Overexpression and purification of transcriptionally competent CREB from a recombinant baculovirus

PROTEIN

EXPRESSION

2,402-411

AND PURIFICATION

(1991)

Overexpression and Purification of Transcriptionally Competent CREB from a Recombinant Baculovirus Chang-You

Chen,*

Daniel

H. Bessesen,t

Stephen

M. Jackson,**$

Department of Medicine, Divisions of *Medical Oncology and tEndocrinology, Genetics, University of Colorado School of Medicine, University of Colorado

Received

August

14, 1991,

and

in revised

form

October

and James P. Hoeffler**t$

and $Department of Biochemistry, Biophysics Cancer Center, Denver, Colorado 80262

and

18, 1991

Signal transduction and viral stimulatory pathways converge ultimately at the level of transcriptional activation to influence the expression of a variety of cellular genes in response to environmental stimuli and developmental signals. Recent studies have implicated the cyclic AMP-responsive element-binding protein (CREB) to be involved in mediating transcriptional activation in response to multiple varied stimuli, including (1) stimulation of the protein kinase A signal transduction pathway; (2) membrane depolarization and increases in intracellular calcium; and (3) viral induced gene expression. In order to study the structure and functional mechanisms of CREB actions in these systems, full-length CREB-327 was expressed in Spodoptero frugiperda (Sf9) cells with the baculovirus expression vector system. The expressed CREB, which is phosphorylated and localized in the nucleus, is capable of enhancing the transcription of a reporter gene containing the CRE sequence in a cell-free transcription assay. Approximately 12.5 mg of purified CREB per liter of infected Sf9 cell culture can be obtained. These large amounts of purified protein will facilitate studies of the structure and functions of this important transcriptional regulatory protien. 0 1991 Academic press. 1~0.

Control of eukaryotic gene transcription is a tightly regulated process mediated by nuclear factors whose availability is determined by cell type, differentiation state, and position in the cell cycle (1,2). Hormonal activation of signal transduction pathways results ultimately in the transcriptional activation of many cellular genes (3-7). Activation of the protein kinases A and C by hormonal stimulation leads to increased transcription of cellular genes bearing related &acting DNA sequences in their promotor regions (8-21). These sequences are represented by the octameric cyclic AMP-

responsive element (CRE, 5’-TGACGTCA-3’) and the closely related heptameric phorbol ester (TPA)-responsive element (TRE, 5’-TGAGTCA-3’) and differ by only a single base insertion or deletion. Multiple studies have demonstrated that the protein products of two protooncogenes, jun and fos, interact and mediate transcription through sequence-specific binding to the TRE motif (22-25). The cloning of human placental CRE-binding protein (CREB (26)) marked the beginning of a long and continuing series of reports describing the cloning of related CREB and activating transcription factor (ATF) proteins, which interact and mediate transcription through sequence-specific binding to the CRE motif (26-32). To date, the only members of this family that have been shown to mediate transcriptional responses to the activation of protein kinase A are the original CREB-327 isolate (26) and an alternatively spliced variant of this factor isolated subsequently (32), CREB-341. A specific member of the ATF group of transcriptional activators, ATF-2, has been shown to mediate adenoviral Ela-stimulated transcription via sequence-specific binding to CRE motifs present in the adenovirus genome (33). Therefore, the CREBIATF proteins have been demonstrated to mediate transcriptional responses to both cellular signalling and viral stimulatory pathways through the common mechanism of sequence-specific binding to the CRE motif. Recently, in collaborration with Dr. Aleem Siddiqui’s laboratory, we have identified a new mechanism of viral-stimulated gene expression mediated by CREB/ ATF proteins (34). We demonstrated that the interaction of either CREB or ATF-2 with the hepatitis B virus transactivator protein, pX, produces a DNA-binding complex with a unique sequence specificity not exhibited by any of these proteins alone. This complex (either CREB:pX or ATF-2:pX) binds specifically to a motif in the HBV enhancer 1 with the sequence B-TGACGCAA-

402

1046-5926191

Copyright 0 1991 All rights of reproduction

hy

$3.00

Academic Press, Inc. in any form reserved.

OVEREXPRESSION BOILED NUCLEI

BOILED CYTOFLABM

OF

403

CREB

MATERIALS

AND

METHODS

Construction

of pVL1393ICREB

Two synthetic oligonucleotides, one containing a BamHI site 5’ to baculovirus translational initiation sequences and the sequences encoding the first 10 amino acids of CREB, another containing the antisense sequences encoding the final 10 amino acids and the stop codon of CREB (26), were used as primers to amplify full-length CREB cDNA by the polymerase chain reaction (PCR) method. The amplified DNA fragment was purified from a 1% low melting agarose gel, the strands were made flush with T4 DNA polymerase, digested with BamHI, and then inserted into the BamHI and SmaI sites within the polylinker of pVL1393. The recombinant plasmid DNA was purified as described (38). BEV-CREB

I INFECTED

Bf-9

CELL

EXTRACTS

FIG. 1.

Localization and heat stability of recombinant CREB-327 in Sf9 cell fractions. Coomassie blue staining of SDS-PAGE separated Sf9 cell nuclei or cytoplasm fractions depicts the recombinant CREB-327 migrating as an apparent 44-kDa species found only in the nuclei of infected cells. Furthermore, the recombinant CREB protein retains the heat stability associated with the native protein from JEG-3 cells as evidenced by its partitioning to the supernatant fraction of the boiled nuclei. No CREB was found in the medium, suggesting that the Sf9 cells correctly translocated the recombinant protein only to the nuclear compartment.

3’. More recently, Sheng et al. (35) have demonstrated activation of CREB transcriptional activity by phosphorylation of CREB by calcium calmodulin-dependent kinases I and II, suggesting that membrane depolarization and increases in intracellular Ca2+ can also activate CREB-mediated transcription. Taken together, these data suggest that the CREB/ATF proteins represent an important family of transcriptional regulatory proteins that normally mediate transcriptional responses to extracellular events transduced to the nucleus via intracellular signalling pathways, but that these proteins are also critical components of the aberrant gene expression programs initiated and maintained by viral infections of normal cells. It is therefore of utmost importance to characterize the structure and functions of these important transcriptional regulatory proteins. In the present study we have utilized the baculovirus expression vector system (BEVS) to overexpress the full-length CREB-327 protein in order to obtain sufficient quantities of this protein for further characterizations of the mechanisms by which it mediates transcriptional activation. The availability of large amounts of CREB-327 will also facilitate crystallographic studies of the tertiary structure of this protein alone, or bound to DNA, and cocrystallization of CREB interacting with other important transcriptional regulatory proteins.

Sf9 Cell Culture

Monolayer

and suspension cell cultures of Spodopteru (Sf9) cells were grown in Grace’s insect medium supplemented with 10% fetal bovine serum, 25 Fg/ ml gentamycin, and 50 pg/ml Fungizone. Pluronic F-68 (0.1%) was added to suspension cell cultures to protect frugiperda

BEV-CREB

INFECTED

CELLS

kD -

200

c

199

C

72

-44

c

28

IblYlYQ-32Pi LABELLIMO

FIG. 2.

Phosphorylation of recombinant CREB-327 in Sf9 cells. Twenty-four hours postinfection, cells were incubated in medium with 32Pi for an additional 24 h. The cell pellet lane depicts total cellular phosphoproteins. Phosphoproteins in whole nuclei or heatfractionated nuclei are depicted in lanes 2,3, and 4. The single heatstable phosphoprotein of 44 kDa present in the supernatant fraction of the boiled nuclei represents the recombinant CREB-327.

404

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-P

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BACTERIAL CREB-BR

BACULOVIRUS CREB-FL P

ET

200 n BEV-CR E% -FL UNLABELLED

I

t- -

Y

Y

CRE I CRE I

HUMAN ALPHA-GONADOTROPIN

PROMOTOR

FIG. 3. DNase I protection of the human a gonadotropin promotor with recombinant CREBs. DNAse I footprinting experiments were carried out utilizing a fragment spanning the sequences between -220 and +l of the human (Y gonadotropin promotor. The fragment was labeled at -220. Incubation of the fragment (left) with full-length recombinant CREB-327 (Baculovirus CREB-FL) or bacterially expressed CREB-254-327 (Bacterial CREB-BR) protected the region from -110 to -150 from DNase I digestion. This region, containing the tandemly repeated CRE motifs, is denoted diagramatically on the outside of the figure. (Right) Reaction mixtures were incubated in the presence of increasing amounts of an unlabeled CRE oligonucleotide corresponding to the CRE and surrounding sequences found in the somatostatin promotor as described previously (26). Note the dose-dependent loss of protection of the CRE sequences as the amount of unlabeled competitor oligonucleotide is increased.

the cells from mechanical shearing. Monolayer cell cultures used for transfections and plaque purification of recombinant viruses were derived from stock suspension cell cultures. All tissue culture media and supplements were obtained from GIBCO (Grand Island, New York). Production

of Recombinant

Recombinant

Virus

plasmid pVL13931CREB and Autogrunuclear polyhedrosis virus (AcNPV) DNA (Invitrogen) were cotransfected into Sf9 cells by calcium phosphate precipitation (39). At 120 h posttransfection, the medium containing wild-type and recombinant virus was collected. The recombinant virus was plaque purified as described (39). phica californica

Expression

and Purification

of CREB

In general, Sf9 cells in monolayer or suspension cultures were infected with the recombinant virus at a m.o.i. of l-10. At 48 h postinfection, the cells were harvested and lysed gently with 0.5% NP-40/hypotonic buffer (10 mM Tris, pH 7.6,l mM EDTA, 10 mM KCl). To separate the nuclei from cytoplasm and plasma membrane, the lysed cell suspensions were spun at 5000 rpm for 10 min. The resulting nuclear pellet was washed three times in PBS and resuspended in 10 packed nuclei volumes of TE. The nuclei were then boiled in l-ml aliquots for 8 min and plunged into ice, and the denatured proteins were pelleted by centrifugation in a microfuge at full speed (12,060 rpm) for 15 min. The heat-stable recombinant, CREB-327, remained in the supernatant and accounted for approximately 98% of total protein.

OVEREXPRESSION

BEV-CREB INFECTED CELLS --

BEV-CREB INFECTED CELLS

VIRUS

2 2

kD

OF

405

CREB

tubes for 8 min and plunged into ice water for 10 min, and the denatured proteins were pelleted as described above. The heat-stable CREB-327 partitions to the supernatant and accounts for 98% of total protein; therefore, it was not purified further. This supernatant was then dialyzed in storage buffer (20 mM Hepes, pH 7.9, 15% glycerol, 100 mM KCl, 0.2 InM EDTA) and frozen in 0.5-ml aliquots at -70°C. The typical yield of purified recombinant CREB-327 from 400 ml of suspension culture (4 X 10’ cells, or 2 ml packed cells) is on average 5 mg. Immunofluorescence

SOUTHWESTERN (LABELLED

CRE)

c

28

f

1s

c

14

FAR-WESTERN (LABELLED

CREB)

FIG. 4. DNA-binding and dimerization of proteins in wild-type and recombinant virus infected extracts. CRE-specific DNA-binding proteins in the wild-type, mock, and BEV-CREB recombinant infected Sf9 cell extracts were analyzed by Southwestern assay (left) using the labeled somatostatin CRE oligonucleotide described previously (26). CRE-binding activity was detected only in the nuclear fraction of BEV-CREB infected St9 cells, and corresponded to the recombinant CREB-327 protein. No CRE-binding proteins were detected under these conditions in mock or wild-type AcNPV infected St9 cell extracts. Dimerization of proteins in these same extracts with iodinated CREB-254-327 peptide was analyzed by Far-Western analysis (right). Protein/protein interactions with the labeled CREB peptide were detected primarily with the recombinant CREB-327 in the nuclear fraction from BEV-CREB infected St9 cells, although a small amount of activity was present in the cytoplasm. No proteins in mock or wild-type extracts interacted with the labeled probe under these conditions. The homologous peptide (bacterial CREB-BR) was utilized as a positive control and formed specific interactions with the labeled probe.

Typically, 400-ml suspension cultures were used for recombinant protein production. Sf9 cells at 1 X lo6 cells/ml were infected with 4 X 10’ pfu/400 ml (m.o.i. = 10 pfu/cell). Forty-eight hours postinfection, the cells were harvested and lysed as described above. The 400ml cultures generally yield 2 ml of packed cells, and the recombinant CREB-327 accounts for approximately 5% of total protein at this point. After hypotonic lysis, the nuclear pellet occupies, on average, a 0.5 ml volume and the recombinant CREB-327 accounts for approximately 50% of total nuclear proteins. Therefore, the nuclear pellet was resuspended in 5 ml of TE, five l-ml aliquots were boiled separately in screw-cap microfuge

St9 cells infected with the wild-type AcNPV or the recombinant AcNPV expressing CREB-327 were grown for 3 days on coverslips coated with triethoxysilane. After the 3-day infection period, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature, washed three times in PBS, and stored at 4°C. Cells from each infection were stained with hematoxylin/eosin and examined with an Olympus BH-2 microscope. Alternate coverslips harboring cells from each group were permeablized with Triton X-100 for 30 s, washed three times with PBS, and blocked with 5% goat serum in PBS with 3% albumin (solution 1) overnight at 4’C. Blocking solution was removed and cells were incubated overnight at 4“C with either primary anti-CREB P-Box serum, or preimmune serum at a dilution of 1:5000 in solution 1. The cells were then washed three times with PBS and incubated with an FITC-conjugated affinity purified goat anti-rabbit Fab-2 secondary antibody (Cappel) at a dilution of 1:lOO in solution 1 for 3 h at room temperature. Finally, the cells were washed in PBS 3 X 10 min, mounted on slides with Aquamount, and viewed with either an Olympus BH-2 microscope equipped with fluorescence optics or a Bio-Rad 2000 confocal microscope. In Vivo Phosphorylation Sf9 cell monolayer cultures in six-well plates were infected with the recombinant virus at a m.o.i. of 1. After 24 h infection, the cells were labeled with [32P]inorganic phosphate for another 24 h. The nuclei were isolated as previously described, boiled for 10 min and spun down to obtain the boiled pellet and supernate, which were separated on a 10% SDS-polyacryamide gel, dried, and exposed to X-ray film at -70°C with an intensifying screen. Southwestern

and Far- Western Analyses

The proteins were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes by electroblotting in Tris-glycine (19 g/liter Tris-0.8

406

CHEN

g/liter glycine) overnight at 4°C with 300 mA current. The blots were then incubated at room temperature in Blotto (40) with gentle shaking for 1 h, followed by incubation with 10 ml ‘251-labeled CREB-binding region (500,000 cpm/ml) in Blotto for Far-Western as described previously (36), or with 32P-labeled CRE oligonucleotides (100,000 cpm/ml) in TNE-50 buffer (40) for Southwestern, respectively, at room temperature for 12 h. The blots were finally washed four times in TNE-50 for total of 30 min before autoradiography. DNase I Footprinting

Assay

The DNA probe represents the sequences from -220 to +l of the human a! gonadotropin gene promotor, which contains two high-affinity CREB-binding sites in tandem from -110 to -150, and was labeled at -220 using T4 polynucleotide kinase. The 220-bp end-labeled fragments were purified from a 4% native polyacrylamide gel. DNase I protection was performed using about 2 fmol(20,OOO cpm) purified DNA fragment. The reaction was carried out as previously described (41). For competition studies, 200 fmol to 1 pmol of the double-stranded oligonucleotides containing the somatostatin CRE-binding sequences (26) were added to the reaction. The probe was sequenced by Maxam and Gilbert chemical modification (42) and used for identification of DNA footprinting sequences.

ET

AL.

min. After ethanol precipitation, the final RNA products were analyzed on an 8% polyacrylamidel7 M urea gel and visualized by autoradiography. Scanning densitometry was used to quantify signals. RESULTS

Expression of CREB-327 in BEVS AcNPV was used to overexpress CREB-327 in SI9 cells. The recombinant CREB-expressing virus (BEVCREB) was produced by recombination of a transfer vector (pVL1393) containing the coding region of a fulllength CREB-327 cDNA (26) flanked by the AcNPV polyhedrin gene sequences with the wild-type AcNPV genome. The polyhedrin gene coding sequences were effectively replaced in the recombinant virus, and the recombinant virus was plaque purified by visual screening and selection of plaques lacking the visible polyhedrin protein. Infection of Sf9 cells with the recombinant virus results in maximal CREB production within 72 h postinfection. Cellular Localization CREB

and Purification

of Recombinant

The cellular localization of the recombinant CREB protein overexpressed in Sf9 cells was analyzed by Coomassie staining of fractionated BEV-CREB infected St9 cell extracts as shown in Fig. 1. Virtually 100% of In Vitro Transcription the recombinant CREB protein was found in the isoPreparation of HeLa whole cell extracts and in vitro lated nuclei, whereas none was detected in the cytotranscription assays were performed according to the plasm or in the medium. Furthermore, the recombinant method of Manley et al. (43). Reactions included 15 ~1 CREB-327 expressed in these Sf9 cells was found to be HeLa whole cell extract (5 pg protein/& 0.4 pg a temheat stable as evidenced by its partitioning to the superplate, 0.1 pg internal control template, 600 PM rNTPs, natant fraction of infected nuclei that had been boiled 10 mM creatine phosphate, and varying amounts of for 8 min. In fact, the recombinant CREB accounted for CREB in a total volume of 25 ~1. Transcription reac- approximately 98% of the total protein in the supernations were carried out at 30°C for 2 h. Templates were tant fraction of boiled nuclei. In repeated cultures, we removed by adding 180 ~1 of transcription stop buffer have found that on average 12.5 mg of the purified re(50 mM NaCl, 1 InM EDTA, 10 mM Tris-HCl, pH 7.5), combinant CREB-327 can be obtained per liter of susvortexing, and centrifugation. The resulting supernapension culture (1 X 10’ cells, or 5 ml packed cell voltant was phenol/chloroform extracted and precipitated ume). The isolation of nuclei and subsequent heat fracwith ethanol. The synthesized RNA was analyzed by Sl tionation of the recombinant CREB-327 allowed nuclease protection. The end-labeled DNA fragment recovery of 90-95% of total recombinant material at (-242 to +44 human Q gonadotropin, plus 78-bp lucifer98% purity and therefore was much more efficient than ase sequences) was hybridized to the RNA for 12 h at previously published protocols for CREB purification 49°C and hybrids were subsequently incubated with Sl employing ion-exchange chromatography and senuclease (200 units in 180 ~1 of Sl buffer) at 37°C for 30 quence-specific affinity chromatography (44). Due to

FIG. 6. Immunocytochemical localization of recombinant CREB-327 in infected Sf3 cells. St9 cells infected with the wild-type AcNPV (A, C, and D) or the recombinant AcNPV expressing CRBB-327 (B, E, F, and G) were grown for 3 days on coverslips coated with triethoxysilane and stained with hematoxylin/eosin (A, B, C, and E) or subjected to immunocytochemical staining with an anti-CREB P-Box antisera and FITC-labeled secondary antibody (D, F, and G). (A, B) High-magnification resolution of nuclear morphology; (C-F) lower magnikation to depict larger numbers of cells. (G) Nuclear localization of the CREB-327 staining as evidenced by high-resolution confocal imaging microscopy on a Bio-Rad 2000 confocal microscope. This image has been digitally magnified.

OVEREXPRESSION

OF

CREB

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CHEN

ET

AL.

A (PKA), because attempts to phosphorylate this residue in vitro with PKA failed unless the purified recombinant CREB was first treated with phosphatase (data not shown). These results indicate that the St9 cells used for the expression of recombinant proteins with the BEVS can correctly modify the PKA activation site on CREB, and further, either that the levels of the modifying enzyme responsible for this phosphorylation are high in these cells, or that the levels of an appropriate phosphatase are low. Integrity of DNA-Binding CREB-327

FIG. 6. Transcriptional activity of purified recombinant CREB327 in a cell-free transcription system. Sl nuclease protection analysis was used to quantitate transcriptional activity of cell-free transcription reactions containing 75 pg of HeLa whole cell extracts alone or in the presence of 50 or 250 ng of purified recombinant CREB-327 produced with the BEVS. The templates utilized included the CMVluciferase plasmid at 100 rig/reaction as an unresponsive internal control, and the human a gonadotropin-luciferase plasmid containing the CREB-responsive sequences and promotor encompassingresidues from -242 to +44. A dose-dependent increase in transcriptional activity was noted from the (Y gonadotropin promotor when increasing amounts of the purified recombinant CREB-327 were added to the reaction mixtures. The CREB-327 additions had no effect on the transcriptional activity of the CMV promotor-driven internal control plasmid. The fold stimulation of transcriptional activity of the (Y gonadotropin promotor-driven plasmid compared to that of the CMV plasmid is denoted below each lane, and was assessed by densitometric scanning of the autoradiogram.

the large amounts of recombinant CREB-327 produced with the BEV system, sequence-specific affinity columns would be less efficient. Phosphorylation

of Recombinant

CREB-327

in Sf9 Cells

To analyze whether the Sf9 cells were capable of phosphorylating the recombinant CREB, we incubated BEV-CREB infected Sf9 cells in medium containing 32Pi for 24 h and analyzed the phosphorylated proteins in whole cell pellets, nuclei, and fractionated nuclei by SDS-PAGE (Fig. 2). Although several phosphoproteins were detected in the total cell pellet, one of these proteins was identified as recombinant CREB because a phosphoprotein of -44 kDa was present in the boiled supernatant fraction of isolated nuclei. We found that the recombinant CREB was fully phosphorylated at the serine residue (residue 119) modified by protein kinase

Specificity of Recombinant

The integrity of the DNA-binding specificity of the recombinant CREB-327 expressed in Sf9 cells with the BEVS was analyzed by DNAse I protection analysis of the human (Y gonadotropin promotor (Fig. 3). The DNase I protection pattern of the human (Ygonadotropin promotor by full-length baculovirus expressed CREB-327 and a bacterially expressed recombinant peptide corresponding to the COOH-terminal74 amino acids of CREB were compared. The expression and purification of the bacterial peptide have been described previously (36). Note that both of these proteins bind specifically to the tandemly repeated CRE elements in the promotor fragment at low concentrations. Furthermore, as shown in the right panel of Fig. 3, addition of increasing amounts of unlabeled CRE-containing competitor oligonucleotide abolishes the protection of the CRE sequences observed with the BEV-CREB. These data demonstrate that the recombinant CREB-327 expressed in Sf9 cells is DNA-binding competent and retains the DNA-binding specificity observed with native CREB protein. Protein/Protein Interactions Infected Sf9 Cell Extracts

and DNA-Binding

in

We utilized Southwestern analysis of DNA-binding and Far-Western analysis (36) of protein/protein interactions (Fig. 4, left and right, respectively) to further characterize the functional properties of the recombinant CREB-327 in BEV-CREB infected Sf9 cell cultures. Again, we noted that the CRE-binding activity was localized to the nuclear fractions and not found in the cytoplasm or in the medium. In fact, CRE-binding activity was not found under these conditions in mockinfected St9 cell extracts or in extracts of the St9 cells infected with the wild-type AcNPV. Similarly, the only protein detected in these extracts that was found to dimerize with a labeled CREB protein by Far-Western assay was the redombinant CREB-327 localized to the nuclear compartment of BEV-CREB infected cells. Mock or wild-type infected cells showed no proteins that interact with the labeled CREB probe.

OVEREXPRESSION

Of interest was the observation that the bacterially expressed CREB peptide corresponding to the DNAbinding and dimerization domain does not bind labeled CRE-containing oligonucleotide in the Southwestern assay, but does dimerize with the homologous labeled CREB probe in the Far-Western assay. These observations have been made repeatedly in the past and deserve consideration. We feel that the small size of the CREBBR peptide together with the charge distribution over the length of this peptide is responsible for this property. In short, we believe that the highly charged DNAbinding domain of this peptide is involved with anchoring the peptide to the nitrocellulose membrane and is therefore not available for DNA-binding. Consequently, the leucine zipper domain of the peptide is available for dimerization and mediates this effect detected by FarWestern assay. We have observed this phenomenon for several of the CREB/ATF peptides containing only the DNA-binding and dimerization domains. Immunocytochmical the Recombinant

Identification and Localization CREB-327 in Infected Sf9 Cells

of

Immunocytochemical localization of recombinant CREB-327 was accomplished with a polyclonal antiserum raised to a synthetic peptide spanning CREB-327 amino acids 92-124 (Kindly provided by Dr. Joel F. Habener). Hematoxylin/eosin staining of wild-type and recombinant CREB-327 infected Sf9 cells shows a distinct nuclear morphology associated with the overexpression of CREB (Figs. 5A vs 5B, and 5C vs 5E). Immunofluorescent analysis of CREB immunoreactivity in St9 cells infected with wild-type AcNPV (Fig. 5D) and recombinant AcNPV overexpressing CREB-327 (Fig. 5F) demonstrated CREB immunoreactivity only in those cells infected with recombinant virus (Fig. 5F). Furthermore, the CREB immunoreactivity was localized to the nucleus of these cells. This is visualized utilizing confocal microscopy in Fig. 5G. Note that the overexpressed CREB-327 protein is not randomly distributed throughout the nuclear compartment, but rather is localized to specific structural components of the nucleus. Transcriptional CREB-327

Activity

of Purified

Recombinant

Sl nuclease protection analysis was used to quantitate the transcriptional activity of the purified recombinant CREB-327 in cell-free transcription assays (Fig. 6). Transcription reactions were performed with 75 pg of HeLa whole cell extracts alone, or in the presence of 50 or 250 ng of recombinant CREB-327. A dose-related increase in transcriptional activity of the human (Ygonadotropin promotor was observed when purified recombinant CREB-327 was added to transcription reactions. The addition of 50 ng of CREB-327 resulted in a 1.5-fold stimulation of transcriptional activity in rela-

OF

CREB

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tion to control CMV activity, whereas the addition of 250 ng of the purified recombinant CREB-327 resulted in a 3.6-fold stimulation of transcriptional activity from the human cxgonadotropin promotor compared to that of the control. The increased transcriptional activity was shown to be specific because transcription from the internal control plasmid, CMV-luciferase, was unaffected by adding purified CREB-327. These results demonstrate that the purified recombinant CREB-327 expressed with the BEVS is functionally competent to stimulate transcription from CRE-driven promotors. DISCUSSION

Studies of the structure and functions of eukaryotic transcriptional regulatory factors require the availability of large quantities of enriched or purified proteins. The baculovirus expression vector system represents a useful tool for producing large quantities of proteins for further characterization. A potential advantage of the BEVS over existing prokaryotic expression systems is the ability of the Sf9 cells utilized to modify the recombinant proteins post-translationally. In the present study it was noted that CREB-327 was constitutively phosphorylated at the native protein kinase A recognition site at serine 119. This modification is required for maximal transcriptional activity of CREB and is necessary for further phosphorylation by other kinases including casein kinase II and glycogen synthase kinase III (37). We have used the BEVS to overexpress full-length CREB-327 and characterized several functional properties of the purified product. We have demonstrated that the overexpressed product is correctly translocated to the nucleus of the Sf9 cells by immunocytochemistry and cellular fractionation. Similarly, we have noted that the serine residue at 119 is constitutively phosphorylated. A recent report by Sheng and co-workers (35) demonstrating phosphorylation of this residue by calcium calmodulin-dependent kinases leaves the identity of the endogenous Sf9 cell enzyme responsible for this modification unknown. High levels of protein kinase A or calcium calmodulin-dependent kinases or low levels of an appropriate phosphatase are all possible mechanisms which could result in the complete modification of this residue. We have also demonstrated that the overexpressed CREB-327 can be efficiently purified by boiling isolated nuclei. DNase I footprinting and Southwestern analyses of the DNA-binding specificity of the boiled CREB protein demonstrate that this treatment does not adversely affect the functional sequence-specific DNA-binding properties of the recombinant CREB-327 protein. Similarly, this treatment does not affect the ability of CREB-327 to interact with itself by Far-Western assay, or other components of the transcriptional machinery necessary to mediate its effects on transcriptional acti-

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vation, because the CREB-327 purified by boiling is able to stimulate transcription of the human CIgonadotropin gene promotor in a cell-free transcription assay in a dose-dependent manner. The recent finding that CREB is phosphorylated at its major transcriptional regulatory site by calcium/calmodulin-dependent kinases (35) in addition to protein kinase A suggests that CREB can mediate transcriptional responses to increases in both intracellular CAMP and free calcium. Similarly, other recent studies (34) have demonstrated a role of CREB in the activation of hepatitis B virus (HBV) transcription via interactions with the transactivating protein, pX, of HBV. An earlier report by Liu and Green (33) has implicated ATF-2 as the cellular factor that mediates adenoviral Ela-mediated transcriptional activation of adenoviral gene expression through sequence-specific binding to the CRE motif. Therefore, it is becoming increasingly clear that the CRE motif, in general, and CREB, in particular, are important targets of multiple signal transduction and viral stimulatory pathways in the cell. The large amounts of purified CREB-327 protein obtained by overexpression in SKI cells with the BEVS will be utilized to investigate the structural properties and post-translational modifications and their consequent structural changes that contribute to the transcriptional activity of this functionally diverse regulatory protein. ACKNOWLEDGMENTS We thank Dr. Max Summers, Texas A&M University, for providing us with the pVL1393 vector; Frank McCormack and Dennis Voellker from National Jewish Hospital and Kurt Christensen and Dean Edwards from the Tissue Culture Core facility of the University of Colorado Cancer Center for help with the St9 cell culture conditions and techniques; and David Kroll for help with the in uiuo laheling studies. We thank Joel Habener for providing the CREB P-Box antisera; Arthur Gutierrez-Hartmann for helpful suggestions about the cell-free transcription assays; and J. Larry Jameson for the human a luciferase plasmid. We also thank Bill Townend from National Jewish Hospital for assistance with the confocal microscopy. This work was supported by grants from the CU Cancer Research Foundation, the Cancer League of Colorado, an ACS Institutional Grant, and a grant from the Biomedical Research Support Fund to J.P.H. These studies were also supported by NIH Grant GM45872 (to J.P.H.) and NIH NRSA DK08329-02 (to D.H.B.).

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at

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