A Potential Role for Protein Kinase C-
in Regulating
Megakaryocytic Lineage Commitment*
Frederick K.
Racke
,
Dongyan
Wang§,
Zan
Zaidi
,
Joshua
Kelley§,
Jane
Visvader¶,
Jae-Won
Soh
, and
Adam N.
Goldfarb§**
From the
Department of Pathology, Johns Hopkins
Medical Institutions, Baltimore, Maryland 21287, the
§ Department of Pathology, University of Virginia Health
Sciences Center, Charlottesville, Virginia 22908, the ¶ Walter and
Eliza Hall Institute of Medical Research, Royal Melbourne Hospital,
Victoria 3050, Australia, and the
Herbert Irving Comprehensive
Cancer Center, Columbia College of Physicians and Surgeons, New York,
New York 10027
Received for publication, June 16, 2000, and in revised form, September 29, 2000
 |
ABSTRACT |
Multiple studies have shown that intracellular
signal transduction by the protein kinase C (PKC) family participates
in the initiation of megakaryocyte differentiation. In this study,
multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel
PKCs, in particular PKC-
. In transfection studies, the isolated
variable domain of PKC-
selectively blocked exogenous activation of
the megakaryocyte-specific
IIb promoter. Constitutively active
mutants of PKC-
, but not of other PKC isozymes, cooperated with the
transcription factor GATA-1 in the activation of the
IIb promoter.
The functional cooperation between GATA-1 and PKC-
displayed
dependence on cellular milieu, as well as on the promoter context of
GATA binding sites. In aggregate, the data suggest that PKC-
specifically participates in megakaryocytic lineage commitment through
functional cooperation with GATA-1 in the activation of megakaryocytic promoters.
 |
INTRODUCTION |
A role for protein kinase C
(PKC)1 signaling in
megakaryocytic differentiation has been established by numerous
experiments over the past two decades. In early studies, the PKC
agonist phorbol diester selectively enhanced megakaryocyte colony
formation by primary mouse bone marrow cells (1). More recent studies
using primary human progenitors confirmed the promegakaryocytic effects of phorbol ester and showed such effects to be inhibitable by the PKC
antagonists GF-109203X and Ro-31-8220 (2). In numerous cell line
models of megakaryocytic differentiation, PKC activation induced an
array of features including the following: cell cycle arrest, secretion
of megakaryocytic cytokines, up-regulation of megakaryocytic surface
antigens, cellular enlargement, polyploidization, development of
proplatelet processes, and appearance of demarcation membranes (3,
4).
The PKC serine/threonine kinase family consists of at least 11 distinct
isozymes organized into three subgroups, based on biochemical,
pharmacologic, and structural properties (5). The classical or
conventional PKCs (cPKCs) require diacylglycerol and
Ca2+ for activation and consist of the
,
I,
II,
and
isozymes. The novel PKCs (nPKCs) require only diacylglycerol
for activation and consist of the
,
,
,
, and µ isozymes.
The atypical PKCs lack responsiveness to diacylglycerol and
Ca2+ and consist of the
and
isozymes. Striking
functional differences exist among PKC isozymes, with divergent
functions noted even for factors with high structural homology
(6-8)
PKC signaling may influence megakaryocytic differentiation through
several isozymes. In K562 cells, PKC-mediated, sustained activation of
the Raf-MEK-ERK signaling pathway is necessary for initiation of
megakaryocytic differentiation (9, 10). Multiple PKC isozymes, in
particular
,
I,
, and
, possess the capacity to activate
the Raf-MEK-ERK pathway (11, 12). However, activation of the
Raf-MEK-ERK pathway appears not to be required for megakaryocytic differentiation of primary progenitor cells, suggesting that PKC signaling plays an additional role in megakaryopoiesis independent of
ERK activation (13).
To examine the contribution of specific PKC isozymes to megakaryocyte
differentiation, we initially employed isozyme-selective pharmacologic
agents in two independent cell line models of megakaryocytic differentiation, K562 and HEL. GF-109203X, an inhibitor of cPKCs and
nPKCs but not of atypical PKCs, potently blocked megakaryocytic induction in both cell lines. By contrast, Gö 6976, an inhibitor only of cPKCs (14), failed to block megakaryocytic differentiation, suggesting a specific requirement for nPKC signaling. For both cell
lines, the PKC-
-selective agonist, ingenol 3,20-dibenzoate (IDB)
(15-17) induced megakaryocytic differentiation, as well as selective
nuclear translocation of PKC-
.
In transfection assays, the isolated variable domain of PKC-
, but
not that of PKC-
, completely blocked exogenous activation of the
megakaryocytic
IIb promoter. Constitutively active mutants of
PKC-
activated the
IIb promoter 3-6-fold. We also addressed whether PKC-
signaling influenced the function of GATA-1, a
transcription factor known to play a critical role in megakaryopoiesis
and in activation of the
IIb promoter (18-20). Indeed, GATA-1 and
constitutively active PKC-
showed synergistic activation of the
IIb promoter. Notably, the ability to synergize with GATA-1
distinguished PKC-
from other PKC isozymes, depended on cellular
milieu, and depended on the context of GATA binding sites within the promoter.
 |
MATERIALS AND METHODS |
Cell Culture--
K562 and HEL, obtained from the ATCC,
were grown in RPMI 1640 with 10% fetal bovine serum at 37 °C, 5%
CO2. C3H10T1/2, obtained from ATCC, was grown in
Dulbecco's modified Eagle's medium with 10% neonatal calf serum at
37 °C, 5% CO2. HEK-293T, provided by Dr. Kevin Lynch
(Department of Pharmacology, University of Virginia School of
Medicine), was grown in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum at 37 °C, 5% CO2. All experiments using K562 and HEL employed mid-log phase cells at a density of 0.5-1.0 × 106 cells/ml. Conditioned media was
obtained as described previously by 72 h of treatment of either
K562 or HEL cells with 25 nM
12-O-tetradecanoylphorbol-13-ester (Sigma) followed by
harvesting and dialysis of supernatant (9).
For megakaryocytic induction, cells were resuspended in conditioned
media and incubated at 37 °C, 5% CO2 for 1-3 days, as indicated. The compounds GF-109203X, Gö 6976, and IDB were
purchased from LC Laboratories. The PKC inhibitors GF-109203X and
Gö 6976 were added, at indicated concentrations, to conditioned
media at the initiation of megakaryocytic induction.
Flow Cytometry--
Staining of cells for surface CD41 employed
the fluorescein isothiocyanate-conjugated antibody PLT1-FITC (Coulter)
at 25 µg/ml. Staining of cells for surface glycophorin A employed the
phycoerythrin-conjugated antibody GA-R2-PE (Pharmingen) at 10 µg/ml.
Appropriate fluorochrome-conjugated, isotype-matched antibody controls
were used at concentrations identical to the corresponding experimental
antibodies. Flow cytometric analysis was performed on a FACScan system
utilizing Lysys II software (Becton Dickinson).
Immunofluorescent Staining for PKC--
Treated cells were
cytospun onto glass slides and fixed for 2 min in ice-cold methanol
followed by 2 min in ice-cold acetone. After blocking for 30 min at
room temperature with 1% normal goat serum in phosphate-buffered
saline, primary antibodies in 0.1% normal goat
serum/phosphate-buffered saline were applied for 1 h at room
temperature. Murine monoclonal antibodies to PKC-
(Santa Cruz
Biotechnology, Santa Cruz, CA) and to PKC-
(Transduction Laboratories, Lexington, KY) were used at 200 ng/ml and at 1.25 µg/ml, respectively. Control murine antibody (NOR 3.2;
BIOSOURCE International) was used at 1 µg/ml.
Secondary antibody, consisting of phycoerythrin-conjugated goat
anti-mouse (Tago, Inc.) diluted 1:100 in 0.1% normal goat
serum/phosphate-buffered saline, was applied for 30 min at room
temperature. For nuclear visualization, 4,6-diamidino-2-phenylindole
was included in the coverslip mounting medium. Cells were visualized by
confocal laser scanning fluorescence microscopy on a Zeiss LSM
410 (Jena, Germany) using Zeiss LSM analysis software.
Plasmid Constructs--
The
IIb-luciferase reporter
constructs were made by polymerase chain reaction amplification of
bases
598 to +32, bases
98 to +32, or bases
348 to +32 from a
human
IIb promoter fragment kindly provided by Dr. Samuel Santoro
(21). The polymerase chain reaction products were co-digested with
XhoI plus HindIII and ligated into the
corresponding sites of pGL3-Basic (Promega). The
-galactosidase
expression vector consisted of pCMV
(CLONTECH). The GATA-1 expression vector employed the EF-1-
-neo expression plasmid and has been previously described (22).
Mammalian expression of a full-length constitutively active (CA) mutant
of PKC-
employed SRD-
-K155A/R156A/A159E (AE3), kindly provided by
Dr. S. Ohno (23). Expression of a full-length CA mutant of PKC-
employed SRD-
R144/145A (DRA), also kindly provided by Dr. S. Ohno
(23). Expression of a full-length CA mutant of PKC-
employed
pRc-CMV-PKC-
A25E, kindly provided by Dr. Gottfried Baier (24).
Expression of a full-length CA mutant of PKC-
employed pRc/CMV-PKC-
R145I/R146W, kindly provided by Dr. J. Anthony Ware (25). Mammalian expression constructs for HA-epitope-tagged, isolated
PKC catalytic and regulatory domains have been previously described
(26). In brief, fragments encoding the catalytic domains of
(amino
acids 326-672) and
(amino acids 395-737) were ligated into
the pHANE vector. The fragment encoding the catalytic domain of
(amino acids 334-674) was ligated into the pHACE vector. Fragments
encoding the variable domains of
(amino acids 2-325) and
(amino acids 2-394) were ligated into the pHANE vector.
Transfections and Reporter Assays--
Transfection of K562 and
C3H10T1/2 cells employed the liposomal reagent DOTAP (Roche Molecular
Biochemicals) using ~6 µg of DNA in 200 µl of HBS (20 mM HEPES, 150 mM NaCl, pH 7.4) combined with 30 µl of DOTAP in 200 µl HBS. The DNA/DOTAP mixture was incubated for
10 min at room temperature and was then added dropwise to 1. 6 × 106 K562 cells in 2 ml of RPMI 1640 with 5% fetal bovine
serum. For C3H10T1/2 cell transfections, the DNA/DOTAP mixture was
added to ~60% confluent cells in 6-well plates. Transfection of
HEK-293T cells was performed as described for C3H10T1/2 cells except
that 4 µg of DNA in 50 µl of HBS was combined with 20 µl of DOTAP
in 100 µl of HBS.
After overnight incubation, cells were changed to fresh complete
medium. Cells were subsequently incubated 24 h prior to harvesting for luciferase and
-galactosidase assays. Luciferase assays were performed using the commercial luciferase assay system (Promega), and
-galactosidase assays were performed using the
O-nitrophenyl
-D-galactopyranoside
(Sigma) colorimetric substrate. All transfections were performed at
least in triplicate, and all luciferase values were normalized
according to
-galactosidase readings.
Immunoblot Assays--
K562, C3H10T1/2, and HEK-293T cells were
transfected and harvested as for the luciferase and
-galactosidase
assays, except that whole cell lysates were prepared by resuspending
cells in 1× SDS polyacrylamide gel electrophoresis loading buffer.
Samples were resolved by SDS polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. Equivalent lane loading was
confirmed by Ponceau staining of membranes. Probing of membranes was
carried out as described previously (27). For detection of HA-tagged PKC regulatory and catalytic domains, the primary antibody consisted of
12CA5, a murine monoclonal directed to the HA epitope tag, employed as
ascites fluid diluted 1:2000. The secondary antibody consisted of
peroxidase-conjugated goat anti-mouse (Sigma) used at a dilution of
1:3000. For detection of GATA-1, the primary antibody consisted of the
rat monoclonal N6 (Santa Cruz Biotechnology) at a final concentration
of 0.4 µg/ml. The secondary antibody consisted of
peroxidase-conjugated goat anti-rat (Sigma) used at a dilution of
1:5000. Signal detection employed enhanced chemiluminescence.
 |
RESULTS |
Pharmacologic Implication of nPKC in Megakaryocytic
Differentiation--
Previous work in our laboratory indicated that
sustained activation of the Raf-MEK-ERK pathway in the K562
hematopoietic cell line resulted in production of autocrine factors
promoting megakaryocytic maturation (9). To identify signaling pathways
triggered by such autocrine factors, we analyzed the effects of
pharmacologic inhibitors on megakaryocytic induction in two independent
cell lines, K562 and HEL. The induction stimulus consisted of
conditioned media from
12-O-tetradecanoylphorbol-13-ester-treated HEL cells, which
show identical activity to that previously reported for K562 cells
(9).
The only compound in our screening that potently and specifically
blocked megakaryocytic induction by conditioned media was GF-109203X.
GF-109203X inhibits both cPKC and nPKC isozymes, as well as
pp90rsk2 (14, 28). Therefore, parallel experiments were
carried out using the compound Gö 6976, known to inhibit cPKCs
and pp90rsk2 but not nPKCs (14, 28). As shown in the flow
cytometric profiles in Fig. 1,
conditioned media alone induced up-regulation of the megakaryocyte
surface antigen CD41 and down-regulation of the erythroid surface
antigen glycophorin A. Whereas GF-109203X completely blocked both
responses, Gö 6976 at similar doses showed no inhibition of
either response.

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Fig. 1.
Effects of the PKC inhibitory compounds,
GF-109203X and Gö 6976, on megakaryocytic differentiation of the
K562 and HEL cell lines. Cells were exposed for 72 h to
conditioned medium either alone or containing the indicated agents at a
concentration of 3 µM. Cells were stained with the
indicated fluorochrome-conjugated antibodies and analyzed by flow
cytometry. The hollow profiles represent uninduced cells,
and the shaded profiles represent conditioned medium-induced
cells. This experiment was performed on three separate occasions, each
yielding similar results. GPA, glycophorin A.
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Analysis of cellular morphology supported the flow cytometric results
in Fig. 1. In particular, HEL cells exposed to conditioned media
undergo spreading and enlargement as part of their megakaryocytic differentiation. As shown in Fig. 2, the
morphologic changes induced in HEL cells by conditioned media were
abrogated by GF-109203X. By contrast, Gö 6976 strikingly enhanced
the cellular spreading and enlargement induced by conditioned media.
These data confirm that biologically active doses of Gö 6976 acted to enhance rather than inhibit features of megakaryocytic
differentiation.

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Fig. 2.
Effects of the PKC inhibitors, GF-109203X and
Gö 6976, on the morphologic differentiation of HEL cells.
Cells were induced by treatment for 48 h with conditioned medium
alone or with the indicated agents at 10 µM. Cells were
visualized by phase contrast microscopy at a magnification of × 200. This experiment was performed on three separate occasions, each
yielding similar results.
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In an alternative approach, the isozyme-selective PKC agonist IDB was
applied directly to K562 and HEL cells in standard growth media.
Multiple previous studies have indicated that IDB is a selective
activator of nPKCs, particularly PKC-
(15-17). As shown in Fig.
3, IDB caused CD41 up-regulation and
glycophorin A down-regulation in K562 and HEL cells. In addition, HEL
cells treated with IDB manifested the standard morphologic changes seen
with megakaryocytic induction (not shown). Immunofluorescent staining
(Fig. 4) showed that treatment of K562
cells with IDB induces rapid nuclear translocation of PKC-
but no
change in the subcellular localization of PKC-
. Rapid nuclear
translocation of PKC-
was also observed in HEL cells treated with
IDB (data not shown). Thus, both agonists and antagonists implicate
nPKC, in particular PKC-
, in the induction of megakaryocytic
differentiation.

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Fig. 3.
Induction of megakaryocytic differentiation
with the nPKC-selective agonist IDB. Cells were treated for 4 days
with 100 nM IDB prior to analysis by flow cytometry.
Hollow profiles indicate control cells treated with
Me2SO carrier, and shaded profiles
indicate cells treated with IDB. This experiment was performed on three
separate occasions, each yielding similar results. GPA,
glycophorin A.
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Fig. 4.
Immunofluorescent subcellular localization of
PKC- and PKC- in K562
cells, untreated and treated with IDB. Cells were treated
for 10 min at 37 °C with or without 100 nM IDB.
Following stimulation, cells were fixed and stained as described under
"Materials and Methods." The images represent the following:
Left column, blue staining indicates
4,6-diamidino-2-phenylindole staining of nuclei. Middle
column, anti-PKC immunoreactivity indicated by red
staining. Right column, composite image superimposing
left and middle columns demonstrating nuclear
localization of PKC- following IDB treatment. This experiment was
performed on three separate occasions, each yielding similar
results.
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Blockade of Megakaryocytic Promoter Activation by the Regulatory
Domain of PKC-
--
The amino-terminal regulatory sequences of
PKCs, when expressed as isolated fragments, function as
dominant-negative PKC inhibitors (29, 30). Accordingly, we examined
whether transfection of isolated regulatory domains from either PKC-
or PKC-
could interfere with exogenous activation of the
megakaryocyte-specific
IIb-598 promoter (31). In cells transfected
with control vector, 24 h of conditioned media caused an
~14-fold up-regulation of the
IIb-598-luciferase reporter activity
(Fig. 5A). Expression in cells
of the isolated
regulatory fragment (
-Reg) minimally inhibited
conditioned media activation of
IIb-598-luciferase. In striking
contrast, expression in cells of the isolated
regulatory fragment
(
-Reg) almost completely eliminated responsiveness to the
conditioned media stimulus. Immunoblotting showed similar expression
levels of the HA-epitope-tagged
and
regulatory domains in
HEK-293T transfectants (Fig. 5B).

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Fig. 5.
A, the effects of PKC regulatory
domains (Reg) on exogenous activation of the megakaryocytic
IIb promoter. Control vector or expression vectors for the or
regulatory domain (3 µg) were cotransfected with the
IIb-598-luciferase reporter plasmid (1 µg), as well as a
-galactosidase expression vector (1 µg), into K562 cells. Cells
were then subjected to 24 h of induction with conditioned media.
Results are shown as -fold increase in luciferase activity relative to
uninduced cells transfected with control vector. All results are
normalized by -galactosidase activity and represent the mean of
three experiments ± S.E. B, immunoblot analysis of
HEK-293T transfectants showing expression of the HA-epitope-tagged PKC
regulatory domains. Positions of molecular mass
markers are indicated to the left of the blot; from
top to bottom they are as follows: 97, 66, 55, 43, 40, and 31 kDa. IB, immunoblot.
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Specific Cooperation of PKC-
with GATA-1 in Activation of a
Megakaryocytic Promoter--
We next tested whether constitutively
active PKC mutants could activate the
IIb megakaryocytic promoter.
Fig. 6A demonstrates the
similar results obtained with two different types of constitutively active mutants, inhibitory domain point mutants (CA mutants in left graph), and isolated PKC catalytic domains (CAT mutants
in right graph). The inhibitory domain point mutants consist
of full-length PKCs with point mutations in the autoinhibitory
regulatory domains (23-25). The isolated PKC CAT completely lack
regulatory domains, which have also been implicated in Ca2+
and lipid binding, interaction with RACKs, and kinase-independent signaling (26). Using the megakaryocyte-specific
IIb-598 reporter, we tested the effects of PKC isozymes alone and in conjunction with
GATA-1, a known positive regulator of the
IIb promoter (18, 19). As
shown in Fig. 6A, constitutively active PKC-
mutants alone modestly activated the
IIb promoter (3-6-fold) but
demonstrated clear functional cooperation with GATA-1. In fact,
coexpression of GATA-1 with either CA or CAT mutants of
led to
levels of reporter activation analogous to those obtained with
conditioned media induction. Functional cooperation with GATA-1 was
clearly isozyme-restricted in that constitutively active mutants of
,
, and
all failed to augment GATA-1-mediated
IIb
activation. In fact,
caused a 2-3-fold inhibition of GATA-1
activation. Immunoblot analysis demonstrated equivalent levels of
GATA-1 expression in all transfectants, indicating that the
differential effects of the PKC isozymes were not because of
differences in GATA-1 levels (Fig. 6B). In addition,
immunoblot demonstrated analogous expression levels of the
HA-epitope-tagged
,
, and
catalytic domains in HEK-293T cells
(Fig. 6C).

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Fig. 6.
Functional cooperation of
PKC- with GATA-1 in the activation of the
megakaryocytic IIb promoter.
A, activity of constitutively active PKC mutants ± GATA-1. CA- represents PKC- K155A/R156A/A159E;
CA- represents PKC- R144/145A; CA-
represents PKC- R145I/R146W; and CA- represents
PKC- A25E. Isolated PKC catalytic domains are designated as
CAT. B, immunoblot (IB) analysis of
transfectants for GATA-1 expression. Positions of molecular
mass markers are indicated to the left of the
blot; from top to bottom they are as
follows: 97, 66, 55, 43, 40, and 31 kDa). C, immunoblot
analysis of HEK-293T transfectants showing expression of the
HA-epitope-tagged PKC catalytic domains. Molecular mass markers are
designated as in B. K562 cells were transfected with 2 µg
of PKC expression vector (or parent vector), 2 µg of GATA-1
expression vector (or parent vector), 2 µg of the
IIb-598-luciferase reporter plasmid, and 0.5 µg of
-galactosidase expression vector. Results are shown as -fold
increase in luciferase activity relative to cells transfected with
reporter plasmid plus control vectors. All results are normalized by
-galactosidase activity and represent the mean of three
experiments ± S.E.
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Cellular Context and Promoter Context Influence PKC-
/GATA-1
Cooperativity--
To determine whether functional interaction between
PKC-
and GATA-1 occurred also in non-hematopoietic cells,
cotransfections were carried out in C3H10T1/2 fibroblasts rather than
in K562 hematopoietic cells. The full-length CA
mutant, as
well as the isolated
CAT, failed to augment GATA-1-mediated
IIb
activation in C3H10T1/2 fibroblasts (Fig.
7A). Immunoblotting
demonstrated equivalent expression of GATA-1 in all of the
transfectants (Fig. 7B). Thus, the functional interaction of
PKC-
with GATA-1 clearly depends on the cell type employed for
transfection.

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Fig. 7.
Influences of cellular milieu and promoter
context on PKC- /GATA-1 cooperativity.
A, C3H10T1/2 fibroblasts were transfected with 2 µg of
GATA-1 expression vector (or parent vector), 2 µg of PKC-
expression vector (or parent vector), 2 µg of the IIb-598 reporter
plasmid, and 0.5 µg of -galactosidase expression vector.
CA- represents the constitutively active regulatory
domain point mutant PKC- K155A/R156A/A159E.
-CAT represents the constitutively active
isolated PKC- catalytic domain. B, immunoblot
(IB) analysis of transfectants for GATA-1 expression.
Positions of molecular mass markers are indicated to the
left of the blot; from top to bottom
they are as follows: 97, 66, 55, 43, 40, and 31 kDa). C,
K562 cells were transfected with the IIb-598 reporter plasmid or
with the 5' truncated reporter plasmids IIb-98 and IIb-348. In
addition, the cells received the constitutively active mutants of
PKC- (versus parent vector) and wild type GATA-1 (or
parent vector). Results are shown as -fold increase in luciferase
activity relative to cells transfected with reporter plasmid plus
control vectors. All results are normalized by -galactosidase
activity and represent the mean of three experiments ± S.E.
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The
IIb promoter contains multiple GATA binding sites, including a
functional site within the promoter-proximal
98 fragment (18, 19). To
determine whether specific promoter regions were required for
PKC-
/GATA-1 cooperativity, 5' truncated reporter constructs,
IIb-98 and
IIb-348, were compared with
IIb-598 for
responsiveness to PKC-
± GATA-1 in K562 cells. Surprisingly, the
IIb-98 and
IIb-348 reporters showed full activation by GATA-1 alone but no evidence of augmentation by PKC-
(Fig. 7C).
Thus, the functional GATA binding sites in the
IIb
348 to +32
fragment were insufficient to mediate PKC-
/GATA-1 cooperativity.
 |
DISCUSSION |
Involvement of PKC signaling in hematopoietic lineage commitment
decisions has been well documented. In progenitors transformed by the
E26 avian leukemia virus, thresholds of PKC activity correlated with
cell fate determinations as follows: (a) no kinase activity was
associated with undifferentiated cells; (b) low activity was associated
with myelomonocytic differentiation; and (c) high activity was
associated with eosinophil differentiation (32). In primary, bipotential granulocyte macrophage colony-forming cells, activation of
PKC-
induced commitment to the macrophage lineage (33). Similarly,
our data suggest that signaling via PKC-
may promote megakaryocytic
lineage commitment of the bipotential BFU-E/MK progenitor, a cell with
capability for either erythroid or megakaryocytic differentiation (34).
Previous studies have notably shown that PKC-
undergoes
down-regulation during erythroid differentiation and that inhibition of
PKC-
specifically enhances erythroid differentiation (35, 36).
Mechanisms by which PKC-
signaling might contribute to the
activation of the megakaryocytic
IIb promoter remain unclear. Earlier studies with isolated catalytic domains have shown PKC-
to
activate multiple pathways that converge on the serum-response element
of the c-fos promoter, c-Raf-MEK1-ERK, MEK kinase
1-stress-activated protein kinase kinase-c-Jun
NH2-terminal kinase, and rhoA (26). However, those studies
showed equivalent activation of the various pathways by the PKC-
catalytic domain. Our results, by contrast, show no activation of the
IIb promoter by the PKC-
catalytic domain. The rapid nuclear
translocation of PKC-
observed with megakaryocytic induction by IDB
(Fig. 4) raises the possibility that PKC-
itself might act directly
upon critical nuclear substrates.
The functional cooperation of PKC-
with GATA-1 raises a number of
mechanistic possibilities. One scenario is that PKC-
signaling targets a transcriptional complex containing GATA-1 and enhances GATA-1
function by phosphorylation of one of the members of this complex, such
as GATA-1 itself or the cofactor friend of GATA-1. The absence
of functional interaction in C3H10T1/2 cells argues against direct
phosphorylation of GATA-1 by PKC-
as a sufficient mechanism. This
scenario also fails to account for the dependence of PKC-
signaling
on promoter context, as illustrated in Fig. 7C. Accordingly
another possibility is that PKC-
signaling targets GATA-1 complexes
binding to specific regions of the
IIb promoter. A recent study
employing embryonic stem cell hematopoiesis has defined within the
human
IIb promoter a 200-base pair critical enhancer region,
398
to
598, that is necessary and sufficient for megakaryocyte-specific
transgene expression (31). Interestingly, our data indicate that a
similar region (from
348 to
598 of the human
IIb promoter) is
required for responsiveness to PKC-
signaling. Future studies will
attempt to correlate PKC-
response elements within the
IIb
promoter with megakaryocyte-specific enhancer function.
A major question in the molecular characterization of hematopoietic
lineage commitment is how two lineages with highly similar arrays of
transcription factors can show non-overlapping, indeed mutually
exclusive, patterns of gene expression. Erythroid and megakaryocytic
cells share expression of the highly restricted factors GATA-1,
GATA-2, Lmo2, NF-E2, friend of GATA-1, and SCL/tal. Most striking among
these factors is GATA-1, which dominantly activates erythroid genes
only in erythroblasts and dominantly activates megakaryocytic genes
only in megakaryocytes. Our current data raise the possibility that
isozyme-specific signaling by PKC may modify GATA function in
accordance with promoter context. In particular, PKC-
signaling
might specifically augment GATA-1 function in the context of
megakaryocytic promoters, thereby redirecting the entire
transcriptional program of a cell from erythroid to megakaryocytic.
 |
ACKNOWLEDGEMENTS |
We thank Kristine Lewandowska for
excellent technical assistance in the early phases of this project. For
generously providing plasmid constructs, thanks go to
Drs. Samuel Santoro, Gottfried Baier, J. Anthony Ware, and
S. Ohno. For helpful discussions and support, thanks go to
Drs. Chi V. Dang, Isa Hussaini, and Julianne J. Sando.
 |
FOOTNOTES |
*
This work was supported in part by the Concern II Foundation
(to A. N. G.), Public Health Service Grant CA-72704 from the NCI,
National Institutes of Health (to A. N. G.), the Johns Hopkins Solo
Cup Clinician Scientist Award (to F. K. R.), and Public Health Service Grant HL-04017 from the NHLBI, National Institutes of Health
(to F. K. R.).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: Dept. of Pathology, HSC
Box 800214, University of Virginia Health Sciences Center, Charlottesville, VA, 22908. Tel.: 804-982-0593; Fax: 804-924-8060; E-mail: ang3x@virginia.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M005236200
 |
ABBREVIATIONS |
The abbreviations used are:
PKC(s), protein kinase C(s);
cPKC(s), classical or conventional PKC(s);
nPKC(s), novel PKC(s);
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
ERK, extracellular
signal-regulated kinase;
IDB, ingenol 3,20-dibenzoate;
HEK, human
embryonic kidney;
CA, constitutively active;
HA, hemagglutinin;
DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
salts;
CAT, catalytic domains;
HEL, human erythroleukemia.
 |
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