From the
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
Erythroid-specific genes are transcriptionally activated when
murine erythroleukemia (MEL)
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
Materials The human
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
COS cells
were transfected with equimolar amounts of p45 and p18 expression
vectors as described previously
(11) .
The following double-stranded oligodeoxynucleotide
probes were 5`-end-labeled with
[
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 protein
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
For alkaline
phosphatase treatment, nuclear extracts were prepared omitting
When p
Since the enhancer function of
the
GATA-1
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-E2
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 R
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
To examine whether p45 (or p18)
phosphorylation might alter NF-E2
We previously found that protein kinase A-deficient MEL cells
are impaired in their ability to transcriptionally activate
erythroid-specific genes including the
The human
The abundance of
NF-E2
p45 and p18 mRNA and protein levels were not significantly changed
under conditions where we observed
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-E2
In conclusion, protein kinase A appears to be
necessary for increased NF-E2
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-E2
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 p
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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-E2
DNA 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-E2
DNA complexes, suggesting
that protein kinase A regulates NF-E2
DNA complex formation
indirectly, e.g. by altering expression of a regulatory
factor(s). Thus, protein kinase A appears to be necessary for increased
NF-E2
DNA complex formation during differentiation of MEL cells
and may influence erythroid-specific gene expression through this
mechanism.
(
)
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.
-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-E2
DNA complexes significantly more than in protein kinase
A-deficient cells, and prolonged activation of protein kinase A
increased NF-E2
DNA complexes in the presence or absence of HMBA.
These changes in NF-E2
DNA 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-E2
DNA complex
formation indirectly, e.g. by altering the expression of an
accessory protein.
-globin promoter and promoter/enhancer chloramphenicol
acetyltransferase (CAT) constructs (p
CAT and p
CAT/LCR,
respectively) were generous gifts of N. J. Proudfoot
(25) ; the
chicken
-actin promoter CAT construct (p
actCAT) 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) .
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
(R
mut) of protein kinase A or the enzyme's specific
peptide inhibitor are protein kinase A-deficient, while cells
overexpressing the wild type R
subunit (R
wt) 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 R
mut/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
R
mut/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 R
wt transfectants)
than in R
mut or PKI transfectants
(4) . Two other
protein kinase A-deficient cell lines (R
mut/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 p
CAT,
p
CAT/LCR, or p
actCAT, 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) .
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 Na
VO
added to the extract
buffer
(33) ; protein was quantitated by the Bradford method
(34) . The yield of nuclear extract protein per 10
cells varied by <20% between different cell lines and culture
conditions.
-
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 (10
cpm) 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. Protein
DNA 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) .
DNA complexes and in the free
probe were quantitated. The sum of both values increased as expected
with increased amounts of probe indicating that the protein
DNA
complexes were stable during electrophoresis
(37) .
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
10
cells 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-E2
DNA 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.
-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 MgCl
in 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) .
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
p
CAT/LCR is identical with p
CAT 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) .
CAT was transiently transfected into MEL cells, CAT
expression was the same in cells with severely reduced protein kinase A
activity (PKI/C2 and R
mut/C3) as in cells with normal
protein kinase A activity (parental cells, R
wt/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 R
wt/C1
transfectant) and protein kinase A-deficient MEL cells (PKI/C2 and
R
mut/C3 transfectants) were co-transfected with
p
CAT/LCR ( open and closed bars) or p
CAT
( 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
p
act-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).
-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
p
CAT/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 protein
DNA 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-E2
DNA 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-1
DNA complex was partially
supershifted in the presence of JunB- or c-Fos-specific
antibodies.
(
)
Thus, the faster migrating
protein
DNA complex contained NF-E2, while the slower migrating
complex contained AP-1
(13) . In the absence of HMBA, the amount
of NF-E2
DNA complexes was similar in all cell lines, but when
cells were treated with HMBA, the NF-E2
DNA complexes increased
3.3-fold in parental cells and in R
wt 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-E2
DNA complexes increased
2.0- to 2.3-fold after HMBA treatment). As shown in
Fig. 4B, the amount of NF-E2
DNA complex increased
linearly with the amount of nuclear extract. Increased NF-E2
DNA
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 R
wt/C1 and R
mut/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-1
DNA 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) .
DNA 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-E2
DNA 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
R
wt/C1), but only a small shift in cells with severely
reduced protein kinase A activity (Fig. 3, closed triangles represent HMBA-treated R
mut/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-E2
DNA 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-E2
DNA
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.
DNA
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 (R
wt/C1 and R
mut/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 R
mut/C3 were
compared directly). EMSAs performed with the same nuclear extracts
showed a >2-fold difference in the amount of NF-E2
DNA 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 protein
DNA complexes would be expected to
migrate differently from the NF-E2
DNA complex containing p45
(48, 49, 50) .
mut/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).
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 (R
mut/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-E2
The amount of NF-E2 dimers capable of forming
NF-E2DNA Complex
Formation
DNA 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-E2
DNA 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
PO
incorporation 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
PO
incorporation
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,
PO
incorporation 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
PO
incorporation 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
PO
incorporation 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,
PO
incorporation 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
PO
incorporation 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.
DNA 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-E2
DNA 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-E2
DNA
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-E2
DNA 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-E2
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
DNA 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-E2
DNA 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-E2
DNA 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-E2
DNA 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-E2
DNA
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-E2
DNA 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-1
DNA
complexes without significant change in the amount of NF-E2
DNA
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).
-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.
-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 p
CAT/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) .
DNA complexes increased during HMBA-induced differentiation
of parental MEL cells; the finding of increased NF-E2
DNA 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-E2
DNA complex formation during HMBA-induced
differentiation. In the absence of HMBA, parental and protein kinase
A-deficient cells exhibited similar basal levels of NF-E2
DNA
complexes; these data correlate with the finding of similar basal CAT
expression from p
CAT/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.
3-fold changes in the amount of
NF-E2
DNA 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-E2
DNA 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-E2
DNA complexes were observed after prolonged activation of
protein kinase A (>12 h), suggesting that protein kinase A may
regulate NF-E2
DNA 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
p45
p18 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-E2
DNA 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-E2
DNA complex formation by altering the interaction of p45
(and/or p18) with this regulatory factor.
DNA 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
Me
SO-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) .
DNA 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
DNA 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.
wt and R
mut, 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.
CAT and
p
CAT/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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.