From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin 3 (IL-3) stimulate the proliferation and maturation of
myeloid progenitor cells following interaction with heterodimeric
receptors that share a common
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
The receptors for GM-CSF and
IL-3 are members of the hematopoietin receptor superfamily, which
includes receptors for interleukins 4 through 9 and the prolactin and
growth hormone receptors(4, 5, 6) . GM-CSF or
IL-3 stimulation of target cells results in tyrosine phosphorylation of
similar sets of proteins(7, 8) . These receptors do not
contain consensus sequence motifs for tyrosine or serine-threonine
kinases(6) . However, nonreceptor tyrosine kinases such as Jak2
form complexes with the GM-CSF and IL-3 receptors(4) . Both
GM-CSF and IL-3 activate pathways, which result in the phosphorylation
of Raf, Vav, Fes, Shc, and microtubule-associated protein kinase and
induce phosphorylation of DNA-binding proteins that recognize the
We demonstrated previously that
signaling by GM-CSF or IL-3 results in rapid and transient induction of
the immediate early response gene (early growth response gene-1, egr-1)(12) . egr-1 encodes a zinc finger
DNA-binding protein which recognizes the sequence
CGCCCCCGC(13, 14) . Like c-fos, this gene is
induced by a variety of stimuli, including serum, tetradecanoylphorbol
acetate (TPA) and other growth factors (e.g. nerve growth
factor (NGF))(15, 16) . egr-1 has also been
demonstrated to be necessary for macrophage but not granulocyte
differentiation(17) . The immediate early genes are believed to
be critical for GM-CSF and IL-3 action, as many of them, including egr-1 and c-fos, encode transcription factors which
participate in the regulation of transcription of late response
genes(18) . Many cytokine-induced late response genes encode
myeloid-specific proteins that function as determinants of myeloid cell
differentiation (18).
The transcriptional activation of egr-1 in response to GM-CSF, IL-3, or serum stimulation in the
factor-dependent human myeloid leukemic cell line, TF-1, requires the
presence of the cAMP response element (CRE) contained within the
-116 nucleotide region of the egr-1 promoter(12) . We found that the signaling pathways
activated by GM-CSF and IL-3 converge upon the CRE-binding protein,
CREB, which specifically binds the CRE in the -116-nt region of
the egr-1 promoter(12) . Many growth factors, including
GM-CSF, stimulate the transcription of specific genes by increasing
intracellular cAMP, which results in the activation of protein kinase A
and phosphorylation of several
proteins(19, 20, 21) . The 43-kilodalton (kDa)
cAMP response element-binding protein, CREB, is one of these factors
phosphorylated by a protein kinase A-dependent
pathway(21, 22) . Phosphorylation of serine 133 is
critical for the activation of CREB and is required for the induction
of specific genes (e.g. c-fos) by growth factors such
as NGF(16, 21) . NGF stimulation of PC12 cells results
in Ras-dependent phosphorylation of CREB, leading to the induction of
c-fos by a protein kinase A-independent pathway(16) .
To characterize the post-translational modification of CREB in
response to signaling pathways activated by GM-CSF and IL-3, we
examined the phosphorylation of CREB in response to growth factor
stimulation in TF-1 cells. We report here that GM-CSF and IL-3 induce
phosphorylation of CREB on serine 133 in TF-1 cells. Moreover, we
determined that phosphorylation of CREB on serine 133 substantially
contributes to the the transcriptional activation of the -116
nucleotide region of the human egr-1 promoter in response to
GM-CSF but not IL-3. Our findings also suggest that transcriptional
events downstream may determine specificity for growth factors with
overlapping activities through phosphorylation of specific nuclear
proteins.
CREB has been
demonstrated to be phosphorylated on serine 133 in response to other
growth factors. We performed electromobility shift assays with antibody
directed against a CREB peptide phosphorylated on serine 133, using
extracts from cells treated with GM-CSF or IL-3 for 10 min. In
electromobility shift assays using anti-CREB antibody, an additional
``super'' shifted band was observed in nuclear extracts
prepared from both unstimulated and GM-CSF-stimulated TF-1 cells (see Fig. 1A). In the presence of antibody directed against
CREB phosphorylated on serine 133, a supershift complex was observed
only with GM-CSF-stimulated nuclear extracts and not with unstimulated
extracts (Fig. 1A). Similar results were seen using
extracts from TF-1 cells stimulated with IL-3, except that a weaker
supershift was observed with anti-phosphorylated CREB antibody (Fig. 1B). We demonstrated previously that CREB
constitutively binds the CRE in the egr-1 promoter (12). CREB
associates with the CRE from a number of heterologous promoters. Our
data confirm these findings (Fig. 1, A and B, Complex III) and suggest that GM-CSF or IL-3 induces
phosphorylation of CREB on serine 133 within 10 min of stimulation of
cells.
GM-CSF- and IL-3-activated pathways may also diverge in the
phosphorylation of specific downstream kinases that can then
phosphorylate CREB at different sites. Previous studies have
demonstrated that CA
In
addition to neuropeptides, peptide growth factors such as fibroblast
growth factor that activate receptor tyrosine kinases also mediate
activation of CREB by a Ras-dependent pathway(16, 34) .
Ginty et al.(16) demonstrated that NGF stimulation of
PC12 cells resulted in phosphorylation of CREB on serine 133 by a novel
CREB kinase. There is further evidence that kinases other than protein
kinase A are capable of activating CREB. The serine/threonine kinase,
p90
The unique role of CREB
phosphorylation in transcriptional activation of target genes by GM-CSF
and IL-3 adds a new perspective to the overlapping but distinct
biological functions of these two cytokines. There may be subtle
differences in the interactions between the GM-CSF and the IL-3
signaling pathways, since both ligands interact with a common receptor
We thank Gayle Baldwin, Judith Gasson, and Karen Yates
for helpful suggestions and critical reading of the manuscript; Anne
O'Shea-Greenfield for technical assistance and Wendy Aft for
preparation of the manuscript. We appreciate the generosity of David
Ginty, who provided the anti-phosphorylated CREB antibody and offered
many helpful suggestions. We are grateful to Larry Souza (Amgen), who
provided rhGM-CSF, and Steve Gillis (Immunex), who provided IL-3.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit required for signal
transduction. Our previous studies have demonstrated that GM-CSF and
IL-3 activate signaling pathways which converge upon a cAMP response
element-binding protein (CREB)-binding site of the human immediate
early response gene (early growth response gene-1, egr-1)
promoter. Using electromobility supershift assays and antibodies
directed against CREB phosphorylated on serine 133, we show that CREB
is phosphorylated on serine 133 in response to GM-CSF or IL-3
stimulation. We demonstrate that phosphorylation of CREB on serine 133
substantially contributes to transcriptional activation of egr-1 in response to GM-CSF but not IL-3. These studies suggest that
phosphorylation of CREB may play different roles during signal
transduction, resulting in unique and overlapping biological functions
in myeloid cells.
(
)and interleukin 3 (IL-3) share many biological
functions which may be mediated by a common receptor
subunit
critical for transmission of signals from the cell surface to the
nucleus of myeloid cells(1, 2, 3, 4) .
It has been shown that GM-CSF and IL-3 exhibit overlapping activities
throughout myeloid cell development; however, the precise mechanisms
leading to the biological activities elicited by these two growth
factors are still unclear (1-3).
-interferon response region (4, 8-10). Recently, GM-CSF and
IL-3 were demonstrated to induce phosphorylation of a novel STAT
protein, STAT5, in the hematopoietic cell line OTT-1(11) . These
findings suggest significant redundancy in the signaling pathways
activated by GM-CSF and IL-3.
Reagents and Cytokines
Antibodies for CREB
(polyclonal antibodies) were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). Anti-phosphorylated CREB, raised against a CREB
peptide containing phosphoserine 133, was generously provided by Dr.
David Ginty(16) . Anti-rabbit IgG was purchased from Sigma. The
enhanced chemiluminescence (ECL) detection kit was obtained from
Amersham Corp. Recombinant human GM-CSF (rhGM-CSF) was provided by
Amgen (Thousand Oaks, CA), and human IL-3 was provided by Immunex
(Seattle, WA). The CREB and mutant CREB (serine 133 mutated to alanine)
constructs were gifts from Dr. M. Montminy(22) . We subcloned
the wild-type and mutant CREBs into the BC12 vector, which contains the
cytomegalovirus (CMV) promoter for transient co-transfection
assays(23) .
Cell Culture and Transfections
TF-1 cells were
cultured in RPMI with 10% fetal calf serum, L-glutamine (2
mM), and penicillin (100 units/ml streptomycin, 100 µg/ml)
at a ratio of 1 unit/ml to 1 µg/ml and either rhGM-CSF (500
pM) or rhIL-3 (500 pM) in nonadherent tissue culture
plates. For transient transfection experiments, cells were factor- and
serum-starved for 24 h and placed in RPMI, 0.5% bovine serum albumin
(BSA) (Sigma). A total of 5 10
cells/sample were
transfected by electroporation at 200 mV with 45 µg of total DNA
(20 µg of egr-1 promoter construct -116cat, 22
µg of wild-type or mutant CREB, and 3 µg of CMV
-galactosidase-containing plasmid). Cells grown in GM-CSF or IL-3
were resuspended in RPMI, 0.5% BSA and stimulated with 500 pM of rhGM-CSF, rhIL-3, or diluent control (phosphate-buffered saline
(PBS), 0.02% BSA) for 4 h, at which time stimulation of the recombinant
construct is maximal (data not shown). Cells were harvested and assayed
for chloramphenicol acetyltransferase or
-galactosidase activity
as described previously(12) . The amount of acetylated and
unacetylated [
C]chloramphenicol was determined
by liquid scintillation counting, and -fold stimulation by GM-CSF or
IL-3 was corrected for transfection efficiency with the luciferase or
-galactosidase assay (Promega). Statistical analysis
(Student's t test) was performed using the STATWORKS
program.
Electromobility Shift Assays and Supershift
Experiments
TF-1 cells (5 10
) cultured in
either GM-CSF or IL-3 were factor- and serum-starved for 24 h and
stimulated for 5 min. Cells were lysed by sonication in buffer with
protease and phosphatase inhibitors, as described
previously(24) . Two microliters of cell lysate were added to
the gel shift reaction. The probe (0.1 ng) CRE from the human egr-1 promoter between nucleotides -57 and -76 was used. A
complementary single-stranded oligonucleotide probe to CRE was
synthesized with an Applied Biosystems synthesizer. The probe was
prepared as described previously (12) and incubated with 2
µl of extract from unstimulated cells or IL-3- or GM-CSF-stimulated
cells. One microgram of poly(di-dc) was added per reaction.
Western and Immunoblotting
TF-1 cells (4
10
) were factor- and serum-starved for 24 h prior to
stimulation. Cells in RPMI and BSA 0.5% were then stimulated for 10 min
with diluent (PBS + BSA, 0.5%), rhGM-CSF (1 nM), rhIL-3
(1 nM), or TPA (50 ng/ml). Cells were lysed in boiling SDS
buffer and boiled again for 5 min, as described previously(16) .
The total cell lysate after centrifugation was loaded on a 10%
SDS-polyacrylamide gel and blotted to nitrocellulose (Hybond-ECL). The
blot was probed with antibody against phosphorylated CREB (0.14
µg/ml). For time course experiments, 4
10
cells
were washed three times with PBS and factor- and serum-starved for 24 h
prior to stimulation. Cells were stimulated with rhIL-3 (1 nM)
or rhGM-CSF (1 nM) for 0, 2, 5, 10, or 15 min or with TPA (50
ng/ml) for 10 min. Cells were lysed with boiling SDS and boiled again
for 5 min(16) . Total cell lysate was loaded onto a 10% SDS-PAGE
and blotted to nitrocellulose in duplicate. The blot was probed with
rat polyclonal anti-CREB antibody (1:7500) or anti-phosphorylated CREB
antibody (0.14 µg/ml).
IL-3 and GM-CSF Activation of Signaling Pathways Induce
Phosphorylation of CREB on Serine 133 in TF-1 Cells
GM-CSF and
IL-3 stimulation of TF-1 cells leads to rapid and transient induction
of egr-1. In transfection assays, deletion of the CRE between
nucleotides -57 and 76 in the context of the surrounding 116
nucleotides results in the abolition of GM-CSF- or IL-3-induced egr-1 expression(12) . The CRE is therefore required
for transcriptional activation of egr-1. CREB binds the CRE of
the egr-1 promoter in TF-1 nuclear extracts from both
unstimulated and GM-CSF- or IL-3-stimulated cells.
Figure 1:
Electromobility shift assays with CRE
probe and TF-1 cells stimulated with GM-CSF or IL-3. A labeled
oligonucleotide probe (0.1 ng) containing the CRE and sequences between
nucleotides -57 to -76 of the egr-1 promoter was
incubated with extracts from TF-1 cells stimulated for 10 min with
diluent control (.02% BSA and PBS) (A and B), 1
nM rhGM-CSF (A), or 1 nM rhIL-3 (B). Total cell lysates from 5 10
cells/sample were prepared by a sonication method. Extracts were
incubated with no competitor (lanes 1 and 6),
500-fold excess specific cold competitor (lanes 2 and 7), no antibody (lanes 3 and 8), anti-CREB
antibody (lanes 4 and 9), and anti-phosphorylated
CREB (lanes 5 and 10). Complex I represents
a supershift complex with anti-CREB antibody, Complex II is
with anti-phosphorylated CREB antibody, and Complex III is a
specific complex. Results were confirmed in three independent
experiments.
We performed Western analysis with TF-1 cell extracts at 10
min post-stimulation with GM-CSF, IL-3, or TPA. A 43-kDa protein was
seen with anti-phosphorylated CREB antibody in response to GM-CSF and
IL-3 (see Fig. 2), and the strongest signal was observed with
TPA. TPA has been previously demonstrated to activate CREB by a protein
kinase C-dependent pathway(15) . We found that the signal in
response to IL-3 stimulation was weaker than that to GM-CSF or TPA
stimulation. These results are consistent with the electromobility
shift assay in which IL-3-stimulated nuclear extracts demonstrated a
weaker supershift complex compared with extracts from GM-CSF-stimulated
cells (Fig. 1B). A lower molecular weight protein also
appears to be phosphorylated in response to GM-CSF, IL-3, or TPA. This
38-kDa band was observed in several experiments and could represent a
short half-life of CREB during IL-3 stimulation or cross-reactivity
with another member of the CREB/ATF family of transcription factors.
The antibody directed against a phosphorylated CREB peptide also
recognizes ATF-1.(
)This observation has been
previously described by Ginty et al.(24) and Hummler et al.(25) . These data suggest that several
independent pathways (i.e. protein kinase A-dependent or
-independent and PKC-dependent pathways) could potentially activate
CREB through phosphorylation on serine 133.
Figure 2:
Western blot with phosphorylation of CREB
on serine 133 induced by GM-CSF, IL-3, or TPA in TF-1 cells. TF-1 cells
(4 10
cells/sample) were starved for 24 h and
stimulated with GM-CSF or IL-3 (1 nM) or stimulated with TPA
(50 ng/ml) for 10 min. Total cell lysates were prepared by the boiling
SDS method, separated on a 10% SDS-PAGE, transferred to nitrocellulose
membranes, and immunoblotted with anti-phosphorylated CREB antibody.
The arrow represents the 43-kDa phosphorylated CREB.
Experiments were repeated and confirmed three
times.
To examine the kinetics
of CREB phosphorylation, we performed a Western analysis at different
time points following GM-CSF or IL-3 stimulation of TF-1 cells.
Although the levels of CREB are constant (see Fig. 3A),
phosphorylation of CREB is seen at 2 min following GM-CSF but not IL-3
stimulation. In contrast, CREB phosphorylation occurs 5-10 min
following IL-3 stimulation. The CREB protein may be less stable in the
presence of IL-3 compared with GM-CSF. The subtle difference in the
time course of CREB phosphorylation suggests that GM-CSF and IL-3
signaling pathways possibly exhibit different kinetic properties which
may contribute to the specificity of their biological activities. Other
proteins with molecular masses of 70, 65, and 35 kDa that appear in
Western blots (Fig. 3, A and B) most likely
represent other cross-reacting proteins or members of the CREB/ATF
family of transcription factors(26, 27, 28) .
Figure 3:
Time
course of CREB phosphorylation in TF-1 cells stimulated with GM-CSF (A) or IL-3 (B). TF-1 cells (4 10
cells/sample) were starved and stimulated with GM-CSF (A) or IL-3 (B) for 0, 2, 5, 10, or 15 min or with
TPA for 10 min. Cells were harvested and lysed with boiling SDS, and
lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose
membranes, and immunoblotted with anti-CREB antibody (left) or
anti-phosphorylated CREB antibody (right). The arrow represents the 43-kDa CREB or phosphorylated CREB. Results were
confirmed in three separate experiments.
In myeloid cells, other examples of differences in the kinetics of
protein phosphorylation following GM-CSF or IL-3 stimulation have been
described. Phosphorylation of the STAT protein, pp93, occurs within
minutes in response to GM-CSF stimulation, whereas phosphorylation in
response to IL-3 was slightly delayed(29) . In addition,
GM-CSF-induced activation of at least one other protein of similar
mobility that was recognized by the phosphorylated STAT91 antibody was
not detected following IL-3 stimulation(29) . Our data are
consistent with these findings and demonstrate that phosphorylation of
CREB, such as with pp93, may represent a delayed response downstream of
the IL-3 signaling pathway. An alternative explanation is that there is
delayed assembly of a functional receptor complex in target cells
stimulated with IL-3 in comparison with cells stimulated with
GM-CSF(30) .
Phosphorylation of CREB on Serine 133 Is Required for
Transcriptional Activation of egr-1 in Response to GM-CSF but Not
IL-3
We examined whether rapid phosphorylation of CREB in TF-1
cells was necessary for transcriptional activation by GM-CSF or IL-3 by
performing co-transfection assays with the -116cat construct and
wild-type or mutant CREB (serine 133 mutated to alanine). When
wild-type CREB or vector is transfected with the -116cat
construct, 3-4-fold stimulation was observed (p <
0.05; see Fig. 4). The -fold induction represents the percent
acetylation of chloramphenicol acetyltransferase using extracts from
GM-CSF- or IL-3-stimulated cells divided by diluent control. Since the
mutation of serine 133 to alanine abrogates egr-1 induction by
GM-CSF, we conclude that CREB phosphorylation on serine 133 plays a
significant role in GM-CSF-induced egr-1 expression. The -fold
stimulation with wild-type CREB was slightly less than vector in every
experiment, which may be due to a squelching effect from the presence
of both endogenous and exogenous CREB, an observation that has been
made by others(31) . When the mutant CREB was co-transfected
with -116cat, there was a statistically significant decrease in
the -fold induction by GM-CSF (p < 0.05; see Fig. 4),
but not IL-3 (p = 0.226; see Fig. 4). These data
suggest that phosphorylation of CREB on serine 133 contributes to
IL-3-induced transcriptional activation of egr-1 but is not
critical. All experiments (n = 3-5) were
performed in triplicate, and the CMV -galactosidase plasmid was
transfected into TF-1 cells as the internal control. Although not
statistically significant, the percent acetylation of diluent control
from TF-1 cells cultured in IL-3 was 2-3-fold of the percent
acetylation of diluent control in GM-CSF-cultured cells (data not
shown).
Figure 4:
Phosphorylation of serine 133 on CREB is
required for GM-CSF- but not IL-3-induced transcriptional activation of egr-1. TF-1 cells (10) were factor- and
serum-starved for 24 h and placed in serum-free media. Twenty
micrograms of reporter construct -116cat and 22 µg of
wild-type or mutant CREB were electroporated into TF-1 cells and
stimulated with diluent control (.02% BSA in PBS), GM-CSF (1
nM), or IL-3 (1 nM) for 4 h. Three micrograms of CMV
-galactosidase plasmid was co-transfected as the internal control
for transfection efficiency. Chloramphenicol acetyltransferase or
-galactosidase assays were performed. -Fold induction by GM-CSF (A) or IL-3 (B) represents percent acetylation of
constructs stimulated with GM-CSF or IL-3, divided by percent
acetylation of constructs stimulated with diluent control. p values were determined by paired t test analysis. Data
represent three to five experiments, with each transfection performed
in triplicate.
These results demonstrate that phosphorylation of CREB on
serine 133 substantially contributes to the transcriptional activation
of egr-1 by GM-CSF, but not by IL-3, supporting the hypothesis
that phosphorylation events of previously existing transcription
factors such as CREB mediate signaling pathways of GM-CSF and IL-3 in
myeloid cells. Although CREB constitutively binds the CRE in the egr-1 promoter, phosphorylation of serine 133 on CREB most
likely results in a conformational change leading to its association
with the transcriptional machinery and induction of egr-1. As
other phosphorylation sites may be modified in response to IL-3, this
would explain the difference between GM-CSF and IL-3 in their
requirements for CREB phosphorylation on serine 133. One possibility
may be that GM-CSF and IL-3 might quantitatively differ in their
abilities to phosphorylate this site. Alternatively, the CREB protein
may be less stable in IL-3-cultured cells. Furthermore, the
stoichiometries of CREB may be different in cells grown in IL-3 than
those in GM-CSF. This would be consistent with the weaker supershift
band with anti-phosphorylated CREB antibody seen in IL-3-stimulated
cells (Fig. 1B). Similarly, in the Western analysis (Fig. 2), the 43-kDa band representing phosphorylated CREB is
weaker following IL-3 stimulation than after GM-CSF stimulation.
/calmodulin-dependent protein
kinase, type 2 stoichiometrically phosphorylates CREB on serine 133 in
addition to a second site in CREB(32) . More recently,
differential activation of CREB by
Ca
/calmodulin-dependent protein kinases types II and
IV has been shown to result in the phosphorylation of a site that
negatively regulates CREB activity(33) . A similar difference in
the activation of CREB may result from GM-CSF and IL-3 stimulation of
myeloid cells. Alternately, other proteins binding to upstream egr-1 promoter elements may be required in addition to CREB to
maximally activate egr-1 induction in response to IL-3.
, was shown to phosphorylate CREB on serine
133 in melanocytes stimulated with various growth factors(35) .
Although NGF and other growth factors have been recently found to
mediate phosphorylation of CREB on serine 133 by a protein kinase
A-independent pathway, the kinases responsible for CREB phosphorylation
by GM-CSF or IL-3 have not been identified. It is possible that GM-CSF
or IL-3 may activate CREB through kinases such as p90
or a novel CREB kinase.
subunit. The specificity of pathways might be initially triggered
by the unique GM-CSF or IL-3
subunits that bind ligand.
Consequently, the kinases or phosphatases which associate with the
subunits may be responsible for the regulation of CREB
activation, thus conferring specificity in the induction of other
target genes containing a critical cAMP response element. The present
studies suggest that GM-CSF and IL-3 modify transcription factors by
potentially distinct mechanisms, indicating that identification of
kinases responsible for CREB activation will be critical in
understanding signaling pathways regulating myeloid cell proliferation
and differentiation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.