©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cAMP-dependent Protein Kinase Is Necessary for Increased NF-E2DNA Complex Formation during Erythroleukemia Cell Differentiation (*)

Arlene D. Garingo (1), Modem Suhasini (1)(§), Nancy C. Andrews (2)(¶), Renate B. Pilz (1)(**)

From the (1) Department of Medicine, University of California at San Diego, La Jolla, California 92093-0652 and the (2) Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

When murine erythroleukemia (MEL) cells are induced to differentiate by hexamethylene bisacetamide (HMBA), erythroid-specific genes are transcriptionally activated; however, transcriptional activation of these genes is severely impaired in cAMP-dependent protein kinase (protein kinase A)-deficient MEL cells. The transcription factor NF-E2, composed of a 45-kDa (p45) and an 18-kDa (p18) subunit, is essential for enhancer activity of the globin locus control regions (LCRs). DNA binding of NF-E2 and -globin LCR enhancer activity was significantly less in HMBA-treated protein kinase A-deficient cells compared to cells containing normal protein kinase A activity; DNA binding of several other transcription factors was the same in both cell types. In parental cells, HMBA treatment and/or prolonged activation of protein kinase A increased the amount of NF-E2DNA complexes without change in DNA binding affinity; the expression of p45 and p18 was the same under all conditions. p45 and p18 were phosphorylated by protein kinase A in vitro, but the phosphorylation did not affect NF-E2DNA complexes, suggesting that protein kinase A regulates NF-E2DNA complex formation indirectly, e.g. by altering expression of a regulatory factor(s). Thus, protein kinase A appears to be necessary for increased NF-E2DNA complex formation during differentiation of MEL cells and may influence erythroid-specific gene expression through this mechanism.


INTRODUCTION

Erythroid-specific genes are transcriptionally activated when murine erythroleukemia (MEL)() cells are induced to differentiate by hexamethylene bisacetamide (HMBA) (1, 2, 3) . We have previously shown that cAMP-dependent protein kinase (protein kinase A)-deficient MEL cells are severely impaired in HMBA-induced differentiation, and that HMBA-induced increases in mRNA expression and transcription rates of several erythroid-specific genes are reduced in protein kinase A-deficient cells in proportion to their residual protein kinase A activity; mRNA expression and transcription rates of several other differentiation-associated and housekeeping genes are similar in HMBA-treated parental and protein kinase A-deficient cells (4, 5) . These data suggest that protein kinase A is involved in regulating genes with erythroid-specific promoters during differentiation of MEL cells.

The erythroid-specific transcription factor NF-E2 recognizes the sequence (T/C)GCTGA(G/C)TCA(C/T) found at DNase hypersensitive sites of the globin locus control regions (LCRs) and in the promoters of several erythroid genes (6, 7, 8, 9, 10, 11, 12) . It is a member of the basic leucine zipper family of transcription factors and binds DNA as a dimer of a 45-kDa tissue-specific protein (p45) and an 18-kDa ubiquitously expressed protein (p18) (8, 9, 11) . Within the NF-E2 recognition site is the sequence TGA(G/C)TCA which is recognized by the transcription factor AP-1 (Fos/Jun); however, several findings indicate that NF-E2 is the functional activator binding at the NF-E2/AP-1 recognition sites of the globin LCRs and the porphobilinogen deaminase promoter, and that p45 is required for globin gene expression during erythroid cell differentiation (13, 14, 15, 16, 17, 18) .

GATA-1 is a zinc finger transcription factor which binds to the consensus sequence (T/A)GATA(A/G) present in the promoters and enhancers of all known erythroid-expressed genes; genetic knock-out experiments have demonstrated that GATA-1 expression is required for erythroid cell differentiation (6, 19, 20, 21) . GATA-1 and NF-E2 appear to cooperate with each other and with other transcription factors, e.g. SP-1, to activate erythroid-specific genes, and both are required for induction of erythroid gene expression during differentiation of MEL cells (7, 13, 14, 20, 22, 23, 24) .

In this work we have examined further the role of protein kinase A in transcriptional activation of erythroid-specific genes. We found in transient transfection experiments that -globin LCR enhancer activity was reduced in HMBA-treated protein kinase A-deficient MEL cells compared with MEL cells containing normal protein kinase A activity. In parental cells, HMBA treatment increased the amount of NF-E2DNA complexes significantly more than in protein kinase A-deficient cells, and prolonged activation of protein kinase A increased NF-E2DNA complexes in the presence or absence of HMBA. These changes in NF-E2DNA complexes did not appear to be due to changes in the amount or phosphorylation of p45 or p18. Rather, our data suggest that protein kinase A regulates NF-E2DNA complex formation indirectly, e.g. by altering the expression of an accessory protein.


EXPERIMENTAL PROCEDURES

Materials The human -globin promoter and promoter/enhancer chloramphenicol acetyltransferase (CAT) constructs (pCAT and pCAT/LCR, respectively) were generous gifts of N. J. Proudfoot (25) ; the chicken -actin promoter CAT construct (pactCAT) was from the American Type Culture Collection (26) , and the luciferase expression vector pRSV-luc was from S. Subramani (27) . The p45, p18, and CHOA cDNA probes and the p45 and p18 expression vectors were described previously (8, 11, 28) .

Rabbit polyclonal antibodies were prepared against bacterially expressed p45- and p18-glutathione- S-transferase fusion proteins as described (8, 11) , and purified C-subunit of protein kinase A was a generous gift of S. Taylor. Double-stranded oligodeoxynucleotides containing recognition sites for NF-E2 and GATA-1 were synthesized with 3` overhangs and provided by D. Brenner; blunt-ended oligodeoxynucleotides containing recognition sites for NF-E2 and GATA-1 were from Santa Cruz Biotechnology and for AP-1 and SP-1 were from Promega. Methods

Cell Lines and Cell Culture

The stable MEL cell transfectants derived from MEL cell line 745 were characterized previously: cells expressing either a mutant Rsubunit (Rmut) of protein kinase A or the enzyme's specific peptide inhibitor are protein kinase A-deficient, while cells overexpressing the wild type Rsubunit (Rwt) of protein kinase A have normal protein kinase A activity (4, 5) . Cells were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10% transferrin-enriched calf serum; unless stated otherwise, experiments were performed in this medium in which parental and transfected cells show similar growth rates. Stock cultures of transfected cell lines were maintained in 400 µg/ml G418 as described previously (4) . Although expression of the transfected genes is under control of the zinc-inducible metallothionein promoter, we did not use zinc in the present studies because of its potential effect on DNA binding of GATA-1 and SP-1 (29, 30) . In the absence of zinc and HMBA, the protein kinase A activity of transfectants Rmut/C3 and PKI/C2 is 26% and 24%, respectively, of the activity found in parental cells (4) . In the presence of HMBA, protein kinase A activity of Rmut/C3 and PKI/C2 is 18% and 15% of the activity found in parental cells because HMBA increases protein kinase A activity to a greater extent in parental cells (and in Rwt transfectants) than in Rmut or PKI transfectants (4) . Two other protein kinase A-deficient cell lines (Rmut/C9.10 and PKI/C16.2) were used as indicated; their protein kinase A activities are 41% and 52% in the absence of HMBA and 30% and 38% in the presence of HMBA, respectively, of parental cells (4) .

Transient Transfection of MEL Cells and COS Cells

Midlog phase cells were washed in serum-free IMDM, and 10cells were incubated for 4 h at 37 °C in IMDM with liposomes containing 1 mg/ml L--phosphatidylethanolamine, 0.3 mg/ml dimethyldioctadecylammonium bromide, 30 µg of pCAT, pCAT/LCR, or pactCAT, and 10 µg of pRSV-luc (31) . The cells were diluted 1:8 into serum-containing IMDM, and, 16 h later, 4 m M HMBA was added to one-half of the cultures. At 48 h, cells were extracted in reporter lysis buffer (Promega), and CAT activity was measured in heat-inactivated (65 °C for 10 min) extracts using 0.06 m M [C]chloramphenicol and 0.75 m M butyryl coenzyme A by mixed phase extraction (32) ; luciferase activity was measured in nonheated extracts using 0.17 m M luciferin and 10 m M ATP in a photon-emission luminometer and served as an internal control for transfection efficiency (27) .

COS cells were transfected with equimolar amounts of p45 and p18 expression vectors as described previously (11) .

Electrophoretic Mobility Shift Assays (EMSAs)

MEL cells were cultured for 3 days in the absence or presence of 4 m M HMBA when nuclear extracts were prepared as described previously with 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin, 0.1 mg/ml soybean trypsin inhibitor, 20 m M -glycerol phosphate, and 0.01 m M NaVOadded to the extract buffer (33) ; protein was quantitated by the Bradford method (34) . The yield of nuclear extract protein per 10cells varied by <20% between different cell lines and culture conditions.

The following double-stranded oligodeoxynucleotide probes were 5`-end-labeled with [-PO]ATP and polynucleotide kinase: ( a) 5`-CCTCCAGTGACTCAGCACAGGTTCC-3` (NF-E2 recognition sequence from the human porphobilinogen deaminase promoter (13) ); ( b) 5`-CCTCCAGTGACTCAGAACAGGTTCC-3` (mutant NF-E2 recognition site (13) ); ( c) 5`-CGCTTGATGAGTCAGCCGGAA-3` (synthetic AP-1 recognition site (35) ); ( d) 5`-ATCTCCGGGCAACTGATAAGGATTCCCTG-3` (GATA-1 recognition site from the mouse -globin promoter (36) ); and ( e) 5`-ATTCGATCGGGGCGGGGCGAGC-3` (synthetic SP-1 recognition site (29) ). Unless stated otherwise, approximately 15 fmol (10cpm) of oligodeoxynucleotide were incubated in 15 µl for 20 min at room temperature with 5 µg of nuclear extract protein in 20 m M sodium HEPES, pH 7.9, 200 m M NaCl, 1 m M MgCl, 0.1 m M EDTA, 0.1 m M EGTA, 1 m M dithiothreitol, 15% glycerol, 100 µg/ml poly(dI-dC), and the above-described protease and phosphatase inhibitors. ProteinDNA complexes were resolved on 6% polyacrylamide gels in 45 m M Tris borate, pH 8.5, 1 m M EDTA (37) . Gels were exposed to x-ray film for variable times and analyzed by laser densitometry scanning (4) .

To assess maximal DNA binding and DNA binding affinity, constant amounts of nuclear extract protein were incubated with increasing amounts of labeled oligodeoxynucleotide, and the amounts of radioactivity present in the proteinDNA complexes and in the free probe were quantitated. The sum of both values increased as expected with increased amounts of probe indicating that the proteinDNA complexes were stable during electrophoresis (37) .

The reaction between a DNA-binding protein (P) and its cognate DNA sequence to form a complex (C) is written as

   

On-line formulae not verified for accuracy

Western Blot Analysis

MEL cells were cultured and nuclear extracts were prepared as described above. Variable amounts of nuclear extract protein were subjected to SDS-PAGE, transferred to polyvinylidine difluoride membranes, and incubated with a p45- or p18-specific antibody as described previously (4, 8) ; bound antibody was detected by enhanced chemiluminescence.

Northern Blot Analysis

MEL cells were cultured as described above, total cytoplasmic RNA was extracted, and Northern blots were prepared and hybridized to radioactively labeled cDNA probes as described previously (4) . The probes were 1.3- or 0.5-kilobase EcoRI fragments containing murine p45 or p18 cDNAs, respectively (8, 11) . Equal loading and transfer of RNA was assessed using a cDNA probe for the structural protein CHOA (28) .

In Vitro Phosphorylation of p45 and p18

Parental and protein kinase A-deficient (Rmut/C3) MEL cells were cultured for 3 days in the presence of 4 m M HMBA; some of the parental cells were treated with 1 m M 8-Br-cAMP for 1 h prior to harvesting to activate protein kinase A in vivo (39) . Nuclear extracts were prepared from 5 10cells as described above; 150 µg (for p45) or 300 µg (for p18) of nuclear extract protein were subjected to immunoprecipitation using a p45- or p18-specific antibody or preimmune serum as described (10) except that extracts were precleared by incubation with Protein A-agarose. Similarly, nuclear extracts were prepared from COS cells transfected with p45 and p18 expression vectors or from untransfected COS cells, and 75 µg of nuclear extract protein were subjected to immunoprecipitation. Washed immunoprecipitates were resuspended in 20 m M Tris-HCl, pH 7.0, 10 m M MgCl and incubated with 1 mCi/ml [-PO]ATP in the presence or absence of 2.5 µg/ml purified C-subunit of protein kinase A for 10 min at 30 °C. After additional washing, immunoprecipitates were analyzed by SDS-PAGE autoradiography.

Treatment of Nuclear Extracts with C-Subunit of Protein Kinase A or Alkaline Phosphatase

Ten micrograms of nuclear extract protein from parental or protein kinase A-deficient cells were diluted to 110 m M NaCl and incubated for 10 min at 30 °C or for 45 min at room temperature in 20 m M Tris-HCl, pH 7.0, 10 m M MgCl, 50 µ M ATP in the presence or absence of 10 µg/ml purified C-subunit of protein kinase A (higher temperatures or longer incubation times resulted in reduced NF-E2DNA complexes). After adjusting the NaCl concentration to 200 m M, poly(dI-dC) and radioactively labeled NF-E2/AP-1 oligodeoxynucleotide probe were added for 20 min at room temperature, and EMSAs were performed as described above.

For alkaline phosphatase treatment, nuclear extracts were prepared omitting -glycerol phosphate in the extract buffer; the extracts were diluted to 110 m M NaCl and incubated in 20 m M Tris-HCl, pH 7.9, 10 m M MgClin the presence or absence of 75 units/ml calf intestinal phosphatase immobilized on agarose beads (Sigma). After incubation for 10 min at 30 °C or 45 min at room temperature, enzyme-bound beads were removed by centrifugation and EMSAs were performed on the supernatants using an NF-E2 oligodeoxynucleotide probe with 3` overhangs internally labeled by reverse transcriptase in the presence of [-PO]dCTP (37) .


RESULTS

Transient Transfection of Erythroid Promoter/Enhancer Constructs into Parental and Protein Kinase A-deficient MEL Cells

Since transcriptional activation of several erythroid-specific genes by HMBA is reduced in protein kinase A-deficient MEL cells compared to parental cells (5) , we examined the activity of erythroid promoter/enhancer constructs in transient transfection assays in these cells. The plasmid pCAT contains the CAT reporter gene under control of the human -globin promoter; this promoter shows low activity in erythroid cells and is minimally inducible during differentiation (25) . The plasmid pCAT/LCR is identical with pCAT except it contains an additional 4-kb DNA fragment with the major tissue-specific enhancer element of the -globin gene cluster (HSS-40 (25) ). This enhancer element is referred to as -globin LCR because of its resemblance to the -globin LCR found upstream of the -globin gene cluster; enhancer activity is dependent upon erythroid differentiation (15, 25, 40, 41) .

When pCAT was transiently transfected into MEL cells, CAT expression was the same in cells with severely reduced protein kinase A activity (PKI/C2 and Rmut/C3) as in cells with normal protein kinase A activity (parental cells, Rwt/C1), and HMBA increased CAT expression 2- to 3-fold in both cell types (Fig. 1, hatched and cross-hatched bars represent cells cultured in the absence or presence of HMBA, respectively).


Figure 1: Transcriptional activity of reporter constructs containing the -globin promoter/enhancer in MEL cells with normal and reduced protein kinase A activity. MEL cells with normal protein kinase A activity (parental cells and Rwt/C1 transfectant) and protein kinase A-deficient MEL cells (PKI/C2 and Rmut/C3 transfectants) were co-transfected with pCAT/LCR ( open and closed bars) or pCAT ( hatched and cross-hatched bars) and pRSV-luc as described under Methods. Cells were cultured in the absence of HMBA ( open and hatched bars) or in the presence of 4 m M HMBA ( closed and cross-hatched bars); after 48 h, CAT activity and luciferase activity were measured as described under Methods. Luciferase activity served to assess transfection efficiency; it was the same in the absence or presence of HMBA and did not differ significantly between the different cell lines. The data are expressed as the ratio of CAT activity (in nmol/h/mg of protein) over luciferase activity (in relative light units/mg of protein) and represent the mean ± S.D. of at least three independent experiments performed in duplicate.



In experiments with pCAT/LCR, CAT expression was approximately the same in cells with normal or reduced protein kinase A activity in the absence of HMBA (Fig. 1, open bars). However, in the presence of HMBA, CAT activity increased 7- to 10-fold in cells with normal protein kinase A activity, but only 2- to 3-fold in cells with severely reduced protein kinase A activity (Fig. 1, closed bars; intermediate results were observed in the cells with moderate protein kinase A deficiency). The difference in CAT expression was not secondary to differences in transfection efficiencies since luciferase activity from the co-transfected plasmid pRSV-luc (27) was similar in all cell lines and was not influenced by HMBA. Moreover, when cells were transfected with pact-CAT (26) , CAT expression from the -actin promoter was the same in all cell lines grown in the absence or presence of HMBA (data not shown).

Since the enhancer function of the -globin LCR is mediated through binding sites for NF-E2, GATA-1, and proteins recognizing the CACCC motif (9, 15, 41) , reduced CAT expression from pCAT/LCR in HMBA-treated protein kinase A-deficient cells suggests that protein kinase A may regulate, directly or indirectly, the activity of one or more of these transcription factors.

DNA Binding of NF-E2, AP-1, GATA-1, and SP-1 in Nuclear Extracts of MEL Cells with Normal or Reduced Protein Kinase A Activity

We cultured parental MEL cells, protein kinase A-deficient transfectants, and transfectants with normal protein kinase A activity (Rwt/C1) in the absence or presence of HMBA and examined the DNA binding of several transcription factors using oligodeoxynucleotide probes containing specific recognition sequences (Fig. 2, only proteinDNA complexes are shown).


Figure 2: DNA binding of NF-E2, AP-1, GATA-1, and SP-1 in nuclear extracts from MEL cells with normal or reduced protein kinase A activity. Nuclear extracts were prepared from the MEL cell lines described in Fig. 1 which were cultured for 3 days in the absence (-) or presence (+) of HMBA. Oligodeoxynucleotide probes contained consensus sequences for the indicated transcription factors ( A, NF-E2/AP-1; B, AP-1; C, GATA-1; and D, SP-1); they were incubated with 5 µg of nuclear extract protein, and EMSAs were performed as described under Methods. Only the proteinDNA complexes are shown; specificity of binding was confirmed by competition with excess unlabeled oligodeoxynucleotide probe (not shown).



Nuclear extracts incubated with an NF-E2/AP-1 probe yielded two proteinDNA complexes (Fig. 2 A); both were eliminated by a 50-fold excess of unlabeled oligodeoxynucleotide, but only the slower migrating complex was eliminated by excess unlabeled oligodeoxynucleotide containing a mutation which abolishes NF-E2 binding but not AP-1 binding ( (13) and demonstrated later in Fig. 8). Moreover, the NF-E2DNA complex was completely abolished when nuclear extracts were preincubated with a p45-specific antibody and partially supershifted in the presence of a p18-specific antibody (8, 11) ; the AP-1DNA complex was partially supershifted in the presence of JunB- or c-Fos-specific antibodies.() Thus, the faster migrating proteinDNA complex contained NF-E2, while the slower migrating complex contained AP-1 (13) . In the absence of HMBA, the amount of NF-E2DNA complexes was similar in all cell lines, but when cells were treated with HMBA, the NF-E2DNA complexes increased 3.3-fold in parental cells and in Rwt transfectants and 1.6-fold in cells with severe protein kinase A deficiency (Fig. 2 A shows a typical experiment and summarizes 4 independent experiments; results with the moderately protein kinase A-deficient clones were intermediate: NF-E2DNA complexes increased 2.0- to 2.3-fold after HMBA treatment). As shown in Fig. 4B, the amount of NF-E2DNA complex increased linearly with the amount of nuclear extract. Increased NF-E2DNA complexes were first detectable 24-36 h after adding HMBA and were maximal at 3 days; increased DNA binding of NF-E2 in differentiating MEL cells has been described previously although the basis of this increase has not yet been examined (6, 7) .


Figure 8: DNA binding of NF-E2 and AP-1 in parental MEL cells treated with 8-Br-cAMP. A, parental MEL cells were grown in the presence of 4 m M HMBA for 3 days ( lanes 1-6), and half of the cells were treated with 1 m M 8-Br-cAMP for 1 h prior to harvesting ( lanes 2, 4, and 6). Nuclear extracts were prepared, and EMSAs were performed with radioactively labeled NF-E2/AP-1 probe as described under Methods. Lanes 3 and 4, addition of 50-fold excess unlabeled oligodeoxynucleotide containing a mutation in the NF-E2 binding site which leaves AP-1 binding intact; lanes 5 and 6, addition of 50-fold excess unlabeled wild type NF-E2/AP-1 oligodeoxynucleotide. B, parental MEL cells were cultured in serum-free media for 24 h prior to adding the following drugs: no addition ( lane 1), 1 m M 8-Br-cAMP ( lane 2), 4 m M HMBA ( lane 3), 1 m M 8-Br-cAMP plus 4 m M HMBA ( lane 4). Serum-free media were used to avoid breakdown of 8-Br-cAMP by phosphatases and phosphodiesterases present in serum during long-term incubation (61). Cells were harvested 16 h later; nuclear extracts were prepared, and EMSAs were performed with wild type NF-E2/AP-1 oligodeoxynucleotide probe as described under Methods. Equal protein loading was demonstrated using an oligodeoxynucleotide probe for SP-1.




Figure 4: Comparison of p45 expression and NF-E2DNA complex formation. A and C, nuclear extracts were prepared and analyzed by Western blotting using a p45-specific antibody and enhanced chemiluminescence detection as described under Methods. B and D, the same nuclear extracts were analyzed by EMSA as described under Methods; increasing amounts of nuclear extract protein were incubated with 2 n M oligodeoxynucleotide probe containing the NF-E2/AP-1 consensus sequence. A and B, parental cells grown in the absence or presence of HMBA; C and D, transfectants Rwt/C1 and Rmut/C3 grown in the presence of HMBA.



AP-1 could compete with NF-E2 for binding at the shared recognition site, and protein kinase A may influence the expression and activity of several members of the AP-1 family ( (13, 17, 42, 43) and Fig. 8). Since the AP-1DNA complex observed with the NF-E2/AP-1 oligodeoxynucleotide probe was faint and appeared slightly less in the protein kinase A-deficient cells (Fig. 2 A), we also examined AP-1 binding to a synthetic AP-1 recognition site (35) . AP-1DNA complexes were the same in parental and protein kinase A-deficient cells and decreased to a similar extent on adding HMBA to both cell types (Fig. 2 B); the results with parental MEL cells are similar to those reported previously (6, 7) .

GATA-1DNA complexes were similar in untreated parental and protein kinase A-deficient cells and did not change when either cell line was treated with HMBA (Fig. 2 C); again, similar results with parental MEL cells have been reported (44) . DNA binding of the ubiquitous transcription factor SP-1 (29) was the same in extracts from parental and protein kinase A-deficient cells with no change on adding HMBA (Fig. 2 D).

DNA Binding Affinity and Amount of NF-E2 in Parental and Protein Kinase A-deficient MEL Cells

Increased NF-E2DNA complexes during erythroid differentiation could be secondary to either increased DNA binding affinity of NF-E2 or an increased amount of NF-E2 dimers which bind to DNA. To distinguish between these two possibilities, we incubated constant amounts of nuclear extract protein from parental and protein kinase A-deficient cells grown in the absence or presence of HMBA with increasing amounts of labeled oligodeoxynucleotide probe and quantitated the amount of probe bound in the NF-E2DNA complex as described under Methods. When we plotted the amount of oligodeoxynucleotide bound versus oligodeoxynucleotide concentration, we found that DNA binding of NF-E2 approached saturation at oligodeoxynucleotide concentrations of >2.5 n M under all conditions in all cell lines tested (Fig. 3). In the absence of HMBA, cells with normal or reduced protein kinase A activity yielded similar results (Fig. 3, open circles represent untreated parental cells, but similar curves were obtained with transfectants with normal or reduced protein kinase A activity). HMBA induced a marked shift in the curve in cells with normal protein kinase A activity (Fig. 3, closed circles represent HMBA-treated parental cells with similar results for Rwt/C1), but only a small shift in cells with severely reduced protein kinase A activity (Fig. 3, closed triangles represent HMBA-treated Rmut/C3 with similar results for PKI/C2). If we assume that 1 mol of NF-E2 is bound to every mole of oligodeoxynucleotide in the NF-E2DNA complex at saturation, we can estimate the amount of NF-E2 present in 16 µg of nuclear extract protein at 1.3 ± 0.3 fmol in untreated parental cells, 3.6 ± 0.3 fmol in HMBA-treated parental cells, and 1.9 ± 0.2 fmol in HMBA-treated protein kinase A-deficient cells. Double-reciprocal plots of 1/oligodeoxynucleotide bound versus 1/oligodeoxynucleotide concentration (Lineweaver-Burk plots) were linear with correlation coefficients >0.99 (Fig. 3, inset). The slope of the plots derived from untreated and HMBA-treated parental cells showed a difference which was explained by the 3-fold difference in the amount of NF-E2 between these two conditions; thus, there did not appear to be a significant change in DNA binding affinity. Similarly, the slope of the plots derived from HMBA-treated parental and protein kinase A-deficient cells showed a difference which was explained by the 2-fold difference in the amount of NF-E2 indicating no significant change in DNA binding affinity. Thus, HMBA did not appear to increase DNA binding affinity of NF-E2 but rather, it appeared to increase the amount of NF-E2; in protein kinase A-deficient cells, this effect of HMBA was significantly attenuated.


Figure 3: Assessment of NF-E2 abundance and DNA binding affinity. Nuclear extracts were prepared from parental cells grown in the absence of HMBA ( open circles) or presence of HMBA ( closed circles) and from Rmut/C3 transfectants grown in the presence of HMBA ( closed triangles). EMSAs were performed after incubating 16 µg of protein with increasing amounts of NF-E2/AP-1 oligodeoxynucleotide probe. The amount of oligodeoxynucleotide bound in the NF-E2DNA complex was quantitated by laser densitometry scanning of autoradiographs within the linear range of exposure; the data are the mean ± S.D. of three independent experiments. Inset, double reciprocal plot of the data.



Since NF-E2 appears to be an obligate heterodimer (45, 46, 47) , an increased abundance of NF-E2 would be seen if: ( a) the concentration of both subunits increased; ( b) the concentration of the subunit normally limiting to dimer formation increased; or ( c) additional proteins were expressed which could bind to p45 or p18 and form a functional dimer similar to the p45p18 dimer.

We examined the amount of p45 protein present in nuclear extracts by Western blotting; on SDS-PAGE, p45 migrates as a doublet, possibly because of the presence of alternative translation initiation sites (8, 45) . We found no change in the amount of either of the two p45 species in parental cells treated for 3 days with HMBA (Fig. 4 A), although EMSAs performed with the same nuclear extracts showed a >3-fold difference in the amount of NF-E2DNA complexes between HMBA-treated and untreated parental cells (Fig. 4 B). We also examined nuclear extracts from HMBA-treated transfectants with normal or reduced protein kinase A activity (Rwt/C1 and Rmut/C3, respectively) and found that both cell types contained the same amount of p45 protein (both species) as parental cells (Fig. 4 C; in other experiments, extracts from parental cells and Rmut/C3 were compared directly). EMSAs performed with the same nuclear extracts showed a >2-fold difference in the amount of NF-E2DNA complexes between HMBA-treated transfectants with normal or reduced protein kinase A activity (Fig. 4 D). Two p45-related proteins were recently isolated on the basis of binding to a tandem NF-E2/AP-1 recognition site, but their apparent molecular sizes are significantly larger than 45 kDa, and proteinDNA complexes would be expected to migrate differently from the NF-E2DNA complex containing p45 (48, 49, 50) .

When we examined the amount of p18 in nuclear extracts of MEL cells, we found no detectable difference between parental and protein kinase A-deficient cells grown in the absence or presence of HMBA; however, due to the low affinity of available p18 antibodies, the level of p18 in MEL cells was at the lower limit of detection, and we cannot exclude small differences in p18 protein expression (Fig. 5 A, lanes 1 and 2 represent parental cells grown in the absence or presence of HMBA and lane 3 represents Rmut/C3 grown in the presence of HMBA; for comparison, COS cells transfected with a p18 expression vector ( lane 4) and untransfected COS cells ( lane 5) are shown).

We examined the expression of p18 and p45 mRNA by Northern blot analysis and found no difference between parental and protein kinase A-deficient cells, and no significant change on exposure to HMBA when the signals were normalized to those obtained with a probe for the structural protein CHOA (Fig. 5 B). p18 is closely related to the avian maf family of small basic leucine zipper proteins, and murine p45 can dimerize with several of the avian Maf proteins in vitro (11, 47) . Immunoprecipitation of NF-E2 from differentiating S-labeled MEL cells demonstrated p18 as the only protein specifically associated with p45; furthermore, p18 co-purified stoichiometrically with p45 during DNA-affinity chromatography (10, 11) . However, other proteins associated with p45 in vivo could have escaped detection by these methods. Under conditions of reduced stringency, the full-length p18 cDNA probe hybridized only to a single 3.2-kilobase band on Northern blots of MEL cell RNA. Thus, we presently have no evidence that other maf-related proteins are expressed in MEL cells.


Figure 5: Western blot analysis of p18 and Northern blot analysis of p45 and p18 mRNA expression. A, parental MEL cells ( lanes 1 and 2) and Rmut/C3 ( lane 3) were cultured for 3 days in the absence ( lane 1) or presence ( lanes 2 and 3) of 4 m M HMBA. Nuclear extracts were prepared and 160 µg of protein per lane were analyzed by SDS-PAGE/Western blotting using a p18-specific antibody and enhanced chemiluminescence detection as described under Methods. Nuclear extracts from COS cells transfected with a p18 expression vector ( lane 4) or from untransfected COS cells ( lane 5) are shown for comparison (20 µg of protein per lane). B, parental cells ( lanes 1 and 2) and protein kinase A-deficient cells (Rmut/C3, lanes 3 and 4) were cultured for 3 days in the absence ( lanes 1 and 3) or presence ( lanes 2 and 4) of HMBA; total RNA was extracted, and duplicate samples were electrophoresed on denaturing agarose gels, transferred to nylon membranes, and hybridized to p45 or p18 cDNA probes as described under Methods. Equal loading and transfer of RNA was assessed by rehybridizing the blots with a probe for the structural protein CHOA.



In Vitro Phosphorylation of p45 and p18 by Protein Kinase A and Effect of Phosphorylation on NF-E2DNA Complex Formation

The amount of NF-E2 dimers capable of forming NF-E2DNA complexes could be regulated by mechanisms other than changes in NF-E2 subunit concentrations: post-translational modification of either subunit could alter dimer formation or other proteins (which might be regulated by post-translational modification) could interact with one or both of the NF-E2 subunits and alter dimer formation or NF-E2DNA complex formation, respectively. Since the amino acid sequence of p45 contains the protein kinase A consensus sequence RRRS at residues 167-170 (8, 51) , we tested whether the C-subunit of protein kinase A phosphorylates p45 in vitro; we immunoprecipitated p45 from nuclear extracts of parental or protein kinase A-deficient MEL cells and incubated the immunoprecipitates with [-PO]ATP and purified C-subunit of protein kinase A (Fig. 6 A). In immunoprecipitates with p45-specific antibody ( lanes 3-8), but not with preimmune serum ( lane 2), we observed POincorporation into a 45-kDa doublet; no signal was observed in reactions lacking the C-subunit of protein kinase A ( lane 1), and the 38-kDa autophosphorylated C-subunit of protein kinase A was clearly separated from p45 ( lane 9). In vitro phosphorylation of a substrate by a purified protein kinase does not necessarily indicate that the enzyme phosphorylates the protein in vivo; however, for the following reasons it appears that protein kinase A may phosphorylate p45 in vivo. First, we observed consistently higher POincorporation into the p45 doublet in immunoprecipitates from protein kinase A-deficient cells ( lanes 7 and 8) compared to parental cells ( lanes 3 and 4) even though the nuclear extracts from both cell types contained the same amount of p45 by Western blotting. Second, in vitro phosphorylation of p45 by protein kinase A was reduced in immunoprecipitates from parental cells treated with 1 m M 8-Br-cAMP to stimulate endogenous protein kinase A activity prior to harvesting (compare lanes 5 and 6 to lanes 3 and 4). Third, when nuclear extracts from 8-Br-cAMP-treated parental cells were incubated with alkaline phosphatase prior to immunoprecipitation, POincorporation into p45 by protein kinase A in vitro was restored (not shown); together, these data suggest that p45 from cAMP-treated parental cells compared to protein kinase A-deficient cells was more highly phosphorylated in vivo at the protein kinase A recognition site(s) decreasing POincorporation in vitro.


Figure 6:In vitro phosphorylation of p45 and p18 by the C-subunit of protein kinase A. A, nuclear extracts were prepared from HMBA-treated parental ( lanes 1-6) or protein kinase A-deficient ( lanes 7 and 8) MEL cells; 150 µg of extract protein were subjected to immunoprecipitation using a p45-specific antibody ( lanes 1 and 3-8) or preimmune serum ( lane 2). The immunoprecipitates shown in lanes 5 and 6 were from parental cells treated with 1 m M 8-Br-cAMP to stimulate endogenous protein kinase A activity for 1 h prior to harvest. Immunoprecipitates were incubated with [-PO]ATP in the absence ( lane 1) or presence ( lanes 2-8) of the C-subunit of protein kinase A and analyzed by SDS-PAGE autoradiography as described under Methods. Lane 9, C-subunit of protein kinase A incubated with [-PO]ATP. B, nuclear extracts were prepared from COS cells transfected with equimolar amounts of p18 and p45 expression vector ( lanes 1 and 3) or from untransfected COS cells ( lanes 2 and 4); 80 µg of extract protein were subjected to immunoprecipitation using a p18-specific antibody ( lanes 1 and 2) or a p45-specific antibody ( lanes 3 and 4), and immunoprecipitates were incubated with [-PO]ATP and the C-subunit of protein kinase A as described above. Proteins were resolved by SDS-PAGE using either 15% ( lanes 1 and 2) or 10% acrylamide ( lanes 3 and 4). The asterisk indicates migration of the autophosphorylated C-subunit of protein kinase A.



Within its basic domain presumed to mediate DNA binding, p18 contains the amino acid sequence RRRT which is a potential protein kinase A consensus sequence although threonine residues in protein kinase A substrates appear to be much poorer phosphate acceptors compared to serine residues (11, 51) . Since the p18 signal on Western blots of MEL cells was low, we transfected COS cells with equimolar amounts of p18 and p45 expression vectors to immunoprecipitate or co-immunoprecipitate p18 using a p18- or p45-specific antibody, respectively (8, 11) . The immunoprecipitates were then incubated with [-PO]ATP and purified C-subunit of protein kinase A as described above. We detected POincorporation into a 18-kDa protein in transfected COS cells with little signal in untransfected COS cells (which contain small amounts of endogenous p18 (11) ). However, POincorporation into p18 was very low compared to p45 phosphorylated under the same conditions (Fig. 6 B, lanes 1 and 3, represent immunoprecipitates from transfected COS cells obtained with p18- and p45-specific antibodies, respectively). Low POincorporation into p18 could indicate that p18 is a poor protein kinase A substrate; however, immunoprecipitation of p18 may not be quantitative. We did not reliably detect p18 phosphorylation by protein kinase A in immunoprecipitates from parental or protein kinase A-deficient MEL cells.

To examine whether p45 (or p18) phosphorylation might alter NF-E2DNA complex formation, we incubated nuclear extracts from parental or protein kinase A-deficient cells grown in the absence or presence of HMBA with purified C-subunit of protein kinase A and ATP; under no condition did we observe an effect of the C-subunit of protein kinase A on the amount of NF-E2DNA complexes (Fig. 7). Similarly, incubation of nuclear extracts with alkaline phosphatase immobilized to agarose beads did not significantly change DNA binding of NF-E2 (data not shown). Thus, protein kinase A phosphorylation of p45 or p18 or of co-factor(s) present in nuclear extracts did not appear to alter NF-E2DNA complex formation.


Figure 7: NF-E2DNA complexes in nuclear extracts treated with C-subunit of protein kinase A. Parental cells ( lanes 1-4) or protein kinase A-deficient cells ( lanes 5-8) were grown for 3 days in the absence ( lanes 1, 2, 5, and 6) or presence of HMBA ( lanes 3, 4, 7, and 8). Nuclear extracts were incubated for 10 min at 30 °C in the presence or absence of purified C-subunit of protein kinase A (C-subunit) as indicated; EMSAs were performed with NF-E2/AP-1 oligodeoxynucleotide probe as described under Methods.



DNA Binding of NF-E2 in Mixing Experiments

A possible reason for fewer NF-E2DNA complexes in the protein kinase A-deficient cells could be the presence of a factor that inhibited complex formation or lack of a factor required for complex formation. To test this possibility, we mixed nuclear extracts from HMBA-treated parental and protein kinase A-deficient cells prior to performing EMSAs. We found that the amount of oligodeoxynucleotide bound in the NF-E2DNA complex was as predicted from the sum of both extracts, suggesting that there was no excess of an inhibitor in the protein kinase A-deficient cells and no excess of a positive regulatory factor in the parental cells. However, important in vivo interactions of NF-E2 with positive or negative regulators may not be detectable under these in vitro conditions.

Effect of Activating Protein Kinase A in Vivo on NF-E2DNA Complex Formation

Certain effects of protein kinase A activation in vivo, e.g. transcriptional or post-transcriptional changes in the expression of proteins regulating NF-E2DNA complex formation, would not be observed when nuclear extracts were incubated with purified enzyme in vitro. To examine whether stimulating protein kinase A activity in vivo may alter NF-E2DNA complex formation in vitro, we treated parental MEL cells with 1 m M 8-Br-cAMP for variable periods of time. When 8-Br-cAMP was added for 1-3 h prior to harvest, NF-E2DNA complexes were unaffected whereas DNA binding of AP-1 increased significantly due to the induction of c-Fos and JunB by 8-Br-cAMP(Fig. 8 A, lanes 1 and 2 show data at 1 h for parental cells pretreated with HMBA for 3 days; similar results were obtained when untreated parental cells were incubated with 8-Br-cAMP or 8-Br-cAMP plus HMBA for 1 h). However, the amount of NF-E2DNA complexes did increase significantly after treating parental cells with 8-Br-cAMP for 12 h and was maximal at 16 h (Fig. 8 B shows data at 16 h in the absence of HMBA ( lanes 1 and 2) and in the presence of HMBA ( lanes 3 and 4)). An increase in NF-E2DNA complexes after prolonged exposure to 8-Br-cAMP was also seen when the HMBA effect on NF-E2 was maximal, i.e. when cells were cultured for 3 days in the presence of HMBA; this increase in NF-E2 DNA binding was observed at saturating oligodeoxynucleotide concentrations without detectable change in DNA binding affinity. The amount of NF-E2DNA complexes was not altered by competition between AP-1 and NF-E2 for the radioactively labeled oligodeoxynucleotide probe, because adding 50-fold excess of unlabeled oligodeoxynucleotide probe containing a mutation in the NF-E2 binding site which leaves AP-1 binding intact (13) eliminated >90% of AP-1DNA complexes without significant change in the amount of NF-E2DNA complexes (Fig. 8 A, lanes 3 and 4; competition with 50-fold excess wild type oligodeoxynucleotide is shown in lanes 5 and 6). Addition of 8-Br-cAMP had no effect on p45 or p18 mRNA levels or p45 protein levels (data not shown).


DISCUSSION

We previously found that protein kinase A-deficient MEL cells are impaired in their ability to transcriptionally activate erythroid-specific genes including the -globin and porphobilinogen deaminase gene (5) . Other genes were normally regulated in these cells, leading us to suggest that protein kinase A may be involved in the regulation of genes with erythroid-specific promoters/enhancers which share common cis-acting regulatory elements. In agreement with this hypothesis, we now have found in transient transfection assays of CAT reporter constructs that the HMBA-inducible enhancer activity of the human -globin LCR was significantly reduced in protein kinase A-deficient MEL cells whereas the activity of the human -globin promoter by itself and the activity of two non-erythroid promoters was normal in these cells.

The human -globin promoter contains multiple binding sites for SP-1 but lacks binding sites for GATA-1 or NF-E2; it is poorly inducible during HMBA-induced differentiation and requires the -globin LCR for high levels of globin gene expression (25, 52) . The -globin LCR is structurally similar to the -globin LCR HSS-2 (15, 53) and acts as a classical tissue-specific enhancer; its major regulatory activity maps to a 350-base pair DNA fragment containing two NF-E2/AP-1 binding sites and several GATA-1 motifs and CACCC boxes which are bound by erythroid-specific proteins in vivo (9, 15, 41) . Deletion analysis suggests that the combination of two NF-E2/AP-1 motifs, one GATA-1 motif, and one CACCC-box are necessary for full enhancer activity (15) . Enhancer activity is diminished by about 70% when site-directed mutagenesis abolishes one of the NF-E2 binding sites leaving AP-1 binding intact; therefore, NF-E2 and not AP-1 appears to be the functional activator (15) . NF-E2, GATA-1, and CACCC-binding proteins appear to cooperate in activating both the - and -globin LCRs during erythroid differentiation (9, 14, 53, 54, 55, 56, 57) ; the finding that HMBA-induced pCAT/LCR expression and endogenous -globin transcription rates were less in protein kinase A-deficient cells compared to parental cells suggests that protein kinase A may directly or indirectly regulate the activity of one or several of these transcription factors (5) .

The abundance of NF-E2DNA complexes increased during HMBA-induced differentiation of parental MEL cells; the finding of increased NF-E2DNA complexes after prolonged activation of protein kinase A in HMBA-treated parental cells and decreased complexes in HMBA-treated protein kinase A-deficient cells suggests that protein kinase A is necessary for increased NF-E2DNA complex formation during HMBA-induced differentiation. In the absence of HMBA, parental and protein kinase A-deficient cells exhibited similar basal levels of NF-E2DNA complexes; these data correlate with the finding of similar basal CAT expression from pCAT/LCR and similar basal transcription rates of endogenous erythroid-specific genes (5) . The protein kinase A-deficient cells may contain sufficient residual protein kinase A activity to express normal basal but not HMBA-induced levels of NF-E2.

p45 and p18 mRNA and protein levels were not significantly changed under conditions where we observed 3-fold changes in the amount of NF-E2DNA complexes (although we cannot exclude small changes in p18 protein levels). Since incubation of nuclear extracts with purified C-subunit of protein kinase A and short-time treatment of intact cells with 8-Br-cAMP did not increase NF-E2DNA complexes, it appears that phosphorylation of a pre-existing protein(s) is not sufficient to explain the difference between HMBA-treated parental and protein kinase A-deficient cells (phosphorylation of protein kinase A substrates is observed within minutes of adding 8-Br-cAMP to intact cells, and nuclear translocation of the C-subunit of protein kinase A occurs within less than 1 h (39, 58, 59) ). Increased NF-E2DNA complexes were observed after prolonged activation of protein kinase A (>12 h), suggesting that protein kinase A may regulate NF-E2DNA complex formation indirectly, i.e. through changes in the expression (and/or stability or nuclear localization) of a ``regulatory factor(s).'' NF-E2 dimer formation could be regulated through the expression of other leucine zipper proteins interacting with p45 or p18 and competing with p45p18 dimer formation; for example, different maf-related proteins can form heterodimers with each other and with proteins of the Fos and Jun families resulting in dimers with variable DNA binding properties (47, 60) . Alternatively, NF-E2DNA complex formation could be regulated by an essential co-factor binding to p45 and/or p18. In either case, p45 (or p18) phosphorylation could contribute to the regulation of NF-E2DNA complex formation by altering the interaction of p45 (and/or p18) with this regulatory factor.

One might speculate that an increase in the intracellular NF-E2 concentration during erythroid differentiation could contribute to the transcriptional activation of erythroid promoters/enhancers containing NF-E2/AP-1 recognition sites. However, the increase in NF-E2DNA complexes in parental MEL cells required >24 h of HMBA exposure, whereas increased globin gene transcription is detectable as early as 12 h after addition of HMBA, indicating that other factors are necessary during the early phase of differentiation (1, 2) . Although we failed to demonstrate a direct effect of protein kinase A phosphorylation on NF-E2 DNA binding, p45 (and/or p18) phosphorylation by protein kinase A could be important for the transactivation properties of NF-E2 including the interaction of NF-E2 with other transcription factors. Since reporter constructs containing single or multimerized binding sites for either NF-E2/AP-1 or GATA-1 with a minimal promoter are not transcriptionally active in MEL cells, it is clear that no single transcription factor is responsible for the activation of erythroid-specific genes during MEL cell differentiation (14, 22) . GATA-1 is phosphorylated by protein kinase A in vitro, and increased phosphorylation of GATA-1 on a serine located within a protein kinase A recognition site is observed during MeSO-induced differentiation of MEL cells; however, phosphorylation of GATA-1 at this site, or on other sites, does not influence DNA binding or transcriptional activation by GATA-1 (44) .

In conclusion, protein kinase A appears to be necessary for increased NF-E2DNA complex formation during HMBA-induced differentiation, and protein kinase A could influence transcriptional activation of erythroid genes through this mechanism. Since activation of erythroid genes requires the cooperation of several transcription factors, it will be important to study whether direct phosphorylation of NF-E2 and/or GATA-1 by protein kinase A affects the way these two transcription factors cooperate with each other and/or with other factors in transactivating erythroid promoters/enhancers.

  
Table: NF-E2DNA complexes in MEL cells with normal or reduced protein kinase A activity

Nuclear extracts were prepared from MEL cells with normal or reduced protein kinase A activity, and EMSAs were performed as described in Fig. 2 with 1 n M oligodeoxynucleotide containing the NF-E2 consensus sequence. The amount of oligodeoxynucleotide found in the NF-E2DNA complex was quantitated by laser densitometry scanning. The amount of oligodeoxynucleotide bound by nuclear extracts from parental cells grown in the absence of HMBA was assigned a value of 1.0; data were normalized to this value and expressed as relative absorption units. Data are the mean ± S.D. of at least three independent experiments.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by a Josephine Especial Leukemia Research Fellowship.

Assistant Investigator of the Howard Hughes Medical Institute.

**
Supported by United States Public Health Service Grant K08 CA01548. To whom correspondence and reprint requests should be addressed. Tel.: 619-534-8805; Fax: 619-534-1421.

The abbreviations used are: MEL, murine erythroleukemia; HMBA, hexamethylene bisacetamide; protein kinase A, cAMP-dependent protein kinase; C-subunit, catalytic subunit of protein kinase A; Rwt and Rmut, wild type and mutant regulatory subunits of protein kinase A, respectively; 8-Br-cAMP, 8-bromo-cAMP; PKI, protein kinase inhibitor; LCR, locus control region; HSS, DNase hypersensitive site; IMDM, Iscove's modified Dulbecco's medium; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.

M. Suhasini and R. B. Pilz, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. S. Taylor for providing the purified C-subunit of protein kinase A, D. Brenner for GATA-1 and NF-E2 oligodeoxynucleotides, N. J. Proudfoot for the pCAT and pCAT/LCR constructs, and S. Subramani for pRSV-luc. We are grateful to Drs. G. Boss, V. Kharitonov, and S. H. Orkin for helpful discussions and to S. Shanks and S. Wancewicz for excellent preparation of the manuscript.


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