©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of the Ca-dependent Phosphatase Calcineurin in Gene Transcription That Is Stimulated by cAMP through cAMP Response Elements (*)

Markus Schwaninger , Roland Blume , Meike Krüger , Gundula Lux , Elke Oetjen (§) , Willhart Knepel (¶)

From the (1) Department of Biochemical Pharmacology, University of Göttingen, 37070 Göttingen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Gene transcription can be induced by cAMP and Cathrough distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Cahas 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.


INTRODUCTION

Much information has accumulated about the pathway by which cAMP stimulates transcription of a number of genes that contain a cAMP-responsive element (CRE)() 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) .

Recently the induction of gene transcription by membrane depolarization and Cainflux 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 Caconcentration 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 Caas 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 Cainflux (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) .

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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 Caconcentration 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 Cahad no significant effect on the subsequent stimulation by forskolin (10 µ M) of insulin secretion (120.5 ± 17.0 ng mlhversus 159.5 ± 26.0 ng mlhin 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 Cachannels, 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 Cainflux through voltage-dependent Cachannels.


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 ICfor 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, 4xCGCRET81Luc) 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 GHcells 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 ICvalues 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.

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.


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 GHcells, 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, GHcells). The concentration of FK506 was 1.35 µ M (JEG cells) or 167 n M (GHcells). 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.




DISCUSSION

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 Cainflux 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.

The cAMP- and Ca-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 dTAF110-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 experimentsto depend on calcineurin phosphatase activity in some but not all cell types, under the experimental conditions used.

Calcineurin is a Ca/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 Caand also lowers basal cytosolic free Caconcentration in these cells (46) . The effect of verapamil suggests that part of the Caneeded for cAMP-induced transcription enters the cell through L-type voltage-dependent Cachannels. This may be accounted for by the stimulation by cAMP of Cainflux through L-type voltage-dependent Cachannels (46, 47) . As the inhibition by EGTA or cyclosporin A and FK506 exceeds that by verapamil, Cafrom other sources may participate (48, 49, 50) .

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.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (Grants Kn 220/4-1 and SFB 236/A25). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: University of Massachusetts Medical Center, 373 Plantation St., Worcester, MA 01605.

To whom correspondence should be addressed: Dept. of Biochemical Pharmacology, University of Göttingen, Robert-Koch-Str. 40, 37070 Göttingen, Germany. Fax: 49-551-399652.

The abbreviations used are: CRE, cAMP-responsive element; CaM kinase, Ca/calmodulin-dependent protein kinase; 8-Br-cAMP, 8-bromo cyclic AMP; CREB, CRE-binding protein.

M. Krüger, M. Schwaninger, and W. Knepel, unpublished results.


ACKNOWLEDGEMENTS

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.


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