choriocarcinoma
cells
(45) . Characterization of the calcineurin-sensitive
phosphorylation event that, as suggested by the present study,
interferes with the transcriptional competence of serine
119-phosphorylated CREB may add to the understanding of the
transactivating mechanism of CREB. The implied inhibitory
phosphorylation offers another mechanism in addition to serine 119
phosphorylation through which CREB/CRE-dependent transcription could be
regulated according to functional, developmental, and cell-specific
cues. Consistent with this view, induction of CRE-directed
transcription by cAMP was found by this study and additional
experiments
to depend on calcineurin phosphatase activity
in some but not all cell types, under the experimental conditions used.
/calmodulin-dependent enzyme
(33) . Consistent with a central role for calcineurin, the
Ca
-chelating agent EGTA reduced the stimulation by
cAMP of CRE-dependent transcription in HIT cells. EGTA treatment
prevents influx of extracellular Ca
and also lowers
basal cytosolic free Ca
concentration in these cells
(46) . The effect of verapamil suggests that part of the
Ca
needed for cAMP-induced transcription enters the
cell through L-type voltage-dependent Ca
channels.
This may be accounted for by the stimulation by cAMP of Ca
influx through L-type voltage-dependent Ca
channels
(46, 47) . As the inhibition by EGTA or
cyclosporin A and FK506 exceeds that by verapamil, Ca
from other sources may participate
(48, 49, 50) .
/calmodulin-dependent protein kinase; 8-Br-cAMP,
8-bromo cyclic AMP; CREB, CRE-binding protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.