(Received for publication, December 5, 1994)
From the
GATA-2 is a member of a family of transcription factors which bind a common DNA sequence motif (WGATAR) through an evolutionarily conserved zinc finger domain. An essential role for GATA-2 in the development of hematopoietic stem cells has recently been shown in gene targeting experiments in mice. Here we show that GATA-2 exists in hematopoietic progenitor cells as a phosphoprotein. Stimulation of progenitors with interleukin-3 (IL-3) results in enhanced phosphorylation of GATA-2 which occurs within 5 min. IL-3 is known to signal in part through mitogen-activated protein (MAP) kinase, and evidence for MAP kinase signaling in the control of GATA-2 phosphorylation was obtained by genetically manipulating the MAP kinase pathway in COS cells using either constitutively activating or interfering mutants of MAP kinase kinase. Furthermore, using an interfering mutant of MAP kinase kinase, we directly demonstrated a critical role for the MAP kinase pathway in the IL-3-dependent phosphorylation of GATA-2 in hematopoietic progenitor cells. Finally, in vitro phosphorylation experiments using recombinant GATA-2 raise the possibility that MAP kinase itself may phosphorylate GATA-2. Our results provide evidence for phosphorylation via the MAP kinase pathway constituting a cytoplasmic link between GATA-2 and growth factor receptors and are consistent with the hypothesis that GATA-2 is involved in the growth factor responsiveness and proliferation control of hematopoietic progenitor cells.
Hematopoietic stem cells are characterized by both their ability to self-renew and their capacity to undergo terminal differentiation down one of at least eight different lineage pathways. Cells in which the balance between self-renewal and differentiation is constitutively dysregulated in favor of self-renewal are thought to be more prone to leukemogenesis(1) . The mechanisms controlling the choice between self-renewal and differentiation are not fully understood, but seem likely to involve receptor-mediated signals producing changes in the functional balance of cellular transcription factors or complexes. In this context, elucidating the signal transduction pathways that link growth factor receptors at the cell surface to transcription factors in the nucleus, is germane to a full understanding of the mechanisms underlying both the normal regulation of hematopoietic stem cells and their pathology.
We have been focusing on the GATA family of
transcription factors which are emerging as key regulators of
hematopoietic cell fate. GATA factors are characterized by their
ability to bind a common DNA sequence motif (WGATAR) by virtue of an
evolutionarily conserved CC
zinc finger DNA
binding domain(2) . To date, four GATA factors have been
described (GATA-1-4); of these only three (GATA-1, -2, and 3) are
expressed in hematopoietic cells, where their pattern of expression is
complex and may show some minor species variation. GATA-1, originally
cloned from erythroid cells(3, 4) , has been shown to
be expressed also in megakaryocytes and mast
cells(5, 6) . In gene targeting experiments, loss of
GATA-1 function results in a block in erythroid differentiation at the
proerythroblast stage(7, 8) . GATA-3 expression in
mammalian hematopoietic cells is restricted to
T-lymphocytes(9, 10) , although it is interesting to
note that GATA-3 was originally cloned from chicken erythroid cells
where it is expressed at a low level(11) . Ectopic expression
of GATA-2 in chicken erythroid progenitor cells blocks differentiation
potential(12) . GATA-2 is expressed not only in early erythroid
cells but also in multipotent hematopoietic progenitor
cells(13, 14, 15, 16) . Recently,
gene targeting experiments in mouse embryonic stem cells have
demonstrated that GATA-2
mice have a severe
deficit in all hematopoietic lineages, suggesting a critical role for
GATA-2 in the biology of the progenitor cells of definitive
hematopoiesis(17) .
A large number of transcription factors
have been shown to exist within cells as phosphoproteins. The
functional consequences of phosphorylation vary but include regulation
of intracellular localization, DNA binding, and transcriptional
regulation(18) . Only recently have the protein kinase cascades
involved in growth factor-induced phosphorylation events begun to be
elucidated. Many receptor tyrosine kinases and cytokine receptors,
including interleukin-3 (IL-3) which is a key cytokine regulator of
progenitor cell self-renewal(19, 20) , have been shown
to activate the ras-MAP kinase ((mitogen-activated protein kinase),
also known as ERK (extracellular signal-regulated kinase))
pathway(21, 22, 23, 24) . MAP
kinases (MAPKs) ()are one element in a series of kinases
that serve to connect the nucleus with cytosolic and plasma membrane
events. The idea that phosphorylation of transcription factors by MAPKs
might provide the cytoplasmic link between receptor-mediated events and
changes in gene expression in the nucleus is supported by the
observation that activated MAPKs can enter the
nucleus(25, 26) . MAPK substrates include cytosolic
phospholipase A
(27) and a number of transcription
factors, e.g. c-myc(28) ,
c-jun(29) , and Elk-1 (30) .
Until recently, little has been known about the phosphorylation status of the GATA family of transcription factors. A very recent report (31) demonstrates that GATA-1 exists as a phosphoprotein in erythroid cells with phosphorylation occurring on serine residues. Both the function of this serine phosphorylation and whether it is regulated by signaling remain unknown. The goal of the studies presented here was to determine if GATA-2 exists as a phosphoprotein within the hematopoietic progenitor cell compartment and, if so, whether this phosphorylation is linked to signal transduction pathways controlling the proliferation status of cells. In this report we present evidence that within hematopoietic progenitor cells, GATA-2 is phosphorylated through the MAPK signal transduction pathway in response to IL-3.
The conditions for co-transfection of the
chloramphenicol acetyltransferase (CAT) reporter with the CA-MAPKK and
pMT2-GATA-2 expression vectors were the same as described above. The
reporter plasmid, p6, which comprises concatamerized
GATA-motif oligomers linked to an
-globin promoter
driving a CAT reporter gene(38) , was a kind gift of G.
Felsenfeld. Cell extracts for CAT assays were prepared and CAT assays
were performed, as described previously(30) . The total amount
of DNA transfected within each experiment was kept constant with the
use of carrier plasmid DNA. Following autoradiography, the regions of
the thin layer chromatography plate containing labeled chloramphenicol
or its acetylated forms were individually cut out and counted by liquid
scintillation to determine the conversion percentage.
Figure 1:
Phosphorylation of GATA-2 in
hematopoietic progenitor cells. A, a 416B nuclear protein
extract was assayed by GMSA using a radiolabeled GATA motif-containing
oligomer derived from the mouse -globin promoter. The positions of
GATA-1 and GATA-2 protein-DNA complexes (arrow in the figure)
were confirmed by the addition of anti-GATA-1 and anti-GATA-2
antibodies; the GATA-2 supershift (bracketed in the figure)
comprises a discrete band as well as slow migrating complexes which
remain in the region of the slot (*S). B,
immunoprecipitations of
[
P]orthophosphate-labeled murine 416B and human
HL-60 cells were performed using an agarose-conjugated, anti-GATA-2
monoclonal antibody. The expected migration position for authentic
human GATA-2 protein is indicated by an arrowhead. The
identity of the faster migrating bands observed in both cell lines
(approximately 38 kDa) remains obscure. The numbers next to
the autoradiographs indicate relative molecular mass in kilodaltons. C, the left lane shows data obtained using an
agarose-conjugated anti-GATA-2 monoclonal antibody for
immunoprecipitation of [
P]orthophosphate-labeled
COS cells which had been transfected with a human GATA-2 expression
vector (pMT2-GATA-2). The right lane shows immunoblot analysis
of the same material using anti-GATA-2 polyclonal antisera. The
SDS-PAGE fractionated material was transferred to PVDF membrane prior
to autoradiography and ECL-based Western analysis. The numbers next to the autoradiographs indicate relative molecular mass in
kilodaltons. D, left lane, material, immunoprecipitated from
pMT2-GATA-2-transfected COS cells using an agarose-conjugated
anti-GATA-2 monoclonal antibody, was fractionated on modified
SDS-polyacrylamide gels that contain a low amount of bisacrylamide,
Western blotted using anti-GATA-2 polyclonal antisera, and visualized
using ECL. Right panel, 5 mg of HL-60 whole cell lysate was
immunoprecipitated with an anti-GATA-2 monoclonal antibody,
fractionated by modified SDS-PAGE, and immunoblotted with anti-GATA-2
polyclonal antisera.
Taken together, these data demonstrate that GATA-2 exists as
a phosphoprotein in proliferating hematopoietic progenitor cells. In
considering the significance of such a result, it is important to
determine what proportion of the total GATA-2 in these cells exists in
the phosphorylated form. Using polyacrylamide gels with a low
percentage of bisacrylamide, the phosphorylated and unphosphorylated
forms of GATA-2 could be resolved (Fig. 1D). Analysis
of the phosphorylation status of these differently migrating forms of
GATA-2 by both P labeling and alkaline phosphatase
treatment (data not shown) demonstrated that the fastest migrating band
represents the unphosphorylated GATA-2 (arrow in Fig. 1D). Using this gel system, over 80% of GATA-2 was
found to be phosphorylated in exponentially growing HL-60 cells (Fig. 1D).
Figure 2: Stimulation of GATA-2 phosphorylation by exposure to IL-3. IL-3-dependent murine hematopoietic progenitor cells were assayed for the phosphorylation status of endogenous GATA-2 and MAPK under conditions of IL-3 deprivation and IL-3 restimulation. The different phosphorylated forms of GATA-2 are labeled a, b, and c, while d and e represent the phosphorylated (activated) and unphosphorylated forms of MAPK. For analysis of GATA-2 phosphorylation, cell lysates were immunoprecipitated with an agarose-conjugated anti-GATA-2 monoclonal antibody prior to SDS-PAGE and subsequent Western blotting using an anti-GATA-2 polyclonal antiserum. For analysis of MAPK activity, cell lysates were directly Western blotted using anti-ERK-2 antibody. Panel A, analysis of BA/F3 cells deprived of IL-3 for 6 h (lane 1) and then stimulated with IL-3 for 5 min (lane 2). Panel B, analysis of Bcl-2-BA/F3 cells deprived of IL-3 for 24 h (lane 1) and then stimulated with IL-3 for 5 min (lane 2); lane 3 is a longer exposure of lane 1.
Although the phosphorylation of GATA-2 appears to be enhanced as a result of IL-3 stimulation, phosphorylated forms of GATA-2 are clearly still present in IL-3-deprived cells. This may reflect the fact that the period of IL-3 deprivation was only 6 h; increasing the period of IL-3 deprivation in BA/F3 cells is not possible since 6 h is the maximum time that these cells can be kept in the absence of IL-3 before initiating an apoptotic program. However, the onset of apoptosis as a result of IL-3 withdrawal is considerably delayed in BA/F3 cells which express the human Bcl-2 gene product (Bcl-2-BA/F3)(34) . In these cells, after 24 h in the absence of IL-3, the b-form of GATA-2 was clearly still present (Fig. 2B, lane 1). In contrast, there was no evidence of the a-form of GATA-2 (Fig. 2B, lane 1) even after longer exposures of the film (see lane 3); the a-form appeared rapidly after stimulation with IL-3 (Fig. 2B, lane 2).
IL-3 is known to signal in part through the MAPK pathway(41, 42) . Like GATA-2 phosphorylation, MAPK activation occurs rapidly in response to IL-3 signaling. We therefore analyzed the status of activation of MAPK in our BA/F3 and Bcl-2-BA/F3 lysates; the phosphorylated and nonphosphorylated forms of MAPK (labeled d and e, respectively, in the lower panels of Fig. 2) can be separated by PAGE where the phosphorylated form migrates more slowly(43, 44) . The results show the absence of phosphorylated MAPK in IL-3-deprived cells and its rapid appearance after 5 min of re-exposure to IL-3.
Figure 3: Manipulation of the MAPK pathway in COS cells. COS cells were co-transfected with human GATA-2 and myc-tagged MAPK expression vectors. MAPK activity in these cells was modulated either by stimulation with EGF or by co-transfection of either constitutively activated or dominant negative MAPKK. A, COS cells transfected with both pMT2-GATA-2 (5 µg) and myc-ERK2 (3 µg) as well as 5 µg of either constitutively active MAPKK (CA-MAPKK) expression vector or the vector alone. B, COS cells transfected with pMT2-GATA-2 (3 µg) and myc-ERK2 (1 µg) as well as 7 µg of either a dominant negative MAPKK (DN-MAPKK) expression vector or the empty vector. 24 h post-transfection, the culture medium was replaced with serum-free DMEM, and the cells were incubated for an additional 24 h. EGF was then added to the indicated samples for 7 min at a concentration of 10 ng/ml. The cells were lysed in S buffer, and GATA-2 was immunoprecipitated with the anti-GATA-2 monoclonal antibody, fractionated by modified SDS-PAGE and immunoblotted using anti-GATA-2 polyclonal antiserum; these results are presented in the upper panels of A and B. An equivalent amount of lysate was analyzed for MAPK activity by direct Western blotting using antibody 9E10 directed against the myc tag; the results of this analysis are presented in the corresponding lower panels of A and B.
These data obtained in COS cells provide strong
evidence for the involvement of the MAPK pathway in the regulation of
GATA-2 phosphorylation. We next addressed the critical issue as to
whether the MAPK pathway actually mediates the IL-3-dependent
phosphorylation of GATA-2 in hematopoietic progenitor cells. BA/F3
cells were stably co-electroporated with eukaryotic expression vectors
containing the DN-MAPKK mutant (pEF-DN-MAPKK) and a selective puromycin
resistance gene (pBABE-puro); DN-MAPKK expression in the stable
transfectants was confirmed by Western blotting (data not shown). The
phosphorylation of GATA-2 in response to IL-3 stimulation in these
DN-MAPKK transfected BA/F3 cells (BAF3/DN-MAPKK) was compared to that
of parental BA/F3 cells. BA/F3 and BAF3/DN-MAPKK cells were labeled
with [S]methionine/cysteine and deprived of IL-3
for 6 h prior to re-exposure to IL-3 for 5 min. GATA-2,
immunoprecipitated from lysates of these cells, was analyzed by
SDS-PAGE; these results are presented in Fig. 4. The uppermost
phosphorylated form of GATA-2 (form a) is not evident in
IL-3-stimulated BAF3/DN-MAPKK cells; the successful impairment of the
MAPK pathway under these conditions of IL-3 stimulation was
demonstrated by the analysis of MAPK phosphorylation presented in the
lower panels. These results thus provide clear evidence for the
involvement of the MAPK pathway in the IL-3-dependent phosphorylation
of GATA-2 in hematopoietic progenitor cells.
Figure 4:
An interfering MAPKK mutant abrogates the
IL-3dependent GATA-2 phosphorylation in hematopoietic progenitor cells. S-Labeled parental BA/F3 cells (lane 1) and
BAF3/DN-MAPKK cells (lane 2) were deprived of IL-3 for 6 h and
then stimulated with IL-3 for 5 min. Cell lysates were
immunoprecipitated with an agarose-conjugated anti-GATA-2 monoclonal
antibody followed by SDS-PAGE. Analysis of MAPK activity was performed
as described in Fig. 2.
Figure 5:
Phosphorylation of GATA-2 by MAPK in
vitro. A, in vitro kinase assays performed using
chemically activated (thiophosphorylated) MAPK. The lanes contain the
following reaction substrates: 1, no substrate; 2, 15
µg of MBP; 3, 2 µg of GST-GATA-2 fusion protein. The
quantity and quality of MBP and GST-GATA-2 used in these in vitro reactions was confirmed by Coomassie Blue staining. The reaction
products were fractionated on a 10% polyacrylamide gel; numbers are in kilodaltons, and the phosphorylated GATA-2 fusion protein
is indicated by an arrowhead. B, phosphoamino acid analysis of
GST-GATA-2 phosphorylated in vitro by MAPK. The positions of
serine (S), threonine (T), and tyrosine (Y)
are marked. C, phosphopeptide fingerprints of GST-GATA-2
labeled by MAPK in vitro (left panel) and
phospholabeled GATA-2 immunoprecipitated from COS cells co-transfected
with CA-MAPKK (right panel). The horizontal dimension (X) was electrophoresis, and the vertical dimension (Y) was chromatography. The data were collected by the
Molecular Dynamics
PhosphorImager.
Figure 6: Analysis of transactivation potential of GATA-2. COS cells were transfected with a GATA-dependent CAT reporter gene along with the combinations of GATA-2 and CA-MAPKK expression vectors indicated in the figure. A, representative data obtained in one of the seven experiments performed. B, the collective data from all seven experiments performed are presented graphically. CAT activities were quantitated by scintillation counting or by scanning on a Molecular Dynamics PhosphorImager. The data are expressed as fold stimulation relative to the basal activity of the GATA-CAT reporter construct, with the standard errors indicated by the vertical bars.
In this report we have shown that GATA-2 is phosphorylated in
proliferating hematopoietic progenitor cells. Interestingly, several
phosphorylated forms of GATA-2 were found; some existing stably in
quiescent cells, others more characteristic of mitogen (IL-3)
stimulated cells. These results obtained in hematopoietic cells were
further supported by mitogen (EGF) stimulation experiments conducted in
GATA-2 transfected COS cells. The mitogen-associated phosphorylated
forms of GATA-2 appear to be associated with activation of the MAPK
pathway. This is particularly clear in our COS cell experiments where
the use of a constitutively activated mutant of MAPKK demonstrated that
activation of the MAPK pathway in vivo is sufficient for
enhanced GATA-2 phosphorylation; that activation of this pathway is
necessary for this event was unequivocally demonstrated using an
interfering MAPKK mutant which acts as a dominant negative in
vivo. Furthermore, using this interfering MAPKK mutant, we have
directly demonstrated a critical role for the MAPK pathway in the
regulation of GATA-2 phosphorylation in response to IL-3 stimulation in
hematopoietic progenitor cells. Taken together, our results are
consistent with the hypothesis that GATA-2 is involved in the growth
factor responsiveness and proliferation control of hematopoietic
progenitor cells. The implications of our results are particularly
interesting in light of the recently reported phenotype of the GATA-2
knockout(17) . In these mice, definitive hematopoiesis is
profoundly impaired with the loss of virtually all hematopoietic
lineages suggesting a major defect at the stem or early progenitor cell
level. Colony formation assays conducted in vitro with
GATA-2 ES cells suggest an impaired
responsiveness to certain cytokines, leading the authors to speculate
that GATA-2 regulates genes controlling the growth factor
responsiveness of early hematopoietic cells. Our data raise the
possibility that GATA-2 itself may directly mediate the cellular
response to growth factor through alterations in its phosphorylation
status.
Our results also raise a secondary, although interesting,
question: does MAPK bring about GATA-2 phosphorylation in a direct or
an indirect fashion? Both our in vitro phosphorylation data
and our two-dimensional phosphopeptide mapping experiments argue that
MAPK may directly phosphorylate GATA-2 in vivo. The GATA-2
protein has at least 14 potential sites for MAPK phosphorylation;
systematic mutagenesis of these positions is currently underway in our
laboratory. The notion that GATA-2 is directly phosphorylated by MAPK
is an attractive one since MAPKs translocate to the nucleus upon
activation and are therefore ideally suited to act as a convergence
point that integrates and transduces diverse cytokine receptor-mediated
signals to transcription factors. It will now be interesting to
determine if stimulation by other cytokines results in GATA-2
phosphorylation, either through MAPK-dependent or independent pathways.
A strong candidate cytokine in this regard is the steel factor
(SLF) (47) . Like IL-3, SLF also activates the MAPK
pathway(49, 50) , and, interestingly, in vitro differentiation experiments with GATA-2 ES cells reveal a profound deficiency in the generation of
SLF-dependent colonies(17) . In our own preliminary experiments
using the human factor-dependent cell line TF-1(48) , we have
found that both IL-3 and SLF effect rapid phosphorylation of GATA-2
(and also MAPK) in factor-deprived cells. These preliminary experiments
lend further support to the notion that phosphorylation of GATA-2 in
response to ligand binding is relevant for hematopoietic progenitor
cell expansion and is closely associated with MAPK activation.
Another member of the GATA family of transcription factors, namely
GATA-1, has recently been shown to exist as a phosphoprotein in
erythroid cells. GATA-1 is phosphorylated exclusively on serine
residues, but the cellular mechanisms regulating this phosphorylation
are not yet known. Systematic mutations of these serine residues do not
appear to alter the transactivation function of GATA-1 as judged by
reporter gene assays conducted in COS cells, nor do they alter the DNA
binding ability of GATA-1 proteins expressed in COS cells. Consistent
with the results obtained with GATA-1, we observed no significant
change in the transactivation potential of GATA-2 when co-transfected
with the CA-MAPKK into COS cells. We suspect that cell fate could prove
a more sensitive and relevant assay than transient transfection
systems. The expression of GATA factors overlaps at certain stages of
hematopoietic cell development (2) and the different GATA
members display subtly different binding site
preferences(49, 50) . Phosphorylation may modulate
both the binding site preferences and the transactivation ability of
GATA factors in the context of a native transcriptional complex.
Modulations such as these could dramatically alter the cellular
transcriptional program elicited by GATA factors and thus ultimately
regulate proliferation versus differentiation decisions.
Testing such a model will require expression of GATA phosphorylation
mutants in stem cell populations which can undergo proliferation and
differentiation in response to cytokine-induced signaling.
GATA-2 ES cells offer one such cellular
model; the IL-3-dependent cell line FDCP-mix A4 which can undergo
multilineage differentiation in response to both stromal components and
hematopoietic growth factors may provide another(51) .