From the Department of Biochemistry, University of
Oslo, N-0316 Oslo, Norway and the
Max-Delbrück-Center
for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, Germany
Received for publication, September 13, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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The viral Myb (v-Myb) oncoprotein of the avian
myeloblastosis virus (AMV) is an activated form of the cellular
transcription factor c-Myb causing acute monoblastic leukemia in
chicken. Oncogenic v-Myb alterations include N- and C-terminal
deletions as well as point mutations. Whereas truncations in Myb cause
loss of various protein modifications, none of the point mutations in
v-Myb has been directly linked to protein modifications. Here we show
that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of
Ser116 differentially destabilizes a subtype of
c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein
abolishes phosphorylation of the adjacent Ser116 residue.
Modification of Ser116 was also detected in live cells in
c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb
failed to activate the resident mim-1 gene. Our data imply
that protein kinase A or a kinase with similar specificity negatively
regulates c-Myb function, including collaboration with C/EBP, and that
the leukemogenic AMV v-Myb version evades inactivation by a point
mutation that abolishes a phosphoacceptor consensus site. This suggests
a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.
The v-myb oncogene and its cellular progenitor
c-myb both encode transcription factors that are implicated
in the switch between growth and differentiation of hematopoietic cells
(reviewed in Refs. 1-4). The c-Myb protein consists of an N-terminal
DNA-binding domain (DBD),1 a
central transactivation domain, and a C-terminal negative
regulatory domain. The DBD of c-Myb is composed of the three imperfect
repeats R1, R2, and R3, each
related to the helix-turn-helix motif (5-7). R1 is deleted
in v-Myb, and the R2 and R3 repeats are
sufficient for sequence-specific DNA binding (hereafter termed the
minimal DNA-binding domain (mDBD)). This prototype Myb mDBD is highly conserved throughout evolution in the animal and plant kingdoms (8).
A large body of evidence supports a central role for c-Myb in
regulating cell growth and differentiation (reviewed in Ref. 4), in
particular in hematopoietic progenitor cells of different lineages
(9-15). This function is consistent with phenotypes observed in mice
with a c-mybnull mutation and in homozygous null
c-Myb/Rag1 chimeric mice (11, 13). The c-Myb
protein has also been reported to be expressed in a variety of tissues
in developing embryos and/or in adult tissues such as tooth buds,
retina, and epithelium of the gastrointestinal and respiratory tracts
and skin (16-19). Consistent with this expression pattern, c-Myb was
found to be required for colon development in mice (20).
Activated forms of c-Myb have been associated with cancer in animal
models (21). In mice, retroviral insertions may result in N-terminal
truncation of the c-Myb protein and deregulated expression, leading to
myeloid leukemia (22). In chickens, the two retroviruses AMV and E26,
which carry truncated Myb proteins, elicit myeloid leukemia and
erythroblastosis/stem cell leukemia, respectively (reviewed in Ref.
23). These viruses encode the v-Myb proteins
p48v-myb (AMV-derived) and the Myb-Ets
fusion protein p135gag-myb-ets (E26-derived), respectively.
Both v-Myb proteins are truncated in the N- and C-terminal ends. In
addition, the p48v-myb protein carries 10 point
mutations relative to c-Myb (23).
AMV v-Myb has been an instructive model for understanding oncogenic
activation and the normal function of c-Myb. AMV v-myb is a
potent oncogene that induces monocytic leukemia after very short
latency (24). A distinctive feature of AMV v-Myb is its cell type
specificity, inducing transformation of the macrophage lineage (23).
Since the oncogenic alterations include both N- and C-terminal
deletions as well as point mutations, AMV v-Myb probably utilizes
multiple mechanisms that synergize in oncogenicity and cell type specificity.
Although the cellular phenotypes induced by AMV v-Myb are well
characterized, much remains to be learned about the molecular mechanisms involved. Several studies have attempted to define oncogenic
determinants of v-myb. These studies have revealed that the
N- and C-terminal deletions remove several sites of protein modification. The v-Myb protein lacks N-terminal CK2 phosphorylation sites (Ser11 and Ser12) present in the wild
type protein. Phosphorylation of these sites reduced specific DNA
binding (25). Substitution of the two serines to alanines increased the
transcriptional activity on both the mim-1 and neutrophil
elastase promoters (26). However, loss of the CK2 sites is not
sufficient to turn myb into an oncogene (27, 28). A
mitogen-activated protein kinase phosphorylation site
(Ser528) in the C-terminal negative regulatory domain is
also deleted in AMV v-Myb. Substitution of this serine to alanine
increased the transcriptional capacity of c-Myb on some promoters but
not on others (29-31). Two studies have described in vitro
phosphorylation sites for PKA in c-Myb. PKA phosphorylation of
Ser8 in the N terminus resulted in partial inhibition of
DNA binding (32), whereas phosphorylation of Ser116 in the
DNA-binding domain was suggested to have no effect on DNA binding (33).
Recently, c-Myb was found to be acetylated within the C-terminal
negative regulatory domain by the histone acetyltransferases p300 and
CREB-binding protein both in vitro and in vivo
(34, 35). The modification sites were mapped to positions
Lys438 and Lys441 in mouse c-Myb and to
Lys471, Lys480, and Lys485 in human
c-Myb. The corresponding residues in chicken c-Myb (Lys443
and Lys446 and Lys472, Lys481, and
Lys486) are all deleted in AMV v-Myb. The truncations in
AMV v-Myb may also cause changes in protein-protein interactions. The
N-terminal deletion permitted cyclin D to inhibit transcriptional
activation in a CDK-independent fashion (36).
The AMV-specific point mutations in the second repeat R2 of
the v-Myb DBD perturb normal Myb functions through altered
protein-protein interactions (37). We have previously shown that
functional aspects of DNA binding are also altered (38). In addition,
the point mutations determine the target genes Myb regulates and the proteins it interacts with (39-41). It therefore appears that the v-Myb DBD has accumulated gain- and loss-of-function mutations to
escape regulation during proliferation and differentiation (40).
Although point mutations in the AMV v-Myb DBD have been linked to
alterations in function and protein-protein interactions, none of them
has been found to affect Myb protein modifications. The fact that one
of the mutations, V117D, is located adjacent to a reported
phosphorylation site for protein kinase A (PKA) (33) led us to
reexamine phosphorylation at Ser116. We found that
Ser116 was phosphorylated by PKA in vitro and
that this modification strongly affected the ability of c-Myb to bind
to a subgroup of Myb recognition elements (MRE) in vitro.
Phosphorylation of the Ser116 site is abrogated in
oncogenic AMV v-Myb, due to the V117D mutation adjacent to
Ser116. We developed a phospho-c-Myb
(Ser116)-specific antibody to demonstrate the presence of
this modification in c-Myb and its absence in AMV v-Myb in cells.
Furthermore, a mutant c-Myb protein (S116D, phosphorylation mimic)
failed to cooperate with NF-M (C/EBP Constructs and Plasmids--
The mutations S116A, S146A, S116D,
S116E, V117D, and S116A/S146A were introduced into the chicken c-Myb
minimal DNA-binding domain (mDBD) R2R3 as
previously described (5) or with the QuikChangeTM mutagenesis kit
(Stratagene) using pBET-R2R3 (5) as a template.
The back-mutation D117V was also introduced into AMV mDBD
(R2R3[AMVNHD]) (38). The
mammalian expression plasmids pCIneo-ccM (encoding the chicken c-Myb
protein, residues 72-443), pCIneo-AMV (encoding the AMV v-Myb protein,
residue 72-440 in chicken c-Myb), and their mutant forms were
constructed as follows. The relevant fragments from the chicken
c-myb gene and the AMV v-myb gene were amplified
by PCR and inserted into the PCIneo (Promega) vector as an
XhoI-NotI fragment behind the cytomegalovirus promoter. All constructs were verified by sequencing. Two effector plasmids expressing shortened c-Myb proteins were used,
pCIneo-ccM-(72-349) and pCIneo-ccM-(72-443), where the encoded
regions are given in parentheses.
Expression of Recombinant Proteins--
Wild type and mutant
minimal DNA-binding domains (R2R3), termed
mDBDs, were expressed in E. coli and purified as previously described (5, 42). The AMV mutant version of
R2R3 has previously been characterized (38).
The protein concentration of each mDBD domain was measured by optical
density at 280 nm (predominantly tryptophan absorption), Bradford
assay, and fluorescence quenching (43) to ensure equal amounts of each
protein in the experimental protocol.
The mDBD domain contains six highly conserved tryptophan residues,
which makes the protein suitable for conformational analysis by
fluorescence emission spectrum analysis. Spectroscopic analyses were
performed as described (38, 43).
In Vitro Phosphorylation of mDBD Proteins--
For
phosphorylation site mapping experiments, recombinant wild type and
mutant mDBD c-Myb proteins (8 pmol) were incubated in PKA buffer (20 mM HEPES, pH 7.9, 60 mM KCl, 5 mM
MgAc, 1 mM dithiothreitol), 5 µCi (3.3 pmol)
[
For the determination of Km of PKA for mDBD, various
amounts of recombinant mDBD (final concentrations 5, 5.6, 5.8, 6.2, 7.6, 10, 15, and 16.6 µM) were incubated in PKA buffer
with 500 µM ATP, 3.3 pmol (5 µCi) of
[
Quantitative phosphorylation of mDBD proteins for EMSA analysis was
performed with 10 µM (200 pmol) mDBD protein in PKA
buffer with 500 µM ATP and 80 units of PKA
C EMSA--
DNA binding was monitored by electrophoretic mobility
shift assay as described in Refs. 5 and 42. In general, recombinant mDBD protein (20 fmol) was incubated with
5'-
The oligonucleotides used for Myb binding are based on the MRE A site
in the mim-1 promoter (46). All oligonucleotide variants of
the Myb recognition element (MRE) have been previously described (see
Ref. 47 and Table I in Ref. 48). The sequence of the basic
mim-1A MRE oligonucleotide (core Myb binding site in italic type) is
5'-ATAACGGTCTTTTAGCGC-3'.
The GG, TG, GT, and TT variants refer to positions 5 and 6 in the core
recognition sequence (underlined), and all carry a C to T substitution
in position 8 (boldface type). The nonspecific oligonucleotide
5'-GCATTATCAAGCTTTTTTAGCGC-3' is a triple mutant variant of the GG MRE oligonucleotide. All oligonucleotides were end-labeled and purified through a G-25 spin
column. Cold double-stranded oligonucleotides for competition were
similarly annealed. Variant MRE oligonucleotides were labeled to the
same specific activity (47) and purified through a G-25 spin column.
Cell Lines--
The c-Myb-expressing erythroid cell line HD3,
quail QT6 fibroblasts, and the HD11 cell line expressing
v-myc have been described (49). All cells were grown in
Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine
serum, 2% heat-inactivated chicken serum, 15 mM HEPES,
penicillin, and streptomycin. Cells were maintained in 5%
CO2 atmosphere at 39 °C.
Immunoprecipitation and Western Analysis--
A synthetic
peptide coding for the human/chicken c-Myb internal 12 amino
acids (LVQKYGPKRWSV), in which the second to last serine was
phosphorylated, was conjugated and used for immunization using the
antibodies service of Eurogentec Bel S.A.
For immunoprecipitation, 6 × 106 QT6 cells were
seeded 5 h before transfection in 10 ml of tissue culture medium.
Cells were subsequently transfected with 10 µg of expression vectors
as indicated, using a calcium phosphate coprecipitation technique (50).
Cells were harvested 16-18 h post transfection. For HD3
immunoprecipitation, 2 × 107 cells were used. Cell
lysates were prepared in 0.3 ml of lysis buffer (20 mM
Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 10% glycerol, 0.8% Nonidet P-40, 0.1% deoxycholate, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of aprotinin, pepstatin, and leupeptin).
Expressed proteins were precipitated with a preimmune rabbit serum, a
Myb-specific rabbit immune serum and a Myb PKA site-specific rabbit
immune serum as indicated for 2 h at 4 °C. A-Sepharose (Amersham Biosciences) was added, incubated for 1 h, and washed extensively with lysis buffer. Immunoprecipitates were separated by
10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp.). Precipitated proteins were revealed with a mouse
monoclonal antibody directed against c-Myb (MYB2-7.77; ATCC) and appropriate peroxidase-conjugated second antibodies and
subsequently revealed using a chemiluminescence kit (ECL; Amersham Biosciences).
Transient Transfections, Reporter Assay, and Resident Gene
Activation Assay--
Plasmids used for transfections were purified
twice on CsCl gradients. QT6 and HD11 cells were transfected using
DEAE-dextran as described previously (51). To monitor reporter gene
activation, cells were harvested after 24 h, whole cell lysates
were prepared, and luciferase activity was determined as previously
described (51, 52). Activation of endogenous genes was determined as previously described (53, 54).
A PKA Phosphorylation Site in the c-Myb mDBD
(R2R3)--
Two putative PKA sites were
identified in c-Myb by a Prosite motif search (55), namely
Ser116 (KRWSV) and Ser146 (KKTSW). Both
residues reside in the R2R3 mDBD and conformed well to the PKA consensus motif RRX(S/T)Y (45). An
alignment of vertebrate Myb proteins (Fig.
1A) shows that both sites are highly conserved. Mapping of these two sites onto the published NMR
structure of mouse mDBD (7) and the recent crystal structure (37)
showed that they are both on the surface of the protein and hence
should be accessible for interaction with other proteins (Fig.
1B). Note the close proximity of Ser116 to the
DNA sugar-phosphate backbone in the model.
A Myb fragment, consisting of the N terminus and all three repeats
(amino acids 1-192) has been shown to be a phosphorylation target of
PKA (56). Pilot biochemical experiments with recombinant c-Myb mDBD
domain corresponding to the AMV-myb version (amino acids 89-192)
showed that this domain was also readily phosphorylated in
vitro by PKA catalytic subunit (data not shown). In order to map
the PKA phosphorylation site(s), mutant mDBD domains carrying the
mutations S116A, S146A, and the double mutant S116A/S146A were
expressed in E. coli and purified by affinity and ion
exchange chromatography (5). To exclude indirect structural effects of
the mutations introduced, the overall conformations of the mutant mDBD
proteins were compared with wild type mDBD by fluoresence quenching
spectroscopy as described (38). We did not detect any gross structural
changes as a consequence of introducing mutations at the
Ser116 and Ser146 positions of mDBD (data not shown).
Wild type and mutant mDBD domains were used in phosphorylation site
mapping experiments with purified PKA C
The Michaelis constant (Km) of mDBD as a substrate
for PKA was determined using purified bacterially expressed mDBD, PKA
C The Oncogenic V117D Mutation in AMV-Myb Abrogates PKA-mediated
Phosphorylation--
In AMV v-Myb, the V117D point mutation located
next to the Ser116 residue abolishes the PKA recognition
motif (as defined by an extended consensus, Fig. 1A). To
examine whether the V117D mutation potentially interferes with
Ser116 phosphorylation, experiments were performed with
recombinant wild type and mutant mDBD proteins and PKA
C Phosphorylation of Ser116 Affects the Stability of
c-Myb/DNA Interaction--
In the NMR and crystal structures of c-Myb
mDBD, the Ser116 residue is in close proximity to the
sugar-phosphate backbone of the DNA helix (see Fig. 1B and
Refs. 7 and 37). A predicted consequence of phosphorylation would
therefore be interference with DNA binding due to electrostatic
repulsion. We tested this idea by phosphorylating wild type mDBD
in vitro with PKA C
We have previously shown that subtle differences in DNA binding for
c-Myb proteins may be detected by analysis of decay of protein-DNA
complexes in EMSA (decay-EMSA) (38, 47, 48). After the formation of a
protein-DNA complex, a large excess of unlabeled specific DNA
oligonucleotide is added, and the time course of complex dissociation
is followed by analyzing the remaining complexes at different time
points by EMSA. Decay-EMSA was performed on wild type mDBD, a S116D
mutant (a phosphorylation mimic), and in vitro
phosphorylated mDBD proteins (n = 4). A typical
experiment is shown in Fig. 5C. Note that 3-fold more
phospho-mDBD protein than wild type or S116D mutant mDBD was loaded
onto the gel in order to visualize the protein-DNA complexes.
PKA phosphorylation of the mDBD protein led to a dramatic increase in
the dissociation rate of the protein-DNA complex (Fig. 5C,
lanes 12-16) compared with the nonphosphorylated
mDBD (lanes 2-6). The dissociation rate of the
mDBD S116D mutant from the complex was also increased compared with
wild type mDBD (compare lanes 7-11). The
protein/DNA dissociation rates were estimated to be approximately >20
min, 2-5 min, and <1 min for wild type, S116D and
phospho-Ser116 mDBD proteins, respectively
(n = 4). Note that the S116D mutation in mDBD did not
fully mimic PKA-phosphorylated mDBD with regard to DNA binding
properties. An alternative mutant, S116E mDBD, exhibited similar
properties as the S116D mutant. The neutral S116A mDBD mutant exhibited
a similar decay rate as wild type mDBD (n = 2, data not
shown), thus excluding a general effect of mutations at
Ser116.
PKA Phosphorylation Differentially Affects c-Myb Binding to Various
MRE Sites--
In previous studies, we have developed an EMSA system
for evaluating the selectivity of both wild type and mutant forms of Myb for different DNA recognition elements. The differences detected with the set of variant MRE oligonucleotides in EMSA studies are reflected both in yeast and in animal cell transactivation systems (38,
47, 48). The effect of PKA phosphorylation of mDBD on MRE site
selectivity was investigated using a set of four variant MRE
oligonucleotides (core sequence TAACGG, TAACGT, TAACTG, and TAACTT) (47, 48). The
oligonucleotides are named after the bases in positions 5 and 6 in the
core sequence (see details in Ref. 48). As shown in Fig.
6A, phosphorylation of mDBD
led to a dramatic decrease in the ability of mDBD to bind to the tested
cognate DNA sequences compared with the wild type protein. The binding
of phospho-mDBD to the GG oligonucleotide (lanes
1-4) was similar to the results obtained with the MRE
oligonucleotide (Fig. 5). Binding to the GT and TG oligonucleotides was
almost completely abrogated (lanes 5-8 and
9-12, respectively). PhosphorImager analysis revealed that
reduction of DNA binding was ~60, 89, and 88% for the GG, GT, and TG
oligonucleotides, respectively (n = 4). The TT
oligonucleotide (lanes 13-16), which is not
bound by c-Myb, was included as a negative control.
DNA-bound Myb Is Barely Accessible to PKA Phosphorylation--
We
have previously reported that the R2 repeat (in which
Ser116 resides) undergoes a disorder-to-order transition
upon binding to DNA (43, 57, 58). Experiments were therefore performed to investigate whether this transition could affect the accessibility of Ser116 in a free mDBD versus in an
mDBD·DNA complex. Phosphorylation reactions with PKA
C c-Myb, but Not AMV v-Myb, Is Phosphorylated at Ser116
in Intact Cells--
An important question to ask was whether a
steady-state pool of Ser116-phosphorylated c-Myb existed in
cells. Antibodies against a Ser116-phosphorylated c-Myb
were generated to elucidate this possibility. As shown in Fig.
7A, the antiserum recognizes
PKA-phosphorylated mDBD but neither nonphosphorylated protein (Fig.
7A, lanes 1 and 2),
S116A-mutated mDBD (lanes 3 and 4),
nor AMV v-Myb (lanes 7 and 8). It did,
however, cross-react with an S116D phospho-mimicking mutant version of
mDBD (lanes 5 and 6), suggesting the
requirement of a negative charge at position 116. Moreover,
PKA-phosphorylated back-mutated AMV v-Myb D117V was recognized
(lanes 9 and 10), indicating that
restoration and phosphorylation of the PKA site restored binding of the
phospho-c-Myb (Ser116)-specific antibody.
The antibody was subsequently used to determine whether endogenous
c-Myb is phosphorylated at Ser116 in vivo. HD3
erythroblasts were chosen, since they express high levels of c-Myb. As
shown in Fig. 7B, the phospho-c-Myb
(Ser116)-specific antibody immunoprecipitated a fraction of
c-Myb in live cells. To analyze whether point mutations in the c-Myb
DBD affect its phosphorylation, we choose quail QT6 cells that lack endogenous c-Myb and that are an established cell line to determine Myb
functions. As shown in Fig. 7C, the phospho-c-Myb
(Ser116)-specific antibody recognized a fraction of
transfected full-length or of truncated c-Myb (lanes
8 and 11) but not of the S116A mutant (lane 14). Similarly, AMV v-Myb was not
recognized by the phospho-c-Myb (Ser116)-specific antibody,
whereas back-mutation of the critical Asp117 in AMV v-Myb
to wild type Val117 that restored the phosphorylation site
also restored antigenicity (lanes 17 and
20). We conclude that c-Myb can be phosphorylated at
Ser116 and that the V117D mutation present in AMV v-Myb
abolishes this phosphorylation site.
Introduction of Charge in Ser116 Affects Resident Myb
Target Gene Expression--
In effector-reporter experiments, we
failed to observe significant differences between wild type c-Myb,
S116A, and phospho-mimicking S116D and S116E mutations (data not
shown). However, different results were obtained when the activation of
the resident mim-1 gene by Myb and C/EBP Here we show that c-Myb is phosphorylated by the catalytic subunit
of PKA on Ser116 in the DNA-binding domain. Phosphorylation
of wild type (Ser116) mDBD severely affects binding of Myb
to DNA. Phosphorylation of Myb at Ser116 by PKA or by a
kinase with similar specificity may link the proto-oncoprotein to a
novel regulatory signal transduction pathway. A specific point mutation
of the AMV oncogene (V117D) abolishes phosphorylation at
Ser116, implying that escape from such a pathway
contributes to leukemogenicity.
AMV v-Myb carries 10 point mutations in comparison with c-Myb (23).
Four of these mutations are conservative replacements (I181V, V267I,
V270I, and R438K) and hence not primary candidates for altered
structure or function. Two mutations (L199P and L207P) introduce
additional prolines in a P-rich putative linker region between the DBD
and transactivation domain. Of the remaining four mutations (I91N,
L106H, V117D, and N285T), those located in the DBD all have been
identified as important for the transformed phenotype (39). The crystal
structures of c-Myb DBD, AMV DBD, C/EBP The concerted function of the three mutations has been linked to both
gain- and loss-of-function phenotypes of AMV v-Myb and to signaling
pathways (40). Biochemical analysis showed that position 117 lies in a
consensus site (RRX(S/T)Y) for PKA phosphorylation and that this consensus site actually represents a high affinity substrate for PKA with a Km of ~10
µM (45, 61). Intriguingly, the PKA consensus sequence and
the phosphoacceptor function are lost in v-Myb, due to the adjacent
V117D mutation. A similar PKA consensus site in A-Myb
(KRWS111L) differs from the c-Myb Ser116 site
(KRWS116V) only in the +1-position and was not efficiently
phosphorylated by PKA (56). Thus, the amino acid present in the
neighboring residues outside of the established consensus PKA site (45) influences the ability to function as a bona fide
PKA target site.
Ramsay et al. (33) located two PKA phosphorylation sites in
c-Myb by phosphopeptide mapping of a C-terminal truncated c-Myb protein, revealing Ser8 and Ser116 as
substrates for PKA in vitro. However, in contrast to our
observations, no functional effects of the phosphorylation of
Ser116 were reported. Our data show that phosphorylation of
Ser116 has biochemical and functional consequences.
Phosphorylation of Ser116 destabilizes Myb binding to DNA,
and a S116D phosphorylation mimic protein failed to activate the
resident mim-1 gene. Whether this effect is due to
destabilization of DNA interaction or loss of cooperativity with C/EBP
is currently difficult to resolve. However, we can infer that the loss
of function of AMV v-Myb to cooperate with C/EBP is at least in part
due to the mutation at V117D. Back-mutation of D117V in AMV v-Myb
regained some of the functional C/EBP/Myb-dependent gene
activation, while constitutive activation of GBX2 was severely
diminished (40). This back-mutation also changed the transformed
phenotype toward the granulocytic lineage and displayed reduced
leukemogenicity in retroviral transformation studies (39). However, the
reciprocal single mutation V117D in the wild type Myb setting was not
sufficient to abrogate C/EBP collaboration entirely or to induce
constitutive GBX2
activation.2 This is in
keeping with the recent structural data, where Ile91 and
Leu106 in wild type c-Myb directly interacted with
C/EBP Our data show that phosphorylation at Ser116 leads to
decreased binding of Myb to MREs. The destabilization of the Myb-DNA
complex upon PKA phosphorylation could be a consequence of
electrostatic repulsion, since the Ser116 residue is in
close proximity to the DNA sugar-phosphate backbone (Fig.
1C). However, additional effects of the introduced phosphate may be postulated, since a substitution of Ser116 with
aspartic acid or glutamic acid to mimic a negative charge in this
position showed a less profound inhibition of DNA binding compared with
phosphorylated mDBD. Additional effects of the phosphate group could be
either steric hindrance and/or an effect on the conformational
transition that occurs upon DNA binding.
PKA has been reported to exert a negative effect on the DNA binding
activity of several transcription factors that contain a
PKA-phosphorylation site located within or close to their DNA binding
domain, such as the transcription enhancer factor TEF-1 (62) and the
Wilms' tumor gene product WT1 (63). A peculiar feature of the negative
effect on complex stability observed with c-Myb was that the severity
of the inhibition depends on the particular MRE sequence. The
experiments were performed with mDBD domains due the inaccessibility of
purified full-length c-Myb. Nevertheless, we have previously reported
that there are only subtle differences observed in in vitro
assays between the DNA sequence preference of the mDBD and full-length
c-Myb expressed in COS cell lysates (48), suggesting that conclusions
drawn from experiments with the mDBD are applicable to the full-length
protein. Hence, our data are consistent with the notion that
Ser116 phosphorylation might down-modulate Myb target genes
in a differential fashion and thus alter the spectrum of genes
activated by c-Myb in vivo. We showed that the accessibility
of the Ser116 phosphorylation site depends on the
interaction of Myb with specific target sites, a phenomenon consistent
with a reported conformational transition of the DBD upon binding to
DNA (43). Thus, it is possible that only the pool of c-Myb that is not
bound to DNA is accessible to specific phosphorylation on
Ser116. This could severely limit the window in which c-Myb
acts as a kinase substrate.
The development of a specific phospho-c-Myb (Ser116)
antibody demonstrated that a fraction of c-Myb is phosphorylated at
this particular site in live cells. In an approach to determine the consequences of Myb Ser116 phosphorylation in cells, we
cotransfected an expression vector encoding the catalytic subunit of
PKA. However, overexpression of PKA has effects on Myb as well as on
C/EBP target genes (data not shown), making such experiments difficult
to interpret. It should be emphasized, however, that although PKA
phosphorylates Ser116 in vitro and although
phospho-Ser116 can be detected in vivo, we do
not imply that PKA is the relevant kinase in vivo. It is
equally conceivable that different kinases with similar recognition
peptide specificities are involved in regulating c-Myb functions
in vivo. Such kinases still have to be identified.
The AMV v-Myb displays both gain- and loss-of-function phenotypes due
to the combined action of the DBD mutations (40). Induction of
GBX2 expression by the c-Myb protein requires activation of
an upstream signaling cascade, whereas the mutations in the DNA binding
domain of AMV-Myb render the protein independent of such signaling
events (40). Although there is currently no evidence that
phosphorylation of Ser116 is involved directly in GBX2
activation, it is possible that stabilization of v-Myb DNA binding
contributes to the activation of distinct Myb target genes.
Alternatively, phosphorylation of Ser116 might affect
protein-protein interactions such as with co-activators or
co-repressors. Finally, this phosphorylation might affect other processes linked to gene regulation such as removal of transcriptional activation to prevent constitutive gene expression (64-66).
What could be the role of PKA regulation of c-Myb in the hematopoietic
system? It is well known that whereas cAMP usually is mitogenic and a
stimulator of hormone secretion in endocrine cells, it generally
delivers an "off"-signal for proliferation in cells of the
immune system (67, 68). Thus, a PKA-induced decrease in the activity of
c-Myb, a factor associated with proliferation, would be a consistent
response in immune cells. Among the targets of c-Myb in T-cells is the
RAG-2 (69), a protein required for recombination of B and T cell
antigen receptor chains. It is intriguing that the rearrangement of the
TCR
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) in the activation of the
resident mim-1 gene. These findings implicate PKA or a
kinase with similar specificity in a novel type of regulation of c-Myb
function from which v-Myb has escaped.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol), 2.5 µg of bovine serum
albumin, and 3.8 units of PKA C
(Roche Molecular
Biochemicals) in a final volume of 120 µl. Reactions were incubated
at 30 °C for 15 min. Reactions were chased with 1.4 µmol of ATP
and placed on ice before loading on a 15% SDS-PAGE gel and
autoradiography. An alternative buffer (10 mM MgAc, pH
~8.5, with or without 4 units of PKA C
(Promega)) was
used for [
-32P]ATP labeling of mDBD proteins for
phosphoamino acid analysis. 250 µM ATP was added for
quantitative phosphorylation of mDBD proteins for mass spectrometry
analysis. Phosphoamino acid analysis of 32P-phosphorylated
wild type and mutant S146A mDBD domains was performed as described
(44). A matrix-assisted laser desorption-ionization time-of-flight mass
spectrometer (Voyager-RP DE; Applied Biosystems) was employed to
measure the molecular mass of PKA-phosphorylated wild type and mutant
mDBDs. All experiments were carried out with the mass spectrometer in
the linear positive ion mode. The total acceleration voltage was 25 kV.
The voltage on the first grid and the delay time between ion production
and extraction were adapted to the mass of the different samples. 100 single scans were accumulated for each spectrum. The matrix,
3,5-dimethoxy-4-hydroxycinnamic acid (D-7927; Sigma), was
dissolved at a concentration of 15 mg/ml in a mixture of 1:1
acetonitrile, 0.1% aqueous trifluoroacetic acid. 0.5 µl of sample
and 1.5 µl of matrix were mixed together on the sample plate and
air-dried. All data were calibrated using an external calibration
standard mixture (Applied Biosystems).
-32P]ATP, and 46 units of PKA C
enzyme
(Promega) in a final volume of 50 µl. The concentration of 500 µM ATP was sufficient for saturating the PKA catalytic
subunit. The reactions were terminated by adding 2 µl of 0.5 M EDTA at 0, 2, 4, and 6 min. Samples were spotted in
triplicate onto Whatman P81 paper, washed in 75 mM H3PO4, and dried in 96% EtOH before
scintillation counting. The 2-min time point (in the linear range) was
chosen for the calculation of Km.
enzyme (Promega) (optimized) in 20 µl at 30 °C
for 30 min. Reactions were terminated by the addition of 2 µl of 0.5 M EDTA. Reproducible quantitative phosphorylation of mDBD
was >75%, as estimated by incorporation of
[32P]phosphate and by mass spectrometry analysis. Note
that the unit definitions for the two PKA enzyme sources used in the
study differ; substrates for activity are casein for the Promega enzyme
and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (45) for the Roche enzyme. One casein unit is equivalent to 1.6 × 105 Kemptide
units (Roche Molecular Biochemicals).
-32P-labeled MRE oligonucleotide probe (10 fmol) in a
total volume of 20 µl for 10 min at 25 °C before electrophoresis.
Binding reactions were run on 6% 0.5× TBE, 5% glycerol PAGE gels at
4 °C. Decay EMSA was performed as described (38). Briefly, protein
and 32P-labeled oligonucleotide were mixed and incubated at
25 °C for 15 min before competition with an excess of unlabeled
competitor oligonucleotide for various times. Samples were then
immediately loaded and run on an EMSA gel. For binding experiments with
phosphorylated proteins, proteins were first phosphorylated in
vitro by PKA C
catalytic subunit (described above)
before they were used in EMSA analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PKA consensus sites in the DNA-binding
domains of Myb family members. A, a comparison of PKA
consensus sites in mammalian c-Myb, B-Myb, and A-Myb and viral AMV
v-Myb proteins. The numbering is according to chicken c-Myb.
B, the putative PKA targets Ser116 and
Ser146 in the mDBD are shown in the published crystal
structure of the c-Myb DBD (space-filling) complexed with
DNA (sticks) (7). C, the AMV v-Myb DBD·DNA
complex (37) is shown, and the three mutant amino acids (I91N, L106H,
and V117D) are highlighted together with Ser116.
B and C were drawn using RASMAC and the 1h89 and
1h8a coordinate files (Protein Data Bank).
catalytic subunit and [
-32P]ATP. The results in Fig.
2A showed that the S116A
mutation (lanes 3 and 5) completely
abolished the mDBD phosphorylation (lane 2), whereas the S146A mutation (lane 4) had no
effect. An SDS-PAGE analysis of the mDBD proteins demonstrated equal
amounts of protein in the phosphorylation reactions (Fig.
2A, lower panel). Phosphoamino acid
analysis of wild type and S146A mutant mDBD domains confirmed phosphorylation on serine residues only (Fig. 2B). Mass
spectrometry analysis showed the addition of one phosphate group in
wild type and S146A mDBD but not in S116A mDBD (Fig. 2C). In
conclusion, of the three serines present in c-Myb mDBD
(Ser116, Ser146, and Ser187), only
Ser116 is a PKA target site.
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Fig. 2.
In vitro mapping of the PKA
phosphorylation site in c-Myb mDBD. A, recombinant wild
type (wt) and mutant mDBD (S116A, S146A, and S116A/S146A)
domains (8 pmol) were incubated with purified PKA C (3.8 units; Roche) and [
-32P]ATP for 15 min. Reactions were
separated on a 15% SDS-PAGE gel and detection by autoradiography. The
arrows denote phosphorylated proteins and free
[
-32P]ATP. Lower panel,
Coomassie staining of an SDS-PA gel with parallel samples of the same
proteins as in the upper panel. Lanes
1-4 contain wild type, S116A, S146A, and S116A/S146A mDBD
proteins, respectively. B, phosphoamino acid analysis of
32P-radiolabeled wild type and S146A mDBD proteins.
Outlines of the ninhydrin staining of unlabeled
phosphoserine (P-Ser), phosphothreonine (P-Thr),
and phosphotyrosine (P-Thr) standards are indicated.
C, molecular mass measurements of wild type, S116A, and
S146A mDBD proteins incubated in the absence or presence of
PKA-C
are given in daltons. Molecular masses calculated
from the primary sequence (calculated), the increase in molecular mass
in the presence of PKA (
), and the calculated mass increase of one
phosphate group addition are also shown.
and [
-32P]ATP as described under
"Experimental Procedures" (Fig. 3).
The Km was calculated to be 10 µM.
This value is in good accordance with other well defined substrates for
PKA. The determination of Ser116 in c-Myb as a high
affinity site for PKA is consistent with conclusions by Ramsay and
co-workers, who reported that Ser116 in C-terminally
truncated recombinant Myb proteins was phosphorylated by PKA in
vitro (33).
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Fig. 3.
Km determination of PKA
for Ser116 in c-Myb mDBD. Recombinant mDBD protein (0, 5, 5,6 6,2, 7,6, 10, 15, 16.6, or 20 µM) was
phosphorylated for 0. 2, 4, and 6 min with 500 µM ATP,
3.3 pmol of [ -32P]ATP, and PKA C
subunit (46 units (Promega)) as described under "Experimental
Procedures." The reactions were stopped by the addition of 2 µl of
0.5 M EDTA and spotted on P81 phosphopaper, washed, and
counted by liquid scintillation counting. The Km of
PKA for R2R3 was calculated in the linear
reaction range at 2 min by linear regression analysis
(n = 3).
, as shown in Fig. 4. In
contrast to wild type mDBD, the AMV mDBD (R2R3
carrying the three AMV mutations I91N, L106H, and V117D) (38) was not phosphorylated by PKA (Fig. 4, lanes 2 and
3). Introduction of the single AMV-Myb point mutation V117D
in c-Myb also abrogated the PKA phosphorylation of the domain
(lane 4). In the reciprocal experiment,
back-mutation of the Asp117 to Val117 restored
PKA phosphorylation of the AMV-Myb mDBD (Fig. 4, lane 7). We therefore conclude that c-Myb but not v-Myb can be
phosphorylated at Ser116.
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Fig. 4.
The effect of the AMV V1117D mutant on
Ser116 phosphorylation in c-Myb mDBD. Upper
panels, recombinant wild type (wt) and mutant
mDBD proteins (AMV, V117D, and AMV/D117V), as indicated, were incubated
with purified PKA C (3.8 units; Roche Molecular
Biochemicals) and [
-32P]ATP for 15 min and analyzed as
in Fig. 2. Lower panel, Coomassie staining of the
same gel as in the upper left
panel.
and assaying DNA binding
on EMSA gels using the mim-1A binding site (MRE) (46) as DNA
probe. Quantitative phosphorylation was consistently >75%, as
calculated by 32P incorporation and mass spectrometry. As
shown in Fig. 5A, the DNA
binding activity of phospho-mDBD was reduced (lanes
1 and 3) compared with nonphosphorylated mDBD
(lanes 2 and 4). Phosphorylated mDBD
migrated also slightly faster in the gel than nonphosphorylated mDBD.
Phosphorylation of mDBD diminished DNA binding but did not totally
abolish complex formation. We confirmed that phospho-mDBD was able to
bind to the cognate MRE sequence by running EMSA reactions with
[
-32P]ATP-labeled mDBD protein and unlabeled DNA
oligonucleotide (Fig. 5B, lanes 2 and
3), side by side with ordinary EMSA reactions with unlabeled
phospho-mDBD and 32P-labeled DNA oligonucleotide
(lanes 4 and 5). Again, note the reduced binding of phospho-mDBD to the MRE oligonucleotide compared with wild type mDBD (lanes 4 and
5).
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Fig. 5.
Effects of phosphorylation on the DNA binding
activity of c-Myb mDBD. A, 25 or 10 fmol of
phosphorylated (+) or nonphosphorylated ( ) mDBD was bound to
32P-labeled MRE and run on an EMSA gel. B,
complexes containing phospho-mDBD/32P-labeled MRE
oligonucleotide or 32P-mDBD/unlabeled MRE oligonucleotide
were compared on an EMSA gel. Lanes 2 and
3, 32P-phosphorylated mDBD alone
(lane 2) or bound to unlabeled MRE
(lane 3). Lanes 4 and
5, nonradioactive phospho-mDBD (lane
4) or nonphosphorylated mDBD (lane 5)
bound to 32P-labeled MRE oligonucleotide. The
arrows show the position of the mDBD·DNA complex, the free
MRE oligonucleotide (lane 1), and free
[
-32P]ATP (lanes 2 and
3). C, decay EMSA, where 20 fmol of wild type
mDBD (lanes 2-6) or S116D mutant mDBD
(lanes 7-11) or 60 fmol of phospho-mDBD
(lanes 12-16) was first bound to the MRE
oligonucleotide (10 fmol) and then competed with unlabeled MRE (750 fmol) for the times indicated before loading on the EMSA gel. Note that
for phospho-mDBD, 3 times the standard amount of protein was used in
each reaction in order to visualize the phospho-mDBD·DNA complexes.
One representative experiment of four is shown.
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Fig. 6.
MRE variants respond differentially to PKA
phosphorylation of c-Myb mDBD. A, the ability of
phospho-mDBD to distinguish between MRE family Myb binding sites. Four
Myb binding site oligonucleotides were used for comparing the
selectivity of phospho-mDBD to nonphosphorylated mDBD. The MRE-GG, -GT,
-TG, and -TT (negative control mutant) oligonucleotides refer to
specific positions in the core MRE sequence (see "Experimental
Procedures"). Recombinant mDBD ( ) or in vitro PKA
phosphorylated mDBD (+) (20 and 35 fmol each) was bound to MRE
oligonucleotides MRE-GG, -GT, -TG, and -TT (10 fmol each) and run on
EMSA gels. The gel was dried and autoradiographed, and signals were
quantified in a PhosphorImager. One representative experiment of four
is shown. B, accessibility of mDBD for phosphorylation by
PKA. Recombinant mDBD alone (mDBD) (0.8 µM)
(lanes 1-6), mDBD in complex with
mim-1A oligonucleotide (mDBD/MRE) (0.8 µM each) (lanes 7-12) or in
complex with nonspecific oligonucleotide
(mDBD/NSO) (0.8 µM each)
(lanes 13-18) was incubated with PKA
C
in the presence of [
-32P]ATP for
0-30 min, as indicated, before SDS buffer was added to terminate the
reactions. Reaction mixtures were separated by SDS-PAGE, and the gel
was autoradiographed.
subunit were performed on free mDBD protein, on mDBD
preincubated with a specific DNA oligonucleotide (MRE), or mDBD
preincubated with a nonspecific DNA oligonucleotide. The preformation
of a mDBD·MRE complex severely reduced the phosphorylation of mDBD
compared with free mDBD protein (Fig. 6B, lanes
7-12 compared with lanes 1-6). In
contrast, preincubation of mDBD with a nonspecific oligonucleotide
barely affected PKA-mediated mDBD phosphorylation (Fig. 6B,
lanes 13-18). This demonstrates that
sequence-specific DNA binding severely reduces the accessibility of the
Ser116 site. This could be due to direct shielding by DNA
or could be a consequence of a DNA-induced conformational change in
R2. Irrespective of the mechanism, the results suggest that
Ser116 can be present in an "open" or "closed"
conformation, being accessible and efficiently modified in the free
protein, but changing into a site with poor accessibility for the
kinase in the protein-DNA complex.
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Fig. 7.
Ser116 phosphorylation occurs
in vivo. A, specificity of a
phospho-c-Myb (Ser116)-specific antibody. Recombinant wild
type (wt) and mutant mDBD (S116A, S146D, AMV, and
AMV(D117V)) purified domains were either subjected to mock ( ,
lanes 1, 3, 5,
7, and 9) or PKA C
(+,
lanes 2, 4, 6,
8, and 10) phosphorylation before loading on a
15% SDS-PAGE gel. Subsequent Western blot analysis was performed using
either a phospho-c-Myb (Ser116)-specific polyclonal
antibody (upper panel) or the 5E11 monoclonal
anti-Myb antibody (lower panel). B,
immunoprecipitation of a fraction of endogenous c-Myb by the
phospho-c-Myb (Ser116)-specific antibody from unstimulated
live HD3 erythroblasts. Proteins were precipitated from cell lysates
with a preimmune rabbit serum, a Myb-specific rabbit immune serum, and
a phospho-c-Myb (S116)-specific antiserum, as indicated.
Immunoprecipitates were separated by 10% SDS-PAGE and transferred to
polyvinylidene difluoride membranes, and proteins were revealed with a
mouse monoclonal antibody directed against c-Myb (MYB2-7.77; ATCC).
C, immunoprecipitation of Myb from QT6 fibroblasts depends
on the integrity of the PKA consensus sequence. QT6 cells were
transfected with the indicated expression constructs. Samples from the
lysates were also loaded as expression control.
was examined.
The chicken macrophage cell line HD11 was chosen for these experiments,
because it constitutively expresses C/EBP
/NF-M but not c-Myb (53,
59). Wild type Ser116 c-Myb constructs induced
transcription of the mim-1 resident gene as assayed by
Northern blotting (lanes 4 and 6). The
activation was only slightly affected by the neutral S116A mutation
(compare lanes 6 and 7) or by the
V117D mutant (lane 9). However, both the
phospho-mimicking mutants S116D and S116E severely inhibited mim-1 mRNA induction (lanes 8 and
10). This suggests that a negative charge at position 116 abrogates expression of a resident chromosomal c-Myb target gene.
Similar observations were made in QT6 cells transfected with both Myb
and C/EBP
expression vectors (data not shown). The latter
experiments also showed that a C/EBP
-only target gene (designated
126; see Ref. 60) was unaffected by the coexpression of
various Myb mutants, excluding indirect effects (data not shown).
Co-expression of the PKA catalytic subunit strongly reduced
mim-1 expression, as shown in Fig.
8 (upper panel,
lane 5). This was not due to reduced c-Myb
expression, as shown in Fig. 8 (middle panel).
However, cotransfection of PKA negatively regulates the induction of
mim-1 also by wild type Ser116 and by the S116A
Myb mutant as well as the expression of Myb independent C/EBP target
genes (data not shown). This suggests pleiotropic effects of the
catalytic subunit of PKA on other factors involved in Myb- and
C/EBP-induced gene activation and, beyond the analysis of Myb mutants
examined, currently precludes further analysis of PKA-specific effects
on Myb.
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Fig. 8.
Ser116 modification affects
resident mim-1 expression in vivo. The HD11 cell
line was transfected with the indicated expression constructs. RNA was
extracted after 24 h. Blots with poly(A)-enriched RNA were
sequentially probed with labeled mim-1 (upper
panel), c-myb (middle
panel), and GAPDH (lower
panel) fragments, the latter serving as loading
control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and DNA complexes confirm
the importance of both the I91N and L106H mutations. These mutations
break specific contacts with the C/EBP
transcription factor,
contribute to a shift in the
2 helix in the
R2 domain, and weaken the interaction with the DNA
phosphate backbone (37). The question about the significance of the
third point mutation at position 117, however, remained to be solved.
, whereas Val117 indirectly affects C/EBP
interaction (37). Accordingly, the point mutation at V117D required the
other R2 v-Myb mutations to achieve the complete v-Myb
phenotype. Taken together, these data suggest that the phosphorylation
at Ser116 or abrogation of this phosphoacceptor site by the
AMV v-Myb-specific mutation has multiple effects on Myb function,
including a direct influence on the interaction of the DBD of Myb with
its co-factors.
locus is completely inhibited upon activation of the cAMP
signaling pathway (70) and that one of the components of the V(D)J
recombinase is also a bona fide Myb target,
having an MRE sequence of the PKA-sensitive type (TAACTG) (69). Whether
PKA inhibition of c-Myb contributes to this effect and whether and how
other genes are affected by Ser116 phosphorylation,
however, remains to be investigated.
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ACKNOWLEDGEMENTS |
---|
We thank A. C. Østvold (Institute for Medical Biochemistry, University of Oslo) for assistance with phosphoamino acid analysis and Dimitrios Mantzilas (Department of Biochemistry, University of Oslo) for help with mass spectrometry measurements.
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FOOTNOTES |
---|
* This work was supported by the Norwegian Cancer Society (to K. B. A., E. M. B., and O. S. G.), the Norwegian Research Council (to O. S. G.), the Anders Jahre Foundation (to O. S. G.), the Blix Foundation for Medical Research (to K. B. A.), and the Deutsche Forschungsgemeinschaft (to E. K. L. and A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Institute for Experimental Medical Research, Ullevål Hospital, 0407 Oslo, Norway.
¶ The two first authors contributed equally to the work.
** To whom correspondence should be addressed: Dept. of Biochemistry, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo, Norway. Tel.: 47-22-85-73-46; Fax: 47-22-85-44-43; E-mail: O.S.Gabrielsen@ biokjemi.uio.no.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M209404200
2 E. Kowenz-Leutz and A. Leutz, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: DBD, DNA-binding domain; AMV, avian myeloblastosis virus; EMSA, electrophoretic mobility shift assay; mDBD, minimal DNA-binding domain; MRE, Myb recognition element; PKA, protein kinase A; v-Myb, viral Myb; C/EBP, CCAAT/enhancer-binding protein; CREB, cAMP-response element-binding protein.
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REFERENCES |
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