MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1306-1312 0270-7306/91/031306-07$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 3

Characterization of Motifs Which Are Critical for Activity of the Cyclic AMP-Responsive Transcription Factor CREB G. A. GONZALEZ, P. MENZEL, J. LEONARD, W. H. FISCHER, AND M. R. MONTMINY* The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037 Received 17 October 1990/Accepted 18 December 1990

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.

structed a series of deletion mutants and tested them by transient assays in F9 teratocarcinoma cells. One critical region consisted of an 87-amino-acid glutamine-rich domain at the N terminus of CREB. Since CREB mutants lacking the N-terminal region were nevertheless efficient substrates of PK-A, these results demonstrate that phosphorylation can indeed be uncoupled from transcriptional stimulation. A second region, consisting of a five-amino-acid motif (DLSSD) C terminal to the PK-A phosphorylation site, also appeared to be indispensable for CREB activity. Since changes in spacing between the DLSSD motif and the PK-A site also rendered CREB inactive, this region may interact with the phosphorylation site to alter CREB structure. To characterize the structural elements predicted by this model, we initiated a series of biochemical experiments using bacterially expressed CREB protein. When added to nuclear extracts depleted of CREB, this protein stimulates transcription of the somatostatin gene. Moreover, the activity of purified CREB in this reconstituted system is inducible by PK-A. Indeed, the ability of antisera directed against the activation domain to specifically block PK-A-stimulated activity further illustrates the importance of this region in stimulating transcription. Limited proteolysis of the purified CREB protein reveals that whereas the DNA-binding domain is almost completely resistant to trypsin digestion, the transactivation domain is highly susceptible in the immediate region surrounding the PK-A phosphorylation site. We propose that PK-A may regulate CREB activity by altering the tertiary structure of the protein and thereby rendering other regions accessible for the interaction with proteins in the RNA polymerase II complex.

A number of growth factors and hormones regulate the expression of target genes by stimulating the phosphorylation of specific transcription factors. We have previously characterized the nuclear factor CREB, for example, which stimulates transcription of genes in response to the secondmessenger cyclic AMP (cAMP) (5, 9, 11, 17). Biochemical and in vitro mutagenesis experiments have revealed that CREB is activated by phosphorylation at a single protein kinase A (PK-A) phosphoacceptor, site Ser-133 (4). Since phosphorylation at Ser-133 activates transcription without changing DNA-binding affinity, it appears that phosphorylation of CREB directly modulates the efficacy of the transactivation domain. Previous work showing that CREB can stimulate transcription of previously unresponsive genes when a cAMP response element (CRE) (11) is attached to these promoters has prompted us to hypothesize a general activating motif in CREB that would interact with ubiquitous proteins in the RNA polymerase II transcription complex. Indeed, two such general activating motifs have been described in a number of nuclear factors, one glutamine rich and the other containing acidic residues (1, 13). Nevertheless, a number of nuclear factors have activation domains that do not fit into either category. The direct increase in negative charge accompanying phosphorylation suggested that proteins like CREB would stimulate transcription by providing an acidic surface. The inability of acidic residues to substitute for the serine phosphoacceptor, however, has argued against this model. Rather, by analogy with other kinase substrates, phosphorylation might activate CREB by triggering a conformational change in the protein which, in turn, would permit a second activating group in the protein to interact with the RNA polymerase II transcription complex (14). To determine whether other domains in addition to the PK-A site were important for CREB activity, we con*

MATERIALS AND METHODS Plasmid constructions. CREB mutants were generated by M13 mutagenesis using the rat CREB cDNA cloned into an M13mpl9 vector (2). Antisense oligonucleotides encoding each mutation were annealed to a uracil-containing CREB

Corresponding author. 1306

template in the sense orientation. Following primer extension with T4 DNA polymerase and ligation with T4 DNA ligase, the double-stranded plasmid was used to transform MV1190 cells. Each CREB mutant was identified by sequencing DNA obtained from bacteriophage plaques. The mutant CREB cDNAs were then inserted into Rous sarcoma virus (RSV) expression vectors and analyzed by transfection assay. Cell lines, transfections, and immunofluorescent studies. F9 teratocarcinoma cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Transfections and immunofluorescence studies were performed as described previously (4). CAT activity was measured from cell extracts after normalization to 3-galactosidase activity derived from the cotransfected RSV-3-galactosidase plasmid. Bacterial expression and purification of CREB protein. A bacterial CREB expression vector was constructed by inserting an NcoI-BamHI fragment encoding amino acids 3 to 341 of CREB into the T7 polymerase plasmid pET-3A (15). BL21 cells transformed with this plasmid were grown to an optical density of 0.6 (A6.), and expression of the CREB protein was induced with 2 mM isopropyl-P-D-thiogalactopyranoside (IPTG) for 3 h. Cells were harvested and lysed with a French press, and extracts were prepared as previously described (4). CREB protein was precipitated from the crude extract by ammonium sulfate fractionation, and the pellets were resuspended in BC50 buffer (50 mM KCl, 10 mM MgC12, 10 mM Tris [pH 8.5], 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride). These fractions were then passed over DNA-cellulose resin equilibrated with the same buffer. CREB protein was eluted from the resin with BC500 buffer (BC50 containing 500 mM KCl). The yield of recombinant CREB protein averaged 1 to 2 mg/liter. Footprinting and in vitro transcription assays. Footprint analysis of the recombinant CREB protein was performed by using an end-labeled 120-bp somatostatin promoter fragment as described previously (9). PC12 nuclear extracts were prepared and depleted of endogenous CREB protein by adsorption to a CREB immunoaffinity resin. This resin was prepared by attaching affinity-purified CREB antiserum 244 to protein A-Sepharose beads. For in vitro transcription assays, somatostatin and a-globin promoter templates (150 ng of each) were incubated with 0.5 ,ug of recombinant CREB protein or buffer for 15 min at 4°C. After addition of immunodepleted PC12 extracts, transcription reactions and primer extensions were performed as described previously

(17). For epitope inhibition experiments, affinity-purified CREB antibodies 220 and 240 were added to reaction mixtures containing DNA template plus purified CREB protein. Antisera were allowed to bind at 4°C for 1 h, and transcription assays were then performed as described above. Partial proteolysis of recombinant CREB protein. Limited proteolysis reactions were performed by using recombinant CREB protein (10 ,ug) plus purified trypsin (50 ng) in 50 mM KCl-2 mM CaCl2-10 mM Tris (pH 8.5). Samples were incubated at room temperature for the times indicated, and the reaction was terminated by addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer. For N-terminal sequencing experiments, 20 ,ug of CREB was used. After electrophoresis, these samples were transferred to polyvinylidene difluoride membranes by electroblotting, and CREB fragments were visualized by staining with Ponceau S. Fragment bands were excised and

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FIG. 1. Analysis of CREB deletion mutants in the N-terminal glutamine-rich domain. (A) Schematic diagram of CREB. Q, Glutamine-rich (20%) motif extending from amino acids 1 to 87; a, alternately spliced oa-helical peptide extending from residues 87 to 101; KID, kinase-inducible domain (amino acids 101 to 160) containing a PK-A phosphorylation site at Ser-133; , DNA-binding and leucine zipper dimerization domains extending from amino acids 284 to 341. (B) Transcriptional activities of N-terminal CREB deletion mutants. RSV expression vectors containing each CREB deletion mutant were analyzed by cotransfection with a CRE-CAT reporter plasmid with (+) or without (-) a PK-A expression construct. Activity represents absolute CAT activity in PK-Astimulated transfections expressed as a percentage of the wild-type value. *, Control transfection without added CREB plasmid. Fold induction represents the ratio of PK-A(+)/PK-A(-) activity for each CREB construct. A67, A87, and A119 are CREB deletion constructs, with the deletion endpoint indicated by the number. All deletion mutants contain identical C termini with intact DNA-binding and dimerization domains. The CRE-CAT reporter plasmid contains a somatostatin promoter fragment extending from -71 to +55 attached to the CAT reporter gene.

subjected to Edman degradation in a gas-phase sequencer (Applied Biosystems 470A) (3, 8). RESULTS To identify domains in CREB which, in addition to the PK-A motif, were important for transactivation, we constructed several N-terminal deletion mutants. Each CREB mutant was inserted into an RSV expression vector and analyzed by cotransfection with a PK-A expression plasmid and CRE-chloramphenicol acetyltransferase (CAT) reporter vector (Fig. 1). We have previously observed that F9 teratocarcinoma cells are ideally suited for these experiments because, in the absence of retinoids, they are almost completely unresponsive to cAMP (4). Cotransfection of wildtype CREB and PK-A expression vectors causes a 200-fold induction in CRE-CAT activity, thereby allowing the activ-

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GONZALEZ ET AL.

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Alternately spliced cc-helical peptide extending from amino acids 87 to 101; cc2, cc-helical region (as predicted by Chou-Fasman rules) extending from amino acids 101 to 120; box putative PK-C phosphorylation site extending from PK-A phosphorylation site extending amino acids 121 to 125; box casein kinase from amino acids 130 to 135; box phosphory16-amino-acid lation site extending from amino acids 151 to 160; region (amino acids 136 to 151) with the predicted a-sheet structure. Activity represents absolute activity in PK-A-stimulated transfections expressed as a percentage of the wild-type value. Fold induction represents the inducibility of each mutant by PK-A, expressed as a ratio of PK-A(+)/PK-A(-) CAT activity. (b to e)

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ities of the N-terminal mutants to be readily gauged relative to the activity of wild-type CREB. Deletion of the N-terminal 67 amino acids of CREB caused a 70% reduction in both basal and PK-A-stimulated activity of the CRE-CAT reporter plasmid. Nevertheless, the A67 CREB mutant remained as responsive to PK-A induction as wild-type CREB (33-fold versus 30-fold induction), suggesting that the activating domain of CREB could be distinguished from its kinase-inducible domain (KID). Deletion to amino acids 87 and 119 further diminished the response to PK-A stimulation. This reduction in CREB activity does not appear to reflect any changes in expression or phosphorylation of the mutant proteins, since each CREB mutant was efficiently targeted to nuclei of transfected cells and was phosphorylated by PK-A in vitro (not shown). The N-terminal domain has a remarkably high glutamine content (20%) which is evenly dispersed throughout the first 87 amino acids. Since transcription factors SP-1, Oct-1, and Oct-2 (1, 16) stimulate transcription through glutamine-rich domains, our results suggest that CREB may also function in part through this general activator motif. To identify additional active regions in CREB, we designed a series of deletion mutants within the KID region, so named because it encodes a cluster of potential phosphorylation sites (5). Box I encodes a potential PK-C phosphorylation site, box II encodes the PK-A motif, and box III encodes a casein kinase II site. An alternately spliced a-helical domain, termed a (amino acids 87 to 101), flanks these sites and strongly enhances PK-A-dependent activity (18). Although deletion of each motif in KID reduced PKA-stimulated activity two- to four-fold, none appeared to be absolutely required for cAMP inducibility (Fig. 2). Most notably, the casein kinase II motif (EEEKSEEE), which constitutes the most acidic region of the CREB molecule, appears to be more inert than the glutamine activator. Only one mutant within KID, spanning amino acids 136 to 160, was completely unable to stimulate CRE-CAT expression in transfected F9 cells. Since A151-160 CREB showed near wild-type activity, we reasoned that the critical region would therefore be restricted to the 16-amino-acid segment in KID spanning residues 136 to 151. In the process of analyzing this region, we noticed that transcription factor ATF-1 (6), sharing about 70% amino acid homology overall, contained a short motif (DLSSE) within the segment from 136 to 151 which was also conserved in CREB (DLSSD). To determine whether this DLSSD sequence was responsible for the loss of activity observed with the A136-160 mutant, we constructed CREB mutant A140-144, in which this motif is removed (Fig. 2b). Although A140-144 was efficiently expressed and targeted to nuclei of transfected cells, it was almost completely unable to stimulate CRECAT expression. Since A140-144 CREB was phosphory-

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Activity of the DLSSD motif mutants in F9 cells. (b) Relative activities and PK-A inducibility of CREB mutants with substitution underlined. *, Control transfection without added CREB plasmid. Mutant 136/137[G]3 contains three Gly residues inserted between amino acids 136 and 137. (c) CAT activities of CREB mutants in the presence (+) or absence (-) of a cotransfected PK-A expression plasmid. (d) Immunofluorescence micrographs of F9 cells transfected with wild-type CREB and mutant A140-144 and stained for CREB expression, using CREB affinity-purified antiserum 244. (e) SDS-PAGE of recombinant CREB or A140-144 mutant protein after phosphorylation in vitro with PK-A. The arrow points to 43-kDa CREB bands. Mr values of markers are in thousands.

GLUTAMINE-RICH DOMAIN IN CREB

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lated by PK-A to the same extent as wild-type CREB (Fig. 2e), we concluded that the DLSSD motif may function cooperatively with the PK-A motif to stimulate transcription. To define more precisely the residues within the DLSSD motif that were critical for CREB activity, we designed several conservative substitution mutants (Fig. 2b). Whereas substitution of acidic Asp-144 with neutral Asn-144 had a minimal effect, substitution of Asp-140 with Asn-140 caused an 80% loss in PK-A-stimulated CREB activity. Mutagenesis of both aspartate residues at positions 140 and 144 further reduced CREB activity to levels comparable to those of the DLSSD deletion mutant. By contrast, substitution of serine residues 142 and 143 with alanine caused only a 40 to 50% drop in CREB activity while maintaining near wild-type inducibility. These results suggest that although other residues within the DLSSD motif contribute to CREB activity, the acidic Asp-140 residue appears to be the most important component. The proximity of the DLSSD sequence (amino acids 140 to 145) to the PK-A motif (amino acids 130 to 136) prompted us to investigate whether the spacing between these regions was critical for activity. Insertion of a three-amino-acid glycine spacer between these motifs in mutant 136/137[G]3 rendered CREB completely inactive, indicating that the acidic residues within the DLSSD motif may interact with basic residues within the PK-A site to stabilize a secondary structure that is important for CREB activity. To understand the structural basis for CREB activation by PK-A, we initiated a series of biochemical experiments using bacterially expressed CREB protein. When the full-length CREB cDNA was inserted into the T7 polymerase bacterial expression vector pET-3a (15), high-level expression of CREB protein was readily detected (Fig. 3a). Using ammonium sulfate fractionated followed by DNA-cellulose chromatography, we were able to purify this protein to approximately 85 to 90% purity. When tested in vitro, the bacterial CREB protein had footprinting activity which was indistinguishable from that of its eukaryotic counterpart. To determine whether the recombinant CREB protein could also stimulate somatostatin transcription, we prepared PC12 nuclear extracts and depleted them of endogenous CREB with an immunoaffinity resin containing affinity-purified CREB antiserum 244 (Fig. 3c). Following removal of CREB from this extract, expression of the somatostatin CRE-CAT vector in vitro was severely diminished. Addition of bacterial CREB protein to immunodepleted extracts (Fig. 3d) specifically induced CRE-CAT activity three- to fourfold but had no effect on at-globin transcription. Addition of purified PK-A to depleted extracts supplemented with recombinant CREB protein further stimulated CRECAT transcription three- to fourfold without affecting ot-globin expression. Since PK-A was unable to stimulate transcription in the absence of detectable CREB protein, these results suggest that other CRE-binding proteins may not be involved in cAMP-responsive transcription. To identify structural elements of the CREB protein that were critical for transcriptional regulation, we performed epitope inhibition experiments (Fig. 4). We tested the ability of two different affinity-purified CREB antibodies to block transcription of the somatostatin gene in vitro. Antibody 220 was raised against a synthetic CREB peptide spanning amino acids 136 to 151, and antiserum 240 was developed against the oa-region peptide spanning residues 87 to 101. We have previously determined, by gel shift assay, that neither antiserum interferes with binding of CREB to a CRE oligonu-

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FIG. 3. DNA-binding activity, in vitro transcriptional activity, and PK-A inducibility of bacterially expressed CREB protein. (a) SDS-PAGE of CREB fractions obtained from Escherichia coli following transformation with a T7 polymerase expression vector. Fraction I, Crude lysate (10 ,ug); fraction II, pellet fraction from ammonium sulfate precipitation (10 p.g); fraction III, eluate fraction following DNA-cellulose affinity chromatography (10 ,ug). Mr values of markers are in thousands. (b) Footprint analysis of purified CREB protein from bacterial extracts, using a somatostatin promoter. C, Control reaction without added extract; +DTT, addition of 1 mM DTT to footprint reactions; -DTT, no DTT added. (c) Immunodepletion of CREB from PC12 nuclear extracts. Shown is a Western immunoblot of fractions, using affinity-purified CREB antibody 244. Lanes: 1, crude extract; 2, extract following depletion with immunoaffinity resin containing CREB antiserum 244; 3, CREB recovered from immunoaffinity resin after incubation with PC12 extract. The arrow points to the 43-kDa CREB protein. Mr values of markers are in thousands. (d) In vitro transcription assay using purified CREB and immunodepleted PC12 extract. Shown is primer extension analysis of somatostatin promoter (SRIF) activity, using ax-globin as an internal control plasmid. Arrows point to products expected for each gene. Addition (+) or absence (-) of CREB (0.5 pRg) or PK-A (1 ,ug) is indicated above the lanes.

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FIG. 4. Inhibition of somatostatin transcription in vitro with CREB antisera. (a) Schematic diagram of the CREB molecule showing the glutamine-rich domain (Q), alternately spliced a peptide (a), PK-A phosphoacceptor (P), and ,B region (a). Antibody (Ab) 240 was developed against a synthetic CREB peptide extending from amino acids 87 to 101, and antibody 220 was raised against a CREB peptide spanning residues 136 to 150. (b) Primer extension analysis of in vitro transcription reactions, using CREB-immunodepleted extracts supplemented with bacterial CREB protein (0.5 ,ug) plus C subunit (1 gxg). Activities of somatostatin (SRIF) and control a-globin plasmids are indicated. Numbers above the lanes indicate microliters of antibody (Ab) 220 or 240 added to each in vitro transcription reaction. Both antibodies contained about 1.4 mg of protein per ml. (c) SDS-PAGE of bacterial CREB protein phosphorylated with PK-A in vitro and then immunoprecipitated with either antibody 220 or antibody 240. Mr values of markers are in thousands. The arrow points to 43-kDa CREB bands.

cleotide (5, 18). Moreover, the two antisera have comparable affinities for the CREB protein, as determined by immunoprecipitation experiments (Fig. 4c). When added to in vitro transcription reaction mixtures containing purified CREB and immunodepleted PC12 extract, antiserum 220 specifically diminished somatostatin transcription in a dose-dependent fashion. Transcription of the endogenous a-globin control plasmid was unaffected by this treatment, however. Surprisingly, antiserum 240, directed against amino acids 87 to 101, had no effect on somatostatin or a-globin expression at any dose. Thus, as predicted by mutagenesis experiments, residues 136 to 151 appear to be important for CREB activity in vitro. As an independent test of CREB structure, we performed

FIG. 5. Partial proteolysis of CREB protein with trypsin. (a) Silver-stained SDS-PAGE of purified bacterial CREB fractions treated with trypsin. The digestion time (in minutes) is indicated above each lane. Positions of molecular weight (MW) standards (in kilodaltons) are shown on the right. (b) SDS-PAGE of heat-denatured CREB treated with trypsin for the time (in minutes) indicated above each lane. (c) Positions of trypsin cleavage sites that generate 30- and 27-kDa CREB fragments, as determined by N-terminal sequencing. Relative positions of these cleavage sites within the KID region are indicated by arrows. (d) Southwestern blot analysis of trypsinized CREB protein, using a double-stranded 32P-labeled CRE oligonucleotide probe. Numbers above the lane indicate the time (in minutes) of trypsin digestion. Mr values of markers are in thousands. The arrow points to the 12-kDa CREB fragment that appears at later times of trypsin digestion. (e) Position of the trypsin cleavage site that generates the 12-kDa CREB fragment (panel d), as determined by N-terminal sequence analysis. Q, Glutamine-rich motif; a, alternately spliced a-helical region; KID, kinase-inducible domain; M, DNA-binding and leucine zipper dimerization domains.

limited proteolysis experiments (Fig. 5). Experiments showing that proteases like trypsin are often useful for identifying active sites of enzymes prompted us to determine whether we could similarly distinguish the transactivation domain of CREB from other regions of the protein. When the recombinant CREB protein was treated with catalytic amounts of purified trypsin, we observed two major proteolytic products of 30 and 28 kDa (Fig. 5a). No such fragments were observed when we used CREB that had been denatured by boiling and addition of SDS to 1% (Fig. 5b), suggesting that the digestion pattern obtained with native CREB protein was indicative of its tertiary structure. Following SDS-PAGE, the 28- and 30-kDa fragments were transferred to polyvinylidene difluoride membranes, visualized with Ponceau S stain, and se-

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quenced. Remarkably, the N-terminal residues of these fragments mapped to amino acids 125 and 135, flanking the PK-A phosphorylation site. To map the C termini of each tryptic CREB fragment, we performed Southwestern blot (DNA-protein) assays (Fig. 5c). Both the 30- and 28-kDa fragments possessed CREbinding activity, suggesting that the DNA-binding domain was protected from trypsin digestion. Moreover, at later times of digestion, we observed a third proteolytic fragment of 11 kDa which also possessed CRE-binding activity and whose N terminus mapped to amino acid 265. Dimerization activity, as assessed by a glutaraldehyde cross-linking assay (not shown), was also intact for each fragment. These results were particularly unexpected because, given the preference of trypsin for basic residues, we had predicted that the CREB DNA-binding domain, with 35% arginine and lysine content, should be far more susceptible to cleavage than the KID region, which has only 10% basic amino acids. In view of the almost complete resistance of the DNA-binding and dimerization domains to proteolysis, our results suggest that these regions may be either relatively sequestered within the CREB molecule or highly structured. Indeed, Hope et al. have observed that the C-terminal DNA-binding domain of the yeast factor GCN4 is also resistant to protease digestion

(7). In contrast, the transactivation domain of CREB, which is sensitive to trypsin digestion, would appear to be differently structured. To determine whether phosphorylation causes detectable changes in CREB structure here, we compared the tryptic digestion patterns obtained with phospho and dephospho forms of the protein (Fig. 6). Surprisingly, phosphorylation of CREB by PK-A caused a significant reduction in the 28-kDa but not the 30-kDa proteolytic fragment. These results suggest that as a consequence of phosphorylation, the structure of CREB near the PK-A motif may be altered. very

DISCUSSION On the basis of these observations, we have developed a working model for transcriptional activation by CREB. Following cAMP stimulation, the catalytic subunits of cAMP-dependent protein kinase appear to migrate to the nucleus (12) and to induce the phosphorylation of CREB at a single phosphoacceptor site, Ser-133. It appears that PK-A directly phosphorylates CREB without any cascade intermediates, since mutagenesis of the basic arginine residues

1311

within the consensus PK-A site (RRXS) renders CREB inactive (10). By what mechanism does phosphorylation of CREB stimulate transcription? The presence of a leucine zipper dimerization motif at the C terminus of CREB had prompted us initially to consider a combinatorial model that involved heterodimer formation with other leucine zipper proteins. Indeed, transcription factor AP-1, which consists of a Jun/Fos heterodimer, requires this leucine zipper-mediated interaction for both transcriptional and DNA-binding activities. Nevertheless, immunoprecipitation and glutaraldehyde cross-linking experiments suggest that CREB monomers do not associate with any other leucine zipper proteins and therefore that CREB stimulates transcription as a homodimer (2). Phosphorylation has been thought to activate a number of enzymes by causing structural changes in these proteins. Glycogen phosphorylase, for example, is a dimeric enzyme whose activity is regulated by phosphorylation of an N-terminal serine residue, Ser-14. Recent crystallographic studies of this enzyme have revealed that phosphorylation allows the N terminus to assume a helical structure which can then bind to the subunit interface (14). Prior to phosphorylation, these subunit interactions are inhibited by the high density of basic residues surrounding the phosphorylation site. The KID of CREB is similarly surrounded by a high density of charged residues that may inhibit close subunit interactions. Phosphorylation may activate CREB initially through a conformational change in the KID region. Our results suggest that the PK-A and DLSSD motifs may be indispensable for this purpose. The spacing between Arg-131 and -135 in the PK-A motif (RRPSYR) is identical to that between Asp-140 and -144 in the DLSSD sequence. The potential formation of salt bridges here may induce a tightturn structure that subsequently alters CREB activity. Indeed, the change in trypsin sensitivity that we observed after phosphorylation of CREB may reflect the formation of such a structure. The N-terminal glutamine-rich (Q) domain in CREB also appears to be important for transcriptional activation by PK-A. That similar glutamine-containing regions are constitutively activated in transcription factor SP-1, however, further suggests that the KID may correspondingly regulate Q-domain function in CREB. The glutamine-rich N terminus of Oct-2, for example, requires the presence of a phosphorylated C-terminal serine/threonine-rich domain for transcriptional activity (16). Thus, like CREB, phosphorylation of this C-terminal region may induce structural changes in Oct-2 which render the glutamine-rich motifs competent for activation. We have previously described an alternately spliced 14amino-acid segment, termed ox, whose absence in ACREB significantly diminishes the transactivation potential of the protein (18). The amphipathic nature of the ot domain had prompted us to suggest that it functions to encourage protein-protein interactions. Indeed, preliminary cross-linking experiments indicate that this region functions to promote interactions between the CREB subunits themselves. Furthermore, the ac region is fortuitously positioned between the Q and KID regions, suggesting that it may orient these regions to promote transcriptional activity. Thus, the inability of antibody 240 to inhibit transcription in vitro may reflect the different mechanisms by which we suspect the ao-region imparts activity compared with the Q or KID region. Our results suggest that CREB can activate a number of different promoters in part through a glutamine-rich activator motif. It

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is possible, therefore, that phosphorylation regulates the transcriptional activity of CREB by causing structural changes in the KID region which render the Q region transcriptionally competent. ACKNOWLEDGMENTS We thank Joan Vaughan for preparing affinity-purified antisera. We also thank Wylie Vale and Jean Rivier for helpful advice and criticism and Bethany Coyne for secretarial assistance. This work was supported by NIH grants GM37828 and DK26741 and by NCI grant CA-14195 and was conducted in part by the Clayton Foundation for Research, California Division. M. M. and W. H. F. are Clayton Foundation Investigators. REFERENCES 1. Courey, A. J., and R. Tjian. 1988. Analysis of Spl in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887-898. 2. Dwarki, V. J., M. Montminy, and I. M. Verma. 1990. Both the basic region and the "leucine zipper" domain of cyclic AMP response element binding (CREB) protein are essential for transcriptional activation. EMBO J. 9:225-232. 3. Fischer, W. H., D. Karr, B. Jackson, M. Park, and W. Vale. 1991. Microsequence analysis of proteins purified by gel electrophoresis. Methods Neurosci., in press. 4. Gonzalez, G. A., and M. R. Montminy. 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675-680. 5. Gonzalez, G. A., K. K. Yamamoto, W. H. Fischer, D. Karr, P. Menzel, W. Biggs III, W. W. Vale, and M. R. Montminy. 1989. A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence. Nature (London) 337:749-752. 6. Hai, T., F. Lin, W. J. Coukos, and M. R. Grren. 1990. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3:2083-2090.

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