OF BIOLOGICAL CHEMISTRY Vol. No. of 25, and Molecular ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 268,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 9, Issue of March 25, pp. 67144720,1993 Printed in U.S.A.

Activating Transcription Factor-1 Can Mediate Ca2+- and CAMP- inducible Transcriptional Activation*

(Received for publication, November 5, 1992)

Fang LiuS, Margaret A. ThompsonQ, Susanne Wagnerli, Michael E. GreenbergQ, and Michael R. Greenll 11 From the $.Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138, the $Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, and the llProgram in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

Increased intracellular cAMP and Ca2+ levels can activate transcription of a number of eukaryotic genes through a common promoter element, the cAMP/Ca2+ response element. This element is the binding site for activating transcription factor (ATF)/CREB proteins, an extensive transcription factor family. Here we report that one member of this family, ATF-1, can mediate both Cas+ and cAMP transcriptional responses, but that the responses to the two pathways differ in magnitude. In contrast, another family member, CREB, has been shown to mediate Ca2+ and cAMP responses to similar levels. Taken together, these results suggest a mechanism that allows cells to integrate and differentiate gene regulation by cAMP and Ca”.

Transcription of a number of eukaryotic genes is activated in response to an increase in the intracellular cAMP and/or Caz+ concentration (reviewed in Refs. 1-4). Transcriptional stimulation by cAMP occurs through a conserved promoter element, the CAMP-responsive element (CRE)’ (5-7). Recent studies have shown that transcriptional stimulation by membrane depolarization and Ca2+ influx can be mediated by a similar DNA sequence. For example, the human c-fos gene contains a regulatory element that mediates a transcriptional response to increased levels of intracellular Caz+ as well as to increased levels of CAMP; mutagenesis studies have shown that its Ca2+-responsive element (CaRE) is indistinguishable from a CRE (8-10). Similarly, depolarization and increased intracellular Ca” concentration activate expression of the human proenkephalin gene through its CAMP-inducible enhancer ( 11).

In addition to the CAMP- and Ca2+-inducible promoters, C/CaRE elements are present in a wide variety of viral and

* This work was supported by grants from the National Institutes of Health (to M. R. G. and M. E. G.), Grant R01 NS 28829 from the National Institute of Neurological Disorders and Stroke, American Cancer Society Faculty Research Award FRA-379 (to M. E. G.), and a scholar’s award from the McKnight Endowment Fund for Neuro- science (to M. E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed Program in Mo- lecular Medicine, University of Massachusetts Medical Center, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-5331; Fax: 508-

The abbreviations used are: CRE, CAMP-response element; CaRE, Caz+-response element; ATF, activating transcription factor; CAT, chloramphenicol acetyltransferase; PKA, protein kinase A; CaM, calmodulin; RSV, Rous sarcoma virus; aa, amino acid; nt, nucleotide.

856-5473.

cellular promoters which are activated, for example, by the adenovirus Ela protein (12) or the type I human T-cell leukemia virus Tax protein (13, and references therein). Ac- cordingly, the C/CaRE is not recognized by a single protein but rather by a class of transcription factors, referred to as the ATF/CREB family (reviewed in Ref. 14). The only sig- nificant amino acid similarity among the members of the ATF/CREB family lies within their DNA-binding domains, comprised of a leucine-zipper dimerization motif and a basic region which interacts with DNA (14). Thus far, at least 14 members of this complex family have been cloned (15-27).

One member of this family, CREB, can activate transcription in response to both increased intracellular cAMP and Ca2+ concentration (10, 28-32). Studies on CREB indicate that its Ser’33 residue and the flanking amino acids constitute a phosphorylation site for CAMP-dependent protein kinase (PKA) and Ca2+-calmodulin-dependent protein kinases (CaM kinases) I and I1 (10, 28, 30, 32-35). Phosphorylation of this site is required for CREB to mediate both cAMP and Ca2+ inducibility (10, 28, 30, 32-35).

In this report, we show that ATF-1, another member of the ATF/CREB family, can also mediate both Ca2+- and CAMP- inducible gene expression. Furthermore, we provide evidence that ATF-1 and CREB respond differently to cAMP and Ca2+.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection-F9 teratocarcinoma cells were grown in 50% Dulbecco’s modified Eagle’s medium, 50% F-12 supple- mented with 10% fetal bovine serum. DNA was introduced into cells by the calcium phosphate precipitation technique (36). In every experiment, the total amount of DNA in each transfection mixture was identical and included 2.5 pg of RSV-pgal or SV-@gal DNA as an internal control. 24 h after transfection, cells were harvested and assayed for @-galactosidase activity. CAT activity was then deter- mined after normalization of p-galactosidase activity. All transfection experiments were carried out in duplicate and were repeated four to six times.

PC12 cells were grown in 10% COz in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated horse serum and 5% fetal bovine serum. DNA was introduced into cells by the calcium phosphate and glycerol shock method as previously described (8). TWO days later, cells were stimulated with different inducing agents. RNA was then isolated and assayed by RNase protection (8).

structed by substituting the RSV-long terminal repeat for the SV40 Plasmid Constructions-An RSV expression vector was con-

enhancer in the pECE expression vector. RSV-ATF-1 and RSV- CREB expression plasmids were constructed by inserting ATF-1 (37) and CREB (16) cDNAs into the RSV vector. pECE-ABATF-1 contains DNA fragments encoding ATF-1 amino acids (1-3) + (57-186) + (226-271). The ATF-1 in vitro expression plasmid was constructed by inserting ATF-1 cDNA encoding amino acids 1-271 into the pGEM3 vector. The CREB in vitro expression plasmid was generated by fusing a DNA fragment encoding CREB (aa 250-341) to an ATG

6714

Ca2+ and CAMP Regulate the Activity of ATF-1 6715

parison between ATF-1 and CREB. FIG. 1. Amino acid sequence com-

The predicted amino acid sequence of ATF-1 is aligned to the 341 amino acids of CREB (16). Identical amino acids between the two proteins are indicated by vertical lines. Asterisks mark recognition sites for PKA and Ca*+/calmodulin-dependent kinases I and 11. PDE-1 and PDE-2 are sequences essential for CREB transcriptional activation (30,42). PDE- 1 can be phosphorylated by casein kinase I1 (30). The two glutamine-rich regions involved in transactivation (21, 22, 42) are indicated as waved lines. The 14-

the CREB-341 isoform. The DNA-bind- amino-acid a-peptide (43) is specific for

ing domain with the leucine residues ( f i k d circles) is indicated.

initiation codon and subsequently cloning into the pGEM3 vector. SomA(-71)CAT and a-hCGA(-169)CAT were constructed by inserting the rat somatostatin gene sequence -71 to +53 and the human a-chorionic gonadotropin gene sequence -169 to +44 upstream of the CAT gene, respectively. The CRE-CAT constructs c-fosA("Il)CAT, pENKAT-12, 5'TH(+27/-272)CAT, and pVIPCAT4 were described in Refs 29, 38, 39, 40, respectively. The plasmids pAF42G9, GAL4- CREB, pSVa-1, and GAL4-ATF1 were described previously (32, 37).

In Vitro TranscriptionlTranshtion and Mobility Shift Assay-The ATF-1 (aa 1-271) and CREB (aa 250-341) in vitro expression plasmids were transcribed and translated (or cotranslated) as previously described (18). For the mixing experiment, equal volumes of the translated product from each reaction mixture were mixed and incu- bated at 30 'C for 40 min.

For mobility shift analysis, 2 pl of the in vitro translated protein were assayed in a 20-4 reaction mixture, containing 0.5 pg of poly(dI- dC), approximately 0.3 ng of a synthetic CRE/ATF oligo probe, and 0.5 X buffer D (10 mM HEPES, pH 8.0, 10% glycerol, 50 mM KCl, 0.1 mM EDTA, and 0.25 mM dithiothreitol). The CRE/ATF oligo (CCCGGGATGACGTCATCCCGGG) was labeled with [y3'P]ATP by T, polynucleotide kinase. DNA-protein complexes were fraction- ated on a 5% native polyacrylamide gel.

Production of Antibody Specifically against ATF-1-A DNA fragment encoding the first 30 amino acids of ATF-1 was fused in frame to the glutathione S-transferase gene (41). This glutathione S-transferase-ATF-1 was expressed in and purified from Escherichia coli essentially as previously described (41). Antibody was raised by injecting the glutathione S-transferase-ATF-1 fusion protein into sheep.

RESULTS

ATF-1 and CREB Are Highly Homologous but Diverge in Their N-terminal Regions-We have previously reported the isolation of multiple ATF cDNA clones (18,37). One of them, ATF-1, is highly homologous to CREB throughout most of its coding region (Fig. 1). Both proteins contain phosphorylation sites for PKA/CaM kinases I and 11, at Sera on ATF-1 and S e P on CREB (Fig. 1). However, ATF-1 and CREB signif-

ATF-1 1

CREB 1

ATF-1 11

CREB 51

ATF-1 31

CREB 101

ATF-1 81

CREB 151

ATF-1 128

CREB 201

ATF-1 178

CREB 251

ATF-1 228

CREB 298

1

icantly diverge from each other at the N terminus. The abrupt N-terminal divergence between ATF-1 and

CREB led us to consider that the ATF-1 N terminus may result from a cloning artifact or alternative splicing. To in- vestigate these possibilities, we performed an RNase protection experiment. We synthesized a 284-nt 32P-labeled antisense RNA probe, which contained 242 nt of ATF-1 sequence spanning both the regions divergent from and homologous to CREB (Fig. 2, bottom). Fig. 2 shows that RNA isolated from HeLa or MG63 cells protected a single species of 242 nt from RNase digestion. Based on this result, we conclude that the ATF-1 cDNA is an authentic cDNA clone and that there is no alternative splicing within the ATF-1 N terminus. These conclusions are also supported by the observation that ATF- 1 cDNA clones independently isolated by several groups show identical sequence (19, 24, 25).

ATF-I Can Mediate cAMP Inducibility-The similarity in amino acid sequence between ATF-1 and CREB prompted us to examine the function of ATF-1 in the cAMP and Ca2+ signaling pathways. To determine whether ATF-1 can mediate CAMP-inducible transcriptional activation, we used the F9 cell transfection assay (28). Undifferentiated F9 cells contain low levels of functional CREB and therefore provide a good system to analyze the activity of an exogenously introduced CRE-binding protein (25, 28, 42, 45). We used a somatostatin (A-71)-CAT fusion gene containing one copy of the CRE as a reporter. As shown in Fig. 3A, the reporter was activated upon cotransfection with plasmids expressing both the catalytic subunit of PKA and ATF-1.

We wished to compare the activity of ATF-1 and CREB under conditions in which both ATF-1 and CREB were in excess relative to the CRE-CAT, so that the comparison would reflect a difference in activity rather than a difference in protein level. We therefore performed several titration curves

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MEDSHKSTTS

KTMDSGADNQQSGDAAVTEAESQQMTVQAQPQIATLAQVSMAAHATSSA I I

ET . . . . . . . . . . APQPGSAVQGAHISHI . . . . . . . . . . . . . . . . . . . . AQ I 1 1 1 I 1

PTVTLVQLPNGQTVQVHGVIQAAQPSVIQSPQVQTVQSSCKDLKRLFSGT a P e p t i d e

QVSSLSESEESQDSSDSIGSSQKAHGILMUWSYRKILKDLSSEDTRGRX I I I l l I I I I I I l l I I I I I I I I I I I I I I I ~ISTIAESEDSQESVDSVTDSQKRREILSRRPSYRKILNDLSSDAPGVPR

PDE-1 ****

PDE-2

GDGENSG . . . VSAAVTSMSVPTPIYQTSSGQYIAIAPNGALQLASPGTDG I I I I I I I I I I I I I I I I I I I I I I I l l I l l 1

IEEEKSEEETSAPAITTVTVPTPIYQTSSGQYIAITQGGAIQLANNGTDG

VQGLQTLTMTNSGSTQQGTTILQYAQTSDGQQILVPSNQVWQQTASGDMQ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I VQGLQTLTMTNAAATQPGTTILQYAQTTDGQQILVPSNQVWQQAASGDVQ

TYQIRTTPSATSLPQTVVMTSPVTLTSQTTKTDDPQLKREIRLMKNREAA I I I I I I I I I l l I I I I l l I I I I I I I I I TYQIRTAPTST. IAPGVVMASSPALPTQP . .AEEAARKiWVRLKKNREAA - RECRRKKKEYVKCLENRVAVLENQNKTLIEELKTLKDLYSNKSV* I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I RECRRKKKEWKCLENRVAVLENQNKTLIEELKALKDLYCHKSD* *

DNA B i n d i n g Region-

10

50

30

100

80

150

127

200

177

250

227

297

271

341

6716 Ca2+ and cAMP Regulate the Activity of ATF-1

62 52

40

309

348 21 7 201 190 180

160 147

122 110

90

76

67

ATF-1 mRNA

antisense RNA probe "O +17*

FIG. 2. RNase protection analysis of ATF-1. A uniformly 32P- labeled ATF-1 antisense RNA probe containing 242 nt of ATF-1 sequences (-70 to +172) and 42 nt of the pGEM 3 vector sequences was synthesized. 30 pg of cytoplasmic RNA isolated from MG63 cells or Hela cells, and 30 pg of control RNA ( tRNA) were analyzed in a RNase protection assay as previously described (44). The protected ATF-1 probe was separated on a 5% denaturing gel. Positions of molecular weight markers (M) are indicated on the left. The struc- tures of ATF-1 mRNA and the antisense RNA probe are diagrammed below the autoradiogram. For ATF-1 mRNA, the lines represent 5'- and 3"untranslated regions; the open and filled rectangles represent coding regions divergent from and homologous to CREB, respectively.

1 01 3

testing different amounts of plasmids expressing PKA (C subunit), ATF-1, and CREB. The transfection experiments in Fig. 3A were carried out under conditions in which both ATF-1 and CREB were at their maximal activity. Under these conditions, ATF-1 has a 4-5-fold lower basal activity than CREB. In the presence of PKA, ATF-1 transactivates the reporter gene -22-fold, whereas CREB transactivates the reporter gene -100-fold.

We also show that ATF-1 can support CAMP-inducible transcription in PC12 pheochromocytoma cells (Fig. 3B). Since PC12 cells contain high levels of endogenous CRE- binding activity, this experiment was carried out using a GAL4-ATF-1 fusion protein and a human c-fos reporter containing GAL4-binding sites. As shown in Fig. 3B, GAL4- ATF-1 stimulates transcription of the GAL4 reporter in response to forskolin, an activator of adenylate cyclase. Acti- vation requires a promoter containing GAL4-binding sites (32) and is dependent on stimulation with forskolin since human c-fos transcript (c-fosH) cannot be detected in unstimulated cells (see Fig. 5). The GAL4 DNA-binding domain itself cannot support cAMP inducibility (32). Consistent with the results in F9 cells, ATF-1 is approximately 4-fold less efficient than CREB in conferring cAMP inducibility (Fig. 3B).

To exclude the possibility that the different transcriptional

activities between GAL4-ATF1 and GAL4-CREB result from a difference in protein level, we examined the expression level of these two fusion proteins in Chinese hamster ovary cells. Immunoblotting experiments with an antibody against the GAL4 DNA-binding domain indicated that GAL4-ATF1 and GAL4-CREB were expressed at comparable levels (data not shown).

The Relative Activities of CREB and ATF-1 Are Similar on Several CAMP-inducible Promoters-As both CREB and ATF-1 can support cAMP inducibility and are expressed in a wide variety of cell lines (19,21, 22, 24, 25, 29,47), we tested the possibility that these two activators might differentially stimulate transcription from various CAMP-inducible promoters. The relative activities of ATF-1 and CREB were analyzed on five well characterized CAMP-inducible promoters: proenkephalin, c-fos, human a-chorionic gonadotropin, tyrosine hydroxylase, and vasoactive intestinal polypeptide. Each of these promoters contains a single CRE, except for the human a-chorionic gonadotropin promoter, which contains two copies of the CRE (1-3,48). Table I shows that the relative activities of ATF-1 and CREB are the same for all these promoters: ATF-1 has a 3-6-fold lower activity than CREB.

ATF-1 Can Mediate Ca2+ Inducibility-We next examined the possibility that ATF-1 is also able to support Ca2+-inducible transcriptional activation. PC12 cells were cotransfected with a GAL4-ATF 1 expression plasmid and a human c-fos reporter which contained GAL4-binding sites. Fig. 4A shows that in response to depolarization by K+/Ca2+, GAL4-ATF 1 stimulates transcription of the reporter. Activation is dependent on the presence of GAL4-binding sites in the promoter and the expression of GAL4-ATF 1 protein (Fig. 4A).

While ATF-1 has a lower activity than CREB in supporting cAMP inducibility, ATF-1 has a nearly equivalent activity as CREB in mediating Ca2+ inducibility (Fig. 4B). These results indicate that ATF-1 and CREB respond differently to cAMP and Ca2+.

ATF-1 -mediated Activation Is Specific for CAMP and Ca2+- We also asked whether ATF-1 can mediate transcriptional activation by other inducing agents. As shown in Fig. 5, the activity of ATF-1 is induced by cAMP and Ca2+ but not by several other stimulating agents, such as 12-0-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. More- over, ATF-1 cannot mediate transcriptional stimulation by adenovirus Ela and human T-cell leukemia virus-I Tax (37, 49, 50). Taken together, these observations suggest that the major role of ATF-1 is to mediate Ca2+- and CAMP-inducible gene expression.

ATF-1ICREB B Is a Substrate for PKA and CaM Kinases- As ATF-1 can mediate cAMP and Ca2+ inducibility, we reasoned that ATF-1 can be phosphorylated by PKA and Ca2+- calmodulin-dependent kinases. A previous study showed that CREB and a 38-kDa protein, referred to as CREB B, were copurified on a CRE DNA affinity column (32). CREB B is phosphorylated in vitro by PKA and CaM kinases I and 11, as is CREB (32). Furthermore, upon PKA and CaM kinases I and I1 phosphorylation, the major phosphopeptide of CREB B comigrates with the CREB phosphopeptide containing Ser133 (32, 51), an important transcriptional regulatory site

In vitro translated ATF-1 migrates at 38 kDa. Moreover, an ATF-1 antibody raised against its unique N terminus, specifically recognizes a 38-kDa protein in crude nuclear extract (data not shown). These observations prompted us to ask whether CREB B was actually ATF-1. We examined this possibility by immunoblotting using the previously purified

(10,28, 30, 32-35).

Ca2+ and cAMP Regulate the Activity of ATF-1 6717

Som Promoter B GAL4- GAL4-

0 ATFl CREB

c-f OSH +

A ..... PKA CREB PKA ATFl PKA

CREB ATFl

globin-, D II

Reporter:

9 GAL4 Sites +

- Human c-fos Gene FIG. 3. ATF-1 can mediate transcriptional activation by CAMP. Panel A, activation by ATE”1. F9 cells were cotransfected with 5

pg of SomA(-71)CAT reporter, 5 pg of expression plasmid for the catalytic subunit of PKA (46), and 7.5 pg of expression plasmids for ATF- 1 or CREB as indicated. Panel B, activation by GAL4-ATF1. PC12 cells were cotransfected with 20 pg of reporter plasmid (pAF42G9) and 5 pg of expression plasmid for GAU-ATF1 or GAM-CREB, as indicated. An a-globin expression plasmid (pSVa-1, 3 pg) was included as a control for transfection efficiency. Cells were stimulated by treatment with 10 p~ forskolin for 60 min. RNA was then isolated and assayed by RNase protection. Arrows indicate the endogenous rat fos transcript (c-fosR), the exogenous globin transcript (globin), and the correctly initiated human c-fos transcript (c-fosH). The two bands above c-fosH are incorrectly initiated transcripts (10, 32). The structure of the reporter is diagrammed below the autoradiogram.

TABLE I Comparison of the relative activity of ATF-1 and CREB on six

CAMP-inducible promoters The table summarizes results from four to six independent trans-

fection experiments in F9 cells. Plasmids expressing the catalytic subunit of PKA and either ATF-1 or CREB were cotransfected with one of the following CRE-CAT reporters: pENKAT-12, SomA(-71)CAT, c-fosA(-’Il)CAT, a-hCGA(-169)CAT, 5’TH(+27/ -272) CAT, and pVIPCAT4 (see “Experimental Procedures”). Num- bers have been normalized to the activity of ATF-1 on each promoter.

Relative activity Promotera CREB Am-1

Proenkephalin 6.0 1 Somatostatin 4.5 1 c-fos 4.0 1 Human a-chorionic gonadotropin 3.0 1 Tyrosine hydroxylase 3.5 1 Vasoactive intestinal polypeptide 4.8 1

protein preparation containing CREB B and CREB. As shown in Fig. 6, the ATF-1-specific antibody detected a single band at 38 kDa, which comigrated with ATF-1 synthesized by in vitro translation. Conversely, a CREB-specific antibody detected only CREB protein at 43 kDa. From these results, we conclude that ATF-1 is identical to CREB B and therefore can be phosphorylated by PKA and CaM kinases I and 11. The major phosphorylation site (Sera) by these enzymes is analogous to the Ser’33 in CREB. These observations support the view that phosphorylation of Sef3 is essential for ATF- 1-mediated cAMP and Ca2+ inducibility.

ATF-1 and CREB Can Form Heterodimers Both in Vitro

and in Vivo-As ATF-1 and CREB contain highly homologous basic/leucine-zipper DNA-binding domains, we reasoned that ATF-1 and CREB can form heterodimers both in vitro and in vivo. To provide in vitro evidence, we synthesized full- length ATF-1 and a partial CREB (aa 250-341) by in vitro translation and analyzed the protein products in a mobility shift assay. As expected, the DNA-protein complex containing the ATF-1 homodimer migrated slower than that containing the CREB(250-341) homodimer. When ATF-1 and CREB (250-341) were cotranslated or mixed after synthesis, a DNA- protein complex with an intermediate electrophoretic mobility appeared (Fig. 7A), indicating that ATF-1 and CREB heter- odimerize in vitro. The direct interaction between ATF-1 and CREB in vitro is supported by studies from other groups (24, 25, 47, 52).

To provide evidence that ATF-1 and CREB also heterodi- merize in vivo, we designed the inhibition experiment shown in Fig. 7B. Previous studies of basic/leucine-zipper transcription factors, such as CREB, have shown that mutations in the basic region abolish DNA binding but not dimerization (53, 54). We therefore constructed an ATF-1 derivative, ABATF-1, which contains an intact leucine-zipper dimerization motif but harbors an in-frame deletion that removes part of the basic region. As expected, ABATF-1 does not bind DNA in a mobility shift assay (data not shown). However, when CREB was cotransfected with ABATF-1, CREB-mediated transcriptional activation was decreased 4-5-fold (Fig. 7B). This inhibition is dependent on the presence of the intact leucine-zipper dimerization motif in ABATF-1, as a

6718 Ca2+ and CAMP Regulate the Activity of ATF-1

A GAL4 Derivative GAL4(1-147) GAL4ATF1

GAL4 Sites 9 9 9 9 0 0

K+/Ca*+ - + - + - +

- -- -globin Reporter:

- I - 9 GAL4 Sites

Human c-fos Gene - B

GAL4ATF1 GAL4CREB

c-fos -+ H

GAL4-ATF1 I

-c-fos"

Reporter:

- I - 9 GAL4 Sites

\/ TATA I -42

-Human c-fos Gene - FIG. 5. Transcriptional activation by ATF-1 is specific for

cAMP and Ca2+. PC12 cells transfected as in Fig. 3B were either unstimulated (-) or stimulated for 60 min with 10 p~ forskolin or 60 mM KC1 and 10 mM CaC12 or for 30 min with 50 ng/ml nerve growth factor (NGF), 3 ng/ml epidermal growth factor (EGF), or 0.3 pg/ml 12-0-tetradecanoylphorbol-13-acetate (TPA ).

lmmunoblot M Pre aATFl aCREB

200 -

R c-fos -+ w w FIG. 4. ATF-1 can mediate transcriptional activation by

Ca'*. Panel A, ATF-1 can support Ca2+ inducibility. PC12 cells were cotransfected with 20 pg of reporter plasmid (pAF42G9 or pAF42), 5 pg of expression plasmids for GAL4(1-147) or GAL4-ATF1, and 3 pg of a-globin control plasmid as indicated. Cells were either unstimulated (-) or stimulated (+) for 60 min with 60 mM KCl. RNA products are detailed in Fig. 3B. Panel B, ATF-1 responds differently to cAMP and Ca2+. PC12 cells were transfected as in Fig. 3B and stimulated for 60 min with either 10 p~ forskolin or 60 mM KC1 and 10 mM CaClz as indicated.

similar construct lacking the leucine-zipper region failed to repress CREB-mediated transcription in the same assay (data not shown). The observation that ABATF-1 acts as a domi- nant negative inhibitor of CREB indicates that ATF-1 and CREB can form heterodimers in vivo.

DISCUSSION

In this report we show that another member of the ATF/ CREB family, ATF-1, can mediate both Ca2+- and CAMP- inducible transcriptional activation. This finding extends the previous observation that CREB can integrate cAMP and Ca2+ signaling pathways (10, 28-35). We further show that ATF-1 and CREB respond differently to cAMP and Ca2+:

97 -

68 -

43 -

29 - FIG. 6. ATF-1 is identical to CREB B. The purified protein

preparation containing CREB B and CREB (32) was separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. CREB B and CREB were detected by incuba- tion with an antibody raised against the first 30 amino acids of ATF- 1 (lane 3 ) and the unique a-peptide of CREB (43) (lane 4 ) , respectively, followed by applying the enhanced chemilumenescence detec- tion reagents (Amersham Corp.). Pre, preimmune serum control for the ATF-1 antibody. Translated =S-ATFI, ATF-1 protein was synthesized by in uitro translation in the presence of [%]Met and transferred to the same membrane. Positions of molecular weight markers are indicated.

ATF-1 has a lower activity than CREB in supporting cAMP inducibility, whereas ATF-1 and CREB are almost identical in conferring Ca2+ inducibility. Such differences could be one

Ca2+ and cAMP Regulate the Activity of ATF-1 6719

A ATFl CREB Co Mix B Som Promoter

ATFl :ATFl +

CREB:CREB

U 4

U 1 ATFl :CREB

Basic L L L L ATF-1 I I

1 271

ABATF-1 0 “ 133 57 186 226 271

FIG. 7. ATF-1 and CREB can form heterodimers. Panel A, in vitro. ATF-1 (aa 1-271) and CREB (aa 250-341) were synthesized by in vitro transcription/translation and analyzed in a mobility shift assay using a 32P-labeled CRE/ATF oligo probe. Co, ATF-1 (aa 1-271) and CREB (aa 250-341) were cotranslated. Mix, the two proteins were separately translated and then mixed after synthesis. The ATF-1 homodimer, ATF-1:CREB heterodimer, and the major CREB homodimer are indicated. Competition experiments have verified that these DNA-protein complexes result from specific interaction with the CRE (data not shown). Panel B, in uiuo. F9 cells were cotransfected with plasmids directing the synthesis of CREB (2.5 pg of DNA), ABATF-1 (10 pg of DNA), and the SomA(-71)CAT reporter (5 pg of DNA) as indicated. 5 pg of plasmid expressing the catalytic subunit of PKA was included in lanes 2-4. The structure of ABATF-1 is diagrammed below the autoradiogram.

mechanism by which cells differentiate cAMP and Ca2+ signals.

The different transcriptional activities of ATF-1 and CREB in the cAMP signaling pathway may result from structural differences. Mutational studies on CREB indicate that in addition to the PKA site, the N-terminal glutamine-rich region, and the two subdomains flanking the PKA site are essential for full activity (30, 42, see Fig. 1). In addition, a stretch of 14 amino acids which can potentially form an a- helical structure contributes to the maximal activity of CREB (42, 43, see Fig. 1). Studies on CREM (CAMP response element modulator), which is composed of several related CRE- binding proteins derived by alternative splicing (21,22), support the view that these structural elements and a glutamine- rich region C-terminal to the PKA site are involved in transcriptional activation (21, 22). The two subdomains flanking the CREB PKA site and the glutamine-rich region C-terminal to the CREB PKA site are well conserved in ATF-1 (Fig. 1). However, ATF-1 lacks the glutamine-rich N-terminal domain and the a region. These structural differences may account for, at least in part, the lower activity of ATF-1 compared to CREB.

Our studies indicate that CREB is more active than ATF- 1 in the cAMP signaling pathway in both F9 and PC12 cells. Similar results have been obtained in JEG3 cells (24). These observations differ from a recent report that ATF-1 has a slightly higher activity than CREB in conferring cAMP inducibility in F9 cells (25). This discrepancy may result from a difference in the F9 cells used by the two groups. Addi- tionally, a greater amount of plasmids expressing the catalytic subunit of PKA and CREB were introduced into F9 cells (25). Overexpression of CREB may have caused an inhibition of transcription (termed “squelching”) and the observed lower activity of CREB compared to ATF-1.

Our results in PC12 cells were obtained using GAL4-ATF1 and GAL4-CREB fusion proteins. These constructs contain the intact leucine-zipper dimerization motifs that can poten-

tially dimerize with endogenous leucine-zipper-containing proteins, such as CREB. These endogenous proteins could add to our measured activity. A previous study using GAL4- CREB suggests that endogenous proteins do not contribute to the activity of GAL4-CREB, as point mutation of the CREB PKA site eliminates the ability of GAL4-CREB to activate transcription of a GAL4-CAT reporter in PC12 cells (55). To test whether endogenous proteins contribute to the activity of GAL4-ATF1, we analyzed a GAL4-ATF1 fusion protein deleted of its leucine-zipper and found that it did not activate transcription. This observation may reflect a require- ment for heterodimerization with endogenous proteins. How- ever, transfection experiments in F9 cells using native ATF- 1 protein indicate that ATF-1 has intrinsic transcription activity. Therefore, it is equally likely that the leucine-zipper may be essential for folding the ATF-1 dimer into a confor- mation required for transcriptional activation. Such a require- ment for the basic/leucine-zipper structure in transcriptional activation has been shown for other basic leucine zipper transcription factors, such as ATF-2 (56); and J u ~ D . ~ Al- though it remains possible that some of the observed activity of GAL4-ATF 1 is contributed by endogenous proteins, this does not preclude the conclusion that ATF-1 responds differently to cAMP and Caz+.

How can cAMP and Ca2+ regulate the activity of ATF-1 differently? Previous studies on other proteins have shorn that differential phosphorylation by the Ca2+ and cAMP signaling pathways occurs on proteins such as synapsin I, tyrosine hydroxylase, and MAP2 (57-59). In particular, phosphorylation of tyrosine hydroxylase by CaM kinase I1 and PKA has different effects on its enzymatic activity (58). The different transcriptional activities of ATF-1 in response to cAMP and Ca2+ signals may also result from differential phosphorylation. The combined results of this study and previous observations (32, 51) indicate that ATF-1 is phos-

F. Liu and M. R. Green, submitted for publication. T. Deng and M. Karin, unpublished results.

6720 Ca2+ and CAMP Regulate the Activity of ATF-1

phorylated on Sera3 by PKA and CaM kinases I and 11. 20. Gaire, M., Chatton, B., and Kedinger, C.(1990) Nucleic Acids Res. 18 ,

Notably, CaM kinase 11 phosphorylates an additional site on 21. Foulkes, N. S., Borrelli, E., and Sassone-Corsi, p. (1991) cell 64,739-749 ATF-1 in uitro (32). phosphorylation of this additional site 22. Foulkes, N. s., Mellstrom, B., Benusiglio, E., and Sassone-Corsi, P. (1991)

in UiUO may account for the different transcriptional activities 23. Hsu, J-C., Laz, T., Mohn, K., and Taub, R. (1991) Pmc. Natl. A c d . Sci. of ATF-1 in response to cAMP and CaZ+. U. S. A. 88,3511-3515

24. Hurst, H. C., Totty, N. F., and Jones, N. C. (1991) Nucleic Acids Res. 19 ,

gene induction through a C/C~RE, C/CaRE-binding 25. Rehfuss R. P. Walton K. M., Loriaux, M. M., and Goodman, R. H. (1991)

proteins display different responses to CAMP and Ca2+. We 26. Abel, T. Bhatt R., and Maniatis, T. (1992) Genes & Deu. 6,466-480 show here that one C/CaRE-binding protein, ATF-l, me- 27. Cowell, i. G., dinner , A., and Hurst, H. C. (1992) MOL cell. B ~ L 12,3070-

diates CAMP and Ca2+ transcriptional responses to different 28. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 69,675-680 magnitudes. contrast, C R ~ B mediates and ca2+

29. Berkowitz, L. A., and Gilman, M. Z. (1990) Proc. Natl. Acad. Sci. U. S. A.

responses to similar levels. Taken together, these results 30. Lee, c. Q., Yun, Y., Hoeffler, J. P., and Habener, J. F. (1990) EMBO J. 9 ,

suggest a mechanism that a h w s cells to integrate and differ- 31. Meinkoth J. L Montminy, M. R., Fink, J. S., and Feramisco, J. R. (1991) entiate gene regulation by cAMP and Ca2+.

32. Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991) Science 2 6 2 ,

Ackmw&gmnts-we gratefully acknowledge M. R. Montminy 33. Yamamoto, K. K., Gonzalez, G. A., Brig@, W. H., 111, and Montminy, M.

for the cDNA and the antibody against CREB a peptide, S. 34. Zhu, Z., Andrisani, 0. M., Pot, D. A, and Dixon, J. E. (1989) J. B ~ Z . Chem. McKnight for PKA expression plasmid, and M. Sheng for the purified 264,6550-6556

Gilman, E. J. Lewis, J. F. Habener, 1. Boime, and K. A. w. Lee for 36. Wigler, M., Pellicer, A,, Silverstein: S., and Axel, R. (1978) Cell 1 4 , 725- materials. We wish to thank members of the Green and Greenberg 731 laboratories, in particular, S. Roberts, G. Denis, D. Ginty, C . Ace, X- ii: ~ ~ , 4 , ~ , d ~ ~ ~ r ~ ~ ~ 1 g ~ ~ ~ ~ ~ ~ ~ , 1 ~ . ~ ~ ~ ~ ~ ~ ~ E,, and Goodman, H. Y. Li, and R. Krauss for critical reading of the manuscript and helpful discussions and T. O'Toole for superb secretarial assistance.

M.(1986) Nature 323,3;3-356 39. Lewis, E. J., Harrington, C. A., and Chikaraishi, D. M. (1987) Pmc. NatL

Acad. Sci. U. S. A. 84,3550-3554 40. Tsukada, T., Fink, J. S., Mandel, G., and Goodman, R. H. (1987) J. BioL

Chem. 262,8743-8747 1. Roesler, W. J., Vandenbark, G. R., and Hanson, R. W. (1988) J. Biol. Chem. ti: ,!!?&~l~; B d . ' ~ d ~ ~ ~ ~ z " e ~ . K ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ E7; g2&ontminy, 2. Goodman, R. H. (1990) Annu. Rev. Neurosci. 13 , 111-127 3, ~ ~ ~ ~ ~ i ~ ~ , M. R,, ~ ~ ~ ~ d ~ ~ , G. A., and yamamoh, K. K, (1990) ped 43. Yam-noto, K. K., GonzaIez, (2. A., Menzel, p., Rivier, J., and Montminy,

4. Sheng, M., and Greenberg, M. E. (1990) Neuron 4,477-485 44. Melton, D. A,, Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and

5. Montminy, M- R., Sevarino* K. J- A.7 Mande17 and Good- 45. Masson, N., Ellis, M., Goodburn, S., and Lee, K. A. W. (1992) Mol. Cell.

6. J. M., Wmshaw-Boris, A.l H. '., and R. w. (1986) 46. Mellon, P. L., Cleg , C H., Correll, L. A., and McKnight, G. S. (1989) Proc.

3467-3473

Nature 366,80-84

In conclusion, although both cAMP and Ca2+ can mediate 460-4609

J. ~ d . cheh. 2 6 6 , i8431-18434

3077

87,5258-5262

4455-4465

Mol. Ctk. Bwl. 11 , 1759-1764

1427-1430

R. (1988) Nature 334,494-498

CREB B-CREB We thank L. A. Berkowitz and M. Z. 35. Dash, p. K., Karl, K. A,, calicos, M. A., PrYWeS, R., and Kendal, E. R.(1991) Pmc. Natl. Acad. Sci. U. S. A. 88 5061-5065

REFERENCES

263,9063-9066 M. R. (1991) Mol. Cell. Bwl. 11,1306-1312

M. R. (1990) Cell 60,611-617

M. R. Green. (1984) Nucleic Ac& Res. 12,7035-7056

Biol. 12,1096-1106

Natl. Acad. Sci. 6. S . A. 86,4887-4892

Neurosci. 13 , 184-188

man, R. H. (1986) Proc. Natl. Acad. Sei. U. S. A. 83,6682-6686

J. Biol. Chem. 261,9721-9726 7. Silver, B. J., Bokar, J. A., Virgin, J. B., Vallen, E. A., Milsted, A., and 47. Hunt, H. c., M ~ ~ ~ ~ , N., Jones, N. c., and h e , K. A. w. (1990) M ~ [ . Cell.

8. Shew, M., Dowan, s. T., McFadden, G.3 and Greenberg, M. E41988) Mol. 4. Lin, y. s.,' and Green, M. R. (1988) Prw. Natl. A C ~ . Sei. u. s. A. 8 5 , Nilson, J. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 6682-6686 Biol. 10 6192-6203

9. S-sone-Corsi, p., Visvader, J., Ferland, L., Mellon, p. L., and Verma, 1. 49. Maekawa T., Matsuab, S., hjisawa, J., Yoshida, M., and Ishii, S. (1991) Cell. Bwl. 8,2787-2796 33963400

10. Sheng, M., Mcfadden, G., and Greenberg, M. E. (1990) Neuron 4,571-582 50. Flint, K. J., and Jones, N. C. (1991) oncogene 6, 2019-2026 M. (1988) Genes & Deu. 2 , 1529-1538 Oncogek 6,627-632

11. Nguyen, T. V., Kobierski, L., Comb, M., and Hyman, s. E (1990) J. 51. Sheng, M. (1990) The Actiuation o Immediate Early Genes Membrane

12. Flint, J., and Shenk, T. (1989) Annu. Reu. Genet. 2 3 , 141-161 Neurosci. 10,2825-2833 Depolurizatwn and Ca2+, Ph.D. tlesis, Harvard University,%oston, MA

13. Giam, C.-Z., and Xu, Y.-L. (1989) J. Bwl. Chem. 2 6 4 , 15236-15241 52. Hoeffler, J. P., Lustbader, J. W., and Chen, C.-Y. (1991) Mol. Endocranol.

14. Habener, J. F. (1990) Mol. Endocriml 4 , 1087-1094 6 ,256266

15. Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L., and Habener, J. F. 53. Dwarki, V. J., Montminy, M. R., and Verma, I. M. (1990) EMBO J. 9 ,

(1988) Science 2 4 2 , 1430-1433 225-232

16. Gonzalez, G. A., Ymamoto, K. K., Fischer, W. H., Karr, K., Menzel, P. 55. Ha wara, M , Alberts, A,, Brindle, P., Melnkoth, J., Feramlsco, J., Deng, 54. Andfisani, 0. M., and Dixon, J. E.(1991) J.,Biol. Chem. 266,21444-21450

Briggs 111. W., Vale, W. W., and Montminy, M. R. (1989) Nature 3 3 7 , f, Karin, M., Shenolikar, S., and Montminy, M. (1992) Cell 70, 105-

17. Maekawa, T, Sakura, H., Kanei-Ishii, C., Sudo, T., Yoshimura,T.,Fujisawa, 56. Zu, Y-L., Maekawa, T., Matsuda, S., and Ishii, S. (1991) J. Bwl. C k m . 749-752 113

18. Hai, T., Liu, F., Coukos, W. J., and Green, M. R. (1989) Genes & Deu. 3 , 57. Czernik, A. J., Pang, D. T., and Greengard, P. (1987) Proc. Natl. Acad. Sci. J., Yoshida, M., and Ishii, S. (1989) EMBOJ. 8,2023-2028 266,21444-21450

2083-2090 19. Yoahimura, T., Fujisawa, J., and Yoshida, M. (1990) EMBO J. 8, 2537- 58. Griffth, L. C. and Schulman, H. (1988) J. BWl. Chem. 263,9542-9549

2542 59. Schulman, H.'(1984) Mol. Cell. Bwl. 4,1175-1178

U. S. A. 8 4 , 7518-7522

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