Regulation of Myeloid Zinc Finger Protein 2A Transactivation
Activity through Phosphorylation by Mitogen-activated Protein
Kinases*
Hironori
Ogawa,
Ayako
Murayama,
Shigekazu
Nagata, and
Rikiro
Fukunaga
From the Department of Genetics B-3, Graduate School of Medicine
and Graduate School of Frontier Biosciences, Osaka University, and
Core Research for Evolutional Science and Technology, Japan Science and
Technology Corporation, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received for publication, July 29, 2002, and in revised form, October 16, 2002
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ABSTRACT |
The myeloid zinc finger protein (MZF)-2 is a
C2H2 zinc finger transcription factor
that is expressed in myeloid cells and involved in the growth,
differentiation, and tumorigenesis of myeloid progenitors. Here we
describe a novel isoform of MZF-2, designated MZF-2A, and show that it
is phosphorylated by the mitogen-activated protein (MAP) kinases. An
in vitro phosphorylation experiment revealed that the
transactivation domain (TAD) of MZF-2A was phosphorylated strongly by
extracellular signal-regulated kinase (ERK) and phosphorylated weakly
by p38 MAP kinase but not by Jun N-terminal kinase. Experiments using
"add-back" mutants showed that three serine residues
(Ser257, Ser275, and Ser295) in the
TAD were phosphorylated in vitro by ERK. In myeloid LGM-1 cells, various extracellular stimuli induced the phosphorylation of
these serine residues, which was differentially inhibited by the
protein kinase inhibitors U0126 and SB203580. Substitution of these
phosphorylation sites with alanines resulted in a strong enhancement of
the ability of MZF-2A to activate transcription in a luciferase
reporter assay. Taken together, these results indicate that
MZF-2A is a novel target for the ERK and p38 MAP kinase signaling
pathways, and its transactivation activity is negatively regulated by
MAP kinase-mediated phosphorylation of the TAD.
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INTRODUCTION |
The development of mature blood cells occurs through the
proliferation and differentiation of hematopoietic progenitor cells and
is controlled by the combined effects of hematopoietic cytokines and
transcription factors (1-4). A set of zinc finger transcription factors, including GATA, EKLF, and Ikaros, plays important roles in the
hematopoietic commitment to specific lineages (1, 4). Myeloid zinc
finger protein (MZF)1 is one
of the candidates in this family for controlling the development of the
myeloid cell lineage. MZF is a Krüppel-like
C2H2 zinc finger protein expressed
predominantly in myeloid progenitor cells, particularly at the
myeloblast and metamyelocyte stage (5-9). Blocking MZF expression with
antisense oligonucleotides inhibits granulocyte colony-stimulating
factor-induced neutrophilic colony formation in vitro,
suggesting that MZF is involved in the proliferation of myeloid
progenitors and/or their differentiation to neutrophilic granulocytes
(6). Furthermore, disruption of the MZF gene in mice results in an
accumulation of Mac-1+ myeloid cells in bone marrow and the development
of neoplasias composed of highly proliferating myeloid blasts in the
liver and spleen, indicating that MZF plays a role in negative
regulation of proliferation and tumorigenesis of hematopoietic
progenitors (10).
We and others have shown that several isoforms of MZF proteins are
produced by alternative transcriptional initiation sites and splicing
from a single gene in both human and mouse cells (5, 7, 11). Human
MZF-1, originally isolated from myeloid leukemia cells, encodes a
485-amino acid protein consisting of 13 zinc finger motifs that bind
specific DNA sequences (5, 12). By the extensive screening of mouse and
human cDNA libraries, we identified an alternative MZF product,
termed MZF-2, which shares the sequence of its C terminus, including
the zinc finger domains, with MZF-1 but also contains a long N-terminal
extension consisting of an acidic domain, a leucine-rich (LeR; also
called SCAN box) domain, and a transactivation domain (TAD) (7).
Functional analysis of the MZF-2 subdomains revealed that the TAD works
specifically in myeloid cells and that the far N-terminal region
spanning the acidic and LeR domains inhibits the neighboring TAD (13).
Recently, Peterson and Morris (11) reported another form of the gene
product, MZF1B/C, whose entire structure is almost identical to that of MZF-2 but differs in the N-terminal region. The consensus MZF-binding sites are found in the promoters of several hematopoietic cell-specific genes, such as CD34, c-myb, lactoferin, and myeloperoxidase (12, 14).
The MZF-binding motif is also found in the promoter of the telomerase
reverse transcriptase gene, where MZF is likely to function as a
negative regulator for gene expression (15). The composite structure of
negative and positive regulatory domains for transcription implies that
the function of MZF could be regulated by posttranslational
modification and/or processing.
The activity of many transcription factors is regulated in a rapid and
reversible manner by specific phosphorylation. Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine protein kinases activated by various extracellular stimuli, including growth
factors, cytokines, and environmental stresses (16-18). Currently,
four distinct MAP kinase pathways are known in mammals. The
extracellular signal-regulated kinase (ERK) and ERK5/BMK1 pathways
mainly convey signals from mitogenic and differentiation stimuli,
whereas the Jun N-terminal kinase (JNK) and p38 MAP kinase pathways
seem to be involved in transducing various stress- and cytokine-triggered signals. Activated MAP kinases in turn phosphorylate a set of cytoplasmic substrates such as MAP kinase-activated protein kinases (18, 19) and various nuclear targets including transcriptional activators, c-Myc, Elk-1, c-Jun, ATF2, and HSF1 (20-29), as well as
transcriptional repressors such as Bcl-6 and the ETS-type repressors (30-32).
Here we first showed that a novel isoform of MZF-2, designated MZF-2A,
is produced by an alternative use of transcription start sites and
subsequent splicing. We then studied the regulatory mechanism of MZF-2A
transcriptional activity and found that three serine residues in its
transactivation domain are phosphorylated by ERK and p38 MAP kinases
in vitro and in vivo. Mutation of the phosphorylation sites to alanines markedly increased the
transactivating potential of MZF-2A, suggesting that MZF-2A activity is
negatively regulated by MAP kinase-mediated phosphorylation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The mouse myeloid cell line LGM-1 (33, 34) and
its transformant cell lines were maintained in growth medium (RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Invitrogen), 45 units/ml recombinant mouse interleukin 3 (IL-3) , 75 µM
2-mercaptoethanol, 100 µg/ml kanamycin, and 100 µg/ml streptomycin).
Plasmid Construction--
The expression plasmid for FLAG
epitope-tagged MZF-2A, pFLAG-MZF-2A, was constructed by inserting
MZF-2A cDNA downstream of the FLAG epitope sequence
(MDYKDDDDKAG) in a pEF-BOS-EX expression vector (13). To
construct the pGL3-4xF113B-G-Luc reporter plasmid, the SV40 promoter
sequence of pGL3-Promoter vector (Promega) was replaced by a DNA
fragment carrying the tetrameric MZF-binding site
(AGTGGGGAgaaaggatctggctGGTGAGGGGGAAtcggatct) (12) and the granulocyte
colony-stimulating factor promoter sequence, derived from the
pBPA-4xF113B-G-Luc plasmid (13). The pRL-SV40 plasmid was purchased
from Promega. The pGAL4-MZF-TAD plasmid was generated by inserting a
fusion construct encoding the DNA-binding domain of yeast GAL4 (amino
acids (aa) 1-146) (35), the FLAG epitope sequence, and a cDNA
fragment corresponding to the MZF-TAD (aa 155-336) in-frame into
pEF-BOS-EX. The deletion mutants of pGAL4-MZF-TAD were constructed by a
combination of restriction enzyme digestion and PCR. The
pGAL4-Pax6 TAD plasmid was made by replacing the MZF-2A sequence of
pGAL4-MZF-TAD with the cDNA fragment corresponding to the
transactivation domain (aa 271-422) of mouse Pax6 (36). pMZF-TAD was
made by inserting the DNA fragment for MZF-TAD into pEF-BOS-EX. The
reporter plasmid pUASG-Luc containing the interferon-
minimal promoter and three GAL4-binding sites was described previously (35). The pGEX-MZF-TAD plasmid was constructed by inserting the DNA
fragment for the MZF-TAD (aa 155-336) into the pGEX-5X1 vector
(Amersham Biosciences). A series of mutants with disrupted phosphorylation sites was generated by recombinant PCR.
In Vitro Phosphorylation of GST-MZF-TAD--
Active human ERK1
was produced in insect Sf9 cells as described previously (37).
To produce active human p38 and JNK1, HeLa cells were transiently
transfected with expression plasmids for FLAG-tagged p38 (38) or
hemagglutinin-tagged JNK1 (39) and stimulated with 0.7 M NaCl for 30 min or 10 µg/ml anisomycin for 30 min,
respectively. Activated kinase was immunoprecipitated using specific
antibodies (anti-FLAG M2 Affinity Gel (Sigma) for FLAG-p38 and
anti-hemagglutinin 16B12 monoclonal antibodies (BAbCO) for
hemagglutinin-tagged JNK1), and the immune complexes were used for
in vitro phosphorylation of GST fusion proteins.
The GST-MZF-TAD proteins were produced in Escherichia coli
BL21-CodonPlus cells (Stratagene) and purified as described previously (40). Two µg of GST, GST-ATF2-(1-253), GST-Elk-1-(307-428), GST-c-Jun-(1-79) (37), or GST-MZF-TAD protein was suspended in 20 µl
of kinase buffer (20 mM Hepes-Na, pH 7.4, 10 mM
MgCl2, 1 mM dithiothreitol, and 25 µM unlabeled ATP) containing 2 µCi of
[
-32P]ATP, and the phosphorylation reaction was
initiated by the addition of each MAP kinase. After a 30-min incubation
at 30 °C, samples were separated by SDS-PAGE and visualized by autoradiography.
Transformation of LGM-1 Cells and Western Blotting--
To
obtain cell lines stably expressing MZF-2A protein, 5 × 106 LGM-1 cells were transfected with 20 µg of
AatII-linearized pFLAG-MZF-2A plasmid together with 1 µg
of XhoI-digested pST-neoB by electroporation and cultured
for 10 days in growth medium containing 0.5 mg/ml G418 (Sigma) as
described previously (41). The G418-resistant clones were tested for
FLAG-MZF-2A expression by immunoprecipitation using anti-FLAG M2
Affinity Gel (Sigma) and subsequent Western blotting using anti-MZF-2A antiserum.
For immunoprecipitation, the cells were lysed in radioimmune
precipitation assay buffer (Hepes-Na, pH 8.0, 150 mM NaCl,
1 mM EGTA, 1.5 mM MgCl2, 10%
glycerol, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate,
0.1% (w/v) SDS, 20 nM calyculin A (Calbiochem), 1 mM dithiothreitol, 2 mM Pefabloc SC (Roche), 1 µg/ml aprotinin, and 1 µg/ml pepstatin), and the supernatant was
recovered after centrifugation at 15,000 × g for 10 min at 4 °C. FLAG-MZF-2A was immunoprecipitated with anti-FLAG M2
Affinity Gel and analyzed by immunoblotting with anti-MZF-2A antiserum.
The Anti-ACTIVE MAP kinase antibodies were purchased from Promega.
Anti-mouse MZF-2A antiserum was prepared by immunizing rabbits with
bacterially produced GSTMZF-TAD.
Luciferase Assay--
LGM-1 cells were suspended at a density of
1 × 107 cells/ml in 500 µl of serum-free RPMI 1640 medium supplemented with 10 mM Hepes (pH 7.2) and mixed
with 20 µg of plasmid DNA. After electroporation at 360 V with a
capacitance of 960 microfarads in a 0.4-cm gap cuvette, the cells were
transferred to 2 ml of growth medium and cultured for 18 h.
Firefly and Renilla luciferase activities were assayed using
the Dual-Luciferase Reporter Assay System (Promega). The firefly
luciferase activity of each sample was normalized to the
Renilla luciferase activity used as an internal standard.
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RESULTS |
Identification of a Novel Form of the Mouse MZF-2 Gene
Product--
Previously, we isolated a murine cDNA for MZF-2 that
encoded a polypeptide of 814 amino acids (Fig.
1A) (7). In the course of
characterizing the MZF-2 transcripts by Northern blotting, however, we
found that the major MZF-2 mRNA species of about 3 kb encoded an
alternative protein product lacking the N-terminal region of the
reported MZF-2 sequence. As shown in Fig. 1B, when poly(A)+ RNA extracted from mouse myeloid cell lines was
analyzed by Northern blotting, the NP2 probe spanning the acidic and
LeR domains detected a major 3-kb transcript and two minor transcripts
of 4 and 8 kb in the RNAs from LG and NFS-60 cells, whereas the NP1
probe derived from the 5' end of the cDNA hybridized only to the
4-kb transcript, indicating that the NP1 sequence was not present in
the 3-kb transcript. A BLAST search of the expressed sequence tag data
base using the NP2 sequence as a query identified an expressed sequence
tag clone (GenBank accession number AA499285) that contained not only the NP2 region but also an additional 5' sequence that was different from the NP1 sequence. Sequencing analysis of the 5'-flanking region of
the MZF genomic clones as well as reverse-transcription-PCR analysis of
the RNA from the myeloid cell lines revealed that the 5' sequence of
AA499285 was derived from an upstream exon (designated exon 0) and
represented the 5'-untranslated region of the 3-kb mRNA species
(Fig. 1A). Consequently, this mRNA would encode a
735-amino acid polypeptide that lacked the N-terminal 79 amino acids of
the previously reported MZF-2 protein. We conclude that the short form
of MZF-2, designated hereafter as MZF-2A (MZF-2 Alternative
form), is the major product of the MZF gene, whereas the long form is
very minor.

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Fig. 1.
Identification of MZF-2A as an alternative
form of the MZF gene products. A, schematic representation
of two mRNA species produced from the mouse MZF gene. The
white and black boxes represent noncoding and
coding regions, respectively, in the exons of the MZF gene. The
arrows indicate putative transcription start sites. MZF-2
mRNA, which is produced from 5 exons, encodes a polypeptide of 814 amino acids (top diagram), whereas the alternative
transcript, for MZF-2A, is produced from 6 exons and encodes a
735-amino acid polypeptide (middle diagram). The coding
region in each mRNA is boxed, and characteristic domains
are indicated as follows: acidic region, A; LeR domain,
LeR; transactivation domain, TAD; and zinc
fingers, Z. The structure of the KM3 mutant is also shown
(bottom diagram). The positions of the NP1 (nucleotide
1-503) and NP2 (nucleotide 500-1021) probes and the AA499285
expressed sequence tag are indicated by thick bars.
B, poly(A)+ RNAs from LG, LGM-1, and NFS-60
cells were analyzed by Northern blotting using the NP1 (left
panel) and NP2 (right panel) probes. C,
LGM-1 cells were transfected with 10 µg of MZF-2, MZF-2A, or KM3
expression plasmid together with 10 µg of pGL3-4xF113B-G-Luc and 20 ng of pRL-SV40 plasmids, and the firefly and Renilla
luciferase activities were measured as described under "Experimental
Procedures." The data are presented as the mean with standard errors
of the relative luciferase units (RLU) obtained from two
independent experiments.
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In our previous study, we showed that the N-terminal region of MZF-2
negatively regulates its transcriptional activity (13). To examine
whether the short form MZF-2A still retains the inhibitory domain,
mouse myeloid LGM-1 cells, which do not express endogenous MZF (Fig.
1B), were co-transfected with a luciferase reporter plasmid
carrying the MZF-binding sites (pGL3-4xF113B-G-Luc) and an effector
plasmid expressing MZF-2, MZF-2A, or an N-terminal deletion mutant, KM3
(Fig. 1A) (13). The level of reporter expression was then
compared. Although MZF-2A showed significant transactivating activity
when compared with MZF-2 or the vector control, its activation level
was much lower than that of the KM3 mutant, which lacked a 155-aa
N-terminal sequence that MZF-2A contained, indicating that the
N-terminal domain of MZF-2A functions as a negative regulatory domain
(Fig. 1C).
Structural Similarity between the Transactivation Domains of MZF-2A
and Pax6--
We previously showed that the TAD of MZF-2A resided in
the middle region of the molecule (Fig. 1A) (13). To
characterize the TAD function independent of the zinc finger domain, we
generated several chimeric proteins in which various regions of the TAD were fused to the DNA-binding domain of yeast GAL4 protein and examined
their ability to activate transcription of the reporter gene carrying a
GAL4-binding promoter. As shown in Fig.
2A, the GAL4-MZF-TAD fusion
protein activated the transcription of the reporter gene in LGM-1
cells. Deletions from the C terminus severely impaired transcriptional
activation (D1 and D2 mutants), whereas N-terminal deletions up to aa
239 did not affect it significantly (D3 and D4). Further combinations
of deletions revealed that the D5 and D6 mutants were able to activate
transcription, but D7 was not, indicating that the 50-aa region from aa
239-288 is the minimal TAD of MZF-2A. This finding was further
supported by the inability of the D8 mutant, which lacked only the D6
region, to activate transcription.

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Fig. 2.
Characterization of the minimal TAD of
MZF-2A. A, various parts of the MZF-TAD were fused to the
DNA-binding domain of yeast GAL4. LGM-1 cells were transfected with 10 µg of the GAL4-MZF-TAD expression plasmid, 10 µg of the
pUASG-Luc reporter plasmid, and 20 ng of the pRL-SV40
reference plasmid, and the cells were harvested for firefly and
Renilla luciferase assays after an 18-h incubation. The
firefly luciferase activities are expressed as percentages of the value
obtained with pGAL4-MZF-TAD, after normalization to the
Renilla luciferase activity. The error bars
indicate standard errors of the mean of two independent experiments.
B, alignment of the amino acid sequences of the MZF-2A D6
region (top sequence) and the TAD of mouse Pax6
(bottom sequence). The serine residue indicated by an
asterisk in the Pax6 sequence corresponds to the site
phosphorylated by ERK/p38 MAP kinases in zebrafish Pax6. C,
effect of MZF-TAD expression on Pax6 TAD-mediated transcriptional
activation. LGM-1 cells were co-transfected with 1 µg of
pGAL4-Pax6-TAD, 3 µg of pUASG-Luc, and increasing amounts
(0, 1, 3, and 6 µg) of pMZF-TAD. The total amount of transfected DNA
was kept at 10 µg by adding the empty expression vector. The
luciferase activities are expressed as percentages of the values
obtained in the absence of pMZF-TAD plasmid.
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To explore the mechanism of transcriptional activation by MZF-2A, we
searched databases for proteins showing any homology with the MZF-TAD
and found that the D6 region showed marked homology with the
transactivation domain of mouse Pax6 (Fig. 2B) (42), which
is a transcription factor that plays a pivotal role in the development
of the central nervous system, eyes, nose, and pancreas (43, 44). This
similarity implies that MZF-2A and Pax6 might utilize common
transcriptional machinery to regulate transcription. In support of this
hypothesis, we found that the expression of the MZF-TAD protein
inhibited GAL4-Pax6-TAD-mediated transactivation in a
dose-dependent manner, presumably through competitive
binding to a shared transcriptional cofactor (Fig. 2C).
The Transactivation Domain of MZF-2A Is Phosphorylated by MAP
Kinases in Vitro and in Vivo--
Mikkola et al. (45)
showed that the transactivation domain of zebrafish Pax6 is
phosphorylated at serine 413 by the ERK and p38 MAP kinases. The
conservation of this serine residue in the MZF-TAD (Fig. 2B)
prompted us to investigate whether MZF-2A was also phosphorylated and
regulated by MAP kinases. For this purpose, we first examined the
phosphorylation of recombinant MZF-TAD by purified ERK, p38, and JNK
MAP kinases in vitro. As shown in Fig.
3A, ERK1 phosphorylated
GST-MZF-TAD more efficiently than GST-ATF2 (left panel), and
p38 MAP kinase weakly phosphorylated MZF-TAD (middle panel).
In contrast, JNK1 did not phosphorylate MZF-TAD at all under the same
conditions in which it phosphorylated ATF2, Elk-1, and c-Jun very well
(right panel). Similarly, JNK2 did not phosphorylate MZF-TAD
(data not shown).

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Fig. 3.
In vitro phosphorylation of
MZF-TAD by MAP kinases. A, 2 µg of GST and GST fusion
proteins of ATF2-(1-253), Elk-1-(307-428), c-Jun-(1-79), and
MZF-TAD-(155-336) were incubated with the indicated MAP kinases in the
presence of [ -32P]ATP at 30 °C for 30 min, resolved
by SDS-PAGE, and detected by autoradiography. B, the
wild-type GST-MZF-TAD protein (WT), A5 mutant, and add-back
mutants (T163, T170, S257, S275, and S295) are schematically
represented (left panel). Each GST fusion protein (2 µg)
was tested for its ability to be phosphorylated by ERK1 as described in
A. In lane WT(-ERK1), GST-MZF-TAD was incubated
without ERK1, as a negative control (right panel).
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Because MAP kinases are proline-directed protein kinases, we focused
our next set of studies on five serine/threonine residues, Thr167, Thr170, Ser257,
Ser275, and Ser295, all of which are followed
by a proline residue and conserved in both human and mouse MZF-TAD
sequences (Fig. 3B, left panel). To determine
which residue(s) is actually phosphorylated, a series of MZF-TAD
mutants in which these serine or threonine residues were replaced with
alanine residues were generated and tested for their ability to be
phosphorylated by ERK1 in vitro. When all five of the sites
were replaced with alanines (A5 mutant), no phosphorylation was
observed (Fig. 3B, right panel). Starting with
this A5 mutant, we constructed "add-back" mutants in which each of
the mutated residues was changed back to the wild-type residue, and we
examined them for in vitro phosphorylation. The S257, S275,
and S295 add-back mutants were phosphorylated efficiently by ERK1,
whereas T163 and T170 were not. These results indicate that ERK1
phosphorylates the MZF-TAD in vitro at three serine residues, Ser257, Ser275, and
Ser295, among the five putative phosphorylation sites.
To investigate whether the serine residues identified above could be
phosphorylated by MAP kinases in vivo, we established LGM-1
transformants that stably expressed the wild-type FLAG-MZF-2A (LGM-1/WT) or the S257A/S275A/S295A mutant (LGM-1/S257/275/295A) and
examined changes in the phosphorylation state of MZF-2A after exposure
of the cells to various extracellular stimuli that are known to
activate MAP kinase pathways. The top panel of Fig.
4A shows changes in
electrophoretic mobility of MZF-2A on SDS-PAGE caused by extracellular
stimulation. The wild-type MZF-2A appeared as two bands in serum- and
IL-3-starved LGM-1 cells. When the cells were stimulated with TPA,
anisomycin, FCS, or IL-3, the lower band disappeared, and the upper
band increased in intensity. In contrast, the S257/275/295A mutant
always appeared as a single band at the same position as the lower band
of the wild-type protein, irrespective of stimulation. When the
FLAG-MZF-2A protein was immunoprecipitated from the TPA-stimulated or
unstimulated cells and treated with calf intestine alkaline phosphatase
(Fig. 4B, CIAP), the upper band of wild-type
MZF-2A was converted into the lower band, whereas the mobility of the
S257/275/295A mutant was not affected (Fig. 4B). These
results indicated that the mobility shift of MZF-2A was caused by
phosphorylation and that one or more of the three serine residues
(Ser257, Ser275, and Ser295) were
phosphorylated by the extracellular stimuli. Western blot analysis
using antibodies specific for the active form of MAP kinases revealed
that a distinct set of MAP kinases was activated by the individual
stimuli (Fig. 4A, bottom panels). To
explore which MAP kinase is responsible for MZF-2A phosphorylation, we used two specific inhibitors for MAP kinase pathways, U0126 and SB203580. U0126 is a potent inhibitor of MEK, an ERK-activating kinase,
and consequently blocks the ERK pathway in vivo (46). SB203580 inhibits some of the p38 members such as p38
/SAPK2a and
p38
/SAPK2b, but not p38
/SAPK3 or p38
/SAPK4 (47-50). As
shown in Fig. 4C, pretreating cells with U0126 strongly
blocked TPA-induced MZF-2A phosphorylation and partially inhibited FCS-
or IL-3-induced phosphorylation. This result, together with the
observation that ERKs were strongly activated by TPA but very weakly
activated by FCS or IL-3 (Fig. 4A), indicates that ERK MAP
kinases phosphorylate MZF-2A in vivo. On the other hand,
SB203580 partially blocked the anisomycin-induced mobility shift,
indicating that p38 MAP kinase members, which are activated by
anisomycin (Fig. 4A), also contributed to the
phosphorylation of MZF-2A. Interestingly, combined treatment with both
U0126 and SB203580 did not completely inhibit the MZF-2A
phosphorylation induced by anisomycin or IL-3, suggesting that other
protein kinases insensitive to these inhibitors may also be involved.
These results indicate that, depending on the types of stimulation, the
ERK and p38 MAP kinase pathways are differentially involved in MZF-2A
phosphorylation.

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Fig. 4.
Phosphorylation of MZF-2A in
vivo. A, top panel, mobility shift of
wild-type MZF-2A upon exposure of cells to various stimuli. LGM-1/WT or
LGM-1/S257/275/295A cells were seeded onto 6-well plates, serum- and
IL-3-starved for 2 days, and stimulated with 100 nM TPA, 25 ng/ml anisomycin, 20% FCS, or 10 units/ml IL-3 for 10 min. As a
control ( ), cells were incubated with 0.1% Me2SO, the
solvent for TPA. After the stimulation, FLAG-MZF-2A protein was
immunoprecipitated with anti-FLAG M2 Affinity Gel, resolved by
SDS-PAGE, and detected by Western blotting with anti-MZF-2A antiserum.
The bottom panels show the activation profiles of each MAP
kinase detected by Anti-ACTIVE MAP kinase antibodies. B, the
wild-type or mutant FLAG-MZF-2A proteins were immunoprecipitated from
unstimulated or TPA-stimulated cells and then treated with calf
intestine alkaline phosphatase (+) or mock-treated ( ) at 37 °C for
1 h. The mobility shift of the FLAG-MZF-2A was examined by
immunoblotting using anti-MZF-2A antiserum. C, effect of MAP
kinase inhibitors on MZF-2A phosphorylation. LGM-1/WT cells were
untreated ( ) or pretreated with 20 µM U0126
(U) and/or 20 µM SB203580 (SB) for
1 h and then stimulated with the indicated reagents or left
untreated ( ) for 10 min. The mobility shift of FLAG-MZF-2A was
examined as described above.
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The Transactivation Activity of MZF-2A Is Negatively Regulated by
Phosphorylation--
To test whether MAP kinase-mediated
phosphorylation regulates the transactivating activity of MZF-2A, we
constructed a series of FLAG-MZF-2A mutants in which one or two of the
three serine residues were replaced with alanine(s) and assessed their
transactivation potentials by a luciferase reporter assay using LGM-1
cells. Western blot analysis of the FLAG-MZF-2A proteins expressed in
LGM-1 cells demonstrated that all the wild-type and mutant MZF-2A
proteins except for the triple mutant (S257/275/295A) migrated to the
position of phosphorylated MZF-2A (Fig.
5, bottom panel). This result
indicates that in the growing LGM-1 cells, the wild-type and mutated
MZF-2A proteins were constitutively phosphorylated at the unmutated
site(s) among the three serine residues. As shown in the top
panel of Fig. 5, when compared with wild-type MZF-2A, each mutant
with a single substitution (S257A, S275A, and S295A) exhibited a
1.4-2.6-fold increase in reporter activity. Introduction of an
additional alanine substitution resulted in a further increase in
reporter expression (S275/295A, S257/295A, and S257/275A). The greatest
augmentation (~6-fold) in transcriptional activity was observed with
the S257/275/295A mutant, in which all three serine residues were
replaced with alanines. These results suggest that phosphorylation of
these serine residues cooperatively reduces the transactivation
activity of MZF-2A. Among the three serine residues, the substitution
of Ser295 showed a greater enhancement in reporter
expression than the others, suggesting that phosphorylation of
Ser295 is the most effective in repressing the
transactivation activity of MZF-2A. The enhancement of reporter
expression was not a result of an increase in the protein level of the
mutants because comparable amounts of MZF-2A protein were expressed in
all of the constructs (Fig. 5, bottom panel). In an attempt
to confirm the negative regulation of MZF-2A activity by
phosphorylation, we examined the effect of the U0126 and SB203580
inhibitors on the luciferase reporter assay. However, we found that the
luciferase assay with these inhibitors could was not a reliable
experiment because treatment of cells with the inhibitors resulted in
strong reduction of gene expression in general (data not shown). We
finally examined the effect of the phosphorylation site mutation on the
activity of the N-terminally deleted KM3 mutant, which showed an
enhanced transcriptional activity (Fig. 1). Interestingly, the
transactivation activity of the S257/275/295A mutant was almost the
same as that of KM3, and disruption of the three phosphorylation sites
of the KM3 mutant (KM3-S257/275/295A) resulted in only a slight
additional enhancement of reporter activation (Fig. 5, top
panel), indicating that the two different mutations, the
N-terminal deletion and the alanine substitution of the phosphorylated
sites, did not work additively to augment reporter expression.
Together, these results demonstrate that the transactivation activity
of MZF-2A is negatively regulated by MAP kinase-mediated
phosphorylation at the three serine residues in the transactivation
domain.

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|
Fig. 5.
Effect of mutations of the phosphorylation
sites on the transactivating activity of MZF-2A. Top panel,
LGM-1 cells were co-transfected with 10 µg of an expression plasmid
encoding wild-type (WT) or mutated FLAG-MZF-2A, 10 µg of
the pGL3-4xF113B-G-Luc reporter plasmid, and 5 ng of pRL-SV40. After
incubation for 18 h, cell lysates were assayed for firefly and
Renilla luciferase activities. The firefly luciferase
activities are expressed as percentages of the value obtained with
wild-type FLAG-MZF-2A, after normalization to the Renilla
luciferase activity. The data are average values with standard errors
of two independent experiments. Bottom panel, Western blot
analysis of the FLAG-MZF-2A mutants. The whole-cell extracts used for
the luciferase assay were separated by SDS-PAGE, and the expressed
FLAG-MZF-2A proteins were detected by Western blotting with anti-MZF-2A
antiserum. Arrowheads indicate the positions of the
unphosphorylated (U) or phosphorylated (P) forms
of FLAG-MZF-2A and FLAG-KM3. NS indicates a nonspecific band
observed even in the vector-transfected cells.
|
|
 |
DISCUSSION |
In this report, we identified and described a novel isoform of
mouse MZF-2, designated MZF-2A, which is generated by the use of an
alternative transcription start site and subsequent splicing. Compared
with MZF-2, MZF-2A lacks 79 N-terminal amino acids and shows a basal
level of transactivation activity. Recently, Peterson and Morris (11)
identified two novel transcripts of the human MZF gene, MZF1B and
MZF1C. These mRNAs differ in their 5'-untranslated region but
encode the same polypeptide of 734 amino acids, designated MZF1B/C
(11). Mouse MZF-2A and human MZF1B/C are highly homologous along their
entire sequences (81.7% amino acid identity) and are likely to
represent the major MZF gene product in myeloid cells.
We showed that the previously identified transactivation domain of
MZF-2A was functional when fused to the heterologous DNA-binding domain
of yeast GAL4 and defined the minimal TAD as a 50-amino acid region (aa
239-288), which is necessary and sufficient to activate transcription.
The amino acid composition of this minimal TAD is very characteristic
of TADs; i.e. it is rich in acidic amino acid and proline
residues. There are five aspartic acid and four glutamic acid residues
but no basic (arginine or lysine) residues, resulting in a low
isoelectric point of 3.8. Similar acidic and proline-rich features are
observed in the TAD of many other transcription factors (51) and are
likely to be important for their interaction with other transcription factors.
Our studies demonstrate that MZF-2A is phosphorylated by ERK and p38
MAP kinases. In vitro phosphorylation experiments showed that MZF-TAD is phosphorylated strongly by ERK, weakly by p38 kinase,
and not at all by JNK1, and further analysis using "add-back" mutants revealed that three serine residues (Ser257,
Ser275, and Ser295) in the TAD could be
phosphorylated by ERK1 in vitro. The mobility shift analysis
of MZF-2A expressed in LGM-1 cells (Fig. 5) demonstrated that any of
the single (S257A, S275A, and S295A) and double (S275/295A, S257/295A,
and S257/275A) serine residue mutants showed the same mobility as
wild-type MZF-2A, indicating that these mutants were still
phosphorylated at the remaining unmutated sites. This result indicates
that all three serine residues are susceptible to phosphorylation by
MAP kinases and that the phosphorylation of any one of them is
sufficient to cause the mobility shift. Therefore, the phosphorylation of any of the three serine residues is likely to cause a similar, repressive effect on the transactivation potential of MZF-2A. Consistent with this, additional alanine substitutions for any of the
serines cumulatively enhanced the transactivation activity of MZF-2A,
and the highest activation was observed when all the phosphorylation
sites were disrupted (Fig. 5). These results indicate that the
phosphorylation of each serine residue functions cooperatively to
repress the ability of MZF-2A to activate transcription.
We have shown that various extracellular stimuli, including TPA,
anisomycin, FCS, and IL-3, induce the phosphorylation of MZF-2A.
Experiments with the protein kinase inhibitors U0126 and SB203580
revealed that distinct MAP kinase pathways are responsible for MZF-2A
phosphorylation, depending on the type of stimulation. TPA activates
ERK MAP kinases strongly, and the MEK inhibitor U0126 blocked
TPA-induced MZF-2A phosphorylation almost completely, clearly
demonstrating that ERKs play a predominant role in phosphorylation of
MZF-2A in response to TPA stimulation. ERKs are also partly involved in
FCS- or IL-3-induced phosphorylation. On the other hand,
anisomycin-induced phosphorylation of MZF-2A was partially inhibited
only by SB203580, suggesting that p38 MAP kinases, but not ERKs, are
involved. Interestingly, combined treatment with both inhibitors mostly
blocked MZF-2A phosphorylation induced by TPA or FCS but still resulted
in partial inhibition of anisomycin- or IL-3-induced phosphorylation,
suggesting that other protein kinase(s) insensitive to these inhibitors
are involved downstream of anisomycin and IL-3. Although the JNK
pathway is known to be resistant to these inhibitors (47, 50), JNK MAP
kinases are unlikely to play this role because neither JNK1 (Fig. 3)
nor JNK2 (data not shown) phosphorylated GST-MZF-TAD in
vitro. Another possibility is that the SB203580-insensitive
p38 isoforms, such as p38
/SAPK3 and p38
/SAPK4 (49, 50),
phosphorylate MZF-2A. These kinases are likely to mediate
anisomycin-inducible and SB203580-resistant phosphorylation of
eukaryotic elongation factor 2 kinase in KB cells (52), and they may
similarly play a prominent role in anisomycin- or
IL-3-dependent phosphorylation of MZF-2A in LGM-1 cells.
These findings demonstrate that the MZF-2A transcription factor
integrates MAP kinase signaling pathways in response to different
extracellular stimuli, such as Elk-1, Sap-1a, and ATF2 (26, 27,
53-55).
Specific phosphorylation events can regulate the activity of
transcription factors via several different mechanisms involving changes in protein stability, subcellular localization, or
protein-protein interactions (19, 28, 30, 32). We showed that protein stability is not significantly affected by alanine substitution of the
phosphorylation sites (Fig. 5). We also found by subcellular fractionation experiments that MZF-2A is present exclusively in the
nuclear fraction, irrespective of its phosphorylation
state.2 The KM3 mutant shows
enhanced transcriptional activity, indicating that the N-terminal
domain inhibits the transcriptional activity of the TAD. We previously
proposed a model for this negative regulation in which an
intramolecular interaction of the N-terminal domain with the adjacent
TAD impedes the recruitment of a transcriptional cofactor(s) (13). The
findings in this report that disruption of all three phosphorylation
sites elevates MZF-2A transactivation activity to a level comparable
with that of the KM3 mutant and that alanine substitution of the
phosphorylation sites in the KM3 construct enhances its activity only
weakly (Fig. 5, top panel) suggest that the alanine
substitution and the N-terminal truncation could cause a similar effect
on TAD function, which may imply that TAD phosphorylation is required
for the N-terminal regulatory domain to repress transactivation
activity. Combining this possibility with the previous model,
dephosphorylation might release the TAD from the inhibitory domain and
allow the TAD to interact with the transcriptional machinery (Fig.
6). Alternatively, because the LeR/SCAN
domain is implicated to function as a module for protein-protein
interaction (56, 57), TAD phosphorylation may cooperate with the
LeR/SCAN domain to bind a repressor protein.

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|
Fig. 6.
A model for
phosphorylation-dependent regulation of MZF-2A
activity. MAP kinase-mediated phosphorylation of MZF-TAD promotes
its intramolecular interaction with the N-terminal inhibitory domain
and consequently prevents the TAD from associating with transcriptional
machinery to activate transcription.
|
|
In summary, this work represents the first report of MZF-2A
phosphorylation by MAP kinases. A challenge for future studies is to
clarify the physiological significance of MZF-2A phosphorylation and
the subsequent repression of its transactivating function. Because
serum and IL-3 are potent growth-promoting factors, inducible or
constitutive phosphorylation of MZF-2A by these factors would contribute to reducing the growth-suppressing function of MZF-2A, and
dephosphorylation and the subsequent activation of MZF-2A may be
involved in cell cycle arrest and terminal differentiation of myeloid
cells. In this regard, it would be intriguing to examine the regulation
of MZF-2A phosphorylation in granulocyte colony-stimulating factor-driven granulocytic differentiation and maturation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Peter Gruss for Pax6
cDNA, Dr. Takashi Fujita for pUASG-Luc, Dr. Roger J. Davis for FLAG-p38 plasmid, and Dr. Michael Karin for
hemagglutinin-tagged JNK1 plasmid.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science, Sports and Technology of Japan.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.
To whom correspondence should be addressed. Tel.: 81-6-6879-3318;
Fax: 81-6-6879-3319; E-mail:
fukunaga@genetic.med.osaka-u.ac.jp.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M207615200
2
H. Ogawa, S. Nagata, and R. Fukunaga,
unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
MZF, myeloid zinc
finger protein;
MAP, mitogen-activated protein;
TAD, transactivation
domain;
ERK, extracellular signal-regulated kinase;
JNK, Jun N-terminal
kinase;
IL-3, interleukin 3;
FCS, fetal calf serum;
GST, glutathione
S-transferase;
TPA, 12-O-tetradecanoylphorbol
13-acetate;
aa, amino acid(s);
LeR, leucine-rich;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
SAPK, stress-activated protein kinase.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.