phosphorylation of cellular proteins regulates their binding to the

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 hy The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264, No. 35, Issue of December 15, pp. 21266-21276,1989

Printed in U. S. A.

Phosphorylation of Cellular Proteins Regulates Their Binding to the cAMP Response Element”

(Received for publication, July 21, 1989)

Alejandro Merino, Leonard Buckbinder$, Fred H. Mermelsteinj, and Danny Reinberglf From the Department of Biochemistry, Robert Wood Johmon Medical School, University of Medicine and Dentistry of New Jersey, Piscatamay, New Jersey 08852-5635

We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region I11 (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72,38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31- 37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus E111 promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the CAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by CAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein.

The transcription of genes by RNA polymerase I1 is con- trolled by a complex array of protein factors that include site- specific DNA-binding proteins (Dynan and Tjian, 1985; McKnight and Tjian, 1987). These factors recognize specific upstream elements and presumably interact with the basic transcription machinery to activate gene expression (Sawa- dogo et al., 1985; Horikoshi et al., 1988). The mechanisms that operate in regulating the action of the site-specific transcription factors are largely unknown.

Adenovirus early gene expression is regulated in part by the products of the immediate early gene, Ela (for review see Nevins, 1981; Berk, 1986). The Ela transcription unit encodes two major proteins, p289 and p243, that differ by an internal

* This work was supported by National Institute of Health Grant GM 37120, National Science Foundation Grant DMB-8819342, New Jersey Commission on Cancer Research Grants 687-035 and 688-026, and a grant from the Foundation of the University of Medicine and Dentistry of New Jersey. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a fellowship from Kirin Beer Company. §Supported by National Institutes of Health Training Grant

T Recipient of American Cancer Society Junior Faculty Award ES07148.

JFRA-205.

block of 46 amino acids unique to the larger protein (Perri- caudet et al., 1979). The p289 protein is known to activate the expression of all six adenovirus early genes (Berk, 1986), while the smaller protein is a repressor of enhancer-driven transcription (Lillie et al., 1986). The mechanism by which Ela activates early gene expression remains unknown, but the protein does not appear to bind specifically to any known promoter element (Chatterjee et al., 1988); thus, it presumably acts by influencing the action of other specific trans-acting factors (Nevins, 1981; Berk, 1986; Jones et al., 1988). Evidence that supports this model of Ela activation includes a protein factor referred to as E2F that interacts with the early I1 promoter (EII) and whose DNA binding activity is increased by the presence of the large Ela product (Kovesdy et al., 1986) in the absence of protein synthesis (Reichel et al., 1988). In addition, a peptide containing the 46 amino acids unique to the Ela p289 product was shown, when microinjected into HeLa cells, to activate the E11 promoter in the absence of protein synthesis (Green et al., 1988).

Most of the adenovirus early promoters contain an element with the consensus DNA sequence 5’-GT(G/T)ACG(T/ A)CA-3’ (for review see Jones et al., 1988). This sequence element is also involved in mediating the cAMP response in some cellular genes and has been termed the cyclic AMP response element (CRE)’ (Montminy et al., 1986). The CRE is recognized by a 43-kDa phosphoprotein, CREB, that was purified from PC12 cells (Montminy et al., 1987). CREB, which binds to the CRE as a dimer, was shown to be regulated by phosphorylation via the CAMP-dependent protein kinase (PKA) in vitro (Yamamoto et al., 1988). A protein factor ATF (activating transcription factor), first identified in HeLa cells (Lee et al., 1987b; Hurst and Jones, 1988) may be identical to CREB based on binding specificity as well as molecular weight (Hurst and Jones, 1988; Hai et al., 1988; Jones et al., 1988).

We have studied the factors that promote transcription through the CRE-like elements in the adenovirus early region 111 (EIII) and early region IV (EIV) promoters. We identified a 65-72-kDa factor, EivF, that was purified from uninfected HeLa cells and shown to promote transcription in vitro from the adenovirus EIV promoter (Cortes et al., 1988). This factor recognized two CRE elements in the adenovirus-EIV promoter [5’-GTGACGT-3], one located at -170 and the other at -60 relative to the CAP site. The ATF protein is thought to interact with these two CRE elements as well as with another related element, 5’-GTGACGA-3’ present a t -147 in the adenovirus EIV promoter (Lee et al., 1987; Hai et al., 1988) and at -55 in the adenovirus E111 promoter (Hurst and

The abbreviations used are: CRE, cyclic AMP response element; PKA, protein kinase A; SDS, sodium dodecyl sulfate; Hepes, 4-(2- hydroxyethy1)-1-piperazineethanesulfonic acid; PKC, protein kinase C; CIP, calf intestine phosphatase; AMP-PNP, adenosine 5’-(/3-7- imino)triphosphate; ATF, activating transcription factor.

21266

Protein Phosphorylation and DNA Sequence Specificity of CRE-binding Proteins 21267

Jones, 1988). An element in the adenovirus EIII promoter, located at -90, is thought to be a recognition site for the factor AP1 (Garcia et al., 1987). AP1, which is similar or identical to the product of the jun oncogene (Angel et al., 1987; Bohmann et al., 1988; Bos et al., 1988; Vogt and Tjian, 1988), has a recognition sequence that is strikingly similar to that of the CRE, but differs by the absence of 1 guanosine residue (5’-TGACTCA-3’). The APl/jun element can confer responsiveness to the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (Lee et al., 198713; Chiu et al., 1987). This effect is thought to be mediated via protein kinase C (Nishizuka, 1984).

We have previously shown that HeLa cells contain multiple factors, with apparent molecular masses in the range of 31- 72 kDa that interact with the CRE. In the present studies we have used preparative polyacrylamide-SDS gel electrophoresis together with DNA affinity chromatography to isolate and characterize the factor interacting with the CRE. Three families of proteins were isolated that included 1) EivF (65-72 kDa), 2) a family of proteins with molecular masses ranging from 38-45 kDa and, 3) another family of factors with molecular masses ranging from 31-37 kDa. Within each family of proteins we have found that there were species that were interrelated by differences in the extent of phosphorylation. We have demonstrated that phosphorylation of all three families of factors is involved directly in DNA binding. Pro- teins dephosphorylated in vitro had diminished DNA binding activity that could be restored by subsequent rephosphorylation with PKA. We have assessed the binding specificity of the “CRE” factors and have shown that EivF specifically recognizes the 5’-GTGACGT-3’ sequence present in the adenovirus EIV promoter, but does not bind to any elements present in the adenovirus E111 promoter. Moreover, we find that the small molecular weight proteins are less discriminat- ing and recognize both the AP1 and ATF sites in the adenovirus E111 promoter, as well as the CRE elements in the adenovirus EIV promoter, but that was dependent on their state of phosphorylation. For simplicity, we refer to the sequence 5’-GTGACGT-3’ as the CRE (Montminy et al., 1986), to the sequence 5’-GTGACG_A-3’ as ATF (Hurst and Jones, 1988) and to the sequence 5’-GTGA(C/G)TCA-3’ as AP1 (Lee et al., 1987b).

EXPERIMENTAL PROCEDURES

Plasmid DNA-The adenovirus EIII promoter construct (pE3 CAT) as well as a deletion mutant (pE3-85) have been described (Weeks and Jones, 1986; Garcia et al., 1987, respectively). Adenovirus EIV promoter constructs and deletions were also described (Leza and Hearing, 1988). The adenovirus E111 promoter construct containing a triple point mutation in the ATF recognition site (5”TTTGTCC- CGCGG-3’) has been described previously (Kornuct et al., 1988). The sequence of oligonucleotides were as follows, ATF: 5”GATCCGGC- TTTCGTCACAGGG-3‘ and its compliment 5”GATCCCCTGTG-

TCAG-3’ and its compliment 5”GATCCTGAGTTAGTCATCTG-

Transcription Reactions in Vitro-Transcription reactions in uitro were as previously described (Cortes et al., 1988) except that the RNA products were analyzed by primer extention and contained the following modifications: RF template replaced linear DNAs, the stop solu- tion contained sonicated salmon sperm DNA (10 pg/ml) instead of RNA, and reaction mixtures were increased in size %fold. The primer consisted of 20 nucleotides of CAT-coding sequence (5”pCTCCA- TTTTAGCTTCCTTAG-3’). Specifically initiated adenovirus EIII and adenovirus EIV transcripts generated cDNAs of 100 and 105 nucleotides, respectively. General transcription factors IIA, IIB, IIE/ IIF, IID, and HeLa RNA polymerase I1 were purified as previously described (Reinberg and Roeder, 1987; Reinberg et al., 1987; Flores et al., 1989).

Cells-HeLa cells grown in Joklick’s modified medium (Hazelton)

ACGAAAGCCG-3‘, AP1: 5“GATCCGAAGTTCAGATGACTAAC-

AACTTCG-3’.

supplemented with 5% calf serum were used for preparation of nuclear extract and purification of transcription factors. Cells were harvested in logarithmic growth (7-10 X lo5 cells/ml).

DNA Mobility Shift Assays and DNase I Protection Experiments- DNA mobility shift assays and DNase 1 protection experiments were performed as previously described by Cortes et al. (1988).

Protein Kinase a d Phosphatase Reactions-Protein kinase A (catalytic subunit) was purchased from Sigma and reconstituted accord- ing to the manufacturer’s directions. Phosphorylation reaction mixtures contained 50 mM potassium phosphate, pH 7.5, 20 p M ATP, 10 mM MgCI,, 10 mM dithiothreitol, 200 units/ml PKA and were incubated at 30 “C for 45 min. Reactions were terminated by bringing the mixture to 10 mM EDTA.

Phosphorylation with protein kinase C (gift of Dr. Ora Rosen, Memorial Sloan Kettering Cancer Center, NY), was performed in reaction mixtures containing 25 mM Hepes-NaOH, pH 7.0, 10 mM MgClz, 0.6 mg/ml phosphoserine, 0.6 mg/ml diacylglycerol, 0.5 mM CaC12, and 25 p~ ATP and were incubated at 30 “C for 45 min. Reactions were stopped by the addition of EDTA to 10 mM final concentration.

Dephosphorylation using calf intestinal phosphatase (Boehringer Mannheim) was performed in reaction mixtures containing 20 mM Hepes-NaOH, pH 7.0, 20 mM MgCl,, 40 mM KCI, and 0.2 mM phenylmethylsulfate fluoride and were incubated at 37 “C for 30 min. Phosphatase was inactivated by heating the reaction mixtures at 65 “C for 15 min in the presence of 5 mM nitrilotriacetic acid.

Labeling of CRE Binding Proteins in Viuo-HeLa cells (1 X 10’) were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 5% calf serum. For labeling studies, the culture medium was removed and the cells were refed with 20 ml of phosphate-free Dulbecco’s Modified Eagle’s Medium supplemented with 1.0 mCi/ml of [32P]orthophosphate (ICN). The cells were then incubated at 37 “C for 8 h. Cells were then harvested and nuclear extract prepared. In 35S-labeling experiments, cells were refed with 20 ml of methionine- free Dulbecco’s Modified Eagle’s Medium containing 10 mCi of [%] methionine and incubated for 1 h as described (Tung et al., 1988). The labeled “CRE-binding proteins” were purified by DNA affinity chormatography as described by Cortes et al., 1988, except that gel filtration chromatography was not performed. DNA affinity chromatography was repeated three times before analysis of the proteins.

RESULTS

Multiple Phosphoproteins Bind to the CRE-We have purified by DNA affinity chromatography several polypeptides, which vary in molecular mass (31-72 kDa), that recognize the CRE. One of these proteins, which we have termed EivF (Cortes et al., 1988), is found in the molecular mass range of 65-72 kDa. EivF stimulated transcription from the adenovirus EIV promoter and bound two CRE sequences present within the promoter (Cortes et al., 1988). We have also identified proteins by SDS-polyacrylamide gel electrophoresis that bind to the CRE and are of smaller molecular mass (31-46 kDa). These proteins were separated from EivF by gel filtration chromatography prior to DNA affinity chromatography (Cortes et al., 1988). Hai et al. (1988) have also identified proteins from HeLa and J Y cells that bound to the CRE with molecular masses of 43 and 47 kDa. Montminy and co-workers (1987) isolated a 43-kDa protein, CREB, from PC12 cells and demonstrated that phosphorylation regulated its DNA binding activity. In order to analyze the relationship of EivF with CREB/ATF and determine if EivF exists as a phosphoprotein in vivo, proteins that bound to the CRE were purified from HeLa cells labeled with [32P]orthophosphate in uiuo. Five major bands derived from a third passage through the CRE affinity column could be resolved by SDS-polyacrylamide gel electrophoresis: major bands migrated with Rf values corre- sponding to a molecular mass range of 31 to 36 kDa, a doublet band at 43 kDa, and three other bands with molecular masses of approximately 56,68, and 100 kDa (see Fig. 1A). The same polypeptides could be resolved when the CRE-binding proteins were isolated from cells that were labeled with [35S] methionine (Fig. lB) , indicating that most or all the proteins

21268 Protein Phosphorylation and DNA Sequence Specificity of CRE-binding Proteins

A B

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97-

68-

43-

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1-68 i

1 -43

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32P 35 s in vivo in vivo

FIG. 1. Identification of phosphoproteins that bind to the CRE from HeLa cells radiolabeled in vivo. CRE-binding proteins labeled in oiuo with ”‘P (panel A ) or [%]methionine (panel E ) were purified as described by Cortes et al., 1988. However, the gel filtration chromatographic step was omitted. Eluates from the DNA affinity column containing the CRE were dialyzed and cycled through the column three times. Proteins were separated by SDS-polyacrylamide gel electrophoresis. Panel A represents samples separated on a gra- dient 8-13s gel, whereas, proteins observed in panel E were separated on a 10% gel.

that bound to the CRE were phosphoproteins. The 56- and 110-kDa phosphoproteins present in these fractions were not always observed, suggesting that they may represent nonspe- cific DNA-binding proteins.

EivF, a Factor Specific for the CRE Present in the Adeno- virus EZV Promoter-In order to examine the relationship between EivF and the small molecular weight proteins that recognize the CRE (5’-ACGTCA-3’) present in the adenovirus EIV promoter and two other related DNA elements, the AP1 (5’-TGAGTCA-3‘) and ATF (5‘-TCGTCA-3’) recognition sites present in the adenovirus E111 promoter, fractions from a gel filtration column derived from the purification of EivF (Cortes et al., 1988) were analyzed for binding to the CRE, ATF, and AP1 sequence elements. Consistent with our previous findings, we observed proteins of different apparent molecular weights that footprinted to the CRE element in the adenovirus EIV promoter (Fig. 2 A ) . Proteins taken from the sizing column fractions (Fig. 2 A ) were pooled and separated by SDS-polyacrylamide gel electrophoresis under reducing conditions after which the polypeptides were eluted from gel slices, denatured, and renatured as previously described (Cortes et al., 1988). The protein samples were assayed for binding to the adenovirus E111 and EIV promoters by the gel mobility shift method. Proteins in the molecular mass range of 62-72 kDa (EivF), and proteins in the range of 31-45 kDa formed complexes with the adenovirus EIV promoter (Fig. 2B). DNA binding activity to the CRE was not observed with renatured proteins in the molecular mass range of 45 to 65 kDa (Fig. 2B).

Discrete patterns of binding were observed within the low molecular weight group (compare complexes in lams 12-15 with those in lunes 16-20). Proteins in the 31-36-kDa range (Fig. 2B) produced two DNA-protein complexes. This may have resulted from a high concentration of factors in these particular fractions as well as the presence of two CRE sites

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FIG. 2. Fractionation of factors that recognize the CRE by preparative SDS-polyacrylamide gel electrophoresis. A, footprinting analysis on the CRE present in the adenovirus EIV promoter was performed with proteins present in fractions derived from the gel filtration S200 column (see Cortes et al., 1988). Aliquots (20 pl ) were analyzed for protection of the CRE site located at -55 on the adenovirus EVI promoter (see “Experimental Procedures”). DNase I footprinting reactions were performed with a DNA fragment containing the adenovirus EIV promoter that was 3’-end labeled using the Hind111 site located 71 nucleotides downstream from the CAP site as previously described (Cortes et al., 1988). Only the region of the EIV promoter containing the CRE site located at -55 is shown. Lanes labeled with the (-) symbol at the top of the panel did not receive protein and represent the unprotected digestion pattern. The lane labeled with the (+) symbol received an aliquot (20 pl) of the gel filtration input material. The relative elution profile of protein standards is indicated above fractions 158 and 186. The bracket on the right denotes protection of the CRE site. E, isolation of proteins that bind to the EIV promoter by preparative SDS-polyacrylamide gel electrophoresis. Fractions derived from the gel filtration chromatographic step that protected the CRE site present in the adenovirus EIV promoter (see panel A ) were pooled and further purified by preparative 10% SDS-polyacrylamide gel electrophoresis. Protein samples were eluted and renatured as previously described. An aliquot (5 p l ) of the renatured samples was analyzed for binding to the adenovirus EIV promoter in a DNA mobility shift assay as described under “Experimental Procedures.” The probe was derived from the adenovirus EIV promoter construct pE4 -227/-46 (Leza and Hearing, 1988) and contained two CRE sites. Molecular weight ranges of the renatured samples that produced protein-DNA complexes are indicated at the top of each lane. Lane 1 received no protein and uncomplexed DNA fragment is observed at the bottom of the gel. Lane 2 (indicated by a (+) symbol at the top of the panel) received an aliquot (1 p l ) of pooled protein derived from the sizing column. C, analysis of the renatured CRE-binding proteins for their ability to bind to the adenovirus E111 promoter. Renatured proteins were assayed for binding to the adenovirus E111 promoter as described under

Protein Phosphorylation and DNA Sequence Specificity of CRE-binding Proteins 2 1269

on the labeled probe. Interestingly, the complexes observed with the large molecular mass proteins (62-72 kDa) were absent when the adenovirus E111 promoter was used in this analysis (Fig. 2C). The binding of all the protein fractions to the adenovirus E111 or EIV promoters could be eliminated by competition with oligonucleotides containing the consensus CRE recognition sequence (5’-ACGTCA-3’) (data not shown, see below). DNase I footprinting demonstrated that proteins from the two different molecular mass ranges (62-72 and 31- 45 kDa) bound the CRE element in the adenovirus EIV promoter (Fig. 3A, see lower bracket), consistent with the oligonucleotide competition experiments. In contrast, only proteins in the 31-45-kDa range bound to the ATF and AP1 sites present in the adenovirus E111 promoter (Fig. 3B). This observation supports our previous findings that EivF, present in fractions of 62-72-kDa molecular mass proteins, did not bind to the AP1 (5’-TGAGTCA-3’) or the ATF (5’-TC- GTCA-3’) element in the adenovirus E111 promoter (Cortes et al., 1988). Renatured proteins in the 31-45-kDa range that hound to the ATF and CRE elements present in the adenovirus E111 and EIV promoters, respectively, did not all recognize the AP1 sequence in the adenovirus E111 promoter. Fig. 3B (lunes 3-7) indicates that proteins in the 33-45-kDa range bound to both that AP1 and ATF elements in the adenovirus E111 promoter, but proteins in the 31-32-kDa range were only able to bind weakly to the ATF element and not to the AP1 site (Fig. 3B, lane 7). Weak binding to the ATF site could not be attributed to a low concentration of the factor in this fraction, since an equivalent concentration was able to protect the CRE element present in the adenovirus EIV promoter (Fig. 3A, lane 7. Also, see Fig. 2B, lane 20 and below). Additional protection was observed on the adenovirus E111 and EIV promoters with proteins in the molecular mass range of 40-42 kDa (Fig. 3, A and B, top bracket). This footprint encompassed sequences that shared similarity with the AP1 recognition site and mapped between nucleotides +6 to +I3 in the adenovirus EIV promoter and sequences that overlapped the TATA box element in the adenovirus E111 promoter (data not shown).

We next tested the renatured proteins for transcriptional activity using the adenovirus E111 promoter and observed that transcriptional stimulatory activity (4-5-fold, Fig. 30 , compare lanes I with lanes 4-7) correlated with binding to both the Apl and ATF sites. Proteins that did not bind to the adenovirus E111 promoter (EivF, 62-75 kDa) or proteins that only bound the ATF element of the adenovirus E111 promoter (31-32 kDa) did not stimulate transcription (Fig. 3 0 , lanes 2, 3, and 8) . In contrast, all the renatured proteins that contained DNA binding activity were capable of stimulating transcription from the adenovirus EIV promoter (Fig. 3C). This correlated with the observation that all of these proteins were capable of binding to the CRE element present in the adenovirus EIV promoter. However, the amount of stimulation varied among the different proteins. This may be because these were renatured proteins and the protein domains required for stimulation of transcription may not renature as well as those required only for DNA binding. The observed stimulation was specific for the adenovirus E111 and EIV promoters. These proteins failed to stimulate transcription

“Experimental Procedures.” The DNA fragment containing the adenovirus E111 promoter was isolated from plasmid DNA pE3 (Garcia et al. 1987) and was 3’-end labeled using the EcoRI site located 238 nucleotides upstream from the CAP site. DNA binding and electrophoresis conditions were as described in panel I?. Only the bound DNA-protein complexes are shown. The molecular masses of the proteins renatured from SDS-polyacrylamide gels are indicated at the top of each lane.

from the adenovirus major late promoter, which lacks any CRE, API, or ATF element (Cortes et al., 1988, and data not shown). Also, proteins in the molecular mass range between 62 and 45 kDa, which failed to form DNA protein complexes with the CRE, AP1, and ATF DNA elements, were unable to stimulate transcription from the adenovirus E111 or EIV promoters (data not shown).

These data suggest that there are three different molecular mass species of renatured proteins that bind uniquely to the CRE, AP1 and ATF sequence elements: 1) proteins in the 62- 72-kDa range (EivF), that bound the CRE site in the adenovirus EIV promoter, but not the AP1 or ATF sites in the adenovirus E111 promoter, 2) proteins in the 34-45-kDa range that bound the CRE present in the adenovirus-EIV promoter and to the AP1 and ATF sites in the adenovirus E111 promoter and, 3) proteins in the 31-32-kDa range that bound to the CRE element present in the adenovirus EIV promoter and weakly to the ATF site in the adenovirus E111 promoter, but failed to bind to the AP1 site. I t is possible that the different DNA binding activities observed within fractions is because of different proteins with a similar molecular weight (see below).

The DNA Binding Actiuity of EivF Is Regulated by Phos- phorylatbn-In the previous section, we described proteins of different molecular weights that bound to the ATF, AP1, and CRE elements present in the adenovirus E111 and EIV promoters. We observed that the complexes formed between these proteins and the adenovirus E111 and EIV promoters migrated differently on native gels (Figs. 2, B and C). The CRE and AP1 elements encompass sequences that have been shown to mediate transcriptional activation in response to cAMP and phorbol esters, respectively (Montminy et al., 1986; Lee et al., 1987c; Chiu et al., 1987 and for a review see Jones et al., 1988). It is also known that the response to cAMP and phorbol esters includes activation of PKA and protein kinase C (PKC), respectively. Furthermore, Montminy and Bilezik- jian (1987) isolated a 43-kDa phosphoprotein from PC12 cells that recognized the sequence element 5”ACGTCA-3’ present in the somatostatin promoter. Also, we have shown that protein able to bind the CRE were phosphorylated in vivo (see Fig. U). Therefore, we further investigated if the differences in electrophoretic mobility of the DNA-protein complexes could be attributed to the phosphorylation state of the bound proteins.

When the renatured proteins were treated with calf intestine phosphatase (CIP), most of the DNA-protein complexes acquired a new migration on polyacrylamide gels, and the yield of some complexes was reduced (Fig. 4). The complexes formed after phosphatase treatment of proteins of large molecular weight (EivF) comigrated (compare lanes 3 and 5), even though their precursors had different mobilities (compare lanes 2 and 4). Phosphatase treatment of the proteins in the 38-45-kDa molecular mass range appeared to reduce the amount of complex formed (Fig. 4, compare lanes 7, 9, 11, with 6, 8, and 10, respectively). Proteins in the 31-37-kDa range responded differently to phosphatase treatment than the proteins in the 38-45-kDa range. After CIP treatment, these complexes exhibited a new and uniform mobility identical to that of the complex formed with the untreated 31-32- kDa proteins (Fig. 4, compare lanes 13, 15, 17, 19, 21, and 23 with lane 22, respectively).

In order to verify that the observed differences were because of dephosphorylation of the proteins and not because of a proteinase contamination in the phosphatase preparation, we rephosphorylated in uitro, using PKA and/or PKC, the proteins dephosphorylated for the experiment shown in Fig. 4.


EIPF TATA Em Promoter

r EN Promoter

I 2 3 4 5 6 7 8 9 1 0 I Z 5 4 3 6 1 8 M

FIG. 3. Analysis of the DNA binding specificity to the adenovirus EIII and EIV promoters of proteins isolated by SDS- polyacrylamide gel electrophoresis. A, aliquots (20 pl) of samples fractioned by SDS-polyacrylamide gel electrophoresis as described in Fig. 2 were analyzed for binding to the adenovirus EIV promoter. The promoter fragment, diagramed at the bottom, was isolated from construct pE4 -330/-103 (Leza and Hearing, 1988) and was 3’-end labeled at the EcoRI site. Footprinting reactions were as described under “Experimental Procedures.” The control pattern of DNase I digestion obtained in the absence of protein is indicated by negative symbols (-) at the top of the lanes. The lane designated by the (+) symbol received an aliquot (20 p1) of the gel filtration pool (as described in Fig. 2 A ) . The top bracket at the left encompasses an AP1-like site located between nucleotides +7 to +13, downstream of the CAP site. The lower bracket represents the CRE site located at -55 relative to the CAP site. Molecular weight ranges of the renatured samples are indicated at the top of each lane. B, proteins renatured from SDS-polyacrylamide gels were analyzed by DNase I footprinting to the adenovirus EIII promoter as described in panel A. The fragment containing the adenovirus-E111 promoter was as described in Fig. 2C. Sites protected from DNase I digestion are outlined by brackets. The unlabeled bracket at the top indicates the position of an AP1 like element that overlaps the TATA box. The molecular weight of the proteins renatured from SDS-polyacrylamide gels are shown at the top of each lane. C and D, aliquots (20 pl) of the samples derived from the SDS-polyacrylamide gels were assayed for transcription activity on the adenovirus EIV (C) and adenovirus E111 (D) promoters. Transcription reactions were carried out as described under “Experimental Procedures” and were reconstituted with general transcription fractors and RNA polymerase 11. The lane labeled EivF (panel C) received an aliquot of gel filtration fraction (described in Fig. 2 A ) enriched in EivF activity. Products of the reactions were analyzed by primer extensions as described under “Experimental Procedures.’’ Two start sites were observed with the plasmid DNA containing the adenovirus EIV promoter. The molecular weight range of the proteins used in the analysis is shown at the top of the gel. The lane labeled with a negative symbol at the top of the panels, received 20 pl of renatured protein in the molecular mass range of

FIG. 4. Treatment of the renatured CRE-binding proteins with CIP results in changes in migration through polyacrylamide gels of the DNA-protein complexes formed with the adenovirus EIV promoter. An aliquot (4 pl) of proteins of various apparent molecular weights, renatured from SDS-polyacrylamide gels as indicated at the top of each lane, were incubated with CIP (0.2 unit, indicated with a + symbol, odd numbered lanes) as described under “Experimental Procedures.” Control samples were treated similarly but with heat inactivated CIP (even numbered lanes) as described under “Experimental Procedures.” The DNA binding activity of both CIP-treated and untreated proteins were analyzed by DNA mobility shift assays using the adenovirus EIV promoter fragment containing two CRE sites as described (Fig. 2B). The lane marked (-) represents uncomplexed DNA fragment.

When EivF (70-72-kDa protein) was treated with a high concentration of phosphatase, the amount of complex formed decreased and migrated faster than that formed with the untreated protein (Fig. 5A, compare lanes 1 and 2, respectively. Also, see Fig. 5B, compare lanes 2 and 1, respectively). However, when the in vitro dephosphorylated protein was treated with PKA, high levels of complex were formed and the DNA protein complex comigrated with the complex formed with the untreated protein (Fig. 5A, compare lane 3 with 2, respectively. Also see Fig. 5B, compare lane 3 with 1, respectively). This result suggested that the binding activity of EivF could be modulated by phosphorylation with PKA in vitro. Interestingly, the DNA binding activity of a dephosphorylated form of EivF could not be recovered as well by rephosphorylation with PKC (Fig. 5B, lane 4 ) . EivF could be phosphorylated by PKC as indicated by the recovery of a labeled protein after incubation of a partially phosphorylated form of EivF (isolated from a SDS-polyacrylamide-gel and containing DNA binding activity) with PKC and [y-32P]ATP (Fig. 5C). The label was in EivF because when the DNA binding activity of the labeled protein was analyzed using an unlabeled DNA fragment (reverse binding), a labeled DNA- protein complex with almost the same electrophoretic mobility as the complex formed with the unlabeled protein and labeled DNA was observed (Fig. 5 0 , compare lane 3 with 2, respectively). The same results were observed when DNA affinity purified EivF was used (data not shown).

The DNA Binding Activity of the Small Molecular Weight Proteins (31-37 kDa) Is Regulated by Phosphorylation with Protein Kinase A-The results presented in Fig. 4 indicated that the DNA binding activity of the proteins in the low molecular mass range (31-45 kDa) was also modified by phosphorylation. However, because of the response of these proteins to CIP treatment, it appeared that there were at least two groups of small molecular mass proteins with DNA binding activity. 1 ) One group extending from 38-45 kDa, showed an overall reduction in DNA binding activity after CIP treatment, and 2) proteins in the molecular mass range of 31-37 ~~

50-54 kDa. This protein fraction failed to produce DNA-protein complexes with the adenovirus EIII and EIV promoters (for details see text).


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42- e

26 - Y -

FIG. 5. Phosphorylation regulates the DNA binding activity of EivF. A and R, EivF (2 pl) was dephosphorylated with CIP (1.0 unit) (panel A, lanes 1 and 3, and panel R, lanes 2-4) as described under “Experimental Procedures.” After 30 min of incubation a t 37 “C, the CIP was inactivated by heating the mixtures a t 65 “C in the presence of nitrilotriacetic acid, see “Experimental Procedures.” Dephosphorylated protein were then incubated with ATP and either PKA (panel A, lane 3, and panel B, lane 3 ) or PKC (panel R, lane 4 ) or with the kinase but in the absence of ATP (panel A, lane 1 and panel B, lane 2) , as described under “Experimental procedures.” Control samples containing EivF were incubated in the presence of inactivated CIP and in the absence of ATP (panel A, lane 2 and p a w l R, lane 1 ) but carried over the same treatment as samples incubated with active CIP (all samples containing EivF were heated prior to the DNA binding assay; this treatment has no apparent effect on EivF activity). All reaction mixtures were then incubated with a fragment containing the adenovirus EIV promoter as described in Fig. 2R. The resulting DNA-protein complexes were separated by electrophoresis through a non-denaturing 4% polyacrylamide gel. C, renatured proteins (2 pl) in the molecular mass range of 70-72 kDa were incubated in the presence of [y-”PIATP with (lane 3 ) and without (lane 2 ) PKC. Autophosphorylation products of the PKC fraction without the addition of exogenous protein substrates are indicated by black dots. Samples were separated on a 10% SDS-polyacrylamide gel. Migration of molecular weight standards are shown in the left margin. D, an aliquot of the labeled EivF generated by incubation of the 70-72-kDa protein renatured from a SDS-polyacrylamide gel with PKC and [y-:’2P]ATP (as indicated in panel C, lane 3 ) was analyzed in a DNA mobility shift assay in which the DNA fragment containing the CRE was unlabeled (lane 3 ) . The migration on a polyacrylamide gel of the resulting DNA protein complex (indicated by an arrow) was compared with the DNA-protein complex formed between a labeled DNA and unlabeled EivF (lane 2). The migration of free labeled DNA is indicated in lane 1. The DNA fragment used in this analysis contained one CRE and was derived from plasmid DNA pE4-330/-104 (Leza and Hearing, 1988). The condition for the formation of the DNA- protein complexes was as described under “Experimental Proce- dures.’’

kDa exhibited a modified mobility on native gels when com- plexed to DNA after CIP treatment. The DNA binding activity of the 38-45-kDa proteins was reduced as a result of dephosphorylation (Fig. 4, lanes 7, 9, and 11). However, it was not possible to reproducibly recover the DNA binding activity by rephosphorylation with PKA and/or PKC (data not shown). On the other hand, treatment with a high concentration of phosphatase of the proteins in the molecular mass ranges of 36-37, 33-34, and 31-32 kDa resulted in a drastic reduction of the DNA binding activity (Fig. 6A, compare lunes 1,4, and 7 with 2,5, and 8, respectively). However, after rephosphorylation with PKA, the DNA binding activity was recovered (Fig. 6A, lunes 3, 6, and 9). Interestingly, the DNA-protein complex formed with the 31-32-kDa proteins phosphorylated in vitro migrated slower than that formed with the untreated proteins (Fig. 6A, compare lune 9 with 7) and comigrated with the complex formed with the 33-34-kDa proteins (Fig. 6A, compare lane 9 with 4 and/or 6) . Further- more, the complex formed with the 36-37-kDa proteins dephosphorylated in vitro also comigrated with the complex formed with the 33-34-kDa proteins after PKA treatment (Fig. 6A, compare lane 3 with 6). Treatment of the 36-37- kDa proteins with increasing amounts of phosphatase yielded DNA-protein complexes displaying the same mobility observed as that formed with untreated proteins in the molecular mass range of 31-37 kDa renatured from an SDS-polyacrylamide-gel (compare Fig. 6B, lanes 2-5 with Fig. 2B, lanes 16- 20). Similarly, treatment of the 31-32-kDa proteins with increasing amounts of PKA resulted in complexes with differential migration (Fig. 6C, lanes 1-4). At higher concentra- tions of PKA, the mobility of this complex (Fig. 6C, band a ) changed to a form that comigrated with that of the 33-34- kDa proteins (Fig. 6C, bund b and data not shown). I t was not possible to convert the complex formed with the 31-32-kDa proteins to one that comigrated with the complex formed with the 36-37-kDa proteins (Fig. 6C, bund c). These results suggested that varying extents of phosphorylation may alter DNA binding activity and account for the differences in mobility on native gels of the DNA-protein complexes formed with the polypeptides in the 31-37-kDa range.

The results obtained with the 36-37-kDa proteins, namely that the DNA-protein complex formed after PKA treatment of the proteins dephosphorylated in vitro migrated faster (Fig. 6A, lane 3 ) than the complex formed with the untreated proteins (Fig. 6A, lane 1 ), suggested that the complex formed with the untreated 36-37-kDA proteins contained two different polypeptides. The DNA binding activity of these proteins was regulated by phosphorylation; however, the activity of only one of these proteins was regained by treatment with PKA. An alternative consideration is the presence of an additional phosphoryl group(s) in the proteins of 36-37 kDA molecular mass range that could not be restored by treatment with PKA in vitro. Phosphoamino acid analysis from in vivo (68, 43, and 31-36-kDa proteins, see Fig. lA) or in vitro labeled proteins (EivF and 31-45 kDa) revealed that only serine residues were phosphorylated (data not shown).

In order to investigate whether the protein-DNA complex formed with the 36-37-kDa proteins was composed of at least two different proteins and whether the complexes formed with proteins in the 31-37-kDa molecular mass range included a common polypeptide that displayed a differential migration on SDS-polyacrylamide gels because of phosphorylation, the experiment described in Fig. 7A was performed. Proteins in the molecular mass range of 31-45 kDa were incubated with PKA and [y”P]ATP (to promote a phosphate exchange reaction) or dephosphorylated and then rephosphorylated


A B 37-36

t~ t t P K A

32-31 34-33

CIp-tt-+t-tt

4 -

1 2 3 4 5 6 7 8 9

Cl I 2 3 4 5 6

l 2 3 4 5 6 7 8 91011

FIG. 6. The DNA binding activity of the 31-37-kDa proteins is regulated by phosphorylation. A, proteins renatured from SDS-polyacrylamide gels, ranging in molecular weight as indicated at the top of the figure, were treated with equivalent amounts of CIP (1.0 unit, lanes 2 ,5, and 8 ) and phosphorylated with PKA subsequent to the inactivation of CIP as described under “Experimental Proce- dures” (lanes 3, 6, and 9). Control lanes, 1, 4, and 7 contain the various molecular weight proteins incubated with inactivated CIP in the absence of PKA. These samples were carried out over the same process as samples that were incubated with active CIP. Reaction mixtures were incubated with a 3’-end labeled DNA fragment containing one CRE element (pE4-330/-104, see legend to Fig. 50) . DNA-protein complexes were separated by electrophoresis through a 4% nondenaturing polyacrylamide gel. B, aliquots (3 pl) of the 36- 37-kDa molecular mass polypeptides were dephosphorylated with increasing amounts of CIP as indicated in the panel (lanes 2-5) and described under “Experimental Procedures.” Untreated sample (lane 2 ) and 31-32-kDa proteins (lane 6 ) were similarly treated, but inactivated CIP was added to the reaction mixtures. Samples were analyzed for their ability to bind to the CRE using the fragment described in Fig. 5C. Products of the reactions were separated by electrophoresis through a 4% polyacrylamide gel. Lane 1 represents the migration of the uncomplexed labeled DNA fragment. C , aliquots (3 pl ) of the 31- 37-kDa proteins were treated with increasing amounts of PKA as shown at the top of the panel (lanes 2,3,4,8,9, and 10) and described under “Experimental Procedures.” Untreated proteins (lanes 1 and 7) and proteins in the 36-37-kDa range (lanes 5 and 1 1 ) were incubated in the absence of PKA. The resulting proteins were analyzed for their ability to bind to the CRE present in the adenovirus EIV promoter. The fragment used was described in Fig. 5C and contained one CRE. Products of the reactions were separated by electrophoresis through a 4% polyacrylamide gel. Lane 6 represents the migration of the uncomplexed labeled DNA fragment. Arrows a, b, and c on either side of the panel represent different DNA-protein complexes.

A Vierent Mdecular Weight Pmteins

fClP I I

PKA + (yW).ATP

pZP)-labeted Pmteins

DNA fragment mntaining !he CRE

Separation of DNA Pmtekl mmplexes

I

I 1

t

B .- L

c

C

E . e -

I . = . - Isolation 01 labeled Pmteins

I I 2 3 * 1 0

( y - 32Pl-ATP SDS-PAGE +

PKA In v i t ro

7 2 ‘

:3

28-

6 4

4 3

2 9

1 2 3 4

. c a -b

I 2 3 4 5

FIG. 7. Differential migration of the small molecular weight proteins (31-37 kDa) through SDS-polyacrylamide gels is because of differing extents of phosphorylation of a common polypeptide. Renatured proteins ranging in molecular mass from 31 to 45 kDa (as indicated at the top of each lane in panels B and C ) were subjected to the analysis described in panel A. Proteins were phosphorylated with PKA and [y-”P]ATP (panel B ) or first dephosphorylated with CIP and then phosphorylated with PKA and [?-”‘PI ATP (panel C ) . Phosphorylation and dephosphorylation reactions, as well as inactivation of the CIP, were as described under “Experi- mental Procedures.” Labeled proteins were incubated with an unlabeled DNA fragment derived from plasmid DNA pE4-330/-104 that contained one CRE. The DNA protein complexes formed were separated by electrophoresis on a 4% polyacrylamide gel. The DNA- protein complexes were excised from the gel and the material was recovered by electroelution. Proteins were then separated by electrophoresis through SDS-polyacrylamide gels (10%). The DEAE-cellulose fraction containing CRE-binding proteins was incubated with PKA and [y3’P]ATP as described under “Experimental Procedures.” Labeled-CRE-binding proteins were further purified by DNA affinity chromatography on a column containing the CRE sequence. The proteins recovered after the third passage were separated by electrophoresis through a 10% SDS-polyacrylamide gel (panel B, lane 1 ) . Panel D, nuclear extracts (50 pg) were treated with or without CIP as described under “Experimental Procedures” (lanes 1 and 2, respectively), or incubated with ATP (lane 4 ) , or AMP-PMP (lane 3) under protein phosphorylation conditions, as described under “Experimen- tal Procedures.” The proteins were separated by electrophoresis through a 10% SDS-polyacrylamide gel. The proteins were then transferred to nitrocellulose and incubated with affinity purified antibodies directed against a peptide (28 amino acids) that contains the PKC domain present in CREB (to be described elsewhere).’ Blots were then incubated with anti-rabbit IgG conjugated to alkaline phosphatase and visualized after incubation with nitro blue tetrazolium (NBT) and 5-bromo-3-indolyl phosphate (BCIP) as previously described (Flores et al., 1988). The molecular mass in kDa of three different protein markers is indicated at the left-hand side of the panel.


with PKA. Labeled proteins were then incubated with an unlabeled DNA fragment that contained the CRE, and the DNA-protein complexes were isolated by electrophoresis as described. Proteins in each complex were eluted and analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. As expected, polypeptides labeled with [y-”PI ATP by the PKA-catalyzed phosphate exchange reaction that were recovered from the DNA-protein complexes, each exhibited a different and distinct mobility after SDS-polyacrylamide gel electrophoresis (Fig. 7B, lanes 2-6). When a crude fraction (DEAE-cellulose) was incubated with PKA and [y- 32P]ATP and the proteins isolated by DNA affinity chromatography through a column containing the CRE, proteins with similar migration on an SDS-polyacrylamide gel could be observed (Fig. 7B, compare lanes 2-6 with I , respectively). This result indicates that the proteins isolated from an SDS- polyacrylamide gel were all contained in fractions isolated by DNA affinity chromatography. A different situation was observed when the proteins were first dephosphorylated and then labeled with PKA and [-p3’P]-ATP before DNA protein complex isolation. Under this condition, the protein initially isolated as 43 kDa still migrated as a 43-kDa specie (Fig. 7C, lane 1 ). However, proteins in the molecular mass range of 37- 31 kDa now comigrated as one specie of approximately 35 kDa (Fig. 7C, lanes 2-4). The protein in the molecular mass range of 37-36 kDa included two polypeptides (lane 2). The larger polypeptide was not observed when the phosphatase concentration was increased (data not shown). These results strongly suggested that the CRE-binding proteins in the 31- 37-kDa range were a single protein whose altered migration through SDS-polyacrylamide gels was due to different phosphorylation states. In order to further study this possibility, we used monospecific antibodies directed against a peptide that contained the consensus PKC phosphorylation motif of CREB (Gonzalez et al. (1989) and Hoeffler et al. (1988b)). Using gel retardation assays, we found that proteins in the molecular mass ranges of 31-37 and 40-43 kDa were further slowed in their migration on native gels (supershift) upon addition of monospecific antibodies while the mobility of the DNA protein complexes formed with EivF or an unrelated DNA binding protein, MLTF (Carcamo et al., 1989), was not affected (data not shown).’ Western blot analysis of nuclear extracts prepared from HeLa cells with these antibodies, identified polypeptides with apparent molecular masses of 47, 43, and a 31-36 kDa (Fig. 70, lanes 1 and 5 ) . That these antibodies did not recognize EivF (65-72 kDa) was expected since the binding activity of the dephosphorylated form of EivF was not affected by phosphorylation with PKC (Fig. 5), suggesting that this protein lacks the PKC domain. Treat- ment of the extract with CIP or ATP did not affect the mobility of the 47- or the 43-kDa polypeptides on an SDS- polyacrylamide gel (compare lanes 1 and 5 with 2 and 4, respectively). However, proteins migrating between 31-36 kDa were converted, after CIP treatment, to a single 31-kDa polypeptide (lane 2, arrow b) . Moreover, when extracts were incubated in the presence of ATP the 31-37-kDa proteins migrated as a single 37-kDa polypeptide (lanes 4, arrow a). This was not observed when AMP-PNP, a nonhydrolyzable analog of ATP, was used. Taken together, these observations strongly suggest that the 31-37-kDa proteins observed by SDS-polyacrylamide gels are the same polypeptide whose mobility varies with the extent of phosphorylation, where the 37-kDa form is the most and the 31-kDa form is the least phosphorylated representative of a common protein.

The DNA Binding Specificity (Affinity) of the 31-32-kDa

A. Merino and D. Reinberg, manuscript in preparation.

Proteins for the APl Site Is Regulated by Phosphovlation- In the previous section we suggested that the 31-37-kDa proteins represent one distinct polypeptide and that the differential migration of this protein on a SDS-polyacrylamide- gel is because of different extent of phosphorylation. Footprint analysis (Fig. 3, A and B) has shown that proteins in the range of 33-37 kDa bound to the CRE present in the adenovirus EIV promoter as well as to the AP1 and ATF elements present in the adenovirus E111 promoter. However, the 31- 32-kDa proteins failed to bind to the AP1 site. These results suggested that phosphorylation could regulate the binding affinity (specificity) of the 31-32-kDa protein for the AP1 recognition site. This possibility was analyzed using the gel mobility shift assays. In agreement with the results presented above (Fig. 3B), proteins in the 31-32-kDa molecular mass range were capable of binding to the EIII-ATF recognition sites (Fig. 8, lane 2 ) . Two DNA-protein complexes were observed which may be the result of dimerization of the proteins. Both complexes were competed by the addition of a 30-fold molar excess of an oligonucleotide containing the ATF rec-

30-3 I kDa protein

phosphorylated 30-3 I

kDa protein - -

AP1 oligo

121314 1011 9 8 7 6 5 4 3 2 I + - - - t - - + - - - t - - ATFoligo

+“+ - - t - - - t - - -

” ”

ATF-2-

ATF- I - L’: - * I !

.d

1 AP1-1 - ATF-2’ - ATF-I’

- f r e e

f ree- I EIII-ATF I Em-APl (Em-ATFI ElU-APll

FIG. 8. PKA-mediated phosphorylation of the proteins in the 31-32 kDa molecular mass weight range modifies the DNA binding affinity for the EIII-AP1 site. Proteins in the molecular mass range of 31-32 kDa were treated with PKA in the absence (lanes 1-8) or in the presence (lanes 9-14) of ATP. The resulting proteins were tested for their ability to bind to the ATF (lanes 2-4 and 9-11) or AP1 (lanes 6-8 and 12-14) recognition sites on the adenovirus EIII promoter as described under “Experimental Procedures.” Competition experiments were carried out using a 30- fold molar excess of oligonucleotides containing the ATF (lanes 3, 7, 10, and 13) or AP1 (lanes 4, 8, 11, and 14) recognition sites. Lanes 1 and 5 represent the migration of uncomplexed “P-labeled DNA fragments containing the EIII-ATF and the EIII-APl sites, respectively. The DNA fragment containing the ATF, but not the AP1 element, was derived by digesting plasmid DNA pE3-85 (g i f t of Dr. R. Gaynor) with restriction endonucleases EcoRI and SmaI. The DNA fragment containing a wild-type APl recognition site but with a triple point mutation in the ATF element was derived from plasmid DNA pE3AII (Kornuc et al., 1988). DNA fragments were 3’-end labeled. DNA-protein complexes were separated by electrophoresis through a nondenaturing 4% polyacrylamide gel.


ognition site (Fig. 8, lane 3). However, these complexes were not or slightly competed by the addition of oligonucleotides containing the AP1 recognition site or by oligonucleotide containing the recognition site for an unrelated DNA binding protein (SP1, see “Experimental Procedures”) (Fig. 8, lanes 4 and 2, respectively). Consistently, the proteins in the 31-32- kDa range failed to form complexes with a DNA fragment that contained the AP1 recognition site present in the adenovirus E111 promoter (Fig. 8, lanes 6-8). When the proteins were first incubated with PKA and ATP, under phosphorylation conditions, and then used in the mobility shift assay, the in vitro phosphorylated 31-32-kDa proteins were now capable of forming complexes with the AP1 site present in the adenovirus E111 promoter (Fig. 8, lane 12). DNA-protein complexes were now competed by oligonucleotide containing the ATF or AP1 recognition site (Fig. 8, lanes 13 and 14, respectively), but not by an oligonucleotide containing the recognition site for SP1 (Fig. 8, lane 12). The migration of the DNA- protein complexes formed with the phosphorylated 31-32- kDa proteins and the EIII-ATF site changed as a consequence of phosphorylation (compare lanes 2 and 9). These complexes were efficiently competed by an oligonucleotide containing the AP1 recognition site (lane 11) but not by an oligonucleotide containing the recognition site for SP1 (lane 9). These results strongly suggest that the binding affinity of the 31- 32-kDa proteins for the AP1 recognition site is regulated by phosphorylation. Phosphorylation by PKA not only affected the affinity of the proteins for the CRE and ATF elements but also may regulate their binding specificity for the AP1 recognition site.

EivF Is Immunologically Related to API-We investigated, using Western blot analysis, whether EivF, a factor that binds exclusively to the cyclic AMP response element, was antigen- icallly related to the other CRE-binding proteins that are themselves immunologically related, AP1 and ATF (Hai et al., 1988). Fractions derived from different steps of the puri-

A

111-

43

1 2 3 4 1 2 3 4 FIG. 9. EivF is antigenically related to APl/c-jun. A, EivF

fractions were separated on an SDS-polyacrylamide gel (8%), transferred to nitrocellulose and incubated with anti-c-jun antibodies (1:2000 dilution of antiserum). Blots were then incubated with anti- rabbit IgG conjugated to alkaline phosphatase and visualized after incubation with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro- 3-indolyl phosphate (BCIP) as previously described (Flores et al., 1988). Samples prepared as described by Cortes et al., 1988, included lane 1.10 pg of protein from a gel filtration fraction enriched in EivF activity; lane 2, 10 pg of protein from a gel filtration fraction enriched in AP1 activity; lanes 3 and 4, 20 p l of affinity purified EivF or an equal amount of buffer B, respectively. B, renatured protein samples (20 pl) of molecular masses ranging from 31 to 43 kDa (shown at the top of each lane) were separated by preparative SDS-polyacrylamide gel electrophoresis (10%) and analyzed for immunoreactivity to anti- c-jun antibody as described above.

fication of EivF were analyzed using antibodies directed against an AP1 fusion protein (Angel et al., 1987). When a partially purified fraction that was enriched in EivF (Fig. 5A, lane 1) or APl/ATF (lane 2) after the gel filtration step (Cortes et al., 1988) was used in this analysis, proteins of approximately 70-72 and 40 kDa were identified. The antibodies were highly specific, since the fraction used in the analysis described above was relatively crude and contained many polypeptides (see Fig. 5 from Cortes et al., 1988)) yet only a few polypeptides were detected by the antibodies. Furthermore, when affinity purified EivF was used, two polypeptides in the molecular mass range of 65-70 kDa reacted with the antibodies (lane 3). In addition, bovine serum albu- min (which is added to preserve EivF transcriptional activity) reacted nonspecifically and was present in the lane containing only the buffer used to elute EivF from the affinity column (lane 4). The small molecular mass proteins (31-43 kDa) reacted with the AP1 antibodies as expected (Fig. 9B). These results indicated that EivF was antigenically related to AP1 and were in agreement with our previous results, confirming that EivF was contained in polypeptides of approximately 65 and 72 kDa (Cortes et al., 1988).

DISCUSSION

We have analyzed the proteins that bind to Ela-responsive DNA sequence elements present in the adenovirus E111 and EIV promoters. Stimulation of transcription from the adenovirus E111 promoter correlated with binding of these proteins to both the AP1 (5’-GTGA(C/G)TCA-3’) and ATF (5’- GTGACGA-3’) recognition sites within this promoter. Tran- scription from the adenovirus EIV promoter correlated with binding of proteins to the CRE sequence (5’-GTGA.CGT-3‘) (Cortes et al., 1988; Lee et al., 1987a; Hai et al., 1988). We found these proteins to be phosphorylated in vivo. Our studies have demonstrated that the DNA binding activity and the ability to stimulate transcription of the proteins recognizing DNA elements, the CRE, ATF, and AP1, present in the adenovirus E111 and EIV promoters and that are known to mediate the response to Ela, was regulated by phosphorylation. Phosphatase treatment of the different isolated proteins resulted in diminished DNA binding activity. However, the binding activity of the proteins dephosphorylated in vitro could be recovered by phosphorylation with the catalytic subunit of protein kinase A.

Our first efforts attempted to directly analyze the effect of phosphorylation of the CRE-binding proteins on transcription. We found that dephosphorylated proteins did not activate transcription. This is not surprising because the DNA binding activity of these proteins was lost as a result of dephosphorylation. The addition of PKA to transcription reactions reconstituted in vitro resulted in the stimulation of transcription. However, stimulation was also observed from promoters without a CRE site, for example the adenovirus MLP, and when the minimum sequences required for transcription, the TATA box and the CAP site, were the only elements present in the promoter. Therefore, PKA-mediated stimulation of transcription lacks specificity in vitro. The studies of Yamamoto et al., 1988, demonstrated that when extracts prepared from PC12 cells were treated with PKA and used to drive transcription from the somatostatin promoter, an increase in activation was observed. They indicated that the stimulation of transcription was dependent on the CRE element. However, it is also of interest that the overall non- specific transcription of a mutant somatostatin promoter missing the CRE sequence was also stimulated to almost the same extent. It is possible that the somatostatin promoter,


known to be a weak promoter, is transcriptionally inactive in the absence of the upstream CRE sequence. These investiga- tors also studied the ability of PKA-treated nuclear extract to simulate transcription from the SV40 promoter, which does not contain the CRE. Nonetheless, transcriptional activation of the SV40 promoter was observed, but to a lesser extent than with the somatostatin promoter. Thus, it is likely that alternative transcription factors can also mediate the response to PKA in uitro. Therefore, analysis of the effects of phosphorylation on transcription requires the use of a system in which the components are better defined.

Previous studies have demonstrated the existence of different proteins that recognize the CRE (Cortes et al., 1988, Hai et al., 1988). Differences in molecular weight, binding specificity, and response to phosphorylation by PKA allowed us to categorize these proteins into three groups: 1) EivF, a 65-72- kDa protein that is specific for binding to the CRE present in the adenovirus EIV promoter. The DNA binding activity of this protein is predominantly regulated by phosphorylation with PKA. 2) A second set of proteins in the molecular mass range of 38-45 kDa that were able to bind to the AP1 and ATF sites present in the adenovirus E111 promoter, as well as to the CREs present in the adenovirus EIV promoter. Our studies demonstrated that phosphorylation regulates the DNA binding activity of these proteins. Treatment of these proteins with phosphatase resulted in a decrease in DNA binding activity. However, we failed to recover binding of the proteins dephosphorylated in uitro after rephosphorylation with PKA and/or PKC. 3) A third set of proteins was identified in the molecular mass range of 31-37 kDa with the same binding specificity as the second set of proteins. How- ever, we were able to recover the DNA binding activity of these proteins after CIP-catalyzed dephosphorylation in uitro by rephosphorylation with PKA.

Our analysis indicated that the proteins in the molecular mass range of 31-37 kDa that recognized the CRE, ATF, and AP1 elements probably represent one polypeptide that migrates differently through SDS-polyacrylamide gels depend- ing on the extent of phosphorylation. All the proteins of the 31-37-kDa molecular mass range displayed the same migration after electrophoresis through SDS-polyacrylamide gels when they were radiolabeled by first dephosphorylation in uitro with CIP, followed by resphosphorylation with [r-”P] ATP and PKA and then purified by elution from native polyacrylamide gels when bound to a DNA fragment containing the CRE. It is important to note that all of the proteins identified by SDS-polyacrylamide gel electrophoresis subsequent to isolation from DNA mobility shift assays were present in fractions purified by DNA affinity chromatography. The advantage of introducing the gel electrophoresis step was to allow the separation and further characterization of each of these species. This analysis permitted us to discover that treatment with PKA not only regulated the ability of the proteins in the 31-37-kDa molecular mass range to specifically bind DNA but also regulated the affinity of these proteins for different sites. Proteins in the molecular mass range of 33-37 kDa bound to the ATF and AP1 recognition sites present in the adenovirus E111 promoter as well as to the CRE element present in the adenovirus EIV promoter. However, proteins in the molecular mass range of 31-32 kDa bound to the CRE and ATF elements but failed to recognize the API site. Phosphorylation of the latter group of proteins with PKA now enabled their binding to the the AP1 recognition site. This was directly demonstrated using the AP1 recognition site present in the adenovirus E111 promoter. Also, the original mobility shift after PKC and/or PKA treatment of the de-

phosphorylated polypeptides in the molecular mass range of 36-37 kDa was not recovered. To explain this result, it is possible that (i) an integral residue in the protein that me- diates binding activity is not phosphorylated by PKA and/or PKC in uitro, (ii) two polypeptides are present in this fraction, each of which possess DNA binding activity that is regulated by phosphorylation. However, it may be that only one of these proteins is a substrate for PKA. Accordingly, it is of interest that the two cDNA clones isolated by Gonzalez et al. (1989) and Hoeffler et al. (1988a), respectively, that encode CREB protein vary by 15 internal amino acids. This may account for the two different polypeptide species observed in the 36- 37-kDa molecular mass range. Alternatively, these clones may be representative of the 38-43 and 31-37-kDa species. How- ever, the 31-37-kDa family of proteins which migrates as one polypeptide after dephosphorylation (31 kDa) followed by exhaustive rephosphorylation (35 kDa) with PKA, is dis- tinctly different than the molecular mass of the 35- and 37- kDa CREB proteins based on the amino acid sequence derived from the cDNA clones. Therefore, the 31-37-kDa proteins we have isolated may represent a subset of CREB entirely. Pro- teins of similar molecular mass (32-37 kDa), as well as a 47- kDa species have been identified in rat ovarian nuclear extracts by Southwestern blotting using the CRE sequence by Kwast et al. (1989). Also, Hai et al., 1988, identified, from HeLa and JY cells, two polypeptide doublets one at 47 kDa and a second at 43 kDa, that bound to a CRE-DNA affinity column and suggested that each doublet consisted of different forms of ATF and AP1 polypeptides. This was determined by the preferential binding of each of the 43- and 47-kDa proteins to columns containing the CRE or AP1 recognition sites. It may also be considered, based on our studies, that the binding specificity of the 43- and 47-kDa proteins to the CRE and AP1 elements could be manifested by their state of phosphorylation. These authors only observed radiolabeled protein using PKA and [ Y - ~ ~ P I A T P in the CRE-enriched fraction and suggested that PKA failed to phosphorylate AP1 in uitro. It is not clear why phosphorylation of the AP1-enriched fraction, which contained ATF as detected by immunoblot analysis, was not observed in this particular experiment. A conclu- sive understanding of the polypeptides comprising AP1 and ATF will require further studies.

The molecular mechanism(s) by which the Ela p289 protein activates transcription of early viral and cellular genes is unknown. Some of the DNA elements capable of mediating the Ela response also participate in regulating the response of CAMP (CRE) as well as to the tumor promoter 12-0- tetradecanoylphorbol-13-acetate (AP1). This, together with the following observations (i) the Ela-p289 protein did not recognize specific sequences on the DNA (Chatterjee et al., 1988), (ii) activation of the early adenovirus E11 promoter required a cellular factor, E2F, whose DNA binding activity increased upon infection by a wild-type virus (Kovesdi et al., 19861, (iii) Ela activation of the E11 promoter could take place in the presence of inhibitors of protein synthesis (Reichel et al., 1988; Green et al., 1988) and, (iv) Ela-mediated activation of the VA I gene, an RNA polymerase I11 transcribed gene, involved the activation of a cellular factor. TFIIIC (Hoeffler et al., 1988b; Hoeffler and Roeder, 1985; Gaynor et al., 1985; Yoshinaga et al., 1986), strongly supports the hypothesis that Ela activation proceeds through the modification of pre-ex- isting cellular factors (Reichel et al., 1988). Our data has demonstrated that there are at least three families of factors that are specific for the CRE, ATF, and AP1 elements. It appears that the state of phosphorylation of these proteins regulates their binding specificity to three related DNA ele-


ments that differ by one nucleotide in sequence (i.e. the CRE, AP1, and ATF elements). Therefore, it is possible that there are at least two modes of action whereby the regulation of gene expression could occur at the CRE. First, the identification of unique amino acid sequences within the CREB protein (Hoeffler et al., 1988a; Gonzalez et al., 1989) that can serve as phosphorylation sites for PKA, PKC, and casein kinase 11, support the hypothesis that the regulation of these proteins can occur internally. Lastly, it is intriguing to con- sider that the three sets of CRE-binding proteins we have identified could confer responsiveness to three different and specific cellular signal transduction pathways induced by CAMP, TPA, and the adenovirus-encoded Ela protein.

Acknowledgments-We wish to thank Dr. L. Vales for helpful discussion and reading of the manuscript. We also thank Drs. K. J . Marians, S. Shuman, and S. Powers for reading the manuscript; Drs. M. Karin, T. Boss, and P. Vogt for providing us with antibodies against different parts of the c-jun protein and Dr. 0. Rosen for providing us with PKC. The expertise of Dr. Nai-Sheng Lin in preparing the nuclear extracts is acknowledged. We also thank the members of the laboratory for active discussions.

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