|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3193-3201
Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic
Adenosine Monophosphate-Dependent Protein Kinase
By
Darren Casteel,
Modem Suhasini,
Tanima Gudi,
Reza Naima, and
Renate B. Pilz
From the Department of Medicine and Cancer Center, University of
California, San Diego, CA.
 |
ABSTRACT |
Activation of cyclic adenosine monophosphate (cAMP)-dependent
protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase-deficient murine
erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by
directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser169; Ala substitution for
Ser169 resulted in a protein that was no longer
phosphorylated by A-kinase in vitro or in vivo. The mutant protein
formed NF-E2 complexes that bound to DNA with the same affinity as
wild-type p45 and functioned normally to restore  -globin gene
expression in a p45-deficient MEL cell line. Transactivation properties
of the (Ser169  Ala) mutant p45 were also
indistinguishable from wild-type p45 when Gal4-p45 fusion constructs
were tested with a Gal4-dependent reporter gene. Transactivation of the
reporter by both mutant and wild-type p45 was significantly enhanced
when A-kinase was activated by membrane-permeable cAMP analogs or when
cells were cotransfected with the catalytic subunit of A-kinase.
Stimulation of p45 transactivation by A-kinase required only the
N-terminal transactivation domain of p45, suggesting that A-kinase
regulates the interaction of p45 with downstream effectors.
| |
INTRODUCTION |
NF-E2 IS A BASIC LEUCINE zipper (bzip)
transcription factor consisting of a hematopoietic cell-specific
subunit, p45, and a ubiquitously expressed subunit that is one of the
small Maf proteins (p18/MafK or MafG).1-4 NF-E2 binds to
the consensus sequence TGCTGA(G/C)TCA(T/C) found in several
erythroid-specific promoters and in the  - and -globin locus
control regions (LCRs).1,5,6 The overall stimulatory
activity of the LCRs depends on the presence of intact NF-E2 binding
sites; an essential role for NF-E2 in remodeling of chromatin
structure, disruption of nucleosomes, and transcriptional activation of
globin genes has been recently demonstrated.6-9 Variant
murine erythroleukemia (MEL) cells lacking p45 expression due to
proviral integration in one allele and loss of the other allele show a
marked reduction of globin gene expression, which is partially restored
by reintroducing p45.9,10 Transgenic mice lacking p45 show
surprisingly mild changes in erythropoiesis with normal globin
switching, suggesting the presence of factors compensating for the lack
of p45 in erythroid precursors; however, thrombopoiesis is severely
impaired in these animals.11,12 Based on their ability to
bind to the NF-E2 recognition sequence, several p45-related proteins
have been identified, including Nrf1/LCR-F1 and Nrf2.13-15
These proteins show significant homology with p45 in the bzip and
surrounding region, but are otherwise distinct; they are strong
transcriptional activators expressed in a wide variety of tissues,
including erythroid cells.13-15 However, despite their
ability to bind to NF-E2 recognition sites, LCR-F1 and Nrf2 cannot
functionally replace p45 in MEL cells.10,16
In MEL cells, induction of erythroid differentiation is associated with
transcriptional activation of erythroid-specific genes and increased
DNA-binding activity of NF-E2.17-19 We found that expression of erythroid-specific genes, activity of the -globin LCR
and NF-E2/DNA complex formation are impaired in cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase)-deficient MEL
cells.18 Prolonged activation of A-kinase in normal MEL cells increases the amount of NF-E2/DNA complexes without significantly changing the expression of p45 or p18.18 The p45 NF-E2
subunit is efficiently phosphorylated by A-kinase in
vitro18; thus, A-kinase could regulate NF-E2 through direct
phosphorylation and/or through the regulation of other factors.
The present work was undertaken to more clearly define the role of
A-kinase in regulating p45 NF-E2. We generated a mutant form of p45
that was no longer phosphorylated by A-kinase; this mutant was
indistinguishable from wild-type p45 with respect to DNA binding and
transactivation properties. We demonstrated that A-kinase strongly
enhanced the transactivation potential of wild-type or mutant p45, an
effect that only required the N-terminal transactivation domain of p45. The transcriptional coactivator CREB-binding protein (CBP) and the
TATA-binding protein-associated factor TAFII130 have been recently shown to bind to the N-terminus of p458,20;
however, we found no significant effect of CBP on p45 transactivation. Our results suggest that A-kinase stimulates p45 transactivation by
changing the interaction(s) of the p45 transactivation domain with
other transcriptional regulatory protein(s). The regulation of p45 by
A-kinase described in this work provides a possible mechanism whereby
cAMP can potentiate erythropoietin-induced erythroid differentiation.21-23
| |
MATERIALS AND METHODS |
Plasmid constructs and site-directed mutagenesis.
The complete murine p45 cDNA clone in pBluescriptKS was provided by N. Andrews.1 To replace Ser169 with Ala, we
performed site-directed mutagenesis using polymerase chain reaction
(PCR) and the principle of unique site elimination.24 The
mutagenesis primer was 5 -GCGGAGGGCCGAGTACGTCGACATGTAC-3 and a second
silent mutation was included to introduce a new Sal I site; the
selection primer was 5-GCCGCTCTAGAACGCGTGGATCCCCC-3, changing the
unique Spe I site in the pBluescriptKS polylinker to
Mlu I. The mutant p45 plasmid was sequenced completely to
confirm the presence of the desired mutation and to exclude the
introduction of other unwanted mutations. To produce pMT2-p45(wt) and
(mut), the wild-type or mutant p45 cDNAs were cloned downstream of the adenovirus major late promoter into the EcoRI site of the
vector pMT2.25 For stable transfection of CB3 cells, the
wild-type and mutant p45 cDNAs were cloned downstream of the chicken
-actin promoter of pRC/-Act using HindIII and Xba
I; pRC/-Act contains a neor transcription unit to confer
G418 resistance.26 To test the transactivation properties
of p45 independently of its DNA binding properties, the N-terminus of
wild-type and mutant p45 was fused in-frame to the DNA-binding domain
of the yeast transcription factor Gal4 in the vector pSG424 (provided
by M. Ptashne27). The resulting vectors pGal4/p45(wt) and
pGal4/p45(mut) were sequenced across the Gal4-p45 fusion; Western blots
developed with a p45-specific antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) detected full-length p45-Gal4 fusion proteins expressed in
baby hamster kidney (BHK) cells (data not shown). Stepwise truncation
of p45 from the C-terminus was performed by digesting pGal4/p45(wt)
with Xba I plus Sal I, Stu I, or Sac I;
the vector containing the remaining portion of p45 was blunted and
religated, generating pGal4/p45 ( 269), pGal4/p45(112), and
pGal4/p45(83) (see Fig 5; the numbers in parentheses
designate the number of N-terminal amino acids of p45 present in the
construct). Digestion of pGal4/p45(mut) with Xba I plus
Sal I removed two C-terminal fragments because of the new
Sal I site at Ser169 introduced during
site-directed mutagenesis; religation of the vector containing the
remaining portion of p45 generated pGal4/p45(171) (see Fig
5). Partial digestion of Gal4/p45(wt) with Pst I
was used to remove all but the first 108 nucleotides of the p45 coding sequence; religation of the 3.4-kb partial digestion product generated pGal4/p45(36). Truncation of p45 in these vectors was confirmed by
restriction analysis and partial DNA sequence analysis.
View larger version (18K):
[in this window]
[in a new window]
| Fig 5.
Transactivation properties of Gal4-fusion constructs
containing truncated versions of p45: effect of A-kinase. The structure of p451 and the Gal4-fusion constructs containing variable
amounts of N-terminal p45 sequences fused to the DNA binding domain of
Gal4 are shown in (A); results of cotransfection experiments using these constructs in BHK cells are shown in (B). The indicated transactivator plasmid (5 ng) was cotransfected with the reporter pGAL4-Luc (100 ng), the control vector pRSV-Gal (50 ng), and either
an expression vector for the C-subunit of A-kinase (pCMV-C, 50 ng,
open bars) or empty vector (pRC/CMV, 50 ng, filled bars). Luciferase
activity was normalized to -galactosidase activity in each sample;
the luciferase/-galactosidase ratio obtained with the parent vector
pSG424, which is lacking p45 sequences, was assigned a value of 1. Results represent the mean ± SD of three independent experiments.
|
|
The p18 expression vector pMT2-p18 was from N. Andrews, the expression
vector for the catalytic (C)-subunit of A-kinase, pCMV-C, was from
S. Taylor, and the reporter plasmid pGAL4-Luc and the pGal4/Fos fusion
vector were from M. Karin.3,28,29 An expression vector for
full-length CBP was from M. Rosenfeld30; an expression
vector for the p45-binding domain of CBP (amino acid residues 451 to
682) was constructed by PCR using previously described
primers.20
Cell culture and transfections.
MEL cells (strain 745 A), A-kinase-deficient MEL cells,31
and the p45-deficient MEL variant CB3 (provided by Y. Ben-David9) were cultured as previously
described.18,31 To generate stable transfectants, 5 × 106 CB3 cells were transfected using 30 µg of Lipofectin
(Life Technologies, Grand Island, NY) liposomes and 10 µg of pRC/Act-p45(wt) and (mut) as described.31 After
2 weeks of growth in 1 mg/mL G418, individual colonies were expanded
and tested for p45 expression by Western blot analysis and
electrophoretic mobility shift assay (EMSA) as described later. For
transient transfections of MEL cells, 1 × 106 cells were
incubated with 6 µg of DMRIE liposomes (Life Technologies), 300 ng of
the pGAL4-Luc reporter, 300 ng of pRSV-Gal (internal control), and
100 to 400 ng of the indicated pGal4 transactivator; control cultures
received 100 to 400 ng of the pSG424 parent plasmid or no
transactivator plasmid and the total amount of DNA was kept constant at
1.2 µg by adding carrier DNA. BHK cells were cultured and transfected
using Lipofectamine (Life Technologies) as
described.32 In some experiments, cells were treated with 1 mmol/L 8-Br-cAMP for 8 hours before harvesting or cells were
cotransfected with 200 ng of the A-kinase C-subunit expression vector
pCMV-C or pRC/CMV (empty vector). Cells were harvested 48 hours
after transfection and luciferase and -galactosidase activities were
measured using luminometer-based assays as described.32
p45 phosphorylation studies.
BHK cells were transfected with 2 µg of pMT2-p45(wt) or
pMT2-p45(mut). For in vitro phosphorylation studies, approximately 106 cells were harvested at 48 hours after transfection and
cell lysates were subjected to immunoprecipitation using either
p45-specific antibody or control rabbit serum as
described.2,18 Washed immunoprecipitates were incubated
with 50 µCi[ -32PO4]adenosine
triphosphate (ATP) (3,000 Ci/mmol) and 100 ng of purified C-subunit of
A-kinase (provided by S. Taylor). For in vivo phosphorylation studies
in BHK cells, approximately 106 transfected cells were
transferred 32 hours posttransfection to phosphate-free medium
containing 10% dialyzed serum and 100 µCi/mL of
32PO4 and were harvested 16 hours later; 1 mmol/L 8-Br-cAMP was added to some cultures during the last hour of
incubation. For in vivo phosphorylation studies in wild-type and
A-kinase-deficient MEL cells (clone
RImut/C331), the cells were incubated in
phosphate-free medium containing 10% dialyzed serum and 300 µCi/mL
of 32PO4 for 16 hours at a density of 2 × 106 cells/mL in 10 mL. Immunoprecipitation was performed
with either p45-specific antibody or control rabbit serum as
described.2,18 All immunoprecipitates were resolved by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and autoradiography.18
For phosphoamino acid analysis, immunoprecipitates were hydrolyzed in
6N HCl, lyophilized, and resuspended with phosphoserine, phosphothreonine, and phosphotyrosine markers. Samples were applied to
cellulose thin-layer chromatography plates and analyzed by two-dimensional electrophoresis as described.33 The plates
were exposed to x-ray films and the phosphoamino acid markers were visualized with ninhydrin.33
EMSAs.
Nuclear extracts were prepared and equal amounts of nuclear extract
proteins were incubated with a radioactively labeled
oligodeoxynucleotide (oligodNT) probe encoding the NF-E2 recognition
sequence from the human porphobilinogen deaminase promoter as described
previously.18 Quality and equal loading of nuclear extracts
was tested by incubation with a probe for the ubiquitous transcription
factor SP-1.18
Western and Northern blot analyses.
Western blots were performed using a p45-specific antibody (Santa Cruz
Biotechnology) and enhanced chemiluminescence detection as described
previously.18 Northern blots were prepared with 8 µg of
total cytoplasmic RNA and probed with a radioactively labeled
-globin probe as described.19
| |
RESULTS |
A-kinase phosphorylates p45 on Ser169 in vitro and in vivo.
Inspection of the amino acid sequence of p45 reveals a consensus
sequence for A-kinase phosphorylation at residues 166 to 169 (Arg-Arg-Arg-Ser); we showed previously that A-kinase phosphorylates p45 in vitro.18 To determine whether Ser169 of
p45 is the site phosphorylated by A-kinase in vitro, we replaced Ser169 by Ala using site-directed mutagenesis and
transfected BHK cells, which do not express endogenous p45, with
expression vectors encoding wild-type and mutant p45. Western blots
demonstrated that the expression levels of wild-type and mutant
proteins were the same (Fig
1A). When p45
immunoprecipitates from cells transfected with wild-type and mutant p45
were incubated with [-32PO4]ATP and
purified C-subunit of A-kinase, there was significant phosphorylation
of the immunoprecipitates from the wild-type but not the mutant p45
transfected cells (Fig 1B).
View larger version (37K):
[in this window]
[in a new window]
| Fig 1.
A-kinase phosphorylates p45 on Ser169 in
vitro and in vivo. (A) BHK cells were transfected with increasing
amounts of expression vector encoding wild-type p45 [lanes 1 to 3, 0.3 µg, 0.6 µg, and 1.2 µg of pMT2-p45 (wt), respectively] or mutant
(Ser169 Ala)p45 [lanes 4 to 6, 0.3 µg, 0.6 µg,
and 1.2 µg of pMT2-p45 (mut), respectively]. Cell extracts were
subjected to Western blotting with a p45-specific antibody as described
in Materials and Methods. The p45 doublet, which is thought to result
from alternative usage of two translational start sites,50
is indicated by a double arrow. (B) BHK cells were transfected with
expression vectors encoding wild-type p45 [1.2 µg and 0.4 µg of
pMT2-p45(wt), lanes 1 and 2, respectively] or mutant p45 [1.2 µg
and 0.4 µg of pMT2-p45(mut Ser169 Ala), lanes 3 and
4, respectively]; lane 5 shows mock-transfected cells. Cell extracts
were subjected to immunoprecipitation with a p45-specific antibody;
immunoprecipitates were incubated with [-32PO4]ATP and purified C-subunit of
A-kinase and applied to SDS-PAGE/autoradiography as described in
Materials and Methods. (C) BHK cells were transfected with expression
vectors encoding wild-type p45 [1.2 µg of pMT2-p45(wt), lanes 1, 2, and 8] or mutant p45 [1.2 µg of pMT2-p45(mut
Ser169 Ala), lanes 4 and 5]; lanes 3 and 6 show
cells transfected with 0.2 µg of wild-type or mutant p45 vector,
respectively, and lane 7 shows mock-transfected cells. Cells were
incubated with 32PO4 and some cultures were
treated with 1 mmol/L 8-Br-cAMP (lanes 2, 3, 5, and 6); cell extracts
were subjected to immunoprecipitation with p45-specific antibody (lanes
1 to 7) or control rabbit serum (lane 8) and immunoprecipitates were
applied to SDS-PAGE/autoradiography as described in Materials and
Methods. (D) Wild-type MEL cells (lanes 1 to 3) and A-kinase-deficient
MEL cells (lanes 4 to 6) were incubated with
32PO4 and some cultures were treated with 1 mmol/L 8-Br-cAMP (lanes 3 and 5); cell extracts were subjected to
immunoprecipitation with p45-specific antibody (lanes 2 to 5) or
control rabbit serum (lanes 1 and 6) as described in (C).
|
|
View larger version (50K):
[in this window]
[in a new window]
| Fig 2.
DNA binding activity of wild-type and mutant
(Ser169 Ala) p45 in BHK cells. BHK cells were
cotransfected with increasing amounts of expression vector encoding
wild-type p45 [lanes 1 to 3, 0.3 µg, 0.6 µg, and 1.2 µg of
pMT2-p45(wt), respectively] or mutant (Ser169 Ala)p45 [lanes 4 to 6, 0.3 µg, 0.6 µg
and 1.2 µg of pMT2-p45(mut), respectively] and equimolar amounts of
p18 expression vector [pMT2-p18]; lanes 7 and 8 show mock-transfected
cells. Ten micrograms of nuclear extract protein was incubated with 10 fmol of end-labeled oligodNT probe encoding a NF-E2 recognition site
(A) or a SP-1 recognition site (B); lane 9 shows probe incubated in the
absence of nuclear extract protein. EMSAs were performed as described
in Materials and Methods. Nuclear extracts incubated with the NF-E2
oligodNT probe yielded 2 protein/DNA complexes (A, lanes 1 to 6); both were eliminated by adding a 50-fold excess of unlabeled oligodNT, but
only the slower migrating complex was eliminated by adding excess
unlabeled oligodNT containing a mutation that abolishes NF-E2 binding,
but not AP-1 binding (data not shown).18 The arrow in (A)
indicates the protein/DNA complex containing p45 and p18 NF-E2.
|
|
To determine whether p45 is phosphorylated by A-kinase in vivo, we
incubated the transfected BHK cells with 32PO4
and treated them for 1 hour with 1 mmol/L 8-Br-cAMP to activate endogenous A-kinase. Immunoprecipitation of p45 from these cells demonstrated a significant amount of 32PO4
incorporation into wild-type p45 in unstimulated cells. Treatment with
8-Br-cAMP induced a mobility shift on SDS-PAGE with a small increase in
total 32PO4 incorporation (Fig 1C, compare lane
2, wild-type p45 from cells treated with 8-Br-cAMP, with lane 1, wild-type p45 from untreated cells). Similar changes in apparent
molecular weight induced by phosphorylation of single amino acid
residues have been observed in other proteins.34,35 Total
32PO4 incorporation into p45 was determined by
quantitating Cerenkov radiation of excised gel slices and increased
25% ± 7% in the presence of 8-Br-cAMP (result of three independent
experiments). Basal phosphorylation of mutant p45 was considerably
lower than that of wild-type p45 and neither the mobility nor the total
32PO4 incorporation into mutant p45 was
influenced by 8-Br-cAMP (Fig 1C, lanes 4 and 5 show mutant p45 from
cells cultured in the absence and presence of 8-Br-cAMP, respectively).
Similar results were obtained when p45 was immunoprecipitated from
32PO4-labeled MEL cells: treatment with
8-Br-cAMP resulted in a mobility shift in p45 immunoprecipitated from
wild-type MEL cells, but not from A-kinase-deficient MEL cells (Fig
1D, compare lanes 2 and 3, wild-type MEL cells cultured in the absence
or presence of 8-Br-cAMP, with lanes 4 and 5, A-kinase-deficient MEL
cells cultured in the absence or presence of 8-Br-cAMP, respectively). Phosphoamino acid analysis of p45 from transfected BHK cells labeled with 32PO4 in vivo demonstrated
32PO4 in p45 associated with phosphoserine, but
not with phosphothreonine or phosphotyrosine.
The lower 32PO4 incorporation into mutant p45
compared with wild-type p45 suggests that the mutation at
Ser169 may influence the phosphorylation of neighboring
sites by other serine/threonine protein kinases; since the
phosphorylation of Ser169 by A-kinase resulted in a
mobility shift on SDS-PAGE, Ser169 does not appear to be
targeted by other protein kinases in unstimulated cells. Thus,
Ser169 is the only site in p45 that is phosphorylated in
vitro and in vivo by A-kinase; p45 phosphorylation in vivo in the
absence of 8-Br-cAMP can be attributed to other serine/threonine
protein kinases.
DNA binding activity of mutant p45(Ser169 Ala) in
BHK cells.
To examine whether A-kinase phosphorylation of p45 Ser169
influences NF-E2/DNA complex formation, we cotransfected BHK cells with
increasing amounts of expression vectors for wild-type or mutant
p45(Ser169 Ala) and equimolar amounts of p18
expression vector. Equal amounts of nuclear extract proteins were
incubated with a radioactively labeled oligodNT encoding the NF-E2
recognition site of the human porphobilinogen deaminase promoter and
protein/DNA complexes were resolved on nondenaturing PAGE: the faster
migrating protein/DNA complex contains the p45/p18 heterodimer and it
is absent in mock transfected cells (Fig
2A, compare lanes 1 through 6, transfected cells, with lanes 7 and 8, mock-transfected cells).10,18
Mutant p45(Ser169 Ala) formed the same amount of
NF-E2/oligodNT complexes as wild-type p45 (Fig 2A, compare lanes 1 to
3, wild-type p45, with lanes 4 to 6, mutant p45). As a control for
equal protein loading, we performed EMSAs with an oligodNT probe
containing a SP-1 recognition site (Fig 2B). EMSAs performed with
variable amounts of NF-E2 oligodNT probe demonstrated that the
DNA-binding affinity of the NF-E2 complex containing mutant
p45(Ser169 Ala) was indistinguishable from that
containing wild-type p45.
View larger version (43K):
[in this window]
[in a new window]
| Fig 3.
Function of (Ser169 Ala) mutant p45 in
the p45-deficient MEL cell variant CB3. P45-deficient CB3 cells were
stably transfected with expression vectors encoding either wild-type
(W) or (Ser169 Ala) mutant (M) p45 and single
G418-resistant colonies were selected as described in Materials and
Methods. Cells were cultured for 72 hours in 4 mmol/L HMBA. (A) EMSAs
were performed with 10 µg of nuclear extract protein and 10 fmol of
NF-E2 oligodNT as described in Fig 2. The amount of NF-E2/oligodNT
complexes formed correlated closely with the amount of p45 detected on
Western blots (not shown). (B) Northern blots prepared with 8 µg of
total cytoplasmic RNA were probed with a -globin cDNA probe as
described in Materials and Methods. Equal loading of the RNA was
confirmed by ethidium fluorescence of the ribosomal RNA bands (not
shown). Several representative clones expressing variable amounts of
wild-type (W1-W6) or (Ser169 Ala) mutant p45 (M1-M5)
are shown; control MEL cells expressing endogenous p45 (C) are shown in
the first lane for comparison.
|
|
DNA binding and transactivation properties of mutant
p45(Ser169 Ala) in CB3 cells.
To examine the DNA-binding and transactivation properties of
the mutant p45(Ser169 Ala) in an erythroid
background, we transfected the variant MEL cell line CB3 with
expression vectors encoding either wild-type or mutant p45
(Ser169 Ala). CB3 cells are completely deficient in
p45 expression, and unlike wild-type MEL cells, they show no increase
in globin gene expression when treated with differentiation-inducing
agents like hexamethylene bisacetamide (HMBA).9,10 Single
clones of stably transfected CB3 cells were tested for NF-E2
DNA-binding activity and -globin mRNA expression in response to
HMBA. Several clones showed no detectable NF-E2/DNA complexes and
little -globin mRNA expression (Fig 3,clones W2, M2, and W4; some globin mRNA was detectable in these clones
on long exposures of the Northern blot). In clones that expressed p45,
the amount of NF-E2/DNA complexes correlated with the amount
of -globin mRNA expression observed in both wild-type and mutant
p45-transfected CB3 cells (Fig 3, eg, clones M1 and W3, which expressed
low amounts of NF-E2/DNA complexes, expressed low amounts of -globin
mRNA). On Western blots, the amount of p45 expressed correlated closely
with the amount of NF-E2/DNA complexes found by EMSA (data not shown). Thus, in p45-deficient CB3 cells, mutant
p45(Ser169 Ala) could restore NF-E2 DNA-binding
activity and -globin mRNA expression to the same degree as wild-type
p45. Only levels of p45 expression that were higher than those found in
wild-type MEL cells restored -globin mRNA expression in the variant
CB3 cells to the amount of -globin mRNA detected in wild-type MEL cells; this finding is in agreement with previous
reports.9,10
View larger version (23K):
[in this window]
[in a new window]
| Fig 4.
Transactivation properties of Gal4-fusion constructs
containing full-length wild-type or (Ser169 Ala)
mutant p45: effect of 8-Br-cAMP. BHK cells were transfected with 100 ng
of the reporter pGAL4-Luc, 50 ng of the -galactosidase expression
vector pRSV-Gal (internal control), and the indicated amounts of
transactivator plasmid [pGal4/p45(wt), triangles; pGal4/p45(mut), squares; pSG424, diamonds]. The total amount of transfected DNA was
kept constant by the addition of carrier DNA. Half of the cultures were
treated with 1 mmol/L 8-Br-cAMP for 8 hours before harvesting to
activate endogenous A-kinase (dashed lines and filled symbols).
Reporter gene activities were determined as described in Materials and
Methods; luciferase activity was normalized to -galactosidase
activity in each sample to correct for transfection efficiencies.
Results represent the mean ± SD of three independent experiments.
|
|
Effect of 8-Br-cAMP on the transactivation properties of p45.
The NF-E2 binding site is recognized by a number of different bzip
proteins, including members of the AP-1 family, which are known to be
regulated by A-kinase.36 To study the transactivation properties of p45 independently of its binding to the NF-E2 recognition site, we fused p45 sequences with the DNA-binding domain of the well-characterized yeast transcription factor Gal4; this allows testing
of the fusion protein's activity on a test promoter bearing GAL4-binding sites.27 When we cotransfected BHK cells with
the reporter pGAL4-Luc and increasing amounts of expression vector encoding Gal4 fusion proteins with either full-length wild-type (wt) or
mutant (mut, Ser169 Ala)p45, we observed the same
modest degree of reporter gene transactivation by wild-type and mutant
p45 (Fig 4, open squares and triangles).
When cells were treated with 8-Br-cAMP to activate endogenous A-kinase,
we observed a fivefold to sevenfold increase in the transactivation of
pGAL4-Luc at each level of pGal4/p45 (wt) or (mut) expression (Fig 4,
filled squares and triangles). When we transfected pGAL4-Luc with the
parent vector pSG424, which contains only the Gal4 DNA-binding domain,
luciferase expression was low and not significantly influenced by the
activation of A-kinase (Fig 4, diamonds). When we cotransfected a
Gal4-fusion protein containing the bzip transcription factor
c-Fos, strong transactivation of pGAL4-Luc-1 was observed,
which was not altered by 8-Br-cAMP (data not shown). Thus, the effect
of A-kinase on p45 transactivation was specific for p45 sequences, but
did not depend on the presence of the A-kinase phosphorylation site
Ser169.
Effect of A-kinase on truncated versions of p45.
To determine which part of the p45 sequence was required for the
stimulation of transactivation by A-kinase, we constructed a series of
p45 deletions in the Gal4-fusion vectors (Fig 5A). We transfected the
Gal4-fusion constructs containing N-terminal p45 sequences of
decreasing length together with the reporter pGAL4-Luc into BHK cells
and compared their activities with the activity of pSG424, the parent
vector lacking p45 sequences (Fig 5B). In the absence of A-kinase,
pGal4/p45 (wt) containing full-length p45 increased luciferase
expression only about twofold over the level found in
pSG424-transfected cells. Removing the bzip domain [pGal4/p45(269)] increased luciferase activity approximately 10-fold, and removing all but the N-terminal 83 amino acids
[pGal4/p45(83)] increased luciferase activity approximately
50-fold when compared with the activity elicited by full-length p45
(Fig 5B, filled bars). Further deletion of the p45 coding sequence
resulted in almost complete loss of transactivation: a construct
containing only the N-terminal 36 amino acids of p45 fused to
the Gal4 DNA-binding domain [pGal4/p45(36)] increased luciferase
activity only about 1.5-fold over the level found in pSG424-transfected
cells (data not shown). The increase in transactivation observed with
the removal of the bzip domain of p45 may be explained by inhibitory dimerization partners of p45 binding to the leucine zipper
domain.4,37 The increase in transactivation seen with the
deletion of sequences between amino acids 83 and 269 of p45 could be
due to the removal of inhibitory protein interactions or to unmasking
of the N-terminal transactivation domain by conformational changes. In
agreement with our results, the main transactivation domain of p45 has
been previously localized to the N-terminal 80 amino acids of the
protein.8,38
When the C-subunit of A-kinase was cotransfected with the Gal4-p45
fusion constructs shown in Fig 5, there was a significant increase in
the transactivation properties of each construct: the increase was
approximately sevenfold to eightfold for most constructs, including
full-length p45(wt) and (mut); the increase was 17-fold for the
construct containing the N-terminal 112 amino acids (Fig 5B, open
bars). These results suggest that A-kinase regulates protein(s) that
interacts with the N-terminal 112 amino acids of p45; this regulation
is obviously independent of Ser169 phosphorylation by
A-kinase.
A similar effect of A-kinase on the activity of pGal4/p45(83) was
observed in MEL cells (Fig 6 shows the
effect of cotransfected C-subunit of A-kinase; similar results were
obtained when endogenous A-kinase was activated with 8-Br-cAMP). When
we transfected pGal4/p45(83) into severely A-kinase-deficient MEL
cells (clones RImut/C1 and RImut/C3, described
previously31), we found that the transactivation potential
of pGal4/p45(83) was reduced by 50% to 70% compared with the
activity of this construct in control MEL cells with normal A-kinase
activity (clones RIwt/Cl31 and parental MEL cells); gene expression from the cotransfected plasmid pRSV-Gal was
similar in all clones. Thus, A-kinase appears to regulate p45
transactivation potential in erythroid as well as nonerythroid cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
Regulation of the p45 transactivation domain by A-kinase
in MEL cells. MEL cells were cotransfected with 300 ng of pGAL4-Luc, 300 ng of pRSV-Gal, the indicated amounts of transactivator plasmid containing the N-terminal 83 amino acids of p45 [pGal4/p45(83)], and 200 ng of either A-kinase C-subunit expression vector ( ) or
empty vector ( ) as described in Materials and Methods. Luciferase activity was normalized to -galactosidase activity; results
represent the mean ± SD of three independent experiments.
|
|
Effect of CBP on p45 transactivation.
Cheng et al20 recently demonstrated that the N-terminal
transactivation domain of p45 binds the transcriptional coactivator CBP
in vitro. Since CBP is known to be regulated by A-kinase
phosphorylation, CBP would be an attractive candidate for the protein
mediating the effect of A-kinase on p45 transactivation. If the
interaction between p45 and CBP were responsible for the ability of
A-kinase to stimulate p45 transactivation, then expression of the
isolated p45-binding domain of CBP (amino acids 451 to 682) would be
expected to dominantly inhibit this effect by competing with endogenous CBP for binding to p45.20 However, coexpression of CBP (451 to 682) had little or no effect on the transactivation potential of p45
in the presence or absence of A-kinase (data not shown). Moreover, when
we cotransfected BHK cells with pGal4/p45(83) and full-length CBP,
transactivation of the Gal4-dependent reporter was stimulated twofold
to threefold by CBP in the presence or absence of A-kinase; this effect
on transactivation appeared to be nonspecific, since CBP also increased
expression of the control plasmid pRSV-Gal twofold (data not shown).
Thus, we could not demonstrate a convincing effect of CBP on p45
transactivation under our experimental conditions.
| |
DISCUSSION |
The importance of NF-E2 for the transcriptional activation of
erythroid-specific genes and in particular for the activity of the -
and -globin LCRs has been recognized, but little is known about the
regulation of NF-E2 activity.5-7,10,39 NF-E2 is expressed
in multipotential hematopoietic progenitor cells before their
commitment to the erythroid lineage and the globin LCR NF-E2
recognition sites are occupied before globin gene expression is
activated.6,39-41 NF-E2 activity may be regulated by
posttranslational mechanisms, such as phosphorylation, or by
interactions with ancillary proteins. Other mechanisms of regulation
may include changes in NF-E2 complex composition through changes in the
expression and activity of maf-related proteins or competition
of other bzip proteins for binding at the NF-E2 recognition
site.4,13-15,37,42
In this study, we found that A-kinase significantly stimulated the
transactivation properties of p45 in erythroid and nonerythroid cells;
in a previous study, we had shown impaired NF-E2/DNA complex formation
and decreased -LCR enhancer activity in A-kinase-deficient MEL
cells.18 Since we could not detect any effect of p45
phosphorylation at the A-kinase recognition site Ser169 on
NF-E2 DNA-binding or transactivation properties, our results suggest
that A-kinase regulates NF-E2 indirectly, through changes in the
activity of ancillary protein(s) present in erythroid and nonerythroid
cells. Since modulation of p45 activity by A-kinase required only the
N-terminal transactivation domain of p45, the effect of A-kinase is not
likely to be mediated through modification of maf-related or
other bzip proteins.
Although the activity of many transcription factors is modulated by
phosphorylation, there are many examples of changes in transcription
factor phosphorylation that have no detectable influence on DNA binding
or transactivation.36,43 The erythroid transcription factor
GATA-1 is a prime example, because careful mapping of seven major
phosphorylation sites demonstrated that phosphorylation at an A-kinase
consensus sequence increases during erythroid differentiation of MEL
cells, but phosphorylation at this site or at the other sites does not
measurably alter GATA-1 functions.43
The positive or negative regulation of transcription factors by
changing protein/protein interactions has been described for several
classes of bzip proteins. An example of negative regulation is the
interaction of AP-1 with steroid hormone and retinoic acid receptors,
which results in repression of AP-1 activity dependent on ligand
binding to the steroid or retinoic acid receptors.30,44,45 An example of positive regulation is the interaction of CREB with the
transcriptional coactivator CBP, which requires several
A-kinase-regulated events, including phosphorylation of CREB and
CBP.46,47 Although CBP has been recently shown to bind to
the transactivation domain of p45 in vitro and appears to mediate the
potentiation of nuclear hormone receptor action by p45,20
we were unable to demonstrate a significant effect of CBP on the
transactivation potential of p45 under our experimental conditions.
However, we cannot exclude that BHK cells express too much endogenous
CBP to demonstrate a positive effect of transfected full-length CBP or
a dominant negative effect of the isolated p45-binding domain of
CBP.
A critical role for the N-terminal domain of p45 for globin gene
expression in MEL cells has been shown previously.10,38 The
main transactivation domain of p45 has been localized to the N-terminal
80 amino acids of the protein; this region is proline-rich and contains
two PXXP motifs and a PPPSY motif that can mediate protein/protein
interactions.8,38,48 We found that this N-terminal domain
of p45 was sufficient to mediate the effect of A-kinase on p45
transactivation. Recently, direct interaction between this p45 domain
and the TATA-binding protein-associated factor TAFII130, as
well as other proteins, has been demonstrated.8,48 More work is necessary to determine whether the interaction of p45 with a
component of the transcription initiation complex or other interactions
between p45 and downstream effector molecules may be regulated by
A-kinase.
The physiologic significance of A-kinase regulation of NF-E2 is
supported by the effect of cAMP on the erythroid differentiation of
various cell lines: stimulation of the cAMP signal transduction pathway
promotes hemoglobin production in the erythropoietin-responsive cell
lines SKT6, TSA8, and J2E.21-23 While binding of
erythropoietin to its receptor does not change intracellular cAMP
concentrations,22 the effect of cAMP on hemoglobin
production in these cell lines is consistent with a role of A-kinase in
the regulation of erythroid gene expression, possibly via changes in
the activity of NF-E2. In MEL cells, we have demonstrated that A-kinase
activity is necessary for erythroid gene expression,18,19
although prolonged treatment of MEL cells with pharmacologic doses of
cAMP analogs results in inhibition of erythroid
differentiation.49 We have recently shown that this
paradoxical effect is due to upregulation of c-myb, mediated at
least in part by NF- B (p50/relB), which is induced by
prolonged activation of A-kinase.49 Thus, in
MEL cells, A-kinase not only produces signals expected to promote
differentiation (increased NF-E2 activity), but prolonged activation of
the kinase can also produce signals that are incompatible with
differentiation (upregulation of c-myb).
| |
FOOTNOTES |
Submitted October 14, 1997;
accepted December 15, 1997.
Supported by National Science Foundation Grant No. MCB-9506327.
Address reprint requests to Renate B. Pilz, MD, Department
of Medicine and Cancer Center, University of California, San Diego, Basic Science Building, 9500 Gilman Dr, La Jolla, CA 92093-0652.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs N. Andrews, M. Karin, M. Ptashne, and M. Rosenfeld for
providing us with plasmids, Dr S. Taylor for the purified C-subunit of
A-kinase, and Dr Y. Ben-David for the variant MEL cell line CB3.
| |
REFERENCES |
1.
Andrews NC,
Erdjument-Bromage H,
Davidson MB,
Tempst P,
Orkin SH:
Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein.
Nature
362:722,
1993[Medline]
[Order article via Infotrieve]
2.
Ney PA,
Andrews NC,
Jane SM,
Safer B,
Purucker ME,
Weremowicz S,
Morton CC,
Goff SC,
Orkin SH,
Nienhuis AW:
Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.
Mol Cell Biol
13:5604,
1993[Abstract/Free Full Text]
3.
Blank V,
Kim MJ,
Andrews NC:
Human MafG is a functional partner for p45 NF-E2 in activating globin gene expression.
Blood
89:3925-3935,
1997[Abstract/Free Full Text]
4.
Igarashi K,
Itoh K,
Motohashi H,
Hayashi N,
Matuzaki Y,
Nakauchi H,
Nishizawa M,
Yamamoto M:
Activity and expression of murine small Maf family protein MafK.
J Biol Chem
270:7615,
1995[Abstract/Free Full Text]
5.
Talbot D,
Grosveld F:
The 5HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites.
EMBO J
10:1391,
1991[Medline]
[Order article via Infotrieve]
6.
Gong Q,
McDowell JC,
Dean A:
Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5 hypersensitive site 2 of the -globin locus control region.
Mol Cell Biol
16:6055,
1996[Abstract]
7.
Armstrong JA,
Emerson BM:
NF-E2 Disrupts chromatin structure at human -globin locus control region hypersensitive site 2 in vitro.
Mol Cell Biol
16:5634,
1996[Abstract]
8.
Amrolia PJ,
Ramamurthy L,
Saluja D,
Tanese N,
Jane SM,
Cunningham JM:
The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the - and -globin gene locus in an erythroid cell line.
Proc Natl Acad Sci USA
94:10051,
1997[Abstract/Free Full Text]
9.
Lu S-J,
Rowan S,
Bani MR,
Ben-David Y:
Retroviral integration within the Fli-2 locus results in inactivation of the erythroid transcription factor NF-E2 in Friend erythroleukemias: Evidence that NF-E2 is essential for globin expression.
Proc Natl Acad Sci USA
91:8398,
1994[Abstract/Free Full Text]
10.
Kotkow KJ,
Orkin SH:
Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer.
Mol Cell Biol
15:4640,
1995[Abstract]
11.
Shidasani RA,
Rosenblatt MF,
Zucker-Franklin D,
Jackson CW,
Hunt P,
Saris CJM,
Orkin SH:
Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.
Cell
81:695,
1995[Medline]
[Order article via Infotrieve]
12.
Shivdasani R,
Orkin SH:
Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.
Proc Natl Acad Sci USA
92:8690,
1995[Abstract/Free Full Text]
13.
Moi P,
Chan K,
Asunis I,
Cao A,
Kan YW:
Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the -globin locus control region.
Proc Natl Acad Sci USA
91:9926,
1994[Abstract/Free Full Text]
14.
Caterina JJ,
Donze D,
Sun C-W,
Ciavatta DJ,
Townes TM:
Cloning and functional characterization of LCR-F1: A bZIP transcription factor that activates erythroid-specific, human globin gene expression.
Nucl Acids Res
22:2383,
1994[Abstract/Free Full Text]
15.
Chan JY,
Han X-L,
Kan YW:
Cloning of Nrfl, an NF-E2-related transcription factor, by genetic selection in yeast.
Proc Natl Acad Sci USA
90:11371,
1993[Abstract/Free Full Text]
16.
Chui DHK,
Tang W,
Orkin SH:
cDNA cloning of murine Nrf2 gene, coding for a p45 NF-E2 related transcription factor.
Biochem Biophys Res Commun
209:40,
1995[Medline]
[Order article via Infotrieve]
17.
Fraser PJ,
Curtis PJ:
Specific pattern of gene expression during induction of mouse erythroleukemia cells.
Genes Dev
1:855,
1987[Abstract/Free Full Text]
18.
Garingo AD,
Suhasini M,
Andrews NC,
Pilz RB:
cAMP-dependent protein kinase is necessary for increased NF-E2/DNA complex formation during erythroleukemia cell differentiation.
J Biol Chem
270:9169,
1995[Abstract/Free Full Text]
19.
Pilz RB:
Impaired erythroid-specific gene expression in cAMP-dependent protein kinase-deficient murine erythroleukemia cells.
J Biol Chem
268:20252,
1993[Abstract/Free Full Text]
20.
Cheng X,
Reginato MJ,
Andrews NC,
Lazar MA:
The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2.
Mol Cell Biol
17:1407,
1997[Abstract]
21.
Taxman DJ,
Wojchowski DM:
Erythropoietin-induced transcription at the murine maj-globin promoter.
J Biol Chem
270:6619,
1995[Abstract/Free Full Text]
22.
Fukumoto H,
Matsui Y,
Obinata M:
Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8).
Development
105:109,
1989[Abstract]
23.
Kuramochi S,
Sugimoto Y,
Ikawa Y,
Todokoro K:
Transmembrane signaling during erythropoietin- and dimethylsulfoxide-induced erythroid cell differentiation.
Eur J Biochem
193:163,
1990[Medline]
[Order article via Infotrieve]
24.
Deng WP,
Nickloff JA:
Site-directed mutagenesis of virtually any plasmid by eliminating a unique site.
Anal Biochem
200:81,
1992[Medline]
[Order article via Infotrieve]
25. Sambrook J: Molecular Cloning: A Laboratory Manual. New York,
NY, Cold Spring Harbor Laboratory, 1989
26.
Helmberg A,
Auphan N,
Caelles C,
Karin M:
Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor.
EMBO J
14:452,
1995[Medline]
[Order article via Infotrieve]
27.
Sadowski I,
Ptashne M:
A vector for expression GAL4(1-147) fusions in mammalian cells.
Nucleic Acids Res
17:7539,
1989[Free Full Text]
28.
Armstrong R,
Wen W,
Meinkoth J,
Taylor S,
Montminy M:
A refractory phase in cyclic AMP-responsive transcription requires down regulation of protein kinase A.
Mol Cell Biol
15:1826,
1995[Abstract]
29.
Deng T,
Karin M:
JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers.
Genes Dev
7:479,
1993[Abstract/Free Full Text]
30.
Kamei Y,
Xu L,
Heinzel T,
Torchia J,
Kurokawa R,
Gloss B,
Lin S-C,
Heyman RA,
Rose DW,
Glass Ck,
Rosenfeld MG:
A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.
Cell
85:403,
1996[Medline]
[Order article via Infotrieve]
31.
Pilz RB,
Eigenthaler M,
Boss GR:
Chemically-induced murine erythroleukemia cell differentiation is severely impaired when cAMP-dependent protein kinase activity is repressed by transfected genes.
J Biol Chem
267:16161,
1992[Abstract/Free Full Text]
32.
Gudi T,
Huvar I,
Meinecke M,
Lohmann SM,
Boss GR,
Pilz RB:
Regulation of gene expression by cGMP-dependent protein kinase.
J Biol Chem
271:4597,
1996[Abstract/Free Full Text]
33. Boyle WJ, Van der Geer P, Hunter T: Phosphopeptide mapping and
phosphorylation site determination, in Hunter T, Sefton BM (eds):
Methods in Enzymol. San Diego, CA, Academic, 1991, p 110
34.
Halbrugge M,
Friedrich C,
Eigenthaler M,
Schanzenbacher P,
Walter U:
Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators.
J Biol Chem
265:3088,
1990[Abstract/Free Full Text]
35.
Hordijk PL,
Verlaan I,
Jalink K,
van Corven EJ,
Moolenaar WH:
cAMP abrogates the p21ras-mitogen-activated protein kinase pathway in fibroblasts.
J Biol Chem
269:3534,
1994[Abstract/Free Full Text]
36.
Angel P,
Karin M:
The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.
Biochim Biophys Acta
1072:129,
1991[Medline]
[Order article via Infotrieve]
37.
Igarashi K,
Kataoka K,
Itoh K,
Hayashi N,
Nishizawa M,
Yamamoto M:
Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins.
Nature
367:568,
1994[Medline]
[Order article via Infotrieve]
38.
Bean TL,
Ney PA:
Multiple regions of p45 NF-E2 are required for -globin gene expression in erythroid cells.
Nucl Acids Res
25:2509,
1997[Abstract/Free Full Text]
39.
Labbaye C,
Valtieri M,
Barberi T,
Meccia E,
Pelosi E,
Condorelli GL,
Testa U,
Peschle C:
Differential expression and functional role of GATA-2 NE-E2, and GATA-1 in normal adult hematopoiesis.
J Clin Invest
95:2346,
1995
40.
Strauss EC,
Andrews NC,
Higgs DR,
Orkin SH:
In vivo footprinting of the human -globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction.
Mol Cell Biol
12:2135,
1992[Abstract/Free Full Text]
41.
Strauss EC,
Orkin SH:
In vivo protein-DNA interactions at hypersensitive site 3 of the human -globin locus control region.
Proc Natl Acad Sci USA
89:5809,
1992[Abstract/Free Full Text]
42.
Oyake T,
Iton K,
Motohashi H,
Hayashi N,
Hoshino H,
Nishizawa M,
Yamamoto M,
Igarashi K:
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
Mol Cell Biol
16:6083,
1996[Abstract]
43.
Crossley M,
Orkin SH:
Phosphorylation of the erythroid transcription factor GATA-1.
J Biol Chem
269:16589,
1994[Abstract/Free Full Text]
44.
Schüle R,
Rangarajan P,
Kliewer S,
Ransome LJ,
Bolado J,
Yang N,
Verma IM,
Evans RM:
Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor.
Cell
62:1217,
1990[Medline]
[Order article via Infotrieve]
45.
Schüle R,
Rangarajan P,
Yang N,
Kliewer S,
Ransone LJ,
Bolado J,
Verma IM,
Evans RM:
Retinoic acid is a negative regulator of AP-1-responsive genes.
Proc Natl Acad Sci USA
88:6092,
1991[Abstract/Free Full Text]
46.
Chrivia JC,
Kwok RPS,
Lamb N,
Hagiwara M,
Montminy MR,
Goodman RH:
Phosphorylated CREB binds specifically to the nuclear protein CBP.
Nature
365:855,
1993[Medline]
[Order article via Infotrieve]
47.
Brindle P,
Nakajima T,
Montminy M:
Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP.
Proc Natl Acad Sci USA
92:10521,
1995[Abstract/Free Full Text]
48.
Gavva NR,
Gavva R,
Ermekova K,
Sudol M,
Shen CKJ:
Interaction of WW domains with hematopoietic transcription factor p45/NF-E2 and RNA polymerase II.
J Biol Chem
272:24105,
1997[Abstract/Free Full Text]
49.
Suhasini M,
Reddy CD,
Reddy EP,
Didonato JA,
Pilz,
RP:
cAMP-induced NF-B (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation.
Oncogene
15:1859,
1997[Medline]
[Order article via Infotrieve]
50.
Chan JY,
Han X-L,
Kan YW:
Isolation of a cDNA encoding the human NF-E2 protein.
Proc Natl Acad Sci USA
90:11366,
1993[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y.-C. Shyu, T.-L. Lee, C.-Y. Ting, S.-C. Wen, L.-J. Hsieh, Y.-C. Li, J.-l. Hwang, C.-C. Lin, and C.-K. J. Shen
Sumoylation of p45/NF-E2: Nuclear Positioning and Transcriptional Activation of the Mammalian {beta}-Like Globin Gene Locus
Mol. Cell. Biol.,
December 1, 2005;
25(23):
10365 - 10378.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Chen, H.-Y. Zhang, K. Pavlish, and J. N. Benoit
Effects of chronic portal hypertension on small heat-shock proteins in mesenteric arteries
Am J Physiol Gastrointest Liver Physiol,
April 1, 2005;
288(4):
G616 - G620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Kronke, V. N. Bochkov, J. Huber, F. Gruber, S. Bluml, A. Furnkranz, A. Kadl, B. R. Binder, and N. Leitinger
Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein
J. Biol. Chem.,
December 19, 2003;
278(51):
51006 - 51014.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Li, S. Zhao, V. Karur, and D. M. Wojchowski
DYRK3 Activation, Engagement of Protein Kinase A/cAMP Response Element-binding Protein, and Modulation of Progenitor Cell Survival
J. Biol. Chem.,
November 27, 2002;
277(49):
47052 - 47060.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Harju, K. J. McQueen, and K. R. Peterson
Chromatin Structure and Control of {beta}-Like Globin Gene Switching
Experimental Biology and Medicine,
October 1, 2002;
227(9):
683 - 700.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Francastel, W. Magis, and M. Groudine
Nuclear relocation of a transactivator subunit precedes target gene activation
PNAS,
September 26, 2001;
(2001)
211444898.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Francastel, W. Magis, and M. Groudine
Nuclear relocation of a transactivator subunit precedes target gene activation
PNAS,
October 9, 2001;
98(21):
12120 - 12125.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract