©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Inactivating Point Mutation Demonstrates That Interaction of cAMP Response Element Binding Protein (CREB) with the CREB Binding Protein Is Not Sufficient for Transcriptional Activation (*)

(Received for publication, January 6, 1995; and in revised form, February 1, 1995)

Peiqing Sun (1) (2) Richard A. Maurer (1)(§)

From the  (1)Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201 and the (2)Genetics Ph.D. Program, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cAMP response element binding protein (CREB) mediates transcriptional activation in response to the cAMP signaling pathway. Several recent studies have suggested that phosphorylation-dependent interaction of CREB with a co-activator designated CREB binding protein (CBP) is a crucial step in mediating transcriptional responses to cAMP. In the present study we have determined that replacement of Ser of CREB with Asp greatly decreases the ability of the cAMP-dependent protein kinase to activate CREB. As Ser is located within the region of CREB that interacts with CBP, it seemed quite likely that mutations at this site might interfere with binding to CBP. However, both in vitro and in vivo protein-protein interaction assays revealed that replacement of Ser with Asp does not interfere with the binding of CREB to CBP. These studies argue strongly that although the binding of CREB to CBP is necessary, it is not sufficient for transcriptional responses to cAMP.


INTRODUCTION

The recent identification of a co-activator that binds to phosphorylated CREB (^1)has provided new insights into the mechanisms that mediate transcriptional responses to cAMP(1) . The co-activator, which is designated the CREB binding protein (CBP), interacts with CREB after CREB is phosphorylated at Ser(2) . Previous studies have shown that phosphorylation of CREB at Ser is crucial for cAMP-activated transcription(3) . An essential role for CBP in mediating transcriptional responses to PKA was recently demonstrated in intact cells by microinjection of neutralizing antibodies to CBP(4) . Interestingly, CBP itself can function as a transcriptional activator protein. When fused to a heterologous DNA binding domain, so that it is brought to a promoter in a phosphorylation-independent fashion, CBP is a strong activator of transcription(1) . It has also been shown that CBP interacts with the basal transcription factor, TFIIB(2) . This suggests a simple model in which phosphorylation of CREB activates transcription by recruiting CBP, which then interacts with basal transcription factors.

In the present paper we have identified a point mutation that greatly reduces the ability of CREB to mediate cAMP-stimulated transcription. The discovery of this inactivating mutation is based on recent studies that demonstrated that phosphorylation of Ser of CREB by Ca/calmodulin-dependent protein kinase II blocks the ability of cAMP to activate CREB(5) . The observation that Ser can function as a negative regulatory site led us to examine amino acid replacements of this residue. We find that replacement of Ser with Asp mimics the effects of phosphorylation and greatly reduces the ability of CREB to mediate cAMP-stimulated transcription. We have tested the ability of CBP to interact with this mutant form of CREB. Surprisingly, CBP interacts with the mutant protein in a manner that is indistinguishable from the wild type protein.


MATERIALS AND METHODS

Cell Culture and Transfections

F9 and PC12 cells were transfected by calcium phosphate precipitation as described previously (5) . Typically cells received 5 µg of the 5xGAL4-TATA-luciferase indicator DNA, 2 µg of an expression vector encoding the GAL4 DNA binding domain fused to the transcriptional activation domain of CREB (GAL4-CREB), and 5 µg of a PKA expression vector. Cells were collected and lysates prepared 48 h (F9 cells) or 24 h (PC12 cells) after transfection. The protein concentration of lysates was determined(6) , and luciferase activities were measured using a constant amount of protein as described elsewhere(7) . Each experiment included three separate transfections for each group, and the experiments have been repeated three to five times. The GAL4CREB expression vector was prepared by polymerase chain amplification of the CREB coding sequence by adding an upstream BamHI site and a downstream XbaI site and substitution of the truncated CREB sequence for the complete coding sequence in the previously described GAL4-CREB vector(8) . A luciferase reporter gene containing five GAL4 binding sites has been described previously(5) . The CREB coding sequence was altered so that Ser was replaced with aspartate, glutamate, or lysine by oligonucleotide-directed mutagenesis(9) . The mutations were confirmed by nucleotide sequence analysis(10) .

Western Blot Analysis of the Expression of Transiently Transfected CREB Mutants

COS-1 cells were transfected with 6 µg of either pBSSK(-) or GAL4-CREB expression vector per 60-mm dish, and cell lysates were prepared as described(5) . Protein concentrations of cell lysates were determined(6) , and 40 µg of total protein from each sample was separated on a 12% polyacrylamide denaturing gel. After electroblotting to a polyvinylidene difluoride membrane, the filter was blocked and incubated with a 1:5000 dilution of a polyclonal antiserum to CREB(8) . The immune complexes were visualized using the ImmunoSelect Photoblot chemiluminescent system (Life Technologies, Inc.) following the manufacturer's protocol.

In Vitro Binding Assay for the Interaction of CREB with CBP

The coding sequence for amino acids 462-661 of CBP (1) was cloned in-frame to the biotin carboxylase carrier protein (BCCP) in the PinPoint Xa-1 bacterial expression vector (Promega). Biotinylated BCCP-CBP fusion protein was expressed in Escherichia coli strain JM109 and purified by affinity chromatography using a monomeric avidin column following the supplier's protocol. Recombinant CREB (5) was phosphorylated with 330 nM purified recombinant catalytic alpha subunit of the cAMP-dependent protein kinase or 300 nM recombinant Ca/calmodulin-dependent protein kinase IIalpha (11) in the presence of [-P]ATP using previously described reaction conditions(5) . After phosphorylation, the reaction mixtures were diluted in binding buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, and 0.5% nonfat dry milk) and then chromatographed on Sephadex G-50 (Sigma) columns to remove free ATP. To prepare immobilized CBP, 40 µg of biotinylated BCCP-CBP were incubated with 100 µl (packed volume) of streptavidin-agarose beads (Life Technologies, Inc.) for 2 h at room temperature with rotation and then washed extensively with the binding buffer. The binding assays were performed in duplicate 80-µl reactions containing binding buffer and 10 µl (packed volume) of BCCP-CBP bound to streptavidin-agarose beads and varying amounts of phosphorylated CREB. After incubation for 2 h at room temperature with rotation, the reaction mixtures were transferred to disposable mini-columns (Kontes) attached to a vacuum manifold. Each column was washed quickly with 3 ml of binding buffer, and radioactivity bound to the column was determined.

In Vivo Analysis of the Interaction of CREB with CBP

A mammalian version of the yeast two-hybrid screen (12) was used to detect the in vivo interaction of CREB and CBP. To prepare a fusion of the VP16 activation domain and the CREB activation domain, the coding sequence for residues 412-490 of VP16 (13) was placed in-frame with an initiation codon. The coding sequence for the transcriptional activation domain of wild type or mutant CREB (amino acids 1-283) was amplified by polymerase chain reaction and subcloned in-frame at the 3` end of the VP16 activation domain and then subcloned into the pcDNA3 mammalian expression vector (Invitrogen). For the mammalian two-hybrid assay, F9 cells were transfected with 5 µg of 5xGAL4-TATA-luciferase(5) , 5 µg of pGAL-CBP3(1) , 5 µg of a wild type or mutant expression plasmid for VP16-CREB, and either 5 µg of pBSSK(-) or 5 µg of an expression vector for the catalytic alpha subunit of PKA(14) . Cells were collected and luciferase activities determined as described above. Each experiment included three separate transfections for each group, and the experiments have been repeated three to five times.


RESULTS AND DISCUSSION

Replacement of Serof CREB with Asp Reduces PKA-activated Transcription

Our recent studies of Ca regulation of CREB activity led to the identification of a phosphorylation site within the CREB activation domain that negatively regulates CREB activity. These studies demonstrated that when Ser is phosphorylated by Ca/calmodulin-dependent protein kinase II, the ability of PKA to activate CREB is greatly reduced(5) . To further assess the functional role of Ser, we replaced this residue with aspartate or glutamate, negatively charged amino acids that might mimic the effects of phosphorylating Ser. Ser was also replaced with lysine to determine if simply placing a charged residue at this position has an effect on CREB activity. The CREB mutants were tested for their ability to mediate PKA-stimulated transcription in a transient transfection assay utilizing GAL4-CREB fusion proteins in both F9 and PC12 cells (Fig. 1). A GAL4-CREB fusion protein was studied in order to eliminate interference from other endogenous proteins that bind to CREs (8, 15, 16, 17) . In both PC12 and F9 cells, the GAL4-CREB fusion was very strongly activated by PKA (please note that for many of the samples, the control values are difficult to distinguish from the x axis). The replacement of Ser with Asp very sharply reduced responses to PKA. Indeed, the Asp mutation had almost as severe an effect as mutation of the PKA phosphorylation site (Ser replaced with Ala). Interestingly, replacement of Ser with Glu or Lys had little or no effect on PKA-stimulated CREB activity. The finding that replacement with Glu has little effect is very interesting and demonstrates that it is not the simple placement of a negative charge at position 142 that blocks activation of CREB. An approximately 1.5-Å difference in the positioning of the negative charge completely changes the effect of the substitution.


Figure 1: Replacement of Ser with Asp reduces activation of GAL4-CREB by PKA. Either PC12 cells (A) or F9 cells (B) were transfected with 2 µg of GAL4-CREB fusion constructs carrying different substitutions at Ser or Ser, 5 µg of 5xGAL4-TATA-luciferase reporter, and 5 µg of either pBSSK(-) or an expression plasmid for the catalytic subunit alpha of PKA (RSV-PKA). Nomenclature for the mutant GAL4-CREB vectors indicates the position of the mutation within the CREB coding sequence followed by the single-letter amino acid code for the mutant residue. Luciferase activity was determined 24 or 48 h after transfection. Values are the means ± S.E. of the mean for three separate transfections.



It is possible that replacement of Ser with Asp results in poor expression or decreased stability of the protein. To examine this possibility, we compared the relative expression levels of GAL4-CREB fusion proteins containing different substitutions at Ser. Immunoblotting analysis of extracts from COS-1 cells transfected with expression vectors for wild type or mutant GAL4-CREB revealed that all of the proteins are expressed at similar levels (Fig. 2). Therefore, the reduced PKA-stimulated activity of the Asp replacement at Ser is not due to low level expression of the mutant. Similar results (although with a considerably weaker signal) were obtained with F9 cells (data not shown).


Figure 2: GAL4-CREB fusion proteins containing different substitutions at Ser are expressed at similar levels in transfected cells. COS-1 cells were transfected with 6 µg of pBSSK(-) or an expression vector for the indicated wild type and mutant GAL4-CREB proteins. Nomenclature for the mutant GAL4-CREB vectors indicates the position of the mutation within the CREB coding sequence followed by the single-letter amino acid code for the mutant residue. Two separate transfections were performed for each construct. Cells were collected 24 h after transfection and lysed by sonication, and 40 µg of protein from each sample were separated on a denaturing polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane, and then GAL4-CREB fusion proteins and endogenous CREB were visualized by immunostaining with a polyclonal antiserum to CREB. The arrows indicate the migration of bands corresponding to the GAL4-CREB fusion produced by the transfected expression vector (upperarrow) and endogenous CREB (lowerarrow).



Replacement of Serwith Asp Does Not Interfere with Binding of CREB to CBP in Vitro

It is possible that replacement of Ser with Asp or phosphorylation of Ser blocks the interaction of CREB and CBP. As binding of CBP to CREB appears to be essential for mediating cAMP-stimulated transcription (4) decreased binding to CBP would offer an explanation for the reduced activity of the Asp replacement. This hypothesis is consistent with the observation that Ser is located within the region of CREB that is required for interaction with CBP(1) . Since the Asp replacement at Ser greatly decreases, but does not completely block, the activation of CREB by PKA, it is possible that the effect of this mutation is a quantitative rather than a complete block of CREB binding to CBP. Therefore, we developed an in vitro binding assay to quantitate the effect of the Asp replacement of Ser on the binding of CREB to CBP. The CREB binding domain of CBP (amino acids 462-661) was expressed in E. coli as a fusion protein with a portion of the BCCP. The BCCP fragment is biotinylated in E. coli(18) . Biotinylated BCCP-CBP was purified, coupled to streptavidin-agarose, and tested for binding to [P]CREB (Fig. 3). When wild type CREB was phosphorylated with PKA and [-P]ATP, the radiolabeled CREB bound to BCCP-CBP streptavidin-agarose in an apparently saturable fashion. Very little binding to the streptavidin-agarose was observed in the absence of BCCP-CBP. Furthermore, although the tissue-specific transcription factor, Pit-1, can be phosphorylated by PKA(19) , phosphorylated Pit-1 did not bind to the immobilized CBP (Fig. 3). Thus, this system offers a quantitative and highly specific assay for analysis of the interaction of CREB with CBP. This assay demonstrated similar concentration dependence for the binding of CBP to wild type CREB or CREB in which Ser was replaced with Asp. We also found that phosphorylation of CREB with Ca/calmodulin-dependent protein kinase II had little or no effect on binding to CBP (data not shown). The preceding experiments tested the interaction of CREB with the 462-661 region of CBP. This region of CBP was used because it is sufficient for PKA-dependent interactions with CREB (1) and because it has been technically difficult to produce full-length CBP. However, it remained possible that substitution of Ser of CREB with Asp might interfere with binding to full-length CBP. To address this possibility we isolated full-length CBP from cultured cells by immunoprecipitation, resolved the immunoprecipitated proteins by gel electrophoresis, and transferred the proteins to a membrane. Binding of P-CREB to the immobilized CBP was then tested using an overlay assay, as described previously(1) . Autoradiographic analysis of the overlay assay demonstrated similar binding of full-length CBP to wild type CREB or CREB in which Ser was replaced with Asp (data not shown). Thus despite the fact that replacement of Ser with Asp severely blunts transcriptional activation, this mutation does not detectably interfere with the interaction of CBP and CREB in vitro.


Figure 3: Replacement of Ser of CREB with Asp does not interfere with the in vitro binding of CREB to CBP. Biotinylated BCCP-CBP was bound to streptavidin beads. Either the BCCP-CBP beads (closedsymbols) or streptavidin beads alone (opensymbols) were incubated with wild type or mutant [P]CREB or [P]Pit-1, which had been previously radiolabeled by incubation with PKA and [P]ATP. Unbound proteins were removed by filtration, and bound radioactivity was determined. For the mutant CREB protein, the position of the mutation within the CREB coding sequence is indicated followed by the single-letter amino acid code for the mutant residue.



Analysis of the Interaction of CREB with CBP in Mammalian Cells Using a Two-hybrid Assay

To further examine interactions between CREB and CBP, we have developed an in vivo protein interaction assay similar to the yeast two-hybrid system(12) . As with the yeast two-hybrid system, the assay involves use of a GAL4-responsive reporter gene and two fusion proteins that interact with each other to form a functional transcription factor. One of the fusion proteins contains the DNA binding domain of the yeast transcription factor, GAL4, fused in-frame to the CREB binding domain of CBP (amino acids 462-661). CBP does not contain a transcriptional activation domain(1) , and by itself, the GAL4-CBP fusion protein did not activate expression of the reporter gene, either in the presence or absence of PKA. The other fusion protein contains the VP16 transcriptional activation domain linked to the CREB transcriptional activation domain (CREBDeltab-zip). As the VP16-CREBDeltab-zip fusion protein does not contain a DNA binding domain, it also cannot activate transcription. GAL4-CBP and VP16-CREBDeltab-zip should not form a competent transcription factor until PKA phosphorylates VP16-CREBDeltab-zip and induces binding to GAL4-CBP. Neither GAL4-CBP nor VP16-CREBDeltab-zip resulted in PKA-stimulated reporter gene expression (Fig. 4). However, when both the GAL4-CBP and the wild type VP16-CREBDeltab-zip expression vector were transfected with a PKA expression vector, a very vigorous activation of the reporter gene was obtained. In several different experiments PKA has yielded a 1000-6000-fold increase in the activity of the two-hybrid reporter system. Replacement of Ser of CREB with Ala within the VP16-CREBDeltab-zip fusion protein drastically reduced PKA-stimulated transcriptional activation. These findings suggest that phosphorylation of CREB at Ser of the VP16-CREBDeltab-zip fusion protein leads to interactions with GAL4-CBP and results in very strong transcriptional activation using this mammalian two-hybrid system. These findings demonstrate that this system provides a very sensitive method for detecting interactions of CREB and CBP in vivo. It is important to note that this activation is very dependent on phosphorylation by PKA, consistent with the fact that there is no detectable interaction between CBP and unphosphorylated CREB(2) . We have used this mammalian two-hybrid system to test the effects of mutations at Ser on interactions between CREB and CBP in vivo. Replacement of Ser with Asp, Glu, or Lys had little or no effect on PKA-induced binding of CREB and CBP, while replacement of Ser with Ala greatly decreased the ability of PKA to induce CREB-CBP binding. Therefore, in vivo, as well as in vitro, the Asp replacement at Ser did not interfere with CREB and CBP interaction.


Figure 4: Replacement of Ser of CREB with Asp does not interfere with the in vivo binding of CREB to CBP. The in vivo interaction of CREB and CBP was assessed using a mammalian version of the two-hybrid assay. F9 cells were transfected with 5 µg of the 5xGAL4-TATA-luciferase reporter gene, 5 µg of GAL4-CBP expression vector, 5 µg of wild type or mutant expression vectors for a VP16-CREBDeltab-zip fusion protein, and either 5 µg of pBSSK(-) or 5 µg of an expression vector for the catalytic subunit of PKA. The designation for the mutant VP16-CREB vectors indicates the position of the mutation within the CREB coding sequence followed by the single-letter amino acid code for the mutant residue. -Fold activation was determined by dividing the luciferase activity for each sample, which was co-transfected with the PKA catalytic subunit expression vector, by the mean basal activity for that group.



The present findings demonstrate that substitution of a negatively charged residue, Asp, at Ser of the transcriptional activation domain of CREB greatly reduces PKA-mediated activation of CREB. Although this mutation severely reduces PKA-mediated transcriptional activation, the mutation does not interfere with the interaction of CREB and CBP either in vitro or in vivo. These finding are rather surprising in view of the observation that CBP appears to function as a constitutive transcriptional activator when brought to a promoter as a GAL4 fusion protein(1) . Indeed, based on the constitutive activity of CBP and the ability of CBP to interact with the basal transcription apparatus, one might consider that CREB simply functions as a scaffold that recruits CBP to the promoter in a PKA-dependent fashion. However, the present findings suggest that such a simple model is probably not correct and provide strong evidence that the binding of CBP to CREB is not sufficient for transcriptional activation. If binding of CBP to CREB is not sufficient for transcriptional activation, what essential step in the PKA-mediated response is disrupted by replacement of Ser of CREB with Asp? It may be that this mutation interferes with binding of an additional factor to CREB. This factor could be another co-activator. Biochemical fractionation experiments and in vitro transcription studies have demonstrated that at least four separable co-activators may be required for maximal responses to several different transcriptional activators(20, 21, 22, 23) . Replacement of Ser with Asp might also interfere with the interaction of CREB and the basal transcription apparatus. A recent study demonstrated that CREB can interact with Drosophila TAF110, a component of the TFIID complex(24) . Alternatively, CBP may need to acquire a particular conformation in order to communicate with the basal transcriptional machinery and activate transcription. Asp replacement of Ser of CREB, although not reducing the affinity for CBP, may alter the conformation of bound CBP and therefore block the interaction of CBP with downstream factors. Further characterization of the structure and function of factors required for transcriptional responses to phosphorylated CREB should help resolve these two alternatives.


FOOTNOTES

*
This research was supported by American Heart Association Grant-in-aid 93006350. 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.

§
To whom correspondence should be addressed: Dept. of Cell Biology and Anatomy, L215, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-7566; Fax: 503-494-4253.

(^1)
The abbreviations used are: CREB, cAMP response element binding protein; CBP, CREB binding protein; PKA, cAMP-dependent protein kinase; BCCP, biotin carboxylase carrier protein. pBSSK(-), pBluescript SK(-).


ACKNOWLEDGEMENTS

We thank Drs. Richard Goodman, Roland Kwok, and Jim Lundblad for valuable reagents, helpful discussions, and critically reviewing the manuscript. We are grateful to Dr. Steven Triezenberg for supplying the VP16 clones used in this study and Paul Howard for providing the recombinant catalytic subunit of the cAMP-dependent protein kinase and recombinant Pit-1. We also thank Bobbi Maurer for aid in preparing this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.