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J. Biol. Chem., Vol. 275, Issue 31, 24032-24039, August 4, 2000
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,,§, and
From the Department of Molecular Pharmacology,
University Medical Center, SUNY/Stony Brook, Stony Brook, New York
11794-8651 and the ¶ Department of Physiology and Biophysics,
Diabetes and Metabolic Diseases Research Program, University
Medical Center, SUNY/Stony Brook, Stony
Brook, New York 11794-8661
Received for publication, March 30, 2000, and in revised form, May 9, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Differentiation of P19 embryonal carcinoma cells
in response to the morphogen retinoic acid is regulated by
G A central role for heterotrimeric G-protein-mediated signaling in
cell proliferation, differentiation, and apoptosis has been established
(1). Various G-proteins have been shown to regulate features of
cellular differentiation. G The P19 embryonal carcinoma cells provide a useful model for murine
pre-implantation development (16, 17). Three germ layers, endoderm,
mesoderm, and ectoderm, can be derived from P19 cells through the use
of appropriate culture conditions and an inducer(s) (18). Retinoic acid
stimulates P19 cells to differentiate into primitive endoderm, as
defined by the loss of the embryonic antigen SSEA-1 and positive
staining with the TROMA antibody, specific for the cytokeratin endo A
protein that is the hallmark of the endodermal phenotype (19).
RA-induced differentiation is accompanied by the expression of
G Cell Culture and Differentiation--
The P19 embryonal
carcinoma cells were purchased from the American Type Culture
Collection (Manassas, VA). Both the stable transfectants and the
wild-type clones were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) in a
humidified atmosphere of 6% CO2. P19 cells cultured as
monolayers on tissue culture plates in the Dulbecco's modified
Eagle's medium with 10% serum were induced to primitive endoderm by
the addition of 10 nM all-trans retinoic acid (Sigma) for 2 to 4 days (21).
Plasmids and Stable Transfection--
The pCMV5 plasmids
harboring no insert (i.e. empty vector), the wild-type (WT),
N-terminal-truncated, constitutively active forms (CA), and
kinase-inactive (substitution of methionine for lysine in the ATP
binding domain) dominant-negative (DN) mutant forms of MEKK1 and MEKK4
(24) were provided generously by Dr. Gary L. Johnson (Department of
Pharmacology, University of Colorado, Denver, CO). Each expression
vector for a mutant form of MEKK1 or MEKK4 was co-transfected with pCW1
empty vector harboring the neomycin resistance gene at a ratio of 5:1
for pCMV5:pCW1. The P19 cells were transfected with the plasmids using
LipofectAMINE (Life Technologies, Inc.). Stably transfected P19 clones
were selected in the presence of the neomycin analog G418 (400 µg/ml). The wild-type and mutant forms of the MEKK1 and MEKK4 had
been epitope-tagged with the hemaglutinin antigen (HA) in order to follow expression of each independent of endogenous MEKKs.
Immunoblotting--
Samples (100 µg of protein/lane) of total
cell lysates were subjected to electrophoresis in SDS on 10%
polyacrylamide gels. The resolved proteins were transferred
electrophoretically to nitrocellulose blots. The blots were stained
with primary antibodies, and the immune complexes were made visible by
the electro-chemiluminescence kit (NEN Life Science Products) in tandem
with a second antibody coupled with alkaline phosphatase. The HA tag
was stained with a polyclonal antibody purchased from BABCO (Berkeley,
CA). Blocking experiments were performed by preincubation of the HA
antibody (1:500 final dilution) with a molar excess (2.5 µg/ml) of
the HA peptide (YPYDVPDYA), as recommended by the commercial supplier. The HA peptide and the JNK1 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The antibodies used to stain MEKK1 were
rabbit polyclonal antibodies raised either to the C-terminal 22 residues of the rat sequence (C-22) or to the N-terminal 1-301 residues of the same (43Y). The antibodies used to stain MEKK4 were
rabbit polyclonal antibodies raised against the N-terminal 19 residues
of the rat sequence (D19). These three antibodies were obtained also
from Santa Cruz Biotechnology.
Immunoprecipitation of MEKKs and JNK and Activity
Assays--
The immunoprecipitation reactions and solid-state assay of
MEKKs and JNK were performed as detailed earlier (20, 25), using the
rGST-JNKK1 and rGST-c-Jun N-terminal fusion protein as substrates for
the MEKKs and JNK activity assays, respectively. The vectors expressing
the rGST-c-Jun and rGST-JNKK1 were provided generously by Dr. Roger
Davis (Howard Hughes Medical Institute, University of Massachusetts
Medical Center, Worcester, MA)
Indirect Immunofluorescence Staining of SSEA-1 and
TROMA--
The staining of the embryonic antigen SSEA-1 with mAb
MC-480 and the endoderm-specific marker antigen cytokeratin endo A by the monoclonal antibody TROMA was performed with antibodies purchased from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa
City, IO). The P19 cells were cultured, stained, and subjected to
analysis by epifluorescence microscopy as described previously (21). As
the differentiated cells often grow from monolayers to whorls of cells
with multiple layers, the indirect immunofluorescence and phase
contrast images may not appear to be "in focus." This artifact is
unavoidable under these conditions of differentiation.
Data Analysis--
For all of the experiments reported, the data
are compiled from at least three independent, replicate experiments
performed on separate cultures on separate occasions with highly
reproducible results. The indirect immunofluorescence and phase
contrast images are of representative fields of interest.
Rat embryonic carcinoma P19 cells were stably transfected with the
expression vectors harboring WT, CA, and DN mutant forms of either
MEKK1 or MEKK4 (23). Endogenous MEKK1 and MEKK4 were identified in
immunoblots of extracts of P19, mouse embryonic fibroblast 3T3-L1, and
human epidermoid carcinoma cells subjected first to SDS-PAGE (Fig.
1A). MEKK1 is subject to ready
proteolysis with a terminal, long lived fragments displaying molecular
masses of ~100 kDa and ~115 kDa, as observed in P19 and 3T3-L1
cells. The full-length form is observed in the blot of A431 cells shown here. MEEK4, likewise, is cleaved to a terminal, long lived fragment displaying a molecular mass of ~100 kDa in blots prepared from P19,
3T3-L1, and A431 cells. Thus, both MEKK1 and MEKK4 are present in P19
cells, and both kinases are sensitive to proteolytic cleavage. These
cleavages of MEKK family members are believed to be a normal and
necessary processing step in their biological activation (26).
12/13 and is associated with activation of c-Jun
N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun
N-terminal kinase was investigated in P19 cells. P19 clones stably
expressing constitutively active and dominant negative mutants of MEKK1
and MEKK4 were created and characterized. Expression of the
constitutively active form of either MEKK1 or MEKK4 mimicked the action
of retinoic acid, inducing these embryonal carcinoma cells to primitive
endoderm. Expression of the dominant negative form of MEKK1 had no
influence on the ability of retinoic acid to induce either JNK
activation or primitive endoderm formation in P19 stem cells.
Expression of the dominant negative form of MEKK4, in contrast,
effectively blocks both morphogen-induced activation of JNK and
cellular differentiation. These data identify MEKK4 as upstream of
c-Jun N-terminal kinase in the pathway mediating differentiation of P19
stem cells to primitive endoderm.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s suppresses adipogenesis of
the NIH 3T3-L1 embryonal fibroblasts, which can be relieved by loss of
Gs in response to inducers of differentiation and mimicked with oligodeoxynucleotides antisense to Gs (2).
Activation of Gs via cholera intoxication, in contrast,
blocks induction of adipogenesis (2). Overexpression of wild-type
Gi2 or its Q205L constitutively activated mutant can
induce differentiation in these 3T3-LI cells (3). Expression of the
highly abundant G-protein subunit Go has been shown to
be obligate for induction of growth cones in developing neurites of
nerve growth factor-treated PC12 cells (4, 5). For Gi2,
which suppresses phospholipase C activity in the F9 teratocarcinoma
cells (6), morphogens stimulate a decline in Gi2, which
de-represses phospholipase C (7) and stimulates the generation of
inositol phosphates and diacyglycerol, which in turn activates protein
kinase C (8). The activation of protein kinase C and the
ERK1/21 members of the
mitogen-activated protein kinase family is the basis for
morphogen-induced differentiation of these totipotent F9 cells (9).
Expression of wild-type or a lesser amount of the constitutively
activated Q205L mutant form of Gi2 blocks induction of
F9 cell differentiation (10). Retroviral infection with a vector
expressing RNA antisense to Gi2, but not one expressing RNA antisense to either Go or Gs,
provokes a decline in Gi2 and differentiation to
primitive endoderm in the absence of morphogen (7). Heterotrimeric
G-proteins have been implicated as mediators of early stages of
vertebrate development in frogs and zebrafish (11-13). Finally,
several inherited diseases that alter the expression or activity of
heterotrimeric G-protein subunits display profound effects on human
development (14, 15).
13 and activation of JNK, but neither ERK1/2 or p38
members of the mitogen-activated protein kinase family (20, 21).
Expression of the constitutively active mutant form of
G13 activates JNK and induces P19 clones to
differentiate into primitive endoderm in the absence of RA induction.
In addition, expression of the dominant negative form of JNK1 blocks
the ability of the morphogen to induce the cells to primitive endoderm
(20). In an effort to elucidate the linkage from G13 to
JNK activation in P19 cell differentiation, we explored the upstream
control of JNK activation, focusing on the potential roles of two
mitogen and extracellularly activated protein kinase kinases, MEKK1 and MEKK4 (22, 23). The results provide compelling evidence that MEKK4
mediates the signaling from retinoic acid to JNK and differentiation in
these embryonic carcinoma cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Expression of wild-type, constitutively
active, and dominant-negative forms of MEEK1 and MEKK4 in P19 embryonal
carcinoma cells. A, whole-cell extracts were prepared
from P19 embryonal carcinoma cells, mouse embryonal fibroblast 3T3-L1
cells, and human epidermoid carcinoma A431 cells. The extracts (0.1 mg
of protein/lane) were subjected to SDS-PAGE and
immunoblotting (IB). The blots were stained with antibodies
to detect endogenous MEKK1 (lanes 1-4) and MEKK4
(lanes 5-7). The blots were stained with two different
antibodies to MEKK1, one an N-terminal epitope (lane 1), the
others stained with an antibody to a C-terminal epitope (lanes
2-4). B, whole-cell lysates of P19 clones stably
transfected with expression constructs harboring WT, CA, or DN forms of
MEKK1 or MEKK4, or the empty vector was subjected to SDS-PAGE and
immunoblotting. All forms of the MEKKs were tagged N-terminally with
the HA peptide. The blots were stained with antibody specific for the
HA antigen (1:500). C, whole-cell lysates of P19 clones
stably transfected with expression constructs harboring WT forms of
MEKK1 or MEKK4, or the empty vector was subjected to SDS-PAGE and
immunoblotting. Both forms of the MEKKs were tagged with the HA
antigen. The blots were stained with antibody specific for the HA
antigen without ( 
) or with prior addition (2 h at room temperature)
of the HA blocking peptide (2.5 µg/ml) to the antibody solution
(1:500), as suggested by the commercial supplier before staining of the
blot. D, whole-cell lysates were prepared from the cells
overexpressing versions of WT, CA, and DN forms of MEKK1 and
MEKK4 that were tagged N-terminally with the HA peptide. The
overexpressed proteins were immunoprecipitated from the cell lysates
using an antibody to HA tag. Solid-phase kinase assay was performed on
the immunoprecipitates using the recombinant GST-JNKK1 protein. The
phosphorylation products were analyzed on a 10% acrylamide gels by
SDS-PAGE. The gel was stained, dried, and subjected to
autoradiography.
HA-tagged forms of MEKK1 WT and CA and DN mutant forms were stably expressed, and extracts of these clones were subjected to immunoblotting with antibodies specific for the HA epitope (Fig. 1B). Much like that observed for the endogenous MEKKs, MEKK1 and MEKK4 wild-type and mutant forms were subject to proteolytic cleavage to a limit digest of ~100 kDa. Multiple clones were prepared and characterized for stable transfection of each construct. The immunoblotting reveals similar levels of expression for wild-type and mutant forms of MEKK1 and for those of MEKK4, although the level of expression for MEKK1 was consistently greater than that observed for MEKK4. The identity of the stained species of wild-types MEKK1 and MEKK4 as HA-tagged was confirmed by use of the HA-blocking peptide (Fig. 1C). In the presence of the HA antigen, the staining of the HA-tagged MEKK1 and MEKK4 forms was virtually abolished. Similar experiments performed with the HA-tagged mutant versions of either MEKK displayed the same sensitivity to blocking by the HA antigen peptide (data not shown).
The various forms of MEKK1 and MEKK4 expressed in P19 cells were examined for their ability to phosphorylate a common substrate. The HA-tagged mutant MEKKs were immunoprecipitated from whole-cell lysates from P19 clones, and their ability to catalyze the phosphorylation of the substrate, rGST-JNKK1 protein, was evaluated for multiple clones from each of the stable transfections (Fig. 1D). The results of representative assays reveal that the expression of the DN forms of either MEKK resulted in a blunted phosphorylation of rGST-JNKK1 in comparison with that of extracts from cells expressing the WT forms of each MEKK. Expression of the constitutively activated forms of MEKK1 and MEKK4 provoked a sharp increase in the phosphorylation of their downstream substrate, JNKK1.
Assay of JNK activity in the whole-cell extracts of the clones
expressing WT, CA, or DN forms of MEKK1 using a GST-c-Jun fusion protein as a read-out confirms the increased activation of JNK1 in the
clones overexpressing WT-MEKK1 or expressing the CA-MEKK1 (Fig.
2A). JNK1 activity increased
~3-fold in the clones overexpressing CA-MEKK1 as well as in the
clones expressing WT-MEKK1. The P19 clones expressing the DN form of
MEKK1 displayed a low level of JNK1 activity, similar to that of the
clones harboring the empty expression vector. Most interestingly,
expression of CA-MEKK1 was sufficient to induce differentiation of P19
cells to an endodermal phenotype in the absence of the morphogen RA, as
evidenced by positive staining with the TROMA antibody (Fig.
2B). The positive staining of P19 clones expressing CA-MEKK1
was similar to that of the P19 clones transfected with empty vector
alone that had been induced to differentiate to primitive endoderm with
RA. The expression of the WT-MEKK1 also induced the P19 clones to
differentiate and stain positive for TROMA. In sharp contrast to the
clones expressing either WT-MEKK1 or CA-MEKK1, those expressing
DN-MEKK1 display no positive staining for TROMA. Staining of the SSEA-1 embryonic antigen was negative for clones expressing WT-MEKK1 or
CA-MEKK1 and positive for the clones expressing the DN-MEKK1 (not
shown).
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The effects of addition of RA to the clones expressing the CA form of
MEKK1 on JNK activation and on the differentiation process was examined
(Fig. 3). RA alone stimulates a 2-3-fold
increase in the activity of JNK1, as measured using the GST-c-Jun
fusion protein as a substrate. The expression of the CA form of MEKK1 yields about the same level of activation of JNK1. Challenging the
clones that express the CA-MEKK1 with the morphogen RA failed to
increase the level of JNK1 activity beyond that observed with either
CA-MEKK1 expression or RA treatment alone, suggesting that the effects
were not additive. In each of the trials, RA treatment of the clones
expressing CA-MEKK1 resulted in a modest (<20%) decline in JNK1
activity. Companion studies of indirect immunofluorescence microscopy
of the clones expressing the CA form of MEKK1 reveal the absence of the
SSEA-1 antigen and the strong, positive staining of the endodermal
marker antigen with TROMA for both the clones expressing CA-MEKK1 in
the absence or presence of RA (Fig. 3B). These data agree
well with the measurements of JNK1 activity and suggest that the
effects for RA and expression of CA-MEKK1 in the differentiation of the
P19 cells are non-additive in nature.
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In view of the ability of overexpression of MEKK1 or expression of the
CA-MEKK1 to induce differentiation much like RA does, the effects of
expression of the DN form of MEKK1 on the ability of RA to induce
differentiation was investigated (Fig.
4). Measurements of JNK1 activity using
the GST-c-Jun fusion protein as a substrate show the ability of the
DN-MEKK1 to suppress JNK activity in the cells unchallenged with
morphogen while not preventing the ability of RA to induce a 4-fold
activation of JNK1 (Fig. 4A). RA induces a loss in staining
of SSEA-1 and an increase in TROMA staining in the clones harboring the
expression vector, which is "empty" (EV), in good
agreement with the results obtained with wild-type P19 cells.
Interestingly, the expression of the dominant negative form of MEKK1
failed to influence the ability of RA to differentiate the P19 clones
to primitive endoderm. The clones expressing DN-MEKK1 lost SSEA-1
staining and displayed positive staining by TROMA in response to RA.
Although overexpression of WT-MEKK1 and expression of CA-MEKK1 can
cause the P19 stem cells to differentiate, mimicking the effects of RA
itself, MEKK1 does not appear to play an obligate role in the signaling
of this process.
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Of the other major upstream regulator of JNKs (23), MEKK4 was explored
for its possible role in RA-induced differentiation of P19 cells.
Analysis of the JNK1 activity of the whole-cell extracts from clones
expressing WT and CA forms of MEKK4 reveals a 2.5-fold and a 4.0-fold
increase in JNK1 activity, respectively, using the GST-c-Jun fusion
protein as a substrate for phosphorylation. The measurements of JNK1
activity agree well with the phenotypic changes in the clones
overexpressing WT-MEKK4 or expressing CA-MEKK4 (Fig.
5A). The clones expressing the
DN mutant of MEKK4 displayed low JNK1 activity, as predicted from the
dominant negative nature of the MEKK4 construct. Much like the effects
of overexpression of the wild-type form and expression of the
constitutively active form of MEKK1, the analogous forms of MEKK4 were
found to provoke differentiation of P19 clones (Fig. 5B).
Based upon positive staining of the endodermal marker TROMA, clones
overexpressing WT-MEKK4 and CA-MEKK4 differentiate in the absence of
the morphogen RA, displaying cell morphologies and staining patterns
not unlike the P19 clones harboring the empty vector upon treatment
with RA. The clones expressing the DN form of MEKK4, in sharp contrast to those expressing either WT- or CA-MEKK4, displayed no TROMA staining.
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The clones expressing the CA-MEKK4 were challenged with RA in an effort
to ascertain if the differentiation process was sensitive to the
morphogen (Fig. 6). The measurements of
JNK1 activity reveal a 2.0-fold increase in phosphorylation of the
GST-c-Jun fusion protein with samples from the P19 clones harboring the
empty vector following challenge with RA (Fig. 6A). The
expression of CA-MEKK4 was accompanied by a 3.5-fold increase in JNK1
activity, correlating well with the differentiation of these cells to
primitive endoderm. When the clones expressing the CA-MEKK4 were
challenged with RA, the amount of JNK1 activity displayed a
reproducible, modest decline. This modest decline in JNK1 activity in
response to RA for the clones expressing CA-MEKK4 was similar to the
decline also noted in the clones expressing CA-MEKK1 following
challenge with RA. Constitutively active MEKK4 provokes differentiation
to primitive endoderm and prominent staining by the TROMA antibody.
SSEA-1 staining is absent in the clones expressing CA-MEKK4, confirming the loss of the embryonic state and differentiation to endoderm (Fig.
6B). When treated with RA, the clones expressing CA-MEKK4 appeared much like the clones not exposed to RA, with the phenotypic morphology and prominent staining with TROMA.
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It was critical to define the effects of the expression of the DN form
of MEKK4 on the ability of the P19 cells to respond to RA. Unlike the
effects observed in the clones expressing the DN form of MEKK1, the
expression of the DN-MEKK4 was associated with a loss in the ability of
the morphogen both to activate JNK and to induce differentiation of the
P19 clones (Fig. 7). Expression of the DN
form of MEKK4 potently suppressed JNK activity, well below the ambient
levels of JNK in the clones harboring the empty vector and in the
absence of morphogen (Fig. 7A). Clones expressing the
dominant negative form of MEKK4 display positive staining with
antibodies for SSEA-1 embryonic antigen (Fig. 7B),
indicating that the clones maintained an embryonic phenotype, even in
the face of a challenge with the morphogen RA. TROMA staining of the clones expressing DN-MEKK4 was negative, also reflecting the embryonic phenotype of these clones made resistant to the actions of RA through
loss of MEKK4-mediated signaling.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Heterotrimeric G-proteins play a pivotal role in control of
differentiation and development (1). Ample examples have accumulated providing a compelling linkage between specific G-protein subunits and
the ability of cells to proliferate, differentiate, and apoptose. Gs acts as a repressor to adipogenesis in a manner that
does not involve the activation of adenylylcyclase (2, 27). Suppression of Gs by classical inducers or by treatment with
antisense oligodeoxynucleotides leads to adipogenesis (2).
Go is expressed at high levels in the leading edge of
neurite growth cones (4, 5), which collapse when Go
levels are suppressed by antisense oligodeoxynucleotides (28).
Expression of the constitutively activated form of Go promotes a doubling in neurite outgrowth in PC12 cells (29). Gi2 is critical in the differentiation of F9
teratocarcinoma stem cells to primitive endoderm (7). Overexpression of
Gi2 blocks the ability of RA to induce differentiation
(9, 10). RA provokes a rapid transient decline in Gi2, which when
mimicked by antisense RNA also leads to differentiation in the absence of the morphogen (8).
In the current work we extended earlier studies that demonstrated a
role for G12/13 in the differentiation of P19 embryonal carcinoma cells (21). The expression of G12/13 increased
dramatically in response to RA in these cells as well as in early mouse
development (21). Expression of constitutively activated
G13 provokes differentiation of P19 cells and downstream
activation to the level of mitogen-activated protein kinases (20). JNK,
but neither ERK1/2 nor p38 kinases, are activated in response either to
RA or to expression of the constitutively active G13. In
addition, expression of DN-JNK1 was able to block differentiation of
these cells to RA (20). Prominent upstream regulators of JNK include
MEKK1 and MEKK4 (23). We demonstrate the ability of overexpression of
the wild-type MEKK1 and MEKK4 or their constitutively active mutant
forms to provoke a robust differentiation of the P19 cells in the
absence of RA. The effects of the CA forms of either MEKK1 or MEKK4 on differentiation of P19 cells was not enhanced further by the addition of RA, suggesting action upon a pathway common to the differentiation.
More revealing were the investigation of the ability of DN forms of
MEKK1 and MEKK4 to influence the action of RA in these stem cells.
Expression of DN-MEKK1 was without effect. Although the DN-MEKK1
displayed the ability to reduce the basal level of JNK activity in the
P19 cells, it failed to block the ability of RA to stimulate activation
of JNK as well as the ability of RA to block primitive endoderm
formation. Despite the fact that the CA mutant form of MEKK1 can both
activate JNK and differentiation, these data provide compelling
evidence that MEKK1 is not a dominant mediator of RA-induced JNK
activation in the pathway controlling cellular differentiation.
Expression of DN-MEKK4, in sharp contrast, blocks the ability of RA to
induce activation of JNK as well as to induce differentiation of P19
cells to primitive endoderm. Interestingly, the level of expression of
the MEKK1 was uniformly greater than MEKK4, as measured by staining for
the HA antigen common to both. Thus, the inability of DN-MEKK1 to block
RA-induced activation of JNK and differentiation cannot be ascribed to
insufficient levels of expression. These studies suggest a central role
for MEKK4 in the ability of RA to induce cellular differentiation via
JNK. Despite the ability of both CA-MEKK1 and CA-MEKK4 to promote
differentiation, only the DN form of MEKK4 specifically blocks the
induction of differentiation in response to RA. The ability of the
DN-MEKK4 to block RA-induced JNK activation and formation of primitive
endoderm might suggest that other upstream regulators of JNK, such as
MEKK2, MEKK3, and the mixed-lineage group of MEKKs (MLK) (30), do not
play a dominant role in RA action to the level of JNK activation or
cellular differentiation in P19 cells. The molecular basis by which the
DN-MEKK4 blocks the action of retinoic acid on cellular
differentiation, however, may include a variety of mechanisms,
including competition for scaffold proteins as well as for molecules
downstream and/or upstream of MEKKs. Further studies will be required
to delineate more precisely the role of MEKK4 and the linkage between
G13 and MEKK4 in this model of early mouse development.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gary L. Johnson (Department of Pharmacology, University of Colorado, Denver, CO) and Roger J. Davis (Program in Molecular Medicine & Howard Hughes Medical Institute, University of Massachusetts Medical Center, Worcester, MA) for invaluable reagents employed in these studies.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant DK30111 (NIDDK, National Institutes of Health).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: Pharmacology-HSC, SUNY/Stony Brook, Stony Brook, NY 11794-8651. Tel.: 631-444-7873; Fax: 631-444-7696; E-mail: craig@pharm.som.sunysb.edu.
Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M002747200
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ABBREVIATIONS |
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The abbreviations used are: ERK, extracellular signal-regulated kinase; CA, constitutively active; DN, dominant negative, JNK, c-Jun N-terminal kinase; JNKK, JNK kinase; MEKK, mitogen and extracellularly activated protein kinase kinase; RA, retinoic acid; SSEA-1, stem cell surface embryonal antigen-1 marker; WT, wild type; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; rGST, recombinant GST.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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